Volume 41 Number 2
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Kasetsart Journal : Natural Science April - June 2007 Volume 41 Number 2
April - June 2007
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Wanchai Chanprasert
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Faculty of Agriculture, Kasetsart University, Thailand
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Associate Professor, Cell and Molecular Biology,
Faculty of Science, Kasetsart University, Thailand
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Roberto Bonoan
Researcher, Department Manager, Soil Science,
National Tobacco Administration, Department of
Agricualture, Philippines
Korchoke Chantawarangul
Assistant Professor, Civil Engineering, Faculty of
Engineering, Kasetsart University, Thailand
Parnjit Damrongkulkamjorn
Assistant Professor, Electrical Engineering, Faculty of
Engineering, Kasetsart University, Thailand
Praparat Hormchan
Associate Professor, Entomology, Faculty of
Agriculture, Kasetsart University, Thailand
Kanapol Jutamanee
Associate Professor, Plant Physiology, Faculty of
Science, Kasetsart University, Thailand
Onanong Naivikul
Professor, Cereal Chemistry and Technology, Faculty of
Agro-Industry, Kasetsart University, Thailand
Eiji Nawata
Associate Professor, Tropical Agriculture, Kyoto
University, Japan
Saran Petpiroon
Associate Professor, Marine Ecology and Pollution,
Faculty of Fisheries, Kasetsart University, Thailand
Witoon Prinyawiwatkul
Professor, Food Science and Technology, Louisiana State
University and LSU Agricultural Center, USA
Hathairat Rimkeeree
Assistant Professor, Product Development, Faculty of
Agro-Industry, Kasetsart University, Thailand
U Ravi Sangakkara
Professor, Crop Ecology, Faculty of Agriculture,
University of Peradeniya, Sri Lanka
Uthaiwan Sangwanit
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Kasetsart University, Thailand
Thongchai Suwonsichon
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Aree Thankijjanukij
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Management, Kasetsart University
Satoru Tsuchikawa
Assistant Professor, Near Infrared Spectroscopy and
Agricultural Science, Graduate School of
Bioagricultural Sciences, Nagoya University, Japan
Sirichai Wongnarkpet
Assistant Professor, Veterinary Epidemiology, Faculty
of Veterinary Medicine, Kasetsart University, Thailand
Kenji Yamane
Associate Professor, Floriculture and Horticulture,
Faculty of Agriculture, Utsunomiya University, Japan
MANAGING EDITORS
Orawan Wongwanich
Senior Researcher, Kasetsart University Research and
Development Institute, Kasetsart University, Thailand
Somporn Maneeprasopsuk
Researcher, Kasetsart University Research and
Development Institute, Kasetsart University, Thailand
EDITORIAL ADVISORY BOARD
Gerald T. Baker
Professor, Entomology, Mississippi State University, USA
A. Bruce Bishop
Professor, Civil and Environmental Engineering, Utah State
University, USA
Samakkee Boonyawat
Associate Professor, Forest Resource Management, Faculty of
Forestry, Kasetsart University, Thailand
Delores Chambers
Assistant Professor, Food Science, College of Human Ecology,
Kansas State University, USA
Edgar Chambers
Professor, Food Science, College of Human Ecology, Kansas State
University, USA
Angsumarn Chandrapatya
Professor, Entomology, Faculty of Agriculture, Kasetsart University,
Thailand
Mauricio A. Elzo
Professor, Animal Breeding and Genetics, University of Florida,
USA
John Hampton
Professor, Seed Science and Technology, Lincoln University,
New Zealand
Parichat Hongsprabhas
Assistant Professor, Food Science, Faculty of Agro-Industry,
Kasetsart University, Thailand
Sathaporn Jittapalapong
Associate Professor, Veterinary Parasitology, Faculty of Veterinary
Medicine, Kasetsart University, Thailand
Helen H. Keenan
Professor, Environmental Science, University of Strathclyde,
Scotland
Saichol Ketsa
Professor, Postharvest Technology, Faculty of Agriculture,
Kasetsart University, Thailand
Jumras Limtrakul
Professor, Physical Chemistry, Faculty of Science, Kasetsart
University, Thailand
Chitochi Miki
Professor, Structural Engineering, Tokyo Institute of Technology,
Japan
Larry Miller
Professor, Agricultural Education College of Food, Agriculture and
Environment, Ohio State University, USA
Tadashi Miyata
Professor, Entomology, Nagoya University, Japan
Punpiti Piamsa-nga
Assistant Professor, Computer Engineering, Faculty of Engineering,
Kasetsart University, Thailand
Wiroj Rujopakarn
Professor, Transportation Engineering, Faculty of Engineering,
Kasetsart University, Thailand
Ed Sarobol
Associate Professor, Crop Physiology, Faculty of Agriculture,
Kasetsart University, Thailand
Narongrit Sombatsompop
Professor, Polymer Processing, School of Energy and Materials,
King Mongkut’s University of Technology Thonburi, Thailand
Peerasak Srinives
Professor, Plant Breeding, , Faculty of Agriculture at Kamphaeng
Saen, Kasetsart University, Thailand
Rungsit Suwanmankha
Professor, Weed Science, Faculty of Agriculture, Kasetsart
University, Thailand
Chanvit Vajrabukka
Professor, Animal Science, Physiology, Animal Behavior, Faculty
of Agriculture, Kasetsart University, Thailand
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KASETSART JOURNAL
NATURAL SCIENCE
The publication of Kasetsart University
VOLUME 41
April - June 2007
NUMBER 2
Changing in TSS, TA and Sugar Contents and Sucrose Synthase Activity in Ethephon-Treated
‘Pattavia’ Pineapple Fruit
...................... Ngarmnij Chuenboonngarm, Niran Juntawong, Arunee Engkagul,
.............................................................. Wallop Arirob and Surin Peyachoknakul
Phylogenetic Analysis of Thai Amomum (Alpinioideae: Zingiberaceae) Using AFLP Markers
............. Wittaya Kaewsri, Yingyong Paisooksantivatana, Uamporn Veesommai,
............................................................. Wichan Eiadthong and Srunya Vajrodaya
Prediction of Sweet Corn Seeds Field Emergence under Wet Soil Condition
............................................ Vichai Wongvarodom and Wikanate Rangsikansong
Modifying Controlled Deterioration for Evaluating Field Weathering Resistance of Soybean
............................................. Ye Changrong, Prapa Sripichitt, Sunanta Juntakool,
.................................................................... Vipa Hongtrakul and Arom Sripichitt
Composite Line Method for the Development of Early Generation Hybrids
of Maize (Zea mays L.)
................. Nguyen Phuong, Krisda Samphantharak and Vatcharee Lertmongkol
Anther Culture of BC1F1 (KDML105//IRBB5/KDML105) Hybrid to Produce Bacterial Blight
Resistance Doubled Haploid Rice
................................. Supanyika Sengsai, Surin Peyachoknagul, Prapa Sripichitt,
.......................................................... Amara Thongpan and Pradit Pongtongkam
Novel PCR Primers for Specific Detection of Xanthomonas citri subsp. citri the Causal Agent
of Bacterial Citrus Canker
Udomsak Lertsuchatavanich, Ampaiwan Paradornuwat, Junlapark Chunwongse,
....................................................... Norman W. Schaad and Niphone Thaveechai
Soil-to-Plant Transfer of Radiocaesium in Thailand
................................................ Thitika Thammavech and Teerasak Veerapaspong
Beta-carotene, Mimosine and Quality of Leucaena Silage Kept at Different Duration
............................ Wanna Angthong, Boonlom Cheva-Isarakul, Somkid Promma
................................................................................ and Boonserm Cheva-Isarkul
Effects of Natural Mineral Soils on Body Weight and Liver Minerals of Black Head Somali
Sheep in Ethiopia
.................................... Sisay Tilahun, Pravee Vijchulata, Pornsri Chairatanayuth
.............................................................................. and Suwapong Swasdiphanich
Protoplast Isolation and Culture of Aquatic Plant Cryptocoryne wendtii De Wit
.................. Kanchanaree Pongchawee, Uthairat Na-Nakorn, Siranut Lamseejan,
.......................................................... Supawadee Poompuang and Salak Phansiri
205
213
227
232
242
251
262
274
282
288
300
Anti HSV-1 Activity of Spirulina platensis Polysaccharide
.... Nattayaporn Chirasuwan, Ratana Chaiklahan, Marasri Ruengjitchatchawalya
.......................................................... Boosya Bunnag and Morakot Tanticharoen
Taura Syndrome Virus Disease in Farm-Reared Penaeus monodon in Thailand
.................................................................... Chalor Limsuwan and Niti Chuchird
Optimization of Docosahexaenoic Acid (DHA) Production and Improvement of Astaxanthin
Content in a Mutant Schizochytrium limacinum Isolated from Mangrove Forest in Thailand
.................... Wassana Chatdumrong, Wichien Yongmanitchai, Savitree Limtong
...................................................................... and Wanchai Worawattanamateekul
Cloning, Expression, Purification and Biological Activities of Recombinant Mouse Interleukin-2
in E. coli M15
........... Sanchai Chantajorn, Ratchanee Hongprayoon and Thaweesak Songserm
Production and Partial Characterization of Chitosanases from a Newly Isolated Bacillus cereus
..... Sutee Wangtueai, Wanchai Worawattanamateekul, Mathana Sangjindavong,
.............................................. Nuanphan Naranong and Sarote Sirisansaneeyakul
Application of Pectin Coating in the Production of Vitamin Fortified Rice
..................................................... Lalita Chatiyanont and Phaisan Wuttijumnong
The effects of starter cultures on biogenic amine and free amino acid contents
in Nham during Fermentation
......... Sasithorn Limsuwan, Wonnop Visessanguan and Jirasak Kongkiattikajorn
Product Development System in Pattern Construction System, Standard Body Measurement
and Suitable Fitting Allowance for Thai Ladies Brand in Fashion Industry
................................................................................. Foengfurad Mungtavesinsuk
A Nonlinear Optimization Problem for Determining Safety Stocks in a Two-Stage
Manufacturing System
............................................................................................. Parthana Parthanadee
Design and Implementation of a Framework for .NET-based Utility Computing Infrastructure
............................................. Thanapol Rojanapanpat* and Putchong Uthayopas
311
319
324
335
346
356
373
380
394
Kasetsart J. (Nat. Sci.) 41 : 205 - 212 (2007)
Changing in TSS, TA and Sugar Contents and Sucrose Synthase
Activity in Ethephon-Treated ‘Pattavia’ Pineapple Fruit
Ngarmnij Chuenboonngarm1, Niran Juntawong2*, Arunee Engkagul3,
Wallop Arirob2 and Surin Peyachoknakul4
ABSTRACT
Exogenous ethylene increases endogenous ethylene which plays a crucial role on ripening in
climacteric fruits. Although pineapple is a non-climacteric fruit, ethylene released from ethephon is
effectively used to hasten the harvesting period. Effects from the use of a high concentration of ethephon
on eating quality, fruit size and the reduction in harvesting period have been reported. In this paper, the
effect of a low concentration of ethephon on pineapple fruit quality and sucrose synthase (SuSy) activity
was investigated. Field experiment was arranged in split plot design. In the main plot, two levels of
ethephon concentrations, i.e. 0 and 500 mg/l, were used by spraying at 110 days after forcing (DAF)
fruits. The sub plot was harvesting time, i.e. 5 times of one-week intervals from 124 to 152 DAF. We
found that the total soluble solid (TSS) was significantly increased in most of harvesting-treated fruits
while the titratable acid (TA) was significantly increased at 131 DAF of harvesting-treated fruits. Only
at 131 DAF harvesting time, the glucose content and SuSy activity of ethephon-treated fruits were
significantly reduced and return to the control level afterward. However, ethephon had no effect on the
fructose and sucrose contents at all harvesting times. In conclusion, fruit quality with shortening of
harvesting time could be improved by applying 500 mg/l ethephon at 110 DAF since TSS content which
is one of the parameter predicting eating quality of pineapple was increased without decreasing fruit
quality.
Key words: ‘Pattavia’ pineapple, ethephon, total soluble solid (TSS), titratable acidity (TA), sucrose
synthase
INTRODUCTION
Ethephon is one of the most effective
inflorescence forcing agents in pineapple [Ananas
comosus L. (Merr.)] that is widely used presently
(Bartholomew et al., 2003). Its function is to
stimulate the respiration rate of fruit while
1
2
3
4
*
chlorophyll remains in shell (Dull et al., 1967).
Moreover, it accelerates the ripening process and
concentrates the harvest peak (Chalermglin, 1979;
Smith, 1991). In other non-climacteric fruit such
as pepper, exogenous ethylene promotes and
increases a cellulase activity (Ferrarese et al.,
1995).
Bioscience Interdisciplinary Graduate Program, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: fscinrj@ku.ac.th
Received date : 19/06/06
Accepted date : 06/10/06
206
Kasetsart J. (Nat. Sci.) 41(2)
To achieve the high fruit quality, high
total soluble solid (TSS) at the range of 12-14%
and relatively low titratable acidity (TA) of citric
acid at the range of 0.4-0.6% in pineapple flesh
are recommended for pineapple production in
Thailand (Thongtham, 1983). Though TSS and
TA are eating quality prediction parameters, TSS
is the only parameter suitable as a year-round index
(Bartolome et al., 1995). Bartolome et al. (1996)
found that TSS in pineapples was positively
correlated with total sugars. Beside reflecting fruit
quality, TA also indicates the sourness. In
pineapples, TA is reported as citric acid, not malic
acid. It varies primarily with fruit developmental
stages but does not relatively respond to short-term
environmental changes, while the malic acid varies
with environmental changes especially the light
(Singleton and Gortner, 1965).
Many factors including ethephon have
affected pineapple fruit quality (Bartholomew et
al., 2003). An application time and the quantity
of ethephon have influences on the quality of fruit.
Too early application causes the reduction in size
and weight of crown and fruit, whereas low TSS
and high TA contents are also found (Audinay,
1970; Chalermglin, 1979). TSS is highly
correlated with test-panel eating quality (Smith,
1988) and with total sugars (Bartoleme et al.,
1996). In pineapple fruits, fructose, sucrose, and
glucose play important roles in flavor
characteristics and are major sugars which vary
according to the stage of fruit development.
Sucrose content is lowest in the flesh during the
early stage of fruit growth but rapidly increases at
6 weeks before harvest and becomes predominant
in mature fruit. In the early stage, glucose is
slightly higher than fructose and remains relatively
constant through development while fructose
slightly increases at 2 weeks before harvest (Chen
and Paull, 2000). The changes in total sugar
contents are affected by the developmental stage
of fruits, climates, and varieties (Bartoleme et al.,
1996), nevertheless the change of each sugar
content in ethephon-treated pineapple fruits has
not been reported.
In a sink organ, sugar accumulation is
related to the presence of sucrose metabolizing
enzymes. One of them is sucrose synthase (SuSy)
(Taiz and Zeiger, 1998) which reversibly converts
sucrose and UDP to fructose and UDP-glucose.
SuSy is important in cell metabolism not only in
sink strength (Nguyen-Quoc and Foyer, 2001) but
also in cell wall synthesis (Nakai et al., 1999; Ruan
et al., 2003), and starch synthesis (D/Aoust et al.,
1999). Furthermore, it accumulates sucrose in
edible tissue of satsuma mandarin fruit (Komatsu
et al., 2002) and saves ATP in glycolysis pathway
(Huber and Azakawa, 1986). Chen and Paull
(2000) reported that in pineapple fruits SuSy
activity was higher at young stage, lower at 6
weeks before harvest, and then constant till
harvesting time. The change of SuSy activity in
ethephon treated fruit has also not been reported.
The objective of this work is to answer the question
if ethephon could increase TSS, TA, sugar content
and SuSy activity in pineapple fruit.
MATERIALS AND METHODS
Plant and fruit materials
Field-grown ‘Pattavia’ pineapple
[Ananas comosus L. (Merr.) cv. smooth cayenne]
planted at Sam Praya district, Petchburi Province,
Thailand, were used. Forcing of pineapple
inflorescence was done in the evening of
November 18, 2002, by spraying 50 ml of 250
mg/l ethephon (a.i. 48% w/v) including 3% (w/v)
urea on shoot. The experimental design used in
this study was split plot design. Main plot was
ethephon concentration of 0 and 500 mg/l by
spraying 50 ml volume per fruit at the age of 110
days after forcing (DAF). Pineapple fruit at this
age is pointed-eyes stage 3 according to the Dole
Company, Thailand, which is the last stage of
pointed-eyes pineapple (immature) and thereafter
the eyes will become flatted. Sub-plot was
Kasetsart J. (Nat. Sci.) 41(2)
harvesting time which started from 124 DAF until
152 DAF. Three replications, 8 fruits each, were
analyzed.
Fruit samples were brought to laboratory
and cut transversely into 3 sections after the size
and weight of crowns and fruits were measured.
Only the flesh of the middle section was used in
this study. A half of the flesh was crushed and the
juice was then used for determination of TSS and
TA. The other half, sliced into small pieces, was
used for the determination of the sugar content
and sucrose synthase activity. These sliced fleshes
of 8 fruits were pooled together as one of three
replications at each harvesting time. The tissues
were then frozen immediately in liquid nitrogen
and stored at -80°C until use.
Soluble sugar content
TSS was determined from extracted juice
using hand sugar refractometer. Soluble sugars in
the form of sucrose, fructose and glucose were
extracted following the method of Chen and Paull
(2000). After extraction, the solution was filtered
through a 0.45 mm filter, and 20 ml was injected
and analyzed with HPLC by using a Waters 2690
Separation Model instrumented with a Waters 410
Differential Refractometer detector, employing a
Sugar-PAK I (Waters Associates, Milford, USA)
column of stainless steel (300 mm length × 6.5
mm internal diameters). The eluting buffer was
0.1 mM calcium EDTA and the flow rate was 0.5
ml/min. Experiments were performed at 90°C.
Soluble sugars were quantified by comparing the
peak areas with external sucrose, glucose and
fructose standard solutions (Sigma Co., Ltd.).
Titratable acidity
TA was analyzed from extracted juice
after the determination of TSS contents and
reported as citric acid according to AOAC (1990).
Sucrose synthase determination
Sucrose synthase (SuSy) in frozen flesh
tissue was extracted as described by Chen and
207
Paull (2000). The extracted solution was desalted
by Hitrap  Desalting column (Amersham
Biosciences) and 50 µl of desalted mixture was
used to determine the enzymatic activity in
synthesis direction according to the method of
Hubbard et al. (1989), as modified by Chen and
Paull (2000).
Statistical analysis
All data were analyzed the variance
(ANOVA) using statistical analysis software of
IRRISTAT version 93-3.
RESULTS AND DISCUSSION
The last harvesting time in this study
(152 DAF) was planned to coincide with
commercial harvesting time. The commercial
harvesting index for cannery fruit industry is
apparent when fruits reach full-size and the shell
color at the basal portion starts to change. The
effects of ethephon and harvesting time on fruit
quality, sugar content and SuSy activity after
treating at 110 DAF are shown in Table 1.
Ethephon concentration did not reduce the size and
weight of the crowns and fruits. The crowns and
fruits continued to develop after the treatment and
the crowns reached a full-size one week (138 DAF)
before the fruits did (145 DAF). Maximum growth
of the crowns indicated that the fruits were nearly
ready for harvest (Paull and Reyes, 1996). The
concentration of ethephon plays a significant role
in increasing the mean of TSS contents (11.02°
Brix) when compared with the mean of untreated
fruits (8.90°Brix). The mean of TA and sugar
contents including SuSy activity did not change,
compared with untreated fruits. The harvesting
time at 145 DAF provided the highest TA, TSS
and sucrose contents of 0.62% citric acid, 12.16°
Brix and 54.12 g/kg FW, respectively (P<0.01).
These indicated that the quality of fruit changes
during fruit development and TSS were related to
sucrose more than glucose and fructose as reported
Mean followed by the same letter within the same column are not significantly different at the 5% level according to LSD. Symbols * and ** indicate significance at the 0.05 and 0.01 levels
analyzed by DMRT, ns indicates no significant.
1/
TA = Tritratable acidity
2/
TSS = Total soluble solid
Table 1 Effects of ethephon concentrations and harvesting times on fruit quality, sugar content and sucrose synthase activity after treated at 110 days
after forcing (DAF).
Crown
Fruit
Flesh
Width Length Weight Width Length Weight
TA1/
TSS2/
Sucrose Glucose Fructose SuSy activity
_____(cm)_____
(g)
_____(cm)_____
(g)
(%citric (°Brix) ________(g/kg FW)________ (mmole/h/
acid)
g FW)
Ethephon
concentration
0 mg/l
12.6
12.6
140.8
11.0
14.0
882.4
0.54
8.90b
31.07
14.78
11.61
2.374
500 mg/l
11.6
11.1
127.1
11.3
14.1
922.9
0.59
11.02a
43.20
13.42
11.40
1.717
Harvesting time
124 DAF
11.4b
9.8b
124.8
11.5
14.2
846.2b
0.42c
8.08b
20.90d
14.46
10.86
2.297
131 DAF
11.3b
10.4b
114.4
10.7
13.4
760.4b
0.52b
8.78b
23.46cd
13.96
10.43
1.984
138 DAF
13.5a
13.4a
139.4
10.8
13.8
849.8b 0.58ab
8.48b
37.08bc
15.22
12.02
1.964
145 DAF
12.2ab
13.0a
143.3
11.8
15.0
1149.8a 0.62a
12.16a
54.12a
14.50
13.08
2.127
152 DAF
12.1ab
12.7a
147.9
11.1
13.8
906.7b
0.67a
12.30a 50.13ab
12.34
11.12
1.864
Ethephon
concentration
ns
ns
ns
ns
ns
ns
ns
*
ns
ns
ns
ns
Harvesting time
*
**
ns
ns
ns
**
**
**
**
ns
ns
ns
Ethephon
concentration X
ns
ns
ns
ns
ns
ns
*
*
ns
*
ns
*
Harvesting time
208
Kasetsart J. (Nat. Sci.) 41(2)
Kasetsart J. (Nat. Sci.) 41(2)
by Chen and Paull (2000). Figure 1 also showed
that sucrose content was low in immature fruit and
the highest content was achieved at 145 DAF while
glucose and fructose contents were relatively
constant during fruit growth as reported by Chen
and Paull (2000).
The interaction of ethephon
concentration with harvesting time significantly
affected TA, TSS and glucose contents at P<0.05
(Table 1). Comparing between the treatments of
ethephon concentration at 0 and 500 mg/l at each
harvesting time, it was found that almost all TSS
of treated fruits were significantly higher than
those of the control (Figure 2B). However only
treated fruits harvested at 131 DAF had TA content
higher (Figure 2A), but glucose content (Figure
2C) and SuSy activity were lower (Figure 2D) than
those of the untreated fruits. The high
concentration of TSS in harvested fruits treated at
131 DAF was affected by high TA rather than sugar
content because TSS does not represent only the
sugar content but also the contents of organic acids,
209
soluble pectins and other dissolved substances
which have different refractive indices from water
(Holcroft and Kader, 1999). This is the reason
why a direct measurement of sugar concentration
by HPLC is carried out. From our results (Figure
2A, 2B), ethephon affected the TA and glucose
contents of treated fruits in a few weeks after
ethephon application because ethephon is an
unstable substance which can be easily degraded
by high temperature and high pH in cytoplasm
(Bartholomew et al., 2003). Changing in TA and
glucose contents in pineapples may also be resulted
from a high respiration rate which is induced by
ethephon (Dull et al., 1967). This is due to the
use of glucose as a first glycolytic substance in a
respiratory pathway (Taiz and Zeiger, 1998) which
enhances organic acid contents (Ulrich, 1970).
High respiration rate also causes high oxygen
admission in tissue and this may be the other
reason for increasing TA.
It was also found that the TSS contents
of harvested fruits treated at 145 and 152 DAF
Figure 1 Sucrose, glucose and fructose contents in pineapple fruits after treated with 0 and 500 mg/l
ethephon at 110 days after forcing (DAF).
Kasetsart J. (Nat. Sci.) 41(2)
210
were higher than that of the untreated fruits (Figure
2B). The exogenous ethylene which was
suggested to increase the lipoxygenase activity by
Yu et al. (2003) might change the permeability of
the membrane and cause the increase of TSS in
these mature fruits. From the results on high TSS
(13.53°Brix) and TA (0.6% citric acid) contents
measured at 145 DAF, the treated fruits which are
in the range of high eating-quality fruit
(Bartholomew et al., 2003) could be harvested one
week earlier. Chalermglin (1979) also reported
that after applying 1,500 mg/l of ethephon at 112
DAF, the treated fruits could be harvested 11 days
18
0.9
a
0.8
a
a
a
0.4
0.3
b
a
a
b
6
0.2
4
0.1
2
0
a
10
8
b
a
12
b
a
b
14
TSS (°Brix)
TA (%citric acid)
16
a
a
a
0.6
a
a
a
0.7
0.5
earlier than those of the control. However, TA was
found to be inereased in treated fruits while fruit
size was reduced and TSS was unchanged. This
study indicates that the application of 500 mg/l
ethephon to 110 DAF fruits hastened the
harvesting time without reducing fruit quality.
Figure 2 also showed SuSy activities
which were affected by a significant interaction
between ethephon concentration and harvesting
time. When harvested at 131 DAF, the SuSy
activity of the treated fruits was significantly lower
than that of the untreated fruits. Chen and Paull
(2000) suggested that the low SuSy activity in
0
124
131
138
145
152 DAF
124
131
138
152 DAF
145
(B)
(A)
4
25
Glucose content (g/kg FW)
20
a
a
a
a
a a
a
a
15
b
10
5
SuSy Activity (µmole/h/g FV)
3.5
a
a
a
3
a
a
a
a
2.5
a
a
b
2
a
1.5
1
0.5
0
0
124
131
138
(C)
145
152 DAF
124
131
138
145
152 DAF
(D)
Figure 2 Changes in tritratable acidity (TA) (A), total soluble solid (TSS) (B) and glucose contents (C)
and sucrose synthase activity (D) in pineapple fruits flesh at various harvesting times after
treated with 500 mg/l ethephon ( ) and without ethephon ( ) at 110 days after forcing
(DAF). Error bars represent standard error of the means of three replications. Bars with the
same letter assigned are not significantly different at 0.05 probability level.
Kasetsart J. (Nat. Sci.) 41(2)
pineapple fruit allowed the accumulation of
sucrose. However, we found that the low SuSy
activity in harvested fruits treated at 131 did not
enhance the sucrose accumulation (no significant
interaction of sucrose was found, Table 1).
Therefore, the SuSy activity was not related to the
accumulation of sucrose in pineapples which is in
contrast to the activity in non-climacteric, satsuma
mandarin fruits (Komatsu et al., 2002). The
decrease of SuSy activity of harvested fruits treated
at 131 DAF might be resulted from the increase
in respiration rate which increases the amount of
ATP in cells. Therefore, SuSy activity which
involves in energy-saving pathway of glycolysis
(Huber and Akazawa, 1986) should be decreased.
SuSy is an important enzyme for synthesizing
UDP-glucose, the cellulose precursor (Nakai et al.,
1999). Thus, exogenous ethylene enhances a
cellulase activity (Ferrarese et al., 1995) which
leads to high production of UDP-glucose that may
act as a negative feedback to the SuSy activity.
The exact mechanisms of the SuSy activity as well
as the effect of ethylene on SuSy activity have still
not been well-defined.
CONCLUSION
We conclude that the ethephon at the rate
of 500 mg/l spraying at 110 DAF could increase
TSS in pineapple fruit, but not TA, sugar contents
and SuSy activity, and the treated fruits could be
harvested at 145 DAF without the decrease of fruit
size and weight.
ACKNOWLEDGEMENTS
The work was partially supported by
Thesis and Dissertation Support Fund, Graduate
School, Kasetsart University. Special thank to
Assoc. Prof. Dr. Napavarn Noparatnaraporn for
her suggestion in preparation of this manuscript.
211
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Kasetsart J. (Nat. Sci.) 41 : 213 - 226 (2007)
Phylogenetic Analysis of Thai Amomum (Alpinioideae: Zingiberaceae)
Using AFLP Markers
Wittaya Kaewsri1*, Yingyong Paisooksantivatana1 , Uamporn Veesommai1,
Wichan Eiadthong2 and Srunya Vajrodaya3
ABSTRACT
The AFLP technique was used to assess the genetic relationships among 45 zingiberaceous
plants including 40 collections of Amomum and 5 outgroup taxa: Alpinia, Etlingera 1, Etlingera 2,
Elettaria and Geostachys. Cluster analysis using unweighted pair group method with arithmetic mean
(UPGMA), based on AFLP data from 122 polymorphic bands generated with five primer combinations,
was performed. The grouping of accessions of most species corresponded with their fruit morphological
characteristics and were found to be consistent with previous studies. The species of Thai Amomum
were classified into 3 groups based on AFLP markers: A. aculeatum group, A. biflorum group, and A.
dealbatum group. The genetic relationships among genus Amomum and other genera in the tribe
Alpinioideae are still incompletely understood.
Key words: phylogenetic, Amomum, AFLP, Thailand
INTRODUCTION
Amomum Roxb. is one of the largest
genera in the ginger family (Zingiberaceae) with
about 150-180 species. As currently recognized,
Amomum occurs from the Himalayas through
Southeast Asia, Northern Australia and extends
into the central Pacific and is widely distributed
in Southeast Asia (Kiew, 1982; Smith, 1985).
Sirirugsa (2001) estimated about 15-20 species to
be found in Thailand. Plants of Amomum are
generally evergreen herbs inhabiting wet forests
in light gaps and at forest margins (Sakai and
Nagamasu, 1998). Many species are used as
medicine, spice, condiment and vegetable. Even
1
2
3
*
though the plants from this genus have been long
utilized, the identification is still confusing because
of the absence of a comprehensive work on the
genus and the much confused taxonomic problems.
These bring about many changes in their
taxonomic status.
Four species of Amomum were first
recognized by Linnaeus (1753) including: A.
cardamomum, A. zingiber, A. zerumbet and A.
grana-paradisi. These species have since been
transferred to Elettaria Maton, Zingiber Boehm
and Aframomum K. Schum. by Burtt and Smith
(1972). Baker (1892), classified Amomum into 5
sections; Geanthus, Achasma, Hornstedtia,
Euamomum and Cenolophon based on
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Department of Forest Biology, Faculty of Forestry, Kasetsart University, Bangkok 10900, Thailand.
Department of Botany, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: wittayakaewsri@yahoo.com
Received date : 30/03/06
Accepted date : 3/10/06
214
Kasetsart J. (Nat. Sci.) 41(2)
morphological characteristics of spike, labellum
and anther crest. Schumann (1904) used the
characteristics of anther crest and classified
Amomum into 2 sections and 4 series. Section
Geanthus was divided into 2 series, series
Oliganthae and Polyanthae, distinguished by the
absence of an anther crest. Section Euamomum
was comprised of series Lobulatae and Integrae,
characterized by an anther crest. Gagnepain (1906)
separated Amomum into 3 groups based on the
characteristics of floral morphology such as anther
crest and lateral staminode. Loesener (1930)
classified Amomum into 2 main groups using
anther crest, Lobulatae and Integrae.
Xia et al. (2004) investigated the
phylogenetic status of Amomum using ITS and
matK DNA sequence data. They indicated that
Amomum as currently defined is polyphyletic with
three major groups of species (A. villosum Group,
A. tsao-ko Group and A. maximum Group) that do
not correspond with any previously recognized
sectional classification of the genus. They also
mentioned that some morphological characters
such as anther crest and fruit type could be useful
for classification.
The AFLP technique has been used to
study genetic diversity and phylogenetic
relationships in a wide range of plant species;
Lubberstedt et al. (2000) studied relationships
among early European maize inbreds, GarciaMass et al. (2000) used AFLP marker for
measuring genetic diversity in melon, Abdalla et
al. (2001) used AFLP marker for estimating
genetic relationships across a wide range of
taxonomic levels and for analyzing the
evolutionary and historical development of cotton
cultivars at the genomic level, Larson et al. (2001)
studied AFLP variation in agamospermous and
dioecious bluegrasses of western North America,
Mizumoto et al. (2003) used AFLP for studying
the diversity of nuclear and chloroplast genome
in wild einkorn wheat (Triticum urartu).
Because the relationships within genus
Amomum and other genera in tribe Alpinioideae
are still incompletely understood, a more detailed
analysis using other molecular techniques is
necessary. Knowledge of the genetic relationships
among them is essential to the classification of
the genus. This study was intended to determine
genetic relationships among species of the
Amomum genus occurring in Thailand using AFLP
markers.
MATERIALS AND METHODS
Plant materials
Forty accessions of Amomum and 5
accessions of outgroup taxa: Alpinia, Elettaria,
Etlingera 1, Etlingera 2 and Geostachys were used
in this study (Table 1). All plant materials were
grown and kept at Department of Horticulture,
Kasetsart University, Bangkok, Thailand.
DNA isolation and AFLP analysis
Total genomic DNAs were extracted
from 100 mg fresh young leaves using Qiagen
DNeasy Plant Mini kit (Qiagen GmbH, Hilden,
Germany).
AFLP analysis was performed following
the method of Vos et al. (1995) with minor
modifications. From each sample, 2 templates
were prepared by digesting 20-50 ng DNA with
the restriction enzyme combination EcoRI-MseI
and by ligating the corresponding oligonucleotide
adaptors in a total volume of 10 µl. Preselective
PCR amplification with primers corresponding to
adaptor core sequences (E+A and M+C) was
performed in a 10 µl reaction containing 3 µl of
AFLP template. PCR contained 10X PCR buffer,
0.5 µmol/L of each primer, 1 µmol/L of each dNTP,
and 1 U Taq DNA polymerase (Fermentas,
Lithuania) and was performed using a Biosystems
Mod. Gene Amp PCR system 9700 (Biosystems,
Montgomeryville, PA). PCR conditions consisted
of 1 cycle of 5 min at 50°C, 1 cycle of 3 min at
94°C, 24 cycles of 30 s at 94°C, 24 cycles of 1
Kasetsart J. (Nat. Sci.) 41(2)
215
Table 1 List of Thai Amomum accessions and outgroup taxa used in AFLP study.
Accessions
Species
Collected number
Collected places(provinces)
1
A. aculeatum Roxb.
Kaewsri-02
Kanchanaburi
2
A. biflorum Jack
Kaewsri-52
Chanthaburi
3
A. dealbatum Roxb.
Kaewsri-110
Chiang Mai
4
A. koenigii 1
Kaewsri-03
Kanchanaburi
5
A. koenigii 2
Kaewsri-29
Nakhon Nayok
6
A. micranthum Ridl.
Kaewsri-63
Chanthaburi
7
A. repoense Gagnep.
Kaewsri-64
Chanthaburi
8
A. rivale1*
Kaewsri-04
Kanchanaburi
9
A. rivale2*
Kaewsri-23
Kanchanaburi
10
A. cf. rivale
Kaewsri-33
Kanchanaburi
11
A. siamense Craib
Kaewsri-14
Tak
12
A. testaceum 1
Kaewsri-15
Tak (Cultivated)
13
A. testaceum 2
Kaewsri-16
Tak (Cultivated)
14
A. testaceum 3
Kaewsri-17
Tak (Cultivated)
15
A. testaceum 4
Kaewsri-96
Tak (Cultivated)
16
Amomum cf. testaceum
Kaewsri-86
Chumphon
17
A. uliginosum1
Kaewsri-30
Nakhon Nayok
18
A. uliginosum2
Kaewsri-92
Tak
19
A. uliginosum3
Kaewsri-32
Trat
20
A. cf. villosum1
Kaewsri-12
Tak (Cultivated)
21
A. cf. villosum2
Kaewsri-13
Tak
22
Amomum sp.1
Kaewsri-01
Kanchanaburi
23
Amomum sp.2
Kaewsri-10
Kanchanaburi
24
Amomum sp.3
Kaewsri-19
Prachuap Khiri Khan
25
Amomum sp.4
Kaewsri-22
Kanchanaburi
26
Amomum sp.5
Kaewsri-24
Kanchanaburi
27
Amomum sp.6a
Kaewsri-113
Chiang Mai
28
Amomum sp.6b
Kaewsri-88
Tak
29
Amomum sp.7
Kaewsri-27
Uthai Thani
30
Amomum sp.8
Kaewsri-35
Ranong
31
Amomum sp.9
Kaewsri-38
Ranong
32
Amomum sp.10
Kaewsri-50
Sakon Nakhon
33
Amomum sp.11
Kaewsri-68
Chumphon
34
Amomum sp.12
Kaewsri-70
Chumphon
35
Amomum sp.13
Kaewsri-81
Ranong
36
Amomum sp.14
Kaewsri-94
Tak
37
Amomum sp.15
Kaewsri-108
Chiang Mai
38
Amomum sp.16
Kaewsri-111
Chiang Mai
39
Amomum sp.17a
Kaewsri-134
Nan
40
Amomum sp.17b
Kaewsri-138
Nan
41
Alpinia nigra
Cultivated at KU
42
Elettaria cardamomum
Tak
43
Etlingera littoralis
Kanchanaburi
44
Etlingera pavieana
Chanthaburi
45
Geostachys sp.
Nakhon Nayok
The number 1, 2, 3 or 4 of each species = Amomum’s specimens that were collected from different places.
216
Kasetsart J. (Nat. Sci.) 41(2)
min at 56°C, and 24 cycles of 1 min at 72°C,
followed by an extension of 5 min at 72°C.
Amplification products were diluted in 100 µl
deionized H2O and 2 µl were used for selective
amplification in a total volume of 10 µl containing
1 µmol/L of 10X PCR Buffer, 5 µmol/L of Especific primer extended by 3 selective
neucleotides (Table 2), 5 µmol/L of M-specific
primer extended by 3 selective nucleotides (Table
2), 1 U of Taq DNA polymerase (Fermentas,
Lithuania) and 1 µmol/L of each dNTPs. PCR was
performed using a touchdown protocol with initial
denaturation of a cycle of 30 s at 94°C, 30 s at 65°
C (decreasing the temperature by 1°C after each
cycle until 57°C) and 1 min at 72°C, followed by
30 cycles of 30 s at 94°C, 30 s at 56°C and 1 min
at 72°C with a final extension of 4 min at 72°C.
Following amplification, 10 µl of formamide
loading dye was added to the PCR products. The
products were electrophoresed on 8% nondenaturing polyacrylamide gel. The bands were
visualized using silver stain.
Data analysis
Each accession was scored (1) for
presence and (0) for absence of each polymorphic
band. AFLP bands within accessions were scored
as missing if they were poorly resolved on the gel
or if the template DNA did not amplify well.
Similarity coefficient was calculated on the basis
of Dice similarity coefficients (Dice, 1945) and is
written as
Cjk = 2a/(2a+b+c)
In which Cjk is similarity coefficient, a
is number of AFLP markers present in both j and
k accessions, b is number of AFLP markers present
only in j accessions and c is number of AFLP
markers present only in k accessions. The
similarity matrix was subjected to cluster analysis
by the unweighted pair-group method with
arithmetic mean (UPGMA) and a dendrogram was
created using the NTSYS-pc version 2.01d
program (Rohlf, 1997).
RESULTS
Five informative AFLP primer
combinations generated a total of 364 reproducible
amplification fragments across all species of
Amomum, among which 122 bands were
polymorphic (Table 2). The number of amplified
AFLP bands per primer pair varied from 66 to 81
with an average of 72.8 bands. The average
number of polymorphic bands detected was 24.4
per primer combination. The fragment sizes were
determined by comparing each one with the
standard DNA ladder, ranging from about 140 to
726 base pairs (bp). Two primer combinations (EAGG, M-CAA (Figure 2) and E-ACC, M-CAA)
produced 30 polymorphic bands, a relatively
higher numbers of polymorphisms compared to
the other primers used in this study.
Table 2 AFLP primer pairs and their number of amplified and polymorphic bands for phylogenetic
study of Thai Amomum.
Primer combinations
Amplified bands
No. of polymorphic bands
(EcoRI+3/MseI+3)
E-AGG, M-CAA
81
30
E-ACC, M-CTA
73
17
E-ACC, M-CAA
66
30
E-AGC, M-CTC
74
25
E-AGG, M-CTC
70
20
Total
364
122
Mean
72.8
24.4
Kasetsart J. (Nat. Sci.) 41(2)
Cluster analysis
Cluster analysis using UPGMA
(unweighted pair group method with arithmetic
mean) was performed to examine genetic
relationships among Thai Amomum species. A
dendrogram was produced from the UPGMA
cluster analysis of genetic similarity (GS) matrix
for 45 accessions, 40 accessions of Amomum
species and 5 accessions of out taxa, based on
AFLP markers varied from 43% to 88% with a
total average genetic similarity of 74.5% (Table
3). Two main clusters (A and B) were separated
at 57% GS. The A cluster was separated into 2
groups: C and D, at 58% genetic similarity. The D
group is subdivided into 2 subgroups (I and II) at
59% GS while the B Cluster generated 2 groups
(E and F) at 59% GS (Figure 3).
The A cluster is characterized by spiny
fruit (rarely smooth fruit). The C group contains
Amomum koenigii 1, A. koenigii 2, A.uliginosum
1, Amomum sp.9, Etlingera littoralis, A.
aculeatum, Amomum sp.12 and Geostachys sp.
while D group is divided into two subgroups (I
and II). Subgroup (I) consists of A. testaceum 1,
A. testaceum 2, A. testaceum 3 and Amomum cf.
testaceum. Subgroup (II) consists of A. testaceum
4, A. cf. villosum2, Amomum sp.5, Amomum sp.7,
A. uliginosum2, A. cf. villosum1, A. rivale1, A.
micranthum, A. rivale2, A. cf.rivale, Amomum sp.
8, Amomum sp.4, A. biflorum and Amomum sp.13.
The B cluster is characterized by smooth, ridged
or wing fruit (rarely spiny fruit). This cluster
contains E and F groups. The E group consists
Amomum sp.16, Amomum sp.3, Amomum sp.2,
Amomum sp.17a, A. siamense, Amomum sp.6b,
Amomum sp.6a, Elettaria cardamomum, Amomum
sp.17b, A.dealbatum, Amomum sp.15, Amomum
sp.10 and Alpinia nigra. The F group contains
Amomum sp.1, Amomum sp.14, A. repoense, and
Etlingera pavieana. Regarding the out group taxa;
Alpinia nigra, Elettaria cardamomum and
Etlingera pavieana were inserted in B group while
Etlingera littoralis and Geostachys sp. were placed
in A group.
217
DISCUSSION
In this study, 40 accessions of Thai
Amomum species were fingerprinted including 5
outgroup taxa. One hundred twenty two
polymorphic AFLP markers were produced from
five primer combinations. UPGMA cluster
analysis (Rohlf, 1997) with genetic similarity of
57% separated Amomum into 2 main clusters: A
consists of C and D groups and B consists of E
and F groups (Figure 1).
Regarding the C group, A. koenigii 1 and
A.koenigii 2 were collected from Kanchanaburi
and Nakhon Nayok provinces, respectively. It is
clear that both collections are closely related
(74%), even though the peduncular lengths vary
greatly. The plants from Nakhon Nayok have a
much shorter peduncle than those found in
Kanchanaburi. The variation in phenotype could
be caused by differences in their respective
habitats. The placement of this species is similar
to morphological analysis that placed it in spiny
fruit group. This result is confirmation of the
paraphyletic relationship between A.koenigii and
the spiny fruit species (A. uliginosum and A.
aculeatum). A.aculeatum and Amomum sp.12 are
placed together at 90% GS. These closely related
species are similar in leafy stem but differ in
peduncular length, colour and size of labellum.
From the results, the species Amomum sp.12
should be established as a new variety. However,
this is difficult to decide from only a single plant.
More collections are needed to solve this problem.
D group is divided into two subgroups (I
and II). Subgroup D (I) consists of A. testaceum
1, A. testaceum 2, A. testaceum 3 and Amomum
cf. testaceum. Regarding A. testaceum species
complex, the dendrogram suggests that this species
can be separated into at least three varieties;
especially A. testaceum 4 which was isolated from
the group. The placement of A. testaceum is rather
close to the spiny fruit species (Amomum sp.1 and
Amomum sp.14). This result does agree with Xia
et al. (2004) whose work was based on ITS and
218
Kasetsart J. (Nat. Sci.) 41(2)
MatK genes. They placed A.testaceum among the
spiny fruit species of A.villosum group. A possible
explanation for this was a paraphyletic origin of
A. testaceum complex. Although its morphological
characteristics are different, its genotype is close
Figure 1 Some species of Amomum
A. A. aculeatum Roxb.
D. A.koenigii Gmelin.
G. A. testaceum Ridl.
to spiny fruit species. The Amomum cf. testaceum
that was collected from Chumphon is also placed
in this group. Its leafy stem is similar to A.
testaceum but dif fers in its hairiness on the lower
surface of leaves.
used in AFLP study.
B. A. biflorum Jack
E. A. repoense Pierre ex Gagnep.
H. A. uliginosum K?nig ex Retz.
C. A. dealbatum Roxb.
F. A. rivale Ridl.
I. A. siamense Craib
Figure 2 AFLP fingerprint of Thai Amomum species and out-groups using E-AGG, M-CAA primer pair. 1. A. koenigii1 , 2. A. koenigii 2, 3. Amomum
sp.16, 4. A. testaceum1, 5. A. testaceum 2, 6. A. testaceum 3, 7. A. testaceum 4, 8. Amomum sp.1, 9. A. aculeatum Roxb., 10. Amomum sp.12,
11. A. rivale1, 12. A. rivale2, 13. A.cf. villosum1, 14. A. cf. villosum2, 15. Amomum sp.4, 16. Amomum sp.5, 17. Amomum sp.7, 18. A.
uliginosum1, 19. A. uliginosum2, 20. A. uliginosum3, 21. A Amomum cf. rivale., 22. Amomum sp.17a, 23. Amomum sp.8, 24. Amomum sp.10,
25. A. biflorum Jack , 26. A. micranthum Ridl., 27. Amomum sp.11, 28. Amomum sp.13, 29. none use, 30. none use, 31. A. siamense Craib,32.
.Amomum sp.3, 33. Amomum sp.2, 34. Amomum sp.6b, 35. A. uliginosum4, 36. Amomum sp.17b, 37. A. repoense Gagnep., 38. Amomum sp.6a,
39. none use, 40. Amomum sp.14, 41. Amomum sp.15, 42. A. dealbatum Roxb., 43. Elettaria cardamomum, 44. Etlingera littoralis, 45.
Etlingera pavieana, 46. Alpinia nigra, 47. Geostachys sp., 48.Amomum cf. testaceum and M=φXHinfI
Kasetsart J. (Nat. Sci.) 41(2)
219
A. uliginosum 3
Amomum cf.rivale
A.momum sp.17a
20
21
22
A. uliginosum 1
A. uliginosum 2
18
19
Amomum sp. 5
Amomum sp. 7
16
17
A. cf. villosum 2
Amomum sp. 4
14
15
A. rivale 2
A. cf. villosum 1
12
13
Amomum sp. 12
A. rivale 1
10
11
Amomum sp. 1
A. aculeatum
8
9
A. testaceum 3
A. testaceum 4
6
7
A. testaceum 1
A. testaceum 2
4
5
A. koenigii 2
Amomum sp. 16
2
3
A. koenigii 1
1
0.53
0.55
0.53
0.53
0.67
0.53
0.57
0.61
0.50
0.54
0.57
0.63
0.58
0.59
0.64
0.50
0.57
0.56
0.56
0.58
0.71
1.00
1
0.52
0.57
0.52
0.57
0.83
0.55
0.60
0.63
0.53
0.56
0.59
0.63
0.63
0.65
0.60
0.55
0.57
0.51
0.54
0.57
1.00
2
0.58
0.69
0.57
0.47
0.61
0.52
0.53
0.58
0.43
0.51
0.65
0.61
0.57
0.60
0.60
0.47
0.51
0.47
0.45
1.00
3
0.53
0.59
0.57
0.65
0.57
0.62
0.60
0.68
0.53
0.57
0.57
0.65
0.64
0.66
0.50
0.59
0.78
0.78
1.00
4
0.51
0.57
0.56
0.68
0.56
0.68
0.58
0.68
0.53
0.52
0.55
0.67
0.62
0.63
0.53
0.62
0.88
1.00
5
0.48
0.62
0.54
0.65
0.59
0.64
0.60
0.71
0.53
0.52
0.63
0.68
0.60
0.64
0.57
0.64
1.00
6
0.53
0.63
0.63
0.69
0.53
0.69
0.65
0.64
0.73
0.64
0.65
0.61
0.60
0.56
0.60
1.00
7
0.56
0.64
0.62
0.57
0.60
0.59
0.57
0.60
0.53
0.52
0.67
0.68
0.64
0.61
1.00
8
0.60
0.62
0.56
0.62
0.68
0.60
0.66
0.67
0.58
0.61
0.60
0.65
0.90
1.00
9
0.60
0.58
0.58
0.64
0.64
0.66
0.65
0.64
0.59
0.57
0.59
0.61
1.00
10
0.63
0.72
0.67
0.64
0.63
0.67
0.64
0.80
0.56
0.65
0.76
1.00
11
0.56
0.85
0.64
0.67
0.60
0.67
0.67
0.74
0.64
0.60
1.00
12
0.60
0.62
0.67
0.71
0.60
0.65
0.77
0.71
0.75
1.00
13
0.48
0.64
0.64
0.74
0.51
0.70
0.83
0.62
1.00
14
0.60
0.78
0.69
0.77
0.63
0.75
0.68
1.00
15
0.53
0.68
0.65
0.74
0.64
0.71
1.00
16
0.60
0.66
0.75
0.94
0.55
1.00
17
Table 3 Dice’s coefficient of similarity matrix from AFLP fingerprints of 40 accessions of Amomom and 5 outgroup taxa.
0.50
0.61
0.58
0.58
1.00
18
0.57
0.67
0.74
1.00
19
0.58
0.67
1.00
20
0.60
1.00
21
1.00
22
220
Kasetsart J. (Nat. Sci.) 41(2)
Alpinia nigra
Geostachys sp.
Amomum cf. testaceum
43
44
45
Etlingera littoralis
Etlingera pavieana
41
42
A. dealbatum Roxb.
Elettaria cardamomum
Amomum sp. 15
38
39
Amomum sp. 14
37
40
A. repoense Gagnep.
Amomum sp. 6a
35
36
A. uliginosum 4
Amomum sp.17b
33
34
Amomum sp. 2
Amomum sp. 6b
31
32
A. siamense Craib
Amomum sp. 3
29
30
Amomum sp. 11
Amomum sp. 13
27
28
A. biflorum Jack
A. micranthum Ridl.
25
26
Amomum sp. 8
Amomum sp. 10
23
24
Table 3 (Continued)
0.53
0.57
0.52
0.58
0.69
0.65
0.61
0.57
0.53
0.59
0.53
0.57
0.59
0.58
0.50
0.52
0.54
0.54
0.50
0.53
0.58
0.55
0.55
1
0.49
0.60
0.55
0.58
0.63
0.56
0.64
0.54
0.53
0.54
0.52
0.56
0.67
0.60
0.53
0.53
0.62
0.67
0.54
0.54
0.58
0.50
0.57
2
0.61
0.59
0.55
0.57
0.64
0.60
0.57
0.56
0.57
0.60
0.58
0.60
0.65
0.58
0.66
0.61
0.57
0.65
0.51
0.62
0.61
0.63
0.66
3
0.70
0.55
0.53
0.54
0.56
0.44
0.56
0.57
0.55
0.50
0.50
0.57
0.58
0.54
0.51
0.53
0.58
0.67
0.64
0.61
0.65
0.54
0.57
4
0.70
0.57
0.53
0.57
0.56
0.52
0.57
0.58
0.57
0.47
0.54
0.63
0.58
0.53
0.51
0.51
0.60
0.61
0.69
0.60
0.65
0.53
0.57
5
0.68
0.53
0.50
0.54
0.59
0.53
0.62
0.55
0.53
0.47
0.54
0.58
0.58
0.51
0.50
0.53
0.57
0.63
0.64
0.63
0.68
0.48
0.59
6
0.58
0.60
0.60
0.57
0.52
0.51
0.52
0.56
0.64
0.45
0.55
0.62
0.60
0.53
0.57
0.55
0.54
0.60
0.71
0.71
0.61
0.57
0.61
7
0.57
0.64
0.56
0.71
0.60
0.74
0.70
0.57
0.67
0.55
0.70
0.69
0.57
0.60
0.67
0.64
0.55
0.60
0.60
0.63
0.65
0.59
0.62
8
0.60
0.69
0.56
0.64
0.59
0.57
0.60
0.61
0.57
0.60
0.56
0.63
0.63
0.57
0.51
0.56
0.60
0.66
0.55
0.63
0.60
0.57
0.59
9
0.60
0.65
0.50
0.63
0.60
0.62
0.53
0.57
0.60
0.57
0.52
0.60
0.62
0.58
0.49
0.52
0.64
0.64
0.57
0.62
0.61
0.58
0.55
10
0.63
0.64
0.61
0.67
0.60
0.62
0.63
0.70
0.65
0.59
0.58
0.68
0.65
0.67
0.60
0.63
0.70
0.73
0.62
0.73
0.71
0.60
0.77
11
0.67
0.57
0.50
0.54
0.62
0.58
0.60
0.66
0.66
0.53
0.59
0.64
0.64
0.60
0.56
0.51
0.57
0.75
0.67
0.75
0.71
0.57
0.85
12
0.47
0.61
0.62
0.51
0.60
0.53
0.64
0.66
0.57
0.50
0.57
0.63
0.60
0.57
0.48
0.51
0.58
0.60
0.61
0.63
0.59
0.60
0.65
13
0.53
0.58
0.56
0.47
0.57
0.50
0.54
0.55
0.61
0.43
0.54
0.52
0.58
0.60
0.47
0.48
0.55
0.60
0.67
0.63
0.56
0.53
0.62
14
0.63
0.65
0.61
0.61
0.60
0.51
0.67
0.65
0.57
0.53
0.57
0.65
0.67
0.63
0.57
0.60
0.62
0.71
0.68
0.64
0.69
0.60
0.77
15
0.56
0.61
0.56
0.50
0.64
0.50
0.57
0.58
0.61
0.49
0.51
0.60
0.64
0.60
0.43
0.47
0.58
0.61
0.71
0.61
0.64
0.51
0.67
16
0.53
0.59
0.55
0.58
0.58
0.56
0.55
0.54
0.59
0.56
0.52
0.60
0.62
0.60
0.53
0.57
0.59
0.68
0.81
0.64
0.67
0.53
0.63
17
0.49
0.67
0.52
0.55
0.71
0.57
0.64
0.54
0.56
0.51
0.52
0.62
0.74
0.57
0.53
0.55
0.64
0.64
0.59
0.57
0.64
0.58
0.61
18
0.53
0.62
0.60
0.58
0.58
0.54
0.60
0.59
0.57
0.54
0.55
0.62
0.62
0.58
0.50
0.55
0.59
0.68
0.81
0.64
0.67
0.58
0.66
19
0.55
0.65
0.58
0.61
0.60
0.59
0.60
0.62
0.60
0.51
0.63
0.59
0.62
0.64
0.58
0.61
0.64
0.70
0.73
0.67
0.67
0.61
0.69
20
0.67
0.65
0.55
0.55
0.63
0.53
0.66
0.67
0.64
0.54
0.63
0.64
0.68
0.63
0.57
0.53
0.60
0.78
0.70
0.76
0.71
0.63
0.92
21
0.57
0.60
0.49
0.61
0.57
0.62
0.60
0.68
0.48
0.60
0.64
0.64
0.57
0.64
0.50
0.52
0.60
0.56
0.53
0.56
0.55
0.61
0.60
22
0.66
0.62
0.58
0.53
0.64
0.56
0.67
0.70
0.62
0.54
0.63
0.67
0.67
0.64
0.55
0.57
0.65
0.78
0.65
0.74
0.71
0.66
1.00
23
Kasetsart J. (Nat. Sci.) 41(2)
221
Etlingera pavieana
Alpinia nigra
Geostachys sp.
Amomum cf. testaceum
42
43
44
45
Elettaria cardamomum
Etlingera littoralis
40
41
Amomum sp. 15
A. dealbatum Roxb.
38
39
Amomum sp. 6a
Amomum sp. 14
36
37
Amomum sp. 17b
A. repoense Gagnep.
34
35
Amomum sp. 6b
A. uliginosum 4
32
33
Amomum sp. 3
Amomum sp. 2
30
31
Amomum sp. 13
A. siamense Craib
28
29
A. micranthum Ridl.
Amomum sp. 11
26
27
Amomum sp. 10
A. biflorum Jack
24
25
Table 3 (Continued)
0.60
0.70
0.67
0.64
0.55
0.60
0.64
0.67
0.54
0.62
0.71
0.70
0.59
0.69
0.66
0.69
0.65
0.64
0.57
0.62
0.60
1.00
24
0.64
0.64
0.55
0.63
0.67
0.57
0.63
0.65
0.70
0.56
0.61
0.70
0.65
0.55
0.58
0.58
0.57
0.74
0.73
0.67
1.00
25
0.65
0.64
0.56
0.59
0.54
0.63
0.54
0.67
0.66
0.57
0.68
0.58
0.66
0.60
0.57
0.57
0.58
0.75
0.63
1.00
26
0.62
0.57
0.54
0.56
0.57
0.49
0.56
0.57
0.61
0.52
0.57
0.67
0.57
0.53
0.53
0.51
0.57
0.64
1.00
27
0.65
0.64
0.59
0.59
0.59
0.53
0.59
0.64
0.64
0.60
0.60
0.63
0.74
0.65
0.60
0.64
0.64
1.00
28
0.51
0.67
0.56
0.60
0.62
0.66
0.59
0.60
0.52
0.67
0.54
0.63
0.64
0.82
0.57
0.60
1.00
29
0.53
0.65
0.61
0.63
0.55
0.59
0.61
0.54
0.60
0.57
0.61
0.62
0.60
0.63
0.88
1.00
30
0.60
0.62
0.57
0.63
0.57
0.59
0.63
0.53
0.64
0.57
0.58
0.64
0.57
0.58
1.00
31
0.55
0.70
0.58
0.57
0.60
0.68
0.64
0.65
0.47
0.71
0.60
0.65
0.62
1.00
32
0.54
0.63
0.54
0.54
0.71
0.55
0.59
0.58
0.61
0.52
0.54
0.52
1.00
33
0.59
0.71
0.54
0.67
0.62
0.61
0.74
0.72
0.60
0.66
0.62
1.00
34
0.63
0.62
0.61
0.69
0.58
0.67
0.66
0.64
0.59
0.56
1.00
35
0.53
0.57
0.54
0.59
0.53
0.69
0.60
0.69
0.43
1.00
36
0.54
0.60
0.54
0.64
0.60
0.53
0.57
0.55
1.00
37
0.65
0.64
0.53
0.60
0.53
0.61
0.70
1.00
38
0.57
0.62
0.60
0.63
0.69
0.59
1.00
39
0.53
0.64
0.51
0.57
0.60
1.00
40
0.55
0.54
0.49
0.52
1.00
41
0.57
0.64
0.64
1.00
42
0.44
0.57
1.00
43
0.54
1.00
44
1.00
45
222
Kasetsart J. (Nat. Sci.) 41(2)
Kasetsart J. (Nat. Sci.) 41(2)
223
Figure 3 Dendrogram depicting the genetic relationship of 45 accessions of Amomum based on AFLP
fingerprint, using similarity coefficient by DICE, clustering with UPGMA.
224
Kasetsart J. (Nat. Sci.) 41(2)
Subgroup D (II) consists of A. testaceum
4, A. cf. villosum, Amomum sp.5, Amomum sp.7,
A. uliginosum 2, Amomum sp.11, A. uliginosum 3,
A. villosum 1, A. rivale 1, A. micranthum, A. rivale
2, A. cf. rivale, Amomum sp.8, Amomum sp.4 and
Amomum sp.13. All members have spiny fruit and
leafy stem less than 1.50 m tall. Regarding
uliginosum 2 and 3 which were collected from Tak
province, they were separated from A. uliginosum
1 and 4 (from Nakhon Nayok and Ranong
provinces, respectively). Their morphological
characteristics differ from the ones in C group in
its shorter leafy stem and smaller inflorescence.
A possible explanation for this is that their
morphological characteristics were the result of
long time adaptation in the surrounding habitats
which resulted in two ecotypes of A. uliginosum.
B cluster consists of E and F groups. It
is characterized by smooth, ridged or winged fruit
(rarely spiny fruit).
E and F groups include Amomum sp.16,
Amomum sp.3, Amomum sp.2, Amomum sp.17a,
A. siamense, Amomum sp.6b, Amomum sp.6a,
Amomum sp. 17b, A. dealbatum, Amomum sp. 15,
Amomum sp. 10, Elettaria cardamomum, Alpinia
nigra, Amomum sp. 1, Amomum sp.14, A. repoense
and Etlingera pavieana.
The dendrogram suggests the placement
of smooth fruit (Amomum sp.16) between spiny
and winged fruit. Similar to the result of Amomum
sp.1 and 10 both of which are spiny fruit but were
placed among winged fruit species. Amomum
sp.17a and 17b from Nan province are similar in
their morphology but were placed in different
clusters. More study is needed to properly identify
the position of these species. A.siamense with fruit
longitudinally ridged is also placed in this group.
This species should be closely related to winged
fruit species. Although the cluster is not completely
separated from the others, all winged fruit species
are clearly placed. Therefore, the results have the
tendency to be consistent with the A.maximum
group of Xia et al. (2004).
The outgroup taxa (Alpinia, Elettaria,
Etlingera and Geostachys) are placed among
Amomum species. The result indicates a closer
relationship among them and the spiny fruit species
of Amomum. This result is similar to Xia et al.
(2004) who found that Etlingera littoralis was
placed in the clade of A.villosum group. The results
then confirmed that Etlingera is related to the
genus Amomum. Furthermore, some species of
Alpinia, Elettaria and Geostachys are also closely
related to the genus Amomum.
Twenty-six representives of Thai
Amomum can be classified into 3 groups by using
AFLP evidence: A. aculeatum, A. biflorum and A.
dealbatum groups.
The A. aculeatum group consists of 4
species: A. koenigii, A. uliginosum, A. aculeatum
and Amomum sp. 12. Species in this group have
smooth and spotted or spiny fruit, anther crest 3
lobes, leafy stem stout and usually more than 1.5
m tall.
The A. biflorum group contains 10
species: A. testaceum, Amomum cf. villosum,
Amomum sp.4, Amomum sp.5, Amomum sp.7,
Amomum sp.8, A.rivale, A. micranthum, Amomum
sp.11 and Amomum sp.13. All members of this
group are defined by smooth or spiny fruit. Most
species of this group are spiny fruit. In the case of
smooth fruit, its fruit shape is usually globular and
fruit colour is white or pale brown. The leafy stem
is usually slender and shorter than 1.5 m.
The A. dealbatum group contains 12
species of Amomum: A. dealbatum, A. repoense,
A. siamense, Amomum sp.1, Amomum sp.2,
Amomum sp.3, Amomum sp.6, Amomum sp.10,
Amomum sp.14, Amomum sp.15, Amomum sp.16
and Amomum sp.17. The species in this group are
characterized by winged, ridged or smooth fruit
(rarely spiny fruit and 3 lobes) and entire, round
or truncate anther crest.
Kasetsart J. (Nat. Sci.) 41(2)
CONCLUSION
AFLP markers classified Thai Amomum
species into three groups (A. aculeatum group, A.
biflorum group, and A. dealbatum group) which
correspond to the fruit and leafy stem
characteristics.
ACKNOWLEDGEMENTS
The authors are thankful to the curators
of Bangkok Herbarium (BK) and Forest
Herbarium (BKF) for their kind permission and
suggestion during this study. Also, this work was
supported by the TRF/BIOTEC Special Program
for Biodiversity Research and Training grant
T_14009.
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Abdalla, A.M, O.U.K. Reddy and K.M. El-Zik.
2001. Genetic diversity and relationships of
diploid and tetraploid cottons revealed using
AFLP. Theor. Appl. Genet. 102: 222-229.
Baker, J.G. 1892. Scitamineae, pp. 198-264. In
J. D. Hooker. Flora of British India vol.VI.
L. Reeve & Co., London.
Burtt, B.L. and R.M. Smith. 1972. Key species in
the taxonomic history of Zingiberaceae. Note
RBG. Edinb. 31: 177-227.
Dice, L.R. 1945. Measures of the amount of
ecological association between species.
Ecology 26: 297-302.
Gagnepain, F. 1906. Du Muséum. Bulletin de La
Societé Botanique de France. 53: 136-145.
Garcia-Mass, J., M. Oliver and H. GomezPaniagua. 2000. Comparing AFLP, RAPD and
RFLP markers for measuring genetic diversity
in melon. Theor. Appl. Genet. 101: 860-864.
Kiew, K. Y. 1982. The genus Elettariopsis
(Zingiberaceae) in Malaya. Notes RBG
Edinb. 42: 295-314.
Larson, S.R., B.L. Waldron, S.B. Monsen, L.St.
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John, A.J. Palazzo, C.L. McCracken and R.D.
Harrison. 2001. AFLP Variation in
agamospermous and dioecious bluegrasses of
Western North America. Crop Sci. 41: 13001305.
Linnaeus, C. 1753. Monandria. In Species
Plantarum-A facsimile of the first edition.
London, Bernard Quaritch Ltd. 560 p.
Loesener, T. 1930. Zingiberaceae (Amomum),
pp.599-602. In A. Engler and K. Prantl, ed.
Die Naturlichen Pflanzenfamilien, Leipzig,
E. Haberland.
Lubberstedt, T., A. E. Melchinger, C. DuBle, M.
Vuylsteke and M. Kuiper. 2000. Relationships
among early European maize inbreds: IV.
Genetic diversity revealed with AFLP markers
and comparison with RFLP, RAPD, and
pedigree data. Crop Sci. 40: 783-791.
Mizumoto, K., S. Hirosawa, C. Nakamura and S.
Takumi. 2003. Nuclear and chloroplast
genome genetic diversity in the wild einkorn
wheat, Triticum urartu, revealed by AFLP and
SSLP analyses. Hereditas 137: 208-214.
Rohlf, F.J. 1997. NTSYS-pc 2.01d: Numerical
taxonomy and multivariate analysis
system, version 2.01. Exeter Software,
Setauket, NY.
Sakai, S. and H. Nagamasu. 1998. Systematic
studies of Bornean Zingiberaceae I. Amomum
in Lambir Hills, Sarawak. Edinb. J. Bot.
55(1): 45-64.
Schumann, K. 1904. Zingiberaceae, pp. 1-458.
In A. Engler, ed. Das Pflanzenreich, Heft 20
(IV, 46) Leipzig.
Sirirugsa, P. 2001. Zingiberaceae of Thailand.
Pp. 63-77. In V. Baimai and R. Kumhom.
BRT Research Reports 2001. Biodiversity
Research and Training Program. Jirawat
Express Co.,Ltd., Bangkok.
Smith, R.M. 1985. A review of Bornean
Zingiberaceae:1(Alpineae). Notes RBG
Edinb. 42: 295-314.
Vos, P., R. Hoger, M.Bleeker, M.Reijans, T. van
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de Lee, M. Hornes, A. Frijters, J. Pot, J.
Peleman, M. Kuiper and M. Zabeau. 1995.
AFLP: a new technique for DNA
fingerprinting. Nucleic Acids Research
23(21): 4407-4414.
Xia, Y.M., W.J. Kress and L.M. Prince. 2004.
Phylogenetic analyses of Amomum
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Kasetsart J. (Nat. Sci.) 41 : 227 - 231 (2007)
Prediction of Sweet Corn Seeds Field Emergence under
Wet Soil Condition
Vichai Wongvarodom* and Wikanate Rangsikansong
ABSTRACT
Field emergence prediction of sweet corn seeds under wet soil conditions was studied using
three different quality seeds of Hawaiian Sugar Super Sweet and Super Sweet Argo Extra MT varieties.
The seeds were subjected to germinate in sand at room temperature. Germination was evaluated for 7
days after planting (DAP). Flooded germination was done by planting the seeds in 1000 g of clay soil in
a plastic basket, flooding at 1 cm above soil level for 5 hours and evaluating at 7 DAP. Field emergence
was studied under daily watering, three times a day, to simulate wet planting soil condition. Field
emergence evaluation was performed at 7 DAP. Results showed that the sweet corn seeds with 79.5091.00% germination of Hawaiian Sugar Super Sweet and Super Sweet Argo Extra MT varieties had
field emergence of 61-79% under wet soil condition. The lower quality seed had the field emergence of
lower than 60%. Sand germination and flooded germination in clay soil did not correspond to field
emergence under wet soil condition. The field emergence under wet soil condition of sweet corn seeds
could be predicted by the polynomial equations which gave better results than sand germination test.
Key words: field emergence, wet soil condition, sand germination, flooded germination, sweet corn
seeds
INTRODUCTION
Germination test is an analytical
procedure to evaluate seed viability under
standardized (favorable) laboratory conditions
(ISTA, 1999; AOSA, 2001). The percentage of
germination reflects the planting value of a seed
lot (Liu et al., 1999). However, it was frequently
found that standard germination did not correspond
to the field performance under stress planting
conditions (Delouche and Baskin, 1973; Vieira et
al., 1999). Sand germination test in room
temperature for corn seeds has been used widely
in many of the developing countries, due to simple
and low cost test, and using less specific
equipment. Most importantly, result of the test has
to be highly accurate with standard germination
(Dungpatra, 1986). Many crop production areas,
including Thailand, are faced with heavy raining
in the planting season which resulted in wet soil
condition in the planting field (Martin et al., 1988;
Jittham, 2002). Some vigor tests have been
develop for more accurate prediction for field
emergence under the stressful planting condition
in corn (Sawatdikarn, 2002), sweet corn (Jittham,
2002) and cucumber (Werakul, 2003) in the humid
Division of Agricultural Technology, Department of Industry and Technology, Faculty of Science and Technology, Prince of
Songkla University, Muang, Pattani 94000, Thailand
* Corresponding author, e-mail: seedinter@yahoo.com
Received date : 08/08/06
Accepted date : 15/11/06
228
Kasetsart J. (Nat. Sci.) 41(2)
tropics. The purpose of this study was to
investigate the relationship between sand
germination and field emergence and flooded
germination in predicting potential to field
emergence under wet planting soil condition.
MATERIALS AND METHODS
Two varieties, namely Hawaiian Sugar
Super Sweet and Super Sweet Argo Extra MT
commercial corn seeds obtained from Songkhla
Field Crop Research Center and a seed company
were used as high quality seeds with germination
of 86.50-91.00%. The seed samples having
different germination percentage (74.50-79.50 and
53.50-66.00%) after accelerated aging at 42°C
(AOSA, 2002) for 48 and 96 hours were used as
medium and low quality seeds, respectively. All
tests were done with four replications.
Sand germination
Fifty seeds per replication were subjected
to germinate in 1,000 g sand in plastic basket at
room temperature and were watered daily. First
and final counts were done at 4 and 7 days after
planting, respectively (AOSA, 2001). Normal
seedlings were averaged as the germination
percentage.
Flooded germination test
Fifty seeds per replication were subjected
to germinate in 1,000 g of clay soil in plastic basket
at room temperature. The planting baskets were
placed in plastic trays and were flooded at 1 cm
above soil level for 5 hours. After the end of
flooding duration, the water was drained (the soil
moisture content was still near to saturation after
drainage) and the seeds were placed for further
germinating. The germination percentage was
evaluated 7 days after planting (Jittham, 2002).
Field emergence under wet soil condition
Fifty seeds per replication were planted
at a depth of 2.5 cm in clay soil of the experimental
field of Division of Agricultural Technology,
Prince of Songkla University, Pattani. Irrigation
was given daily for three times a day, morning,
noon, and evening, to simulate wet planting field
condition. Also in the each irrigation time, the soil
was watered till wet. The normal seedlings were
counted at 4 and 7 days after planting, respectively.
Field emergence percentage was calculated using
the same procedure as described in AOSA (2001).
Analysis of variances for a completely
randomized design among sand germination, field
emergence, and flooded germination was
performed. The statistical significance of
differences among means was tested by Duncan,s
Multiple Range Test (DMRT). The relation
between sand germination and field emergence
were plotted as well as mathematical equations
for predicting the field emergence which are also
presented as polynomial.
RESULTS AND DISCUSSION
Comparison of the sweet corn seed
germination among sand, flooded condition, and
wet field planting condition was undertaken (Table
1). The sweet corn seeds with 79.50-91.00%
germination of Hawaiian Sugar Super Sweet and
Super Sweet Argo Extra M.T. varieties had field
emergence of 61-79%. The lower quality seeds
had the field emergence of lower than 60%.
Seeds of Hawaiian Sugar Super Sweet
and Super Sweet Argo Extra MT varieties in sand
test showed significant higher germination
percentages than field emergence under wet
planting field condition. The seeds germinated in
soil in baskets under the flooded condition gave
lower germination percentage than those in both
sand test and under the field condition. This is not
in agreement with the earlier report by Jittham
(2002) that the flooded germination gave the same
germination percentage as field emergence in rainy
season planting. This is probably due to the
Kasetsart J. (Nat. Sci.) 41(2)
229
loam soil, and as a consequence sweet corn seed
germinability dramatically reduced.
Sand germination showed a significant
correlation with field emergence both in Hawaiian
Sugar Super Sweet corn (r=0.694*) (Figure 1) and
Super Sweet Argo Extra MT corn (r=0.919**)
difference of soil texture used in the flooded
germination test causing different results. The high
amount of water holding after drainage in clay soil
used in this study might cause more reduction of
oxygen diffusion and become more compact
during the germination period comparing to sandy
Table 1 Germination of Hawaiian Sugar Super Sweet and Super Sweet Argo Extra MT corn seeds
with three quality classes tested in sand, flooded condition and under wet field condition.
Test methods and
Germination (%)
field conditions
High
Medium
Low
Hawaiian Sugar Super Sweet
Sand
91.00 A
74.50 A
66.00 A
Flooded condition
13.00 C
11.00 C
7.50 C
Wet field condition
60.50 B
55.00 B
46.50 B
F-test
**
**
**
C.V.(%)
12.73
12.31
14.05
Super Sweet Argo Extra MT
Sand
86.50 A
79.50 A
53.50 A
Flooded condition
48.00 C
27.00 B
29.00 C
Wet field condition
79.00 B
77.00 A
41.50 B
F-test
**
**
**
C.V.(%)
6.47
12.75
17.26
** = significant at P <0.01.
Means not sharing the same letter in each column of each variety are significantly different by DMRT.
Field emergence under wet soil
condition (%)
100
2
y = -0.0348x + 6.0133x - 198.64
2
R = 0.4815
80
60
40
20
0
0
20
40
60
80
100
Sand germination (%)
Figure 1 Relation between sand germination and field emergence under wet soil condition of Hawaiian
Sugar Super Sweet corn seeds, r=0.694*.
Kasetsart J. (Nat. Sci.) 41(2)
230
(Figure 2). This is in agreement with the previous
report by Kulik and Schoen (1982) that emergence
of sweet corn seeds in sand bench was highly
correlated with field emergence. However, sand
germination could not predict seedling emergence
of sweet corn under wet soil condition or in rainy
planting season as data shown in Table 1. The
results of this study showed that percentage of field
emergence calculated using the mathematical
equation, polynomial, is very closely to field
emergence (Figure 3). The data suggested that in
sweet corn, the calculated field emergence is
superior to sand germination and flooded
germination tests in predicting field emergence
Field emergence under wet soil
condition (%)
100
80
2
y = -0.0231x + 4.2883x - 119.05
2
R = 0.8443
60
40
20
0
0
20
40
60
80
100
Sand germination (%)
Figure 2 Relation between sand germination and field emergence under wet soil condition of Super
Sweet Argo Extra MT corn seeds, r=0.919**.
3
Germination difference (%)
2.5
2
1.5
High
1
Medium
0.5
Low
0
-0.5
HSSS
SAEMT
-1
-1.5
Figure 3 Germination differences between the predicted field emergence and field emergence under
wet soil condition of different quality seeds of Hawaiian Sugar Super Sweet (HSSS) and
Super Argo Extra MT (SAEMT) corn.
Kasetsart J. (Nat. Sci.) 41(2)
under wet soil condition planting.
Additional evaluations of other varieties
and lots as well as hybrid varieties are needed to
confirm the present results and to investigate more
optimum mathematical equation which could be
widely used in most sweet corn. Future study
should also be conducted to relate field emergence
results to drought planting condition.
CONCLUSION
1. Sweet corn seeds with 79.50-91.00%
germination of Hawaiian Sugar Super Sweet and
Super Sweet Argo Extra MT varieties had field
emergence of 61-79% under wet soil condition.
The lower quality seed had the field emergence of
lower than 60%.
2. Sand germination and flooded
germination in clay soil did not correspond to field
emergence under wet soil condition.
3. The field emergence under wet soil
condition of sweet corn seeds could be predicted
by the polynomial equations obtained from
relationship between sand germination and the
field emergence which gave better results than
sand germination test.
ACKNOWLEDGEMENTS
Thanks are due to the Faculty of Science
and Technology, Prince of Songkla University, for
financing this research. We wish to thank the
Songkhla Field Crop Research Center for
supplying the seeds.
LITERATURE CITED
Association of Official Seed Analysts. 2001. Rules
for Testing Seeds. The Association of Official
Seed Analysts. 126 p.
231
Association of Official Seed Analysts. 2002. Seed
Vigor Testing Handbook. Contribution
No.32 to the Handbook on Seed Testing. 105
p.
Delouche, J. C. and C. C. Baskin. 1973.
Accelerated aging techniques for predicting
the relative storability of seed lots. Seed Sci.
& Technol. 1: 427-452.
Dungpatra, J. 1986. Seed Testing and Analysis.
Agri Book Group. Bangkok. 194 p. (In Thai).
International Seed Testing Association. 1999.
International Rules for Seed Testing. Seed Sci.
& Technol. 27: Supplement.
Jittham, O. 2002. Germination test under water
stress conditions for sweet corn seed vigor
evaluation. MS. Thesis. Prince of Songkla
University. Songkhla.
Kulik, M.M. and J.F. Schoen. 1982. Germination,
vigor and field emergence of sweet corn seeds
infected by Fusarium moniliforme. Seed Sci.
& Technol. 10: 595-604.
Liu, H., L. O. Copeland, O. Schabenberger and
D. Jamieson. 1999. Variability of germination
tests of corn and soybeans. J. Seed Technol.
21: 25-33.
Martin, B.A., O.S. Smith and M.O. Neil. 1988.
Relationships between laboratory germination
tests and field emergence of maize inbreds.
Crop Sci. 28: 801-805.
Sawatdikarn, S. 2002. Germination test of corn
seed under water stress conditions. MS.
Thesis. Prince of Songkla University.
Songkhla.
Vieira, R. D., J. A. Paiva-Aguero, D. Perecin and
S. R. M. Bittencourt. 1999. Correlation of
electrical conductivity and other vigor tests
with field emergence of soybean seedlings.
Seed Sci. & Technol. 27: 67-75.
Werakul, S. 2003. Germination test under water
stress conditions for cucumber seed vigor
evaluation. MS. Thesis. Prince of Songkla
University. Songkhla.
Kasetsart J. (Nat. Sci.) 41 : 232 - 241 (2007)
Modifying Controlled Deterioration for Evaluating Field
Weathering Resistance of Soybean
Ye Changrong1,2, Prapa Sripichitt2*, Sunanta Juntakool2,
Vipa Hongtrakul3 and Arom Sripichitt4
ABSTRACT
To develop practical methods for testing field weathering resistance of soybean varieties, pods
and seeds from CM60 (susceptible) and GC10981 (resistant) were tested by seven treatments. Among
the treatments, modified incubator weathering (yellow pods were incubated at 30°C under 90-100%
relative humidity for 7 days) and the controlled deterioration (dry seeds were soaked in distilled water
for 60 minutes and then incubated at 41°C under 90-100% relative humidity for 3 days) showed widerange differences in seed germination and viability between CM60 and GC10981. These two treatments
were then tested on 11 soybean varieties comparing with a field weathering treatment. The germination
of seeds treated by controlled deterioration was highly correlated to the germination of seeds subjected
to field weathering treatment (r=0.964**, n=11). The viability of seeds submitted to both incubator
weathering and controlled deterioration were also correlated to the viability of seeds exposed to field
weathering (r=0.697* and 0.716*, n=11). The modified incubator weathering and controlled deterioration
methods were further used to evaluate the field weathering resistance of 139 F2 progenies derived from
the cross CM60/GC10981. There was a significant correlation between the incubator weathering and
the controlled deterioration by considering the germination and viability of seeds (germination r=0.331**,
viability r=0.425**, n=139). Both the modified incubator weathering and controlled deterioration were
efficient for evaluating the field weathering resistance of soybean varieties. Particularly, controlled
deterioration method was found to be a useful way for evaluating the field weathering resistance of
soybean seeds.
Key words: Glycine max (L.) Merr., field weathering resistance, incubator weathering, controlled
deterioration
INTRODUCTION
Soybean [Glycine max (L.) Merrill] is
one of the world’s leading sources of vegetable
1
2
3
4
*
oil and plant protein. As the world demand for
vegetable oil and protein meal continues to
increase, soybean production has spread rapidly
from the temperate zone into the hot and humid
Present address: School of Land and Food Science, The University of Queensland, Brisbane, Queeensland, Australia.
Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Plant Production Technology, Faculty of Agricultural Technology, King Mongkut’s Institute of Technology
Ladkrabang, Bangkok 10520, Thailand.
Corresponding author: e-mail: agrprs@ku.ac.th
Received date : 24/07/06
Accepted date : 26/01/07
Kasetsart J. (Nat. Sci.) 41(2)
tropics. Following the expansion, weather
conditions have become the major factor affecting
the soybean seed quality and production in tropical
and subtropical regions (Tekrony et al., 1980).
Weather conditions (mainly high temperature and
relative humidity) during the post-maturation and
pre-harvest period increase the difficulty in
producing soybean seed with high quality in the
tropics. This obstacle in producing good quality
seed is also the most important factor that limits a
distribution of soybean production in the tropics.
Soybean seed attains its highest vigor,
viability and potential quality at physiological
maturity (maximum seed dry weight). However,
due to high moisture content at physiological
maturity (about 55%), the seed cannot be harvested
commercially at this stage and must remain on the
plant through a desiccation period till the seed
reaches a harvestable moisture level. This period
may vary from a few days to over 3 weeks
depending on the environmental conditions in the
field. The seeds deteriorate rapidly during this
period (Delouche, 1980). Deterioration of seed
vigor, as well as viability, due to high temperature
and high relative humidity between the stages of
seed physiological maturity and harvesting is
referred to as field weathering (Tekrony et al.,
1980). Improving the field weathering resistance
of new varieties is an important objective for
soybean breeding programs in the tropics.
To evaluate the field weathering
resistance of soybean varieties, various methods
have been developed (Kueneman, 1982; Dassou
and Kueneman, 1984; Horlings et al., 1994).
Among these methods, delayed harvest and
incubator weathering have been widely used for
field weathering evaluation. However, it is difficult
to unify the maturation time of different varieties
to make them suffer the same weather damage by
delayed harvest (Kueneman, 1982). Dassou and
Kueneman (1984) compared three weathering
treatments and concluded that the incubator
weathering treatment (incubated at 30°C under
233
90-95% relative humidity for 10 days) minimized
intraplant variability and environmental effects
among genotypes with different maturity. The
incubator weathering method promised a practical
screening procedure for identification of resistance
to field weathering of soybean seeds and has been
widely used since then. However, the incubator
conditions are conducive to the rapid growth of
pathogens which is likely to encourage
deterioration (Balducchi and McGee, 1987).
Horlings et al. (1994) modified the treatment to
incubate the pods at 27°C under 90-100% relative
humidity for 4 days and indicated that the modified
incubator treatment had the most detrimental effect
on seed quality. But this treatment is probably too
gentle since the temperature in tropical soybean
fields is normally hotter and the duration is longer.
The field weathering resistance of
soybean is usually evaluated by the germination
and vigor of the weathered seeds. Controlled
deterioration method has been widely used to
evaluate seed vigor and viability of seeds
(Matthews, 1980; Powell and Matthews, 1981),
but the relationship between field weathering and
controlled deterioration has not been studied. The
purpose of this study is to evaluate the possibility
and efficiency of using controlled deterioration
treatment for evaluating the field weathering
resistance of soybean seeds.
MATERIALS AND METHODS
Plant materials
Twelve soybean varieties/lines, namely
Chiangmai 60 (CM60), Yodson, TGX814-26D,
Kalitor, 9520-21, 9519-1, Jakapan-1, Lee, CM
9501-3-17, MK-35, SSR 8502-14-1 and GC10981,
and 139 F2 progenies from the cross CM60 /
GC10981 were used in this study. Soybean CM60
and GC10981 were employed as susceptible and
resistant control for field weathering resistance,
respectively (Kaowanant, 2003).
234
Kasetsart J. (Nat. Sci.) 41(2)
Methods
Experiment A
Soybean CM60 and GC10981 were
grown in a field at the National Corn and Sorghum
Research Center, Nakhon Ratchasima Province.
Water, fertilizer, pesticide and fungicide were
applied when necessary. The yellow pods at
physiological maturity were harvested for field
weathering test. The field weathering resistance
was evaluated by seven treatments as follows.
1. Incubator weathering: Fresh yellow
pods (36 pods for each treatment) were placed
upright in the cells of a grid to avoid pod contact,
and then sealed in a plastic box with 1 cm water
under the grid to ensure a high relative humidity
(90-100%) during the incubation. The boxes with
pods inside were incubated by three treatments as
follows:
• 30°C for 10 days
• 35°C for 7 days
• 30°C for 7 days
After the incubation, the pods were dried,
threshed and the seeds were used for germinating
evaluation.
2. Accelerated ageing test and
controlled deterioration: Fresh yellow pods were
dried and threshed. The seeds (50 seeds for each
treatment) were subjected to the following four
treatments:
• Seeds were put in a wire-mesh tray.
The trays were then sealed in a plastic box with 1
cm water under the trays to ensure a high relative
humidity (90-100%) during the incubation. The
boxes with seeds inside were incubated at 41°C
for 3 days (standard AA test).
• Seeds were soaked in distilled water
for 15 minutes, and then put in wire-mesh tray
and sealed in a plastic box with 1 cm water under
the trays. The boxes with seeds inside were
incubated at 41°C for 7 days.
• Seeds were soaked in distilled water
for 30 minutes, and then put in wire-mesh tray
and sealed in a plastic box with 1 cm water under
the trays. The boxes with seeds inside were
incubated at 41°C for 4 days.
• Seeds were soaked in distilled water
for 60 minutes, and then put in wire-mesh tray
and sealed in a plastic box with 1 cm water under
the trays. The boxes with seeds inside were
incubated at 41°C for 3 days.
After the treatment, the seeds from each
treatment and 50 non-treatment seeds (control)
were germinated between wet papers at 25°C for
5 days. The normal seedlings, abnormal seedlings,
fresh ungerminated seeds, hard seeds and dead
seeds were counted (AOSA, 2000). The field
weathering resistance of the variety was evaluated
by germination (percentage of normal seedlings
and hard seeds) and viability (percentage of normal
seedlings, abnormal seedlings, fresh ungerminated
and hard seeds) of the treated seeds.
Experiment B
Soybean seeds of CM60, Yodson,
TGX814-26D, Kalitor, 9520-21, 9519-1, Jakapan1, Lee, CM 9501-3-17, MK-35, and SSR 850214-1 were grown in a greenhouse at the
Department of Agronomy, Kasetsart University,
Bangkok. Water, fertilizer, pesticide and fungicide
were applied when necessary. At physiological
mature stage, the yellow pods were subjected to
the following treatments:
1. Control (no treatment): The yellow
pods were harvested, dried and threshed, and then
50 seeds of each variety/line were germinated and
investigated as described in experiment A.
2. Field weathering: The yellow pods
were left on the plant (green and brown pods were
cut out) for 2 weeks with water spraying twice a
day. Then the pods were harvested, dried and
threshed. Fifty seeds of each variety/line were
germinated and investigated as described in
experiment A.
3. Incubator weathering: The yellow
pods (36 pods for each variety/line) were harvested
and placed upright in the cells of a grid, and then
Kasetsart J. (Nat. Sci.) 41(2)
sealed in a plastic box with 1 cm water under the
grid. The boxes with pods inside were incubated
at 30°C for 7 days. After the treatment, the pods
were dried and threshed, and fifty seeds of each
variety/line were germinated and investigated as
described in experiment A.
4. Controlled deterioration: The
yellow pods were harvested, dried and threshed.
After measuring the moisture content (MC, wet
weight basis) of the seeds, fifty seeds from each
variety/line were weighed and soaked in distilled
water for 60 minutes, and then the seeds were
quickly dried by tissue paper, weighed again and
put in a wire-mesh tray. The trays with seeds inside
were sealed in a plastic box with 1 cm water under
the trays to ensure a high relative humidity (90100%) during the incubation. The boxes were then
incubated at 41°C for 3 days. After the treatment,
the seeds were weighed, germinated and
investigated as described in experiment A.
Experiment C
Soybean seeds of CM60, GC10981 and
their F2 progenies were grown in a field at the
National Corn and Sorghum Research Center,
Nakorn Rachasima Province. Water, fertilizer,
pesticide and fungicide were applied when
necessary. The pods at physiological maturity were
harvested from each plant for field weathering
evaluation by incubator weathering and controlled
deterioration test:
1. Incubator weathering: Fresh yellow
pods (18 pods for each progeny) were placed
upright in the cells of a grid, and then sealed in a
plastic box with 1 cm water under the grid. The
boxes with pods inside were incubated at 30°C
for 7 days. After the treatment, the pods were dried
and threshed, and then the seeds were germinated
and investigated as described in experiment A.
2. Controlled deterioration: The
yellow pods were harvested, dried and threshed.
Twenty-five seeds from each progeny were soaked
in distilled water for 60 minutes, and then the seeds
235
were put in a wire-mesh tray. The trays with seeds
inside were sealed in a plastic box with 1 cm water
under the trays. The boxes were then incubated at
41°C for 3 days. After the treatment, the seeds
were germinated and investigated as described in
experiment A.
Data analysis
The frequency distribution, paired t-test
and Pearson correlation test of germination and
viability data were carried out following the
procedure of Komez and Komez (1984).
RESULTS
The efficiency of different treatments
To determine the optimum treatment for
evaluating the field weathering resistance of
soybean varieties, the seed quality of CM60 and
GC10981 was evaluated by seven treatments. The
germination and viability of soybean seeds of
CM60 and GC10981 after being subjected to seven
treatments (experiment A) are shown in Table 1.
For incubator weathering, after the pods were
incubated, the germination of CM60 and GC10981
seeds were 0-9.2% and 0-42.9%, and the viability
of CM60 and GC10981 seeds were 19.5-41.5%
and 27.3-72.5%. The most serious seed
deterioration was caused by incubating the pods
at 30°C for 10 days due to a serious pathogen
infection. The seeds of both varieties/lines had lost
their germinability (0% germination). Decrease in
both treating temperature and time could increase
the germination and viability of the treated seeds.
After incubating the pods at 30°C for 7 days, the
difference between CM60 and GC10981 in
germination (33.7%) and viability (31.0%) were
greater than the other two treatments. Therefore,
this treatment was considered to be more efficient
to distinguish the seed weathering of CM60 and
GC10981 than the other two treatments.
For the standard accelerated aging (AA)
test, the germination of CM60 and GC10981 were
236
Kasetsart J. (Nat. Sci.) 41(2)
Table 1 The germination and viability of the soybean seeds of CM60 and GC10981 after being subjected
to various treatments.
Treatment1
Germination (%)2
Viability (%)
CM
GC
Dif.
CM
GC
Dif.
IW: 30°C, 10 days
0.0
0.0
0.0
19.5
27.3
7.8
IW: 35°C, 7 days
6.6
15.2
8.6
35.6
46.8
11.2
IW: 30°C, 7 days
9.2
42.9
33.7
41.5
72.5
31.0
CD: Water 15 min. + 41°C, 7 days
8.0
32.0
24.0
52.0
60.0
8.0
CD: Water 30 min. + 41°C, 4 days
28.0
48.0
20.0
64.0
76.0
12.0
CD: Water 60 min. + 41°C, 3 days
32.0
70.0
38.0
68.0
85.0
17.0
AA: 41°C, 3 days
60.0
84.0
24.0
92.0
100.0
8.0
Control (no treatment)
92.0
94.0
2.0
100.0
100.0
0.0
1
2
IW= incubator weathering, CD= controlled deterioration, AA= accelerated ageing
CM= CM60, GC= GC10981, Dif.=difference= GC-CM.
decreased from 92% to 60% and from 94% to 84%,
respectively, but the viability was only decreased
slightly from 100% to 92% for CM60 comparing
to the control. For controlled deterioration
treatments, soaking the seeds in distilled water
prior to incubation further decreased the
germination and viability of the treated seeds. The
lowest germination and viability were caused by
soaking the seeds in distilled water for 15 minutes
and incubating at 41°C for 7 days. Soaking the
seeds in distilled water for 60 minutes and then
incubating at 41°C for 3 days showed the widest
difference in seed germination (38.0%) and
viability (17.0%) between CM60 and GC10981.
Thus this treatment was considered to be an
optimum one to distinguish the seed weathering
of CM60 and GC10981.
If ignore the fault treatment (incubator
weathering at 30°C for 10 days), by comparing
the means of the other 6 treatments (paired t-test),
there was significant difference between CM60
and GC10981 (t5=5.825, p=0.002 for germination
and t5=4.083, p=0.01 for viability). It was clear
that the field weathering resistance of CM60 and
GC10981 was significantly different.
Relationship among field weathering, incubator
weathering and controlled deterioration
To confirm the possibility of using the
modified controlled deterioration method for
evaluating the field weathering resistance of
soybean seed, field weathering (delayed harvest
with water spraying) along with incubator
weathering and controlled deterioration were
carried out on 11 soybean varieties/lines. The seed
characters and the changes in moisture content
during controlled deterioration treatments are
shown in Table 2. The seed moisture content of
all the varieties/lines increased (3.12 to 31.1%)
after soaking in distilled water for 60 minutes. The
moisture content continued to increase during
incubation. After soaking and incubation, the seed
moisture content increased from 15.72 to 31.25%
depending on the variety/line. The final moisture
content of all the treated seeds varied from 23.45
to 37.95%. Different varieties absorbed water and
moisture at different speeds. The seed moisture
content after soaking was highly correlated to the
final moisture content (r=0.951**, n=11) and the
final moisture content increase (r= 0.950**, n=11).
The increase in moisture content after soaking was
also correlated to the final moisture content
increase (r=0.952**, n=11). The treated seeds that
absorbed water faster also showed the higher
increases in moisture content finally. There was a
significant negative correlation between the
Kasetsart J. (Nat. Sci.) 41(2)
moisture content increment after soaking and the
moisture content increase after incubation (r=
-0.958**, n=11). The treated seeds that absorbed
more water during soaking absorbed less moisture
during incubation. In contrast, the treated seeds
that absorbed less water during soaking absorbed
more moisture during incubation.
237
The germination, viability and
hardseedness of 11 soybean varieties/lines after
being subjected to 3 different treatments (field
weathering, incubator weathering and controlled
deterioration) are shown in Table 3. The
germination and viability of seeds decreased after
being subjected to all the three treatments
Table 2 The seed characters and changes in moisture content during controlled deterioration treatment
of 11 soybean varieties/lines.
Variety/line
Seed
100 seeds
MC* before
MC after
MC after
color
weight (g)
soaking (%)
soaking (%)
incubation (%)
(increase)
(increase)
CM60
Yellow
15.11
7.04
30.33 (23.29)
31.63 (24.59)
Yodson
Black
12.13
7.20
15.47 ( 8.27)
26.76 (19.57)
TGX-814-26D
Yellow
9.47
7.16
22.29 (15.13)
30.83 (23.67)
Kalitor
Black
7.47
7.73
10.85 ( 3.12)
23.45 (15.72)
9520-21
Yellow
15.74
7.02
32.51 (25.49)
34.09 (27.07)
9519-1
Yellow
11.73
6.70
37.80 (31.10)
37.95 (31.25)
Jakapan-1
Yellow
11.84
7.05
15.95 (8.91)
25.78 (18.73)
Lee
Yellow
8.62
7.21
33.64 (26.43)
35.20 (27.99)
CM9501-3-17
Yellow
8.44
7.18
11.87 ( 4.69)
28.09 (20.91)
MK35
Yellow
10.25
7.40
21.66 (14.26)
29.94 (22.54)
SSR8502-14-1
Black
13.28
7.13
13.90 ( 6.77)
24.84 (17.71)
*
MC = moisture content
Table 3 The germination, viability and hardseedness of 11 soybean varieties/lines after being subjected
to 3 different treatments.
Variety/line
Germination (%)*
Viability (%)
Hard seeds (%)
CK FW IW
CD CK FW IW
CD CK FW IW
CD
CM60
86
44
30
50
90
70
46
56
4
12
0
0
Yodson
96
66
56
82
98
86
78
86
6
10
6
4
TGX-814-26D
90
62
82
76
96
78
98
88
0
0
0
0
Kalitor
98
76
68
96
100
90
94
100
24
52
12
34
9520-21
96
58
80
72
98
86
96
90
0
0
0
0
9519-1
96
54
56
64
98
84
82
68
0
0
0
0
Jakapan-1
98
56
58
70
98
76
64
80
22
26
0
8
Lee
90
56
68
66
92
80
94
86
0
0
0
0
CM9501-3-17
94
72
64
90
96
88
78
92
14
38
0
8
MK35
92
50
42
68
96
74
62
82
0
0
0
0
SSR8502-14-1
90
62
78
84
100
86
94
96
4
14
2
4
*
CK = check (no treatment), FW = field weathering, IW = incubator weathering, CD = controlled deterioration.
238
Kasetsart J. (Nat. Sci.) 41(2)
comparing to the control. There was a significant
correlation between the germination of seeds
treated by field weathering and by controlled
deterioration (r=0.964**, n=11), as well as the
viability of seeds subjected to these two treatments
(r=0.716*, n=11). The correlation between the
germination of seeds treated by field weathering
and by incubator weathering was not obvious.
However, there was a significant correlation
between the viability of seeds treated by field
weathering and by incubator weathering
(r=0.697*, n=11). To a certain extent, the
controlled deterioration treatment was more
efficient for indicating the field weathering
resistance of soybean than the incubator
weathering treatment. The correlation between the
germination of seeds treated by incubator
weathering and by controlled deterioration was not
obvious. There was only a correlation between the
viability of seeds treated by incubator weathering
and by controlled deterioration (r=0.739**, n=11).
All the tested varieties/lines showed higher
germination and viability than CM60. Among
these varieties/lines, Kalitor, a variety with black
seed coat and high percentage of hardseedness,
showed high germination and viability in every
treatment. It is a useful resource for future breeding
programs of soybean field weathering resistance.
Application of incubator weathering and
controlled deterioration on F2 progenies
The field weathering resistance of 139
F2 plants was investigated by incubator weathering
and controlled deterioration treatment. For
incubator weathering treatment, the seed
germination of the F2 progenies ranged from 21.3
to 81.6%, whereas those of their parents, CM60
and GC10981, were 34.7% and 75%, respectively.
The viability of the F2 progenies varied from 47.8
to 95.6%, whereas those of CM60 and GC10981
were 59.7% and 93.4%, respectively. For
controlled deterioration test, the germination of
the F 2 progenies extended from 20 to 82%,
whereas those of CM60 and GC10981 were 32%
and 72%, respectively. The viability of the F2
progenies ranged from 44 to 90%, whereas those
of CM60 and GC10981 were 54% and 94%,
respectively. The distribution of the germination
and viability of the F2 progenies are shown in
Figure 1. The germination and viability of the
seeds under both treatments showed normal
distribution (skewness < ± 0.5, kurtosis < ± 0.1).
There was a significant correlation between the
incubator weathering and the controlled
deterioration as assessed by germination
(r=0.331**, n=139) and viability (r=0.425**,
n=139). Both treatments could be used for
evaluating the field weathering resistance of
soybean seeds.
DISCUSSION
Since the field weathering occurs under
the hot and humid conditions in the field after the
seeds are physiologically mature, the most
common procedure for evaluating seed resistance
to field weathering is to leave the plants in the
field beyond the normal harvest period and then
assess the quality of the seed by visual score,
examining seed-borne fungi, seed vigor, or use a
combination of these assessment methods. This
delayed harvest technique for evaluating the field
weathering has several limitations, for example,
genotypes matured at different times are subjected
to different environmental weathering stresses and
different periods of weathering. It is difficult to
apply the same environmental stress conditions to
cultivars of different maturities by delayed harvest.
In an attempt to overcome the limitations of
delayed harvest, Kueneman (1982) developed
spreader row and overhead irrigation techniques
to accelerate weathering based on the delayed
harvest method and found the cultivar differences
were highly significant. Artificial seed weathering
methods, such as incubator weathering, can
minimize the effects of variable pod maturity. In
Kasetsart J. (Nat. Sci.) 41(2)
239
CD germination
IW germination
30
30
GC10981
20
10
GC10981
CM60
Frequency
Frequency
CM60
20
10
0
0
20.0
30.0
40.0
50.0
60.0
70.0
20.0
80.0
30.0
40.0
50.0
60.0
70.0
80.0
Germination (%)
Germination (%)
IW viability
CD viability
40
40
GC10981
GC10981
CM60
CM60
30
Frequency
Frequency
30
20
10
20
10
0
0
50.0
60.0
70.0
80.0
90.0
Viability (%)
40.0
50.0
60.0
70.0
80.0
90.0
Viability (%)
Figure 1 The distribution of seed germination and viability of the F2 progenies after being subjected
to incubator weathering (IW) and controlled deterioration (CD) test. The skewness and
kurtosis for each distribution are as follows: IW germination (0.079, -0.523), CD germination
(-0.319, -0.742), IW viability (0.199, 0.656), CD viability (-0.395, 0.721).
experiment A of this study, three incubator
weathering treatments were carried out to identify
the difference between susceptible variety CM60
and resistant variety GC10981. After the fresh
yellow pods were incubated at 30°C and 90-100%
relative humidity for 10 days, a serious pathogen
infection occurred, and some seeds even
germinated during the incubation. The remaining
seeds had lost their germinating ability (0%
germination) and showed a very low viability
(19.5% and 27.3%). Increasing the temperature
and shortening the incubating time (35°C, 7 days)
reduced the pathogen infection and germination
during incubation, but the treatment still caused
serious damage to the seeds. This treatment had
been successfully used to identify the field
weathering difference between CM60 and
GC10981 by Kaowanant (2003). However, the
constant temperature at 35°C practically does not
occur in the soybean field. By reducing the
temperature (30°C, 7 days), the pathogen growth
and seed germination during incubation were
controlled. The results showed obvious differences
in germination and viability between CM60 and
240
Kasetsart J. (Nat. Sci.) 41(2)
GC10981 which could be used to identify the field
weathering resistance of these varieties.
On the other hand, since the ability of
seed coat to absorb moisture from the environment
is a decisive factor in field weathering, the faster
the seed absorbs moisture from the environment,
the more serious the weathering that occurs. Thus,
the controlled deterioration method developed by
Matthews (1980) was modified to evaluate the
seed weathering. The modified treatments
emphasized the relationship between seed
moisture absorbing speed and seed weathering.
The original controlled deterioration method was
modified into three combinations of water soaking
time and incubating time to compare with the
standard accelerated aging test. Soaking the seeds
in distilled water for 60 minutes and incubating at
41°C under 90-100% relative humidity for 3 days
showed a wide-ranging difference in germination
and viability between CM60 and GC10981. Since
the difference in field weathering resistance of
these two soybean varieties had been stated by
Kaowanant (2003), the treatments which showed
more difference between these varieties should be
more efficient for distinguishing the field
weathering resistance of soybeans. Thus, this
treatment was considered to be efficient for testing
field weathering resistance of soybean.
The efficiency of the modified incubator
weathering and controlled deterioration were
further confirmed on 11 soybean varieties/lines in
experiment B. Highly significant correlation was
found between the germination of seeds treated
by field weathering and by controlled deterioration
(r=0.964**, n=11), as well as the viability of seeds
subjected to these two treatments (r=0.716*,
n=11). It is possible to use this controlled
deterioration method to predict the field
weathering resistance of soybean varieties. There
was highly significant negative correlation
between the water absorbing speed and the final
germination of the treated seeds (r=-0.785**,
n=11), it was confirmed that the seeds absorbing
water faster would suffer more serious
deterioration during the incubation resulting in a
lower percentage of germination. The incubator
weathering and controlled deterioration methods
were further applied to evaluate the field
weathering resistance of F2 progenies derived from
the cross CM60/GC10981. The germination and
viability of seeds subjected to both treatments were
continuous with a normal distribution. There was
highly significant correlation between incubator
weathering and controlled deterioration by
considering germination and viability of the seeds
(germination r=0.331**, viability r=0.425**,
n=139). Both incubator weathering and controlled
deterioration may be used to determine the field
weathering resistance of soybean varieties.
However, it is difficult to treat a great number of
pods at the same time in incubator weathering test
due to the laboratory limitations, especially in
large-scale breeding programs. Controlled
deterioration method makes it possible to harvest
the pods at physiological maturity, dry to a similar
moisture content level, and then store for testing.
This will be very beneficial to large-scale
screening in breeding programs.
CONCLUSION
The modified controlled deterioration
(soaking seeds in distilled water for 60 minutes
and incubating at 41°C under 90-100% relative
humidity for 3 days) was confirmed to be useful
for evaluating field weathering resistance of
soybean seeds based on the hypothesis of a
correlation between the water absorbing speed and
field weathering resistance of seeds, especially for
large-scale soybean breeding programs that focus
on seed quality.
ACKNOWLEDGEMENTS
This study was partly supported by the
Graduate Research Scholarship from the Graduate
Kasetsart J. (Nat. Sci.) 41(2)
School, Kasetsart University. The authors also
would like to express thanks Mr. Adrian Hillman
for editing the English grammar of this paper and
to Miss Sirikwan Sawatsitung, Miss Peeraya
Thanarog, Miss Phan Thi Thanh, and Miss
Supaporn Dechkrong for their helps in the
experimental field.
LITERATURE CITED
Association of Official Seed Analysts. 2000. Rules
for testing seeds. Proc. Assoc. Off. Seed Anal.
60(2): 1-39.
Balducchi, A.J. and D.C. McGee. 1987.
Environmental factors influencing infection
of soybean seed by Phomopsis and Diaporthe
species during seed maturation. Plant Disease
71: 209-212.
Dassou, S. and E.A. Kueneman. 1984. Screening
methodology for resistance to field weathering
in soybean seed. Crop Sci. 24: 774-779.
Delouche, J.C. 1980. Environmental effects on
seed development and seed quality.
HortScience 15(6): 775-780.
Horlings, G.P., E.E. Gamble and S.
Shanmugasundaram. 1994. Weathering of
soybean in the tropics as affected by seed
characteristics and reproductive development.
Trop. Agric. (Trinidad): 71(2): 110-115.
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Kaowanant, R. 2003. Varietal Differences of
Soybean in Quality and Physical
Characteristics of Seeds in Resistance to
Field Weathering. M.S. thesis. King
Mongkut’s Institute of Technology
Ladkrabang. Bangkok.
Komez, K.A. and A.R. Komez. 1984. Statistical
Procedures for Agriucltural Research. 2nd
ed. International Rice Research Institute.
John Willey & Sons, Inc., New York.
Kueneman, E.A. 1982. Genetic differences in
soybean seed quality screening methods for
cultivar improvement, pp 31-41. In J.B.
Sinclair and J.A. Jackobs (eds.). Soybean
Seed Quality and Stand Establishment.
International Agriculture Publications.
University of Illinois, Urbanna-Champaign,
IL.
Matthews, S. 1980. Controlled deterioration: A
new vigor test for crop seeds, pp 647-660. In
P.D. Hebblethwaite (ed.). Seed Production
Butterworths, London.
Powell, A.A. and S. Matthews. 1981. Evaluation
of controlled deterioration, a new vigor test
for crop seeds. Seed Sci. and Tech. 9: 633640.
Tekrony, D.M., D.B. Egli and A.D. Phillips. 1980.
Effect of field weathering on the viability and
vigor of soybean seed. Agron. J. 72: 749-755.
Kasetsart J. (Nat. Sci.) 41 : 242 - 250 (2007)
Composite Line Method for the Development of Early Generation
Hybrids of Maize (Zea mays L.)
Nguyen Phuong, Krisda Samphantharak* and Vatcharee Lertmongkol
ABSTRACT
Six commercial single crosses were used for the improvement of composite and inbred lines.
Modified S1-full sib selection was applied to improve the three sister line composite. Lines were visually
selected under low-competition environment in honeycomb arrangement with equilateral triangular
side of 0.866 m. Testcross as well as diallel cross were applied to identify high combining lines. All
yield trials were conducted in randomized completed block design with 4 replications, 1 row plot of 5 m
long and 0.75 × 0.25 m plant spacing. Standard cultural practices were regulated and irrigation was
applied as needed.
Statistically, there was no clear advantage of yield between composite and inbred lines in early
generation testcrosses. Besides, the diallel sets of both groups of lines gave similar results. However,
the top hybrids of overall trials came from composite crosses even though it was not significant. In
addition, composite lines were superior to S3 lines in yield, earliness and plant height. Modified S1-full
sib selection is a flexible breeding method but its merit for the construction of early generation hybrids
must be thoroughly investigated even though the positive results were observed.
Key words: maize breeding, testcross, honeycomb, composite line
INTRODUCTION
Development of single cross hybrid of
maize is the ultimate goal of most of maize
breeding programs. However, finding stable high
yield inbred lines to ensure the high level of
economic return for commercial hybrid seed
production is the main obstacle of small and new
emerged single cross development programs.
Combined line selection and testing for combining
ability is time and space consuming processes.
Instead of five or six generations of selfing usually
practiced in the development of inbred lines,
composite-sibbing lines from individual of S1
progenies have been proposed (Kinman, 1952).
The method fixed the composite-sibbed lines since
the first selfing and therefore improvement in the
combining ability or other characteristics of
composite-sibbing lines can not be made after
several generations of mass sibbing unless
effective selection is practiced. In other way, line
selection from cross between closely related
parents has been proved to be an effective method
for inbred line development (Rasmusson and
Phillips, 1977; Troyer, 1999). Selection for high
and low yield lines effectively separated lines into
high and low combining ability groups but yield
of lines within group cannot be used as criterion
Department of Agronomy, Faculty of Agriculture, Kasetsart university, Bangkok 10900, Thailand.
* Corresponding author, e-mail: agrkrs@ku.ac.th
Received date : 10/04/06
Accepted date : 04/09/06
Kasetsart J. (Nat. Sci.) 41(2)
for combining ability of lines (Lamkey and
Hallauer, 1986). In addition, for effective
differentiation of lines, Fasoula and Fasoula (1997)
proposed line selection under nil-competition
environment in honeycomb designs. In order to
improve yield and combining ability of population,
Landi and Frascaroli (1993) applied full-sib
selection in F2 population of single cross. The
method proved to be very effective for several
cycles of selection. However, the previous study
of Genter (1976) which applied the same method
suggested that using S1 instead of S0 to form fullsibs was more effective to identify high yielding
full-sibs as well as in improvement of population
per se. This finding agreed well with suggestion
of Lonnquist (1950) that testing for combining
ability after one generation of selfing is desirable
when the composite sib-breeding method is used.
The above finding suggested that
alternate selfing and full sibbing among few
closely related lines under low-competition
environment should lead to uniform, high yield
and high combining composite lines as high level
of homozygosity is approached and provide a
chance for continuous improvement of composite
lines in the successive cycles.
The present study therefore aim to
formulate the effective breeding method for the
development of composite lines and evaluate its
merit as compared to the conventional line
selection with early generation testing for
combining ability. The modified S 1 -full sib
selection within related lines is proposed.
MATERIALS AND METHODS
Six commercial single cross hybrids
comprised Monsanto 949, Monsanto 919, Pioneer
A33, Pioneer 3012, Pacific 984 and Syngenta NK
48 were planted in normal plant spacing (0.75 ×
0.25m) and selfed to obtain S1 ears. Nine S1 ears
within each family were randomly grouped in to
3 ear sets, 3 sets per family and therefore resulted
243
in 18 sets of 3 S1 and 54 individual S1 lines. They
were separately ear-rowed in honeycomb
arrangement (HC) with equilateral triangular side
of 0.866m. Three best S1 plants within each set
were intercrossed (full sibbing) to form 18 intraset diallel crosses which will be refered to as full
sib sets while 3 best S1 plants from each family
were also selfed to obtain 18 S2 lines.
Consequently, they were ear-rowed in
HC, the 18 S 2 plants were selfed as well as
testcrossed to the inbred tester, KRi 208 to obtain
18 S3 lines and 18 testcrosses, S2 × KRi 208
hybrids. The best S2 lines by visual selection, one
from each family, were also intercrossed to form
15 diallel crosses of 6 S2. In the meantime, the
best 3 F1 plants from each full sib set were crossed
in all possible combinations to form 18 composite
lines and they will be referred to as composite line
cycle-1 (C#1). The method is essentially similar
to S1 and full-sib selection of which it will be
referred to as modified S1-full sib selection for
composite line development. Afterward, C#1 were
testcrossed to KRi 208. As a result, 18 C#1
testcrosses were obtained. In addition, the best C#1
by visual selection, one from each family, were
intercrossed to form 15 diallel crosses of 6 C#1.
Yield trials of 18 S3 lines, 18 C#1, 18
testcrosses of S2 × KRi 208, 18 testcrosses of C#1
× KRi 208, 15 diallel crosses of 6 C#1 and 15
diallel crosses of 6 S2 lines were conducted in
separate trials in adjacent areas in randomized
completed block design with 4 replications, 1 row
plot of 5m long and 0.75 × 0.25m plant spacing.
Five original hybrids were included as common
checks in all hybrid yield trials. Pacific 984 was
excluded and replaced by Suwan 4452 because
the former was dropped out from the market and
there was no seed available.
All experiments were conducted from
September 2004 to March 2006 at National Corn
and Sorghum Research Center, Suwan Farm, in
Nakhon Ratchasima province (14030’N, 101030’
E, and 356m asl.), Thailand under standard cultural
Kasetsart J. (Nat. Sci.) 41(2)
244
demonstrated a superior shelling percentage over
other lines including the checks.
In comparison S3 with C#1 lines, the C#1
lines were consistently superior in the
characteristics used as measures of vigor; grain
yield, earliness of anthesis and silking, plant and
ear height. They were earlier, taller and had higher
yield regardless of germplasm sources. Moreover,
better distribution of germplasm sources of top10 C#1 was evident. All six germplasm sources
were present in the top-10 C#1 while in the top10 S 3 lines, visual selection leaned toward
Monsanto 949 and Pacific 984. The results
indicated that C#1 was more stable by outcrossing.
On the other hand, inbred lines from each
germplasm source should have different level of
inbreeding depression and thus selection for
performance per se was biased toward the less
inbreeding depression germplasm. In this case,
Pioneer 3012 was lost from the top-10 S3 lines.
The present results agreed well with
report presented by Kinman (1952) of which
practices. Basal fertilizers were applied at planting
time at the rate of 75 kg ha-1 of N and 100 kg ha-1
of P2O5. Top-dressing was done at the 6 to 8 leaf
stages with the rate of 75 kg N ha-1. Pre-emergence
herbicides, Atrazine and Alachlor were used by
mixing at the rate 1.5 and 1 kg a.i. per ha,
respectively. Thinning was done at 14 days after
sowing. Irrigation was applied when necessary.
RESULTS AND DISCUSSION
Mean grain yields and other agronomic
traits of top-10 S3 lines are presented in Table 1.
All selected lines were statistically not different
except line 406-3 and only line 401-6 showed
significant difference over the inbred check, KRi
208. However, the KRi 208 had higher level of
homozygosity and therefore, if further inbreeding
was applied, all lines were expected to be similar
in yield level. There was no clear evidence for the
advantage or disadvantage of other agronomic
traits among the top ten lines but line 403-5
Table 1 Grain yields at 15 percent moisture and other agronomic traits of top 10 S3 lines and KRi 208
at Suwan Farm, Thailand in November 2005 (dry season).
S3 lines
Source of
Grain
Days to
Days to
Moisture
Plant
Ear
Shelling
germplasms
Yield
Anthesis
Silking
Content
Height
Height
(%)
(days)
(days)
(%)
(cm)
(cm)
401-6
Pac.984
(ton/ha)
4.21 a
68.7 a-d
69.0 bcd
21.9 a-d
131.5
54.2 bc
76.6 a-d
402-6
Mon.949
3.77 ab
68.3 a-e
67.3 edf
25.6 a
140.2
60.3 abc
73.6 bcd
404-4
Pio.A33
3.71 ab
67.7 b-f
67.3 edf
23.7 abc
140.3
63.5 abc
73.9 bcd
402-8
Mon.949
3.63 ab
66.7 ef
67.0 ef
24.9 ab
134.3
60.7 abc
75.6 bcd
401-9
Pac.984
3.40 ab
70.0 a
68.8 b-e
23.4 a-d
131.3
54.3 bc
79.1 ab
402-7
Mon.949
3.38 ab
66.3 f
66.3 f
23.9 abc
138
52.3 c
75.7 bcd
405-4
Syn. 48
3.36 ab
67.0 def
67.7 c-f
22.8 a-d
134.7
60.5 abc
71.8 cd
403-5
Mon.919
3.21 abc
68.0 b-f
68.0 b-f
20.2 cd
126
52.3 c
82.2 a
401-7
Pac.984
3.08 a-d
70.0 a
69.0 bcd
22.0 a-d
131.5
58.7 abc
78.0 ab
406-3
Pio.3012
2.89bcd
70.0 a
68.8 b-e
24.5 ab
151.5
68.2 ab
74.6bcd
KRi 208
Pio.3012/3013
3.01bcd
69.0 abc
69.7 ab
25.5 a
120.3
50.2 c
73.5 bcd
Mean
3.48
68.2
68.0
23.4
132.8
56.7
76.0
F-value1/
**
**
**
**
ns
**
**
19.568
1.338
1.353
8.489
10.561
13.461
3.973
CV(%)
1/
ns: non significant, * : significant, ** : highly significant
Kasetsart J. (Nat. Sci.) 41(2)
selective mass sibbing within individual S 1
progenies was used. In Kinman’s words, the
population is closed at the time of first sibbing, it
should not be expected that improvement in the
combining ability or other characteristics of
composite-sibbed lines will be made even after
several generations of mass sibbing unless
effective selection is practiced. Unlike Kinman’s
method, the modified S 1 -full sib selection
employed in the present study provided a more
flexible approach. Selection for S1 performance
per se alternate with diallel cross of individual of
3 selected S1 lines (full sibbing) should improve
general combining ability as well as specific
combing ability of S1 lines from successive cycles.
In the meantime, the newly emerged individual
S1 as well as full sib of each cycle can be fixed by
mass sibbing method and used in early generation
hybrid combinations while the successive cycles
of composite sets move slowly toward higher level
of homozygosity and hence more uniform lines
and hybrids in later stages.
Lamkey and Hallauer (1986) found that
245
inbred line performance per se can be used as a
criterion to differentiate combining ability between
high and low yield inbreds. However, yield per se
within high or low yielding groups cannot be used
to predict line performance in hybrid
combinations. Yielding ability of line per se in
Table 1 and their testcross performance in Table 3
clearly supported the above finding. Since all 18
inbred lines came from the top-3 high yield lines
of each original hybrid therefore they should be
considered high yield lines. However, their
yielding ability did not represent the combining
ability of lines in the testcross combinations with
the inbred tester (KRi 208), line 403-4 which was
excluded from the top-10 lines gave the highest
yield in the testcrosses while the top yield line,
401-6 ranked 9th in testcrosses. Besides, only two
Pioneer lines, 406-1 and 404-4 were present in the
top-10 testcrosses. This is not unexpected because
the tester line, KRi 208 derived from Pioneer 3012/
Pioneer 3013. Therefore, genetic background of
tester played an important role in the combinations
with tested lines. However, 406-1/KRi 208 is
Table 2 Grain yields at 15 percent moisture and other agronomic traits of composite lines of cycle 1st
at Suwan Farm, Thailand in November 2005 (dry season).
Compos
Source of
Grain Yield
Days to
Days to
Moisture
Plant
Ear
Shelling
_ite lines
germplasms
(ton/ha)
Anthesis
Silking
Content
Height
Height
(%)
(days)
(days)
(%)
(cm)
(cm)
Set 4
Mon.949
6.13 a
67.3 abc
67.3 bc
25.5 a
168.7
82
77.2
Set 5
Mon.949
5.53 ab
65.0 d
67.0 bc
25.6 a
165.6
71.2
79.2
Set 18
Mon.919
5.26 abc
63.0 e
66.0 c
21.4 cd
157.4
63.9
80.8
Set 10
Syn. 48
4.91 bc
66.0 cd
66.7 bc
21.9 bcd
173.3
80.3
79.6
Set 11
Syn. 48
4.87 bcd
66.0 cd
67.0 bc
22.9 bcd
161.2
67.7
72.9
Set 2
Pac.984
4.81 bcd
68.3 ab
67.7 bc
23.8 abc
171.8
75.2
79.4
Set 8
Pio.A33
4.66 bcd
66.7 a-d
67.0 bc
22.8 bcd
165.8
80.8
80.2
Set 14
Pio.3012
4.64 bcd
68.0 abc
67.7 bc
22.0 bcd
158.1
69.2
80.2
Set 3
Pac.984
4.63 bcd
68.7 a
68.3 ab
23.8 abc
159.3
65.5
79.7
Set 7
Pio.A33
4.58 bcd
66.3bcd
67.0 bc
22.6 bcd
150.3
67.7
79.5
Mean
5.00
66.5
67.2
23.2
163.2
72.4
78.9
F-value1/
*
**
*
**
ns
ns
ns
14.076
1.73
1.575
5.815
7.111
15.454
3.299
CV(%)
1/
ns: non significant, * : significant, ** : highly significant
Kasetsart J. (Nat. Sci.) 41(2)
246
Top S 2 lines, one from each of six
original hybrids were intercrossed and the top-10
interfamily hybrids are presented in Table 4. As
expected, the average of top-10 S2 interfamily
diallel hybrids was lower than that of top-10 S2
testcrosses because both parental lines of S2interfamily hybrids were more heterogeneous than
the tester line, KRi 208 in S2 testcrosses. Therefore,
the specific combining ability of lines were more
pronounced. Seven out of 10 S 2-interfamily
hybrids were involved with Pioneer 404-6 and
Pioneer 406-1 and 6 out of 10 were crosses
between Pioneer and Monsanto lines. Evidently,
both germplasm sources complimented each other
of which they showed a good heterotic pattern.
essentially a backcross to sister line and ranked
6th in the top-10 testcrosses indicated a strong
additive effect in this hybrid combination. Since
different testers gave different performance with
the same group of lines (Castellanos et al., 1998),
all high yield lines should be tested for their hybrid
combinations directly to their counterpart parental
lines to identify the best hybrid combination.
Statistically, all top-10 testcrosses
yielded as high as the top-4 checks but somewhat
better than Monsanto 949 and Monsanto 919.
However, 403-4/KRi 208 gave an outstanding
feature of yield and earliness even though it was
taller and lower in shelling percentage than the
average.
Table 3 Grain yields at 15 percent moisture and other agronomic traits of top 10 testcrosses between
selected S2 × KRi 208 and original hybrids conducted at Suwan Farm, Thailand in November
2005 (dry season).
Lines ×
KRi
2082/
Source of
Grain Yield
Days to
Days to
Moisture
Plant
Ear
Shelling
germplasms
(ton/ha)
Anthesis
Silking
Content
Height
Height
(%)
(days)
(days)
(%)
(cm)
(cm)
403-4
Mon.919
8.96 a
61.3 h
61.7 j
22.6 def
165.8 b-f
81.7 a-f
75.9 h-k
405-5
Syn. 48
8.82 ab
62.3 e-h
64.0 d-i
23.0 c-f
153.3 fgh
70.3 fg
77.1 f-i
402-6
Mon.949
8.40 a-d
62.0 fgh
62.7 hij
24.9 abc
161.0 d-h
80.3 a-f
75.3 k
405-4
Syn. 48
8.14 a-e
61.7 gh
62.3 hij
23.2 c-f
158.0 d-h
79.0 b-g
77.5 efg
405-6
Syn. 48
8.08 a-f
63.3 b-g
64.3 b-g
24.3 bcd
157.6 d-h
82.7 a-f
75.6 ijk
406-1
Pio.3012
8.07 a-f
63.0 c-h
63.7 e-i
24.1 bcd
164.2 b-f
81.7 a-f
78.3 def
402-7
Mon.949
8.00 a-f
61.3 h
62.0 ij
24.0 bcd
150.0 gh
73.7 d-g
77.0 f-i
404-4
Pio.A33
7.89 a-f
65.0 ab
65.3 c-f
23.6 b-f
159.8 d-h
76.8 b-g
75.4 jk
401-6
Pac.984
7.70 a-g
62.7 e-h
63.3 f-j
23.4 c-f
156.3 e-h
72.7 d-g
79.5 cd
403-5
Mon.919
7.66 a-g
62.0 fgh
62.7 hij
21.9 ef
162.1 c-h
89.8 a-g
81.1 bc
Check
Pio.A33
8.47 abc
64.7 bc
66.0 bcd
21.9 ef
182.0 a
91.9 a
79.8 bc
Check
SW 4452
8.24 a-e
66.7 a
68.7 a
25.0 abc
177.3 ab
84.2 a-e
76.3 g-k
Check
Pio.3012
8.08 a-f
66.7 a
68.0 ab
22.4 def
165.2 b-f
89.1 ab
77.2 fgh
Check
Syn. 48
7.86 a-f
64.0 b-e
66.0 bcd
22.7 def
171.3 a-d
74.4 dfg
77.9 ef
Check
Mon.949
7.57 b-g
62.7 e-h
64.3 d-g
26.7 a
178.3 ab
81.6 a-f
77.9 ef
Check
Mon.919
7.32 c-g
63.3 b-g
65.0 c-g
23.9 b-e
176.1 abc
88.1 abc
82.9 a
10 topcrosses
8.17
62.5
63.2
23.5
158.8
78.9
77.3
F-value1/
**
**
**
**
ns
*
**
11.006
1.836
1.985
5.198
5.296
9.685
1.179
Mean of top
CV(%)
1/
2/
ns: non significant, * : significant, ** : highly significant
Pedigree of KRi 208 is Pio.3012/Pio.3013
Kasetsart J. (Nat. Sci.) 41(2)
Although most of S2-interfamily hybrids were
significantly not different from the checks, 4046/402-6 (Pioneer A33/Monsanto 949) gave
outstanding features for yielding ability, earliness,
plant and ear height while retained good shelling
percentage. Therefore, beside the conventional
testcross program, diallel cross between the top
high yield lines is necessary for thorough use of
germplasms and identification of new unique
hybrid combination.
The numbers of original germplasm
sources involved in top-10 S2 and C#1 testcrosses
in Table 3 and 5 were almost the same; 4:5
(Monsanto), 3:3 (Syngenta), 2:1 (Pioneer) and 1:1
(Pacific) indicated that they responded similaly to
247
the same tester, even though each S2 line derived
from visual selection within each composite set.
However, average yield of S2 testcrosses was
higher than that of C#1 testcrosses but top testcross
yields of both groups as well as the best check
were more or less the same.
The average yield of S2 diallel crosses
in Table 4 and that of C#1 diallel crosses in Table
6 were almost the same but with the higher trend
toward the C#1 lines. Evidently, general
combining ability of S2 and C#1 were somewhat
the same even though the C#1 were more
heterogeneous. Surprisingly, the top-2 hybrids of
C#1 gave higher yield over other hybrids and
checks tested in the present studies eventhough
Table 4 Grain yields at 15 percent moisture and other agronomic traits of interfamily diallel hybrids
of selected S2 lines and original hybrids at Suwan Farm, Thailand in November 2005 (dry
season).
S2 × S 2
Source of
Grain
Days to
Days to
Moisture
Plant
Ear
Shelling
germplasms
Yield
Anthesis
Silking
Content
Height
Height
(%)
(cm)
(cm)
(ton/ha)
(days)
(days)
(%)
25.1 ab
171.9 de
404-6×402-6
Pio.A33 × Mon.949 8.86 a
62.3 d
63.3 g
404-6×403-6
Pio.A33 × Mon.919 7.82 abc
62.7 cd
64.3 efg
23.1 b-f 170.3 de
81.7 c-g 77.2 efg
406-1×402-6
Pio.3012 × Syn.48
67.3 abc
24.0 b-e 202.3 a
106.9 a
406-1×405-5
Pio.3012 × Syn.48
7.53 b-e
64.0 bcd
65.3 def
24.3 bcd 197.5 ab
96.7 ab
77.4 efg
403-6×401-9
Mon.919 × Pac.984
7.51 b-e
62.3 d
63.7 fg
21.8 efg 169.2 de
77.0 fg
80.6 b
406-1×404-6
Pio.3012 × Pio.A33
7.47 b-e
65.7 ab
66.7 bcd 23.8 b-f 194.7 abc 97.2 ab
78.2 c-f
404-6×401-9
Pio.A33 × Pac.984
7.11 cde
64.0 bcd
66.3 bcd 23.3 b-f 175.4 de
80.0 bc
406-1×403-6
Pio.3012 × Mon.919 6.59 de
63.7 bcd
65.0 d-g
22.1 def 183.5 bcd 93.3 bc
405-5×402-6
Syn.48 × Mon.949
63.3 cd
65.3 def
24.6 abc 178.3 de
7.61 bcd 66.3 a
6.55 de
80.5 d-g 76.1 gh
84.1 c-f
74.8 h
77.8 efg
79.9 efg 75.0 h
401-9×402-6
Pac.984 × Mon.949
6.32 e
63.0 cd
64.3 efg
23.4 b-f 180.9 cde 77.3 efg 78.2 c-f
Check
Pio.A33
8.47 ab
64.7 abc
66.0 cde
21.9 ef
182.0 bcd 91.9 bcd 79.8 bcd
Check
SW 4452
8.26 abc
66.7 a
68.7 a
25.0 ab
177.3 de
Check
Pio.3012
8.08 abc
66.7 a
68.0 ab
22.4 def 165.2 e
89.1 b-e 77.2 efg
Check
Syn. 48
7.86 abc
64.0 bcd
66.0 cde
22.7 c-e 171.3 de
74.4 g
Check
Mon.949
7.57 bcd 62.7 cd
64.3 efg
26.7 a
81.6 c-g 77.9 d-g
Check
Mon.919
7.32 b-e
65.0 d-g
23.9 b-e 176.1 de
63.3 cd
178.3 de
84.2 c-f
76.3 fgh
77.9 d-g
88.1 b-f 82.9 a
Mean of top 10
interfamily cross
7.34
63.7
65.2
23.6
182.4
87.5
77.5
F-value1/
**
**
**
**
**
**
**
10.322
1.9
1.839
5.706
5.327
8.393
1.494
CV(%)
1/
ns: non significant, * : significant, ** : highly significant
Kasetsart J. (Nat. Sci.) 41(2)
248
they were statistically not different.
Evidences from previous studies (Genter,
1976; Landi and Frascaroli, 1993; Rasmusson and
Phillips, 1997 and Troyer, 1999) showed that
selections in a very narrow base populations were
very effective for the improvement of the
populations as well as inbred lines per se. The
method for composite line improvement used in
the present studies is very similar to that suggested
by Genter (1976) for population improvement but
only 3 S1 lines were used to form new population
of each cycle, aiming to get uniform, high yield
and high combining ability composite lines for
better hybrid combinations. The method is simply
a modification of S1 and full-sib selection and
therefore it will be referred to as modified S1-full
sib selection method. Data presented in this study
did not show any clear advantage of line selection
over the composite line method. More advanced
cycles of S1-full sib selection are underway to
prove the merit of the method as compared to the
conventional line selection by pedigree method.
The composite-sibbed lines as proposed
by Kinman (1952) is clearly had an advantage over
line selection method when time and space are
involved. Composite-sibbed lines are ready for
final testing without five or six generations of
selfing usually practice in the development of
inbred lines. In the modified S1-full sib selection,
composite-sibbed lines can be derived from
Table 5 Grain yields at 15 percent moisture and other agronomic traits of top 10 testcrosses between
composite lines of cycle 1 × KRi 208 and original hybrids at Suwan Farm, Thailand in
November 2005 (dry season).
Set
Source of
Grain Yield
Days to
Days to
Moisture
Plant
Ear
Shelling
numbers
germplasms
(ton/ha)
Anthesis
Silking
Content
Height
Height
(%)
(days)
(%)
(cm)
(cm)
set 4
Mon.949
8.74 a
62.7 fg
(days)
63.3 ghi
24.9 abc
172.5 a-d
84.4
76.1 def
set 3
Pac.984
8.31 abc
65.7 a-d
67.0 a-d
23.8 b-g
165.5 c-g
84.9
78.9 bc
set 11
Syn. 48
7.80 a-d
64.0 c-f
65. d-h
22.9 b-h
163.9 d-g
78.5
74.6 fg
set 5
Mon.949
7.65 a-d
61.7 g
62.7 i
23.8 b-g
168.8 b-g
80.5
76.5 c-f
set 12
Syn. 48
7.44 b-d
63.3 efg
64.7 e-h
22.9 b-g
160.7 efg
77.2
76.3 def
set 17
Mon.919
7.37 b-f
63.0 efg
65. d-h
21.1 h
161.9 d-g
81.9
76.2 def
set 10
Syn. 48
7.34 b-f
62.7 fg
63.0 hi
22.2 fgh
160.5 fg
81.2
77.1 cde
set 15
Pio.3012
7.24 c-g
66.0 abc
67.0 a-d
24.0 b-g
189.9 g
82.3
77.2 cde
set 16
Mon.919
7.20 c-g
63.3 efg
64.7 e-h
21.7 gh
161.9 d-g
83.7
75.3 efg
set 18
Mon.919
7.20 c-g
63.7 efg
65.3 c-g
23.9 b-g
169.3 b-g
85.4
77.3 cde
Check
Pio.A33
8.47 ab
64.7 b-f
66.0 b-f
21.9 gh
182.0 a
91.9
79.8 b
check
SW 4452
8.26 abc
66.7 ab
68.7 a
25.0 ab
177.3 ab
84.2
76.3 def
Check
Pio.3013
8.08 a-d
66.7 ab
68.0 ab
22.4 e-g
165.2 d-g
89.1
77.2 cde
Check
Syn. 48
7.86 a-d
64.0 c-f
66.0 b-f
22.7 c-g
171.3 a-e
74.4
77.9 bcd
Check
Mon.949
7.57 a-d
62.7 fg
64.3 ghi
26.7 a
178.3 ab
81.6
77.9 bcd
Check
Mon.919
7.32 b-f
63.3 efg
65. d-h
23.9 b-g
176.1 abc
88.1
82.9 a
topcrosses
7.63
63.6
64.8
23.1
167.5
82.0
76.6
F-value1/
**
**
**
**
**
ns
**
10.175
1.923
1.921
5.964
3.873
7.455
1.93
Mean of top 10
CV(%)
1/
ns: non significant, * : significant, ** : highly significant
Kasetsart J. (Nat. Sci.) 41(2)
composite sets as used in this study or using the
individual S1 and full-sib of each successive cycle.
In addition, S1 lines may be selfed for one or two
additional generations in order to eliminate the
undesirable alleles and several desirable sister lines
may then be composited to establish the
composite-sibbed lines.
CONCLUSION
Line selection combined with early
generation testing for combining ability is an
effective method. It gave higher average yield of
top-10 S 2 testcrosses over the composite
249
testcrosses. However, statistically, there was no
clear advantage of yield between both groups of
lines in early generation testcrosses. Besides, the
selected S2 and composite lines showed similar
results in diallel cross sets. Visual selection under
low-competition environment proved to be a very
effective method to identify good combining and
relatively high yield lines. However, testcross and
diallel cross should be applied for thorough test
of combining ability of lines.
Composite lines had clear advantages
over S3 lines in yield, earliness and plant height.
The modified S 1 -full sib selection for the
improvement of composite lines is a flexible
Table 6 Grain yields at 15 percent moisture and other agronomic traits of interfamily diallel hybrids of
composite lines (cycle 1) and original hybrids at Suwan Farm, Thailand in November 2005
(dry season).
Composite2/ Source of germplasms
×
composite
Grain
Days to
Days to
Moisture
Plant
Ear
Shelling
Yield
Anthesis
Silking
Content
Height
Height
(%)
(ton/ha)
(days)
(days)
(%)
(cm)
(cm)
65.0 c-h
66.3 b-e
22.9 b-e
190.1
95.3 abc
2×4
Pac.984 × Mon.949
4×7
Mon.949 × Pio.A33
9.18 ab
63.7 g-j
64.6 def
24.4 bc
181.4
92.9 a-d
78.5 b-e
2 × 15
Pac.984 × Pio.3012
7.98 c-f
65.7 a-f
66.3 b-e
19.3 f
182.9
91.9 b-e
78.9 b-e
4 x 11
Mon.949 × Syn. 48
7.98 c-f
63.0 ij
64.0 f
23.0 b-e
184.9
89.3 b-f
77.5 de
7 × 17
Pio.A33 × Mon.919
7.62 c-h
63.7 g-j
65.0 def
21.4 ef
177.6
95.7 ab
77.9 cde
11 × 15
Syn. 48 × Pio.3012
7.20 d-i
65.3 b-g
66.3 b-e
22.8 c-e
187.5
100.5 a
77.6 de
2 × 17
Pac.984 × Mon.919
7.06 e-i
66.0 a-e
66.7 a-d
20.5 ef
189.5
86.3 e-g
77.3 de
7 × 15
Pio.A33 × Pio.3012
6.97 e-i
66.7 abc
68.3 ab
21.4 ef
169.7
87.1 c-g
77.8 de
4 × 15
Mon.949 × Pio.3012
6.86 f-i
64.3 e-j
65.0 def
23.1 b-e
186.5
92.4 a-e
77.8 de
9.33 a
78.8 b-e
15 × 17
Pio.3012 × Mon.919
6.75 ghi
63.3 i-j
65.0 def
21.0 ef
175.6
89.1 b-f
81.2 abc
Check
Pioneer A33
8.47 abc
64.7 d-i
66.0 d-f
21.9 def
182
91.9 b-e
79.8 a-d
Check
Suwan 4452
8.26 a-d
66.7 abc
68.7 a
25.0 ab
177.3
84.2 efg
76.3 e
Check
Pioneer 3012
8.08 b-e
66.7 abc
68.0 abc
22.4 c-f
165.2
89.1 b-g
77.2 de
Check
Syngenta NK 48
7.86 c-g
64.0 f-j
66.0 d-f
22.7 c-f
171.3
74.4 h
77.9 cde
Check
Monsanto 949
7.57 c-h
62.7 j
64.3 ef
26.7 a
178.3
81.6 fgh
77.9 cde
Check
Monsanto 919
7.32 c-i
63.3 hij
65.0 def
23.9 bcd
176.1
88.1 b-g
82.9 a
Mean of top 10
interfamily cross
7.69
64.7
65.8
22.0
182.6
92.1
78.3
F-value1/
**
**
**
**
ns
**
*
9.449
1.825
1.941
5.827
7.565
5.779
2.557
CV(%)
1/
2/
ns: non significant, * : significant, ** : highly significant
Crosses between two sets of composite lines.
Kasetsart J. (Nat. Sci.) 41(2)
250
method which can be applied to improve the
composite as well as inbred lines. However, further
investigation is required to prove its merit for the
construction of early generation hybrids as well
as for the improvement of inbred lines.
ACKNOWLEDGMENT
We are grateful to Higher Education
Project of Nong Lam University, Vietnam for
financial support and to the staff of National Corn
and Sorghum Research Center, Nakhon
Ratchasima, Thailand for their kind helps during
the time we did experiments.
LITERATURE CITED
Castellanos, J.S., A.R. Hallauer and H.S. Cordova.
1998. Relative performance of testers to
identify elite lines of corn (Zea mays L.).
Maydica 43: 217-226.
Fasoula, D.A. and V.A.Fasoula. 1997. Competitive
ability and plant breeding. Plant Breeding
Review 14: 89-138.
Genter, C.F. 1976. Recurrent selection for yield
in the F2 of maize single cross. Crop Sci. 16:
350-352.
Kinman M.L. 1952. Composite-sibbing versus
selfing in development of corn inbred lines.
Agron. J. 44: 209-241.
Lamkey, K.R. and A.R. Hallauer. 1986.
Performance of high x high, high x low and
low x low crosses of lines from the BSSS
maize synthetic. Crop Sci. 26: 1114-1118.
Landi, P. and E. Frascaroli. 1993. Responses to
four cycles of full-sib family recurrent
selection in an F2 maize population. Maydica
38:31-37.
Lonnquist, J.H. 1950. The effect of selection for
combining ability within segregating lines of
corn. Agron. J. 42: 503-508.
Rasmusson, D. C. and R. L. Phillips. 1997. Review
and interpretation: plant breeding progress and
genetic diversity from de novo variation and
elevated epistasis. Crop Sci. 37: 303-310.
Troyer, A.F. 1999. Review and interpretation:
background of U.S. hybrid corn. Crop Sci.
39: 601-626.
Kasetsart J. (Nat. Sci.) 41 : 251 - 261 (2007)
Anther Culture of BC1F1 (KDML105//IRBB5/KDML105) Hybrid to
Produce Bacterial Blight Resistance Doubled Haploid Rice
Supanyika Sengsai1, Surin Peyachoknagul1, Prapa Sripichitt2,
Amara Thongpan1 and Pradit Pongtongkam1*
ABSTRACT
Maltose was found to be a better carbon source for callus induction in BC1F1 (KDML 105//
IRBB5/KDML105) anther culture compared with sucrose. Statistical analysis, however, showed that
increasing maltose or sucrose concentrations had no differential promotive effects on callus formation.
One-step plantlet formation was found when maltose and NAA were supplemented together in the
induction media. Adding 2 mg/l 2,4-D to the medium further increased the percentage of callusing
anthers from 5.57% to 10.19%. However, the highest percentage of green plant regeneration was obtained
(1.29%) from calli induced on N6 medium without 2,4-D and subsequently cultured on regeneration
medium containing MS supplemented with 2 mg/l BAP, 0.2 mg/l NAA, 300 mg/l casein hydrolysate,
15% coconut water, and 30 g/l sucrose. AFLP analysis of all six anther-derived plants showed 57.3%
to 67.12% recurrent parental alleles. After planting, seeds were detected in two out of six anther
culture-derived plants indicating the occurrence of spontaneous chromosome doubling in these plants.
Unfortunately, none of these six plants contained bacterial blight resistant gene (xa5) as detected by
specific PCR-based RG556 marker and pathogen inoculation.
Key words: KDML 05, anther culture, maltose, 2,4-D, AFLP, RG556, bacterial blight
INTRODUCTION
The production of haploid plants and
doubled haploid plants from anther culture offers
a rapid achievement of homozygous lines for early
release of new crop varieties. Many desirable traits
such as high grain weight, disease resistance, dwarf
plant type and abiotic stress tolerance were
introgressed into rice breeding population by
culturing of anthers. Unfortunately, low
percentages of both callus induction and plant
regeneration are the principal constraints in
1
2
*
establishing successful anther culture in some rice
varieties especially in indica rice since these
critical culturing responses are genotype
dependent (Roy and Mandal, 2005). Consequently,
the effective culture medium used for some rice
varieties may not be appropriate for others, and
the composition of culture media should be
carefully selected when the anthers of particular
rice variety was subjected to culture.
Sucrose is generally added in rice anther
culture media to serve as the standard carbon
source and the osmotic regulator. However, many
Department of Genetics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: fscipdp@ku.ac.th
Received date : 19/07/06
Accepted date : 22/01/07
252
Kasetsart J. (Nat. Sci.) 41(2)
reports revealed that the percentages of callus
induction and plant regeneration could be
increased by using maltose instead of sucrose
(Lentini et al., 1995). In addition, the type of auxin
in anther culture medium has been proposed to
regulate the formation of rice callus. It was found
that culturing on 2,4-D supplemented-media
stimulated callus induction and cell proliferation
in rice anther culture whereas having NAA resulted
in direct androgenesis (Ball et al., 1993).
This work aimed at investigating the
effects of maltose, sucrose and 2,4-D on the anther
culture response of the BC1F1 hybrid of two
recalcitrant genotypes, Khao Dawk Mali 105
(KDML105) a well-known aromatic rice variety
and IRBB5 a bacterial blight resistant rice variety
containing xa5 resistant gene. In addition, the
regeneration ability of the anther calli was also
observed. After anther culture-derived plants (ACderived plants) were obtained, the Amplified
Fragment Length Polymorphism (AFLP) was used
to assess the contribution of the two parental
genomes in these plants, and subsequent detection
of xa5 resistant gene for bacterial blight resistance
was done using PCR-based marker.
MATERIALS AND METHODS
BC1F1 seeds culturing and panicles collection
BC 1 F 1 seeds (KDML105//IRBB5/
KDML105) were obtained from the crossing
between KDML 105 (a bacterial blight susceptible
variety) and IRBB5 (the donor parent containing
xa5 bacterial blight resistant gene). The BC1F1
seeds were cultured on a modified MS medium
(Murashige and Skoog, 1962) supplemented with
2-3 mg/l BAP, 1 g/l yeast extract, 15% coconut
water, 30 g/l sucrose and 0.8% agar. The BC1F1
plants having xa5 gene were selected by PCRbased RG556 marker (Huang et al., 1997). Healthy
tillers from the selected BC 1 F 1 plants were
separated and grown in pots. The panicles were
collected from the primary, secondary and also
tertiary tillers of plants when the microspores in
anther were at the mid-to-late uninucleate stage
as seen from the distance between the auricles of
the two last leaves that reached 6-12 cm.
Anther culture
The panicles covered with flag-leaf
sheaths were wrapped in a moist-soft paper, sealed
in a plastic bag, and kept in the dark for 8-10 days
at 12°C. After this cold-pretreatment, the panicles
were surface-sterilized by spraying with 70%
ethanol and the flag-leaf sheaths were removed.
Then the anthers were cut off and cultured on
callus induction media containing N6 salts and
vitamins (Chu, 1978), supplemented with 2 mg/l
NAA, 1 mg/l kinetin, 500 mg/l casein hydrolysate,
0.7% agar and also different concentrations of
maltose or sucrose (40 , 50 , 60 g/l) at the adjusted
pH of 5.8. The cultures were maintained under
alternate 16/8 h light/dark at 25 ± 2°C for 45-50
days. The numbers of anther calli formation were
recorded and the percentages of calli induction
were calculated. The experiment was set as 2×3
factorial in completely randomized design with
three replications.
An addition of 2 mg/l of 2,4-D to callus
induction medium of the same formular described
above having 50 g/l maltose was also performed
to determine the effect of 2,4-D on callus
induction.
Callus differentiation
Anther calli of 1-2 mm diameter were
randomly collected and transferred to the
regeneration media consisted of MS salts and
vitamins, supplemented with different
concentrations of kinetin (1, 2, 3 mg/l), 300 mg/l
casein hydrolysate, 1 g/l L-proline, 15% coconut
water, 30 g/l sucrose and 2.5 g/l phytagel. An
addition of regeneration medium having 2 mg/l
BAP and 0.2mg/l NAA to replace kinetin and Lproline was set. The cultures were kept under
alternate 16/8 h light/dark at 25 ± 2°C for 15-20
Kasetsart J. (Nat. Sci.) 41(2)
days. The numbers of calli producing complete
plantlets were recorded and the percentage were
calculated.
AFLP analysis
To assess the contribution of two parental
genomes in AC-derived plants, AFLP was
performed as described by Vos et al. (1995) with
some modification. Fifteen combinations of primer
(synthesized by KU Vector), set E primer (EcoRI
end) and set M primer (MseI end), were used. Only
clear AFLP bands were scored as present or absent.
AFLP fingerprints of KDML105, IRBB5 and also
AC-derived plants were analyzed.
Detection of xa5 resistant gene for bacterial
blight resistance by PCR-based marker
The DNA of the resistant donor parent
(IRBB5), recurrent susceptible parent
(KDML105), and AC-derived plants were
extracted from the leaves using the method
described by Agrawal et al. (1992) and subjected
to PCR amplification using synthesized primers
(KU Vector). The RG556 primer linked to the xa5
resistant gene was used to detect the presence of
resistant gene. The sequence of RG556 F is 5′
TAGC TGCTGCCGTGCTGTGC 3′ while RG556
253
R is 5′ AATATTTCAGTGTGCATCTC 3′ (Huang
et al., 1997). PCR products were digested with
Hpy CH4 IV restriction enzyme to detect the
polymorphic DNA bands from bacterial blight
resistant and susceptible plants (Sanchez et al.,
2000).
RESULTS AND DISCUSSION
Anther culture
Ten days after incubation on callus
induction media, approximately 90% of anthers
turned brown (data not shown). This result,
however, was not surprising because Guzman and
Zapata-Arias (2000) also reported this changing
of anther colors which is possibly due to the
transition of gametophytic phase to sporophytic
phase during androgenesis. In addition, our results
showed that anther calli asynchronously emerged
through the split lobes of these browning anthers
after 45-50 days of culturing (Figure 1A). Most
of the responding anthers produced multiple calli
which ultimately became yellowish in color having
both campact and friable callus types (Figure 1B).
The calli were all in satisfactory condition. This is
the first report on anther culture of BC1F1 seeds
(KDML105//IRBB5/KDML105).
Figure 1 Calli formation: (A) calli emerged through the split lobes of anther (arrow), (B) the anther
calli (arrow), after 45-50 days of culturing on callus induction media containing maltose.
Kasetsart J. (Nat. Sci.) 41(2)
254
The effects of maltose and 2,4-D on callus
induction
Using different concentrations of matose
and sucrose in the culture medium, it was found
that the percentages of calli induction ranging from
4.61% to 6.11% in maltose but only 3.33% to
3.43% in sucrose (Table 1). Callus formation was
significantly affected by the type of sugar (P=0.01)
but not significantly affected by the concentration
of sugar itself. The highest percentage of callus
induction was obtained by culturing BC1F1 anthers
on N6 medium supplemented with 2 mg/l NAA, 1
mg/l kinetin, 500 mg/l casein hydrolysate, 60 g/l
maltose and 7 g/l agar. Maltose, therefore, seemed
to be a preferred carbon source for prolific callus
formation in rice anther culture as also reported
by Lentini et al. (1995). The beneficial effect of
maltose has been ascribed to its slow degradation
which results in stabilization of medium
osmolarity (Kuhlmann and Foroughi-Wehr, 1989).
In contrast, sucrose is rapidly hydrolysed to
glucose and fructose, thereby, the osmolarity of
the medium became double causing the negative
effect on callus formation (Xie et al., 1995).
Although callus induction rate gradually
increased as the concentration of maltose or
sucrose increased (Table 1), statistical analysis
showed that rising of sugar concentration (40, 50,
60 g/l) had no differential promotive effects on
callus formation. This result did not agree with
Table 1 Effects of maltose, sucrose and 2,4-D on anther callus formation of BC1F1 (KDML105//
IRBB5/KDML105) hybrid.
Types of sugar
Concentration
Number of
Number of
Percentage of callus
in callus induction
of sugar
cultured
callusing
induction (%)
medium
(g/l)
anthers
anther
Sucrose
40
1,140
38
3.33
50
1,808
62
3.43
60
848
29
3.42
Maltose
40
4,252
196
4.61
50
9,979
557
5.57
60
7,695
470
6.11
50a
4,378
446
10.19
Analysis of variance: ANOVA table
Source
Treatment
Factor A
Factor B
A×B
Error
Total
Df
SS
5
1
2
2
12
17
22.16
18.68
1.69
1.52
15.12
37.28
MS
18.68
0.98
0.76
1.26
** indicated highly significance at 1%, ns indicated no significance
50a referred to 50 g/l maltose supplemented medium + 2 mg/l 2,4-D
Factor A was types of sugar, viz. maltose and sucrose.
Factor B was sugar concentration, viz. 40, 50 and 60 g/l.
F(cal)
value
14.83**
0.78 ns
0.60 ns
F(table)
value
5%
1%
4.75
3.88
3.88
9.33
6.93
6.93
Kasetsart J. (Nat. Sci.) 41(2)
the report of Ching (1982) showing the increase
of sugar concentration (30, 60, 90 g/l) to promote
the higher percentage of callusing anthers as well
as plantlet formation. The contradictory results
may be due to the narrow range of sugar
concentrations used which could not cause
distinctive effects on callus induction in this study.
It is also interesting to find that some of
yellowish compact calli grown in 50 g/l or 60 g/l
maltose containing media could differentiate to
complete plantlets. The development of complete
plantlets while they are culturing on induction
medium is known as “one-step plantlet formation”.
This result agreed with other reports showing that
addition of maltose to NAA containing callus
induction media promoted the formation of
complete plantlet in rice anther culture (Zhao et
al., 1999). Since the formation of plantlets by one
step did not frequently occur in anther culture of
KDML105 hybrids (Lertvichai, 1995; Boonintara,
2004), three complete green plantlets (2.63%, data
not shown) obtained from this experiment is
considered a positive and satisfactory result. It
should be noted here that genotype of anther donor
plants, the type and concentration of sugar as well
as the type of auxin have some effect on one-step
plantlet formation (Zhao et al., 1999).
255
To further increase the percentage of
callus formation, 2 mg/l of 2,4-D was added to 50
g/l maltose supplemented-medium. The results
showed that the percentage of callus formation was
increased from 5.57% to 10.19% (Table 1).
Although the addition of 2,4-D favoured callus
initiation and proliferation, the organogenesis of
the calli might be inhibited hence one-step plantlet
formation was not obtained. This result did not
agree with those obtained by Datta et al. (1990)
showing the combination of NAA and 2,4-D
supplemented in callus induction medium
promoted green plantlet formation in rice. This
opposing result is possibly due to the powerful
influence of genotype of anther donor plants on
the response of rice anthers to these auxins.
Callus differentiation
After culturing on regeneration media,
differentiation of the calli was observed at 15-20
days. The percentages of calli development are
shown in Table 2. The results indicated that the
highest percentage of green plantlet formation
(1.29%) was obtained when the calli grown on
induction media without 2,4-D were transferred
onto regeneration medium containing MS salts and
vitamins supplemented with 2 mg/l BAP, 0.2
Table 2 Development of calli on regeneration media.
Callus
Regeneration Number of
Browning
induction
media
cultured
calli
media
anthers
(%)
without
SR1
140
14.29
2,4-D
SR2
123
18.71
SR3
180
10.00
MR1
77
9.09
with
SR1
15
13.33
2 mg/
SR2
82
2.44
2,4-D
SR3
93
0.00
MR1
92
4.34
Proliferated
calli
(%)
74.29
65.85
58.33
12.89
80.00
96.34
92.47
92.39
Plantlet formation (%)
Green
Albino
plantlets
plantlets
0.71
4.29
0.81
1.63
0.00
13.89
1.29
12.99
0.00
0.00
0.00
12.12
0.00
4.30
1.09
0.00
SR1, SR2, SR3 = MS + different concentration of kinetin (1 mg/l, 2 mg/l, 3 mg/l) +300 mg/l casein hydrolysate + 1 g/l L-proline
+ 15% coconut water + 30 g/l sucrose + 2.5% phytagel
MR1
= The same formular as SRs media but having 2 mg/l BAP and 0.2 mg/l NAA to replace kinetin and L-proline.
256
Kasetsart J. (Nat. Sci.) 41(2)
mg/l NAA, 300 mg/l casein hydrolysate, 15%
coconut water, 30 g/l sucrose and 2.5% phytagel
(MR1 medium). It was also found that the
percentage of green plantlet formation of calli
cultured on the media without BAP and NAA was
increased when the concentration of kinetin
increased. By two steps culturing (callus induction
and subsequent regeneration of calli), the total of
four green plantlets were obtained (Figure 2A,B).
However, one of these plantlets died during
subculturing leaving only three plantlets for further
investigation.
It is interesting to find that regeneration
medium highly supported callus proliferation
(80%-96.34%, Table 2) of those previously grown
in the medium containing 2 mg/ 2,4-D but the
complete plantlets formation was better formed
in the media without 2,4-D. These results implied
that regeneration response of calli was possibly
affected by the interaction between the
composition, particularly the types of auxin, of
induction media and regeneration media.
Although the percentages of callus
induction (3.33-6.11%) and green plantlet
formation (0.00-1.29%) of BC1F1 in this study
were lower than those of Lemont/KDML 105
hybrid (2.22%-44.46% and 0.00%-20%
respectively) as reported by Lertvichai (1995), they
were comparable to those of KDML 105/Chainat1
hybrid (Boonintara, 2004). The low outcome of
callus formation and recovering plants from anther
culture of indica rice are known to be genotypic
dependent (Khanna and Raina, 1998).
A large percentage of albinos (more than
90%, data not shown) obtained in this study is
considered unsatisfactory but similar to other
reports of albinos ranging from 5% to 100% in
rice anther culture, especially in indica rice
(Bhojwani et al., 2001). Several factors, including
pre-treatment, culture medium and culturing steps
considerably affected the frequency of albinism.
However, high sugar concentration might be
another cause of albino plant formation as seen in
the increase percentage of albino plantlet of
japonica rice (Tainan 5) in the increased sugar
culture (Chen, 1978).
AFLP based background analysis in anther
culture-derived plants
AFLP analysis was performed on six ACderived plants (three plants obtained by one-step
plantlet formation and the others from two-step
plantlet formation) to assess the contribution of
two parental genomes in these plants. Out of 373
Figure 2 Development of calli: (A) green spot producing callus (arrow), (B) green plantlet regeneration,
after 15-20 days of culturing on regeneration media containing MS supplemented with 2
mg/l BAP, 0.2 mg/l NAA, 300 mg/l casein hydrolysate, 15% coconut water, and 30g/l sucrose.
Kasetsart J. (Nat. Sci.) 41(2)
clearly amplified bands generated by 15 primer
combinations, 73 bands showed polymorphisms
between the parents (Figure 4) of which 50 bands
were specific for KDML105 and 23 specific bands
for IRBB5 (data not shown). By assuming random
distribution of AFLP markers in rice genome, it
was found that the percentage of recurrent parental
alleles (KDML105) recovered in AC-derived
plants population was found ranging from 57.3%
to 67.12% (Table 3). This result is somewhat
narrower than the anticipated distribution of
chromosomes containing recurrent parental alleles
(50%-100%) in plantlets obtained from anther
culture of BC1F1 plants, which is possibly due to
the fact that AFLP primers used in the present study
did not cover the whole genome. Furthermore,
only six AC-derived plants were obtained and
could not, thereby, represent the broad distribution
of the overall AFLP alleles in the population. In
addition, Guiderdoni (1991) reported that
androgenesis of microspores containing more
genetic make-up of recurrent parent may be
masked by gametic selection. This selection
resulted in the segregation distortion of alleles and
preventing AC-derived plants from being truly
257
BC1F1 gametic array as also shown in the anther
culture of japonica/indica and indica/indica rice
hybrids.
After planting six healthy AC-derived
plants, seeds were obtained in two out of these six
plants (33.33%, data not shown) indicating the
occurrence of spontaneous chromosome doubling
which resulted in two homozygous lines from
anther culture of BC1F1 (KDML105//IRBB5/
KDML105). This result was not surprising since
the mechanisms to double chromosome can occur
at various stages in vitro, including callus
formation, callus re-differentiation and
embryogenesis in rice anther culture, and even in
tillers (Bishnoi et al., 2000). Although the
percentage of chromosome doubling obtained
from this study (33.33%) was lower than the anther
culture of KDML105/RD23 (86.1%) reported by
Pakdeechanuan (1997), it was higher than that of
KDML105/Lemont (25%) (Lertvichai, 1995) and
comparable to those of KDML105/Chainat1
(33.33%) (Boonintara, 2004). Genotypic
dependence was suspected to be the main cause
affecting the frequency of chromosome doubling
in rice anther culture (Sopory et.al. 1996).
Table 3 The percentage of recurrent parental alleles (KDML105) recovered in AC-derived plants
population as detected by AFLP using 15 primer combinations.
Individual ACNumber of specific
Number of specific
Percentage of
derived plants
bands presented
bands presented
recurrent parental
only in KDML105
only in IRBB5
alleles (KDML105)
recovered in
AC-derived plants
(%)
1
49
24
67.12
2*
47
26
64.38
3
42
31
57.53
4*
47
26
64.38
5
45
28
61.64
6
43
30
58.90
Average percentage of recurrent parental alleles recovered in
62.33
AC-derived plants
*
indicated spontaneous double haploids
258
Kasetsart J. (Nat. Sci.) 41(2)
Detection of xa5 resistant gene for bacterial
blight resistance by PCR -based marker
Since the anther donor plants (BC1F1)
were confirmed to be heterozygous for the xa5
resistant gene (data not shown), AC-derived plants
from BC1F1 were also tested for the xa5 gene using
PCR-based marker RG556. The PCR product gave
monomorphic amplification products of 1,600 bp
(Figure 3A). However, after digesting with Hpy
CH4 IV, polymorphism of DNA bands between
resistant and susceptible plants was detected. Two
bands of 1,000 bp and 300 bp (doublet) were found
in IRBB5 (bacterial blight resistant variety) while
non-digested DNA band (1,600 bp) was shown in
susceptible variety of KDML105 (Figure 3B).
Genotyping by PCR-based method revealed none
of these six AC-derived plants contained the xa5
gene for bacterial blight resistance (Figure 3B).
This result was confirmed by pathogen inoculation
test on 45 days old plants grown from healthy seeds
of two homozygous lines (obtained by
spontaneous chromosome doubling as described
in AFLP analysis section) which showed bacterial
blight susceptability (data not shown).
Sanchez et al. (2000) reported that the
distance between RG556 marker and the xa5 gene
was 0.8 cM, then the loss of the xa5 gene in six
AC-derived plants in the present study possibly
Figure 3 PCR analysis of the bacterial blight susceptible variety (KDML105), resistant variety (IRBB5)
and AC-derived plants (the samples number 1, 2, 3, 4, 5, 6): (A) Monomorphic bands amplified
with primer RG556, (B) PCR products digested with Hpy CH4 IV. M= 1 kb plus DNA marker.
Kasetsart J. (Nat. Sci.) 41(2)
caused by either the recombination of RG556
marker and the xa5 resistant gene or the
segregation of gene during gametogenesis.
CONCLUSION
Two homozygous lines were rapidly
achieved by anther culture of BC 1F 1 hybrid
(KDML105//IRBB5/KDML105) and subsequent
spontaneous chromosome doubling. In the present
study maltose has proven to be a preferred carbon
259
source compared to sucrose as seen from the
significant effect on callus formation. Furthermore,
one-step plantlet formation was promoted when
callus induction medium supplemented with
combination of maltose and NAA was used.
Although the percentages of callusing anther and
callus proliferation were increased by adding
2,4-D to NAA containing induction medium, the
percentages of organs formation as well as
complete plantlet formation were very low,
moreover one-step plantlet formation did not
Figure 4 AFLP fingerprint of the bacterial blight susceptible variety (KDML105), resistant variety
(IRBB5) and AC-derived plants (the samples number 1, 2, 3, 4, 5, 6) generated by different
primer combinations: (A) E-AGG / M-CTC primer (B) E-AAG / M-CAA primer (C) EAAG / M-CAG primer, M = 25 bp DNA size marker (Life Technologies); arrows indicate
polymorphic bands specific for KDML105.
Kasetsart J. (Nat. Sci.) 41(2)
260
occur. By two-steps culturing, the highest
percentage of green plant regeneration was
obtained (1.29%) from calli induced on N 6
medium without 2,4-D and subsequently cultured
on regeneration medium containing MS
supplemented with 2 mg/l BAP, 0.2 mg/l NAA,
300 mg/l casein hydrolysate, 15% coconut water,
and 30g/l sucrose. The contribution of recurrent
parental genome in AC-derived plants was
revealed by AFLP analysis. Even though these ACderived plants did not contain a bacterial blight
resistant gene (xa5) when screened by PCR-based
RG556 marker, other desirable traits such as dwarf
plant type, photoperiod insensitive response and
aroma, characteristic of the parents could be
obtained from them.
ACKNOWLEDGEMENTS
This research was financially supported
by Kasetsart University Research and
Development Institute (KURDI) and Thesis and
Dissertation Support Fund, Graduate School,
Kasetsart University. The authors also would like
to thank Dr. Kanchana Klakhaeng, Patumtani Rice
Research Center for supplying rice seeds, and Dr.
Nongrat Nilpanit, Division of Plant Pathology and
Microbiology, Department of Agriculture, for
providing pathogen inoculation test.
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Kasetsart J. (Nat. Sci.) 41 : 262 - 273 (2007)
Novel PCR Primers for Specific Detection of Xanthomonas citri
subsp. citri the Causal Agent of Bacterial Citrus Canker
Udomsak Lertsuchatavanich1, Ampaiwan Paradornuwat1, Junlapark Chunwongse2,
Norman W. Schaad3 and Niphone Thaveechai1*
ABSTRACT
The new primers were developed for specific detection of Xanthomonas citri subsp. citri (Hasse)
(Xcc) [syn. X. axonopodis pv. citri (Xac)], the causal agent of Asiatic citrus canker disease. Twenty
three strains of Xcc and 34 strains of other xanthomonads including X. fuscans subsp. aurantifolii, X.
alfalfae subsp. citrumelonis, X. campestris pv. campestris, X. campestris pv. glycines, X. citri subsp.
malvacearum and X. fuscans subsp. fuscans were tested for specificity of new primers by classical PCR.
The results showed that these 354 F/R primers specifically amplified all of Xcc strains but not other
xanthomonad strains. The 354-bp PCR fragment was sequenced and its nucleotide sequences were
compared for similarity with Genbank database. The 354-bp nucleotide sequences were 99.7% similar
to gene XAC2443 of Xac strain 306 (Accession AE011881). The sensitivity of these specific primers
for detection of viable cells and total DNA of Xcc were 70 CFU/µl and 50 pg/µl, respectively. Therefore,
these novel primers can be used as an alternative application for rapid and specific detection of Xcc.
Key words: Xanthomonas, bacterial citrus canker, detection, polymerase chain reaction
INTRODUCTION
Bacterial canker of citrus is a serious
disease of most citrus species and cultivars in many
citrus-producing areas worldwide. Five forms of
the disease have been described, cankers A, B, C,
D, and E. Canker A or A-strain (Asiatic canker) is
the most common and most damaging of the citrus
canker strains (Schubert et al., 2001). It was
originally found in Asia and is by far the most
widespread. Recently, information based upon
DNA sequences comparison or alignment of 16S23S internal transcribed spacers (ITS) regions with
1
2
3
*
amplified fragment length polymorphism (AFLP)
analysis of the five recognized forms of citrus
canker was demonstrated by Schaad et al. (2005,
2006). Citrus pathogens were reclassified into
three pathovars of Xanthomonas campestris (or
X. axonopodis): pathovars citri for strain “A”,
aurantifolii for strains “B/C/D” and citrumelo for
strain “E”, which were revealed as taxon I
including all “A” strains; taxon II containing all
“B”, “C”, and “D” strains; and taxon III containing
all “E” strains. The taxa I, II and III citrus strains
were reinstated with new names, respectively as
Xanthomonas citri subsp. citri (Hasse, 1915),
Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Department of Horticulture, Faculty of Agriculture, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140,
Thailand.
USDA-ARS FDWSRU, 1301 Ditto Avenue, Fort Detrick, MD 21702-5023, USA.
Corresponding author, e-mail: agrnpt@ku.ac.th
Received date : 31/05/06
Accepted date : 24/10/06
Kasetsart J. (Nat. Sci.) 41(2)
Xanthomonas fuscans subsp. aurantifolii (Gabriel
et al., 1989), and Xanthomonas alfalfae subsp.
citrumelonis (Riker et al., 1935). A new strain of
X. citri subsp. citri, designated A*, was identified
in southwest Asia and has a restricted natural host
range to Mexican lime (Verniere et al., 1998).
Another Aw-strain, which behaves similarly, has
recently been discovered in Florida. This strain
has a restricted host range that includes Mexican
lime and alemow (Citrus macrophylla) (Sun et al.,
2004). The A-strain is the target of international
quarantine efforts, in which the development of
rapid and reliable procedures for the diagnosis of
this pathogen has been a priority.
The polymerase chain reaction (PCR) is
a principle for plant disease diagnosis (Henson and
French, 1993). However, routine application of
PCR for detection of plant pathogens can result in
false-positive diagnosis when PCR primers are
non-specific to the pathogen. Several sets of
primers have been developed for diagnosis of Xcc.
Non-specific amplification of Hartung’s primers
(Hartung et al., 1993) for detection of Xcc were
reported by Zaccardelli and Mazzucchi (1997).
Miyoshi et al. (1998) studied similarity of the
intergenic spacer region between 16S-23S rRNA
genes among X. citri subsp. citri, X. campestris
pv. glycines, X. alfalfae subsp. alfalfae (X. c. pv.
alfalfae), X. c. pv. physalidicola, X. c. pv. pisi, X.
c. pv. pruni, X. c. pv. cucurbitae and X. c. pv.
vesicatoria, and designed primers XCF and XCR
based on the data from this study. Primers XCFXCR were not only for detection of Xcc but also
for X. c. pv. glycines. Kingsley et al. (2000)
developed fluorogenic PCR assay for specific
detection of Xcc A and A*-strain using the
forward/reverse primers and probes designed from
unique RAPD fragment to target 126 bp amplicon.
Mavrodieva et al. (2004) designed primers, VM3
and VM4, for real-time PCR by selecting to
amplify the pthA gene family and run experiments
to compare with Kingsley’s primers, KF and KR.
The results showed that Xcc (A, A* and Aw) and
263
X. fuscans subsp. aurantifolii (B and C) reacted
and gave expected product sizes with VM3-VM4
primers. On the other hand, Kingsley’s primers
gave prominent band with Xcc A and A*-strain
but the reaction with Aw-strain and X. fuscans
subsp. aurantifolii (B and C) were inconsistent and
also gave more primer-dimer products when
compared with VM3-VM4 primers. The purpose
of this study was to design specific PCR primers
of Xcc from genomic DNA especially gene
XAC2443 (Accession AE011881) in order to
apply them for detection of this international
quarantine bacterial pathogen of citrus.
MATERIALS AND METHODS
Bacterial strains
To obtain original local strains to be used
in this study, canker lesions on lime (Citrus
aurantifolia), mandarin (C. reticulata), sweet
orange (C. sinensis) and leach lime (C. hystrix)
were collected from leaves, twigs, and fruit from
each major citrus growing area in Thailand. The
corky-like raised surface lesions, surrounded by a
yellow halo were washed in running water for 1-2
minutes, sprayed with 70% ethyl alcohol, and airdried. Each lesion was removed from the leaf and
cut into 4-5 pieces then soaked in 0.85% NaCl for
20 minutes. A loop of the suspension was streaked
onto Fieldhouse and Sasser (FS) agar (Schaad et
al., 2001) and incubated at 30°C. After 3-4 days,
plates were examined for small green-colored
starch hydrolyzing colonies typical of Xcc.
Promising colonies of Xcc were transferred onto
nutrient agar (NA) (Schaad et al., 2001) twice and
stored either in sterile distilled water at room
temperature or on NA slants at 4°C, and in 50%
glycerol at -80°C. Several bacterial strains from
Japan and the United States of America were
included in this experiment (Table 1).
PCR primers
The new primer pair namely 354F-354R
264
Kasetsart J. (Nat. Sci.) 41(2)
Table 1 Geographical origin, host and year of isolation of strains of Xanthomonas species used in this
study
Bacterial strain
Geographical origin
Host
Year
X. citri subsp. citri
T1
Kamphaeng Phet
Thailand
Citrus sinensis
2003
T3
Chiang Mai
Thailand
Citrus grandis
2003
T4
Chiang Mai
Thailand
Citrus reticulata
2003
T5
Kamphaeng Phet
Thailand
Citrus aurantifolia
2003
T7
Chiang Mai
Thailand
Citrus reticulata
2003
T8
Chiang Mai
Thailand
Citrus reticulata
2003
T10
Chiang Mai
Thailand
Citrus grandis
2003
T13
Kamphaeng Phet
Thailand
Citrus sinensis
2003
NT14
Kamphaeng Phet
Thailand
Citrus reticulata
2003
NT18
Chiang Mai
Thailand
Citrus aurantifolia
2003
NT20
Sukhothai
Thailand
Citrus aurantifolia
2003
NT22
Chiang Mai
Thailand
Citrus grandis
2003
NT25
Kamphaeng Phet
Thailand
Citrus reticulata
2003
OCr1.1
Chiang Rai
Thailand
Citrus reticulata
2002
OCr1.2
Chiang Rai
Thailand
Citrus reticulata
2002
LCp2.1
Chumphon
Thailand
Citrus aurantifolia
2002
LCp2.2
Chumphon
Thailand
Citrus aurantifolia
2002
SWRb
Ratchaburi
Thailand
Citrus sinensis
2003
Fp1-2
Chiang Rai
Thailand
Citrus grandis
2003
XCC-32
Shimizu
Japan
Citrus natsudaidai
1998
XCC-131
Yui
Japan
Citrus unshiu
1998
1258 (Hartung, Xc-322)
Saudi Arabia
Citrus sp.
ND
1270 (Hartung, Xc-328)
Saudi Arabia
Citrus sp.
ND
X. fuscans subsp. aurantifolii
1415 (IBSBF 392)
Brazil
Citrus limon
1981
1416 (IBSBF 423)
Uruguay
Citrus limon
1981
1417 (IBSBF 1583)
Argentina
Citrus limon
1990
1418 (IBSBF 380)
Brazil
Citrus aurantifolia
1981
1419 (IBSBF 434)
Brazil
Citrus aurantifolia
1982
X. fuscans subsp. aurantifolii
1420 (IBSBF 1473)
Brazil
Citrus aurantifolia
1999
1421 (IBSBF 1495)
Brazil
Citrus aurantifolia
2000
1460
ND
ND
ND
1461
ND
ND
ND
1463
ND
ND
ND
X. alfalfae subsp. citrumelonis
1267 (X-85, J. Miller)
Florida
Citrus sp.
1985
1274 (4600, D. Gabriel)
Florida
Citrus sp.
ND
Kasetsart J. (Nat. Sci.) 41(2)
Table 1 (continued)
Bacterial strain
Geographical origin
X. citri subsp. malvacearum
1318 (ATCC 14982)
Uganda
317
Sukhothai
Thailand
579
ND
Thailand
584
Sukhothai
Thailand
1034
Nakhon Sawan
Thailand
1035
Nakhon Sawan
Thailand
1037
Lop Buri
Thailand
1051
Loei
Thailand
1232
Prachin Buri
Thailand
X. fuscans subsp. fuscans
1316 (NCPPB 381)
Canada
X. campestris pv. campestris
657
Phetchaburi
Thailand
X. campestris pv. glycines
NKR21
Nakhon Ratchasima
Thailand
CM 60-1
Nakhon Ratchasima
Thailand
No.21-1
Chiang Mai
Thailand
RE 07
Khon Kaen
Thailand
239
Chachoengsao
Thailand
241
Phitsanulok
Thailand
281
Phitsanulok
Thailand
X. campestris pv. glycines
285
Phitsanulok
Thailand
728
Chiang Rai
Thailand
1204
Songkhla
Thailand
1324
Songkhla
Thailand
265
Host
Gossypium hirsutum
Gossypium hirsutum
Morus sp.
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Gossypium hirsutum
Year
ND
1984
1986
1986
1990
1990
1990
1990
1993
Phasolus vulgaris
ND
Brassica oleracea
2004
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
2001
2002
2002
2002
1983
1982
ND
Glycine max
Glycine max
ND
Vigna radiata
ND
1987
1992
1994
Abbreviations: IBSBF, Phytobacteria Culture Collection of Instituto Biological, Campinas, Brazil; ATCC, American Type Culture
Collection, Manassas, VA; NCPPB, National Collection Plant Pathogenic Bacteria, York, England; ND, not determined.
was designed from Xanthomonas axonopodis pv.
citri strain 306 at section no. 259 from 469 sections
of complete genome. This target region resulted
from subtractive hybridization (Schaad et al.,
unpublished). The target at position 4411 to 5228
(partial gene XAC2443) was used for designing
new primers for classical PCR by using
DNASTAR software (LASERGENE, Version5.1).
The sequences of the primers were 354F at position
4675-4693 (5’-GACGGCGCGGCTCAGGATG3’) and 354R at position 5006-5028 (5’-
CAGCCCAGCCAACTCAGCACCAG-3’).
Other primer pairs also evaluated in this
experiment were designed by Kingsley et al.
(2000), KF (5’-TCCACTGCATCCCACAT CTG3’), and KR (5’-CAGGTGTACTGCGCTC
TTCTTG-3’); Mavrodieva et al. (2004), VM3 (5’GCATTTGATGACGCCATGAC-3’), and VM4
(5’-TCCCTGATGCCTGGAG GATA-3’); and
Hartung et al. (1993), 2 (5’-CACGGGTGCAAAA
AATCT-3’), and 3 (5’-TGGTGTCGTCGCTT
GTA T-3’) which are respectively referred to as
266
Kasetsart J. (Nat. Sci.) 41(2)
Kingley’s, Mavrodieva’s and Hartung’s primers
in the article.
PCR reaction
PCR was carried out in a 25 µl reaction
that consisted of 1x PCR buffer, 3mM MgCl2 for
354 F/R and 2-3 primers and 2mM MgCl2 for KFKR and VM3-VM4 primers, 0.1 mM dNTPs, 0.6
unit Taq DNA polymerase, DNA template 1 µl (50
ng), 0.4 pmole of each primer for 354 F/R, KFKR and VM3-VM4 primers and 1 pmole for 2-3
primer.
The PCR profiles were designed for each
primer as follow: 1.) 94°C for 10 min and 30 cycles
of 94°C for 30 sec, 60°C for 30 sec, 72°C for 60
sec and 72°C for 10 min for 354 F/R primers, 2.)
94°C for 10 min and 30 cycles of 94°C for 30
sec, 57°C for 30 sec, 72°C for 60 sec and 72°C for
10 min for KF-KR and VM3-VM4 primers and
3.) 95°C for 10 min and 35 cycles of 95°C for 70
sec, 60°C for 70 sec, 72°C for 60 sec and 72°C for
10 min for 2-3 primers.
Primers specificity tests
Strains of Xanthomonas species in Table
1 were used for specificity assay by comparing
354 primers with the Kingsley’s, Mavrodieva’s and
Hartung’s primers. Ten microliters of PCR product
of each primer was determined by gel
electrophoresis on agarose gels in 0.5x TBE buffer
at concentration of 1% for 354 bp-PCR primers
and 1.5% for Kingsley’s, Mavrodieva’s and
Hartung’s primers.
Sensitivity tests
Genomic DNA of Xcc strain T7 was
calculated from the absorbance at 260 nm with
UV-Visible Spectrophotometer (UV-1601,
SHIMADZU) and adjusted by ten-fold dilution
with sterile distilled water from 50 ng to 50 fg for
sensitivity tests. Cell suspension of Xcc strain T7
at 0.2 OD of wavelength 600 nm which was about
108CFU/ml was also used for sensitivity tests with
the ten-fold serial dilutions.
Cloning and sequencing of target DNA
fragment
Taq polymerase-amplified PCR products
using primer pair 354 F/R were purified and
recovered with commercial silica spin column
(Promega). Cloning reactions were according
to pCR 8/GW/TOPO  TA Cloning  Kit
(Invitrogen). Briefly, the mixture was incubated
at room temperature for 5 min, mixed with One
Shot Mach1TM-T1R Chemically Competent E.
coli and incubated on ice for 5 min. The cells were
transformed for 30 sec at 42°C without shaking
and immediately transferred on ice. A 250 µl
aliquot of S.O.C. medium were added and
incubated on a rotary shaker for 1 hr at 37°C.
The transformed cells were centrifuged and
suspended in new S.O.C. medium and then 50µl
was spread onto LB agar containing 100 µg/ml
spectinomycin.
Recombinant clones were screened by
PCR amplification with 354 primers, as described
above. Sequencing of target DNA product was
commercially provided by BSU (Bioservice Unit)
using GW1 and GW2 as sequencing primers. The
nucleotide sequences were analyzed by the Vector
NTI Advance 9.0 software (Invitrogen).
Southern blot hybridization: The 354bp PCR fragment was amplified by using 354 F/R
primers and used as the target DNA probe. The
method for recovery the DNA fragment from gel
was modified from Yue and Orban (2001). The
DNA fragment was excised from 0.7% agarose
gel in 0.5x TBE with a razor blade. The gel slice
was ground with a sterile pestle in a microtube
and 300 µl of phenol was added. After vigorously
mixing with a vortex, the suspension was
centrifuged at 10,000 rpm for 10 min and then 200300 µl of the supernatant was collected and added
to 0.5 volume of 7.5M ammonium acetate and 2.5
volume of absolute ethanol. The supernatant was
centrifuged at 10,000 rpm for 15 min and the pellet
Kasetsart J. (Nat. Sci.) 41(2)
was collected and washed with 70% ethyl alcohol.
After centrifuging at 10,000 rpm for 10 min, the
pellet was dried and suspended in 20-30 µl of
sterile distilled water.
The purified target fragment from the
previous experiment was labeled with
digoxigenin-11-dUTP (Dig-11-dUTP) by using
10xDIG-11-dUTP mixs. The procedure was as
follows: the DNA template was diluted to 50 ng
and prepared for the 50 µl labeling reaction
containing 1µl of DNA template, 5µl of 10x PCR
buffer (200mM TrisHCl, 500mM KCl, 20mM
MgCl2), 5µl of 10x PCR DIG labeling, 2µl of
each 20 pmole/µl primer and 1µl of Taq DNA
Polymerase (5 units/µl). The labeling PCR
product was separated as described above. The
labeled DNA probe was stored at -20°C and
denatured by heating in boiling water for 10 min
and immediately chilled on ice for 5 min before
use.
The agarose gel containing PCR products
was depurinated in 0.25% HCl for 30 min and
neutralized in 0.4M NaOH for 15 min, and
transferred to Highbond N+ nylon membrane by
alkaline 0.4N NaOH. DNAs were fixed under UV
transilluminator for 2.5 min to crosslink the DNA
to the membrane, and washed as suggested by its
manufacturer (Roche ). The membrane was
placed into a hybridization bottle containing 3 ml
hybridization solution containing 1% blocking
solution. After incubating for 1 hr at 65°C the
hybridization solution was replaced with a new
hybridization solution containing labeled DNA
probe and incubated at 65°C for additional 18-24
hr. The membrane was removed and washed on a
rotary shaker in solution I (2xSSC, 0.1% SDS) at
65°C for 5 min. The solution was replaced and
the membrane was washed for additional 15 min.
Finally the membrane was washed twice
consecutively with solution II (1xSSC, 0.1%SDS)
and solution III (0.5xSSC, 0.1%SDS) for 15 min
each at 65°C.
267
After being washed briefly in washing
buffer and 30 min in 1% blocking buffer, the
membrane was transferred to anti-digoxigenin
alkaline phosphatase conjugated (Roche) and
incubated on a rotary shaker for 45 min at room
temperature. The membrane was washed two
times with washing buffer for 15 min before being
transferred to plastic bag. After adding 500 µl
CDP-Star solution, the bag was sealed, placed into
a Kodak x-ray cassette and moved to the dark
room for the detection step. X-ray film was cut to
the proper size and placed over of the membrane
and the closed cassette for 30-60 sec. The film
was transferred to the developer solution until the
band was visible. After washing briefly in water,
the film was removed to the fixer solution until
the background was clear. Finally the film was
washed briefly in water and dried at room
temperature before being photographed.
RESULTS
Bacterial strains
X. citri subsp. citri strains of Thailand
were isolated from different kinds of Citrus spp.,
namely, mandarin, (C. reticulata), lime (C.
aurantifolia), pummelo (C. grandis) and sweet
orange (C. sinensis) from major citrus producing
provinces of Thailand (Table 1). Total X. citri
subsp. citri strains in this study were 19 strains
from Thailand, 2 strains from Japan, and 2 strains
from Saudi Arabia. Other xanthomonads included
in this study consisted of 10 strains of X. fuscans
subsp. aurantifolii, 2 strains of X. alfalfae subsp.
citrumelonis, 1 strain of X. campestris pv.
campestris, 11 strains of X. campestris pv. glycines,
9 strains of X. citri subsp. malvacearum and 1
strain of X. fuscans subsp. fuscans.
PCR specificity
The specific 354-bp PCR fragment was
amplified with 354 F/R primers from all 23 strains
of Xsc (Table 2 and Figure 1A). No fragment of
268
Kasetsart J. (Nat. Sci.) 41(2)
Table 2 Comparison of specificity test of 354 F/R, VM3-VM4, KF-KR and 2-3 primers by classical
PCR. Specific amplification product of each primer pair was determined on agarose gel at
concentration of 1% for 354 F/R primers and 1.5% for VM3-VM4, KF-KR and 2-3 primers in
0.5x TBE buffer.
Xanthomonas species
PCR primers
354F-354R
VM3-VM4
KF-KR
2-3
y
z
X. citri subsp. citri (23)
23
23
23
23
X. fuscans subsp. aurantifolii (10)
0
5
0
0
X. alfalfae subsp. citrumelonis (2)
0
0
0
0
X. citri subsp. malvacearum (9)
0
9
0
9
X. fuscans subsp. fuscans (1)
0
0
0
0
X. campestris pv. campestris (1)
0
0
0
0
X. campestris pv. glycines (11)
0
9
0
0
y
z
Total number of Xanthomonas species in specificity test.
Total number of classical PCR positive results of each Xanthomonas species and 0 = negative result.
Figure 1 A) PCR amplification products of 354 F/R primers on 1% agarose gel 0.5x TBE buffer. B)
Southern blot hybridization with 354 bp probe of Xanthomonas species. Lane 1) DNA
marker 1 kb (Biolab); 2-5) X. citri subsp. citri: T7, J131, 1258, 1270; 6-11) X. fuscans
subsp. aurantifolii: 1415, 1416, 1419, 1420, 1360, 1361; 12-13) X. alfalfae subsp. citrumelonis:
1267, 1274; 14) X. campestris pv. glycines: NKR 21; 15) X. citri subsp. malvacearum: 1318;
16) X. fuscans subsp. fuscans: 1316; 17) X. campestris pv. campestris: 657.
Kasetsart J. (Nat. Sci.) 41(2)
expected size was amplified with 354 F/R primers
from 34 strains of other xanthomonads including
10 strains of X. fuscans subsp. aurantifolii, 2
strains of X. alfalfae subsp. citrumelonis, 9 strains
of X. citri subsp. malvacearum, 1 strain of X.
fuscans subsp. fuscans, 1 strain of X. campestris
pv. campestris and 11 strains of X. campestris pv.
glycines (Table 2).
Other primer pairs, VM3-VM4, KF-KR
and 2-3, also amplified expected fragment of PCR
product from all strains of Xcc (Table 2).
However, VM3-VM4 primers still provided the
expected fragment from 5 strains of X. fuscans
subsp. aurantifolii including 1 strain of B-strain
Xsc(T7)
1
269
and 4 strains of C-strain, 9 strains of X. citri subsp.
malvacearum and 9 strains of X. campestris pv.
glycines. The KF-KR primers were not crossreacted to other xanthomonads. The 2-3 primers
also gave expected fragment from 9 strains of X.
citri subsp. malvacearum.
PCR sensitivity
Sensitivity of 354 F/R primers for
detection of viable cells of Xcc and purified total
DNA of Xcc strain T7 were 70 cells per µl and 50
pg per µl (Figure 3), respectively by PCR reaction
and amplification program were followed as
previously.
GACGGCGCGGCTCAGGATGCTGCTAAGGGAGCTGGACGCGCGAAAGGTAATCTGGAAGAC
60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AE011881.1
Xsc(T7)
4675
61
GACGGCGCGGCTCAGGATGCTGCTAAGGGAGCTGGACGCGCGAAAGGTAATCTGGAAGAC
CAGCTGCGTGTTGCCAACGAGCTACTGCGTGGC*TTGCAAATCCTTGGCATTAGCGACGAA
||||||||||||||||||||||||||||||||
AE011881.1
Xsc(T7)
4735
121
4734
120
|||||||||||||||||||||||||||
CAGCTGCGTGTTGCCAACGAGCTACTGCGTGGT*TTGCAAATCCTTGGCATTAGCGACGAA
GCCGAAGCGTTGGAGCAGGACCTCACCGGGATCTTAAATGCCTTTTCAAAGTCGATTCTG
4794
180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AE011881.1
Xsc(T7)
4795
GCCGAAGCGTTGGAGCAGGACCTCACCGGGATCTTAAATGCCTTTTCAAAGTCGATTCTG
4854
181
CAAAGTGAAAGAGGGATCGCGACTGCTGAGGAGGCTAGACGCGAGCAGGCTCTCAATACG
240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AE011881.1
Xsc(T7)
4855
241
CAAAGTGAAAGAGGGATCGCGACTGCTGAGGAGGCTAGACGCGAGCAGGCTCTCAATACG
4914
CTTGTTGCATTTCTAATGAGCTTCGCGAGCCGAAGCGGCGTACGTGATCGACTGAACATC
300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AE011881.1
Xsc(T7)
4915
301
CTTGTTGCATTTCTAATGAGCTTCGCGAGCCGAAGCGGCGTACGTGATCGACTGAACATC
4974
TTTACCACCAACTATGACAGGCTAATCGAAGCTGGTGCTGAGTTGGCTGGGCTG
354
||||||||||||||||||||||||||||||||||||||||||||||||||||||
AE011881.1
4975
TTTACCACCAACTATGACAGGCTAATCGAAGCTGGTGCTGAGTTGGCTGGGCTG
5028
Figure 2 Comparison of nucleotide sequences of PCR product fragment of 354 F/R primers from
Xanthomonas citri subsp. citri (T7 strain) and Xanthomonas axonopodis pv. citri strain 306
gene XAC 2443 (Accession AE011881) with BlastN program showed 99.7% similarity
* non-similar nucleotide
Kasetsart J. (Nat. Sci.) 41(2)
270
Southern blot hybridization
The amplified PCR products from all
strains of Xcc (Table 2) were hybridized with 354bp probe but not with other xanthomonads (Figure
1B).
easily misidentified as Xcc (Schoulties et al.,
1987).
Effective control and eradication of citrus
canker needs a rapid, specific, and sensitive
detection techniques. The polymerase chain
Sequencing of target PCR product
The 354 bp, expected PCR fragment
from Xcc was amplified by 354 F/R primers. The
sequences obtained from 354 F/R cloned were
blast(N) in Genbank database at National Center
for Biotechnology Information (http://www.ncbi.
nlm.nih.gov/BLAST/). Searching results showed
that sequences of 354 bp of expected product were
99.7% similar to sequence of Xac strain 306 gene
XAC 2443 (Accession AE011881) (Figure 2).
DISCUSSION
Xanthomonas citri subsp.citri (Xcc) is
the causal agent of citrus bacterial canker disease,
an important pathogen of Citrus species, and it is
important to international phytosanitary quarantine
in many citrus producing countries worldwide
(OEPP/EPPO, 2005). Other bacterial citrus
pathogens, X. fuscans subsp. aurantifolii and X.
alfalfae subsp. citrumelonis are closely related to
Xcc and X. alfalfae subsp. citrumelo and have been
Figure 3 PCR amplification products of 354 F/
R primers on 1% agarose gel 0.5x TBE
buffer. Lane 1) marker DNA 1 kb
(Fermentas?), 2-7) chromosomal DNA
of X. citri subsp. citri at concentration
from 50 ng to 50 fg per microliter by
ten-fold dilution.
Table 3 Sensitivity of classical PCR for detecting viable cells and purified DNA of Xanthomonas citri
subsp. citri strain T7.
Dilutiona
Cell/µl
PCR resultsc
DNA concentrationb
PCR resultsc
0.1 OD600 nm
7.0×104
+
50 ng/µl
+
-1
3
10
7.0×10
+
5 ng/µl
+
10-2
7.0×102
+
500 pg/µl
+
-3
10
7.0×10
+
50 pg/µl
+
10-4
7.0
5 pg/µl
-5
10
0
500 fg/µl
50 fg/µl
a
b
c
Cell suspension of Xcc was adjusted to turbidity 0.1 O.D. of wavelength 600nm which gave 7.0×104 cell/µl and ten-fold
serially diluted to 10-5. The number of cell per microliter was counted by haemacytometer.
DNA of Xcc was adjusted by ten-fold dilution from 50 ng/µl to 50 fg/µl.
PCR specific amplification with 354 F/R primers were performed following the reaction mix and amplification program in
methods. Presence (+) or absence (-) of unique predicted PCR product size after agarose gel electrophoresis.
Kasetsart J. (Nat. Sci.) 41(2)
reaction technique has been used for rapid and
reliable detection for many plant pathogens
(Henson and French, 1993). Thus, this PCR
technique is suitable for routine assay in
international quarantine which requires a rapid and
sensitive method for routine assay. Plasmid (pthA
gene family) and chromosomal DNA of Xcc have
been used to design specific PCR primers (Hartung
et al., 1993, Kingsley et al., 2000, Mavrodieva et
al., 2004) for detection of Xcc.
In this experiment, new specific primers,
354 F/R primers, were designed from a fragment
in chromosomal DNA of Xcc by substractive
hybridization that translated to conserved
hypothetical protein (gene XAC2443). The results
were that 354 primers showed specific DNA
amplification of all strains of Xcc. These Xcc
strains were isolated from different hosts and
geographical areas in Thailand including strains
from Japan and Saudi Arabia which gave the
expected 354 bp PCR fragment but not from other
xanthomonads (Table 2). This is the first report
of using sequences from a conserved hypothetical
protein in chromosomal DNA to design specific
PCR primers for detection of Xcc. The PCR
primers from conserved hypothetical protein
region showed more specificity than primers from
plasmid DNA (VM3-VM4 and 2-3 primers, Table
2).
The primers designed from chromosomal
DNA, KF-KR, had specific amplification with all
strains of Xcc in this experiment. The primers
also produced prominent band with Xcc A and A*strain but the reactions with Aw-strain and X.
fuscans subsp. aurantifolii (B and C-strain) were
inconsistent and also gave more primer-dimer
products (Mavrodieva et al., 2004). At present,
PCR product fragment of KF-KR primers still
cannot be identified when searching with Genbank
database by using BLAST program provided by
National Center for Biotechnology Information
(NCBI).
The primers designed from plasmid
271
DNA, 2-3 primers and pthA gene family, VM3VM4 primers, amplified not only all strains of Xcc
but also other xanthomonad strains. Primers 2-3
cross-reacted with X. citri subsp. malvacearum and
primers VM3-VM4 cross-reacted with X. fuscans
subsp. aurantifolii, X. citri subsp. malvacearum
and X. campestris pv. glycines (Table 2) because
these primers were designed for detection of
avirulence or pathogenicity genes which are
commonly found in the genus Xanthomonas
(Gabriel, 1997).
The plasmid DNA has been reported as
being easily cured, frequently mutants within the
internal sequence and not present in all pathogens
(Miyoshi, 1998). The propose of VM3-VM4
primers is to develop universal detection of Xsc
and X. fuscans subsp. aurantifolii. However,
results in this experiment showed that the primers
did not completely detect all target strains of
xanthomonad. They detected one third from X.
fuscans subsp. aurantifolii (B-strain), all 4 strains
of X. fuscans subsp. aurantifolii (C-strain) but did
not detect any of X. fuscans subsp. aurantifolii (Dstrain). Nine strains of X. citri subsp. malvacearum
and X. campestris pv. glycines also reacted with
VM3-VM4 primers. The pthA, pthB and pthC,
members of pthA gene family, belong to a family
of avirulence or pathogenicity genes found in the
genus Xanthomonas (the avrBs3/pthA gene family;
Leach and White, 1996; Gabriel, 1997) and these
may be transferred horizontally on plasmids
between Xcc and X. fuscans subsp. aurantifolii
(Brunings and Gabriel, 2003). The results of this
experiment also confirmed that the avrBs3/pthA
gene family is distributed in X. citri subsp.
malvacearum and X. campestris pv. glycines.
The assay of 354 F/R primers with
classical PCR had the ability to detect a lower limit
of about 70 CFU/µl of viable cells and the lower
limit of detection of 50 pg/µl of purified Xcc total
DNA. Sensitivity of other primers to detect viable
cells of Xcc and purified Xcc total DNA were 10
CFU/µl and 25 pg/µl for 2-3 and 10 CFU/µl and 1
Kasetsart J. (Nat. Sci.) 41(2)
272
pg/µl for VM3-VM4 primers. The target PCR
product of 354 primers was located in
chromosomal DNA of which Xcc carries a single
copy per cell, lower than plasmid that Xcc carries
multiple copies per cell (Mavrodieva et al., 2004).
However, the novel 354 F/R primers gave more
specific and reliable detection of Xcc than other
primers. Real-time PCR techniques, which are
based on hybridization of specific probe
sequences, are faster and have higher sensitivity
and specificity than classical PCR (Schaad et al,
2002). Combination of the new specificity primers
(354 F/R) with real-time PCR technique will
improve the efficacy of Xcc detection to be more
specific, sensitive, accurate, reliable, and faster in
the future work.
ACKNOWLEDGEMENTS
The authors are thankful for the financial
support from the Thailand Research Fund under
the Royal Golden Jubilee Ph.D. Program and
Kasetsart University Research and Development
Institute. The authors are also thankful to the kind
support of bacterial cultures from Ms. Nuttima
Boonwatana, Plant Pathology Research Group,
Plant Protection Research and Development
Office, Department of Agriculture and Dr. Srimek
Chowpongpang, Department of Plant Pathology,
Kasetsart University, for his kind suggestions and
support for cloning and sequencing of Xcc.
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Kasetsart J. (Nat. Sci.) 41 : 274 - 281 (2007)
Soil-to-Plant Transfer of Radiocaesium in Thailand
Thitika Thammavech and Teerasak Veerapaspong*
ABSTRACT
Soil-to-plant transfer factors (TF) of radiocaesium-137 were estimated by considering soil
properties of 51 provinces in Thailand, and by using the model of Absalom. According to our study, the
Absalom model could estimate average TF values to be 0.0852 ± 0.0475. Compared with average
measured TF values which was 0.1289 ± 0.0529, it was found that calculated TF values decreased with
increasing pH, clay contents and exchangeable K+. The corresponding calculated TF values increased
with increasing organic matter contents and NH4+ concentrations. Statistical analysis showed that Relative
Euclidean Difference (RED) was 0.238, reliability index (k) was 0.661 and geometrically intuitive
reliability index (kg) was 1.97, which confirmed that the Absalom model was reasonably accurate.
Calculated TF values by the Absalom model were in good agreement with the measured ones. However,
calculated TF values were found to be significantly different from the measured ones for some provinces
in Thailand. The parameters used in the Absalom model needed to be modified to suitably match soil
properties in Thailand.
Key words: transfer factor, Absalom model, radiocaesium; soil properties
INTRODUCTION
Radionuclides produced by nuclear
explosion and nuclear facilities have the potential
to be released into the atmosphere. These nuclides
are part of the fallout which is deposited on the
ground and reach human bodies via food chain
(Eisenbud, 1973). Among deposited radionuclides,
radiocaesium (137Cs, half life is 30 years) is the
dominant fission product which has a high relative
mobility in the soil–plant system, long term
bioavailability, high radiotoxicity, continuing to
cycle through the soil plant-animal system, and is
longlived. The plant uptake of deposited 137Cs
from soil, commonly expressed as soil to plant
transfer factor (TF) is widely used while
calculating the radiological humus dose via the
ingestion pathway.
Absalom et al. (2001) presented a model
which predicted the radiocaesium soil to plant
transfer factor (TF) on the basis of easily measured
soil characteristics (pH, clay content, organic
+
matter content, exchangeable K + and NH 4
concentration). In the present work, data of soil
properties and 137Cs activity concentrations in soil
and grass of some selected provinces in Thailand
were collected and were used as input parameters
to calculate transfer factor (TF) in the Absalom
model. Finally, the calculated TF values were
compared with the measured TF values to test
whether the Absalom model could be applied to
the soil characteristics in Thailand.
Department of Physics, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
* Corresponding author, e-mail: fscitsv@ku.ac.th
Received date : 06/11/06
Accepted date : 01/02/07
Kasetsart J. (Nat. Sci.) 41(2)
MATERIALS AND METHODS
Model descriptions
Absalom et al. (1999) presented a semimechanistic model, which predicted activity
concentrations of 137Cs in plants. The model
utilized as input soil characteristic parameters
including clay content and exchangeable K+. In
2001, Absalom et al. (2001) developed the model
which accounted for the effect of organic matter
on 137Cs adsorption by soil and uptake by plants.
Therefore, radiocaesium bioavailability is strongly
influenced by soil properties such as pH, clay
content, organic matter and exchangeable K+
(Cremers et al., 1988). This model can be applied
to mineral and organic soils simultaneously to
provide a more generally applicable simulation of
137Cs dynamics. The model of Absalom et al.
(2001) assumed that 137Cs adsorption occurred
exclusively on both clay and humus surfaces,
however, fixation only occured on clay, and the
radiocaesium adsorbed on the organic fraction was
not subject to fixation. The relationship between
adsorbed and solution of 137Cs was described by a
labile 137Cs distribution coefficient (kdl, dm3 kg-1)
which was estimated as a function of clay content
and exchangeable K + . Plant uptake of
radiocaesium was described by a concentration
factor (CF, Bq kg-1 plant/Bq dm-3 soil solution)
which was related to solution K+ concentration
([mK], moles dm-3).
Data sources
According to input parameters, the data
referred to six different regions in Thailand.
Samples were collected from several provinces in
the north, northeast, east, west, middle and south
of Thailand. Each soil sample consisted of
subsamples collected from an area of 100 m2. The
samples were taken from 0 to 10 cm upper soil
layer. Specific soil parameters in each province
were available for comparison with 137 Cs
concentration in the grass samples.
275
Five independent soil properties (pH,
clay content, organic matter, exchangeable K+ and
NH +4 concentration) and initial 137Cs activity in
soil were required as the model input parameters
in the Absalom model assuming certain days after
a deposition of 137Cs in soil for the prediction of
TF values in the selected regions. Organic matter
(OM) content was calculated as OM = organic
carbon × 1.724 (Nelson and Sommers, 1982). The
five values (pH, clay content, organic matter,
+
exchangeable K+ and NH 4 concentration) in Table
1 (LLD, 1988) are used as the input parameters to
calculate transfer factor of soil-to-plant (here, it
was grass) in the model. The soil and grass were
dried and homogenized before being analysed.
137Cs activities in soil and grass, measured by a
Hyperpure Germanium gamma-ray detector
(HPGe), are also shown in Table 1
(Itthipoonthanakorn).
RESULTS AND DISCUSSION
Since the Absalom model takes into
account the time-dependent changes in TF due to
radiocaesium fixation, the calculations were
performed assuming 365 days after uniform
deposition of a certain amount of 137Cs (Bq m-2)
in soil. The same parameters as in the model were
used in the calculations.
Predicted and observed 137Cs transfer
factor (TF) values for grass are given in Table 2
and Figure 1.
Calculated TF values of 137Cs from soil
to grass grown in tropical Thailand are shown in
Figures 2-6 compared to different functions of soil
properties. It can be seen from Figures 2-4 that
the calculated TF values decrease with increasing
pH, clay content and exchangeable K +. The
corresponding calculated TF values increase with
increasing organic matter content and NH +4
concentration, as shown in Figures 5-6.
Kasetsart J. (Nat. Sci.) 41(2)
276
Table 1 Soil properties and 137Cs activities in soil and grass of some selected provinces in Thailand.
Region
North
Central
North-East
Province
pH
Clay
Organic
content
matter
Ex-K+
(cmolc
kg-1)
[NH4+]
137Cs
activity
137Cs
activity
(×10-5)
concentration in
concentration in
(%)
(%)
(mol dm-3 )
soil (Bq kg-1)a
grass (Bq kg-1)a
1.Chaiang Rai
4.3
8.0
0.914
0.10
2.40
1.669
0.108 ± 0.018
2.Chaiang Mai
5.3
15.6
1.810
0.20
7.70
0.989 ± 0.235
0.086 ± 0.022
3.Nakhon Sawan
8.2
30.7
2.879
0.20
34.30
0.813 ± 0.182
0.068 ± 0.021
4.Phayao
5.7
9.5
1.379
0.10
5.00
1.171 ± 0.269
0.066 ± 0.024
5.Phichit
4.5
45.0
2.689
0.20
18.50
0.685 ± 0.223
0.118 ± 0.061
6.Phetchabun
5.9
6.0
1.672
0.10
4.00
0.619 ± 0.140
0.060 ± 0.031
7.Phrae
5.1
12.0
2.069
0.10
6.00
1.033 ± 0.252
0.081 ± 0.028
8.Uthai Thani
4.8
13.5
3.448
0.10
5.60
1.150 ± 0.304
0.155 ± 0.024
9.Bangkok
4.2
61.5
0.879
0.60
25.50
1.197 ± 0.480
0.078 ± 0.023
10.Kanchanaburi
4.7
36.5
0.759
0.10
8.40
0.684 ± 0.158
0.088 ± 0.033
11.Chai Nat
6.0
19.8
0.345
0.10
6.30
0.676 ± 0.345
0.050 ± 0.009
12.Nakhon Nayok
5.1
44.9
0.172
0.20
10.60
0.734 ± 0.218
0.097 ± 0.030
13.Nakhon Pathom
5.0
65.4
4.241
0.50
29.40
0.997 ± 0.327
0.073 ± 0.037
14.Nonthaburi
7.2
52.3
0.721
0.49
5.31
0.953 ± 0.284
0.141 ± 0.051
15.Pathum Thani
4.2
46.0
1.569
0.20
20.30
0.898 ± 0.312
0.111
16.Ratchaburi
4.8
65.1
9.775
0.30
36.50
0.474
0.104 ± 0.019
17.Lop Buri
7.8
52.0
2.534
0.70
84.00
0.969 ± 0.242
0.172 ± 0.048
18.Samut Prakan
5.3
74.5
1.827
1.00
28.10
0.519
0.068 ± 0.026
19.Saraburi
6.6
86.0
1.327
0.30
54.30
0.977 ± 0.244
0.162 ± 0.048
20.Sing Buri
5.9
44.5
2.155
0.30
27.00
0.630 ± 0.046
0.079 ± 0.043
21.Ang Thong
5.0
79.6
3.827
0.50
32.20
0.902 ± 0.327
0.127 ± 0.034
22.Ayuthaya
5.0
65.1
1.207
0.30
25.40
0.955 ± 0.268
0.139 ± 0.064
23.Kalasin
6.6
10.0
0.034
0.03
0.60
0.834 ± 0.167
0.097 ± 0.027
24.Khon Kaen
6.0
5.8
1.379
0.10
5.20
1.456 ± 0.113
0.170 ± 0.032
25.Chaiyaphum
4.7
8.7
0.241
0.03
2.80
0.643 ± 0.157
0.075 ± 0.024
26.Nakhon Phanom
5.4
6.1
2.862
0.20
5.30
0.963 ± 0.150
0.106 ± 0.033
27.Maha Sarakham
5.4
2.5
0.931
0.10
2.90
0.791 ± 0.139
0.099 ± 0.024
28.Mukdahan
5.0
3.6
3.069
0.10
4.80
0.497 ± 0.158
0.101 ± 0.034
29.Yasothon
5.2
10.8
0.162
0.42
3.58
0.541 ± 0.170
0.082 ± 0.022
30.Roi Et
5.3
6.6
0.103
0.03
1.00
0.769 ± 0.176
0.132 ± 0.028
31.Loei
6.1
6.3
0.345
0.07
1.63
0.705 ± 0.133
0.070 ± 0.026
32.Si Sa Ket
5.0
17.0
0.914
0.03
3.30
0.329
0.098 ± 0.029
33.Sakon Nakhon
5.9
11.0
8.068
0.40
23.50
0.906 ± 0.243
0.106 ± 0.024
34.Surin
4.3
10.7
1.862
0.10
7.40
0.762 ± 0.181
0.131 ± 0.029
35.Nong Bua Lam Phu
4.1
7.9
0.197
0.19
0.92
1.140 ± 0.211
0.127 ± 0.030
36.Ubon Ratchathani
4.9
2.0
0.414
0.10
1.58
0.681 ± 0.122
0.070 ± 0.016
37.Chachoengsao
5.5
2.8
0.793
0.10
1.60
0.731 ± 0.216
0.108 ± 0.041
38.Chon Buri
5.1
6.6
0.707
0.10
1.60
1.659 ± 0.265
0.105 ± 0.028
39.Prachin Buri
5.8
4.8
0.707
0.05
1.90
0.646 ± 0.184
0.124 ± 0.029
40.Sa Kaeo
4.6
7.5
0.271
0.53
5.30
0.5989 ± 0.182
0.095
West
41.Prachuap Khiri Khan
7.3
1.5
1.741
0.10
4.20
0.403
0.109 ± 0.030
42.Phetchaburi
7.1
4.0
0.155
0.10
1.40
0.994 ± 0.288
0.098 ± 0.026
South
43.Krabi
4.3
8.6
2.327
0.10
3.40
0.876 ± 0.350
0.122 ± 0.025
44.Trang
6.0
11.0
2.638
0.10
5.40
0.726 ± 0.252
0.058 ± 0.025
45.Nakhon Si Thammarat
4.7
19.0
2.276
0.10
4.70
0.811 ± 0.230
0.071 ± 0.055
46.Narathiwat
4.3
14.2
8.448
0.30
34.30
1.341 ± 0.288
0.204 ± 0.027
47.Pattani
6.3
8.3
0.586
0.10
1.60
0.742 ± 0.083
0.163 ± 0.035
48.Phangnga
5.9
7.0
1.879
0.10
2.60
1.210 ± 0.164
0.121 ± 0.026
49.Phuket
4.6
18.5
3.293
0.10
4.50
1.132 ± 0.285
0.151 ± 0.053
50.Songkhla
4.6
8.0
1.017
0.10
2.20
1.132 ± 0.262
0.070 ± 0.023
51.Satun
4.8
14.5
a
(average value ± standard error)
Source: Land Development Department or LDD (1988)
4.207
0.30
6.30
1.076 ± 0.301
0.040 ± 0.018
East
South
Kasetsart J. (Nat. Sci.) 41(2)
Table 2 Measured and calculated TF values of some provinces in Thailand.
Region
Province
Measured TF value
North
1.Chaiang Rai
0.0647
2.Chaiang Mai
0.0871
3.Nakhon Sawan
0.0830
4.Phayao
0.0562
5.Phichit
0.1725
6.Phetchabun
0.0975
7.Phrae
0.0783
8.Uthai Thani
0.1347
9.Bangkok
0.0655
10.Kanchanaburi
0.1289
central
11.Chai Nat
0.0743
12.Nakhon Nayok
0.1318
13.Nakhon Pathom
0.0735
14.Nonthaburi
0.1482
15.Pathum Thani
0.1237
16.Ratchaburi
0.2195
17.Lop Buri
0.1772
18.Samut Prakan
0.1320
19.Saraburi
0.1663
20.Sing Buri
0.1248
21.Ang Thong
0.1410
22.Ayuthaya
0.1459
North-East
23.Kalasin
0.1159
24.Khon Kean
0.1166
25.Chaiyaphum
0.1161
26.Nakhon Phanom
0.1101
27.Maha Sarakham
0.1253
28.Mukdahan
0.2032
29.Yasothon
0.1513
North-East
30.Roi Et
0.1713
31.Loei
0.0989
32.Si Sa Ket
0.2979
33.Sakon Nakhon
0.1168
34.Surin
0.1713
35.Nong Bua Lam Phu
0.1114
36.Ubon Ratchathani
0.1027
East
37.Chachoengsao
0.1476
38.Chon Buri
0.0633
39.Prachin Buri
0.1927
East
40.Sa Kaeo
0.1587
West
41.Prachuap Khiri Khan
0.2697
42.Phetchaburi
0.0981
277
Calculated TF value
0.0677a
0.0448b
0.0790
0.0738a
0.1124
0.0837
0.0918a
0.1053
0.0350b
0.1242
0.0640
0.0550b
0.0479
0.0095b
0.1256a
0.1527b
0.0329b
0.0160b
0.1173
0.0613b
0.0543b
0.0787b
0.0792
0.0856
0.1876a
0.0635b
0.1093
0.1683
0.0185b
0.1174
0.0661
0.2344
0.0772
0.1139
0.0358b
0.1001
0.0915
0.0601
0.1096b
0.0199b
0.2134
0.0525b
Kasetsart J. (Nat. Sci.) 41(2)
278
Table 2 (continued).
Region
Province
South
43.Krabi
44.Trang
45.Nakhon Si Thammarat
46.Narathiwat
47.Pattani
48.Phangnga
49.Phuket
50.Songkhla
51.Satun
a
b
Measured TF value
0.1398
0.0806
0.0871
0.1523
0.2202
0.0998
0.1331
0.0611
0.0370
Calculated TF value
0.0876
0.0856a
0.0830
0.1506
0.0473b
0.0721
0.0894
0.0651a
0.0370
The calculated TF values were found to be overestimating compared to the measured TF values for most provinces in
Thailand.
The calculated TF values were found to be significantly different from the measured TF values for several provinces in
Thailand.
0.35
measured TF value
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
calculated TF value
Figure 1 Measured and calculated TF values of 137Cs for grass in Thailand. The solid line indicates 1:1
relationship for measured and calculated values.
calculated TF value
0.25
0.20
0.15
0.10
0.05
0.00
0.0
2.0
4.0
6.0
8.0
10.0
pH
Figure 2 Calculated TF values are shown as a function of pH. The solid line is a curve fitted to the data
in the graph.
Kasetsart J. (Nat. Sci.) 41(2)
279
calculated TF value
0.25
0.20
0.15
0.10
0.05
0.00
0.0
20.0
40.0
60.0
80.0
100.0
% Clay
Figure 3 Calculated TF values are shown as a function of clay content. The solid line is a curve fitted
to the data in the graph.
calculated TF value
0.25
0.20
0.15
0.10
0.05
0.00
0.00
0.20
0.40
0.60
0.80
1.00
1.20
-1
+
Ex-K (cmolc kg )
Figure 4 Calculated TF values are shown as a function of exchangeable K+. The solid line is a curve
fitted to the data in the graph.
calculated TF value
0.25
0.20
0.15
0.10
0.05
0.00
0.00
2.00
4.00
6.00
8.00
10.00
12.00
% OM
Figure 5 Calculated TF values are shown as a function of organic matter (OM) content. The solid line
is a curve fitted to the data in the graph.
Kasetsart J. (Nat. Sci.) 41(2)
280
calculated TF value
0.25
0.20
0.15
0.10
0.05
0.00
0.00
10. 00
20. 00
30. 00
40. 00
50. 00
60. 00
[NH4 ] × 10-5 (mol dm-3)
+
Figure 6 Calculated TF values are shown as a function of NH4+ concentration. The solid line is a
curve fitted to the data in the graph.
Measured TF values of 137Cs for grass
were observed to be 0.037 - 0.298 in the north,
northeast, east, west, middle and south, with an
average of 0.129 ± 0.053. These values were
relatively high compared to the corresponding
values (0.010 - 0.234 in the north, northeast, east,
west, middle and south, with an average of 0.085
± 0.048) predicted by the Absalom model.
These calculated values differed
significantly from the measured values. This is due
to the differences in soil, the types of grass and
the environmental conditions. In addition, soil
management such as plough, cultivation method
and fertilization, microbial process, root density,
soil moisture and 137Cs uptake may decrease with
increasing 137 Cs-soil contact time after the
deposition on soil (Bell et al., 1988; Kirk and
Staunton, 1989; Noordijk et al., 1992; Ehlken and
Kirchner, 2002; Rahman and Voigt, 2004).
Simple statistical analysis (Williams and
Leggett, 1984) showed that the agreement between
model and measured values (Relative Euclidean
Difference, RED) was 0.238, the value of the
reliability index (k) was 0.661 and the
geometrically intuitive reliability index (kg) was
1.97, which confirmed that the Absalom model
was reasonably accurate. Calculated TF values by
the Absalom model were in good agreement with
the measured ones. However, calculated TF values
were found to be significantly different from the
measured ones for some provinces. As a result,
the parameters used in the Absalom model needed
to be suitably modified to the characteristics of
soils in Thailand.
CONCLUSION
In this work, the uptake of deposited
has been predicted based on the soil
properties, such as pH, clay content, organic matter
+
content, exchangeable K+ and NH 4 concentration
valid for the tropical environment in Thailand, and
using the Absalom model. It has been found that
the calculated TF values differ significantly from
the measured values for some provinces in
Thailand, which implies that the soil properties in
these provinces differ from those used in the
Absalom model and they need to be measured
practically in order to validate the model.
Furthermore the parameters (k3, k4, k5, k6, kfast,
kslow, Pslow and CECclay) could be re-evaluated for
the tropical environment of Thailand.
137 Cs
ACKNOWLEDGEMENTS
The authors would like to express their
Kasetsart J. (Nat. Sci.) 41(2)
sincere gratitude and deep appreciation to Dr.
Kanokrat Tiyapun at the Bureau of Technical
Support for Safety Regulation, Office of Atom for
Peace (OAP), Thailand, for her initiative idea and
guidance for utilizing the Absalom model, and also
fruitful discussions. For supporting data of 137Cs
activities in soil and grass, they would like to thank
Mr. Thawatchai Itthipoonthanakorn at the Bureau
of Technical Support for Safety Regulation, OAP.
LITERATURE CITED
Absalom, J.P., S.D. Young and N.M.J. Crout.
1995. Radiocaesium fixation dynamics:
Measurement in six Cumbrian soils. Eur. J.
Soil Sci. 46: 461-469.
_____, _____, _____, A.F. Nisbet, R.F.M.
Woodman, E. Smolders and A.G. Gillett.
1999. Predicting soil to plant transfer of
radiocaesuim using soil characteristics.
Environ. Sci. Technol. 33: 1218-1223.
_____, _____, _____, A. Sanchez, S.M. Wright,
E. Smolders, A.F. Nisbet and A.G. Gillett.
2001. Prediction the transfer of radiocaesium
from organic soils to plant using soil
characteristics. J. Environ. Radioact. 52: 3143.
Bell, J.N.B., M.J. Minski and H.A. Grogan. 1988.
Plant uptake of radionuclides. J. Soil Use
Manage. 4 (3): 76-84.
Cremers, A., A. Elsen and P. DePreter. 1988.
Quantitative analysis of radiocaesium
retention in soil. Nature 335: 247-249.
Ehlken, S. and G. Kirchner. 2002. Environmental
processes affecting plant root uptake of
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radioactive trace elements and variability of
transfer data: a review. J. Environ. Radioact.
58: 97-112.
Eisenbud, M. 1973. Environmental Radioactivity.
Academic Press, New York. p. 118-136.
Itthipoonthanakorn, T., at the Bureau of Technical
Support for Safety Regulation, Office of Atom
for Peace (OAP). private communication.
Kirk, G.L.D. and S. Staunton. 1989. On predicting
the fate of radioactive caesium in soil beneath
grassland. J. Soil Sci. 40: 71-84.
LDD. 1988. Soil group database search. Land
Development Department. Ministry of
Agriculture and Cooperatives. Available
Source: http://www.ldd.go.th/dinthai/,
October 9, 2005.
Nelson, D.W. and L.E. Sommers. 1982. Total
carbon, organic carbon and organic matter, p.
539-577. In A.L. Page, R.H. Miller and R.
Keeney, (eds.). Methods of soil analysis.
Part 2. Chemical and microbiological
properties. American Society of Agronomy,
Madison, Wisconsin.
Noordijk, H., K.E. Bergeijk, J. Lembrechts and
M. Frissel. 1992. Impact of ageing and
weather conditions on soil to plant transfer of
radiocesium and radiostrontium. J. Environ.
Radioact. 15: 277-286.
Rahman, M.M. and G. Voigt. 2004. Radiocaesium
soil to plant transfer in tropical environments.
Environ. Sci. Technol. 71: 128-138.
Williams, L.R. and R.W. Leggett. 1984. A measure
of model reliability. Health Phys. 46 (1): 8595.
Kasetsart J. (Nat. Sci.) 41 : 282 - 287 (2007)
Beta-carotene, Mimosine and Quality of Leucaena Silage Kept
at Different Duration
Wanna Angthong1, Boonlom Cheva-Isarakul2*, Somkid Promma3
and Boonserm Cheva-Isarkul2
ABSTARCT
Leucaena leucocephala leaves (LL) were ensiled by mixing with 20% rice bran and 20%
water (fresh LL basis). The material was kept for 21, 51, 81 and 111 days in vacuumed double layer
plastic bags, each containing 26 kg. Five bags were randomly taken at each interval for quality evaluation
by organoleptic test as well as by organic acid and chemical analysis. It was found that the ensiling
period did not have much influence on most of the chemical compositions. All samples of leucaena leaf
silage (LLS) had pH of 4.4-4.5 and 35.22-35.65% DM (dry matter). The compositions on DM basis
were 21.49-22.29% CP (crude protein), 7.76-8.22% EE (ether extract), 31.18-33.68% NDF (neutral
detergent fiber), 2.0-2.9% acetate and 6.9-9.7% lactate (DM basis). DM loss was 10.35-12.32% which
was in the normal range for good quality silage. The most interesting points were the increment of βcarotene after ensiling from 88.50 to 99.92-120.25 mg/kg DM while mimosine content decreased over
90% (from 1.79 to 0.12-0.16% of DM) which were superior to a drying method. It indicated that LLS
is a good alternative for preserving LL and for reduction of mimosine.
Key words: leucaena, β-carotene, mimosine, silage, organic acids
INTRODUCTION
Leucaena leucocephala is a legume
plant, commonly found in Thailand and many
other tropical countries. The nutritive value is
comparable to alfalfa. The leaf contains around
24% CP and 116-161 mg β-carotene/kg DM
(Lamchoun, 1998). It is widely used in animal
feed for monogastrics and ruminants as a source
of CP, vitamins and minerals. In addition, it also
provides pigment for skin and egg yolk. However,
there is a limitation of using leucaena leaves (LL)
as animal feed because of its high mimosine
1
2
3
*
content. This toxic substance is a non-protein
amino acid. The chemical name is β-N-(3hydroxy-4-pyridone)-α-amino propionic acid.
After ingestion it converts to 3-hydroxy-4(1H)pyridone (DHP), that can induce goiter (Jone,
1994). And since the structure of mimosine is
similar to that of tyrosine, it becomes an antagonist
to this amino acid and inhibits protein synthesis.
Therefore, it reduces growth and production
performance. In addition, it interferes with B6
activity which is necessary for cystathionine
synthetase and cystathionase in converting
methionine to cystine, thus causes hair loss (Liener,
Department of Livestock Development, Payathai, Bangkok 10400, Thailand.
Department of Animal Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand.
Chiang Mai Livestock Research and Breeding Center, Sanpatong, Chiang Mai 50120, Thailand.
Corresponding author, e-mail: agibchvs@chiangmai.ac.th
Received date : 15/08/06
Accepted date : 05/02/07
Kasetsart J. (Nat. Sci.) 41(2)
1989). In monogastrics, the inclusion of LL above
5-10% of the ration depresses growth rate and
induces alopecia, cataract, paralysis, infertility, low
production efficiency and finally death (Norton,
1994).
In Thailand, mimosine content in LL is
1-5% and in commercial LL meal is 1-2% (Panja,
1983; Pharkwiwat, 1984). Ruminants in South
East Asia can tolerate higher mimosine level due
to gram negative rod shape rumen microbes
“Synergists jonesii” which can degrade mimosine
and its derivative DHP into non toxic substances
(Jone, 1994). However, these microbes are not
found in monogastrics, therefore it is necessary
to reduce this toxic substance before use. The most
popular method is drying under the sun. Although
other methods such as soaking in ferrous sulfate
or in running water or the supplement of amino
acids, Fe, Al and Zn are reported to be effective
(Panja, 1983; Pharkwiwat, 1984; Wee and Wang;
1987), there are some practical limitations.
Ensiling may be an appropriate method
for preservation and toxic reduction because LL
is surplus in wet season during which drying is
rather difficult. Hongo et al. (1986) and Sunagawa
et al. (1989) reported that around 90% of mimosine
is destroyed after 14-21 days of ensiling. Since
LL is a legume, it contains low soluble
carbohydrate and high buffering capacity.
Therefore silage additives such as rice bran (RB)
should be added (Thepprapakarn, 2001).
However, the report on mimosine residue
and β-carotene content in leucaena leaf silage
(LLS) is still limited, therefore this study aimed
283
to determine the quality and chemical composition
of LLS during 111 days of ensiling.
MATERIALS AND METHODS
Fresh LL leaves with green petioles are
chopped into 2-5 cm length and mixed with rice
bran and water at the ratio of 100:20:20 (w/w).
Then the mixture was filled in 2 layer plastic bags
(26 kg each), vacuumed, closed tightly and kept
for 21, 51, 81 and 111 days. At the end of each
period, 5 bags were randomly opened and the
samples were taken for Proximate and Detergent
analysis (AOAC, 1984; Georing and Van Soest,
1970). Physical quality of the silage was evaluated
by organoleptic test (Gross, 1982). The pH was
determined according to Bal et al. (1997). The
concentrations of lactic, acetic and butyric acids
were analysed by distillation (Zimmer, 1966).
Mimosine and β-carotene content were analysed
by the modified methods of Hegarty et al. (1964).
RESULTS AND DISCUSSION
Chemical composition of ingredient and LLS
Chemical compositions of LL, RB and
LLS ensiled with RB are shown in table 1. Crude
protein of fresh LL in this experiment is higher
than that reported by Göhl (1975) but lower than
that reported by Cheva-Isarakul (1982) who
reported 21.0 and 26.0% CP, respectively. It might
be due to the age of plant, the ratio of leaves, stems
and pods as well as season and cultivation
condition. NDF and ADF were similar to those
Table 1 Chemical compositions (DM basis) of fresh leucaena leaves (LL), rice bran (RB) and leucaena
silage (LLS).
DM
OM
CP
EE
Ash
NDF1/ ADF1/
ADL
NFC
pH
(% DM)
LL
30.49
92.01
23.50
3.08
7.99
39.14
23.70
8.67
26.29
6.03
RB
91.01
90.72
12.70
10.55
9.28
19.75
9.71
1.52
47.72
2/
LL
37.07
91.89
22.82
7.95
8.11
34.76
16.54
5.01
26.36
5.08
1/
ash free
2/
ensiled with 20% rice bran
284
Kasetsart J. (Nat. Sci.) 41(2)
reported by Halim (1992).
LLS had lower CP, ADF and ADL but
higher EE and ash than those of fresh LL. This
might be due to the inclusion of RB as a silage
additive.
Physical and chemical property of LLS
The quality of LLS, as evaluated by
organoleptic test, was good to fairly good (Table
2). Although the precision of this test method may
not be high due to the experience and the
sensitivity of test panels, it is practical and popular
since it needs no equipment. It was found that the
scent of lactic acid in LLS was milder than that in
corn silage. The odour of RB was also noticed
but no smell of fungi or rotting was detected. The
odour of LLS was similar to that of tea leaf silage
which is a local product for human consumption
in Northern Thailand.
The colour of LLS was darker than that
of good quality grass silages. It might be owing
to the loss of Mg in chlorophyll when reacted with
organic acids and became phaeophytin which has
brown color (Watson and Nash, 1960). In addition,
legume leaves have more pigments than grass,
therefore legume silage had darker colour than
grass silage. However, the colour intensity also
depends on other factors such as oxygen amount
in silo and temperature during ensiling. LLS in
this study had good texture and had no mold. Only
small amount of fungi was found at the opening
point of some bags. It might be due to oxygen
remaining at this point after closing these bags.
Organic acids of LLS ensilaged at
different ages, determined by distillation
techniques, are shown in Table 3. Although lactic
acid of the silage kept for 81 days was significantly
higher than that of the others, no significant
differentce was found on quality scores because
light smell of butyric acid was also noticed. All
samples were considered good grade silage even
though pH were higher than 3.7-4.2 which is
generally found in good quality grass silage. It is
owing to the fact that leucaena is leguminous plant,
therefore it contains high buffering capacity, thus
inhibits pH change. However, pH and acid content
Table 2 Quality of leucaena silage ensiled at different durations.
Ensiling period (days)
21
51
81
Odor
11.06ab
10.10a
10.92a
Color
2.00b
1.68a
1.92b
Texture
3.40
3.06
3.60
b
a
Score
16.48
14.84
16.48b
111
11.90b
1.96b
3.82
17.68b
Values in a row with different superscripts differ significantly (p<0.05)
Score: 16-20 = grade 1 (good – very good), 10-15 = grade 2 (fairy – good), 5-9 = grade 3 (fair), 0-4 = grade 4 (poor)
Table 3 Organic acids and pH of leucaena silage at different ensiling periods.
Ensiling period
pH
Acids (% of DM)
Acid (mEq/100 gDM)
(days)
Acetic Butyric
Lactic
Acetic Butyric
Lactic
21
4.5
2.00
0.00
6.86a
33.35
0.00
76.11a
a
51
4.4
2.10
0.00
7.62
34.95
0.14
85.89a
81
4.4
2.79
0.00
9.65b
46.53
0.21
107.09b
a
111
4.4
2.88
0.00
8.09
47.97
0.23
89.82a
Values in a column with different superscripts differ significantly (p<0.05)
Score: 81-100 = (very good), 61-80 = (good), 41-60 = (fair), <40 = (bad)
Score
94.00
92.10
84.80
85.30
Kasetsart J. (Nat. Sci.) 41(2)
of all LLS samples in this study were in the normal
ranges of good quality silage according to the
report by Watson and Nash (1960); i.e. ≤65%
moisture, pH < 4.8, lactate 3-14% and butyrate <
0.2% (DM basis).
Chemical compositions of LLS at
different ensiling periods are shown in Table 4. It
was found that the length of ensiling period had
no influence on DM loss and most of the chemical
compositions. The loss of DM of 10.35-12.32%
found in this experiment was in a normal range.
McDonald et al. (1991) reported that the
unavoidable loss of silage due to the action of plant
enzymes, microbial enzymes, plant respiration and
ensiling technique were 1-2, 2-4 and 2-5%,
respectively.
No significant difference was found on
CP content between prior and after ensiling with
the exception of the lower CP content of the group
kept for 111 days. The unremarkable protein loss
was due to the good ensiling condition since the
bags were vacuumed, thus only minute amount of
oxygen remained in the bags. In addition, moisture
level of the ensiling material was optimal (63%)
therefore no excess heat was produced. These
conditions led to the low dry matter and nutrient
285
loss (Watson and Nash, 1960; McDonald et al.,
1991).
Furthermore, the low CP loss might be
due to the fact that LL has condensed tannin (46% DM basis; Balogun et al., 1998). This
substance is able to inhibit protein degradation by
microbial and animal enzymes (Albrecht and
Muck, 1991). Moreover, trypsin inhibitor in RB
may also inhibit protein degradation. Most (8590%) of this inhibitor was found in embryo. The
other part of RB (without embryo) had 5-10%
while polished rice had less than 1% of this
inhibitor (Juliano, 1985).
The concentration of ash and that of EE
were not affected by ensiling period. Even though
the forms of minerals and the pattern of fatty acids
may change during ensiling process, their amount
should not decrease because no seepage was found
due to low moisture content (<65%) of the ensiling
materials. Non fiber carbohydrate (NFC) tended
to decrease after ensiling due to the conversion of
starch to lactic acid (McDonald et al., 1991;
Jaurena and Pichard, 2001) even though the
efficiency may be lower than that of sugar.
Hemicellulose tended to decrease and lactic acid
increased significantly after ensiling but no change
Table 4 Composition (DM basis) and dry matter loss of leucaena silage in various ensiling periods.
Ensiling period (days)
0
21
51
81
111
DM
37.07
35.56
35.65
35.22
35.65
Dry matter loss (%)
10.35
10.92
11.69
12.32
OM
91.89
91.93
91.75
91.57
91.66
c
c
ab
bc
CP
22.82
22.29
21.56
22.22
21.49a
EE
7.95
8.03
8.22
7.76
8.02
Ash
8.11
8.07
8.43
8.25
8.34
NFC
26.36a
28.80ab
28.09ab
30.60b
28.47ab
NDF*
34.76b
32.81ab
33.70ab
31.18a
33.68ab
a
b
b
b
ADF*
16.54
18.45
18.37
18.84
19.02b
Hemicellulose
18.22b
14.36a
15.33ab
12.34a
14.65a
Cellulose
11.53
11.87
11.86
12.22
12.11
a
b
b
b
Lignin
5.01
6.59
6.51
6.63
6.91b
Values in a row with different superscripts differ significantly (p<0.05), * ash free
Kasetsart J. (Nat. Sci.) 41(2)
286
Table 5 β-carotene and mimosine content in leucaena mixed with rice bran and ensiled at
durations.
Ensiling period
β-carotene
Mimosine
(days)
mg/kg DM
% increment
% DM
0
88.50a
0.00
1.79b
21
99.92ab
12.90
0.13a
bc
51
116.29
31.40
0.14a
81
120.28c
35.91
0.16a
abc
111
105.21
18.88
0.12a
different
% lost
0.00
92.74
92.18
91.06
93.30
Value in a column with different superscripts differ significantly (p<0.05)
was found on cellulose. These results were similar
to those reported by Jaurena and Pichard (2001).
Beta-carotene and mimosine content in
leucaena silage
β-carotene of LLS increased significantly
(Table 5). Even though no clear explanation can
be given, the result was in agreement with that
reported by Peterson et al. (1935; cited by Watson
and Nash, 1960) who found the increment of
carotene in alfalfa ensiling with acid. However,
Hellbery (1945; cited by Watson and Nash, 1960)
reported that 11-75% of carotene was loss by
oxidation during fermentation. The extent of loss
depended on oxygen content and temperature in
the silo.
Fermentation decreased 91-93% of
mimosine. The length of ensiling had no effect
on mimosine loss. The result was in agreement
with that reported by Hongo et al. (1986) and
Sunagawa et al. (1989) who found mimosine
reduction over 90% in LLS either with or without
additives. The reduction of mimosine by ensiling
being higher than by sun drying (14.5-51.1% of
the original samples) was reported by Panja
(1983), Parkwiwat (1984) and Wee and Wang
(1987). These results indicated that LLS is an
interesting alternative for feed preservation.
CONCLUSION
The ensiling of LL by mixing with 20%
rice bran and 20% water (fresh LL weight basis)
in airtight containers gave a good quality silage.
It can be kept for a long time without deterioration.
In addition, it increased β-carotene and decreased
mimosine content over 90% which was much
better than the preservation and detoxification by
sun drying. It is expected to be a good feed for
ruminant and monogastric animals.
LITERATURE CITED
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in ensiled forage legumes that vary in tannin
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AOAC. 1984. Official Methods of Analysis. 14th
ed. Association of Official Analytical
Chemists. Inc., Virginia.
Bal, M. A., J. G. Coors and R. D. Shaver. 1997.
Impact of the maturity of corn for use as silage
in the diets of dairy cows on intake, digestion,
and milk production. J. Dairy Sci. 80: 24972503.
Balogun, R. O., R. J. Jones and J. H. G. Holmes.
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Sci. Technol. 76: 77-88.
Cheva-Isarakul, B. 1982. The composition, intake
and digestibility of legume tree leaves in North
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Residues as Animal Feeds. School of
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Melbourne, Parkville, Victoria.
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fibre analysis (apparatus, reagents,
procedure and some application). USDA/
ARS Agricultural Handbook No. 379,
Washington, D.C.
Göhl, B. 1975. Tropical feeds. FAO Feed
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Futterkonservierung. pp. 6-42. In Tierische
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of six fodder tree species. pp 54. In
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Science Congress, vol. III, Bangkok.
Hegarty, M. P., R. D. Court and P. M. Thorne. 1964.
The determination of mimosine and 3,4dihydroxypyridine in biological material.
Aust. J. Agric. Res. 15: 168-179.
Hongo, F., S. Tawata, Y. Watanabe and S. Shiroma.
1986. Mimosine degradation as affected by
ensiling of Leucaena leucocephala de Wit.
Japanese J. Zootech Sci. 57(3): 223-230.
Jaurena, G. and G. Pichard. 2001. Contribution of
storage and structural polysaccharides to the
fermentation process and nutritive value of
Lucerne ensiled alone or mixed with cereal
grains. Anim. Feed Sci. Technol. 92: 159173.
Jone, R. J. 1994. Management of anti-nutritive
factors-with special reference to leucaena. pp.
216-231. In R.C. Gutteridge and H.M. Shelton
(eds.). Forage, Tree Legumes in Tropical
Agriculture. CAB International, Wallingford.
Juliano, B. O. 1985. Rice bran. pp. 647-687. In
B.O. Juliano (ed.). Rice Chemistry and
Technology. 2nd ed. American Association of
Cereal Chemists Inc., Minnessota.
Lamchoun, W. 1998. Seasonal variation and
effect of sources of beta-carotene on plasma
beta-carotene concentration of dairy cattle.
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Mai.
Liener, I. E. 1989. Antinutritional factors. pp. 339382. In R.H. Matthews (ed.). Legumes
Chemistry, Technology and Human
nutrition. Marcel Dekker, Inc., New York.
McDonald, P., N. Henderson and S. Heron. 1991.
The Biochemistry of silage. 2 nd ed.
Chalcombe Publications, UK. 340p.
Norton, B. W. 1994. The nutritive value of tree
legumes. pp. 177-191. In R.C. Gutteridge and
H.M. Shelton (eds.). Forage Tree Legumes
in Tropical Agriculture. CAB International,
Wallingford.
Panja, P. 1983. Determination of mimosine
content and toxic reduction in Leucaena
leucocephala leaves. M.Sc. Thesis, Kasetsart
University, Bangkok.
Pharkwiwat, S. 1984. Study on nutritive values
and detoxification methods of mimosine
reduction in Leucaena leucocephala leaves.
M.Sc. Thesis, Kasetsart University, Bangkok.
Sunagawa, L., F. Hongo, Y. Kawashima and S.
Tawatana. 1989. The effect of mimosinereduced leucaena feed on sheep. Japanese J.
Zootech Sci. 60(2): 133-140.
Thepprapakorn, R. 2001. Research and
development on method for production and
using giant leucaena leaves silage for local
farmer dairy cattle feeding. M.Sc. Thesis,
Chiang Mai Rajabhat University, Chiang Mai.
Watson, S. J. and M. J. Nash. 1960. The
Conservation of Grass and Forage Crops.
2nd Ed. Oliver and Boyd Ltd., Edinburgh.
Wee, K. L. and S. S. Wang. 1987. Effect of postharvest treatment on the degradation of
mimosine in Leucaena leucocephala leaves.
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Kasetsart J. (Nat. Sci.) 41 : 288 - 299 (2007)
Effects of Natural Mineral Soils on Body Weight and Liver Minerals
of Black Head Somali Sheep in Ethiopia
Sisay Tilahun1, Pravee Vijchulata2*, Pornsri Chairatanayuth2
and Suwapong Swasdiphanich3
ABSTRACT
The effects of different mineral soils on body weight and liver mineral concentration were
investigated using 48 Black Head Somali Sheep in Jijiga Somali region, Ethiopia. The soil samples
collected from 4 different sites were compared with a complete mineral mix and a control non supplement
treatment. Chemical composition of the soils indicated that they all are alkaline. Arabi, Jair and Hermokale
soils from different localities had adequate amount of Ca, K and Mg whereas Mn, Fe and Zn were below
the recommended standard by 76 to 95%, 87 to 97% and 68 to 88%, respectively. The mean daily
mineral intakes of sheep supplemented with Jair, Hermokale, Arabi and Bole soil were 18.14, 16.51,
16.02 and 11.86 grams/sheep/day, respectively. No significant differences were observed in mineral
intake among Jair, Arabi and Hermokale groups. When compared to other treatment the daily weight
gain (mean 74.79 g), and total weight gain (mean 8.98 kg) were recorded highest (p<0.05) for sheep fed
on complete mineral mixture. Based on liver analysis sheep in the study area did not suffer from Mn, Cu
and Zn deficiencies. However, concentration of sheep fed on complete mineral mixture was significantly
higher (p < 0.05) in Ca and Fe concentration when compared to those from the non supplemental animals.
With the exception of Mg, there was no significant difference (p>0.05) in liver minerals of sheep provided
with different mineral soils. Liver Mg in sheep from Bole treatment group was significantly different
when compared to those receiving Jair, Hermokale and Arabi soils. In addition, when compared to
animals fed on different minerals soils, mineral concentration in the liver of sheep fed Bole soil was
lower (p>0.05) in Mn (5.49 ppm) and Zn (92 ppm) than those from the other groups.
Keywords: Mineral soils, weight gain, Black Head Somali sheep, liver minerals
INTRODUCTION
In the lowland parts of Ethiopia, sheep
rearing has been hampered over the years primarily
due to non-availability of good quality and
adequate feeds. During the dry season when the
available forage is low in both quantity and quality
1
2
3
*
what usually occurs is loss of live weight, low birth
weight, lower resistance to disease and poor
fertility.
In Somali region, sheep usually suffer
from diseases resulting from shortage of feed and
mineral deficiencies. Mineral imbalance
(deficiencies or excesses) in soil and forages were
Somali Pastoral and Agro Pastoral Research Institute P. O. Box 398, Jijiga, Ethiopia.
Department of Animal Science, Faculty of Agriculture, Kasetsart University, Bangkok 10900,Thailand.
Department of Agronomy, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: agrpvv@ku.ac.th
Received date : 10/01/07
Accepted date : 19/03/07
Kasetsart J. (Nat. Sci.) 41(2)
considered responsible for low productive and
reproductive performance of grazing ruminants in
the tropics (McDowell et al., 1997). Mineral
deficiencies are considered to be one of the
nutritional constraints to animal productivity. Poor
body conditions, slow live weight gain, low
fertility and high mortality are normally observed
in mineral-deficient animals (McDowell et al.,
1983; Vijchulata, 1995).
The main sources of mineral for animals
in the Somali region are salty water, shrub plants
and natural mineral soils. The soil mineral known
as Carro is found in vast area of Afder,
Degehabour, Gode, Jijiga, Liben and Shinile
zones. It is commonly observed that pastoralists
in these zones feed natural mineral soils to animals.
Considerable use is being made of the natural
mineral licks since they are relatively free and are
easily obtained as compared to complete mineral
mixture. Supplementation with multi-nutrient
blocks and local mineral soils such as Bole and
Megadua in some parts of Ethiopia may provide
an adequate or even excess amount of most of the
essential minerals except phosphorus (Tolera and
Said, 1994).
Studies regarding mineral supplementation have not been conducted in the region.
Moreover, attention has not been paid to its effect
on Black Head Somali (BHS) sheep. The main
objective of this study was, therefore, to determine
the mineral composition of these soils, and to
evaluate the effect of their supplementation in
comparison to Bole soil and complete mineral
mixture on body weight and liver mineral
concentrations of BHS.
MATERIALS AND METHODS
Animals and management
The study was conducted in Jijiga Somali
Region from July to October 2004. Forty eight
males BHS sheep about 12 months of age
weighing 20–25 kg were randomized by weight
289
assigned to six groups of eight sheep each. Prior
to the commencement of the experiment, the
animal were kept for 15 days for adaptation and
to observe their health status. All animals were
ear-tagged. They were also provided with neck
strips of six different colours for group
identification. All the animals were drenched with
a broad-spectrum antihelmentic and vaccinated
against Anthrax, Pasteuriolosis and Blackleg
diseases.
The natural mineral soil Arabi soil, Jair
soil and Hermokale soil were collected from Jijiga
and Shinile districts, Somali region, Bole soil was
collected from Zeway district, Oromiya region and
complete mineral lick from Thailand (Phosrich
Rockie: Phillips International Co. Ltd.). Six
treatment groups were randomly assigned to
mineral supplementation. Group I (control) was
not supplemented. Group II, III, IV, V were
supplemented with Arabi soil, Jair soil, Hermokale
soil and Bole soil, respectively. Group VI was
provided with complete mineral lick.
Sheep barn was constructed using
eucalyptus wood with 19 m × 5 m dimension and
was divided into 48 equal pens (1m × 0.8m) for
individual feeding of the minerals and to
accommodate the animals at night. Sheep were
allowed to graze together in flock on the same
pasture from 8:00 am to 6:00 pm. Between 12:30
pm and 1:30 pm and 6:00 pm to 8:00 am the
animals were driven into their pens where they
were fed individually with their respective mineral
supplements. Mineral soils were offered ad libitum
in the boxes which were fixed at the corner of
individual sheep pen. Mineral residues were
weighed on weekly basis and intakes for each
sheep were calculated. The animals were weighed
on monthly basis through out the experimental
period. All the experimental animals were
provided ad libitum with water in the pens.
Soil sampling and analysis
To study the mineral content of different
290
Kasetsart J. (Nat. Sci.) 41(2)
natural mineral soils, the soil samples were
collected from different sites of Somali region and
from Zeway area of East Showa zone, Oromiya
Regional State. The sites in Somali region were
selected from Jijiga and Shinile zones based on
the suggestion of Somali Regional Pastoral and
Agro Pastoral Research Institute (SoRPARI) and
the clan leaders. Soil samples were collected from
the sites (Arabi, Jair, Hermokale and Bole) where
pastoralists mostly use for mineral supplementation to their animals. Individual natural mineral
soil was collected using hoe to the depth of 30 cm
from three different spots which fall in the radius
of 50 to 70 m. Soil samples were mixed together
and then filled into a plastic collection bag, labeled
and stored at room temperature. A total of four
composite soil samples from the study sites were
taken and analyzed for physical and chemical
properties at National Soil Laboratory for Research
Center (NSLRC). Analysis of each sample was run
in duplicate.
The pH and electrical conductivity of the
soils were measured according to the procedure
of Landon (1984). Ca, Mg, Fe, Mn, Zn, and Cu
were determined by atomic absorption
spectrophotometer (Lindsay and Norvell, 1978).
Sodium and potassium were analysed by flame
photometer (Black, 1965). Organic carbon was
determined following the wet digestion method
of Walkley and Black (1934). Available
phosphorus was determined following Olsen
methods (Olsen et al., 1954). Total nitrogen was
determined using the modified Kjeldahl method
(Jakson, 1958).
Liver tissue collection and analysis
A total of sixteen sheep (two sheep from
each treatment group) were randomly selected and
slaughtered to determine liver mineral contents.
Liver tissue samples of 50 to 100 g were taken
from the right lobule of liver of individual animal.
The samples were stored frozen until analysed for
Ca, Mg, Fe, Mn, Cu, Zn and Co using flame atomic
absorption spectrophotometer (Perkins-Elmer,
Model 2380).
Statistical analysis
Data were analyzed using the PROC
ANOVA procedure of SAS (1999). Differences
among treatment means were evaluated using
Duncan Multiple Range Test (Cody and Smith,
1997). The statistical model used was:
yij = µ+ Ti + εij
Where, Yij = Response variable (body
weight gain, mineral intake and mineral in liver
tissue)
µ = Overall mean
Ti = The effect of ith treatment
(i = 1, 2, 3, 4, 5, 6)
εij = Residual effect
RESULTS AND DISCUSSION
Physical properties of the mineral soils
Most naturally occurring mineral
deficiencies in livestock are associated with
specific regions, and they are related to both soil
mineral concentration and soil characteristics
(McDowell 1986). Physical properties of mineral
soils collected from different sites are presented
in Table 1. pH of the soil samples ranged from 8.0
for soils from Arabi to 9.5 for soils from Bole sites
indicating that they were alkaline in nature. The
pH values soils was higher than the pH values
(ranged from 7.86 to 8.05) of natural mineral soil
from Southern low lands of Ethiopia (Kabaija and
Little, 1987; Fikre, 1990). Colours of the soils
varied from site to site ranging from dark brown
to light brownish colours. The soil textures varied
from silt to loam. The soils in Jair and Hermokale
areas were predominantly silt loam but Arabi soil
were silt in texture whereas Bole soils were sandy
clay. The color and texture of individual natural
soils are as shown in Figure 1.
Kasetsart J. (Nat. Sci.) 41(2)
291
Figure 1 Mineral soil collected from different localities
A) Arabi soil, B) Jair soil, C) Hermokale soil, D) Bole soil, E) Complete mineral lick.
Table 1 Physical properties of soil licks collected from different locations in Ethiopia.
Items
Jair
Hermokale
Arabi
pH
9.3
8.6
8.0
Sand,%
11
17
9.0
Silt, %
64
70
84
Clay, %
25
13
7.0
Texture
Silt loam
Silt loam
Silt
Chemical composition of the mineral soils
Minerals and certain chemical
characteristics of mineral soils are shown in Table
2 and 3. The organic carbon in the soils ranged
from 0.28 % for soils from Jair to 1.07 % for soils
from Hermokale area. Bole soils were less in
nitrogen than soils from any other sites in the
Somali region. Based on the critical levels set by
Bole
9.5
49
10
41
Sandy clay
Mtimuni (1982) and McDowell (1983) for tropical
soils, Arabi, Jair and Hermokale soils are sufficient
in Na, Ca, K and Mg. Therefore, the soils can be
used as supplements for certain macro element.
Arabi, Jair and Hermokale soils had lower K and
P and higher Ca, Mg, Mn, and Cu, than Bole soil.
The macro mineral composition of Bole reported
by Mohammed et al. (1989) and Adugna (1990)
Kasetsart J. (Nat. Sci.) 41(2)
292
Table 2 Macro mineral compositions of soils collected from different sites.
Sites
Ca
P
Mg
Jair, ppm
39.42
2.06
16.20
Hermokale, ppm
52.20
8.00
6.91
Arabi, ppm
67.86
6.30
8.42
Bole, ppm
4.10
2.54
0.98
Complete mineral, %
8.19
10.01
0.50
K
5.07
5.26
6.31
3.35
-
Na
191.00
179.00
60.32
84.73
20.78
Table 3 Micro mineral composition (ppm), Total Nitrogen and Organic Carbon of soils collected from
different sites.
Sites
Fe
Cu
Co
Mn
Se
Zn
I
TN (%) OC (%)
Jair
2.42
2.22
-
2.36
-
0.36
-
0.05
0.28
Hermokale
1.78
1.50
-
4.58
-
0.64
-
0.07
1.07
Arabi
0.78
0.66
-
2.96
-
0.50
-
0.09
0.98
Bole
1.00
0.28
-
0.90
-
0.50
-
0.04
0.67
3,000.00
-
50.00
300.00
300.00
2.07
-
Complete mineral
2,500.00 100.00
was similar to what was found in the present study.
However, in contrast to their studies Mn (0.90
ppm) and Zn (0.50 ppm) were found extremely
low in the present study. This could be due to the
difference in soil sampling site. In the present
study, all of the mineral soils collected from four
sites in the region could not be used as phosphate
supplement for sheep as they contain relatively
low amount of this mineral than the 10 ppm critical
level suggested by McDowell (1997). This is in
agreement with the works of (Kabaija and Little,
1987; Mohamed et al., 1989; Fikre, 1990) who
found that soils from Central and Eastern parts of
Ethiopia low in phosphorus. Phosphorus
deficiency results in reduced growth and feed
deficiency, decreased appetite, impaired
reproduction and weak fragile bone (Underwood,
1981). The Fe, Mn and Zn concentrations in all
the soil samples were below critical level at 19
ppm for Fe set by Mtimuni (1982) and at 19 ppm
and 2 ppm for Mn and Zn by McDowell (1997).
Comparing to the stipulated critical levels, Fe, Mn
and Zn were found to deficit by 87 to 96%, 76 to
95% and 68 to 82%, respectively.
The fact that the Bole soils in this study
had lower Mn, Zn and Cu than the suggested
critical values. This was on the contrary to what
was reported by Kabaija and Little (1987) and
Fikre (1990) for Southern parts of Ethiopia. This
may be attributed to the difference in the time and/
or the specific area the soil samples were collected.
The Cu content of the soils from Jair and
Hermokale was higher than the critical level of
0.6 ppm (Mtimuni, 1982) but there was a high
degree of variation among samples. Apart from
Ethiopia, Cu deficient soils have also been reported
in several other African countries (Sillanpää,
1982). The current study is in agreement with the
result of Faye et al. (1983) who reported that Cu
and Zn were deficient in forage taken from
rangelands in Southern part of Ethiopia. Blood et
al. (1983) also reported that if the amount of copper
in the diet is inadequate and copper deficiency may
occur. Composition of the natural mineral soil in
the area indicated that Na was the dominant
mineral. According to NRC (1985) recommendation, an appropriate mineral supplement should
contain at least 0.04% to 0.10% Na. The current
study indicated that Jair soil was found the highest
in Na concentration compared to other mineral
soils (Table 2). Although, macro minerals in the
soil are high compared to the requirement of
Kasetsart J. (Nat. Sci.) 41(2)
mineral supplementation suggested by NRC
(1985) but it may not be sufficient to fulfill the
mineral requirement. Moreover, micro minerals
in the soil were below sufficient levels for mineral
supplementation. Individual minerals should be
adjusted to meet a minimum of 50% of the daily
intake requirement of the sheep while formulating
mineral supplement. If the imbalance minerals
were rectified to meet standard mineral mixture
requirement, the soils would be more beneficial
to animals.
Mineral intake and live weight of the sheep
The mineral intake and live weight
change of sheep during the experimental
period are presented in Table 3. The mean daily
mineral intake of sheep supplemented with
mineral soils of Jair, Hermokale, Arabi, Bole and
complete mineral were 18.14, 16.51, 16.02, 11.86
and 15.00 gram/head, respectively. There was no
significant difference (p>0.05) in mineral
intake among the sheep fed with soils from Arabi,
Jair and Hermokale areas. The lowest mineral
intake was recorded for sheep fed with Bole soil
(11.86 g/day) from Zeway area. Bole soil had
lower Cu (0.28 ppm), Mn (0.80 ppm) and Zn (0.50
ppm) than the suggested critical values set by
Mtimuni (1982) and McDowell (1983). When
compared to all other mineral soils the level of K
in Bole soil was higher. However, it had lower
concentration of Na, Ca and Mg. The imbalance
293
of minerals of Bole soil may attribute to the lower
mineral intake by the animals. Khalili, (1993)
reported that sodium deficiency which was
evident in central parts of Ethiopia usually
causes increased soil ingestion among grazing
livestock.
The mean daily weight gains and total
weight gain of sheep fed complete mineral mixture
were significantly higher (p<0.05) than those fed
natural mineral soils. While mean daily weight
gain for sheep supplemented with complete
mineral was 74.79 g/day, sheep receiving Bole
soils and the control treatments yielded lowest
daily gain (53.02 and 53.54 g/head, respectively).
This could be attributed to the well balanced nature
of the mineral mixture including cobalt, iodine and
selenium which were not available in any of the
natural mineral soil. Van Eys et al. (1985) reported
that complete mineral supplementation increased
(p<0.01) the weight gain of both pre and post
weaning animals. The total weight gain of the
animals supplemented with different mineral
sources, in general followed the trend of the daily
weight gains. The highest total weight gain per
animal was recorded for sheep fed with complete
mineral mixture while the lowest was observed
for sheep fed with Bole soil. The total weight gains
of sheep on Arabi, Jair and Hermokale soils were
significantly higher (p<0.05) than the gains of
those sheep on bole soil and the control group.
This was in agreement with the works of
Table 4 Average mineral intake and live weight change of sheep fed the different mineral soils
(Means ± SD).
Treatment
Control
Daily mineral
Initial wt.
Final weight
Total weight
Daily weight
intake (g)
(kg)
(kg)
gain (kg)
-
22.78±1.56a
29.20±0.91b
6.43±0.78c
53.54±6.51c
gain (g)
Jair
18.14±0.00a
22.58±2.09a
29.88±1.67ab
7.30±0.89bc
60.83±7.45bc
Hermokale
16.51±2.45ab
22.43±1.59a
29.68±1.05ab
7.25±0.84bc
60.42±7.00bc
Arabi
16.02±2.85ab
22.15±1.45a
29.85±1.94ab
7.68±1.59b
63.96±12.33b
Bole
11.86±0.57c
23.38±1.87a
29.73±1.19ab
6.36±1.19c
53.02±9.97c
Complete mineral
15.00±0.00b
22.23±2.00a
31.20±1.76a
8.98±0.83a
74.79±6.92a
Means within the same column with different superscripts are significantly different (p<0.05).
Kasetsart J. (Nat. Sci.) 41(2)
294
Mohammed et al. (1989) who reported that
weight gains of Arsi sheep improved by an
average of 19±8 g/day when fed with natural
mineral lick as free choice. The low body
weight gain observed might be due to the fact
that Bole soil had lower Mn, Zn and Cu but
higher in pH, sand and clay than the other soils
(Table 1). The imbalance mineral content of the
soil might result in depressing effect on
feed intake which in turn affects the body weight
gain. Allen et al. (1986) observed a decrease
in weight gain when 1000 mg/kg of Fe were
included in the diet of sheep. Hodgson (1962)
reported that because of large quantities in the
soil, animals are also likely to augment their
Fe supplies through direct ingestion of soil or from
soil contaminated herbage Moreover, in
New Zealand it was reported that the amount of
soil ingested annually reach 75 kg for sheep
(Healy, 1978).
The average monthly intake of different
mineral soils is illustrated in Figure 2. During the
first month of the experimental period, there was
a steady increase in mineral intake of sheep in all
the treatment groups. This was followed by a
nearly constant intake over the period from second
to third month. This decline coincided with
seasonal summer rain and emergence of green
grasses after which an increase was observed until
the end of the fourth month. According to
McDowell (1997), energy and protein supplies
from emerging forages during the wet season are
higher, livestock gain weight more rapidly
resulting in high mineral requirements.
Mean monthly body weight gain of the
sheep fed different mineral sources during the
experimental period is shown in Figure 3. Sheep
supplemented with the complete mineral mixture
tended to gain more live weight than others
through out the experimental period. Sheep
receiving complete mineral lick were less heavy
than that of control animals during the 1st and 2nd
month. However, it was observed that they attained
2 kg weight more than the control group by the
4th month. Sheep fed with Bole soil were found
to loose weight during the 2nd to the 3rd months
Mineral intake (gram/month)
700
650
600
550
500
450
400
350
300
July
August
September
October
Experimental period (months)
Jair
Hermokale
Arabi
Bole
Figure 2 Average monthly intakes of different mineral soils over the four months period
Kasetsart J. (Nat. Sci.) 41(2)
295
Body weight chane (kg/head)
32
30
28
26
24
22
20
July
August
September
October
Experimental period (months)
Control
Jair
Hermokale
Arabi
Bole
Complete
Figure 3 Average monthly body weight change of sheep supplemented with different mineral soils
and complete mineral.
of the experimental period followed by gain in the
later period. This could be due to lower preference
and hence lower intake of this soil by the animals.
Animals in control group had slower increase in
body weight. Sheep in the control group showed
compensatory gain from second to fourth month
but grew slower than that of supplemented
animals. Animals in the remaining treatment
groups showed a sharp increase in body weight
after third month of the experiment. This could be
partly due to the availability of good pasture as
rain started during the second month of
experiment.
Liver mineral concentration
Mineral concentration in liver of sheep
fed on different mineral sources is presented in
Table 5. McDowell (1992) reported that liver is
the organ that often represents the status of several
elements in animals. Liver minerals varied from
0.04 to 0.08 ppm for Ca, 0.04 to 0.05 ppm for Mg,
160 to 282 ppm for Fe, 5.49 to 11.79 ppm for Mn,
113 to 229 ppm for Cu and 92 to 110 ppm for
Zn. With exception to Cu and Zn, all the liver
minerals varied significantly (p<0.05) among
treatments. Compared to the other groups,
concentration of mineral in the liver of sheep
receiving complete mineral lick were highest
(p<0.05) in Ca (0.08 ppm), Fe (282 ppm), Mn
(11.79 ppm) and Zn (110 ppm). On the contrary,
liver mineral in the control group were lowest in
Ca (0.04 ppm) and Fe (160 ppm) than the
remaining treatment groups.
However, no significant differences (p
>0.05) were observed in liver Mn, Cu and Zn of
sheep from the control and other groups. This may
be due to the fact that, before the experiment,
animals might have accumulated sufficient
amounts of certain minerals in the tissues while
the duration of the experiment was too short to
cause major changes in the status of the minerals
in the liver. Additionally, the sheep might have
consumed sufficient amounts of these minerals
from available forage and water.
Kasetsart J. (Nat. Sci.) 41(2)
296
Table 5 Mineral concentration (ppm) in the liver of sheep supplemented with different sources of
minerals (Means ± Standard Deviation).
Treatment
Ca
Mg
Fe
Mn
Cu
Zn
Control
0.04±0.00b
0.050±0.01a
160.00±27.57b
8.69±2.53ab
139.80±1.14a
97.85±4.38a
Jair
0.05±0.00b
0.040±0.01b
244.15±62.30ab
10.30±1.59ab
179.62±119.37a
94.84±1.28a
Hermokale.
0.06±0.01ab
0.040±0.01b
194.59±37.29ab
9.43±0.14ab
228.48±47.49a
106.52±9.5a
Arabi
0.05±0.00b
0.040±0.01b
162.42±24.99b
9.11±1.29ab
112.90±60.61a
96.44±5.19a
Bole
0.06±0.02ab
0.050±0.01a
170.47±27.98b
5.49±3.21b
192.80±60.74a
92.10±8.19a
Complete minerals
0.08±0.01a
0.045±0.01ab
282.20±7.49a
11.79±0.43a
167.01±7.85a
109.63±8.93a
-
-
< 180**
6*
25-75 *
< 84 **
Critical. Level
* Georgievskii (1982)
** Mtimuni (1982)
Means within the same column with different superscripts are significantly different (p<0.05)
With the exception of Mg, there was no
significant difference (p>0.05) in liver minerals
of sheep provided with different mineral soils.
Liver Mg in sheep from Bole treatment group was
significantly different (p<0.05) when compared to
those receiving Jair, Hermokale and Arabi soils.
In addition, when compared to animals fed on
different minerals soils, mineral concentration in
the liver of sheep fed Bole soil was lower (p>0.05)
in Mn (5.49 ppm) and Zn (92 ppm) than those
from the other groups. The low Mn and Zn
concentration in the liver might be induced by the
low soil Mn (0.90 ppm) and Zn (0.50 ppm) mineral
content in Bole soils (Table 3).
Although no significant difference
(p>0.05) was observed in liver Fe among mineral
soil groups, when compared to the suggested Fe
standard deficiency status at 180 ppm by Mitimuni
(1982), Fe concentration was lower in the Control
(160 ppm), Arabi (162 ppm) and Bole (170 ppm)
treated sheep. The animals fed on Jair soil showed
higher Fe concentration in the liver than
Hermokale, Arabi and Bole treatment groups. This
might be due to the fact that Jair soil had higher
Fe (2.42 ppm) content than the other mineral soils.
Similarly, sheep provided with Arabi soil showed
low Fe concentration in the liver. This agrees with
the low Fe (0.78 ppm) content in this soil. Besides
liver Fe, sheep fed on Bole soil was lower in liver
Mn (5.49 ppm) than the suggested deficiency
standard at 6 ppm by Georgievskii et al. (1982).
This corresponds to the low Mn level found in Bole
soil.
According to Georgevskii et al. (1982)
and Mtimuni (1982), the suggested standard
deficiency levels, for liver Cu and Zn ranges from
25 to 75 ppm and <84 ppm, respectively. In the
current study, Cu and Zn concentrations in the liver
of sheep from all treatments were at sufficient
levels (Table 5). Animals assigned to Arabi, Jair
and Hermokale treatment groups received
sufficient amount of Cu from the supplemented
mineral soils. When compared to suggested levels
for mineral soils set by McDowell (1997) for Cu
of 0.6 ppm, Arabi, Jair and Hermokale soils had
Cu content at 0.66 ppm, 2.22 ppm and 1.50 ppm,
respectively (Table 3). Contrary to this, animals
fed on Bole soil and complete mineral lick had
higher liver Cu than the suggested standard
deficiencies even though the Cu content in the
supplemented soils were below the suggested
deficiencies level (Table 5). The discrepancy might
be due to the variation from forage and water Cu
intake, low Cu requirement relatively the maturity
of the sheep and/or the experimental duration.
Intraraksa et al. (1998) reported that copper
deficiency is much more likely to occur in young
sheep than adults and clinical signs are most severe
in yearlings.
Kasetsart J. (Nat. Sci.) 41(2)
CONCLUSION
AND RECOMMENDATION
With the exception of Bole soils, the
present study revealed that supplementation with
the three remaining mineral soils improved the
total weight gain over the negative control sheep.
Moreover, the daily mineral soil intake of sheep
fed on Jair soil was higher than sheep received
different mineral sources. Sheep in all soil mineral
treatments except Bole soil, consume minerals at
the same level as complete mineral treated group.
However, the daily weight gain of animals fed
complete mineral lick was highest when compared
to all the remaining treatment sheep. Based on liver
analysis, the present study reveals that all treatment
group animals do not require additional micro
minerals such as Mn, Cu and Zn. Hence,
pastoralists can use natural mineral soils as mineral
supplement sources to their animals. Though, there
is a need to correct the deficiencies of certain
minerals. In order to achieve the desire result in
sheep production, phosphorus should be adjusted/
corrected in mineral supplementation. For
improved mineral feeding, the provision of salt
licks together with mineral soils and bone meal
would provide a convenient and effective means
of ensuring adequate mineral supplementation.
This could be beneficial to the pastoralists and in
return would have a national benefit in having
sustainable sheep production. Pastoralists should
be made aware of the possible incidence of mineral
deficiencies as parts of range land are lacking in a
number of mineral elements that are essential in
animal nutrition. Therefore, it is recommended that
planned mineral surveys must be conducted in
wide areas of the region in order to detect mineral
inadequacies for formulating balanced mineral
mixture to the animals.
ACKNOWLEDGEMENTS
The authors would like to express their
297
gratitude to the management of Somali Pastoral
and Agro Pastoral Research Institute (SoRPARI)
staff for their endless support during the
experimental period. We gratefully acknowledge
EARO/ARTP for funding this study. Moreover,
we appreciate Assistance Professors Dr. Sakron
Koonawootrittriron and Dr. Panwadee
Sopannarath for their valuable assistance in
statistical analysis.
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Kasetsart J. (Nat. Sci.) 41 : 300 - 310 (2007)
Protoplast Isolation and Culture of Aquatic Plant
Cryptocoryne wendtii De Wit
Kanchanaree Pongchawee1*, Uthairat Na-Nakorn2, Siranut Lamseejan3,
Supawadee Poompuang2 and Salak Phansiri4
ABSTRACT
The optimum conditions for protoplast isolation and culture of Cryptocoryne wendtii
De Wit were investigated. Protoplasts were successfully isolated from in vitro four-week-old leaves
using an enzyme mixture comprising 2% Cellulase Onozuka R-10, 0.2% Pectolyase Y-23, 0.5 M mannitol,
2.5 mM CaCl2.2H2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES), pH 5.6. Approximately
1.04±0.06 × 107 protoplasts per gram fresh weight with 90.79±4.80% viability were obtained after
incubating in enzyme solution for 4 hours in the dark and purified with 16 % sucrose gradient
centrifugation. Protoplasts were cultured on modified MS medium supplemented with 0.2 mg/l 2,4dicholorophenoxyacetic acid (2,4-D), 1 mg/l α-naphthalene acetic acid (NAA), 0.5 mg/l zeatin, 0.15 M
sucrose and 0.3 M mannitol by agarose-bead with thin layer liquid culture. The protoplasts regenerated
cell walls within 24 hours. First cell division was observed after culturing for 2-3 days, and microcolonies were formed within 4 weeks. Enzyme mixture, osmotic solution, incubation time, age of leaves,
and sucrose solution concentration were found to influence both yield and viability of protoplasts. Culture
media, plant growth regulators and method of culture affected protoplast division.
Key words: aquatic plant, Cryptocoryne wendtii De Wit, protoplasts isolation, protoplasts culture
INTRODUCTION
The Cryptocoryne genus is a member of
Araceae with more than 50 different species. They
are distributed throughout Southeast Asian coastal
zones. Some species are commercially cultivated
as aquarium plants (Mühlberg, 1982).
Cryptocoryne wendtii De Wit is an important
species used in the aquarium plant trade (Rajaj
and Horeman, 1977). It is a medium-sized species
with thin rhizomes and runners, able to grow
1
2
3
4
*
emerged or submersed and is propagated by
runners (Mühlberg, 1982). The aerial leaves are
oblong with round or heart shaped base, 8 to 10
cm long by 2 to 3 cm wide and below water. The
blade are narrower (Allgayer and Teton, 1986).
In order to increase the value of exports
and to cope with international market demand, the
improvement of new aquatic plant varieties for
desirable traits such as variable leaf color and form
are the key to success. Related or relevant genera
of cultivated crops contain a large reservoir of
Aquatic plant and Ornamental Fish Research Institute, Bangkok 10900 , Thailand.
Department of Aquaculture, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
Gamma Irradiation Service and Nuclear Technology Research Center, Kasetsart University, Bangkok 10900, Thailand.
Scientific Equipment Center, KURDI, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: kanchanp@fisheries.go.th
Received date : 19/09/06
Accepted date : 22/01/07
Kasetsart J. (Nat. Sci.) 41(2)
genes covering a variety of desirable traits (Liu et
al., 2005). However, reproductive incompatibility
generally prevents simple hybridization between
taxa. Somatic cell fusion enables nuclear and
cytoplasmic genomes to be combined, fully or
partially, at the interspecific and intergeneric levels
to circumvent naturally occurring sexual
incompatibility barriers (Davey et al., 2005). There
have been many reports of the transfer of useful
agronomic traits by protoplast fusion for
production of triploid (Fu et al., 2003) and
polyploid (Mizuhiro et al., 2001) plants and
increasing plant vigour (Cheng et al., 2003). This
technique may be a possible alternative for the
genetic improvement of Cryptocoryne. For
successful protoplast fusion, a reliable procedure
for protoplast isolation and culture is a prerequisite.
Up to date, there are a few reports of protoplast
isolation and culture of aquatic plants such as
seagrass (Balestri and Cinelli, 2001).
In this study, the procedures for isolation
and culture of C. wendtii protoplasts were
established for the first time. The information
obtained from this study will greatly benefit further
genetic improvement of Cryptocoryne.
MATERIALS AND METHODS
Plant materials
Shoot tip explants of C. wendtii were
surface-sterilized by immersion in 50% (V/V)
301
ethanol for 1 min and 1.05 % NaOCl containing 1
drop of Tween-20 per 100 ml for 12 min, followed
by rinsing three times with sterile distilled water
(Kane et al., 1999). Explants were cultured on
semi-solid MS medium (Murashige and Skoog,
1962) supplemented with 2 mg/l 6-benzyladenine
(BA), 0.25 mg/l NAA, 30 g/l sucrose and 1.6 g/l
gelrite (Sigma, USA). The cultures were incubated
under 16/8 h light/dark photoperiod at 25°C.
Plantlets derived from shoot tips were subcultured
into the same medium every four weeks. Leaves
of plantlets were used as the explants for protoplast
isolation.
Factors affecting the protoplast isolation
1. Enzyme mixtures
Five enzyme mixtures (Table 1) were
examined for the suitable protoplast isolation. The
tested enzyme mixtures were dissolved in 0.5 M
mannitol, 2.5 mM CaCl2.2H2O and 5 mM 2-Nmorpholino-ethanesulfonic acid (MES) pH 5.6.
One gram of in vitro four-week-old leaves were
cut transversely into 1-2 mm wide strips in a
washing solution (0.45 M mannitol, 2.5 mM
CaCl2.2H2O and 5 mM MES, pH 5.6). The sliced
tissues were plasmolysed by immersion in washing
solution for 30 minutes. The plasmolysis solution
was pipetted off, replaced with 5 ml of the filtersterilized (Satorius, pore size 0.20 µm) enzyme
mixtures and incubated in the dark on a gyratory
shaker (40 rpm) at 25°C for 4 hr. The protoplasts
Table 1 Components of enzyme mixtures used for protoplast isolation of C. wendtii
Enzyme
Enzyme concentration (% w/v)
mixtures
Cellulase
Pectinase
Cellulase R-10a
Cellulase RSa
Macerozyme R-10a Pectolyase Y-23b
E1
2
2
E2
2
0.2
E3
2
2
E4
2
0.2
E5
2
2
0.1
a
b
Yakult, Tokyo.
Seishin, Tokyo.
302
Kasetsart J. (Nat. Sci.) 41(2)
were then gently filtered through a 60 and 40 µm
nylon mesh to remove undigested tissue and
debris. The filtrate was centrifuged for 5 min at
750 rpm. The same process was repeated once
more. The protoplast pellets were purified by
floating on 20 % sucrose solution and centrifuged
at 800 rpm for 10 min. The purified protoplasts
were further washed twice with washing solution.
Protoplast yield was estimated by a
hemocytometer (Gleddie, 1995). Viability of
protoplasts was assessed using 0.01% (w/v)
fluorescein diacetate staining (FDA) (Sigma,
USA) followed by observation with a UV
fluorescence microscope (Widholm, 1972).
2. Concentration of osmoticum
solution
The best result of experiment 1 was used
in experiment 2. One gram of four-week-old in
vitro leaves was incubated in 5 ml of filtersterilized enzyme mixture, 2% (w/v) Cellulase
Onozuka R-10 (Yacult Honsha, Japan), 0.2% (w/
v) Pectolyase Y-23 (Kyowa Chemical, Japan) in
washing solution of four varied mannitol
concentrations; 0.4, 0.5, 0.6 or 0.7 M. The
protoplasts were isolated and purified as
previously described. Protoplast yield and
viability were determined.
3. Incubation period
The best result of experiment 2 was used
in experiment 3. One gram of four-week-old in
vitro leaves was incubated in 5 ml of enzyme
mixture, 2% Cellulase Onozuka R-10, 0.2%
Pectolyase Y-23, 0.5 M mannitol, 2.5 mM
CaCl2.2H2O and 5 mM MES. The digestion was
performed for 3, 4, 5 or 6 hr in the dark. The
protoplasts were then harvested and purified as
previously described. Protoplast yield and
viability were determined.
4. Age of leaves
One gram of four-, six-, eight- and ten-
week-old leaves was isolated using enzyme
mixture, 2% Cellulase Onozuka R-10, 0.2%
Pectolyase Y-23, 0.5 M mannitol, 2.5 mM
CaCl2.2H2O and 5 mM MES, and incubated in
the dark on a gyratory shaker (40 rpm) at 25°C for
4 hr. The protoplasts were then harvested and
purified as previously described. Protoplast yield
and viability were determined.
5. Sucrose concentrations for
purification
One gram of four-week-old in vitro
leaves was incubated in 5 ml of enzyme mixture,
2% Cellulase Onozuka R-10, 0.2% Pectolyase Y23, 0.5 M mannitol, 2.5 mM CaCl2.2H2O and 5
mM MES. The protoplasts were harvested and
purified with varying levels of sucrose solution;
16, 18, 20 and 22 % and centrifuged at 800 rpm
for 10 min. Protoplast yield and viability were
determined.
Factors affecting the protoplast culture
1. Culture medium
The purified protoplasts at the density of
5
5 × 10 protoplasts/ml were cultured in two kinds
of liquid culture media; MS (Murashige and
Skoog, 1962) and KM8P (Kao and Michayluk,
1975) containing 0.2 mg/l 2,4-D, 1 mg/l NAA,
0.5 mg/l zeatin, 0.15 M sucrose and 0.3 M
mannitol incubated at 25°C in the dark. The cell
division was observed periodically with an
inverted microscope. The plating efficiency (% of
plated protoplasts which were under cell division)
and the survival rate of protoplasts were
determined after 10 days of culture.
2. Plant growth regulators
The protoplasts were cultured in liquid
MS medium containing various combinations of
growth regulators. Three culture media tested for
protoplast culture were M1 (1.5 mg/l NAA and
0.4 mg/l BA), M2 (0.2 mg/l 2,4-D, 1 mg/l NAA
and 0.5 mg/l zeatin) and M3 (0.2 mg/l 2,4-D, 2
Kasetsart J. (Nat. Sci.) 41(2)
mg/l NAA and 0.5 mg/l zeatin) incubated at 25°C
in the dark. The plating efficiency and percentage
of survival were evaluated after 10 days of culture.
3. Culture method
Protoplasts were cultured using two
methods, namely, the liquid thin layer and agarose
bead methods. For the liquid thin layer method,
protoplasts in liquid MS medium at the density of
5 × 105 protoplasts/ml were poured into a 6 cm
Petri dish. For agarose bead method, one volume
of the protoplast suspension was gently mixed with
one volume of modified MS medium containing
0.2 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D),
1 mg/l NAA and 0.5 mg/l Zeatin with 1.2 % (w/v)
agarose (SeaPrep, FMC BioProducts, U.S.A.).
The protoplast suspension was dropped into a 6
cm Petri dish. The droplets were covered with 3
ml of modified liquid MS medium and incubated
at 25°C in the dark for 10 days, dim light for 10
days, and then in the light for 30 days. Cell wall
regeneration was observed using 0.01% (w/v)
calcofluor white staining under a fluorescence
microscope (Phansiri et al., 1992). The plating
efficiency and percentage of protoplast survival
were examined after 10, 30 and 50 days of culture.
Statistical analysis
All data were assessed by one-way
analysis of variance (ANOVA), and the means
were compared by the Turkey test at 95% interval
of confidence (*P<0.05). The significance of
difference in plating efficiency and survival rate
as influenced by the culture media and culture
methods were assessed by independent sample ttest. All statistical analysis were carried out using
SPSS 11.0 software (SPSS, Chicago, IL, USA).
RESULTS
Factors affecting the protoplast isolation
1. Enzyme mixtures
Among five enzyme mixtures tested, E2
303
(2% Cellulase Onozuka R-10, 0.2% Pectolyase Y23, 0.5 M mannitol, 2.5 mM CaCl2 and 5 mM
MES) was most appropriate for protoplast
isolation, since it produced the highest yield of
81.87 × 105 protoplasts/g FW with the highest
viability of 91.78 % (Figure 1). This was
significantly different from other enzyme solutions
(*P<0.05). The protoplasts isolated with E1 and
E3 showed the lowest yield and viability.
2. Concentration of osmoticum
solution
The concentration of mannitol in the
enzyme solution significantly affected the yield
and viability of the protoplasts (Figure 2). A 0.5
M mannitol solution was found to be most efficient
for regulation of the osmotic pressure in protoplast
isolation. It gave the highest yield of 80.56x105
protoplasts/g FW with the highest viability of
85.01 %, and was significantly different from other
concentrations (*P<0.05). In addition, there was
a significant decrease (*P<0.05) in protoplast
viability as the mannitol concentration increased.
3. Incubation period
The incubation period during enzyme
digestion significantly affected (*P<0.05) the yield
and viability of the protoplasts. The highest yield
of 84.36×105 protoplasts/g FW, with the highest
viability of 85.10 % was obtained at the incubation
period of 4 hr (Figure 3). The viability of
protoplasts decreased with prolonged incubation
period. The lowest viability of 59.27 % (*P<0.05)
was recorded in protoplasts incubated in enzyme
solution for 6 hr.
4. Age of leaves
The age of leaves also influenced the
viability and yield of protoplasts. It was found that
four- (Figure 5A) and six-week-old leaves were
more suitable for protoplast isolation than eightand ten-week-old leaves. The isolated protoplasts
were spherical and contained many chloroplasts
Kasetsart J. (Nat. Sci.) 41(2)
(Figure 5B). Their viability was 87.14 % and 82.76
% for the four- and six-week-old leaves,
respectively, as determined by FDA staining
(Figure 5C). Protoplast viability decreased
significantly with the increase in leaf age (Figure
4). It was also found a remarkable number of
raphids when using the leaves as a source of
protoplasts.
100
bc
80
70
80
80
70
ab
a
60
90
90
60
c
50
50
40
40
b
30
30
20
10
a
0
E1
E2
E3
E4
d
60
50
50
40
40
30
30
a
20
10
10
10
0
0
0
0.4
E5
c
60
50
b
40
30
40
20
a
20
10
0
0
5
6
Incubation time (hours)
Yield (×105 prtoplasts/ g FW)
a
100
100
70
c
60
Viability
b
Viability (%)
Yield (×105 prtoplasts/ g FW)
ab
80
Yield
0.7
120
80
4
0.6
Figure 2 Effect of mannitol concentrations in the
enzyme mixture on yield and viability
of C. wendtii protoplasts. Data
represent mean ± standard error of
three replicates.
90
b
3
0.5
Yield
Figure 1 Effect of different enzyme mixtures on
yield and viability of C. wendtii
protoplasts. Data represent mean ±
standard error of three replicates.
c
60
b
Viability
100
a
c
Mannitol concentration (M)
Yield
b
70
ab
70
Enzyme mixtures
120
80
b
20
20
a
90
c
90
ab
ab
80
c
a
80
70
60
60
50
b
40
40
Viability (%)
Yield (×105 prtoplasts/ g FW)
c
d
Yield (×105 prtoplasts/ g FW)
c
90
100
Viability (%)
100
Viability (%)
304
30
a
20
20
10
0
0
4
6
8
10
Leaf age (weeks)
Viability
Figure 3 Effect of incubation time on yield and
viability of C. wendtii protoplasts.
Data represent mean ± standard error
of three replicates.
Yield
Viability
Figure 4 Effect of leaf age on yield and viability
of C. wendtii protoplasts. Data
represent mean ± standard error of three
replicates.
Kasetsart J. (Nat. Sci.) 41(2)
305
Figure 5 Isolation, culture and cell division of Crytocoryne wendtii protoplasts. Four-week-old plantlets
suitable for the isolation of leaf protoplasts (A), protoplasts after purification with 16 %
sucrose solution (B), vigorous protoplasts fluoresce a yellow-green color when stained with
FDA (C), first cell division of protoplast culture in agarose bead after a few days of culture
(D), second cell division after 10 days of culture (E), small cell colonies after culturing for 30
days (F). Bar = 20 µm.
5. Purification by various sucrose
concentrations
There was a significant difference
between the yield of protoplasts centrifuged in the
four sucrose concentrations tested, but no
significant difference in the viability (Table 2).
Purification with 16 % sucrose solution gave the
highest yield of 103.62×105 protoplasts/g FW with
the viability of 90.79 %, and without cell debris.
Factors affecting the protoplast culture
1. Culture medium
MS medium was found to be more
effective than KM8P medium. The first cell
306
Kasetsart J. (Nat. Sci.) 41(2)
division was found within 2-3 days in MS medium
supplemented with 0.2 mg/l 2,4-D, 1 mg/l NAA
and 0.5 mg/l Zeatin, 0.15 M sucrose and 0.3 M
mannitol. The plating efficiency and survival rate
at 10 days after culture were 21.27 % and 60.44
%, respectively (Table 3). In contrast, the
protoplasts cultured in KM8P medium with the
same growth regulator as MS medium did not
divide but turned brown and died after 10 days of
culture. This indicates that MS medium was
suitable for culturing mesophyll protoplasts of C.
wendtii.
2. Plant growth regulators
Protoplasts did not divide after being
cultured in M1 (1.5 mg/l NAA, 0.4 mg/l BA) for
10 days. The highest plating efficiency (21.27 %)
and cell survival (57.11 %) were observed in M2
(0.2 mg/l 2,4-D, 1 mg/l NAA and 0.5 mg/l Zeatin),
which was statistically similar to that in M3 (0.2
mg/l 2,4-D, 2 mg/l NAA and 0.5 mg/l Zeatin)
(Table 4).
3. Culture method
The freshly isolated protoplasts cultured
in liquid and agarose bead culture regenerated cell
walls within 24 hr. The first division of protoplasts
was observed in 2-3 days (Figure 5D). After 10,
30 and 50 days there were no significant
differences within the plating efficiency and
survival rate of both culture methods (Table 5, 6).
The plating efficiency and survival rate decreased
Table 2 Effect of sucrose concentration on yield and viability of C. wendtii protoplasts.
Sucrose (%)
Yield (×105 protoplasts/g FW)
Viability (%)
16
103.62±5.63 c
90.79±4.80ns
b
18
80.38±1.78
84.74±3.23 ns
20
81.04±1.78 b
80.27±4.52 ns
a
22
58.79±2.96
76.96±1.50 ns
Data represent mean ± S.E. of three replicates. Means in the same column sharing the same superscript letter are not significantly
different as determined by Turkey’s test (*P>0.05).
Table 3 Effect of culture medium on cell division and survival of C. wendtii protoplasts after culturing
for 10 days.
Culture media
Plating efficiency (%)
Survival rate (%)
MS
21.27 ± 1.32b
60.44 ± 3.61b
K8
0.00 ± 0.00a
0.00 ± 0.00a
Data represent mean ± S.E. of three replicates. Means in the same column not sharing the same superscript letter are significantly
different as determined by Turkey’s test (*P<0.05).
Table 4 Effect of plant growth regulator on plating efficiency and survival of C. wendtii protoplasts
after culturing for 10 days.
PGRs combinations
Plating efficiency
Survival rate
(mg/l)
(%)
(%)
a
M1:1.5 mg/l NAA + 0.4 mg/l BA
0.00 ± 0.00
0.00 ± 0.00a
M2:0.2 mg/l 2,4-D + 1 mg/l NAA + 0.5 mg/l Zeatin
22.71 ± 3.02b
57.11 ± 4.89b
b
M3:0.2 mg/l 2,4-D + 2 mg/l NAA + 0.5 mg/l Zeatin
18.09 ± 2.82
48.62 ± 5.71b
Data represent mean ± S.E. of three replicates. Means in the same column not sharing the same superscript letter are significantly
different as determined by Turkey’s test (*P<0.05).
Kasetsart J. (Nat. Sci.) 41(2)
as the culture period increased. Some protoplasts
survived, divided and developed to small colonies
only in agrarose bead (Figure 5F). However, callus
was not formed, they turned brown and finally
died.
DISCUSSION
The success in protoplast isolation of C.
wendtii was influenced by the enzyme mixture,
osmoticum solution, incubation period, age of
leaves, and sucrose concentration. The
combination of enzyme solution has been reported
to be an important factor on yield and viability of
protoplasts in many plant species such as Artemisia
judaica L. and Echinops spinosissimus Turra (Pan
et al., 2003) and Echinacea augustifolia (Zhu et
al., 2005). Cellulase Onozuka R-10 was a preferred
enzyme for leaf protoplast isolation of C. wendtii
rather than Cellulase RS which had higher
cellulase activity (Marchant et al., 1997). Cellulase
Onozuka 10 combined with Pectolylase Y-23 was
the most efficient for protoplast isolation of C.
wendtii. Pectolyase Y-23 was efficient for digestion
of mesophyll protoplast (Nagata and Ishii, 1979;
Eriksson, 1985) due to Pectolyase Y-23 having
endo-polygalacturonase activity about 50 times
307
stronger than Macerozyme R-10 (Nagata and Ishii,
1979).
In isolating protoplasts, the wall pressure
must be replaced by osmotic pressure in the
isolation mixture. Mannitol is considered to be
relatively inert metabolically and infuses slowly
into the protoplast (Eriksson, 1985). The
concentration of mannitol in the enzyme solution
was another important factor affecting C. wendtii
protoplast release. The yield and viability of
protoplasts were shown to decrease with the
increasing of mannitol concentration due to the
protoplasts being plasmolyzed (Sinha, 2003). The
prolonged incubation period decreased the yield
and viability of protoplasts because of the over
digestion (Zhu et al., 2005).
The ages of the leaves were also critical
for the successful protoplast isolation of C. wendtii.
The younger leaves gave the maximum of both
viability and yield because less pectic substances
accumulate in young cell walls than in the old cells
(Babaoǧ lu, 2000), and the cell wall of a rapidly
expanding leaf is thinner (Marchant et al., 1997).
There were many calcium oxalate needles found
when leaves were used as the source of protoplasts.
These crystals are able to puncture and burst
protoplasts during isolation (Price and Earle, 1984;
Table 5 Effect of culture methods on plating efficiency of C. wendtii protoplasts in MS medium.
Culture method
Plating efficiency (%)
Day 10
Day 30
Day 50
ns
ns
Liquid
28.61 ± 4.72
18.77 ± 3.50
13.60 ± 1.80 ns
ns
ns
Agarose bead
25.82 ± 2.46
20.66 ± 4.67
14.76 ± 2.14 ns
Data represent mean ± S.E. of three replicates. Means in the same column sharing the same superscript letter are not significantly
different as determined by Turkey’s test (P>0.05)
Table 6 Effect of culture methods on survival rate of C. wendtii protoplasts in MS medium.
Culture method
Survival rate (%)
Day 10
Day 30
Day 50
Liquid
67.25 ± 5.46ns
57.47 ± 4.65 ns
33.67 ± 4.06 ns
Agarose bead
68.12 ± 5.37 ns
54.93 ± 5.65 ns
36.57 ± 3.08 ns
Data represent mean ± S.E. of three replicates. Means in the same column sharing the same superscript letter are not significantly
different as determined by Turkey’s test (P>0.05)
Kasetsart J. (Nat. Sci.) 41(2)
308
Kunasukdakul and Smitamana, 2003). However,
all raphids and debris could be successfully
removed by centrifugation of protoplasts with 16
% sucrose solution.
For the protoplasts culture of C. wendtii,
the culture media, culture method and plant growth
regulators were important factors affecting plating
efficiency and survival rate. The protoplasts could
divide in liquid as well as in agarose bead culture.
However, microcolonies were formed only in
agarose bead culture. The agarose bead culture
methods have been found to be an efficient method
for cell division and microcolony formation in
many crop species including Lavatera thuringiaca
(Vazquez-Tello et al., 1995); Rosa hybrida
(Marchant et al., 1997) and Cucumis melo ‘Green
Delica’ (Sutiojono et al., 1998). The enhanced
protoplast division observed in bead culture was
due to the dilution of substances having inhibitory
effects on protoplast division which are secreted
from the cell to the medium (Mizuhiro et al.,
2001).
Colony formation was observed after
culturing protoplasts in MS medium supplemented
with 0.2 mg/l 2,4-D, 1 mg/l NAA, 0.5 mg/l Zeatin,
0.3 M mannitol, and 0.15 M sucrose for 30 days.
However, it did not form a callus but turned brown
and finally died. It has been reported that the
protoplasts isolated directly from leaves of
monocotyledons, except rice, was very difficult
to culture (Kuehnle and Nan, 1990). It was
suggested that leaf cells rapidly lose totipotency
thus preventing cells from dedifferentiating and
reentering the cell cycle (Krautwig and Lörz,
1995). Plant regeneration has been found possible
when callus and cell suspension were used as the
source of protoplast isolation and culture
(Kobayashi et al., 1993; Pauk, et al., 1994).
CONCLUSION
The procedure for simple and reliable
isolation and culture of C. wendtii protoplasts has
been described for the first time. It might lead to
the improvement of the Cryptocoryne through
somatic hybridization, somaclonal variation and
genetic engineering by using the protoplast
technique. Even though the viable protoplasts of
C. wendtii could form microcolonies, further
research is needed to develop the efficient
procedure for the protoplast regeneration.
ACKNOWLEDGMENTS
This research was financially supported
by the Department of Fisheries, Ministry of
Agriculture and Co-operatives, Bangkok,
Thailand. We thank Dr. Sureeya Tantiwiwat,
Department of Botany, Kasetsart University, Dr.
Yuphin Khentry and Mr. Adrian Hillman,
Graduate School, Kasetsart University for editing
this manuscript and their helpful suggestions.
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Kasetsart J. (Nat. Sci.) 41 : 311 - 318 (2007)
Anti HSV-1 Activity of Spirulina platensis Polysaccharide
Nattayaporn Chirasuwan1*, Ratana Chaiklahan1, Marasri Ruengjitchatchawalya2
Boosya Bunnag2 and Morakot Tanticharoen3
ABSTRACT
Aqueous extracts of Spirulina platensis were precipitated by cetyltrimethylammonium bromide
(CTAB). The hot water extract was found anti Herpes simplex virus type 1 (HSV-1) activity at 50%
inhibitory concentration (IC50) values of 21.32 µg/ml. Partial purification by gel filtration of the crude
extract on Sepharose 6B column gave two fractions, SHP-F1 and SHP-F2, which revealed about 4 and
2 times higher activity than that of the crude hot water extract, respectively. The crude hot water extract
was a polysaccharide with rhamnose as the main sugar component. Calcium ion and sulfate groups in
this polysaccharide had major roles in antiviral activity. However, the crude hot water extract
polysaccharide contained approximately 42% carbohydrate and 31% protein. Decreasing the amount
of protein by precipitation with trichloroacetic acid (TCA) resulted in higher purity of the crude hot
water extract polysaccharide.
Key words: Spirulina platensis, Herpes simplex virus type 1 (HSV-1), polysaccharide
INTRODUCTION
Spirulina platensis is one of the edible
microalgae that has been used as health food and
feed for a long time. There is an increased interest
in components of S. platensis because of their
potential properties such as anti thrombin activity
(Hayakawa et al., 1996), lowering cholesterol level
and blood pressure (Kato et al., 1984; Nakaya et
al., 1988). Herpes simplex virus type1 is a
common human pathogen causing infections of
the orofacial mucosal region (Whitley and
Roizman, 2001). Over the past decade, the
1
2
3
*
incidence and severity of HSV infection have
increased due to the increase in number of
immuno-compromised patients produced by
aggressive chemotherapy treatments, organ
transplant and human immunodeficiency
infections. Acyclovir, a synthetic drug which has
remarkable effect against HSV-1 infection, inhibits
virus replication by acting on viral DNA synthesis
(Elion et al., 1977; Schaeffer et al., 1978).
Acyclovir-resistant HSV infections have emerged
due to the increase in drug use frequency (Field
and Biron, 1994). Therefore, many researchers
have attempted to search for effective and
Pilot Plant Development and Training Institute King Mongkut’s University of Technology Thonburi, Bangkhuntien,
Bangkok 10150, Thailand.
School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkhuntien,
Bangkok 10150, Thailand.
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency,
Pathum Thani 12120, Thailand.
Corresponding author, e-mail: nattayaporn@pdti.kmutt.ac.th, nattayaporn1@yahoo.com
Received date : 02/11/06
Accepted date : 04/12/06
312
Kasetsart J. (Nat. Sci.) 41(2)
inexpensive anti-viral agents from natural sources.
The inhibitory effects of polysaccharides from
marine algae on virus replication were first
reported almost four decades ago.
Gerber et al. (1958) reported that algal
polysaccharides exhibited antiviral activity toward
mumps and influenza B virus. Further, Hayashi et
al. (1993) reported the anti HSV-1 activity of
aqueous extracts from S. platensis. Our
preliminary study revealed that both water soluble
and non-polar extracts of S. platensis exhibited
antiviral activity (HSV-1). This study investigated
the anti HSV-1 activity of polysaccharides (water
soluble compound) extracted from S .platensis.
The isolation, partial purification, and composition
determination of the anti HSV-1 activity extracts
are described.
MATERIALS AND METHODS
Extraction of polysaccharides
The lipid component was extracted from
freeze-dried powder of S. platensis with
CHCl 3 :MeOH (2:1). Then, the residue was
extracted with distilled H2O. After fillration, the
filtrate was precipitated by 1% CTAB
(cetyltrimethylammonium bromide). After
centrifuging, the precipitant was washed stepwise
with saturated sodium acetate in 95% EtOH, 95%
EtOH, absolute EtOH and diethyl ether,
respectively. The resulting in cold water extract
polysaccharide was obtained. For the extraction
of hot water extract polysaccharide, the same
method was performed except using boiling H2O.
Partial purification of polysaccharide
The hot water extract polysaccharide was
dissolved in 0.01 M citrate buffer, pH 7.0
containing 0.1 M NaCl. The soluble portion was
applied on to a Sepharose 6B (Pharmacia) column
(3×30 cm) and eluted with the same citrate buffer.
Fractions of 5 ml were collected and monitored
using the phenol-sulfuric method with detection
by spectrophotometer (Bausch&Lomb, Spectronic
21) at an absorbance of 485 nm (Hayashi et al.,
1996a). The collected fraction was concentrated
using an evaporator (below 40°C under reduced
pressure), dialyzed with deionized water and
lyophilized.
Preparation of sugar derivatives for GC
analysis
One milligram of sugar was treated with
1 ml of 20 g/l sodium tetraborohydride and cooled
down to nearly 0°C. After standing over night,
amberite IR-120 (H+) was slowly added until no
bubble. The solution was filtered through filter
paper (Whatman #541). After filtration, the
solution was evaporated under reduced pressure
to thick syrup. The syrup was repeatedly dissolved
in methanol and evaporated to remove boric acid.
The syrup was further treated with 0.5 ml of acetic
anhydride and 0.5 ml of pyridine at 80°C for 2 h.
The solution was then immersed in an ice bath
and 1 ml of methanol was added to the solution.
The solution was then evaporated to remove
methyl acetate. Then, 1 ml of heptane was added
and evaporated to remove the remaining pyridine.
Dried sample was dissolved in 200 µl of
dichloromethane and analyzed by GC (Shimadzu,
17A) using Rtx-2330 capillary column (Blakeney
et al., 1983).
Hydrolysis of hot polysaccharide solution
A 5 mg sample from partial purification
of polysaccharide and 1 mg of internal standard
(inositol) were mixed and treated with 2 N H2SO4
at 100°C for 8 h. The hot solution was neutralized
with barium carbonate to pH 5 and filtered through
filter paper (Whatman #541). Barium was
eliminated from the supernatant using Amberite
IR-120 (H+) acidic cation-exchange resin. The
solution was then applied to a Dowex 1-X8 anionexchange column and eluted with distilled H2O.
The fraction was evaporated and converted to an
alditol acetate derivatives form and analyzed by
Kasetsart J. (Nat. Sci.) 41(2)
GC.
Analytical methods Total carbohydrate
content was estimated by phenol sulfuric acid
assay (Dubois et al., 1956). Total protein content
and lipid content were determined according to
the methods of Lowry et al. (1951) and Folch
Folch et al. (1957), respectively. Calcium content
was carried out by Inductive Couple Plasma
Spectroscopy (ICP, Model JY 124) and
quantitative analysis of sulfate was performed by
precipitation with 10% BaCl2 (Burns, 1995).
Removal of calcium was achieved using
a cation exchange column on Dowex 50W (X-8,
H+ form) (Hayashi et al., 1993).
Desulfation pH of the polysaccharide
solution was adjusted to pH 7.6 with pyridine and
the pyridinium salt was eliminated with dimethyl
sulfoxide (containing 10% of MeOH) at 80-100°
C (Nagasawa et al., 1977).
Antiviral activity was detected by using
a colorimetric method modified from Skehan et
al. (1990). Herpes simplex virus type 1 (HSV-1)
was maintained in a Vero cell line (kidney
fibroblasts of an African green monkey), which
was cultured in Eagle’s minimum essential
medium (MEM) with the addition of 10% heat
inactivated fetal bovine serum (FBS) and
antibiotics. The test samples were put into wells
of a microtiter plate at final concentrations ranging
from 20-50 µg/ml. The viral HSV-1 (30 PFU) was
added into a 96-well microplate, followed by
plating of Vero cells (1 × 105 cells/ml); the final
volume was 200 µl. After incubation at 37°C for
72 h, under 5% of CO2 atmosphere, cells were
fixed with 50% trichloroacetic acid (TCA) and
stained with 0.05% sulforhodamine B in 1% acetic
acid and optical density was measured at 510 nm
using a microplate reader. Acyclovir was used as
the reference compound.
Determination of cytotoxicity assay
Compounds were tested for their
cytotoxicity against Vero cells (African green
313
monkey kidney fibroblasts in 96-well tissue culture
plates). One hundred and ninety µl of Vero cell
suspension containing 1 × 105 cells/ml and 10 µl
of tested compound were added to each well in
triplicate. Elliptine and 10%DMSO were used as
positive and negative control, respectively. The
cells were incubated at 37°C for 72 h in 5%CO2.
After incubation, the cytotoxicity was determined
by the colorimetric method as described by
Skehan et al. (1990). The cytotoxicity was
expressed as IC50, i.e., the concentration of the
compound which inhibits cell growth by 50%,
compared with untreated cell.
RESULTS
Crude cold and hot water polysaccharides
were obtained from extraction of dried S. platensis
by distilled water (room temperature) and boiling
water, respectively. The extracts were precipitated
by CTAB solution. The freeze-dried extracts as
fine creamy powder were shown in Figure 1. The
yields of the cold and hot water extracts were 1.2
and 0.3 % (W/W), respectively. The hot water
extract polysaccharide showed an IC50 value
against HSV-1 at 21.32 µg/ml whereas no activity
was detected in the cold water extract
polysaccharide.
Figure 1 Cold water polysaccharide (pale color)
and hot water polysaccharide (dark
color).
Kasetsart J. (Nat. Sci.) 41(2)
314
The crude hot water polysaccharide was
partially purified by gel-filtration on Sepharose 6B
column. Two fractions, SHP-F1 and SHP-F2, were
collected (Figure 2). Attempts to completely
separate the two fractions by decreasing flow
rate from 1.2 to 0.8 ml/min was not successful.
The curde hot water polysaccharide comprised of
approximately 40% of fraction 1 (SHP-F1) and
60% of fraction 2 (SHP-F2). The fractions of SHPF1 and SHP-F2 were repeatedly run using the same
method at a lower flow rate of 0.5 ml/min.
4.5
Absorbance (OD485)
4
3.5
SHP-F2
3
2.5
SHP-F1
2
1.5
1
0.5
0
0
10
20
30
40
50
60
70
80
Fraction number
Figure 2 Elution profile of hot water
polysaccharide by Sepharose 6B
column chromatography.
After the pool fraction of SHP-F1 was
repeatedly applied on Sepharose 6B column, the
purified SHP-F1 was obtained (Figure 3A).
However, SHP-F2 still exhibited 2 peaks of
absorbances, a small peak and a bigger one,
designated as SHP-F2/1 and SHP-F2/2,
respectively (Figure 3B). When the partially
purified fractions of the hot water polysaccharide
(SHP-F1 and SHP-F2) were subjected to
cytotoxicity and anti HSV-1 assays, both fractions
exerted non-toxicity on the growth of Vero cells
at the maximum concentrations tested and had
significantly higher anti HSV-1 activity than the
crude hot water polysaccharide (about 4 and 2
times, respectively) (Table 1).
The analysis of monosaccharide was
performed by GC. It was found that SHP-F1
fraction contained only three sugars; rhamnose,
ribose and arabinose, whereas, the SHP-F2 fraction
contained rhamnose, ribose, arabinose, glucose,
mannose, galactose and xylose. Both fractions
contained rhamnose as the main sugar component
(Table 2).
Table 3 showed the comparison of
proximate analysis of dried cells of Spirulina and
the crude hot water extract polysaccharide. Results
showed that dried cells consisted of 21.9%
carbohydrate, 61.4% protein, 7.2% lipid and 7.2%
4.5
4.5
4
A
Absorbance (OD485)
Absorbance (OD485)
4
3.5
3
2.5
2
1.5
3
2.5
SHP- F2 / 2
2
1.5
1
1
0.5
0.5
0
B
3.5
SHP- F2 / 1
.
0
0
10
20
30
40
50
Fraction number
60
70
80
0
10
20
30
40
50
60
70
80
Fraction number
Figure 3 Elution profile of SHP-F1 (A) and SHP-F2 (B) by Sepharose 6B column chromatography.
Kasetsart J. (Nat. Sci.) 41(2)
ash. The crude hot water polysaccharide contained
42.5% carbohydrate, 31.0% protein, 12.9% ash
and trace of calcium and sulfate.
Table 4 shows the remaining
carbohydrate and protein precipitated by various
concentrations of trichloroacetic acid (TCA).
These data suggested that at the highest
concentration of TCA (50% TCA), 41% of the
protein in dry cells was eradicated (a decrease from
31% to 18%), while the percentage of carbohydrate
increased about 25% (from 42.5% to 53.1%). After
315
the treated crude hot water polysaccharide
(precipitated with 50% TCA) was tested for anti
HSV-1 activity, results showed that the activity
was not significantly different from the untreated
crude hot water polysaccharide (data not shown).
To determine the role of calcium ion and
sulfate groups of the hot water polysaccharide in
antiviral activity, calcium ion and sulfate groups
in the polysaccharide were eliminated before
testing for cytoxicity and anti HSV-1 activity. The
results indicated that all of the calcium-free
Table 1 Cytotoxicity and Anti HSV-1 activity of the hot water polysaccharide fractions from S. platensis.
Fractions
Cytotoxicitya
Anti HSV-1b
(IC50: µg/ml)
(IC50: µg/ml)
SHP-F1
> 50
5.25
SHP-F2
> 50
9.61
concentration of compound for cytotoxicity test was 50 µg/ml
compound was non-toxic to the growth of Vero cells when IC50 >50 µg/ml
(if compound was toxic on the growth of Vero cells, the compound will be subjected to the serial dilution
for determination of IC50 value)
a maximum
b%
inhibition of HSV-1; < 25%= inactive, 25-35%= weakly active, >35-50% = moderately active,
> 50%= active (the compound will be subjected to the serial dilution
for determination of IC50 value)
Table 2 Sugar composition of fractions of the hot water extract polysaccharide.
Fractions
% Sugar composition
Rhamnose
Ribose
Arabinose
Glucose
Mannose
Galactose
SHP-F1
75.6
13.4
11.0
SHP-F2
30.4
27.1
10.0
18.2
7.5
4.5
Xylose
2.3
Table 3 Composition of S. platensis powder and crude hot water polysaccharide.
Composition
Dry weight (%)
Spirulina powder
Crude hot water polysaccharide
Carbohydrate
21.9 ± 0.8
42.5 ± 0.3
Protein
61.4 ± 1.1
31.0 ± 0.8
Lipid
7.2 ± 1.3
0
Calcium
-*
0.123 ± 0.0006
Sulfate
-*
1.44 ± 0.03
Ash
7.2 ± 0.1
12.9 ± 0.4
Mean ± standard deviation (n = 3)
* It was not determined
Kasetsart J. (Nat. Sci.) 41(2)
316
compound exerted weak anti HSV-1 activity when
compared with that of the crude hot water
polysaccharide, whereas, in the absence of sulfate
groups in polysaccharide, no significant anti
HSV-1 activity was detected in this compound
(Table 5).
DISCUSSION
This study found that the hot water
extract polysaccharide exhibited anti HSV-1
activity, while the cold extract of the
polysaccharide did not. Previous studies found that
the majority of potential antiviral algal
polysaccharides were extracted from tissues by hot
water, dilute acid or alkali solution (Damonte et
al., 1994; Hoshino et al., 1998). Crude hot water
polysaccharide still contained a high level of
protein which may co-precipitate when CTAB is
used for polysaccharide precipitation (Tomanee et
al., 2004).
Partial purification of the hot water
polysaccharide by gel-filtration on Sepharose 6B
column gave 2 fractions (SHP-F1 and SHP-F2),
both fractions effectively inhibited HSV-1 activity.
Results reported by Hayashi et al. (1996a) revealed
3 fractions (SP-H-1, SP-H-2 and SP-H-3) but only
a SP-H-2 fraction had anti HSV-1 activity. The
sugars found in SHP-F1 and SHP-F2 fractions in
this study are almost the same as previously
reported by Hayashi et al. (1966a) except for
arabinose which was found in this study instead
of fructose which was reported by the same
researchers.
Calcium ion and sulfate groups in the hot
water polysaccharide were important for the anti
HSV-1 activity. This result was supported by
Hayashi’s study that when the calcium-free
Table 4 Carbohydrate and protein content of crude hot water polysaccharide which was precipitated
by trichloroacetic acid (TCA).
TCA concentration (%)
% w/w of crude hot water polysaccharide
Carbohydrate
Protein
0
42.5 ± 0.3
31.0 ± 0.8
10
41.8 ± 1.5
28.1 ± 2.1
20
45.3 ± 1.8
25.4 ± 1.3
30
49.2 ± 2.3
22.3 ± 1.5
50
53.1 ± 2.0
18.0 ± 0.8
Mean ± standard deviation (n = 3)
Table 5 Anti HSV-1 activity in the crude hot water polysaccharides from S. platensis.
Sample
Cytotoxicitya (IC50 : µg/ml)
Anti HSV-1b (IC50 : µg/ml)
Polysaccharide
> 50
21.3
2+
Polysaccharide (-Ca )
> 50
38.4
Polysaccharide (-SO42-)
> 50
Inactive
concentration of compound for cytotoxicity test was 50 µg/ml
compound was non toxic on the growth of Vero cells if IC50 >50 µg/ml
(if compound was toxic on the growth of Vero cells, the compound will be subjected to the serial dilution
for determination of IC50 value)
a maximum
b%
inhibition of HSV-1; < 25%= inactive, 25-35%= weakly active, >35-50% = moderately active,
> 50%= active (the compound will be subjected to the serial dilution
for determination of IC50 value)
Kasetsart J. (Nat. Sci.) 41(2)
spirulan (H-SP), and a desulfated compound from
Ca-SP were subjected to cytotoxicity and antiviral
assay, both compounds exerted strong toxicity to
the growth of host cell (HeLa cells) and weakly
inhibited HSV-1 (Hayashi et al., 1996a). Ca-SP
was found to inhibit replication of several
enveloped virus and selectively inhibited the
penetration of virus into host cell (Hayashi et al.,
1996b). Loya et al. (1998) postulated that the
negatively charged (e.g., sulfonate vs. sulfate) may
interact with the positively charged side chains on
the DNA polymerase and Witvrouw et al. (1994)
assumed that sulfated polysaccharides disruption
of ionic interactions between positively charged
regions of viral surface glycoproteins and cellular
membrane phospholipids.
CONCLUSION
Results from this study demonstrated the
significant potential of S. platensis polysaccharide
for activity against HSV-1. The hot water extract
polysaccharide which contained rhamnose as the
main sugar component showed anti HSV-1 activity
at IC50 21.3 µg/ml. Calcium ion and sulfate groups
in the polysaccharide had major roles in the anti
HSV-1 activity. S. platensis, is a possible source
for new drugs in the treatment of HSV-1 and other
viral diseases.
ACKNOWLEDGEMENTS
This study was supported by TRF (The
Thailand Research Fund, RDG4330032).
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Kasetsart J. (Nat. Sci.) 41 : 319 - 323 (2007)
Taura Syndrome Virus Disease in Farm-Reared Penaeus monodon
in Thailand
Chalor Limsuwan and Niti Chuchird*
ABSTRACT
Taura syndrome virus (TSV) has caused major economic losses to shrimp aquaculture throughout
the world. TSV has been reported to infect a number of penaeid species as hosts. In this study, we
reported the natural infection of TSV in farm-reared Penaeus monodon from eastern provinces of Thailand
between June to September 2004. There were different degrees of disease outbreak severity. In some
cases large number of shrimp died and caused great losses to farmers. However, in most cases only
small number of shrimp died and the farmers could control the situation enough to raise the majority to
marketable size. Diseased shrimp varied in size from aged 40-50 days (4 g) to 20 g. Infected shrimp was
characterized by black cuticular lesions and loose shell. Histopathological changes in infected shrimp
showed multifocal to extensive areas of necrosis in the sub-cuticular epithelium, connective tissue and
adjacent striated muscle. Affected cells often displayed an increased cytoplasmic eosinophilia, nuclear
pyknotic and karyorrhexis. In situ hybridization tests gave positive results with the tissues of shrimp
collected from the TSV outbreaks. In addition to TSV infection, most moribund shrimp also had dual
infections with microsporidians in the hepatopancreas and/or gregarines in the gut.
Key words: Taura syndrome virus, Penaeus monodon
INTRODUCTION
Taura syndrome was first recognized as
a shrimp disease in farms near the mouth of the
Taura river, Ecuador, in June 1992 (Jimenez, 1992;
Rosenbery, 1993; Lightner et al., 1994). The
infectious agent was named Taura syndrome virus
or TSV in 1994 (Hasson et al., 1995; Lightner et
al., 1995). TSV was first isolated from Litopenaeus
vannamei and characterized as a non-enveloped,
icosahedral particle, 31-32 nm in diameter, with a
density of 1.338 g/ml in CsCl. Its genome consists
of a linear, positive sense ssRNA molecule of
approximately 10.2 kb and it is classified as a
Picornavirus (Bonami et al., 1997; Brock et al.,
1997). From nucleotide sequence data, TSV is
more closely related to the cricket paralysis- like
viruses (Mari et al., 2002).
The occurrences of TSV outbreaks in L.
vannamei include cultured shrimp stocks in
Hawaii, Peru, Ecuador, Colombia, Panama, Costa
Rica, Nicaragua, El Salvador, Honduras,
Guatemala and Mexico (Lightner 1996). The
outbreaks were reported for P. stylirostris, P.
setiferus and P. schmitti in Ecuador and Peru
(Lightner et al., 1995; Brock et al., 1997). In Asia,
TSV was first reported from Taiwan in 1999 (Tu
et al., 1999).
Department of Fishery Biology, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
* Corresponding author, e-mail: ffisntc@ku.ac.th
Received date : 17/09/06
Accepted date : 25/12/06
320
Kasetsart J. (Nat. Sci.) 41(2)
In Thailand, since early 2000, the
cultivation of black tiger shrimp, Penaeus
monodon, had suffered slow growth, leading
shrimp farmers to shift to the cultivation of L.
vannamei. Most of the nauplii were illegally
imported from China and Taiwan. Alarmed by the
possibility of TSV introduction, the Thai
Department of Fisheries permitted legal
importation of L. vannamei from March 2002February 2003, if the imported stocks were
certified free of TSV by RT-PCR testing. However,
in early 2003, TSV outbreaks occurred in inland
farm-reared L. vannamei (Limsuwan, 2003;
Nielsen et al., 2005). Since then more TSV
outbreaks were reported in L. vannamei in most
areas of cultivation. Shortly thereafter, in early
2004, mortalities were observed in P. monodon
intensive culture ponds. Diseased shrimp were
PCR-negative for both white spot syndrome virus
(WSSV) and yellow-head virus (YHV) but
positive for TSV. This disease was widespread and
caused heavy mortalities to some farms.
This paper describes an epizootic of TSV
including gross signs, histopathology and in situ
hybridization in intensively reared P. monodon in
Thailand.
MATERIALS AND METHODS
Penaeus monodon samples were
collected from TSV-affected farm ponds in the
eastern provinces of Thailand during June to
September 2004. The shrimp samples weighing
of 4-20 g were preserved in Davidson’s fixative
solution and then transferred to 70% ethanol after
48 h. All histological materials were prepared
using standard histological procedures for shrimp
and stained with haematoxylin and eosin (H&E)
as described in Bell and Lightner (1988). A
commercially available in situ hybridization probe
for TSV (Diagxotics Inc.) was used according to
the manufacturer’s instructions. The protocols
have been outlined by Lightner (1996) and Mari
et al. (1998).
RESULTS AND DISCUSSION
Taura syndrome virus has been reported
to infect a number of penaeid species as hosts.
However, only Litopenaeus vannamei appears to
be highly susceptible to the disease (Lightner
1996). Overstreet et al. (1997) and Lightner (1996)
reported natural TSV infections in P. setiferus and
experimental infections have been reported in
P. schmitti, P. aztecus, P. duoraram, P. chinensis,
P. monodon, and P. japonicus. TSV may be
transmitted horizontally by co-habitation or
cannibalism (Lotz et al., 2003). In Thailand, TSV
was first reported from intensive farm-reared P.
monodon in June 2004. Moribund shrimp aged
40-50 days were found in scattered areas around
the edges of the pond. Although in some farms it
could be found in younger or older shrimp as well.
Diseased shrimp was characterized by black
cuticular lesions and loose shell. Shrimp with these
black lesions are at some risk of mortality during
the succeeding molt, but if they survive, lesions
disappear from the cuticle and shrimp look normal.
However, affected shrimp did not display signs of
red body or tail (Figure 1 and 2) which was
different from the report of Lightner et al. (1995)
indicated that the expansion of red chromatophores
in the appendages, especially of the uropods,
telson, and pleopods of L. vannamei infected with
TSV. There were different degrees of disease
outbreak severity in cultured P. monodon. In some
cases large number of shrimp died quickly and
caused great losses to farmers. However, in most
cases only small number of shrimp died and the
farmers could control the situation enough to raise
the majority to marketable size and harvest them
for sale.
Histopathology of moribund shrimp
showed multifocal to extensive areas of necrosis
in the sub-cuticular epithelium, connective tissue
and adjacent striated muscle (Figure 4). Affected
Kasetsart J. (Nat. Sci.) 41(2)
cells often displayed an increased cytoplasmic
eosinophilia, nuclear pyknosis and karyorrhexis
(Figure 5). Some samples also showed necrosis
in the cells of haematopoietic tissue corresponded
to those previously described for TSV infections
(Lightner et al., 1995). In situ hybridization tests
also gave positive results with the tissues of shrimp
collected from the TSV outbreaks (Figure 6). In
addition to TSV infection, most moribund shrimp
also had infections with microsporidians in the
hepatopancreas (Figure 7) and gregarines in the
gut (Figure 8). These protozoans are highly
pathogenic and frequently cause epizootics in
Figure 1 Moribund shrimp with TSV during the
first 2 months of culture with multiple
melanized cuticular lesions.
Figure 3 Normal subcuticular epidermal and
connective tissue (H&E).
321
crustacean populations (Overstreet,1973;
Sindermann, 1990). Sprague and Couch (1971)
indicated that in addition to microsporidians,
shrimps in the ponds often harbor cephaline
gregarines, similar to the results in this report.
Brock et al. (1997) reported experimental infection
of P. monodon with TSV and indicated that P.
monodon was susceptible to TSV but suffered few
mortalities. To avoid TSV infections or a
significant outbreak of the disease, farmers must
have sufficient reservoir ponds available and only
refill the shrimp ponds or stocking postlarvae into
the pond with water that has been left to rest for at
Figure 2 Affected shrimp at harvest with
multiple black melanized cuticular
lesions.
Figure 4 Typical TSV lesion showing area of
extensive subcuticular epidermal and
connective tissue necrosis (H&E).
Kasetsart J. (Nat. Sci.) 41(2)
322
least 15 days (Chuchird and Limsuwan, 2005). It
will then be less likely that the virus will be alive
in the water and the farmers will have a greater
chance of rearing a good harvest of shrimp.
eosinophilic to densely basophilic inclusions and
gave the tissue a kind of “buck-shot” appearance.
Most moribund shrimp had dual infections with
microsporidians in the hepatopancreas and/or
gregarines in the gut.
CONCLUSION
ACKNOWLEDGEMENTS
Gross sign of TSV in P. monodon was
characterized by black cuticular lesions and loose
shell. Histologically, sub-cuticular lesions were
characterized by large numbers of spherical
Figure 5 Higher magnification of TSV lesion
with numerous nuclear pyknosis (P)
and karyorrhexis (K), (H&E).
Figure 7 Microsporidians (arrows) infection in
the hepatopancreas of TSV infected
shrimp (H&E).
The authors would like to thank the
National Research Council of Thailand (NRCT)
for financial support.
Figure 6 Tissue section of cuticular epithelium
with positive in situ hybridization
reaction for TSV (arrows).
Figure 8 Gregarine (arrow) in the gut of TSV
infected shrimp (H&E).
Kasetsart J. (Nat. Sci.) 41(2)
LITERATURE CITED
Bell, T.A. and D.V. Lightner. 1988. A Handbook
of Normal Shrimp Histology. World
Aquaculture Society.
Bonami, J.R., K.W. Hasson, J. Mari, B.T. Poulos
and D.V. Lightner. 1997. Taura syndrome of
marine penaeid shrimp: characterization of the
viral agent. J. Gen. Virol. 78: 313-319.
Brock, J.A., R.B. Gose, D.V. Lightner and K.W.
Hasson. 1997. Recent developments and an
overview of Taura Syndrome of farmed
shrimp in the Americas, pp. 267-283. In T. W.
Flegel and I.H. MacRae, eds. Diseases in
Asian Aquaculture III. Fish Health Section,
Asian Fisheries Society, Manila, Philippines.
Chuchird, N. and C. Limsuwan. 2005. The
viability of Taura syndrome virus on lowsalinity water. Kasetsart J. (Nat. Sci.) 39:
406-410.
Hasson, K.W., D.V. Lightner, B.T. Poulos, R.M.
Redman, B.L. White, J.A. Brock and J.R.
Bonami. 1995. Taura syndrome in Penaeus
vannamei : Demonstration of a viral etiology.
Dis. Aquat. Org. 23: 115-126.
Jimenez, R. 1992. Syndrome de Taura (Resumen).
Aqucultura del Ecuador 1: 1-16.
Lightner, D.V. 1996. A Handbook of Pathology
and Diagnostic Procedures for Diseases of
Penaeid Shrimp.World Aquaculture Society.
Lightner, D.V., R.M. Redman, B.T. Poulos, J.L.
Mari, J.R. Bonami and M. Shariff. 1994.
Distinction of HPV-type virus in Penaeus
chinensis and Macrobrachium rosenbergii
using a DNA probe. Asian Fisheries Science
7: 267-272.
Lightner, D.V., R.M. Redman, K.W. Hasson and
C.R. Pantoja. 1995. Taura syndrome in
Penaeus vannamei (Crustacea: Decapoda):
gross signs, histopathology and ultrastructure.
Dis. Aquat. Org. 21: 53-59.
Limsuwan, C. 2003. Diseases of Pacific White
Shrimp (Litopenaeus vannamei) in Thailand.
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AAHRI Newsletter 12(1): 1-4.
Lotz, J.M. , A.M.Flowers and V. Breland. 2003. A
model of Taura syndrome virus (TSV)
epidemics in Litopenaeus vannamei. J.
Invertebr. Pathol. 83: 168-176.
Mari, J., J.R. Bonami and D.V. Lightner. 1998.
Taura syndrome of penaeid shrimp:cloning of
viral genome fragments and development of
specific gene probes. Dis. Aquat. Org. 33:
11-17.
Mari, J., B.T. Poulos, D.V. Lightner and J.R.
Bonami. 2002. Shrimp Taura syndrome virus:
genomic characterization and similarity with
members of the genus Cricket paralysis-like
viruses. J. Gen. Virol. 83: 915–26.
Nielsen, L., W. Sang-oum , S. Cheevadhanarak
and T.W. Flegel. 2005. Taura syndrome virus
(TSV) in Thailand and its relationship to TSV
in China and the Americas. Dis. Aquat. Org.
63(2-3): 101-106.
Overstreet, R.M. 1973. Parasites of some penaeid
shrimps with emphasis on reared hosts.
Aquaculture 2: 105-140.
Overstreet, R.M, D.V. Lightner, K.W. Hasson, S.
McIIwain and J.M. Lotz. 1997. Susceptibility
to TSV of some penaeid shrimps native to
the Gulf of Mexico and Southeastern US.
J. Invertebr. Pathol. 69: 165-176.
Rosenbery, B. 1993. World Shrimp Farming 1993.
Annual Report Shrimp News International.
52 p.
Sindermann, C.J. 1990. Principle Diseases of
Marine Fish and Shellfish, 2nd ed. Academic
Press.
Sprague, V. and J.A. Couch. 1971. An annotated
list of protozoan parasites, hyper-parasites and
commensals of decapod Crustacea. J.
Parasitol. 18: 526-573.
Tu, C., H. Huang, S. Chuang, J. Hsu, S. Kuo, N.
Li, T. Hsu, M. Li and S. Lin. 1999. Taura
syndrome in Pacific white shrimp Penaeus
vannamei culture in Taiwan. Dis. Aquat. Org.
38: 159-161.
Kasetsart J. (Nat. Sci.) 41 : 324 - 334 (2007)
Optimization of Docosahexaenoic Acid (DHA) Production and
Improvement of Astaxanthin Content in a Mutant Schizochytrium
limacinum Isolated from Mangrove Forest in Thailand
Wassana Chatdumrong1, Wichien Yongmanitchai1*, Savitree Limtong1
and Wanchai Worawattanamateekul2
ABSTRACT
Polyunsaturated fatty acids including DHA are essential dietary fatty acids. At present, fish
oils are a major source, but an alternative supply is needed because of increasing demand and fish
dwindling stocks. This need might be satisfied using a thraustochytrids found in mangrove forests of
Thailand and identified by 18S rDNA sequencing as either Schizochytrium limacinum or Thraustochytrium
aggregatum. S. limacinum was tested in various culture conditions to find the optimal yield of DHA.
This culture medium contained 7.5% glucose, 0.5% peptone, 0.5% yeast extract (with either 0.25%
soybean meal or 1% skimmed milk) and 0.75% sea salt at 20-30°C. The C:N ratio was about 15:1. The
culture was mutated using NTG and one isolate showed high DHA content and also a red pigment
identified as astaxanthin by TLC and HPLC. Astaxanthin synthesis peaked on day 6 - 10 of incubation
in medium containing 2% glucose using shaking flasks at 180 rpm, 25°C, 2 kLux light intensity with a
18:6 h light:dark periods. Six days of incubation yielded the highest yields of both DHA (224.6 mg/l)
and astaxanthin (8.9 µg/ml of medium). These results suggested that this microorganism could provide
a commercial source of this valuable lipid and pigment.
Key words: astaxanthin, Schizochytrium, DHA, mutation, mangrove forest, Thailand
INTRODUCTION
Thraustochytrids such as Schizochytrium
and Thraustochytrium are aquatic heterotrophic
microorganisms commonly found in marine and
estuarine environment (Barr, 1992). The capacity
of thraustochytrids to accumulate large amounts
of polyunsaturated fatty acids (PUFAs), especially
omega-3 fatty acids including docosahexaenoic
acid (C22:6, DHA), is well recognized (Lewis et
al., 1999; Huang et al., 2001). They are important
1
2
*
in preventing and treating pathologies such as
coronary heart disease, stroke and rheumatoid
arthritis (Kinsella, 1987), provide protection
against asthma, dyslexia, depression and some
forms of cancer (Simopoulos, 1989; Takahata et
al., 1998). DHA is an essential fatty acid for
neuronal development (Yongmanitchai and Ward,
1989). Demand of these fatty acids as a dietary
supplement has increased and the major supply is
presently derived from fish oil. But dwindling fish
stocks and increasing demand has created a need
Department of Microbiology, Faculty of Science, Kasetsart University, Bangkok 10900, Thailand.
Fishery Product Department, Faculty of Fishery, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: fsciwcy@ku.ac.th
Received date : 25/10/06
Accepted date : 14/02/07
Kasetsart J. (Nat. Sci.) 41(2)
for alternative sources of supply.
Astaxanthin (3,3’-dihydroxy-β,βcarotene-4,4’dione) is a carotenoid found
especially in marine crustaceans. It is added to
food products (Vazquez et al., 1997) and use as a
colorant for cultured fish, poultry (Johnson and
An, 1991) and shrimp. It also acts as a scavenger
of free oxygen radicals which damage DNA and
oxidizes proteins (Schroeder and Johnson, 1993).
Astaxanthin used as an animal feed is often
produced commercially by chemical synthesis.
However, the public have a preference for
additives coming from natural source (Fang and
Cheng, 1993) such as algae, fungi and small
crustaceans. When in the food chain, they lead to
pigmentation of larger animals including fish
(especially salmon), lobsters, krill and small
marine and freshwater organisms (Johnson and
Lewis, 1979).
Recently, microbial production of
astaxanthin pigment has been improved through
isolated or combined strategies, i.e., mutagenesis
and media fermentation (Fontana et al., 1996). The
thraustochytrids, Schizochytrium aggregatum
(Valadon, 1976) and Thraustochytrium CHN-1
(Marvelisa et al., 2003), have both been found to
contain this pigment. This study aims to improve
both the astaxanthin and DHA production by the
creation of mutations of Schizochytrium sp.
BR2.1.2 and also by optimizing the media and
conditions in small scale cultures and then
applying to larger vessels.
325
streaking on the agar medium, pure culture was
obtained for this study.
Identification of microorganism by
18S rDNA sequencing
Morphological characteristics of
thraustochytrid
BR2.1.2
resembled
Schizochytrium. Identification was further
confirmed by 18S rDNA sequencing. Two primers
of NS1 and NS8 were used for amplification of
18S rDNA by PCR technique using a thermal
cycler (Perkin Elmer GeneAmp PCR system
2400). The amplification program was carried out
following the protocol of Mo et al. (2001). Purified
PCR product of 18S rDNA was analyzed by DNA
autosequencer with NS1-8 primers set according
to White et al. (1990)
Mutagenesis
Increased expression of astaxanthin was
sought by mutagenesis of the wild type BR2.1.2
using N-methyl-N’-nitro-N-nitrosoguanidine
(NTG) (Fluka Chem, AG) modified from Chaunpit
(1993). The initial concentration of 1-9×106 cells/
ml was treated with NTG (0.1 mg/ml) for 20 min
with shaking. NTG was removed from suspension
by centrifugation and cells pellet washed by 0.5
M phosphate buffer pH 7.0 and spreaded on GPY
agar plate. The treated culture contained 0.05 –
0.1 % of the initial cells. Red colonies indicating
accumulation of astaxanthin were collected for
further assessment of growth, astaxanthin and
DHA contents.
MATERIALS AND METHODS
Microorganisms
Wild type strain
The wild type of thraustochytrid selected
strain BR2.1.2 was isolated from mangrove forest
at Bang-rong area, Amphur Thalang in Phuket
province, Southern Thailand. The isolation was
carried out in GPY agar medium (Huang et al.,
2001) by baiting technique. After a series of
Optimization of growth and DHA production
by wild type BR2.1.2
Culture conditions for thraustochytrid
BR2.1.2 were optimized for growth and DHA
production. The following conditions were applied
throughout unless otherwise stated. Thirty
milliliters of GPY medium (Huang et al., 2001)
composed of 3% glucose, 1% peptone, 0.5% yeast
extract and 50% of natural sea water was used as
326
Kasetsart J. (Nat. Sci.) 41(2)
the basal medium and placed in a 125 ml
Erlenmeyer flask. All experiments were carried
out in triplicate flasks. Cultivation was initiated
by addition of 1 ml of inoculum (adjusted cells
concentration to 1.0 at OD 600 nm). Incubation
was done on a rotary shaker (Sac Science-ENG
LTD, Part) at 140 rpm at room temperature for 4
days. To test different media, the basal media was
modified in the following manner:
1. The carbon source replaced by either
glucose, fructose, sucrose, glucose syrup and
agricultural products i.e. molasses, and sugar cane
juice (Sahakarnnamtan Co. Ltd., Chonburi,
Thailand).
2. The nitrogen sources replaced by
peptone, soybean meal, skimmed milk,
ammonium sulfate, potassium nitrate, sodium
nitrate, monosodium glutamate (MSG).
3. Sea salt concentration (salinity) 0200% of sea water.
The effect of temperature on growth and
DHA production were also determined by using
temperature gradient incubator (Model TN-3,
Toyo, Kagaku Sangyo Co., Ltd., Tokyo, Japan)
set at 15, 20, 25, 30 and 35°C.
Optimization of growth and astaxanthin
production by thraustochytrid mutant
The mutant was cultivated in a 125 ml
Erlenmeyer flask containing 30 ml of GYC
medium (Marvelisa et al., 2003) and kept in an
incubator shaker at 180 rpm for 10 days at 25°C.
Light was provided by fluorescent lamps at the
intensity of 2 kLux with light:dark periods at 16:8
hrs. Effects of carbon sources such as sugar cane
juice, molasses and maltose:glucose (1:1, w/w)
contained the same carbon equivalent as 2%
glucose were studied. Environmental conditions
such as light intensity at 0, 5 and 10 kLux and
temperature (as described above) were also
determined.
Analytical procedures
Growth was determined as the dry weight
of the cells (drying conditions).
Lipid was extracted by the modified
method of Bligh and Dyer (1959), followed by
transmethylation according to Holub and Skeaff
(1987). Fatty acid methyl esters were analyzed in
a gas-liquid chromatography (GC-14B; Shimadzu,
Tokyo, Japan) equipped with flame ionization
detector and a split injector at 1:40 ratio using
capillary column in 30 m length, 0.25 mm internal
diameter, 0.25 mm. film thickness (AT-WAX,
Alltech Associates Inc, USA). Fatty acids were
identified by comparing retention times with
authentic standards from Sigma by using C-R6A
Chromatopac Data Integrator (Shimadzu, Japan).
The astaxanthin content was determined
by the method modified from Fontana et al. (1996).
The concentration was quantified by using
absorbance values at 479 nm calculated with the
specific absorption coefficient a(1cm,1%) = 1600 as
proposed by Anderwes et al. (1976). Isomers of
astaxanthin were identified by thin layer
chromatography according to Donkin (1976)
compared with reference standards extracted from
Haematococcus pluvialis. These determinations
were confirmed by HPLC (model HP1100, Agilent
Technology) following the procedure of Marvelisa
et al. (2003).
RESULTS AND DISCUSSION
Identification of thraustochytrid BR2.1.2 by
18S rDNA sequence analysis
The corrected partial sequence of 18S
rDNA of thraustochytrid BR2.1.2 was 912 bases
in length after gaps, inserts and ambiguous
positions had been removed and was deposited in
DDBJ as accession number 794133. A
phylogenetic tree was constructed from an
alignment of the BR2.1.2 sequence with those
from related species obtained from GenBank by
the NJ method (Figure 1). It was clearly seen that
Kasetsart J. (Nat. Sci.) 41(2)
BR2.1.2 formed the same clade with
Thraustochytrium aggregatum and Schizochytrium
limacinum but with slight distance. Hence the
strain BR2.1.2 was finally identified as
Schizochytrium limacinum.
Effect of culture conditions on growth and DHA
production by S. limacinum BR2.1.2
1. Carbon source
Among the various carbon sources
tested, S. limacinum BR2.1.2 exhibited highest
growth rates in 3% fructose and glucose with 14.3
and 13.4 g/l of CDW, respectively (Figure 2A).
DHA yields were 392.5 and 362.1 mg/l with DHA
327
contents at 49.1 and 49.7% of TFA, respectively.
Although, relatively good growth rates were
obtained in complex carbon sources, i.e. molasses
(10.5 g/l) and sugar cane juice (11.5 g/l), DHA
production was low. Sucrose and glucose syrup
were poorer carbon source for this organism. The
results coincided with those of Wu et al. (2005) as
glucose syrup contained mainly oligosaccharides
that could not support growth for many
microorganisms. Although glucose was slightly
inferior compared to fructose, it is considered to
be the good carbon source, because it was ready
available and substantially cheaper.
Figure 1 Phylogenetic tree reconstruction based on 18S rDNA sequence by neighbour-joining (NJ)
method. The number at each branch shows bootstrap values 1000 replications.
Kasetsart J. (Nat. Sci.) 41(2)
328
that among complex nitrogen sources (1%
peptone) was the best in supporting growth for
both CDW (20.9 g/l) and DHA (828.2 mg/l).
Soybean meal and skimmed milk although
relatively good nitrogen source for CDW but DHA
production was considerably lower at 441.8 and
545.9 mg /l, respectively. In the medium
containing 0.2% MSG, BR2.1.2 grew at 20.3 g/l
and produced DHA 768.5 mg/l, almost the same
levels as supported by 1% peptone. This result
agrees with those using with Thraustochytriun
aureum ATCC 34304 that grew well in medium
containing glucose, peptone, yeast extract and
Figure 2B demonstrates the effect of
glucose concentration. Cell mass depended on
glucose concentrations and was maximal with 7%
(cell mass 28.3 g/l). However, the highest DHA
production was obtained in 5% glucose (732
mg/l) making up 50.6% of TFA. When glucose
concentration was increased to 7%, the proportion
of DHA (641.1 mg/l) was 44.6% of TFA.
2. Nitrogen source
Further experiments used 5% glucose
and 0.5% yeast extract to determine the effect of
the nitrogen source in Figure 3A. Results revealed
450
(A)
400
50
350
300
40
250
30
200
150
20
100
10
DHA Production (mg/l))
CDW (g/l); DHA (% of TFA))
60
50
0
0
Fructose
Glucose
Glucose
Syrup
Molasses
Sucrose
Sugar Cane
Juice
Carbon source
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
60
800
700
50
600
40
500
30
400
300
20
200
10
DHA Production (mg/l))
CDW (g/l); DHA (% of TFA))
(B)
100
0
0
2
3
4
5
6
7
Glucose concentration (%)
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
Figure 2 Effect of (A) carbon sources and (B) glucose concentration on growth and DHA production
by S. limacinum BR2.1.2 cultivated at room temperature in shaker 140 rpm.
Kasetsart J. (Nat. Sci.) 41(2)
329
which was expensive and economically unsuitable
for large scale production. Figure 3B shows the
effect of various peptone and soybean meal
mixtures on growth and DHA production by S.
limacinum BR2.1.2. In this experiment, the base
medium consisted of 5% glucose, 1% skimmed
milk and 0.2% MSG and 0.5% yeast extract.
Clearly, treatment with 0.5% peptone and 0.25%
soybean meal produced highest DHA contents at
1,170.9 mg/l which was 45.3% of TFA.
supplement with glutamate (Iida et al., 1996).
Although, soybean meal and skimmed
milk were slightly inferior compared to peptone
and probably not suitable as sole nitrogen source,
they are agricultural products that are less
expensive and readily available in Thailand.
Moreover, soybean meal not only provided protein
but also carbohydrate, fat, mineral and vitamins
which was likely to support growth and DHA
production of thraustochytrids (Fan et al., 2002).
Therefore, they could partially replace peptone
60
900
(A)
800
700
40
600
500
30
400
20
300
200
10
DHA Production (mg/l))
CDW (g/l); DHA (% of TFA))
50
100
0
0
Ammonium Potassium
sulfate
nitrate
MSG
Sodium
nitrate
Peptone
Soybean
meal
skimmed
milk
Nitrogen source
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
50
(B)
45
1400
1200
1000
35
30
800
25
600
20
15
400
10
DHA Production (mg/l))
CDW (g/l); DHA (% of TFA))
40
200
5
0.
5
M
0.
5
0.
25
P1
.5
+S
B
P1
.5
+S
BM
0.
25
1.
0+
SB
M
P
0.
5
M
P1
.0
+S
BM
P0
.5
+S
B
P0
.5
+S
BM
0.
25
0
P0
.5
0
Nitrogen source
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
Figure 3 Effect of (A) single nitrogen source and (B) combined nitrogen source on growth and DHA
production by S. limacinum BR2.1.2 cultivated at room temperature in shaker 140 rpm (P =
peptone; SBM = soybean meal).
Kasetsart J. (Nat. Sci.) 41(2)
CDW (g/l); DHA (% of TFA))
3. C/N ratio
Lipid accumulation in oleaginous
microorganisms can be enhanced by providing
excess carbon while limiting nitrogen (Ratledge,
2004). Figure 4 showed that optimum C/N ratio
at 15:1 was suitable for S. limacinum BR2.1.2 in
terms of growth and DHA production of 2,416.7
mg/l. Although, the cell concentration was
improved (27.6 g/l) the highest biomass of 38.0
g/l was achieved in medium with C/N ratio of 20:1.
4. Salinity
Seawater was used as the source of
salinity in this study. It should be noted that
although S. limacinum BR2.1.2 was isolated from
marine environment, it could grow and produced
DHA at all levels of salinity (Figure 5). The results
coincided with Yokochi et al. (1998) who reported
that S. limacinum SR21 could grow in condition
at zero salinity or without salt. However, a salinity
equivalent to 25% of natural sea water appeared
60
3000
50
2500
40
2000
30
1500
20
1000
10
500
0
DHA Production (mg/l))
330
0
0:1
10:1
15:1
20:1
25:1
30:1
C/N ratio
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
Figure 4 Effect of C:N ratio on growth and DHA production in S. limacinum BR2.1.2 cultivated at
room temperature in shaker 140 rpm.
1200
40
1000
35
30
800
25
600
20
15
400
10
DHA Production (mg/l))
CDW (g/l); DHA (% of TFA))
45
200
5
0
0
0
25
50
75
100
150
200
Salinity (as % of seawater)
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
Figure 5 Effect of salinity (as % of seawater) on growth and DHA production in S. limacinum BR2.1.2
cultivated at room temperature in shaker 140 rpm.
Kasetsart J. (Nat. Sci.) 41(2)
331
optimal for S. limacinum BR2.1.2 for DHA
production (975.4 mg/l , 41.1% of TFA). At the
highest salinity (200%), the organism showed
good growth but DHA production was lowest at
277.5 mg/l This contrasts to T. aureum which
fails to grow at zero salinity and also completely
inhibited at 200% salinity of sea water (Iida et al.,
1996).
by TLC and HPLC confirmed that it was
astaxanthin. Hence, it was considered to be
appropriate to improve the content of this pigment
in S. limacinum BR2.1.2 by mutation. If successful
this organism would provide two important
nutrients, i.e., DHA and astaxanthin making it
suitable for animal and human consumption
5. Effect of temperature
In this study cultures were grown in Lshaped tubes containing 10 ml medium and
incubated in a temperature gradient incubator. S.
limacinum BR2.1.2 grow well and produced fairly
constant DHA levels at a wide range of
temperature between 20-30°C. Growth of culture
varied from 8.7-10.3 g/l, and DHA contents were
220-236 mg/l (Figure 6).
mutants
1. Isolation of S. limacinum BR2.1.2
From an initial S. limacinum BR2.1.2
concentration of 8.75×106 cells/ml, the culture was
treated with NTG for 20 minutes which yielded a
0.05% cell survival rate. The treated culture was
then plated on GYP medium but only one colony
showed a distinctive red color. After sub-culturing
for several times the deep red color persisted which
showed that it was stably expressed. The mutant
was then used for further investigation.
The mutant grew rapidly for the first 2
days with cell concentration of 7.8 g/l. Maximum
cells mass was obtained on the 6th days at 10.8
g/l and declined gradually (Figure 7). Astaxanthin
contents in cell mass increased corresponding with
growth and reached highest value at 8.9 µg/ml and
remained relatively constant towards the end of
fermentation. This result coincided with Marvelisa
Improvement of astaxanthin content by
mutation
Although, culture of S. limacinum
BR2.1.2 in liquid GPY medium was creamy white
color, it developed orange colonies on agar plate
after several weeks of incubation. This might be
explained by an accumulation of carotenoid
pigments. Preliminary analysis of the pigments
CDW (g/l); DHA (% of TFA))
50
200
40
150
30
100
20
50
10
0
DHA Production (mg/l))
250
60
0
15
20
25
30
35
Temperature (C)
CDW (g/l)
DHA (% of TFA)
DHA Production (mg/l)
Figure 6 Effect of temperature on growth and DHA production in S. limacinum BR2.1.2 cultivated in
L-shaped tubes.
Kasetsart J. (Nat. Sci.) 41(2)
332
Under dark condition, the mutant
accumulated 5.6 µg/ml of astaxanthin at 25°C after
incubation for 8 days. However, when fluorescent
light source of 5 kLux was provided, the culture
produced higher pigment yield of 13.1 µg/ml.
Further increase of light intensity to 10 kLux had
adverse effect on astaxanthin production (10.7
µg/ml) (Figure 8). Therefore, moderate light was
an important bioinduction for carotenogenesis as
it was also shown by Phycomyces blaksleeanus
and several species of Rhodotolula (Goodwin,
1984). Yamaoka et al. (2004) also demonstrated
that Thraustochytrium sp. CHN-1 grown under
et al. (2003) who reported that, carotenoid contents
of Thraustochytrium CHN-1 paralleled the
biomass and cell growth. The mutant S. limacinum
BR2.1.2 could produce both DHA and astaxanthin
at moderate amounts. However, DHA production
decreased from 224.6 mg/l day 6 to only 29.8
mg/l at day 10. Hence it seemed that we have to
sacrifice either DHA or astaxanthin production
depending on the degree of necessity.
2. Effect of light intensity on
astaxanthin accumulation by S. limacinum
BR2.1.2 mutant
250
10
200
8
150
6
100
4
50
2
0
DHA production ((mg/l)
CDW (g/l); Astaxanthin (ug/ml)
12
0
2
4
6
8
10
DHA production (mg/l)
CDW (g/l)
Time (days)
Astaxanthin (ug/ml)
Figure 7 Growth, astaxanthin and DHA production by S. limacinum BR2.1.2 mutant strain in GYC
broth at 25°C with 2 kLux light intensity and light:dark 16:8 hrs.
14
Astaxanthin (ug/ml)
12
10
8
6
4
2
0
0
5
10
Light intensity (kLux)
Figure 8 Effect of light intensity for astaxanthin production by S. limacinum BR2.1.2 mutant strain in
GYC broth at 25°C, 180 rpm for 8 days.
Kasetsart J. (Nat. Sci.) 41(2)
fluorescent lamp at 1.5 kLux developed orange to
red color.
CONCLUSIONS
A thraustochytrid strain BR2.1.2 was
isolated from mangrove forest in Thailand. The
strain showed an ability to grow rapidly while
accumulating large amounts of DHA.
Identification of the strain based on morphological
characteristics and 18S rDNA sequence revealed
that it belonged to Schizochytrium limacinum
species. Under optimal culture conditions, i.e., 5%
glucose, combined nitrogen source (0.5% peptone,
0.2% MSG, 0.25% soybean meal and 1% skimmed
milk) and C/N ratio at 15:1, the DHA yield was
2,416.7 mg/l from a cell dry weight of 27.6 g/l.
Furthermore S. limacinum BR2.1.2 had a unique
feature of growing in media having a wide range
of salinity equating to 0-200% seawater. When the
strain was cultivated in liquid GPY medium the
culture appeared creamy white color. But on agar
medium with prolong incubation, color of the
colony developed into typical orange color of
carotenoid pigment which was identified as
astaxanthin. Improvement of S. limacinum
BR2.1.2 for astaxanthin content by mutation with
NTG was carried out and resulted in a colony with
intense red color. This mutant produced
astaxanthin in liquid medium even without light.
Optimization of culture conditions in liquid GYC
medium, particularly high light intensity at 5 kLux
at 25°C caused the mutant to accumulate the
pigment at 13.1 µg/ml.
ACKNOWLEDGEMENTS
The authors would like to thanks The
Graduate School of Kasetsart University,
Bangkok, Thailand for providing research grant.
Special thank to Dr. C. Norman Scholfield, Queen
University, UK, for his English editing was also
acknowledged.
333
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Kasetsart J. (Nat. Sci.) 41 : 335 - 345 (2007)
Cloning, Expression, Purification and Biological Activities
of Recombinant Mouse Interleukin-2 in E. coli M15
Sanchai Chantajorn1, Ratchanee Hongprayoon2* and Thaweesak Songserm3
ABSTRACT
Molecular cloning, sequencing and expression of recombinant mouse interleukin 2 (rmIL-2)
were described. The interleukin-2 (IL-2) cDNA, 450 base pairs in length with repeating CAG, was
amplified using specific primers. The IL-2 cDNA showed high homology at 100%, 100%, 91%, 96%
and 94% with five strains of mice previously reported (GeneBank accession number AY147902.1,
MMU41494, MMU41504, MMU41505 and MMU41506). The IL-2 gene was cloned into the pDrive
cloning vector and consequently expressed using pQE30 expression vector which provided high
expression level of the recombinant protein. The predicted rmIL-2 sequence is 161 amino acids with a
molecular weight of 19 kDa. The expressed protein was then purified by Ni-NTA column under denaturing
condition. Analysis of the rmIL-2 by SDS-PAGE demonstrated two bands of 19 and 38 kDa representing
monomeric and dimeric forms of this protein. The biological activity in stimulating T-cell proliferation
was also described and the binding signal to the receptor was easily observed by immunofluorescence.
Key words: mouse interleukin-2, cloning, protein expression, protein purification, immunofluorescence
INTRODUCTION
Interleukin-2 (IL-2) is a growthpromoting activator for bone marrow-derived T
lymphocytes (Smith, 1989), and was among the
first cytokines to be characterized at the molecular
level. Interleukin-2 was the major autocrine
growth factor for T lymphocytes, and the quantity
of IL-2 synthesized by activated CD4+ T cells was
an important determinant of the magnitude of
immune response. The action of IL-2 on T-cells
was mediated by binding to IL-2 receptor proteins.
1
2
3
*
This system was perhaps the best understood
mechanism of all cytokine receptors (Morgan et
al., 1976). Interleukin-2 exerts its effects on many
cell types, the most prominent of which is the T
lymphocyte. Indeed, one of the most rapid
consequences of T cells activation through its
antigen receptor is the de novo synthesis of IL-2.
This was quickly followed by expression of a high
affinity IL-2 receptor on surface membrane of
CD4+ T cells, CD8+ T cells, B cells and natural
killer (NK) cells (Lenardo et al., 1999).
Interleukin-2 induce cells proliferation via pro-
Centre for Agricultural Biotechnology, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom 73140, Thailand.
Department of Plant Pathology, Faculty of Agriculture at Kamphaeng Saen, Kasetsart University, Kamphaeng Saen Campus,
Nakhon Pathom, 73140, Thailand.
Department of Veterinary Pathology, Faculty of Veterinary, Kasetsart University, Kamphaeng Saen Campus, Nakhon Pathom
73140, Thailand.
Corresponding author, e-mail: agrrat@ku.ac.th
Received date : 15/08/06
Accepted date : 13/02/07
336
Kasetsart J. (Nat. Sci.) 41(2)
proliferative signals through the proto-oncogenes
c-myc and c-fos, in combination with antiapoptotic signals through an essentially identical
receptor (Ma, 2000; Carson et al., 1997; Giri et
al., 1994; Giri et al., 1995). Interleukin-2 promotes
production of NK-derived cytokines such as TNF·,
and granulocyte macrophage colony stimulating
factor (GMCSF). Furthermore, IL-2 acts
synergistically to enhance NK cytotoxic activity
(Khatri, 1998). A number of functions for IL-2 in
B cells have been identified, mostly pertaining to
antibody secretion. In IgM expressing B cells, IL2 (in synergy with IL-5) upregulates expression
of heavy and light chain genes as well as inducing
de novo synthesis of the immunoglobulin J chain
gene (Blackman et al., 1986). The latter is required
for oligomerization of the IgM pentamer, and
represents a tightly controlled stage in B cells
activation (Koshland, 1985). As in T cell, IL-2
increases expression of IL-2Rα in B cells, thus
enhancing their responsiveness to IL-2 (Gaffen et
al., 1996). Therefore, IL-2 is one of the key
cytokines in immunology-base studies.
Measurement of IL-2 by ELISA method has been
widely used in clinical investigations and research.
There are quite a number of commercially
available IL-2 ELISA kits which are very
expensive. Production of the ELISA system is
necessary for our use to investigate the effect of
plant extract on mouse immune response. The
objectives of this study were, therefore, to produce
mouse recombinant IL-2 (rmIL-2) by cloning its
gene into pDrive cloning vector and express the
rmIL-2 in pQE30 expression vector to investigate
its biological activities in vitro for the use in
immunization.
MATERIALS AND METHODS
Media and reagents
Complete culture medium was RPMI
1640 (Hyclone, Utah, USA) supplemented with 2
mM L-glutamine, 0.05 mM 2-mercaptoethanol
and 10% fetal calf serum (Hyclone, Utah, USA),
100 U/ml penicillin and 100 µg/ml streptomycin
(Sigma-Aldrich, St. Louis, USA). Concanavalin
A (Con A) (Sigma-Aldrich, St. Louis, USA) was
used for stimulation of splenocytes.
Preparation of mouse splenocytes
A BALB/c male mouse weighing 25-30
g, 12 weeks of age was purchased from the
National Laboratory Animal Centre, Mahidol
University, Nakhon Pathom, Thailand and used
in the experiments. Mouse spleen was removed
aseptically, homogenated and cells were washed
with cold RPMI 1640 medium (HyClone, Utah,
USA) and resuspended in complete medium.
Viability and number of splenocytes were
determined microscopically by staining with
trypan blue (Gibco, New York, USA). Splenocytes
(5 × 106 cell/ml) were activated with Con A
(2 µg/ml) in complete medium and transferred into
24 well, flat-blottom tissue culture plate (Costar,
New York, USA). Cells were incubated for 36 h
at 37°C in 5% CO2 and were harvested for mRNA
extraction.
Primers design
The PCR primers were designed
specifically to mouse IL-2 gene by the FastPCR
program. Briefly, primers for the amplification of
IL-2 mRNA were selected from the conserved
nucleotide sequences of the five strains of mice
(GeneBank accession numbers AY147902.1,
MMU41494, MMU41504, MMU41505,
MMU41506). Additionally, primer sequences for
the detection of mouse β-actin gene were taken
from the literature (Deng et al., 2000) (Table 1).
RNA extraction and cDNA library synthesis
Splenocytes were harvested after the
incubation period (36 h). Total RNA was extracted
from mitogen-stimulated cells (1 × 107 cells) and
non-stimulated controls according to the methods
of RNA purification kit (Epicentre, Madison,
Kasetsart J. (Nat. Sci.) 41(2)
337
Table 1 Primer sequences used for the amplification of IL-2 and house keeping gene transcripts in
lymphocytes.
Gene
primer sequences (5′-3′)
IL-2
GCACCCACTTCAAGCTCCACTTC
TTATTGAGGGCTTGTTGAGATGATGC
S
AS
Nucleotide
position
61-83
485-510
IL-2
GGATCCGCACCCACTTCAAGC*
GTCGACTTATTGAGGGCTTGTTGAG**
S
AS
61-75
478-510
462
-
S
AS
87-105
555-577
491
(Deng et al., 2000)
β-actin TGTATTCCCCTCCATCGTG
GGATCTTCATGAGGTAGTCTGTC
Direction
Length
(bp)
450
Reference
-
bp: base pair, IL-2: interleukin-2; β-actin: mouse beta-actin; S: sense strand; AS: antisene strand
* sense strand primer with restriction site GGATCC (BamHI)
** antisense strand primer with restriction site GTCGAC (SalI)
Wisconsin, USA). Total extracted RNA was
applied to 1.2% agarose gels and electrophoresed
in TBE buffer following standard procedures
(Sambrook et al., 1989). A cDNA library was
generated from total RNA using oligo (dT)15 (DNA
Technology Laboratory, Kasetsart University,
Nakhon Pathom, Thailand) as primers.
SuperScript III reverse transcriptase (Invitrogen,
California, USA) was used for cDNA synthesis
following the standard procedures.
Cloning of the interleukin-2
The synthesized cDNA library was used
as a template for the amplification of IL-2 gene
by two steps RT-PCR. Briefly, PCR master mixture
consisted of 1X PCR buffer, 1.6 mM MgCl2, 0.5
mM dNTP, 0.25 µmol of specific primers (Table
1) and 1U Platinum Taq DNA polymerase
(Invitrogen, California, USA). The PCR samples
were then denatured at 94°C for 2 min and
continually cycled for 30 times at 94°C for 45 s,
60°C for 45 s and 72°C for 1 min. For complete
amplification, an additional extension step at
72°C for 7 min was included. The PCR products
were analysed in 1.2% agarose gel electrophoresis
and visualized by ethidium bromide staining. The
PCR products were cloned into pDrive cloning
vector (Qiagen, Valencia, USA) and transformed
into Escherichia coli (DH5α). The transformants
were easily observed by blue/white screening and
the PCR was applied for conformation.
DNA sequence analysis
The DNA from positive clones were
sequenced by the automated DNA sequencer ABI
377 (GMI, Minnesota, USA). Comparison and
multiple alignment of BALB/c nucleotide and
amino acid sequences with those of other mice
strains were carried out using ClustalW version
1.83 with additional manual adjustments.
Expression of rmIL-2 and purification
IL-2 forward and reverse primers
including the restriction site were used for the
amplification of IL-2 gene from the positive
clones. The PCR products were cloned into a
pDrive cloning vector and then transformed into
E. coli (DH5α). The IL-2 gene was amplified and
digested with BamHI/SalI, the IL-2 gene was
ligated into the same sites of the expression vector
pQE30 (Qiagen, Valencia, USA) and then
transformed into E.coli M15 strain by heat shock
method (Sambrook et al., 1989). Screeninng of
the transformants was carried out for ampicillin
and kanamycin resistance. The positive clones
were induced by culturing at 37°C for 5 h in 2YT
338
Kasetsart J. (Nat. Sci.) 41(2)
medium containing 100 µg/ml ampicillin, 25 µg/
ml kanamycin and 1 mM isopropyl-1-1-thio-β-Dgalactoside (IPTG). Cells were harvested and
extracted by denaturing condition and then the
recombinant IL-2 was purified with Ni-NTA resin
affinity column chromatography according to the
recombinant protein purification procedures
(Qiagen, Valencia, USA). The rmIL-2 was allowed
to refold in native conformation by dialysis in PBS
and the protein concentration was determined by
Bradford protein assay (Bradford, 1976). The
protein purity was determined by SDS-PAGE
(Laemmli, 1970).
Western blotting
Twenty micrograms of the rmIL-2 was
loaded in a mini-gel apparatus and resolved on a
12% SDS-PAGE gel and transferred to
nitrocellulose membrane by electroblotter (BioRad, California, USA). The blot was blocked with
5% skim milk, incubated with rat anti-mouse
interleukin 2 IgG monoclonal antibody (Serotech,
North Carolina, USA) (1 µg/ml) for 30 min at
room temperature. After washing, it was incubated
with goat anti-rat IgG conjugated with alkaline
phosphatase at 1:10,000 dilution (Sigma-Aldrich,
St. Louis, USA). Detection was performed using
the 5-bromo-4-chloro-3-indolyl phosphate/
nitroblue tetrazolium (Zymed, South San Fancisco,
USA) as substrates.
Cell proliferation assay
The rmIL-2 was investigated for the
ability to stimulate cell proliferation which was
quantified by the colorimetric assay based on the
2,3-bis (2-Methoxy-4-nitro-5-sulfophenyl)-5[(phenylamino)-carbonyl]-2H-tetrazolium
hydroxide (XTT) assay as previously described
(Scudiero et al., 1988).
Splenocytes were transferred into 96 well
microtitre plates (Costar, New York, USA) at a
density of 1 × 105 cells/well for 48 h in complete
medium. The XTT (Sigma-Aldrich, St. Louis,
USA) solution was prepared freshly at 1 mg/ml in
prewarmed balance salt solution without phenol
red. Then, 5 mM phenazine methosulfate (PMS)
(Sigma-Aldrich, St. Louis, USA) solution was
prepared in PBS, stored at 4°C until use and
protected from light. Culture medium was
removed from each well, after that a 50 µl of XTT
solution with 0.025 mM phenazine methosulfate
was added. After 5 h of incubation, the absorbance
at 450 nm was determined by a Multiskan EX
(Labsystems, Finland).
Receptor binding assay
A New Zealand white rabbit was first
immunized with a mixture of rmIL-2 (1 mg/ml)
and Freund’s Complete adjuvant (Sigma-Aldrich,
St. Louis, USA) at 1:1 ratio following by three
injections at weekly intervals with the same
antigen and Freund’s Incomplete adjuvant (SigmaAldrich, St. Louis, USA). The antiserum with the
highest titre was used for immunofluorescent
detection of IL-2 receptor binding. The splenocytes
were stimulated with Con A mitogen at 5 µg/ml
final concentration compared with non-stimulated
control. Cells were incubated for 6 h at 37°C in
5% CO2 and harvested for receptor binding assay.
They were washed twice with PBS and then
resuspended in 50 µl of PBS, and fixed with 100
µl of 4% paraformaldehyde for 30 min in the dark
at 4°C. The experiment was done on a glass slide.
Proteins on cell surface were stained with 500 µM
sulforhodamine B (SRB) (Sigma-Aldrich, St.
Louis, USA), washed with 1% acetic acid and
PBS. Cells were then incubated with recombinant
IL-2 for 1 h at 37°C, washed with PBS and reacted
with rabbit anti-rmIL-2 polyclonal antibody
(1:500). After washing step, FITC goat anti-rabbit
IgG (H+L) conjugate (Zymed, South San
Fancisco, USA) (1:50) was added and incubated
for 1 h at 37°C and then washed with PBS. Cells
were analyzed by reflected light fluorescence
illuminator BH2-RFL (Olympus, New York, USA)
within 5 h.
Kasetsart J. (Nat. Sci.) 41(2)
RESULTS
Cloning and sequencing of mouse IL-2
The cDNA library of a BALB/c mouse
was synthesized from total RNA and amplified by
using the specific primers for IL-2. The cDNA
synthesis was compared by using β-actin primers
with two steps RT-PCR. The PCR product of IL-2
gene showed a band of 450 bp and 491 bp for the
β-actin gene which was a positive control (Figure
1). The PCR product was cloned into the pDrive
cloning vector and its sequence was analysed
(Figure 2). The IL-2 cDNA sequence consisted of
eight codons of CAG which differs from the
previous reports of other mouse strains including
C3HeB/FeJ (AY147902.1), RF (MMU41494),
C57BL6/J (MMU41504), CZECHII/Ei
339
(MMU41505), and BKL (MMU41506). The other
mouse strains showed CAG codon with 8, 8, 12,
21 and 21 contiguous codons, respectively. The
IL-2 cDNA shows 99%, 99%, 96%, 97% and 96%
high homology with C3HeB/FeJ (AY147902.1),
RF (MMU41494), C57BL6/J (MMU41504),
CZECHII/Ei (MMU41505), and BKL
(MMU41506). The deduced mouse IL-2 protein
included 149 amino acid with a predicted
molecular weight of 17,101 Da. Amino acid
alignment of mouse IL-2 to those of the other
strains showed high homology at 100%, 100%,
91%, 96% and 94% with C3HeB/FeJ
(AY147902.1), RF (MMU41494), C57BL6/J
(MMU41504), CZECHII/Ei (MMU41505), and
BKL (MMU41506), respectively.
Figure 1 Agarose gel electrophoresis of the amplified IL-2 cDNA products in non-stimulated, mitogenstimulated splenocytes and β-actin cDNA for house keeping gene after an incubation period
of 36 h. A: Positive control (β-actin gene 491 bp); B: Non-stimulated Control and C: Con A
stimulated cells (IL-2 450 bp). The sizes of PCR products were compared with φX174 DNA
- Hae III markers (M).
Kasetsart J. (Nat. Sci.) 41(2)
340
(A)
(B)
Figure 2 The 450 base pairs of IL-2 cDNA sequence of a BALB/c mouse (A). Alignment of the predicted
protein sequences of BALB/c mouse (DQ836354), C3HeB/FeJ mouse (AY147902.1), RF
mouse (MMU41494), C57BL6/J mouse (MMU41504), CZECHII/Ei mouse (MMU41505),
and BKL mouse (MMU41506) which were analyzed by GeneDoc (B). The homology of
amino acid was marked by black stripes.
Kasetsart J. (Nat. Sci.) 41(2)
Expression of rmIL-2 and purification
The recombinant plasmid containing
pQE30/mouse IL-2 cDNA was induced with IPTG
to produce rmIL-2. The bacterial extracts
containing the rmIL-2 gene could be obtained
according to the protein pattern from SDS-PAGE.
The expressed protein tended to aggregate with
the cell debris as observed in the insoluble fraction
by SDS-PAGE. However, under denaturing
condition during the protein extraction, the rmIL2 could be resolved and then purified by Ni-NTA
column chromatography. The rmIL-2 obtained
from Ni-NTA resin column was highly purified
(Figure 3). The bands of fusion proteins on SDSPAGE showed a molecular weigh of 19,000 Da
and 38,000 Da for monomeric and dimeric forms,
respectively. In addition, these two bands were
positively reacted with the IL-2 specific
monoclonal antibody by Western blotting (Figure
4).
Cell proliferation assay
The purified rmIL-2 was analyzed for
341
their biological activities by measuring the XTT
colorimetric assay. The splenocytes were
stimulated with serial dilutions of the purified
rmIL-2 compared with non-stimulated cell culture
as a negative control. The result showed the
increasing values of OD450 nm and proliferative
response after adding rmIL-2 at the concentration
range of 5-2,560 ng/ml. When 40 ng/ml of the
rmIL-2 was added, the increasing rate was
distinctively seen and reached the plateau at the
concentrations of 640-2,560 ng/ml (Figure 5).
Receptor binding assay
Mouse splenocytes were used in this
study to determine the ligand-receptor binding
activity. Cell surface membrane was stained red
with sulforhodamine B (SRB) while the rmIL-2
bound to the cell receptor showed fluorescence
green of the FITC conjugate. No or less signal was
observed in non-stimulated cells. However, the
splenocytes stimulated by mitogen exhibited
increasing signal with high expression of IL-2
receptors on the cell surface (Figure 6).
Figure 3 Ni-NTA purification of the recombinant mouse IL-2, analyzed by SDS-PAGE, demonstrated
the two bands 19 kDa monomeric and 38 kDa dimeric forms in the eluate fractions (E1-E3)
compared with non-induced transformant (C/-), IPTG-induced transformant (C/+), washing
fractions (W1-W3) and molecular weight markers (M).
Kasetsart J. (Nat. Sci.) 41(2)
342
DISCUSSION
The ability to produce and purify large
quantities of biologically active interleukin-2 has
been made possible by the use of recombinant
DNA technology. The mouse cDNA library of
BALB/c strain was cloned and characterized for
its activity. The IL-2 cDNA consisted of 450 base
pairs, repeating CAG and showed high homology
at 100%, 100%, 91%, 96% and 94% with five
strains of mice previously reported (GeneBank
accession number AY147902.1, MMU41494,
MMU41504, MMU41505 and MMU41506). The
rmIL-2 has been intensively studied and found that
Figure 4 Detection of the recombinant mouse IL-2 (rmIL-2) by SDS-PAGE (A) and Western blotting
(B) by probing with mouse IL-2 specific monoclonal antibody. M; molecular weight markers.
Figure 5 The effect of various concentrations of the recombinant IL-2 on cell proliferation of the
lymphocytes determined by XTT colorimetric assay while non-incubated cells with the
recombinant IL-2 showed the OD450 nm = 0.248 (data not shown).
Kasetsart J. (Nat. Sci.) 41(2)
other strains of mice have different effects on the
biological activity of IL-2 (Matesanz and Alcina,
1996). The IL-2 cDNA did not contain the
hydrophobic leader sequence of a 20 amino acid
peptide and the expressed rmIL-2 was purified
from the cells later. According to Robb et al.
(1981), even though the IL-2 exhibited O-linked
glycosylation at threonine 3 of N-terminus and the
E. coli system did not provide the posttranslational
glycosylation, it did not affect the IL-2 activity
nor change its activity in standard bioassay. The
functional significance of glycosylation of IL-2
was not known but it was likely that it enhances
solubility in aqueous environments. Thus an Nterminal 20 amino acid sequence was reported to
be essential for the interaction with the IL-2
receptor (Eckenberg et al., 2000). The
polymorphism of the CAG sequence has been
reported among different strains of mice including
C3HeB/FeJ mouse (AY147902.1), RF mouse
(MMU41494), C57BL6/J mouse (MMU41504),
CZECHII/Ei mouse (MMU41505), and BKL
343
mouse (MMU41506) (GeneBank data base) which
contained the sequences of 8, 8, 12, 21 and 21
codons, respectively. Characterization of the rmIL2 by ProtParam program (ExPASy) showed that it
consisted of 149 amino acids of mature IL-2
protein and 12 amino acids of protein tag from
the expression vector. The expressed protein was
estimated to weigh 18,489 Da, with the isoelectric
point (pI) at 5.87 with good solubility. However,
the protein bands observed on the SDS-PAGE
were found to be 19 and 38 kDa which were
predicted as a monomeric and dimeric forms of
the protein. The increased molecular weight from
the data obtained by program analysis may due to
the phosphorylation of the rmIL-2 (Adachi et al.,
1997; Brennan et al., 1997; Gesbert et al., 1998;
Justement, 2001; Cook and Unger, 2002; Michelle
et al., 2003; Stoker, 2005). The rmIL-2 were
applied to the cell culture with the following
addition of XTT to examine its biological activity.
The result showed that the activity was raised
according to the increasing concentration of rmIL-
Figure 6 The binding of the recombinant mouse IL-2 to the IL-2 receptors was analyzed by
immunofluorescence. Cells stained with sulforhodamine B (SRB) illustrating red color and
the signal for IL-2 binding showed greenish fluorescence (arrows). (A) Non-stimulated cells
and (B) mitogen-stimulated cells, after 6 h of incubation.
Kasetsart J. (Nat. Sci.) 41(2)
344
2 fusion proteins. In the receptor binding assay,
the rmIL-2 bound to its receptor showing green
fluorescence on the cell surface. This experiment
confirmed the rmIL-2 biological activity in binding
to its receptor and leading to cell proliferation by
XTT assay. The future plan of our project will be
the use rmIL-2 as an antigen to raise anti-IL 2
polyclonal antibody and develop the ELISA
method for the measurement of mouse IL-2 for
further investigation.
ACKNOWLEDGEMENTS
The authors would like to acknowledge
the Centre for Agricultural Biotechnology,
Kasetsart University. Kamphaeng Saen Campus,
Nakhon Pathom for the facility and financial
support throughout this study.
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Kasetsart J. (Nat. Sci.) 41 : 346 - 355 (2007)
Production and Partial Characterization of Chitosanases from a
Newly Isolated Bacillus cereus
Sutee Wangtueai1, Wanchai Worawattanamateekul1*, Mathana Sangjindavong1,
Nuanphan Naranong2 and Sarote Sirisansaneeyakul3
ABSTRACT
The production of chitosanases by a newly isolated Bacillus cereus TP12.24 was studied both
in shake flask and fermenter cultures. The M9-chitosan medium was found most suitable with 0.5%
chitosan as a sole carbon source optimized under aerobic growth conditions at pH 6.0 and 30°C. The
specific rates of growth, substrate consumption, and enzyme production were improved using controlled
completely aerobic conditions in 2-l fermenter. While the yield of biomass was considerably increased,
the enzyme yield was on the contrary decreased. As a result, the volumetric chitosanases productivity
was 43.55 U/l h, which was 1.2 times that obtained from shake flask culture due to higher specific rates
of chitosan consumption and chitosanases production. In this work, the crude chitosanases from Bacillus
cereus TP12.24 showed their optimal pH and temperature at 6.5 and 55°C, while the stabilities to pH
and temperature were found at 3.0-8.0 and 30-50°C, respectively. The Bacillus cereus chitosanases
could be used for preparing the chitosano-oligosaccharides under mild temperature.
Key words: chitosanases, chitosan, Bacillus cereus, optimization, batch culture
INTRODUCTION
Chitosan (poly-β-(1→4)-2-amino-2deoxy-D-glucose) is a long chain polymer derived
from chitin by deacetylation (Kumar et al., 2000).
Mostly, the sources of chitin in Thailand are solid
wastes derived from the shrimp processing
industries. Chitosan has been utilized as multipurpose products in food, semi-food and non-food
industries. Whereas the production of chitosanderived oligosaccharides shows its potential as
high value added food product, the enzymatic
hydrolysis rather than chemical degradation that
1
2
3
*
provides an attractive process is obviously limited.
Chitosanase (EC 3.2.1.132) is exploited for the
production of chitosano-oligosaccharides. Various
sources of enzyme could be obtained from soil
fungi and bacteria, such as Bacillus circulans MHK1 (Yabuki et al., 1988), Bacillus sp. No.7-M
(Uchida and Ohtakara, 1988), Bacillus
licheniformis UTK (Uchida et al., 1992), Bacillus
cereus S1 (Kurakake et al., 2000), Streptomyces
N-174 (Boucher et al., 1992), Streptomyces sp.
No.6 (Price and Storck, 1975), Amycolatopsis sp.
CsO-2 (Okajima et al., 1994), and Burkholderia
gladioli strain CHB101 (Shimosaka et al., 2000).
Department of Fishery Products, Faculty of Fisheries, Kasetsart University, Bangkok 10900, Thailand.
Department of Applied Biology, Faculty of Science, King Mongkut’s Institute of Technology Ladkrabang, Bangkok 10520,
Thailand.
Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
Corresponding author, e-mail: ffiswcw@ku.ac.th
Received date : 21/04/06
Accepted date : 14/12/06
Kasetsart J. (Nat. Sci.) 41(2)
The chitosano-oligosaccharides are
water-soluble and possess versatile bioactivities
such as immunopotentiating, bacteriostatic
activities which have their advantages in food
materials, agricultural and medical, and antitumor
activity (Tominaga and Tsujisaka, 1975; Price and
Storck, 1975; Suzuki et al., 1984; Papineau et al.,
1991; Somashekar and Joseph, 1996; Jeon and
Kim, 1998, 2000). The purpose of the present work
was to optimize the production of chitosanases
from the newly isolated Bacillus cereus TP12.24
(Wangtueai et al., 2006). The crude chitosanases
were also partly characterized for their optimal and
stability based on pH and temperature.
MATERIALS AND METHODS
Microorganism
Bacillus cereus TP12.24, a newly
isolated soil bacterium (Wangtueai et al., 2006)
was used throughout the experiments. The stock
culture was maintained on the chitosanasedetection agar medium (CDA) (Cheng and Li,
2000) and freshly transferred every 2 weeks.
Factors affecting enzyme production in shake
flask culture
All experiments were carried out in shake
flask cultures using 500-ml Erlenmeyer flask
containing 250-ml M9-chitosan medium at 250rpm for 72 h. Samples were taken every 6 h for
determining total viable cells, dry cell weight,
residual chitosan and enzyme activity. The culture
conditions and all analyses have been described
previously (Wangtueai et al., 2006).
Effect of pH
The study on pH optimum for enzyme
production was carried out by varying the pH
values of M9-0.5% chitosan medium at 4.0 to 8.0
at 30°C.
347
Effect of chitosan
The M9-chitosan media containing 0.1,
0.5, 1.0 and 2.0 % chitosan were used for the
production of chitosanases under optimized initial
pH 6.0 at 30°C.
Effect of temperature
The temperatures of 30, 40 and 50°C
were investigated for growth and enzyme
production under optimized initial pH 6.0 and
0.5% chitosan concentration.
Enzyme production in 2-l fermenter
The 2-l fermenter (EYELA Mini jar
fermenter, Model M-100, Tokyo Rikakikai Co.,
Ltd.) which contained 1.5-l M9-chitosan medium
with 0.5% chitosan was used for the production
of chitosanases from Bacillus cereus TP12.24. The
fermentation conditions were controlled
automatically at 30°C, pH 6.0, 1 vvm aeration rate
and 400 rpm agitation rate for 58.5 h of cultivation
time. The samples were taken every 6 h for
determining the total viable cells, dry cell weight,
residual chitosan and enzyme activity (Wangtueai
et al., 2006). The fermentation kinetics of bacterial
growth and chitosanases production were studied
based on the experimental results.
Characterization of crude chitosanases
The crude chitosanases were prepared by
growing cells in 2-l fermenter under the optimal
conditions obtained in this work. The enzyme
supernatant was collected from the culture broth
after centrifugation at 8,000 rpm, 4°C for 20 min.
This supernatant as crude chitosanases was used
for the determination of enzyme optimal and
stability on the basis of pH and temperature.
Optimal pH
The crude chitosanase activity was
measured at various pH values, using 80%
deacetylated chitosan as a substrate. The reaction
mixtures consisting of 1.0 ml of 1% soluble
348
Kasetsart J. (Nat. Sci.) 41(2)
chitosan and 1.0 ml of the crude enzyme solution
were incubated for 10 min at 30°C. The extended
pH ranges of 3.0-7.5 and 8.0-9.0 were monitored
by 0.05 M citrate phosphate buffer and 0.05 M
carbonate bicarbonate buffer, respectively.
Optimal temperature
The temperatures were varied from 3070°C for optimizing the enzyme activity for 10
min at the optimal pH 6.5 obtained in this work.
The reaction mixture prepared was the same as
mentioned above.
pH stability
The prepared crude enzyme was diluted
5 times with buffers at various pH’s (crude
enzyme:buffer, 1:4) using 0.05 M citrate phosphate
buffer for pH 3.0-8.0 and 0.05 M carbonatebicarbonate buffer for pH 9.0-11.0. The diluted
enzyme solutions at these various pH’s were
incubated at 40°C for 60 min. Then the residual
activities of chitosanases were determined under
the specified conditions modified from Shimosaka
et al. (1995) and Cheng and Li (2000).
Temperature stability
The diluted crude enzyme solutions were
prepared with 0.05 M citrate phosphate buffer pH
6.5 and incubated at different temperatures varying
from 30-80°C for 30 min. The residual enzyme
activities of chitosanases were determined under
the specified conditions modified from Shimosaka
et al. (1995) and Cheng and Li (2000).
Analyses
Determination of growth
The total number of viable cells was
determined by spread plate technique and the dry
cell weight was calculated from the prepared
standard curve of dry cell weight and total viable
cells.
Determination of chitosan
The concentration of chitosan in culture
broths was measured by the procedure described
by Kobayashi et al. (1988).
Chitosanase assay
The 1% soluble chitosan was prepared
by dissolving one gram of chitosan in 40 ml of
deionized water and 9 ml of 1.0 M acetic acid.
The solution was stirred for 2 h and the pH was
adjusted to 6.0 with 1.0 M sodium acetate. This
solution was finally made up to 100 ml by adding
0.05 M acetate buffer pH 6.0.
Chitosanase activity was analyzed by
estimating the reducing ends of chitooligosaccharides produced from the catalytic
hydrolysis of chitosan. The assay was performed
by mixing 1.0 ml of 1 % chitosan solution at pH
6.0 and 1.0 ml of suitably diluted enzyme. After
10 min incubation at 30°C, the reaction was
stopped by boiling the mixture for 3 min. A 1.0 ml
sample of the reaction mixture was taken for
determining reducing sugar by the procedure
described by Miller (1959). One unit chitosanase
activity was defined as the amount of enzyme
required to release 1 µmol of detectable reducing
sugar at 30°C in 1 min.
RESULTS AND DISCUSSION
Optimizing chitosanases by shake flask culture
Effect of pH
Bacillus cereus TP12.24 grown in the
M9-chitosan medium with varying initial pH 4.0,
5.0, 6.0, 7.0 and 8.0 at 30°C, gave the highest
enzyme activities of 336.24 U/l in 24 h, 503.31 U/
l in 30 h, 2,040.64 U/l in 54 h, 428.71 U/l in 30 h
and 567.40 U/l in 48 h, respectively. While the
maximal dry cell weights were 0.421 g/l at 24 h,
0.503 g/l at 30 h, 2.125 g/l at 54 h, 1.246 g/l at 30
h and 1.733 g/l at 30 h, respectively. Mostly, the
production of chitosanases was associated with the
bacterial growth, in which the concentrations of
Kasetsart J. (Nat. Sci.) 41(2)
enzyme and cells were maximized by using the
initial pH of 6.0. The maximal specific growth rate
obtained was 0.260 h-1 at the initial pH 6.0 (Table
1). Higher or lower initial pH’s gave less favorable
specific growth rates. At this optimal initial pH
6.0, the specific rates of chitosan consumption and
chitosanases production were 0.091 g/g h and
31.99 U/g h, respectively. As a result, the yield
and volumetric productivity of chitosanases were
247.59 U/g and 35.29 U/l h, respectively (Table
1). The optimal pH obtained in this work was quite
similar to the results reported by Yoshihara et al.
(1990) culturing Pseudomonas sp. at pH 6.3 and
Tominaka and Tsujisaka (1975) producing Bacillus
R-4 chitosanases at pH 6.0. Moreover, at higher
pH 6.5 chitosan was difficult to dissolve and could
not provide a useful carbon source for the bacterial
growth. Especially, at initial pH 7.0 and 8.0,
chitosan appeared in large particle sizes, which
was barely consumed by the bacterial cells. The
solubility of commercial chitosan being most
excellent in diluted organic acids has been also
reported (Kim et al., 2001). In particular, it is clear
that the specific rate of chitosan consumption was
enhanced 2.3-7.0 times higher at pH 4.0 than those
349
at elevated pH’s. Nevertheless, the specific growth
rate maximized at pH 6.0 dictated the production
yields of both cells and enzymes, so that the better
substrate consumption could no longer monitor the
production of enzymes.
Effect of chitosan
With 0.1 % chitosan, the highest
concentrations of cells and enzymes were 0.474
g/l and 475.70 U/l at 66 and 54 h, respectively.
The cell and enzyme concentrations were
increased to 2.125 g/l and 2,040.64 U/l at 54 h,
respectively, when using 0.5% chitosan as the main
substrate. No bacterial growth was found at 1.0
and 2.0% chitosan because high viscosity of the
culture medium limited oxygen availability for the
bacterial growth. It was also reported that high
chitosan concentration can inhibit the bacterial
growth (No et al., 2001). In this study, the chitosan
concentration of 1.0 and 2.0% could not be used
as appropriate substrate concentration for the
production of chitosanases. Therefore, 0.5%
chitosan was finally selected for the optimal
growth and chitosanases production from Bacillus
cereus TP12.24. The specific growth rate and the
Table 1 Factors affecting growth and chitosanases production by Bacillus cereus TP12.24 using shake
flask culture.
Factors
Variables
µ
YX/S
YP/S
qS
qP
QP
(h-1)
(g/g)
(U/g)
(g/g h)
(U/g h)
(U/l h)
pH
4.0
0.043
0.112
122.19
0.218
36.13
9.81
5.0
0.155
0.046
37.74
0.094
45.91
13.06
6.0
0.260
0.352
247.59
0.091
31.99
35.29
7.0
0.138
0.395
39.89
0.031
13.46
5.64
8.0
0.126
0.739
107.27
0.068
25.28
8.91
Chitosan (%)
0.1
0.111
0.221
222.15
0.253
57.39
7.59
0.5
0.260
0.352
247.59
0.091
31.99
35.29
Temperature
30
0.260
0.352
247.59
0.091
31.99
35.29
(°C)
40
0.175
0.032
168.62
1.388
285.06
23.01
50
0.208
0.025
167.59
2.428
441.38
22.14
Note: Specific growth rate (µ) obtained from plotting the graph between log dry cell weight and culture time, the yields (YX/S, YP/
S) obtained from plotting the graph of dry cell weight or enzyme activity with substrate, and the specific rates (qS, qP)
calculated at the maximal enzyme production with culture time using average dry cell weight.
Kasetsart J. (Nat. Sci.) 41(2)
Although the specific rates of substrate
consumption were much higher at elevated
temperatures (Table 1), these higher temperatures
inhibited the bacterial growth and resulted in lower
specific growth rate and the yields of cell and
enzyme production. In conclusion, the factors that
maximized the bacterial growth affected the
production of both cells and enzymes. This
revealed that chitosanases from the newly isolated
Bacillus cereus TP12.24 was the growth associated
enzymes.
volumetric enzyme productivity were 2.3 and 4.6
times higher, respectively, as compared to 0.1%
chitosan (Table 1). As discussed above, the more
chitosan consumption, shown as the higher
specific rate of chitosan consumption, did not favor
the production of cells and enzymes even at
optimal pH 6.0. Here, the limiting substrate at
0.5% chitosan which maximized the bacterial
growth played an important role instead, for the
production of chitosanases.
Effect of temperature
The maximal concentrations of dry cell
weight and chitosanases were 2.125, 0.169 0.154
g/l and 2,040.64, 1,433.09 and 1,444.13 U/l at 30,
40 and 50°C in 54 h culture, respectively. The
bacterial growth was clearly retarded at higher
temperatures of 40 and 50°C, in which the cell
concentrations decreased markedly after 18
and$12 h of culture times, respectively (data not
shown). Both specific growth rate and the
volumetric enzyme productivity decreased when
increasing the growth temperatures beyond 30°C.
Therefore, growth and enzyme production were
found optimum at 30°C, as shown in Figure 1.
The production of chitosanases in 2-l fermenter
Optimal conditions obtained from the
shake flask culture were applied for kinetic study
of the production of chitosanases in a laboratory
fermenter, using the M9-chitosan medium
containing 0.5% chitosan. The conditions were
controlled at 30°C and pH 6.0 under completely
aerobic cultivation (1 vvm aeration and 400 rpm
agitation). Bacillus cereus TP12.24 produced
highest dry cell weight at 0.904 g/l in 21 h, enzyme
activity at 1,562.12 U/l in 28.5 h (Figure 2).
However, the enzyme was harvested at 58.5 h at
the end of cultivation for studying the properties
8.0
6
pH
Dry cell weight
Total viable cells
Chitosan
Enzyme activity
6.0
5
6
4
2000
1500
1000
Chitosan (g/l)
6.5
Chitosanase activity (U/l)
pH
7.0
2500
4
3
2
4
2
2
1
500
5.5
7
6
7.5
8
Dry cell weight (g/l)
3000
0
Total viable cells x 10 (CFU/ml)
350
0
0
0
6
12
18
24
30
36
42
48
54
60
66
0
72
Time (h)
Figure 1 The production of chitosanases by Bacillus cereus TP12.24 in shake flask culture controlled
at 30°C.
Kasetsart J. (Nat. Sci.) 41(2)
of crude chitosanases. Fortunately, the enzyme
activity was found stable after its maximal at
28.5 h.
The kinetic parameters for growth and
enzyme production were summarized in Table 2.
The bacterial growth was promoted noticeably in
fermenter cultivation, resulting in rapid production
of chitosanases. As the specific growth rate
increased, the high yield of cells provided higher
cell concentration with higher specific rates of
chitosan consumption and chitosanases
production. As a result, the volumetric productivity
of chitosanases was 1.2 times increased under
aerobic conditions in fermenter. This indicated that
3.0
351
oxygen plays a very important role in promoting
the bacterial growth and the production of
chitosanases. More or less, any suitable parameters
for monitoring the supply of oxygen during
cultivation, such as DO or KLa might be a key
strategic optimization for scaling up the production
of chitosanases in a large scale fermenter.
Moreover, when compared to the shake
flask culture, the lag period of bacterial growth in
fermenter culture was reduced to 6 h from 18 h
(Figure 1 and 2). Substrate was also rapidly
consumed under aerobic condition in fermenter.
Chitosanases were produced in 18-36 and 6-20 h
in shake flask and fermenter cultures, respectively.
6
1.00
1800
5
0.80
1.5
1.0
4
0.60
Dry cell weight
Total viable cell
Chitosan
Chitosanase activity
0.40
3
Chitosan (g/l)
2.0
1200
1000
800
2
600
0.5
0.20
Chitosanase activity (U/l)
1400
Dry cell weight (g/l)
7
Total viable cells x 10 (CFU/ml)
1600
2.5
1
400
0.0
0
0.00
0
10
20
30
40
200
50
Time (h)
Figure 2 The production of chitosanases by Bacillus cereus TP12.24 in fermenter culture controlled at
1 vvm aeration, 400 rpm agitation, pH 6.0 and 30°C.
Table 2 Fermentation kinetics of Bacillus cereus TP12.24 from shake flask and fermenter cultures.
Culture
µ
YX/S
YP/S
qS
qP
QP
conditions
(h-1)
(g/g)
(U/g)
(g/g h)
(U/g h)
(U/l h)
Flask
0.260
0.352
247.59
0.091
31.99
35.29
Fermenter
0.304
0.447
181.01
0.682
154.37
43.55
Note:
(1) Flask culture referred to optimized conditions at initial pH 6.0, 0.5% chitosan and 30°C.
(2) The optimal conditions for fermenter culture were pH 6.0, 30°C, 400 rpm and 1 vvm.
(3) Calculations were done at maximal chitosanase activity obtained.
Kasetsart J. (Nat. Sci.) 41(2)
352
The enzymes were also increased at stationary
growth phase to show the non-growth associated
enzyme production. However, enzyme was quite
stable in fermenter culture. Oxygen might confirm
its important role during declining growth phase
in promoting enzyme stability. Further
investigation will be conducted on optimizing an
effect of oxygen for the production of chitosanases.
The properties of crude chitosanases
The crude chitosanases after cell
removal, prepared from the 2-l fermenter
mentioned earlier were used without any further
treatment for studying the pH and temperature
optimum and stability of enzyme.
Effect of pH
The optimal pH of crude chitosanases
was at pH 6.5 (Figure 3). At lower pH 3.0 and
higher pH 9.0, the relative enzyme activities were
47.18 and 56.64%, respectively. This optimal pH
was comparable to Bacillus cereus S1 chitosanases
(pH 6.0) (Kurakake et al., 2000) and similar to
chitosanases from Bacillus circulans MH-K1
(Yabuki et al., 1988) and Bacillus sp. No. 7-M
(Uchida and Ohtakara, 1988). This, however,
differed totally from those produced by Bacillus
subtilis IMR-NK1 (Chiang et al., 2003) and
Bacillus megaterium P1 (Pelletier and Sygusch,
1990) which were optimized at pH 4.0 and 4.56.5, respectively. As previously reported, the
optimal pH’s for various chitosanases were in a
broad range of 4.0-8.0 (Somasheka and Joseph,
1996) depending on the bacterial strains.
Bacillus cereus TP12.24 chitosanases
were found stable at a wide pH range of 3.0-8.0
retaining more than 70% activity after
preincubation at 40°C for 60 min. However, at pH
9.0 and 11.0, the relative activities were decreased
to 47.14 and 34.29%, respectively. Different
chitosanases showed different pH stability, such
as pH 6.0-11.0 for Bacillus cereus S1 chitosanases
(Kurakake et al., 2000) and pH 5.0-9.0 for Bacillus
subtilis IMR-NK1 chitosanases after preincubation
at 25°C for 1 h (Chiang et al., 2003).
Relative activity (%)
100
pH optimum
pH stability
80
60
40
20
3
4
5
6
7
8
9
pH
Figure 3 Optimal pH and pH stability of Bacillus cereus TP12.24 chitosanases.
10
11
Kasetsart J. (Nat. Sci.) 41(2)
353
Relative activity (%)
100
80
60
Temperature optimal
Temperature stability
40
20
30
40
50
60
70
80
Temperature (°C)
Figure 4 Optimal temperature and temperature stability of Bacillus cereus TP12.24 chitosanases.
Effect of temperatures
The activity of crude chitosanases from
Bacillus cereus TP12.24 was found optimal at
55°C (Figure 4). At lower or higher temperatures,
the relative activities were reduced to 86.36 and
85.86% at 30 and 70°C, respectively. This optimal
temperature was slightly lower than that of
Bacillus cereus S1 (60°C) (Kurakake et al., 2000),
but higher than those of Bacillus subtilis IMR-NK1
(45°C) (Chiang et al., 2003) and Bacillus
megaterium P1 (50°C) (Pelletier and Sygusch,
1990).
Bacillus cereus TP12.24 chitosanases
were stable at temperature of 30-50°C showing
74.26-81.19% activity. However the enzyme
activity was decreased at temperature higher than
50°C. Chitosanases from Bacillus cereus S1 were
ever reported to be stable at temperature higher
than 60°C at pH 5.0 for 30 min (Kurakake et al.,
2000). Therefore, Bacillus cereus TP12.24
chitosanases were not the thermostable enzyme.
The enzyme could be used at moderate
temperature and neutral pH under the wide pH
range of stability.
ACKNOWLEDGEMENTS
The present study was financially
supported by the National Center for Genetic
Engineering and Biotechnology (Biotec), Thailand
and partially supported by DNA Technology
Laboratory, Kasetsart University Kamphaeng Saen
Campus in association with the Commission on
Higher Education, Thailand. The laboratory at the
department of Biotechnology, Kasetsart University
Bangkhen Campus was greatly acknowledged for
providing the fermentation facilities.
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Kasetsart J. (Nat. Sci.) 41 : 356 - 362 (2007)
Application of Pectin Coating in the Production of
Vitamin Fortified Rice
Lalita Chatiyanont* and Phaisan Wuttijumnong
ABSTRACT
The quantity of vitamins in rice grain is decreased by milling, washing and cooking process.
Therefore, the production of vitamin fortified rice using edible coating was investigated. Three types of
low methoxyl pectin (36% degree of methoxyl, 31% degree of methoxyl with 21% degree of amidation
and 28% degree of methoxyl with 18% degree of amidation) and control (no pectin coating) were
studied. The results showed that L* a* b* values and moisture contents of rice premix were not
significantly different (p > 0.05). Their values were 71.67-73.00, 13.07-14.32, 78.97-80.92 and 8.018.93%, respectively. Rice premix coated with pectin at 36% degree of methoxyl showed the lowest loss
of thiamine, riboflavin and niacin during washing. However, pectin coating could not prevent the
significant loss of thiamine and riboflavin during cooking in excess water (p > 0.05). The suitable ratio
of rice premix to milled rice was 1:70. The cooked vitamin fortified rice at this ratio had 0.17 mg/100 g
of thiamine and 27.89 mg/100 g of niacin content. The results of consumer acceptance test using Central
Location Test (CLT) and Home Use Test (HUT) were similar. It was found that vitamin fortified rice
was accepted by consumers at 95% (CLT) and 98% (HUT), respectively.
Key words: low methoxyl pectin, edible coating, vitamin fortified rice
INTRODUCTION
Rice is a staple food of Thai population.
Estimated consumption in 2004 was 10.24 million
tons (Organization of Agricultural Economics,
2005). Rice is eaten in 2 forms, brown and white
rice. But the trend of eating white rice is still
upward. Causes of nutrient loss especially soluble
vitamins such as thiamine, riboflavin and niacin
are milling, washing and cooking process.
However, rice can be enriched to restore those lost
in milling, washing and cooking by using edible
coating. Peil et al. (1981) reported that rice coated
with combined hydroxypropylmethylcellulose and
methylcellulose (3:1 ratio) retained 70, 100, 18,
18 and 21% of vitamin A, iron, niacin, thiamine
and riboflavin, respectively. Shrestha et al. (2003)
reported that rice premix coated with low methoxyl
pectin retained 9% and 31% of folic acid during
washing and cooking in excess water, respectively.
The objectives of this study were to study
the effects of edible coating on qualities of rice
premix, to determine the suitable ratio of rice
premix to milled rice in order to attain desired
enrichment levels in the final product and to
determine the consumer acceptance of edible
coated vitamin fortified rice.
Department of Product Development, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
* Corresponding author, e-mail: nan_foodtu@hotmail.com
Received date : 25/09/06
Accepted date : 22/01/07
Kasetsart J. (Nat. Sci.) 41(2)
MATERIALS AND METHODS
1. Materials
Milled rice (Khao Dauk Mali 105) was
purchased from Tesco Lotus. Purple Ribbon Pure
pectin (from yellow apple and citrus peel, Degree
of methoxyl 36% and pectin content 85-100%) was
obtained from Nutrition Partnership Limited. 7210
pectin (from citrus peel, 28% Degree of methoxyl
with 21% degree of amidation and 63% pectin
content) and 7220 pectin (from citrus peel, 31%
Degree of methoxyl with 18% degree of amidation
and 63% pectin content) were obtained from The
East Asiatic (Thailand) Public Company Limited.
Thiamine hydrochloride, riboflavin and
niacinamide were obtained from DSM Nutritional
Product Co.,Ltd.
2. Effects of edible coating on qualities of rice
premix
2.1 Preparation of mixed vitamin
solution
Mixed vitamin solution was prepared by
dissolving 95 mg of the thiamine hydrochloride,
52.6 mg of the riboflavin and 559 mg of the
niacinamide in 8 ml of the distilled water.
2.2 Preparation of low methoxyl pectin
solutions
Pectin solutions were prepared followed
Rolin et al. (1998) by dissolving 1% of Purple
Ribbon Pure pectin, 2% of 7220 and 7210 pectins
in hot water (60-80°C) in a high-speed mixer. The
viscosity of these solutions was 31-36 cP.
2.3 Preparation of rice premix
Rice grain (100 g) was coated with mixed
vitamin solution and then with pectin solution
followed by calcium chloride solution in a tablet
coating pan (model SPKR, MITSUBISHI). The
coating of pectin solution followed by calcium
chloride solution was repeated and finally, the
coated rice was dried in a dryer at 50 degree celcius
for 2 hours.
2.4 Washing and cooking of rice premix
357
Washing test was carried out in a 250 ml
erlenmeyer flask by rinsing 20 g rice premix with
60 ml distilled water and gently swirling for
exactly 60 s. In cooking test, 5 g of rice was cooked
in 125 ml erlenmeyer flask with 100 ml distilled
water for 30 min in a water bath (97 ± 3 °C) and
cooled immediately. (Shrestha et al., 2003).
2.5 Quality measurements
2.5.1 Structure images analysis
Unwashed, washed and cooked rice
premixes were viewed on a Laser scanning
confocal microscope (model AXIO, ZEISS Laser
LSM 5 PASCAL). He/Ne laser at 488 nm was used
as a light source to excite the riboflavin (Gue et
al.,1999). The images acquired with a 5x, 0.15NA.,
dry objective and 512 × 512 pixel resolution. They
were individual placed in a glass slide without
further preparation.
2.5.2 Color measurement
The color characteristics (L* a* and b*
values) of rice premixes were quantitatively
measured using spectrophotometer (model CM3500d , MINOLTA). L*, a* and b* values indicate
lightness, red to green and yellow to blue,
respectively.
2.5.3 Moisture contents of rice premixes
were analyzed by using hot air oven (model
FD115, WTB binder) at 105 ± 1°C until the weight
was constant (A.O.A.C., 2000).
2.5.4 Determination of vitamin loss after
washing and cooking
Unwashed, washed and cooked rice
premixes were analyzed for thiamine, riboflavin
and niacin. Thiamine and riboflavin contents were
determined by fluorometric method (A.O.A.C.,
2000). Niacin contents was determined by the
Food Quality Assurance Service Center, Kasetsart
University.
3. The suitable ratio of rice premix to milled
rice to attain desired enrichment level in the
final product
3.1 Preparation of fortified rice
358
Kasetsart J. (Nat. Sci.) 41(2)
The rice premix coated with the low
methoxyl pectin was obtained from part 2
according to the highest vitamins retained after
washing and cooking. The rice premix was blended
with milled rice with different ratios (1:100, 1:85
and 1:70) in a cubic mixing tank for 10 min. The
milled rice was used as control sample.
3.2 Washing and cooking of fortified
rice
Milled and fortified rice were washed the
same way as in 2.4 using rice to water ratio 1 : 3.
Cooking was done in automatic rice cooker (model
SR-D10HN, Panasonic) using rice to water ratio
of 1:1.25.
3.3 Quality measurements
3.3.1 Color measurement
The color (L*, a* and b*) values of
cooked fortified rice were measured by
spectrophotometer.
3.3.2 Determination of vitamin contents
Unwashed, washed and cooked rice were
analyzed for thiamine, riboflavin and niacin.
Thiamine and riboflavin contents were determined
by fluorometric method (A.O.A.C., 2000). Niacin
contents was determined by the Food Quality
Assurance Service Center, Kasetsart University.
3.3.3 Sensory evaluation
The likina scores of cooked rice were
evaluated by 50 untrained panelists using 9-points
hedonic scale (1 = dislike extremely to 9 = like
extremely) for appearance, color, odor, flavor and
overall liking.
4. Consumer acceptance test
Consumer acceptance test was carried
out using Central Location test (CLT) and Home
Use Test (HUT). 100 consumers were used in CLT
at two locations (Kasetsart University cafeterias
1 and 2). The samples (before and after cooking
fortified rice) and questionnaires were provided
for the consumers. For HUT, 100 consumers were
provided with samples (fortified rice 142 g for 1
meal) and questionnaires. The 9-points hedonic
scale was used to score the consumers’ liking. The
acceptability of fortified rice was also evaluated
by consumers.
RESULTS AND DISCUSSION
1. Effect of edible coating on qualities of rice
premix
1.1 Physical properties
The appearances of unwashed, washed
and cooked rice premix coated with pectin viewed
by confocal laser scanning microscopy were
shown in Figure 1. It was found that there were
cracks in washed rice and the kernel shape seems
to be lost in cooked rice. This may cause a heavy
losses of vitamins in rice premix. The color
characteristics (L* a* and b* values) of rice premix
Figure 1 Rice premix as viewed in the CLSM at 5X (a) before cooking (b) after washing (c) after
cooking in excess water and draining.
Kasetsart J. (Nat. Sci.) 41(2)
without coating and coated with pectins were not
significantly different (p>0.05). Their values were
between 71.67-73.00, 13.07-14.32 and 78.9780.92 for L*, a* and b* values, respectively. The
rice premix has yellow color (high b* value) due
to addition of riboflavin.
1.2 Chemical properties
Moisture contents of all rice premix
samples were not significantly different (p>0.05).
359
Their values were between 8.01-8.93%. Table 1-3
showed the loss of thiamine riboflavin and niacin
in washed and cooked rice premix without coating
and coated with pectins in excess water and
draining. The rice premix coated with pectins
showed lower vitamin losses after washing than
those without coating. The higher degree of
methoxyl pectin showed the lower vitamin losses
in washed rice premix than lower degree of
Table 1 Thiamine contents in rice premix, washed and cooked rice in excess water and draining,
washing and cooking losses.
Rice premix
Thiamine contents (mg/100 g)
Washing loss
Cooking
(%)
loss (%)
1.68 ± 0.20
79.03 ± 1.14 a
92.84 ± 0.88
11.41 ± 0.02
1.37 ± 0.32
55.86 ± 0.29 c
94.69 ± 1.28
8.19 ± 0.93
1.46 ± 0.05
56.90 ± 5.23 c
92.15 ± 2.09
7.57 ± 0.15
0.84 ± 0.00
68.78 ± 0.99 b
96.67 ± 0.23
Rice premix
Washed rice
premix
premix
23.47 ± 0.13
4.92 ± 0.24
25.84 ± 0.12
with 18 % degree of amidation 19.28 ± 4.50
- No pectin coating
Cooked rice
(ns)
- Coated with pectin
36 % degree of methoxyl
- Coated with pectin
31 % degree of methoxyl
- Coated with pectin
28 % degree of methoxyl
with 21% degree of amidation
24.25 ± 0.27
Note: alphabets a-c were different within column mean values were significantly different (p≤0.05)
ns means values within column were not significantly different (p>0.05)
Table 2 Riboflavin contents in rice premix, washed and cooked rice in excess water and draining,
washing and cooking losses.
Rice premix
Riboflavin contents (mg/100 g)
Rice premix
Washing loss
Cooking
(%)
loss (%)
Washed rice
Cooked rice
premix
premix
41.98 ± 2.76
8.19 ± 1.41
2.82 ± 0.20
80.54 ± 2.07 a
93.28 ± 0.50
38.68 ± 3.46
12.52 ± 1.46
3.01 ± 0.25
67.66 ± 0.90 c
92.17 ± 1.34
with 18 % degree of amidation 45.48 ± 0.30
12.34 ± 0.33
3.32 ± 0.01
72.86 ± 0.55 b
92.69 ± 0.08
10.91 ± 0.50
2.90 ± 0.02
71.28 ± 2.60 bc
92.53 ± 0.29
- No pectin coating
(ns)
- Coated with pectin
36 % degree of methoxyl
- Coated with pectin
31 % degree of methoxyl
- Coated with pectin
28 %degree of methoxyl
with 21 % degree of amidation 38.81 ± 1.79
Note: alphabets a-c were different within column mean values were significantly different (p≤0.05)
ns means values within column were not significantly different (p>0.05)
Kasetsart J. (Nat. Sci.) 41(2)
360
methoxyl pectin. This may be due to the fact that
the presence of calcium ion was not enough to
strengthen gel (Thakur et al., 1997).
However, pectin coatings were not good
enough to prevent leaching of these vitamins from
rice premix when boiled in excess water. The
preparation process of rice premix consisted of
many steps of coating and drying which had an
effect on rice cracking. During boiling, water can
easily access into the interior of the cracked grain,
this causes increasing of hydration and
subsequently leaching of vitamin into the cooking
water. (Shrestha et al., 2003)
Since, the rice premix coated with pectin
(36% degree of methoxyl) had the lowest vitamin
loss during washing and cooking, it was selected
for the next experiment.
2. The suitable ratio of rice premix to milled
rice to attain desired enrichment level in the
final product
2.1 Physical and chemical properties
Cooked fortified rice had light yellow in
color, due to the leaching out of vitamins from
surface of rice premix during washing and
cooking. The a* and b* values increased with
increasing rice premix to milled rice ratios (Table
4).
The amount of vitamins (thiamine,
riboflavin and niacin) in unwashed, washed and
cooked milled rice and fortified rice were shown
Table 3 Niacin contents in rice premix, washed and cooked rice in excess water and draining, washing
and cooking losses.
Rice premix
Niacin contents (mg/100 g)
Rice premix
Washing loss
Cooking
(%)
loss (%)
Washed rice
Cooked rice
premix
premix
429.28 ± 11.28
92.46 ± 14.67
22.87 ± 0.83
78.41 ± 3.99 a
94.67 ± 0.06
473.71 ± 0.68
211.14 ± 2.82
25.87 ± 0.37
55.43 ± 0.54 b
94.54 ± 0.08
with 18 % degree of amidation 405.07 ± 23.81 182.41 ± 16.54
22.53 ± 0.07
54.77 ± 6.74 b
94.43 ± 0.35
24.88 ± 0.25
62.08 ± 3.03 b
94.79 ± 0.28
- No pectin coating
(ns)
- Coated with pectin
36 % degree of methoxyl
- Coated with pectin
31 % degree of methoxyl
- Coated with pectin
28 % degree of methoxyl
with 21 % degree of amidation 478.86 ± 30.57
181.14 ± 2.93
Note: value in the same column with different superscripts differ significantly (p≤0.05)
ns means values within column were not significantly different (p>0.05)
Table 4 L* a* and b* values of cooked vitamin fortified rice.
Rice premix to milled rice
L*
Milled rice
77.49 ± 0.10 a
Fortified rice
(rice premix to milled rice)
1 : 100
77.46 ± 0.12 a
1 : 85
77.47 ± 0.10 a
1 : 70
76.64 ± 0.02 b
a*
-2.11 ± 0.06 b
b*
8.94 ± 0.22 d
-2.59 ± 0.06 a
-2.63 ± 0.06 a
-2.57 ± 0.07 a
9.93 ± 0.17 c
11.43 ± 0.07 b
11.66 ± 0.19 a
Note: value in the same column with different superscripts differ significantly (p≤0.05)
Kasetsart J. (Nat. Sci.) 41(2)
in Table 5-7. It was found that the amount of
vitamins in unwashed, washed and cooked rice
increased with increasing ratios of rice premix to
milled rice). The ratio of rice premix to milled rice
at 1:70 met the requirement for thiamine and niacin
fortification of rice, according to Thai Reference
Daily Intake (Thai RDI) in which the cooked
fortified rice should have thiamine and niacin
contents more than 10% of cooked milled rice.
But the riboflavin content was less than 10% of
cooked milled rice.
361
2.2 Sensory evaluation
The liking score for each attribute of
cooked milled rice and fortified rice were not
significantly different (p>0.05) (Table 8). This
indicates that the fortification of rice with vitamins
by mixing rice premix with milled rice had no
effects on the panelists preference.
Therefore, the ratio of rice premix to
milled rice at 1:70 was selected for study on
consumer acceptance.
Table 5 Amount of thiamine in unwashed, washed and cooked rice and fortified rice.
Rice premix to milled rice
Amount of thiamine (mg/100 g)
Unwashed
Washed rice
Cooked rice
Milled rice
0.05 ± 0.01 c
0.04 ± 0.01 c
0.01 ± 0.03 c
Fortified rice
(rice premix to milled rice)
1 : 100
0.28 ± 0.01 b
0.17 ± 0.00 b
0.10 ± 0.00 b
1 : 85
0.30 ± 0.01 b
0.19 ± 0.01 b
0.12 ± 0.01 b
1 : 70
0.39 ± 0.21 a
0.25 ± 0.03 a
0.17 ± 0.01 a
Note: value in the same column with different superscripts differ significantly (p≤0.05)
Table 6 Amount of riboflavin in unwashed, washed and cooked rice and fortified rice.
Rice premix to milled rice
Amount of riboflavin (mg/100 g)
Unwashed
Washed rice
Cooked rice
Milled rice
0.04 ± 0.00 b
0.03 ± 0.00 c
0.01 ± 0.00 b
Fortified rice
(rice premix to milled rice)
1 : 100
0.43 ± 0.04 a
0.12 ± 0.01 b
0.04 ± 0.01 a
1 : 85
0.52 ± 0.04 a
0.18 ± 0.02 a
0.05 ± 0.01 a
1 : 70
0.55 ± 0.01 a
0.20 ± 0.00 a
0.05 ± 0.00 a
Note: value in the same column with different superscripts differ significantly (p≤0.05)
Table 7 Amount of niacin in unwashed, washed and cooked rice and fortified rice.
Rice premix to milled rice
Amount of niacin (mg/100 g)
Unwashed
Washed rice (ns)
Cooked rice
Milled rice
27.86 ± 3.72 b
26.17 ± 1.39
14.70 ± 1.50 b
Fortified rice
(rice premix to milled rice)
1 : 100
49.25 ± 0.12 a
30.10 ± 0.80
12.09 ± 4.23 b
1 : 85
49.77 ± 0.52 a
28.83 ± 0.21
11.34 ± 4.57 b
1 : 70
48.33 ± 0.97 a
32.12 ± 8.07
27.89 ± 0.44 a
Note: value in the same column with different superscripts differ significantly (p≤0.05)
ns means values within column were not significantly different (p>0.05).
Kasetsart J. (Nat. Sci.) 41(2)
362
Table 8 Liking scores of cooked rice and fortified rice.
Attributes
Rice premix to milled rice
Milled rice
1 : 100
1 : 85
Appearance (ns)
6.7 ± 1.4
6.7 ± 1.3
6.4 ± 1.4
Color (ns)
7.0 ± 1.3
6.7 ± 1.4
6.5 ± 1.3
Odor (ns)
5.9 ± 1.1
6.1 ± 1.5
6.1 ± 1.5
Flavor (ns)
6.1 ± 1.2
6.3 ± 1.2
6.2 ± 1.3
Texture (ns)
6.1 ± 1.5
6.0 ± 1.4
5.9 ± 1.4
Overall liking (ns)
6.3 ± 1.5
6.3 ± 1.4
6.1 ± 1.3
1 : 70
6.7 ± 1.3
6.6 ± 1.1
6.2 ± 1.7
6.5 ± 1.2
6.4 ± 1.4
6.6 ± 1.3
Notes: ns means values within row were not significantly different (p>0.05).
3. Consumer acceptance test
The results showed that the vitamin
fortified rice was significantly accepted by the
consumers at 95% (Central Location Test) and
98% (Home Use Test). The overall liking scores
of the vitamin fortified rice before and after
cooking were 6.9 and 7.5 (CLT) ; 7.4 and 7.8
(HUT), respectively.
CONCLUSION
Rice premix coated with pectin (36%
degree of methoxyl) had a minimal loss of vitamins
during washing. But pectin coating could not
prevent vitamins from cooking loss. The vitamins
fortified rice at ratio of 1:70 was suitable. It was
significantly accepted by consumers at 95% and
98% with overall liking scores 7.5 and 7.8 for
Central Location Test and Home Use Test,
respectively.
For further study, we recommend to
focus on the protein-based films because they are
better in mechanical and barrier properties than
polysaccharide based films. Therefore it might
protect vitamin loss in washing and cooking
process.
ACKNOWLEDGEMENTS
This research was kindly supported by
The Thailand Research Fund (TRF) in major
Science and Technology, 2005.
LITERATURE CITED
A.O.A.C. 2000. Official Method of Analysis. 17th
ed., The Association of Official Analytical
Chemists Gaithersburg, Maryland.
Guo, H.X., J. Heinämäki and J. Yliruusi. 1999.
Characterization of particle deformation
during compression measured by confocal
laser scanning microscopy. International
Journal of Pharmaceutics 186: 99-108.
Organization of Agricultural Economics Ministry
of Agriculture and Cooperative. 2005. Data
Base of Economic. Organization of
Agricultural Economics Ministry of
Agriculture and Cooperative, Bangkok.
Peil, A., F. Barrett, C. Rha and R. Langer. 1981.
Retention of micronutrients by polymer
coatings used to fortify rice. J. Food Sci. 47:
260-262, 266.
Rolin, C., B.U. Niclsen and P.E. Glahn. 1998.
Pectin, pp. 377-431. In S. Dumitriu (ed.).
Polysaccharide, structural diversity and
functional versatility, Marcel Dekker, Inc.,
New York.
Shrestha, A.K., J. Arcot and J.L. Paterson. 2003.
Edible coating materials-their properties and
use in the fortification of rice with folic acid.
Food Reseach International 36: 921-928.
Thakur, B.R., R.K. Singh and A.K. Handa. 1997.
Chemistry and Uses of Pectin-A Review.
Critical Reviews in Food Science and
Nutrition 37: 47-73.
Kasetsart J. (Nat. Sci.) 41 : 363 - 372 (2007)
The Effects of Starter Cultures on Biogenic Amine and
Free Amino Acid Contents in Nham during Fermentation
Sasithorn Limsuwan1*, Wonnop Visessanguan2 and Jirasak Kongkiattikajorn1
ABSTRACT
Fermented pork sausage, or Nham, is a Thai-style fermented food which relies mainly on
adventitious microorganisms, normally found in raw materials. The fermented foods usually contain
biogenic amines produced by the microorganisms which caused the reaction of amino acids
decarboxylation. These compounds are associated with toxicological symptoms. The objective of this
study was to study the influence of two decarboxylase negative starter cultures in the presence of biogenic
amines and free amino acid contents in Nham. Derivative biogenic amines by dansyl chloride were
determined by high performance liquid chromatography (HPLC). Cadaverine and tyramine were
determined during ripening process. The highest concentrations of biogenic amines were found at the
end of the ripening process in the control sausage with no starter culture. Starter cultures test showed
that Lactobacillus sakei BCC102 and Debaryomyces hansenii BCC 106 were efficient in reducing the
amine production since these strains caused a quick pH drop during sausage fermentation. Total free
amino acids after fermentation process decreased and the high decreases in the contents were glutamine
and arginine while tyrosine and lysine, precursors for tyramine and cadaverine, respectively, increased
in all batches. This study suggested that the use of decarboxylase negative lactic acid bacteria as starter
cultures, which produced a rapid decrease on the pH of the meat mixture, was important factor to be
considered to reduce the levels of biogenic amines in Nham.
Key words: starter culture, Nham, fermentation, biogenic amine, amino acid
INTRODUCTION
Biogenic amines (BAs) are naturally
present in many foods and relatively high contents
of some BAs can be present in fermented foods.
BAs are organic molecules with low molecular
weight. These compounds are usually generated
by microbial decarboxylation of amino acids
present in foods. The aromatic BAs (histamine,
tyramine, serotonin, β-phenylethylamine,
1
2
*
tryptamine) have been reported as vasoactive or
psychoactive amines and they have been
associated with food histaminic intoxications,
food-induced migraines, and severe hypertensive
crisis due to monoamine oxidase inhibitor (MAOI)
drug interactions. Moreover, diamines such as
putrescine and cadaverine could generate
carcinogenic nitrosamines in the presence of
nitrites (Scanlan, 1983). Interest in cadaverine,
putrescine, tyramine and histamine also lies in their
School of Bioresources and Technology, King Mongkut’s University of Technology Thonburi, Bangkok 10140, Thailand.
National Center for Genetic Engineering and Biotechnology, Phathum Thani 12120, Thailand.
Corresponding author, e-mail: jirasak.kon@kmutt.ac.th
Received date : 08/12/05
Accepted date : 22/01/07
364
Kasetsart J. (Nat. Sci.) 41(2)
potential as spoilage indicators of food. In addition,
they may have unpleasant odours and it was also
found that putrescine and cadaverine could inhibit
the activity of muscle aminopeptidases (Flores et
al., 1996).
Nham is a Thai traditional fermented
pork sausage. Nham fermentation generally takes
3-5 days. The microorganisms involved in the
fermentation process can yield much higher BA
amounts than those found in the corresponding raw
materials, because some BAs are the result of
amino acid decarboxylation by microbial enzymes.
BAs may represent a food-poisoning hazard in
conjunction with additional promoting factors such
as MAOI antidepressant drugs, alcohol, other food
amines, gastrointestinal diseases and genetically
deficient detoxification systems (Vidal-Carou et
al., 1990).
Meat fermentation offers favourable
conditions for BA formation, since the main
required factors are present, i.e. there is growth of
microorganisms over several days, a certain degree
of proteolysis takes place giving rise to the
presence of free amino acids as precursors of BA
and, finally, the existence of an acidic environment
can favour the amino acid decarboxylase activity
of microorganisms. It has been reported that
bacteria could be encouraged to produce
decarboxylase enzymes in such acidic
environments as part of their defense mechanisms
against adverse conditions (Eitenmiller et al.,
1978). Since microbial flora naturally present in
the raw materials seem to have a strong influence
on BA formation during ripening, the choice of
good quality raw materials helps to minimize the
number of amine-producing bacteria (Halasz et al.,
1994). An important factor suggested for
preventing amine accumulation is the addition of
adequate starter cultures to complete the
fermentation. Starter cultures usually consist of
one or several strains such as lactic acid bacteria
(LAB). LAB are being widely used as starter
cultures in fermented sausages. The absence of BA
formation of LAB was proposed as a selection
criterion for starter cultures (Buckenhuskes, 1993).
Proteolysis during the fermentation of
meat products is favoured by the denaturation of
proteins as a consequence of the acidity,
dehydration and the action of sodium chloride
(DeKetelaere et al., 1974). During the
fermentation, production of free amino acids from
proteolysis might occur. Therefore, the
determination of free amino acid contents can be
useful in studying the potential relationship
between proteolysis and BA formation in
fermented sausages.
The objectives of the present study were:
(1) to study the changes in BA levels during the
fermentation processes (2) to examine the effect
on BA formation of L. sakei BCC 102 and D.
hansenii BCC 106 which are nondecarboxylase
activity used as starter cultures added naturally
fermented Nham and (3) to determine the effects
of starter culture on the formation of free amino
acid during the fermentation of Nham (4) to
compare the formation of BAs in control Nham
(naturally fermented) and in Nham fermented with
starter microorganisms.
MATERIALS AND METHODS
Microorganisms
Two starter culture strains (Lactobacillus
sakei BCC 102 and Debaryomyces hansenii BCC
106) isolated from Nham were chosen after
screening for nondecarboxylase activities. Both
strains were gift from the Culture Collection
Laboratory, National Center for Genetic
Engineering and Biotechnology (BIOTEC),
Patumthani, Thailand. Cultures were stored at 80°C in 20% glycerol. One loopful of a stock
culture was cross-streaked on Man, Rogosa and
Sharpe (MRS) agar and then incubated at 30°C
for 48 h. A single colony on MRS agar was grown
in MRS broth at 30°C for 18-24 h. Cell-free
supernatants were obtained after centrifugation of
Kasetsart J. (Nat. Sci.) 41(2)
the cultures at 10,200 × g for 15 min at 4°C. The
starter culture was prepared to obtain an
approximate cell concentration of 107 CFU/ml in
sterile deionized water.
Preparation of Nham
Nham was prepared to make a total 100
g Nham by mixing 52 g minced pork, 35g cooked
pork rind, 1.9 g curing salt, 0.2 g sodium
erythrobate, 0.2g sodium tripolyphosphate, 4.3 g
minced garlic, 4.3 g minced cooked rice, 2 g chilli,
0.4 g sucrose, 0.2 g monosodium glutamate, 0.01
g potassium nitrite and 0.6 g sodium chloride. The
ingredients were thoroughly mixed and divided
into three fractions for three different batches, i.e.
control or naturally fermented without the addition
of starter cultures (NC) and batches I and II
processed through a starter-mediated fermentation,
with a single starter culture of 104 CFU/g L. sakei
BCC 102 (LS batch) and mixed starter culture
consisting 104 CFU/g L. sakei BCC 102 combined
with 106 CFU/g D. hansenii BCC 106 (LSY
batch), respectively. Approximately 200 g of Nham
was stuffed into a plastic casing 3 cm diameter
and sealed tightly prior to incubation at 30°C for
120 h. Samples were taken every 24 h for chemical
and microbiological analysis.
365
prepared. LAB were enumerated on MRS agar
adding with 0.5 % calcium carbonate and
incubated anaerobically at 30°C for 48 h.
Biogenic amine determination
HPLC determinations were performed
with a LC 10 AD Shimadzu LC using a 20 µl loop.
Detection was at 254 nm with UV detector. LC
column C18-Hypersil BDS (200 mm.× 4.6 mm, 5
µm particle size) was used. Amine standard
solutions were prepared in water to a final
concentration of 5 mg/ml for each biogenic amine.
Tyramine, putrecine, cadaverine, tryptamine,
phenylethylamine and histamine were used.
Biogenic amine concentrations in the working
standard solutions chosen for the calibration curve
were 0.005, 0.01, 0.05, 0.1, 0.5 and 1 mg/mL.
These working solutions were made by further
dilution of the stock solution with water. Internal
standard solution was prepared by diluting 15 mg
of 1, 7-diaminoheptane in 5 ml of water. The
gradient-elution system was methanol as solvent
A and water as solvent B. The system was
equilibrated for 5 min before the next analysis.
The flow rate was 1.5 ml/min.
pH Measurement
pH of Nham samples of 0-5 days of
fermentation were measured directly using a
microcomputerized pH meter (Mettler Teledo 320,
UK; Mettler Toledo, Inlab 427) by inserting the
electrode into the centre of Nham and recorded as
the mean value of three measurements.
Sample preparation and extraction
Four grams of sample was mixed with
10 ml of 5% trichloroacetic acid and extracted
using homogenizer. The homogenate was
centrifuged at 17,212 × g for 10 min at 4°C, the
supernatant was collected and the precipitate was
extracted again with 10 ml of 5% trichloroacetic
acid. After centrifugation, the supernatant was kept
at -20°C.
Microbiological analysis
For microbial analysis, 25 g of Nham was
aseptically removed from the casing, cut into small
pieces, placed in sterile Stomacher bag and
homogenized using a Stomacher (IUL Instrument,
Spain) with 225 ml of sterile diluent containing
0.1% peptone. Serial decimal dilutions were
Derivatization of sample extracts and mixed
standards
A 100 µl of 2 N NaOH and 150 of µl
saturated NaHCO3 were added to 0.5 ml of the
extract, mixed with 1 ml of dansyl chloride (10
mg/ml in acetone) and incubated at 40°C in a water
bath for 45 min. To remove residual dansyl
Kasetsart J. (Nat. Sci.) 41(2)
366
distilled water and filtered through 0.45 µm
(Minisart RC4, Sartorious), then 10 µl aliquot of
filtrate was transferred into a vial, and 70 µl of
Waters AccQ Fluor borate buffer was added. A
20 µl of AccQ Fluor reagent was added and the
mixture was incubated at 55°C for 10 min before
HPLC analysis.
chloride, 50 µl of 100% ammonia was added and
the solution was centrifuged at 500 × g for 30 min
and the supernatant was filtered through a 0.45
mm filter. Dansyl derivatives of the calibration
standards were mixed with the samples as
previously described (Eerola et al., 1994).
Free amino acid (FAA)determination
Free amino acids were determined
according to the method of Benjakul and
Morrissey (1997) using an amino acid analyzer
(Waters 2690 Alliance with 280 nm Fluorescent
detector). The column was an AccQ-TagTM C18,
4 µm. The solvent system consisted of three
eluents: (A) AccQ Tag Eluent pH 5.02; (B) HPLCgrade acetonitrile and (C) Nanopure distilled water.
The flow rate was set at 1.0 ml/min. Five g of
Nham blend was mixed with 20 ml of 5%
trichloroacetic acid, then stomachered at 200
rpm/min for 8 min and centrifuged at 12,000 × g
for 15 min. A 100 µl of supernatant was mixed
with 20 µl of 2.5 mM ABAA and 800 ml nanopure
Statistical analyses
The differences between the results of
physical, chemical and microbiological analysis
of Nham fermented by different strains were tested
using one-way analysis of variance (ANOVA).
RESULTS AND DISCUSSION
pH determination
Changes of pH in Nham during ripening
are shown in Figure 1. The pH of the control
sample and the batch with L. sakei and D. hansenii
decreased after 4 h of incubation while that of
batch with L. sakei decreased after 8 h of
6.5
6
5.5
pH
5
4.5
4
3.5
3
0
20
40
60
80
100
120
140
Time (h)
NC
LS
LSY
Figure 1 Changes in pH values during the ripening of Nham from different batches. Nham without
added cultures (NC), Nham fermented with L. sakei BCC102 (LS), L. sakei BCC102 and D.
hansenii BCC106 (LSY), for 120 h at 30°C (each data point represents the mean and standard
deviation of three independent trials).
Kasetsart J. (Nat. Sci.) 41(2)
367
(LAB) counts in the starter added samples were
higher than in the control samples. LAB counts
increased during fermentation for both control and
starter added samples. Initial counts of LAB (7.08
± 0.24 log, 8.04 ± 0.32 log and 8.11 ± 0.21 log
CFU/g for control, L. sakei added samples and
mixed cultures of L. sakei and D. hansenii added
samples, respectively) increased during
fermenting, till the microorganism being at 9.48 ±
0.22 log, 9.45 ± 0.15 log and 9.66 ± 0.19 log CFU/
g in the control, L. sakei added samples and mixed
culture of L. sakei and D. hansenii added samples,
respectively. This was an increase due to the
environmental conditions which made gramnegative bacterial grow. After 16 h of fermentation,
no significant differences were observed in total
LAB counts in all batches. LAB increased during
the ripening process, reaching maximum levels at
day five in all types of sausages.
Changes in microbial counts in Nham
inoculated with single starter culture of L. sakei
and mixed starter cultures of L. sakei and D.
incubation and reached the final pH values of 4.5,
4.2, and 4.2 for the control, batch with L. sakei
and batch with L. sakei and D. hansenii,
respectively. The initial pH of all batches of Nham
was pH 6.1, thereafter it decreased rapidly in the
batch with starter cultures to pH 4.7, at 28 h. The
pH of the control Nham was decreased to 4.9 at
28 h of fermentation. After 28 h, the pH in all
batches gradually decreased throughout of
incubation and pH values of the control slightly
decreased less than that of the batches with starter
cultures. The pH reduction during processing
probably due to organic acid production by the
inoculated starter cultures as well as the lactic flora.
Statistical analysis of pH values recorded
throughout ripening revealed significant
differences between treatments at 24 h.
Microbiological analyses
Microbial counts increased in both the
controls and starter added samples during
fermentation (Figure 2). Initial lactic acid bacteria
10
Log total LAB (CFU/g)
8
6
4
2
0
0
20
40
60
80
100
120
140
Time (h)
NC
LS
LS Y
Figure 2 Changes in the population levels of LAB in Nham without added cultures (NC), Nham
fermented with L. sakei BCC102 (LS), L. sakei BCC102 and D. hansenii BCC106 (LSY), for
120 h at 30°C (each data point represents the mean and standard deviation of three independent
trials).
368
Kasetsart J. (Nat. Sci.) 41(2)
hansenii were similar to that of naturally fermented
Nham (Figure 1). This microbial group rapidly
increased after casing and reached the values of
about 109 CFU/g in all the sausages, even in the
samples to which starter cultures were not added.
These high values remained relatively constant
during ripening. Due to relatively high microbial
load in Nham raw mix(107 CFU/g), inoculation
of starter cultures at levels of 104 and 106 CFU/g
had no significant effects on the LAB counts
during fermentation. Similar to the results of
Khieokhachee et al. (1997), initial flora of the
Nham derived mainly from the raw materials. The
number of LAB increased drastically to a
maximum of 109 CFU/g within 18 h and remained
the same until the fermentation was completed at
24 h for all batches . Thus, fermentation of Nham
involving successive growth of different groups
of microorganisms was dominated by LAB.
Various metabolic products of LAB, such as shortchain organic acids, carbon dioxide, hydrogen
peroxide, diacetyl, and bacteriocin, are known as
antimicrobial agents (Rowan et al., 1998).
Accumulation of organic acids also resulted in a
decrease of pH. Thus, the dominance of LAB is
likely to contribute to the inhibition of other
microorganisms.
The addition of L. sakei and D. hansenii
as starters might prevent the development of flora
LAB that were present naturally in the initial
mixture, and L. sakei might be able to dominate
the whole ripening period in the batches while the
control predominated with LAB flora.
Biogenic amines determination
Tyramine and cadaverine were present
exclusively in 24 h fermented samples in typical
quantitative sequence: cadaverine content was
more than tyramine content (Table 1), other amines
were not detectable. Both biogenic amines
increased after 24 h of ripening until the final
ripening of 120 h.
The differences between Nham
elaborated with and without starter culture were
observed, and the control Nham had significantly
higher values of cadaverine and tyramine than the
Nham inoculated with starter cultures. The L. sakei
had significantly lower concentrations of biogenic
amines than L. sakei and D. hansenii. Cadaverine
and tyramine in fermented sausages were produced
Table 1 Biogenic amine contents (mg/kg) in Nham.
Time (h)
Control
L. sakei
CAD
TYR
CAD
TYR
0
ND
ND
ND
ND
24
135.47
74.28
174.54
57.28
± 14.85aA
± 5.71aB
± 18.22aC
± 7.96aD
48
197.32
95.72
204.69
68.46
± 18.92bA
± 8.42bB
± 23.85bA
± 4.85bC
72
216.41
97.61
225.87
75.37
± 21.56cA
± 7.48bB
± 19.51cC
± 6.29bcD
96
218.82
102.32
218.34
77.16
±12.85bB
± 25.18cA
± 7.28cC
± 24.63cA
120
235.95
138.59
224.74
82.23
± 17.44dA
± 14.74cB
± 21.82cC
± 5.45dD
L. sakei and D. hansenii
CAD
TYR
ND
ND
197.41
67.91
± 16.34aF
± 7.14aG
201.36
94.85
± 14.89aA
± 5.54bB
223.52
96.47
± 26.25bAC
± 8.20bB
227.28
104.62
± 21.94bA
± 7.87bcB
232.86
107.58
± 25.17cA
± 6.41cE
Mean values and standard deviations with different letters (a, b, c) in the same column indicate significant differences (P<0.05)
during fermentation, and different letters (A, B, C) in the same row indicate significant differences (P<0.05) among Nham formula.
(ND = Not detectable, CAD = cadaverine and TYR = tyramine)
Kasetsart J. (Nat. Sci.) 41(2)
by lysine- and tyrosine-decarboxylase activities,
of Enterobacteriaceae, respectively. So, L. sakei
and D. hansenii in Nham might inhibit the growth
of Enterobacteriaceae resulting in decrease the
lysine- and tyrosine-decarboxylase activity and
biogenic formation (Bover-Cid et al., 2001b).
The main factors seemed to be a suitable
starter culture and good quality raw materials
(Bover-Cid et al., 2001a). However, in the present
study, the high quality raw materials used were
not effective in preventing the production of
cadaverine and tyramine in control Nham (Table
1), and low contents of these amines were obtained
only when a starter culture was included in sausage
formulation.
In conclusion, to avoid the presence of
high concentrations of biogenic amines in Nham,
it was advisable to use a competitive starter culture
such as L. sakei, a negative-decarboxylase strain,
which might be predominant throughout the
process, thus it would prevent the growth of
bacteria which could produce biogenic amines.
Low occurrence of biogenic amines in
raw pork meat: usually tyramine did not exceed a
few mg kg-1 (tyramine less than 3.5 mg kg-1 as
observed by Hernandez-Jover et al., 1997) while
ripened and cured meat showed a general increase
of amines (Bover-Cid et al., 1999). The choice of
starters could be useful tool to control and reduce
the development of some Enterobacteriaceae
strains. However, the presence of biogenic amines
in ripened dry uncooked fermented meat was
fundamentally a consequence of the activity of
decarboxylase-positive strains of Lactobacillaceae
and Enterococcaceae.
Biogenic amines content depended also
on an equilibrium between the decarboxylating and
amine oxidizing activity of microflora (Gardini et
al., 2002).
Therefore, to obtain Nham with low
amine concentrations besides the high quality raw
materials and good manufacturing practices, it is
necessary to employ highly competitive amino
369
acids decarboxylase negative starters cultures and
the starter culture should be able to compete and
grow well at the temperature intended for
processing of the product (Maijala et al., 1995).
Analysis of FAA
The main differences in the content of
total FAA among batches were detected at the end
of the processing (5 days), where lower quantities
were detected in all batches. From Table 2, after 5
days of incubation at 30°C, NC caused decrease
of 36.8 % in total FAA while Nham with L. sakei
and the Nham with mixed cultures of L. sakei and
D. hananii caused decrease of 13.3% and 19.73%,
respectively, in the concentration of FAA. The total
FAA of Nham with starter culture was higher than
that of the control (Table 2).
This suggested that the starter cultures
batches might have higher proteolytic activity than
the non-inoculated control batch and /or
catabolized free amino acids to be the other
products such as biogenic amines less than the
control due to the batches added with starters
lagged of amino acid decarboxylase.
Free amino acids precursors of biogenic
amines were detected by HPLC. During
fermentation step, the increases of tyrosine and
lysine which were the precursors of tyramine and
cadaverine, respectively were obsereved in all
batches. However, after 5 days of ripening, the
concentrations of tyrosine and lysine in Nham with
starter cultures were more than that of the control,
while the biogenic amines, tyramine and
cadaverine in the control were more than that of
the batches with starter cultures.
Evaluation of FAA during the ripening
of fermented Nham sausages indicated an increase
in most FAA over the 0-5 day fermentation period.
The main changes observed in the
decrease of free amino acids at the end of
processing showed a higher decrease proportion
of glutamic acid and arginine in the control than
that of the batch with L. sakei and the batch with
370
Kasetsart J. (Nat. Sci.) 41(2)
L. sakei and D. hansenii after 5 days of ripening.
The decreases in glutamic acid and arginine
contents might be due to these amino acids were
used for the growth of the microorganisms and
might be metabolized to flavours. The quantities
of alanine, aspartic acid, glycine, isoleucine,
leucine, methionine, phenylalanine, tyrosine,
valine and lysine in the control after 5 days of
ripening were higher than those before ripening.
Some of these amino acids in the batches with L.
sakei and the batches with L. sakei and D. hansenii
also increased after ripening.
Some amino acids, especially those
branched-chain amino acids, have been
metabolised to generate volatile compounds (Dura
et al., 2004). The contents of alanine, isoleucine,
histidine and proline were similar in the three
batches at the end of processing. Alanine,
contributors of sweet taste was found in higher
contents after ripening of fermented sausages.
Therefore, the balance of these free amino acids
would affect the sensory characteristics of the
product (Ordonez et al., 1999). The addition of
starter culture produced a limited effect on the free
amino acid generation although the effect was
different depending on the quantity of
microorganisms inoculated. Many factors could
affect the generation of free amino acids such as
the presence of different substrates, the pH, the
presence of different microorganisms and their
evolution during processing. The significant (P <
0.05) reduction in the concentration of free amino
acids could be produced by a more intense
microorganism metabolism than their production
Table 2 Total and free amino acid contents (mg/100 g) in Nham during fermentation.
Amino
Starter culture
acid
NC, 0 h
NC, 120 h
L. sakei
L. sakei
L. sakei and
0h
120 h
D. hansenii
0h
Ala
1.59 ± 0.16a 3.13 ± 0.29b 1.98 ± 0.04c 3.59 ± 0.12b 1.96 ± 0.05c
Asp
0.28 ± 0.04a 0.49 ± 0.04b 0.27 ± 0.01a 0.81 ± 0.10c 0.25 ± 0.01a
Gly
0.84 ± 0.10a 1.47 ± 0.07b 0.95 ± 0.02c 1.87 ± 0.18d 0.93 ± 0.01c
Ile
0.14 ± 0.01a 0.51 ± 0.04b 0.16 ± 0.00a 0.59 ± 0.04b 0.16 ± 0.00a
Leu
0.22 ± 0.01a 1.35 ± 0.13b 0.23 ± 0.00a 0.11 ± 0.01c 0.26 ± 0.01a
Met
0.04 ± 0.02a 0.39 ± 0.03b 0.61 ± 0.01c 0.53 ± 0.03c 0.05 ± 0.00a
Phe
0.10 ± 0.01a 0.55 ± 0.04b 0.12 ± 0.01a 0.73 ± 0.04c 0.12 ± 0.01a
Tyr
ND
0.14 ± 0.01a 0.14 ± 0.01a 0.29 ± 0.03b 0.15 ± 0.01a
Val
0.27 ± 0.02a 0.72 ± 0.04b 0.33 ± 0.01a 0.24 ± 0.02a 0.33 ± 0.00a
Arg
12.89 ± 1.71a 3.66 ± 0.51b 13.63 ± 0.28c 7.71 ± 0.04c 13.66 ± 0.06d
Cys
1.26 ± 0.20a 0.32 ± 0.07b 1.45 ± 0.05c 1.43 ± 0.05c 1.49 ± 0.03c
Glu
7.65 ± 0.99a 2.57 ± 0.49b 8.62 ± 0.27c 5.37 ± 0.39d 7.57 ± 0.04a
His
0.79 ± 0.11a 0.40 ± 0.05b 0.93 ± 0.02a 0.53 ± 0.02c 0.93 ± 0.01a
Ser
0.96 ± 0.16a 0.40 ± 0.12b 1.08 ± 0.02a 0.67 ± 0.09c 1.08 ± 0.02a
Lys
0.55 ± 0.07a 1.01 ± 0.05b 0.61± 0.00c 1.48 ± 0.02d 0.62 ± 0.01c
Pro
0.28 ± 0.04a 0.22 ± 0.01a 0.29 ± 0.01a 0.33 ± 0.02ab 0.37 ± 0.01b
Thr
0.98 ± 0.13a 0.28 ± 0.01b 1.05 ± 0.02a 1.15 ± 0.02c 1.04 ± 0.00a
Total
28.84 ± 3.66a 17.59 ± 1.79b 31.83 ± 0.71a 27.43 ± 0.90a 30.97 ± 0.20a
L. sakei and
D. hansenii
120 h
3.15 ± 0.36b
0.76 ± 0.03c
1.69 ± 0.08d
0.56 ± 0.02b
1.70 ± 0.07d
0.49 ± 0.02c
0.61 ± 0.03c
0.17 ± 0.01a
0.66 ± 0.05b
5.93 ± 0.20e
1.34 ± 0.04a
4.37 ± 0.15e
0.44 ± 0.05b
0.40 ± 0.09b
1.34 ± 0.04d
0.28 ± 0.01a
0.99 ± 0.05a
24.86 ± 1.13c
Mean values and standard deviations with different letters (a, b, c) in the same column indicate significant differences (P<0.05)
during fermentation among Nham formula. (ND = Not detectable)
Kasetsart J. (Nat. Sci.) 41(2)
during the stages of ripening as suggested by
Hughes et al. (2002) and Ordonez et al. (1999).
The changes of free amino acid contents
represented the degradation of protein and the
conversion of these free amino acids to the other
compounds such as biogenic amines and flavours
as well as growth metabolism of the
microorganisms.
In conclusion, this study determined the
effect of the starter cultures on biogenic amine
formation in fermented Nham sausages. In
addition to these, amino acid contents were
analyzed to note changes of amino acids in Nham
sausages. The starter culture, L. sakei BCC102,
decreased pH quickly and suppressed the
accumulations of tyramine. To avoid the formation
of high concentration of biogenic amine in Nham,
it is advisable to inoculate starter culture with
negative-decarboxylate activity such as L. sakei
BCC102 and use to top-quality raw meat materials
for the manufactured food products.
CONCLUSIONS
The production of biogenic amines is
dependent of several variables, such as the growth
of the microorganisms, their proteolytic and
decarboxylase activities, which interact with each
other. Furthermore, there is not a univocal rule
linking these variables with the different metabolic
mechanisms necessary for the formation of
biogenic amines. The results indicated that
inoculation of starter cultures with decarboxylase
negative should be carried out to initiate
fermentation process. Inoculation with appropriate
starter may lead to the decrease of biogenic amine
as fermentation progressed. This study suggests
that the use of L. sakei as starter culture was
effective to reduce the accumulation of biogenic
amine; cadaverine, during the ripening of
fermented Nham.
371
ACKNOWLEDGEMENTS
This study was supported by a grant of
the National Center for Genetic Engineering and
Biotechnology (BIOTEC), Thailand.
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Kasetsart J. (Nat. Sci.) 41 : 373 - 379 (2007)
Product Development System in Pattern Construction System,
Standard Body Measurement and Suitable Fitting Allowance for
Thai Ladies Brand in Fashion Industry
Foengfurad Mungtavesinsuk
ABSTRACT
Concept of the brand, theme of the design is the spirit of the collection in the fashion branding.
But the Pattern Construction System, standard body measurement and suitable fitting allowance are the
sustainable part for the branding in the market.
The study found that most brands in Thailand did not correct and less detailed, about body
measurement, standard sizing with appropriate fitting allowance for pattern construction and the pattern
construction system.
The objectives of this research, firstly the author used the Germany Pattern System and
appropriate German standard body measurements to make pattern construction. The results showed that
humans with different figures (and it does not matter in which country), the body type selected and the
size range of body measurement are almost similar.
Secondary, German Standard fitting allowance was applied to the Thai fashion industry. The
results showed that the tight fit should be used for the first or second step (of fitting allowance) and the
blouse item in second or third step of fitting allowance, etc.
Additionally, the study found that through systemization, productivity increased and the cost
of the product development was reduced.
Key words: pattern construction, body measurement, fitting, fashion brand, fashion industry
INTRODUCTION
The fashion business is an exciting,
stimulating, fascinating, ever changing, never the
same. In fashion, as in everything else, there are
always ups and downs, stops and starts. The
movement of fashion is always forward, never
backward. Its movements depend on the
environment. From the designer to the consumer,
everyone involved in the movement of fashion.
As a business, fashion was once
considered an art form controlled by designers who
dictated its content. But fashion has now evolved
into a science that can be measured and evaluated.
Modern fashion manufacturing was born during
the industrial revolution and has matured in the
age of technology. Without machines, clothing
could never be mass-produced. Technology has
revolutionized the way fashion is made. Almost
all stages of clothing production from design to
delivery rely to same extent on technology. (Stone,
1990)
Department of Textile Science and Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
e-mail: ofrm@ku.ac.th
Received date : 02/08/06
Accepted date : 29/01/07
374
Kasetsart J. (Nat. Sci.) 41(2)
A fashion retailer is in the business of
selling fashion products and not art. The modem
merchandiser is able to plan to supply very unique
local demand patterns. For example, people in
different sized, immigration into local areas can
often fundamentally change the sizing patterns
required in a local shop.
For this reasons, the product
development process needs to have fitting sample.
The fitting sample is checked on models that are
the “base size” (a medium or a size 12 in women’s
wear). Most fashion retailers have a limit to the
number of fit sample amendments they will accept,
the comment being up to three. After this, the style
is at risk of being cancelled. (Jackson et al., 2001)
The first pattern is important in the
process and has to be carefully and methodically
produced. And pieces need to match in the right
places. (Shreeve et al., 2004)
Bangkok Fashion city project guide the
Thai Fashion Industry into the global market. The
global market is highly competitive and needs
really professional and knowledgeable team.
As the experience in the global fashion
business , business will suffer, no matter how good
the design is or the accountants in the back office,
without the right goods they will be not be able to
generate enough sales, and ultimately enough
profit . That means, fashion business needs the
whole, healthy team and the product development
is part of it.
In the Fashion Industry, product
development covers the material and sampling. It
needs very strong knowledge about apparel
technology and management. And for the
sampling, it needs detail of styles with correct
information about fitting allowance from designer
and request standards body measurement from the
item for the first sample.
Thailand has limited information about
the standard body measurement and sizing. The
fitting allowance for pattern construction system
is mostly by experience but not methodical
workers. So, how and what can the fashion brands
in Thailand fashion industry do?
In this research, the standard body
measurement used was from Hohenstein Institue
Germany; the fitting step and pattern methodology
used was from University of Applied Science
Niederrhein Germany; applied to Thai Fashion
Industry. Through this research, the most
appropriate body measurement, sizing, fitting
allowance and suitable pattern system for the Thai
fashion industry was found especially for ODM
(Original Design Manufacture) in Ladies Fashion.
MATERIALS AND METHODS
Document and samples data
Hohenstein Institute made the research
about body measurement and sizing for the
Germany and EU people, and set up standards for
body measurements in different figure groups. This
basic data is used by us as reference in standard
body measurement. The fitting step and pattern
methodology used as reference came from the
document of University of Applied Science
Niederrhein Germany.
The target group for this case study is
from companies in the fashion industry with local
ladies in Thai market. All together, 25 companies
with 28 Ladies outwear brandings joined the
research as a case study. For full scale period of
the fashion collection and market feed back, a long
term study is needed to repeat the process and get
the correct result. So the 25 companies were
divided into three groups and three phases, each
phase running for one year and the process of each
group was the same. The first phase: 5 companies
as pilot group, second phase: 10 companies
repeated the process and the third phase: 10
companies repeated the process and to get
confirmation of the results, it was done by the first
two phases.
The process design
Kasetsart J. (Nat. Sci.) 41(2)
As the 25 companies were divided into
three phases for the case study, each group
followed the process and the methodology to get
the data and the result as standardized and
systemized. We analyzed the problems of the
existing products by fitting samples in the body
measurement, fitting allowance and pattern
construction system from the 28 brands and design
process for this study.
Firstly, we analyzed the suitable sample
size and tried the standard size 38 and size19 for
medium size.
Secondly, as this is the rainy season in
Thailand we used the fitting steps 1-5 as reference
for the fitting allowance in different items of
product. As the standards: step 1-2 for the tight fit
or tank top, step 2-3 for the blouse and step 3-5
for jacket, all the fitting allowance data are with
percentage by calculation instead of by experience
data.
Thirdly, we took the medium size body
measurement with suitable fitting step to calculate
and apply it to the pattern construction in German
methodically system.
All three processes were transferred to
the product development and made the samples
by each brand, then we did the sample fitting to
analysis the data and the methodology. After first
sample fitting, we corrected or adjusted the
reference data necessary, we remade the samples
and rechecked it again to fix the standards body
measurement, fitting step and construction
methodology for each brand.
As soon as the standard body
measurement, sizing, fitting step and pattern
construction methodology is confirmed, the
product development section of the brand done in
the collection will be put into market for sale.
When the feed back from market are good that
means the system is going in the correct direction
then the standardization of the product
development system is fixed and each brand can
set up the standard basic block of pattern for each
375
season following the fashion trend.
RESULTS AND DISCUSSION
After three years study, we grouped the
problems from 28 different Thai Ladies Brands as
found out in this research and divided them into
three parts to show the result and for discussion
1. Standard body measurement and
sizing
2. Fitting allowance in different steps
3. Pattern construction system
Standard body measurement and sizing
After three years study, we grouped the
28 different Thai Ladies Brands by market segment
and items of product, and then found out the
results: the young generation group is mostly fixed,
the sample size or medium size in size 38 and the
older generations mostly are in size 19. This also
shows the human body development of the
different generations and the development of the
social environment.
Thailand has very limited information
about body measurement; in cases there are some
but still not the full scale of body measurement
for the pattern construction.
In Germany, research of standard body
size specification is made every 10 years and
divided size group as the figure in normal high
group around 168 cm, short group around 160 cm
and extra high group around 176 cm.
(Mungtavesinsuk, 2005) As the grouping
compared to Thai peoples figure we can use the
normal and short group to apply in the Thailand
market.
Group, in normal high will take size 38
as standard medium size for sample fitting and in
short group will take size 19 as standard medium
size for sample fitting. After the size is selected,
detail of the body measurement placed in the size
table will be used for pattern construction. Through
all three phases as market segment for carrier
376
Kasetsart J. (Nat. Sci.) 41(2)
women will be in normal group and is fixed with
the Germany body size, in old generation group
will be in German size 19 but need shorter in back
waist length and in young attitude will be in
German size 18. As soon as the results come up,
all the sample making will follow the standards
body measurement and sizing for branding.
Fitting allowance in different step
Most in the local brand during product
development has not fixed the standard fitting
allowance by percentage but with experience data
added into the finishing garment measurement.
Those experience data to make the samples, can
be good for this sample and this size but not sure
for next sample. That means the reprocess in
product development are uncountable and the cost
of product development is higher. In the study, we
were gave the fitting allowance in German system
as reference for the pattern construction to made
the first sample. After the first fitting, maybe some
have a bit adjustment but mostly almost fix as
request. Through the try out, we set up the suitable
fitting step for each item and each brand to make
the pattern construction and then get the standard
basic block for whole collection. It means, during
the product development, there should be the
standards medium size and the fitting allowance
should be fixed in same level for the same item in
same collection and it depends on market request
and fashion trend too.
For Thai local market we have only
summer item, so the fitting allowance do not need
the whole range from step 1 to step 7. We just
need the fitting allowance from step 1 to step 5.
That means the garment is more fit on bodies.
Pattern construction system
In the fashion industry, the first sample
is very important. But it needs the most correct
information for pattern construction. As the
standards body measurement, sizing, and the step
of fitting allowance are fixed, it should get the best
fit sample. But why it still has problems in the
sample fitting?
Using the German pattern construction
system, formula is calculated methodically. All
formula is based on the standard body
measurement with fitting allowance step to
calculate in percentage. The pattern construction
actually needs very strong mathematical back
ground especially nowadays with the computer.
Table 1 The German standard sizes 38 as medium size apply into Thai local market in medium size as
38, 19, or 18.
Group
S
M
L
XL
Normal – carrier women
36
38
40
42
Short – old generation
18
19
20
21
Short – young generation
17
18
19
20
Note: S = Small size; M = Medium size; L= Large size; XL = Extra large
Table 2 Fitting allowance for different items such as body suit, blouse, jacket, etc.
Fitting allowance in step
Standard German allowance
Thailand and new fashion trend
(add % in chest)
1st step (6%)
For body suit
For tight fit knit wear
nd
2 step (9%)
For tight fit knit wear
For tight fit blouse or with elasticity
3rd step (12%)
For blouse and shirt
For lose blouse
4th step (15%)
For tight fit jacket or suit
For suit some with 3 1/2 step
5th step (18%)
For lose fit jacket or suit
For Jacket some with 4th step
Kasetsart J. (Nat. Sci.) 41(2)
All the data comes up with informative system.
But before computerizing the correct pattern
construction system is needed otherwise the
sample making still has problems.
During research it was found that all
points which happen frequently is mostly from
basic pattern construction problems. This means
the fundamental pattern construction system has
problems.
During the study most problems in
trousers were with wrinkles in the crotch position
and leg twist. The top items: center front rides up
and center backs are too loose. Those problems
are all from the basic pattern construction system,
Figure 1 The problem of crotch in trousers.
Figure 2 The problem with leg twist.
377
which the pattern for samples are by experienced
but not systemized.
Wrinkle in crotch of trousers
In Figure 1: the sample piece is in “A”
and the standard one is in “B”. We can see the
width in crotch position especially in part “A” is
too narrow as part “B”. That means the proportion
in crotch position in back rise should be 1/8 of
Hip circumference of body measurement.
Leg twist in trousers
In Figure 2 the standard piece is in “A”
and the sample piece is in “B”. The middle line
378
Kasetsart J. (Nat. Sci.) 41(2)
for the trousers must be in the middle, like back
piece “A” the width of leg must be b1=b2, b3=b4
and front piece”B”b5=b6, b7=b8, but in back piece
“C” t1>t2, t3>t4 and front piece “D” t5<t6, t7<t8.
That means the back piece “C” and front piece
“D” this leg is going to twist due to the unbalance
leg construction.
Center front rides up in top
In figure 3, the standard piece is in “A”
and the sample piece is in”B”. We can see the
Figure 3 Problem with center front rides up.
Figure 4 Problem with back part too lose.
center front in piece “B” be shortened that is the
reason cause the central front hop up. Or some
make front waist length and back waist length in
same length, but actually the front waist length
and back waist length in body measurement is
different due to the bust length concern.
Center back too loose in top
In Figure 4, the standard pieces are in
“A” & “C” and the sample pieces are in “B” &
“D”. As body figure center back should be in curve
Kasetsart J. (Nat. Sci.) 41(2)
as piece “A” or straight as piece “C”. We can see
the center part in pieces “B” & “D” that the center
back line is not straight in follow the body figure
like piece “C” but in straight down like pieces
“B” & “D”. That means the pieces “B” & “D” are
wider then pieces “A” & “C”, this cause too loose
in center back.
CONCLUSION
Systemization is the successful key for
the fashion industry, it does not matter in which
section, even the product development. As the
result we found through this research that we need
to have a standard body measurement, standard
fitting allowance step and systemization of pattern
construction. The human figure actually can be
dividing into different groups and different sizes.
As soon as the data base is there, that can be
applied to almost everybody’s figure. Thailand
does not have the standard body measurement. But
the competitiveness and the chance would not wait.
The best solution for the Thai ladies fashion brands
is to follow the German Pattern construction
system as base to get the best of the technology
for product development. Through this research
we also confirm the system from Germany is
workable for Thailand too. Everyone who is
concerned about product development for the
fashion industry must follow this format to running
the fashion business.
379
Additionally, as soon as the product
development systems are running smoothly, then
quick response and cost goes down which is
happening for the fashion business too, because
everyone is working in the same direction and
speaks the same language. The high technology is
introduced to the fashion industry. With strong
fundamental knowledge will bring high quality
through high technology.
ACKNOWLEDGEMENT
The author would like to thanks the
Department of Industry Promotion from Industry
Ministry to support this research and the 25
companies cooperated to get the valuable data for
this research.
LITERATURE CITED
Mungtavesinsuk, F. 2005. Industrial Pattern
Construction of Lady’s wears in German
system. 2 nd ed. Kasetsart University.
Bangkok, Thailand. 115p.
Shreeve, A. and C. Kelly. 2004. Developing
communication skills through fashion
design. IFFTI 2004: 51 – 63
Stone, E . 1990. Fashion Merchandising. 5 th ed.
Merchandising management. MACMILLAN
PRESS LTD. London. 454p.
Kasetsart J. (Nat. Sci.) 41 : 380 - 393 (2007)
A Nonlinear Optimization Problem for Determining Safety Stocks
in a Two-Stage Manufacturing System
Parthana Parthanadee
ABSTRACT
Safety stock is the inventory which is used to buffer against the uncertainties in business
operations. Managers must decide how much safety stock of each raw material and each finished product
should be maintained. Determining appropriate safety stock levels is an important decision. Too much
safety stock would incur extra inventory carrying costs, whereas too less safety stock would increase
the risk of having product stockouts and lost sales. In this paper, a nonlinear programming problem for
determining safety stock levels in a two-stage manufacturing system, was presented. Instead of using
the well-known search algorithms, simple decision rules for determining safety stock levels were derived
from an analysis of the derivatives of cost functions, with respect to the delivery performances of suppliers
and prior manufacturing process. Two algorithms based on those decision rules were proposed and
tested on seventy-five problem instances. The results showed that the proposed algorithms provided,
within 1 second, the solutions with less than 3% deviations, on average, from the known integer solutions
or the best lower bounds. The algorithms also performed better than the pattern search algorithm, which
was the method applied in the previous research.
Key words: safety stock, inventory, nonlinear programming problem, two-stage manufacturing systems
INTRODUCTION
Safety stock or buffer stock is the amount
of inventory held in a short run to protect against
demand and supply uncertainties and forecasting
errors in business operations. When demands are
underestimated, or supplies are insufficient or
backordered, product stockouts may occur and
cause the company some lost sales, especially
when the degree of product substitutability is high.
On the other hand, if too many safety stock
quantities are held, high inventory costs would be
charged to the company. The two types of costs:
opportunity costs and inventory costs must be
traded off to find the appropriate safety stock
levels.
The classical approach for determining
safety stock is to specify a desired service level or
a stockout probability and use it to identify a safety
factor, k. If the demand during lead time is assumed
normally distributed, the safety factor is usually
set to z and the safety stock is set to z⋅σL, where z
denotes the z-score to achieve the desired service
level and σL denotes the standard deviation of the
probability distribution of demand during lead time
(Vollmann et al., 1997). The other choices of safety
factor, demand deviation, and safety stock
calculations can be found in Krupp (1997); Silver
Program of Agro-Industry Technology Management, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand.
e-mail: fagiptp@ku.ac.th
Received date : 22/06/06
Accepted date : 22/01/07
Kasetsart J. (Nat. Sci.) 41(2)
et al. (1998); Zeng (2000); and Talluri et al. (2004).
Maia and Qassim (1999) derived
optimum safety stocks for a one-stage
manufacturing system, in which a finished product
was produced from a number of raw materials.
The problem was formulated as a nonlinear
program (NLP), which minimized the total of
inventory and opportunity costs. From the analysis,
Maia and Qassim (1999) found that it was
economical to either hold every safety stock at its
maximum level or not hold it at all. A set of
decision rules for finding optimum safety stocks
was provided and illustrated through a small
numerical example.
Siribanluoewut (2006) extended the
work by Maia and Qassim (1999) to determine
safety stocks for a two-stage manufacturing
system. The problem was solved using three
optimization heuristics, which were genetic
algorithm, pattern search algorithm, and the hybrid
genetic algorithm with pattern search. All the
optimization heuristics performed efficiently on
the test problems and the qualities of solutions
reported were found not statistically different from
each other. However, the pattern search algorithm
provided good solutions in significantly shorter
time than other heuristics did.
Inderfurth and Minner (1998) formulated
an optimization problem of determining safety
stocks in multi-stage manufacturing systems with
normally distributed demands. The system was
assumed to be under a periodic review, base-stock
control policy, in which inventories were reviewed
every fixed period of time and replenished up to a
specified level. The safety factor in this study was
found to be depending on service level, type of
service level, and coverage time. The service level
and coverage time for different types of multi-stage
manufacturing systems were derived to establish
the optimal policy for determining safety stocks
in these multi-stage systems.
In this paper, the problem for determining
safety stocks in the two-stage manufacturing
system, as presented in Siribanluoewut (2006), was
381
considered. Instead of using the optimization
heuristics, which required the users to understand
their mechanisms, a set of simple decision rules
for finding optimum safety stocks was developed,
and tested on the number of test instances as shown
in the following sections.
MATERIALS AND METHODS
Problem description
A two-stage manufacturing system, as
presented in Siribanluoewut (2006), was
considered in this study. In such system, a
manufacturer ordered m raw materials (RMs) for
its stage-1 manufacturing process and n raw
materials for its stage-2 manufacturing process.
Each raw material was ordered from a single
supplier. The stage-1 process produced a workin-process (WIP) from those m raw materials. The
WIP and the n other raw materials were then fed
to stage 2 to produce a final product. Figure 1
illustrated this two-stage manufacturing system.
The model formulation of this system was
modified from that of the one-stage manufacturing
system by Maia and Qassim (1999). The notations
used in the formulation were as follows.
Stage 1
i
index of raw materials in stage 1; i = {1,
2,…, m}
p1,i
the on-time delivery performance of
supplier i
q1,*i
q1,i
x1,i
k1,i
c1,i
the quantity of stage-1 raw material i that
is delivered on time
the quantity of stage-1 raw material i that
is ordered
the safety stock of stage-1 raw material i
the delivery performance to manufacture
of stage-1 raw material i
the unit inventory cost of stage-1 raw
material i
ps1
the stage-1 manufacturing performance
qw
xw
the quantity of WIP that is required
the safety stock of WIP
382
Kasetsart J. (Nat. Sci.) 41(2)
Figure 1 The two-stage manufacturing system.
kw
cw
the WIP delivery performance to stage2 manufacturing process
the unit inventory cost of WIP
Stage 2
j
index of raw materials in stage 2; j = {1,
2,…, n}
p2,j
the on-time delivery performance of
supplier j
*
q2,
j
q2,j
x2,j
k2,j
c2,j
the unit inventory cost of stage-2 raw
material j
ps2
the stage-2 manufacturing performance
qp
the quantity of finished product that is
required
the safety stock of finished product
The finished product delivery
performance to customer
the unit inventory cost of finished
product
the unit opportunity cost of finished
product that is not delivered on time
xp
kp
cp
the quantity of stage-2 raw material j that
is delivered on time
the quantity of stage-2 raw material j that
is ordered
the safety stock of stage-2 raw material j
the delivery performance to manufacture
of stage-2 raw material j
co
As in Maia and Qassim (1999), the ontime delivery performance of supplier i and the
on-time delivery performance of supplier j could
be calculated from the past data records, using
Equations (1) and (2), respectively.
Kasetsart J. (Nat. Sci.) 41(2)
383
q*
p1,i = 1,i
q1,i
(1)
q*
p2,i = 2,i
q2 , i
(2)
If the manufacturer held safety stocks for every raw material, the delivery performances to
manufacture of stage-1 raw material i and stage-2 raw material j could be defined as in Equations (3)
and (4), respectively.
q * + x1,i
x
k1,i = 1,i
= p1,i + 1,i
q1,i
q1,i
k2 , j =
q2*, j + x2, j
x 2, j
= p2, j +
q2 , j
q2 , j
∀i = {1, 2, K, m}
(3)
∀j = {1, 2, K, n}
(4)
The inventory cost of the safety stock of each raw material could be computed from Equations
(5) or (6) as follows.
C1,i = c1,i x1,i = c1,i q1,i ( k1,i − p1,i )
∀i = {1, 2, K, m}
(5)
C2, j = c2, j x2, j = c2, j q2, j ( k2, j − p2, j )
∀j = {1, 2, K, n}
(6)
The manufacturing performance of stage-1 process, ps1 , was defined as the ratio between ontime and planned production, accounting for all delays that may occur, but excluding those caused by
material stockouts. The ps1 could be found from Equation (7). The WIP delivery performance to stage2 manufacturing process, kw, was given in Equation (8).
q*
ps1 = w
qw
(7)
m
x
kw = ps1 ∏ k1,i + w
qw
i =1
(8)
The inventory cost of the WIP safety stock could be calculated from Equation (9).
m
Cw = cw x w = cw qw ( kw − ps1 ∏ k1,i )
i =1
(9)
Similarly, the manufacturing performance of stage-2 process, ps2 , the product delivery
performance, kp, and the product inventory cost could be calculated as follows.
ps2 =
q *p
(10)
qp
n
xp
j =1
qp
k p = ps2 kw ∏ k2, j +
(11)
n
C p = c p x p = c p q p ( k p − ps2 kw ∏ k2, j )
j =1
(12)
Kasetsart J. (Nat. Sci.) 41(2)
384
Finally, the opportunity cost, defined as the cost incurring whenever the finished product failed
to be delivered to the customers on time, was given in Equation (13).
Co = co q p (1 − k p )
(13)
Mathematical model
A nonlinear programming (NLP) model, for determining the delivery performances k1,i, kw,
k2,i, and kp was formulated in this section. The objective of this NLP model was to minimize the total of
the inventory costs charged for holding all the safety stocks and the opportunity costs, subject to the
bounds on the delivery performances. The model was formulated as follows.
m
m


Min C = co q p 1 − k p + ∑ c1,i q1,i k1,i − p1,i + cw pw  kw − ps1 ∏ k1,i 


i =1
i =1
(
)
(
)
n
n


+ ∑ c2, j q2, j k2, j − p2, j + c p q p  k p − ps2 kw ∏ k2, j 


j =1
j =1
(14)
p1,i ≤ k1,i ≤ 1
∀i = {1, 2, K, m}
(15)
p2, j ≤ k2, j ≤ 1
∀j = {1, 2, K, n}
(16)
(
)
Subject to
m
ps1 ∏ k1,i ≤ kw ≤ 1
(17)
i =1
n
ps2 kw ∏ k2, j ≤ k p ≤ 1
(18)
j =1
Solution analysis
It was known that the optimal solution of the NLP is necessarily on the border of the feasible
region, if the Hessian matrix of the objective function is indefinite, as in this problem (see Marsden and
Tromba (1981), for example). Therefore, the optimal delivery performances k1,i, kw, k2,j, and kp in the
presented NLP must be either on their lower bounds or upper bounds. In this paper, the analysis followed
*
the method in Maia and Qassim (1999) by defining reference costs, c1,i for the stage-1 raw material
*
*
for the WIP, and c2,
i, cw
j for the stage-2 raw material j, as shown in Equations (19) - (21). The upper
bounds of these reference costs were found from the derivatives of the cost function with respect to the
delivery performances k1,i, kw, k2,j, and k2,j.
c1*,i ≤
c1,i q1,i
*
cw
≤
∀i = {1, 2, K, m}
m
qw ps1
∏
i 2 = i +1
cw q w
n
q p ps2 ∏ p2, j
j =1
(19)
p1,i2
(20)
Kasetsart J. (Nat. Sci.) 41(2)
c2*, j ≤
c2, j q2, j
∀j = {1, 2, K, n}
n
q p ps2 kw
∏
385
j 2 = j +1
(21)
p2, j2
*
*
*
The reference costs, c1,i cw
and c2,
j were then analyzed against all the unit costs in the model
to identify when the corresponding delivery performances and safety stocks should be set to their lower
or upper bounds. If the opportunity cost was high, the manufacturer should hold safety stocks to prevent
the products shortages. In contrary, it would not be economical to stock the materials, when the inventory
costs (and hence the reference costs) were costly. The optimal solution of the presented optimization
model could be derived as follows:
Stage-1 raw materials:
c1*,i
co ≤ c1*,i then k1,i = p1,i and x1,i = 0

≤ min(cw , c p ) and 
*
co > c1,i then k1,i = 1 and x1,i = q1,i 1 − p1,i
(i)
If
(ii)
If c1*,i > min(cw , c p ) then k1,i = p1,i and x1,i = 0
(
Work-in-process:
(iii)
m

*
co ≤ cw then kw = ps1 ∏ k1,i and x w = 0
i=1

*
If cw
≤ c p and 
m



*
1
1
c
>
c
then
k
=
and
x
=
q
−
p
w
w
w
w
s1 ∏ k1,i 
 o


i=1

(iv)
*
If cw
> c p then kw = ps1 ∏ k1,i and x w = 0
m
i=1
Stage-2 raw materials:
c2*, j
co ≤ c2*, j then k2, j = p2, j and x2, j = 0

≤ c p and 
*
co > c2, j then k2, j = 1 and x2, j = q2, j 1 − p2, j
(v)
If
(vi)
If c2*, j > c p then k2, j = p2, j and x2, j = 0
(
Finished product:
n
(vii)
co > c p then k p = ps2 kw ∏ k2, j and x p = 0
j=1
(viii)
n


co > c p then k p = 1 and x p = q p 1 − ps2 kw ∏ k2, j 


j=1
)
)
386
Kasetsart J. (Nat. Sci.) 41(2)
Proposed algorithms
Since the exact values of the reference
costs were not known, they could be initially set
to their upper bounds in which all other rawmaterial delivery performances, besides the one
corresponding to the considered raw material, (k1,i
raw-material reference costs. The delivery
performances and safety stocks of WIP and
finished product, including the total costs, were
recalculated and recorded at every step. Finally,
the minimum total cost and the best solution were
identified.
: ∀i ≠ i’ and k2,j : ∀j ≠ j’) were set at their lower
bounds. The estimated reference costs of the raw
materials in every stage were sorted in a nondecreasing order and the values were recalculated
as in Equations (19) and (21). This solution finding
algorithm was specified as Algorithm 1.
From the preliminary testing, it was
found that when the estimated values of reference
costs were not much different from each other or
from the opportunity cost, Algorithm 1 may not
always provide the optimal solutions. Algorithm
2 was then proposed. Again, the reference costs
of the raw materials in every stage were sorted as
in Algorithm 1. At the initial step, the delivery
performances and safety stocks of all raw materials
were set to their lower bounds. The delivery
performances and safety stocks of WIP and
finished product were found from the decision
rules presented in the previous section,
accordingly. The total cost was calculated and
recorded. Then, the delivery performance and
safety stock of each raw material in each stage
were increased to their upper bounds, one by one,
corresponding to the non-decreasing order of the
A numerical example
In this section, a small example,
consisting of three raw materials in stage 1 and
two raw materials in stage 2, was presented. The
data for this example was given in Table 1. The
opportunity cost was assumed to be 8.44 baht.
Algorithm 1:
The initial reference costs for stage-1 raw
materials 1, 2 and 3 were found to be 2.1778,
5.4652 and 9.2014 baht, respectively. Thus, the
stage-1 raw material order followed the natural
order. The reference costs for RM 1, RM 2 and
RM 3 were recalculated and their values became
2.1778, 5.1868 and 8.1043 baht, respectively.
Following the proposed decision rules, the safety
stocks of RM 1 and RM 2 should be set to their
upper bounds, which were 8 and 10 units,
respectively. The safety stock for RM 3 and WIP
were found unnecessary.
Next, the initial reference costs for stage2 raw materials 4 and 5 were found to be 7.5490
and 5.5337 baht, respectively. Hence, the
Table 1 Data for a small example with three raw materials in stage 1 and two raw materials in stage 2.
Materials
q
q*
p
c
Initial ref. Ref. cost
Algorithm 1
Algorithm 2
cost (baht) (baht)
k
x
k
x
RM 1
157
149 0.9490 2.86
2.1778
2.1778 1.0000
8 1.0000
8
RM 2
139
129 0.9281 8.29
5.4652
5.1868 1.0000
10 0.9281
0
RM 3
244
242 0.9918 7.44
9.2014
8.1043 0.9918
0 0.9918
0
WIP
232
224 0.9655 9.40
16.5918 0.9576
0 0.8887
0
RM 4
117
107 0.9145 8.88
7.5490
6.9549 1.0000
10 0.9145
0
RM 5
216
199 0.9213 3.50
5.5337
5.5337 1.0000
17 1.0000
17
Product
173
156 0.9017 7.20
1.0000 23.61 1.0000 46.21
Total cost
424.1001
415.0962
(baht)
Kasetsart J. (Nat. Sci.) 41(2)
387
algorithm would consider RM 5, prior to RM 4.
The reference costs of RM 4 and RM 5 were
recalculated and found to be 6.9549 and 5.5337
baht. Thus, the safety stocks of RM 4 and RM 5
were set to their upper bounds, which are 10 and
17 units, respectively. Finally, the product safety
stock was computed and set to 23.61 units. The
corresponding total cost is 424.10 baht.
shown in Table 2. From the Table, the sixth
solution provided the minimum total cost of 415.10
baht, with the safety stock levels set to 8 units for
RM 1, 17 units for RM 5, and 46.21 units for the
finished product. Algorithm 2 provided a superior
solution to Algorithm 1 for this test instance.
Algorithm 2:
Following the initial reference costs
found in Algorithm 1, the priority for increasing
raw-material safety stock levels would be in the
orders of RM 1 – RM 2 – RM 3 and RM 5 – RM
4. Twenty-four solutions were evaluated and
To facilitate the implementation,
Algorithms 1 and 2 were coded in MATLAB
6.5. Both algorithms were tested on 75 test
instances (from 5 test problem sets, each with 15
instances) in Siribanluoewut (2006). Table 3
presented structures of the test instances and the
RESULTS
Table 2 The twenty-four solutions evaluated by Algorithm 2.
No.
Safety Stocks (units)
RM 1
RM 2
RM 3
WIP
RM 4
1
0
0
0
0
0
2
8
0
0
0
0
3
8
10
0
0
0
4
8
10
2
0
0
5
0
0
0
0
0
6
8
0
0
0
0
7
8
10
0
0
0
8
8
10
2
0
0
9
0
0
0
0
10
10
8
0
0
0
10
11
8
10
0
0
10
12
8
10
2
0
10
13
0
0
0
36.33
0
14
8
0
0
25.82
0
15
8
10
0
9.84
0
16
8
10
2
8.00
0
17
0
0
0
36.33
0
18
8
0
0
25.82
0
19
8
10
0
9.84
0
20
8
10
2
8.00
0
21
0
0
0
36.33
10
22
8
0
0
25.82
10
23
8
10
0
9.84
10
24
8
10
2
8.00
10
RM 5
0
0
0
0
17
17
17
17
17
17
17
17
0
0
0
0
17
17
17
17
17
17
17
17
Product
62.14
56.19
47.13
46.09
52.67
46.21
36.38
35.25
41.43
34.36
23.61
22.38
41.56
41.56
41.56
41.56
30.33
30.33
30.33
30.33
17.00
17.00
17.00
17.00
Total cost
(baht)
447.42
427.44
445.15
452.54
438.73
415.10
427.23
433.98
446.56
418.58
424.10
430.09
640.70
564.82
497.48
495.10
619.36
543.48
476.14
473.76
612.16
536.28
468.94
466.56
388
Kasetsart J. (Nat. Sci.) 41(2)
average percentage of deviations from the optimal
NLP total costs, including the solution times, by
Algorithms 1 and 2. The result showed that
Algorithm 2 did outperform Algorithm 1.
As aforementioned, the optimization
model presented in this paper was an NLP model.
Therefore, the levels of safety stocks in the final
solution may be reported as non-integers. This
safety stock determination problem could also be
modeled as a mixed integer nonlinear program
(MINLP) for minimizing the total of the
opportunity costs and the inventory costs charged
for holding all the safety stocks, subject to the
bounds on the safety stock levels. The safety stocks
x1,i, xw, x2,j, and xp, which were required to be
integers, would be sought from the MINLP, in lieu
of the delivery performances k1,i, kw, k2,j, and kp in
the NLP. However, the MINLP was a much more
complex problem. It may not be solved in
reasonable computation times with regular
optimization methods, even for small-size problem
instances. Thus, it was suggested that the safety
stock levels should be found by applying
Algorithm 2 and then rounding down the noninteger safety stocks to their nearest integers. The
rounded solutions were compared with true
optimal integer solutions found from the
enumeration method, in which all possible integer
solutions were enumerated and evaluated.
However, the enumeration method could not be
implemented on the large problem instances, due
to their long computation times. Therefore, only
the true optimal integer solutions of test problem
sets 1 and 2 could be identified. The qualities of
these rounded solutions were presented in Tables
4 and 5. For test problem sets 3, 4 and 5, the
rounded solutions were compared with the
corresponding MINLP lower bounds (i.e. the
optimal NLP solutions) instead. The qualities of
these solutions were given in Tables 6-8.
Furthermore, the pattern search algorithm (using
a complete search, a mesh expansion factor of 1.0
and a mesh contraction factor of 0.5) was also
investigated. The details of this algorithm can be
found in Kolda et al. (2003). The qualities of the
integer solutions found from the pattern search
algorithm were also presented in Tables 4-8, for
comparison purpose.
From Tables 4 and 5, Algorithm 2 with
solution rounding provided high-quality results.
The rounded solutions were 2.10% deviating from
the known MINLP optimum on average (with a
maximum deviation of 10.20%) for problem set
1, and 3.86% deviating from the known MINLP
optimum on average (with a maximum deviation
of 20.23%) for problem set 2. Algorithm 2 with
solution rounding provided the good solutions in
much shorter times (i.e. less than 1 second) than
the enumeration method did (i.e. more than 7
minutes for problem set 1 and more than 35
minutes for problem set 2, on average). For larger
test problem sets, the average deviation of the
Algorithm-2 solutions from the corresponding
MINLP lower bounds were less than 2.5%, with
Table 3 Structures of the test instances and the average percentage of deviations from the true optimal
total costs of Algorithms 1 and 2.
Set
No. of RMs
No. of % Deviation from true optimum Average solution time (sec.)
Stage 1 Stage 2
instances
Algorithm 1 Algorithm 2
Algorithm 1 Algorithm 2
1
3
1
15
0.00%
0.00%
0.0013
0.0047
2
3
2
15
0.47%
0.00%
0.0013
0.0033
3
7
2
15
0.24%
0.00%
0.0020
0.0047
4
12
2
15
0.98%
0.00%
0.0033
0.0073
5
15
2
15
2.79%
0.00%
0.0013
0.0087
Average
0.90%
0.00%
0.0019
0.0057
Kasetsart J. (Nat. Sci.) 41(2)
Table 4 The quality of the rounded solutions for test problem set 1.
No.
Enumeration
Algorithm 2
Total
Time
Total
Time % Dev.
costs
(seconds)
costs
(sec)
from
(baht)
(baht)
opt.
1
226.2863
557.00
226.2863
0.03
0.00
2
223.0773
454.98
245.4778
0.00 10.04
3
188.9893
574.17
191.7372
0.00
1.45
4
174.6790
233.69
179.7564
0.00
2.91
5
422.4267
616.30
434.7873
0.00
2.93
6
538.7574
805.59
538.7574
0.00
0.00
7
85.4226
753.52
86.9119
0.00
1.74
8
194.1343
40.50
196.3468
0.00
1.14
9
53.3469
9.66
58.7888
0.00 10.20
10
240.7639
16.64
240.7639
0.00
0.00
11
396.7343
419.02
398.7735
0.00
0.51
12
173.3962
692.55
173.4065
0.00
0.01
13
359.3905
51.55
359.3905
0.00
0.00
14
225.1412
171.69
226.3883
0.02
0.55
15
399.7456 1182.19
399.7456
0.00
0.00
Average
438.6033
0.0033
2.10
Table 5 The quality of the rounded solutions for test problem set 2.
No.
Enumeration
Algorithm 2
Total
Time
Total
Time % Dev.
costs
(seconds)
costs
(sec)
from
(baht)
(baht)
opt.
1
231.96
388.13
231.96
0.05
0.00
2
231.15
310.00
267.04
0.00
15.52
3
195.83
843.94
196.89
0.00
0.54
4
286.68
1640.67
291.76
0.00
1.77
5
448.69
3548.72
461.05
0.00
2.75
6
623.79
3839.44
623.79
0.00
0.00
7
95.46
3736.39
99.51
0.00
4.24
8
233.65
252.59
235.87
0.00
0.95
9
63.30
22.25
76.10
0.00
20.23
10
353.81
161.06
353.81
0.00
0.00
11
415.36
1184.09
415.36
0.00
0.00
12
137.41
1197.42
150.03
0.00
9.18
13
110.35
9400.22
111.73
0.00
1.25
14
171.36
5738.92
173.87
0.00
1.47
15
210.21
930.83
210.21
0.02
0.00
Average
2212.98
0.00
3.86
0.62
4.82
389
Pattern search
Total
Time % Dev.
costs
(sec)
from
(baht)
opt.
226.2863
0.656
0.00
223.3209
0.547
0.11
190.3275
0.89
0.71
174.6790
0.453
0.00
442.0500
0.656
4.65
538.7574
0.343
0.00
89.8888
0.484
5.23
199.1684
0.578
2.59
58.6819
0.344 10.00
240.7639
0.391
0.00
453.2415
0.453 14.24
176.3493
0.61
1.70
359.3905
0.562
0.00
244.0558
0.422
8.40
399.7456
0.484
0.00
0.5249 3.18
Pattern search
Total
Time % Dev.
costs
(sec)
from
(baht)
opt.
231.96
0.70
0.00
232.33
0.58
0.51
198.17
0.55
1.20
286.68
0.63
0.00
468.31
0.84
4.37
623.79
0.48
0.00
97.66
0.61
2.30
245.51
0.53
5.08
73.49
0.42
16.11
353.81
0.59
0.00
415.36
0.72
0.00
142.33
0.64
3.58
110.35
0.63
0.00
238.33
0.69
39.08
210.21
0.64
0.00
390
Kasetsart J. (Nat. Sci.) 41(2)
Table 6 The quality of the rounded solutions for test problem set 3.
No.
LB of
Algorithm 2
Pattern search
Total costs Total costs
Time
% Dev.
Total costs
Time
% Dev.
(baht)
(baht)
(sec)
from LB
(baht)
(sec)
from LB
1
341.4262
349.9014
0.05
2.48
381.6719
0.532
11.79
2
698.4675
699.5436
0.00
0.15
698.7817
0.641
0.04
3
306.6030
313.5858
0.00
2.28
327.0063
0.469
6.65
4
782.9640
789.0657
0.00
0.78
791.1078
0.516
1.04
5
141.2319
145.3478
0.00
2.91
147.0369
0.625
4.11
6
248.9744
252.0126
0.00
1.22
250.6189
0.5
0.6
7
418.7751
418.7751
0.00
0.00
418.7751
0.313
0.00
8
870.7950
871.3350
0.00
0.06
871.3350
0.516
0.06
9
188.2021
189.3901
0.00
0.63
189.3901
0.563
0.63
10
607.2085
611.1003
0.02
0.64
618.0523
0.766
1.79
11
310.3231
317.2456
0.00
2.23
313.7932
0.578
1.12
12
767.5441
779.7637
0.00
1.59
775.4064
0.687
1.02
13
796.4144
811.3657
0.00
1.88
872.1025
0.765
9.50
14
216.6816
220.6952
0.00
1.85
226.5848
0.563
4.57
15
329.8299
331.9408
0.00
0.64
348.9675
0.453
5.80
Average
0.0047
1.29
0.5658
3.25
Table 7 The quality of the rounded solutions for test problem set 4.
No.
LB of
Algorithm 2
Pattern search
Total costs Total costs
Time
% Dev.
Total costs
Time
% Dev.
(baht)
(baht)
(sec)
from LB
(baht)
(sec)
from LB
1
781.7861
781.7861
0.05
0.00
937.4422
0.875
19.91
2
116.0982
128.7529
0.00
10.90
118.4608
1.125
2.04
3
250.6759
253.0172
0.00
0.93
276.9489
1.281
10.48
4
863.7852
866.8405
0.02
0.35
932.2620
0.844
7.93
5
416.318
425.6585
0.00
2.24
434.4976
1.282
4.37
6
769.3648
784.0514
0.00
1.91
775.9314
0.906
0.85
7
831.0751
834.5863
0.00
0.42
861.3861
0.875
3.65
8
732.6523
737.9136
0.00
0.72
769.4482
1.157
5.02
9
171.9773
180.0650
0.02
4.70
175.0647
0.765
1.80
10
356.2244
366.1220
0.00
2.78
359.5120
1.062
0.92
11
883.9080
883.9080
0.02
0.00
883.9080
0.39
0.00
12
197.3155
217.3515
0.00
10.15
206.1805
0.672
4.49
13
532.1986
533.0392
0.00
0.16
632.9300
0.703
18.93
14
905.1188
910.7604
0.00
0.62
921.6216
1.125
1.82
15
805.6426
807.9736
0.00
0.29
845.8867
0.781
5.00
Average
0.0073
2.41
0.9229
5.81
Kasetsart J. (Nat. Sci.) 41(2)
the maximum deviation of about 10%. The solving
times were still less than 1 second for all test
instances.
The qualities of solutions and the
computation times from Algorithm 2 and from
pattern search seemed to be competitive, especially
for the small-size test problems. The differences
between the total costs found from Algorithm 2
and from pattern search were compared using the
paired t-test and the signed rank test (Montgomery
and Runger, 2004). The former was tested whether
391
or not the average of the differences in total costs
equaled zero. The latter was a non-parametric
hypothesis test on the median of the differences
in total costs. Under the normality assumption of
data, the paired t-test was more powerful than the
signed rank test. However, the signed rank test was
less sensitive to the outliers. Herein, the signed
rank test was applied because the distributions of
the total costs showed significant departures from
normal distributions. The summary of the
statistical tests was presented in Table 9.
Table 8 The quality of the rounded solutions for test problem set 5.
No.
LB of
Algorithm 2
Pattern search
Total costs Total costs
Time
% Dev.
Total costs
Time
% Dev.
(baht)
(baht)
(sec)
from LB
(baht)
(sec)
from LB
1
335.2583
341.7339
0.05
1.93
338.9744
1.204
1.11
2
448.8165
457.5385
0.02
1.94
468.6784
1.078
4.43
3
241.1376
243.6205
0.00
1.03
243.0759
1.313
0.80
4
689.6022
692.9453
0.00
0.48
708.7569
1.641
2.78
5
977.388
980.5293
0.02
0.32
1010.1633
1.391
3.35
6
326.5053
328.5352
0.00
0.62
328.1805
1.078
0.51
7
750.4167
751.3261
0.00
0.12
751.2409
1.000
0.11
8
810.929
813.4720
0.00
0.31
858.0251
1.766
5.81
9
763.4033
764.7848
0.00
0.18
777.3780
1.250
1.83
10
1047.4942 1047.4942
0.02
0.00
1047.4942
1.734
0.00
11
964.0926
972.7866
0.00
0.90
1626.5400
1.532
68.71
12
600.9621
612.3057
0.00
1.89
685.6725
0.656
14.10
13
819.0913
820.9975
0.00
0.23
1098.4323
1.265
34.10
14
966.8934
968.9883
0.00
0.22
1042.7315
1.360
7.84
15
730.5132
730.5132
0.02
0.00
730.5132
1.172
0.00
Average
0.0087
0.68
1.2960
9.70
Table 9 The statistical results from the paired t-test and the signed rank test.
Problem set
Average of
Median of
p-value
the difference
the difference
Paired t-test
Signed rank test
in total costs
in total costs
1
3.9592
0.0000
0.3578
0.7109
2
1.9540
0.0000
0.7095
< 1.0000
3
8.6375
1.6891
0.3865
0.1180
4
27.9770
10.8612
0.0372*
0.0438*
5
79.2191
12.5932
0.0999
0.0287*
*
indicates a significant difference in total costs found from both methods
Kasetsart J. (Nat. Sci.) 41(2)
392
From the statistical tests, the qualities of
solutions found from both methods were not
significantly different for test problem sets 1, 2
and 3. However, Algorithm 2 became superior to
the pattern search for larger problem sets. Notice
on the test results, the total cost obtained from the
pattern search could be as poor as 68% deviating
from the MINLP lower bounds in large problem
instances, while those from Algorithm 2 would not
be worse than 20% from the lower bounds.
Algorithm 2 was hence the most efficient method
for solving this safety stock determination
problem, in terms of both solution quality and
computation time.
comparisons. The algorithms were found to work
very efficiently on the test problems. They could
provide high-quality solutions for every test
instance in less than 1 second. The deviations from
the known integer solutions or the lower bounds
were less than 3% on average. The algorithms also
outperformed the pattern search algorithm, which
was presented in the previous research.
DISCUSSION
LITURATURE CITED
It had been shown in the previous section
that Algorithm 2, which was based on a basic NLP
theorem, could provide high quality solutions in
short computation times for the safety stock level
determination problem in the considered two-stage
manufacturing system. The algorithm utilized only
a set of simple decision rules, in contrast to the
pattern search heuristic, which required the users
to comprehend its mechanisms. The decision rules
for finding optimum safety stocks also matched
the common managerial logics that when the
opportunity cost was high, the safety stocks should
be held to prevent the product deficiency, but they
should not be stocked when the inventory costs
were high. Moreover, the search heuristic such as
pattern search would terminate the search after
some stopping criteria had been satisfied.
Therefore, in some cases, it might not thoroughly
search the solution space for the solutions.
Inderfurth, K. and S. Minner. 1998. Safety Stocks
in Multi-Stage Inventory Systems under
Different Service Measures. Eur. J. Oper.
Res. 106: 57-73.
Krupp, J.A.G. 1997. Safety Stock Management.
Prod. Inventory Manag. J. 38(3): 11-18.
Kolda, T.G., R.M. Lewis and V. Torczon. 2003.
Optimization by Direct Search: New
Perspectives on Some Classical and Modern
Methods. Siam Rev. 45(3): 385–482
Maia, L.O.A. and R.Y. Qassim. 1999. Minimum
Cost Safety Stocks for Frequent Delivery
Manufacturing. Int. J. Prod. Econ. 62: 233236.
Marsden, J.E. and A. Tromba. 1981. Vector
Calculus. 2 nd ed. W.H. Freeman. San
Francisco. 591 p.
Montgomery, D.C. and G.C. Runger. 2004.
Applied Statistics and Probability for
Engineers, 3rd ed. John Wiley & Sons. New
Jersey. 706 p.
Silver, E.A., D.F. Pyke and R. Peterson. 1998.
Inventory Management and Production
Planning and Scheduling. 3rd ed. John Wiley
& Sons. New Jersey. 754 p.
Siribanluoewut, Y. 2006. Determining Safety
CONCLUSION
In this research, two algorithms for
determining safety stocks in a two-stage
manufacturing system were proposed by analyzing
the derivatives of cost function and the cost
ACKNOWLEDGEMENT
This project was funded by faculty of
Agro-Industry, Kasetsart University. The author
gratefully acknowledges this support.
Kasetsart J. (Nat. Sci.) 41(2)
Stock Quantities Using Heuristic
Optimization. M.S. Thesis. Kasetsart
University, Bangkok.
Talluri, S., K. Cetin and A.J. Gardner. 2004.
Integrating Demand and Supply Variability
into Safety Stock Evaluations. Int. J. Phys.
Distrib. Logist. Manag. 34: 62-69.
393
Vollmann, T.E., W.L. Berry and D.C. Whybark.
1997. Manufacturing Planning and Control
Systems, 4th ed. McGraw-Hills. New York.
836 p.
Zeng, A.Z. 2000. Efficiency of Using Fill-Rate
Criterion to Determine Safety Stock: A
Theoretical Perspective and a Case Study.
Prod. Inventory Manag. J. 41(2): 41-44.
Kasetsart J. (Nat. Sci.) 41 : 394 - 405 (2007)
Design and Implementation of a Framework for .NET-based Utility
Computing Infrastructure
Thanapol Rojanapanpat* and Putchong Uthayopas
ABSTRACT
Future organizations must handle a very large and complex IT infrastructure that consists of
very diverge and highly heterogeneous computing systems. Moreover, the future generation applications
must access services and resources regardless of the geographical location, access methods, and domain
of authorization. In order to meet these challenging requirements, a very high degree of virtualization
has to be implemented using a smart middleware. This is a very challenging problem for both theory
and practice.
This paper presents a new framework called OpenUCI (Open Utility Computing Infrastructure).
The OpenUCI project aims to explore the innovative design of scalable and flexible software infrastructure
that manages large scale heterogeneous distributed system ranging from large Server, PC, and Mobile
Devices. OpenUCI exploits a well established technology such as Grid, Web services and .NET technology
to build a virtualized and unify access to resources. Basic services that need to be presented will be
discussed. The prototype system has been implemented along with the prototype financial engineering
application. The results are presented along with the discussion of the experiences learned. With OpenUCI,
users can easily harness computing and storage of large distributed system.
Key words: utility computing, .NET technology, web services
INTRODUCTION
The competition in business causes
organizations to be ready to handle a large amount
of demand of users, which need more high
performance computing system. It is a risk for the
small and medium organizations to invest in the
high performance computing system, because they
have to pay for the system maintenance cost. There
are two solutions. Firstly they can outsource the
computing power. The other solution is to create
the supercomputing system by utilizing the already
existing personal computers (PC) in their
company. Building a supercomputing system from
personal computers or desktop PCs now is not an
imagination, because the speed and performance
of PCs has been increasing as well as the speed
and bandwidth of network. From this advantage,
it emerges many new computing systems; one of
them is the utility computing system.
Utility computing (Eilam et al., 2004) is
a computing model that involves the use of many
diverge technology such as grid computing (Foster
et al., 2002) and autonomic computing (Ganek and
Corbi, 2003). Utility computing system focuses
on the creating of virtual computing environment
High Performance Computing and Networking Center, Faculty of Engineering, Kasetsart University, Bangkok 10900, Thailand,
* Corresponding author, e-mail: thanapolr@hpcnc.cpe.ku.ac.th, pu@ku.ac.th
Received date : 03/10/06
Accepted date : 25/12/06
Kasetsart J. (Nat. Sci.) 41(2)
which dynamically and automatically virtualizes,
provisions and manages resources and services on
users’ demand. The major benefits of utility
computing are
• better utilization – the resources in
utility computing system can be shared and used
in efficient ways,
• more flexibility – utility computing
system provides flexibility in the creation of the
dynamic computing environment which can
automatically increase or decrease the computing
resources corresponding to users’ demand, and
• lower total cost of ownership – utility
computing can provide IT and business process
outsourcing which help reducing cost of investing
in resources such as hardware assets, maintenance
cost, training cost, etc.
The design and building of utility
computing infrastructure is still a complex and
challenging task. Figure 1 shows the concept of
resources virtualization and resources provisioning
in a modern IT infrastructure. Utility computing
system must consist of a way to provide both
features. Firstly, resources virtualization is a
feature of system that can make resources
transparent to application. Since the resources that
we use for build a utility computing infrastructure
are PCs, these systems have a high dynamism e.g.
resources can be available and unavailable from
time to time. The utility computing system must
have mechanisms for collecting resources and
monitoring its status. Furthermore, resource
virtualization should have mechanisms for
discovering and accessing resources. Secondly,
resources provisioning is a feature of a system to
perform an on-demand resources allocation to
application. A good utility computing system must
have mechanism for creating the automatic
adjustable virtual computing environments which
consist of hardware resources and utility services
in order to keep responsiveness when users’
demand increases. Moreover, the utility computing
system must provide friendly-used interfaces to
395
user, which may be business manager for
accessing and managing this virtual computing
environment. These interfaces can be Windows
applications, Web applications, command line, and
API.
Most utility computing systems are based
on current distributed system technology. Thierry
(2006) provides a good survey of platform
technology that is available. There are three
commonly used distributed systems and
technologies. The first one is distributed
information system that focuses on sharing
knowledge such as the web. Secondly, a distributed
storage system for sharing data such as peer-topeer files sharing. Finally, distributed computing
or metacomputing (Smarr and Catlett, 1992)
frameworks for sharing computing power. The
systems that are classified in this area and related
to OpenUCI framework are the followings.
Business
Processes/
Appications
Business
Processes/
Appications
Virtual
Computing
Environment
Virtual
Computing
Environment
Resources Provisioning
Virtualized Resource
Resources Virtualization
Resources/
Services
Resources/
Services
Resources/
Services
Figure 1 The relationship of
viortualization
and
provisioning.
resource
resource
396
Kasetsart J. (Nat. Sci.) 41(2)
Grid computing (Foster et al., 2002)
focuses on integrating geographically distributed
resources into a unified system. Grid computing
provides concept of Virtual Organization (VO)
which is an integrated resources shared by real
organizations, and it also has a well-defined
architecture, services and protocols such as
resource discovery, job submission, system
monitoring and accounting, which are good
patterns for designing and developing the utility
computing system. The most well-known project
in this area is Globus (The Globus Alliance,
2005a).
Peer-to-Peer (P2P) computing is a class
of applications that takes advantage of resources
such as storage, CPU cycles, and content that are
available on the Internet. There are two major
categories of P2P system, P2P networking (file
sharing) and P2P computing (CPU sharing), The
P2P networking is a communication model in
which each node (peer) has the same capabilities
and either node can directly initiate a
communication session. The P2P computing is a
processing power sharing rather than a files
sharing.
Volunteer computing (Sarmenta, 2001)
focuses on making computers to be a part of
metacomputer dynamically when computing
power is available. The topology of volunteer
computing is usually similar to the third generation
of peer-to-peer computing. The peer can be both
client, who submits jobs to server (super-peer),
and can be worker who dedicates itself to execute
jobs. This includes system such as SETI@home
(Anderson et al., 2002), Bayanihan (Sarmenta et
al., 2002), and Alchemi (Luther, 2005).
In this paper, we present a design and
implementation of a framework called OpenUCI
(Open Utility Computing Infrastructure) which is
for constructing the utility computing
infrastructure from Windows-based personal
computers, because the most of computers in the
organization are Windows-based operating system
and the most of users are familiar to Windows. To
solve the resource virtualization and resource
provisioning problems, OpenUCI framework
provides many services such as resource
collecting, resource monitoring, resource
discovering, resource invocation, and etc. In
addition, we use Microsoft’s .NET technology for
implementing the OpenUCI system because it
provides a powerful and comfortable development
environment and it also provides ASP.NET Web
service, a standard way for communication
between systems. So, we can ensure that all
OpenUCI’s components can work together and can
communicate to other systems seamlessly.
MATERIALS AND METHODS
1. Hardware and software requirements
This paper develops and tests a
framework on Windows-based system. The
computers used in this development comprise one
manager node, 32 worker nodes, and one user
node. All nodes are connected with Fast Ethernet
switch. The system configuration is shown in
Figure 2.
The software for developing and testing
the framework is as follows:
• Microsoft Windows Server 2003
• Microsoft Windows XP Professional
• .NET framework redistributed 1.1
and 2.0
• Microsoft Visual Studio .NET 2003
and 2005
Figure 2 The windows cluster.
Kasetsart J. (Nat. Sci.) 41(2)
2. Framework architecture and components
In this paper, utility service is a function
provided by any computers. The utility service
must depand on the Service Oriented Architecture
(SOA) technology such as .NET web services, and
Grid services. The example of utility service is
such web service for calculating risk of trading
stock (VaR) (Rojanapanpat et al., 2005). The
resource is an entity shared by a computer and can
be computing power (CPU), storage, files and
utility services.
According to the utility computing
system development problems mentioned before,
Resources Virtualization and Resources
Provisioning, the proposed framework, OpenUCI,
must be designed to solve these problems.
To deal with Resources Virtualization
problem, OpenUCI must have mechanism to
support the dynamism, heterogeneity, scalability,
interoperability of resources. The mechanisms are
such resource collecting for gathering resources
and track its status, resource discovery used to find
and select the resources, resource accessing which
defines a unite way to use and interoperate
resources and etc.
In the Resources Provisioning problem,
OpenUCI must provide mechanisms for creating
virtual computing environments that can be
automatically adjustable depending on demand of
users. Moreover, OpenUCI must provide userfriendly interfaces and tools using OpenUCI
system and accessay resources to users.
The architecture of the OpenUCI
framework is shown in Figure 3. There are four
layers of the OpenUCI framework, i.e. resources,
.NET platform, core services, and applications.
2.1 Resources layer
Resources layer is the layer of shared
resources distributed on the network. The shared
resources consist of CPU, storage and utility
services.
2.2 .NET platform layer
.NET platform layer provides a runtime
397
environment, .NET framework, which OpenUCI
system relies on. This layer also provides
technologies for implementing OpenUCI system,
and sharing resources. These technologies are
.NET web services, .NET remoting and
WSRF.NET. The resources can be shared via these
technologies.
2.3 Core layer
This layer provides a set of necessary
services for building the utility computing
infrastructure and supporting the basic functions
of the application running on the utility computing
infrastructure. The core services are classified into
two groups according to our requirements.
The core services that solve the resources
virtualization problem consist of resource
management service, data management service
and execution management service.
1. Resources Management Service
(RMS) is responsible for gathering resources
distributed on the network and tracking the
existence and status of resources. Moreover, RMS
also provides mechanisms for resource discovery,
resource reservation and etc.
2. Data Management Service (DMS) is
responsible for transferring files and sharing files
APPLICATIONS & TOOLS
VIRTUAL
USER
COMPUTER
MANAGEMENT
MANAGEMENT
RESOURCES PROVISIONING
JOB
MANAGEMENT
RESOURCES
MANAGEMENT
EXECUTION
MANAGEMENT
DATA
MANAGEMENT
RESOURCES VIRTUALIZATION
CORE SERVICES
.NET WEB
SERVICES
.NET WEB
REMOTING
WSRF .NET
.NET PLATFORM
CPU
STORAGE
SERVICE
RESOURCES
Figure 3 The OpenUCI architecture.
Kasetsart J. (Nat. Sci.) 41(2)
398
among computers in the OpenUCI system.
3. Execution Management Service
(EMS) is used to start and controls processes.
Furthermore, EMS also supports the invocation
of web and grid service jobs.
The core services that address the
resources provisioning problem consist of user
management service, virtual computer
management service and job management service.
1. User Management service (UMS)
handles authentication, authorization, accounting
and users profiles.
2. Virtual Computer Management
Service (VCMS) is used for managing and
controlling the virtual computing environment
created by users.
3. Job Management Service (JMS) is
used for creating jobs and supporting job
submission from users. JMS also provides job
queuing and scheduling mechanisms.
2.4 Applications and tools layer
Applications and tools layer is the layer
of user applications developed for using facilities
of OpenUCI system. OpenUCI system also
provides basic command-line tools and web
application interfaces for login, logout, virtual
computer creation, resources discovering, job
submission and etc.
There are three main components in
OpenUCI system as shown in Figure 4.
1. Manager is a computer that provides
core services used for managing shared resources
and supporting incoming requests of users.
2. Workers are computers that share its’
resources such as computing power, files, storage
and utility services. There are two worker types in
the OpenUCI system, dedicated and non-dedicated
workers. Dedicated workers are always online and
cannot reject jobs assigned by managers. For nondedicated workers, they can be online or offline
all the time and they will request for a job and
execute it when they are not busy.
3. Users are the people who need to
access resources. They can discover resources,
create job, submit job, download and upload files
and any services provided by managers.
RESULTS AND DISCUSSION
1. Proof of concept application
Currently, the high performance
computing is widely needed and not limited to the
computer research field anymore. The financial
engineering (FE) is a field that requires the high
computing power because it has to handle and
analyze a large amount of data in order to reduce
or keep turn around time constantly as number of
users increased. We evaluated the performance of
OpenUCI system by applying the existing
financial engineering application named Value-at-
Manager
Core Services
Workers
Agent
Users
Applications
Figure 4 The interaction of manager, worker and user.
CPU
Storage
Services
Kasetsart J. (Nat. Sci.) 41(2)
Risk (VaR) calculation which was implemented
in .NET web services. The VaR measures the
maximum loss money which may be occurred in
portfolio at a given time horizon (time of holding
portfolio) and at a given level of confidence. The
formula for calculating VaR has high complexity.
Then, we will show the general form of formula.
VaR = –Vp* (µp – Q*σp)
The Vp is the portfolio value, and the µp
and the σp are the expected return and the standard
deviation, respectively. The Q is the quantile value
of %confidence level. For example, the 99%
confidence level gives ~2.326 quantile value and
the 95% confidence level gives ~1.645 quantile
value.
In this test, we used the VaR calculation
web service as a utility service of OpenUCI system
which was installed to all worker machines and
then we developed VaR client program with
Microsoft Excel. The VaR Excel program uses the
OpenUCI API to connect to manager, discover
VaR web services and then invoke them.
2. Test configuration
The topology of test system is shown in
Figure 2. The software that was installed on each
machine is shown in Table 1.
3. Test assumptions
• Each worker executes only one job
at a time. Since the test application is a compute
intensive application, the execution of more than
399
one job on each worker will not give a better
performance due to the overhead of task switching.
• The input data is already in the
workers. This can be done by preloading fixed data
and table to worker prior to the execution. Thus,
the communication can be minimized which yield
a better performance for the system.
4. OpenUCI throughput test
We evaluated the throughput of
OpenUCI by submitting jobs to OpenUCI system
that has 1, 2, 4, 8, 16, and 32 workers, and the run
times used for testing are changed from 10, 30,
60, 90, 120, 180, 240, and 300 seconds. Figure 5
shows the procedure of this testing.
1. The client application discover URLs
of web service located on the worker nodes from
the manager.
2. The manager runs the resource
selection algorithm and returns the URLs of the
chosen worker node to requested client
application.
3. The client application uses the
returned URLs for connecting and invoking web
service on worker nodes. After that, the client
application will wait until there are some available
workers.
4. The worker node executes the service
and then it returns a result to client application.
5. The client program invokes web
service on an available worker
Table 1 Hardware and software configuration for testing OpenUCI system.
Machines
Hardware
Operating system
1 Manager
AMD Athlon 2.0GHz, 512
Windows server 2003
MB RAM
32 Workers
Intel Celeron 2.53GHz, 512
MB RAM
Windows XP
Professional
1 User
Intel Pentium M 1.5GHz,
768 MB RAM
Windows server 2003
Software
OpenUCI Broker, MS
SQL 2005 for
OpenUCI database
OpenUCI Worker,
MS SQL 2005
Express for VaR
database
VaR client application
Kasetsart J. (Nat. Sci.) 41(2)
400
VaR Client
(OpenUCI User)
OpenUCI Manager
OpenUCI Worker1
OpenUCI Worker n
1) Discovery for VaR web
service
2) Return the suitable
VaR web service URLs
Wait for available
worker
3) Invoke VaR web
service on all workers
4) Return the result
5) Continue invoking
Figure 5 The throughput test procedure.
The result of throughput test is shown in
Table 2 and Figure 6. Figure 7 shows average of
throughput of OpenUCI system based on the
different number of workers.
From these results, it shows that
OpenUCI system gave a good throughput when
the number of workers increased and the
increasing of throughput was nearby the increasing
of number of workers. For example, the average
throughput of 32 workers system was ~6.4 jobs/
sec and the average throughput of 1 worker system
was ~0.214 jobs/sec. The throughput was
increased about 30 times.
Table 2 The throughtput of OpenUCI.
Time
1 Worker
2 Workers
10
0.20
0.30
30
0.23
0.43
60
0.22
0.42
90
0.21
0.41
120
0.22
0.43
180
0.21
0.42
240
0.21
0.42
300
0.21
0.42
5. OpenUCI speed up test
In this test, we observed the run time used
to finish jobs when the number of workers was
changed from 1, 2, 4, 8, 16, to 32 workers. The
procedure of the speed up testing was similar to
the throughput testing, but the speed up test
changed the number of jobs submitted to system
and observed the run time instead of fixing the
run time and observed the number of finished jobs.
Table 3 and Figure 8 show the run time
of this testing. Table 4 and Figure 9 show the speed
up. Table 5 and Figure 10 show the efficiency.
4 Workers
0.70
0.73
0.82
0.79
0.84
0.83
0.84
0.84
8 Workers
1.40
1.63
1.57
1.64
1.67
1.63
1.67
1.65
16 Workers
3.10
3.17
3.18
3.24
3.31
3.30
3.31
3.31
32 Workers
6.00
6.27
6.33
6.44
6.47
6.59
6.56
6.60
Kasetsart J. (Nat. Sci.) 41(2)
401
Throughput
7
6
Throughput (job/sec)
5
1 Worker
2 Workers
4
4 Workers
3
8 Workers
16 Workers
2
32 Workers
1
0
0
30
60
90
120
150
180
210
240
270
300
330
Time (second)
Figure 6 The throughput of OpenUCI system.
Average Throughput
Average Throughput (job/sec)
7.00
6.40
6.00
5.00
4.00
3.2
4
3.00
2.00
1.61
0.80
1.00
0.21
0.41
0.00
1
2
4
8
Number of Workers
Figure 7 The average throughput of OpenUCI system.
16
32
Kasetsart J. (Nat. Sci.) 41(2)
402
Table 3 The run time of testing (second).
Worker
100 Jobs
500 Jobs
1
476.33
2359.17
2
248.03
1191.59
4
122.14
596.77
8
61.82
303.70
16
33.30
157.66
32
19.88
76.25
1000 Jobs
4726.77
2400.58
1185.05
609.31
308.28
151.92
2000 Jobs
10083.33
4734.84
2386.19
1216.19
619.43
301.97
3000 Jobs
14794.66
7106.32
3566.53
1825.01
923.29
451.55
Run time
100000
100 Jobs
500 Jobs
1000 Jobs
Time (second)
10000
2000 Jobs
3000 Jobs
1000
100
10
1
0
2
4
6
8
10 12 14 16 18
20 22 24
26
28 30 32
34
Number of workers
Figure 8 The run time plot.
Table 4 The speed up of testing.
Worker
100 Jobs
1
1.00
2
1.92
4
3.70
8
7.71
16
14.31
32
23.97
500 Jobs
1.00
1.98
3.95
7.77
14.96
30.94
1000 Jobs
1.00
1.97
3.99
7.76
15.33
31.11
2000 Jobs
1
2.13
4.23
8.29
16.28
33.39
3000 Jobs
1
2.08
4.15
8.11
16.02
32.76
Kasetsart J. (Nat. Sci.) 41(2)
403
Speed up
40
35
30
Speed up
25
20
15
100 Jobs
500 Jobs
10
1000 Jobs
5
2000 Jobs
3000 Jobs
0
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Number of workers
Figure 9 The speed up plot.
Table 5 The efficiency of testing.
Worker
100 Jobs
500 Jobs
1
1.00
1.00
2
0.96
0.99
4
0.97
0.99
8
0.96
0.97
16
0.89
0.94
32
0.75
0.97
1000 Jobs
1.00
0.98
0.99
0.97
0.96
0.97
The speed up (S) of n-workers system is
defined by the run time of 1-worker system
(sequential run time, Ts) divided by the run time
of n-workers system (parallel run time, Tp), and
the efficiency (E) is defined as the speed up (S)
divided by number of workers (P). From Figure 9
and Figure 10, we found that there were three
interesting characteristic results.
1. The speed up and efficiency were
decreased when the number of workers increased,
for example, 100 jobs testing. This characteristic
happened because all workers in system are not
fully utilized. For example, in 32-workers system,
it had to use 4 iterations to finish 100 jobs
(32+32+32+4 = 100). So, in the last iteration, there
were 28 workers free. Assume that 1 job used 1
second for executeing. The speed up was 25 (Ts/
Tp = 100/4 = 25), and the efficiency was 0.78 (S/
P = 25/32 = 0.78). If we submitted 128 jobs
(32+32+32+32) to this system, the speed up and
efficiency would be 32 (128/4) and 1, respectively.
2. The speed up and efficiency were
almost perfect. The perfect speed up was the speed
up that was equal to number of workers in system.
The perfect efficiency was the efficiency that is
equal to 1. Basically, the communication overhead
2000 Jobs
1.00
1.06
1.06
1.04
1.02
1.04
3000 Jobs
1.00
1.04
1.04
1.01
1.00
1.02
Kasetsart J. (Nat. Sci.) 41(2)
404
Effciency
1.2
Effciency
1
0.8
100 Jobs
500 Jobs
0.6
1000 Jobs
2000 Jobs
3000 Jobs
0.4
0
2
4
6
8
10
12
14
16
18
20
22
24
26
28
30
32
34
Number of workers
Figure 10 The efficiency plot.
such as input data transfer time makes the speed
up and efficiency dropped. In this test, we reduced
the data transfer time by replicating VaR database
to all workers. So, the efficiency and speed up were
nearly perfect.
3. The super speed up and the over
efficiency. This characteristic happened because
the overhead time before calling web services of
client application makes the run time of client
application increased. The high number of jobs
made the total overhead time grower. However,
the total overhead time was reduced by the
increasing of number of workers. So, at the large
amount of jobs such as 2000 and 3000 jobs, the
run times of 2, 4, 8, 16, and 32 workers system
were decreased more than the number of workers
in system.
CONCLUSION
The demand of using super computing
system in organizations has been increasing. They
need the system that has more dynamicity and
flexibility in order to support the various types and
large amount of demand of customers. Moreover,
this system must provide an easy and familiar
mechanism for customers to use the power of
system. This paper proposed the design and
implementation of framework used for building
the computing environment that can achieve these
requirements. This framework is called OpenUCI
(Open Utility Computing Infrastructure) which
works on Microsoft .NET platform. OpenUCI will
gather resources distributed on the network, and
automatically adjust and provisioning resources
to users. The prototype of OpenUCI has already
been implemented and evaluated with a financial
engineering application named VaR calculation.
The result of evaluation showed that OpenUCI can
give a good performance and high utilization when
the number of computers and demand of users
increased
The prototype version of OpenUCI has
only a few modules such as resource collecting
Kasetsart J. (Nat. Sci.) 41(2)
and discovery, resource selection and broker
mechanism. There are still many necessary
modules that should be implemented, for example,
web and grid services invoker, job queue manager
and virtual computer management. The following
is the list of future work. There are many possible
works in the future such as integrating the
executable file launcher implemented in another
related project to OpenUCI system, implementing
the job queue management module, implementing
the web and grid services invoker module,
implementing the virtual computer management
service, implementing the user authentication and
accounting modules, implementing the data
transfer service, exploring the mechanisms for
handling fault of machines and jobs and
investigating a proper workload distribution
scheme and study using simulation.
All these works will make OpenUCI
more useful in the modern computing
environments.
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