Journal
Journal of Applied Horticulture, 14(2): 88-91, 2012
Appl
Ginger juice enhanced growth of aromatic chilli during in vitro
culture and acclimatization
K. Bodhipadmaa, S. Noichindaa, P. Luangsriumporna, C. Meenapaa, K. Nathalangb
and D.W.M. Leungc*
a
Department of Agro-Industrial Technology, Faculty of Applied Science, King Mongkut’s University of Technology North
Bangkok, Bangsue, Bangkok 10800 Thailand. bDepartment of Biology, Faculty of Science, Mahidol University, Rama VI
Rd., Rajathevi District, Bangkok 10400, Thailand. cSchool of Biological Sciences, University of Canterbury, Private Bag
4800, Christchurch 8140, New Zealand. *E-mail: david.leung@canterbury.ac.nz
Abstract
Stem explants excised from seedlings of aromatic chilli (Capsicum frutescens L) grown under aseptic conditions were cultured on
basal medium alone (control), and basal medium supplemented with 5, 10 or 20 mL/L juice of ginger rhizome of 6 or 10 months old
(herein referred to as YGE and OGE, respectively). At the end of 6 weeks of culture, the average number of roots formed per stem
explant was higher when cultured on media supplemented with the three different levels of YGE or OGE (except 5 mL/L) compared
to the control. Roots, formed in stem explants cultured on media containing the different levels of YGE (except 20 mL/L) and OGE,
were longer than those cultured on basal medium. Particularly notable was that the average length of roots formed in stem explants
cultured on medium supplemented with 5 mL/L OGE was more than double that of the control. Prior culture on media containing
the different levels of YGE had no promotive effect on the number of leaves per exflasked plantlet compared to the control at the end
of three weeks of acclimatization but the plantlets cultured previously on 5 or 10 mL/L YGE were taller than the control. The best
performance of plantlets regarding leaf number and stem height after acclimatization was exhibited by those cultured previously on
medium containing 10 mL/L OGE as they had at least 20% more leaves and were taller than the control.
Introduction
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Key words: Acclimatization, aromatic chilli, ginger juice, root elongation, root induction
substances that can be sourced locally such as ginger rhizomes
in Thailand for micropropagation of plants. Chemical analysis of
ginger juice has shown that it has antimicrobial potential and it is
a good source of antioxidants, carbohydrates and some minerals
(Shirin and Prakash, 2010). Here, we evaluated the hypothesis
that different concentrations of ginger juice had some beneficial
effect not only on in vitro culture but also on acclimatization of
exflasked aromatic chilli plantlets. In addition, the effect of juice
from ginger rhizome of two different ages was compared as it
might be possible that biological activity could vary with different
ages of ginger rhizomes.
Many complex food extracts have been added to plant tissue
culture medium for different purposes. For example, there
are reports showing use of banana and apple juice to promote
growth of cymbidium protocorms (Kusumoto and Furukawa,
1977), pineapple juice for plantlet development of several orchid
species (Kitsaki et al., 2004), coconut water and tomato juice
for shoot multiplication from encapsulated buds of Pogostemon
cablin (Swamy et al., 2009), banana extract for rapid plant
regeneration of Dendrobium lituiflorum (Vyas et al., 2009), yeast
extract, casein hydrolysate and potato extract for callus induction
of Stevia rebaudiana (Das and Mandal, 2010) and sugarcane
juice for shoot proliferation and growth of Pogostemon cablin
and bananas (Swamy et al., 2010; Buah et al., 2011). Rooting
of shoots derived from juvenile stem explants of avocado was
promoted in medium supplemented with peptone and an auxin
(NAA or 1-naphthlene acetic acid) but not with the auxin alone
(Nhut et al., 2008). There is no report on use of complex natural
substances to improve performance of exflasked plantlets during
acclimatization, particularly, if these substances are commonly
available at a relatively low cost. This is an important practical
objective of micropropagation of plants.
Materials and methods
In our preliminary work, a protocol was developed for
regenerating plantlets from stem explants of aromatic chilli, an
important ingredient in many Thai cusine. It is of interest to the
plant production industry to find low-cost natural organic complex
Preparation of ginger juice: Ginger rhizomes (Zingiber
officinale Rosc. Cv. Yai) of about 6- and 10-months old, herewith
referred to as young and old ginger rhizomes, respectively, were
purchased from a local market in the Nonthaburi province,
Plant materials and surface-sterilization: Seeds of aromatic
chilli (Capsicum frutescens L.) were purchased from Thai Seed
and Agriculture Co., Ltd., Bangkok, Thailand. The following
sequence of steps was carried out to surface-sterilize the seeds:
(1) soaking in distilled water overnight, (2) washing briefly with
tap water and liquid soap, (3) immersing in 15% (v/v) Clorox
(a commercial bleach solution containing 5.25% (w/w) sodium
hypochlorite as available chlorine) for 15 min, (4) 10% (v/v)
Clorox containing 3-4 drops of Tween-20 for 5 min, and (5)
rinsing 3 times (1 min each time) with sterile distilled water.
Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization
Germination of seeds, explant preparation and culture:
Surface-sterilized aromatic chilli seeds were placed onto basal
MS (Murashige and Skoog, 1962) medium gelled with 0.9% agar
(w/v) and kept under 16 h of illumination with white fluorescent
lamps (47.31 μmol/m2/s light intensity) and 8 h of darkness in a
growth room at 25±2 °C. After 3 weeks of culture, stem explants
(2 cm long comprising the shoot tip and 2-3 nodes) were excised
from aromatic chilli seedlings which were approximately 3 to 4
cm tall. These explants (one per culture jar and there were six
replications) were transferred to MS medium supplemented with
different concentrations (5, 10 and 20 mL/L ) of juice from young
and old ginger rhizomes. The pH of all the media was adjusted
to 5.6 before they were autoclaved. The cultures were kept for 6
weeks under the same conditions as already described for aseptic
germination of the surfaced-sterilized seeds.
Plantlet acclimatization: Plantlets were taken out of culture jars
and the agar adhered to the roots were removed by shaking gently
in running tap water for a couple of minutes before they were
placed in plastic pots (5.8 cm tall, 3.2x4 cm on top and 2x2.5 cm
at the base of a pot) containing a soil mixture (soil:sand:chopped
rice straw in 5:4:1 ratio). One plantlet was placed in each pot
(there were six replications) filled to the top with the soil mixture
which was kept watered to capacity throughout the experiment.
Each pot was covered loosely with a clear plastic bag and lifted
briefly daily throughout the experiment so that the leaves were
sprayed with water using a hand-held water spray bottle. At
the beginning of the second week of acclimatization, two holes
(1.5 cm diameter each) were made on the side of the bag and six
more holes of the same size were made at the beginning of the
third week. All the plantlets were grown in a plant nursery under
a plastic shade net with 12 hours of natural sunlight a day and
average day temperature around 30-32oC and night temperature
around 22-23oC. Number of leaves and stem height (measured
from top of a pot to the shoot apex) per plantlet were determined
after 4 weeks of acclimatization under ex vitro conditions.
Data analysis: Means of root number, root length, leaf number and
stem height ± SE per plantlet were analyzed. One-way ANOVA
(STATISTIX for Windows 2.0 analytical software) was first performed
at the significance level of 0.05. After this, when appropriate, Duncan
comparison of means was carried out at P<0.05.
endogenous auxin or hormonal balance within these explants was
sufficient for adventitious root organogenesis (Bodhhipadma et
al., 2010). However, compared to control more roots per explant
were induced when the explants were cultured on basal MS
medium supplemented with different concentrations of YGE or
OGE with the exception of 5 mL/L OGE (Fig. 1). Interestingly,
YGE or OGE at the concentrations used in this study had no effect
on root formation in stem explants of Kaffir lime (Citrus hystrix
DC.), another plant that rooted in medium without exogenous
plant growth regulators (data not shown).
With the exception of 20 mL/L of YGE, different concentrations
of YGE or OGE added to basal MS medium promoted root
elongation in aromatic chilli stem explants compared to control
(Fig. 2). The promotive effect of 5 mL/L of YGE on elongation
of roots formed in explants was more than that of the higher
concentrations of YGE (Fig. 2). This was also observed among
different concentrations of OGE. Overall, the most effective
treatment was 5 mL/L of OGE as root length was more than double
a
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Thailand. Juice was extracted from 300 g of ginger rhizome (of
a particular age) which had been peeled and chopped into small
pieces using a juicer blender (Severin Entsafter Juicy 300 ES
3557). Young and old ginger rhizomes yielded 167 and 151 mL
of juice, respectively, which was added to plant tissue culture
medium as described below.
89
Fig. 1. Number of roots formed per stem explant (n=6) of aromatic chilli
cultured for 6 weeks on different media supplemented with different
concentrations of juice of young or old ginger rhizome (YGE or OGE
respectively). Values are mean number of roots±SE and those assigned
with different letters are significantly different (P<0.05). Different media:
1 = basal MS; 2= MS + 5 mL/L YGE; 3= MS + 10 mL/L YGE; 4= MS
+ 20 mL/L YGE; 5= MS + 5 mL/L OGE; 6= MS + 10 mL/L OGE; 7=
MS + 20 mL/L OGE.
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Results and discussion
Root formation and growth in vitro: Adventitious root
formation is a crucial step in micropropagation from stem
explants. Many studies have been carried out to investigate the
factors influencing rooting in vitro. An auxin such as IAA, IBA,
NAA or 2,4-D is generally added to plant tissue culture media
for root initiation in vitro (Saxena et al., 2000; Ashrafuzzaman et
al., 2009). In the present study, root formation occurred in stem
explants of aromatic chilli cultured on basal MS medium without
any exogenous plant growth regulators (control), suggesting that
Fig. 2. Elongation of roots formed by stem explants (n=6) of aromatic
chilli cultured for 6 weeks on different media supplemented with different
concentrations of juice of young or old ginger rhizome (YGE or OGE
respectively). Values are mean root length±SE and those assigned with
different letters are significantly different (P<0.05). Different media: 1
= basal MS; 2= MS + 5 mL/L YGE; 3= MS + 10 mL/L YGE; 4= MS
+ 20 mL/L YGE; 5= MS + 5 mL/L OGE; 6= MS + 10 mL/L OGE; 7=
MS + 20 mL/L OGE
Ginger juice enhanced growth of aromatic chilli during in vitro culture and acclimatization
Table 1. Per cent survival (A), number of leaves (B) and stem height (C)
of aromatic chilli plantlets from different media at the time of exflasking
(day 0) and after acclimatization for 4 weeks
(A) Survival of ex flasked aromatic chilli plantlets (%)
MS medium
Basal
+5 mL/L YGE
+10 mL/L YGE
+20 mL/L YGE
+5 mL/L OGE
+10 mL/L OGE
+20 mL/L OGE
Day 0
100+0a
100+0a
100+0a
100+0a
100+0a
100+0a
100+0a
4 weeks
100+0a
100+0a
100+0a
100+0a
100+0a
100+0a
100+0a
(B) Number of leaves per exflasked aromatic chilli plantlet
MS medium
Day 0
4 weeks
Basal
18.5 ± 0.922a
24.66 ± 1.406c
+5 mL/L YGE
15.83 ± 0.543b
25.0 ± 1.693c
+10 mL/L YGE
15.83 ± 0.401b
25.0 ± 0.730c
+20 mL/L YGE
14.66 ± 0.494bc
25.16 ± 0.401c
+5 mL/L OGE
18.33 ± 0.882a
29.5 ± 1.258b
+10 mL/L OGE
19.16 ± 0.307a
31.5 ± 0.719ab
+20 mL/L OGE
20.0 ± 1.0a
33.5 ± 1.408a
(C) Stem height (mm) per exflasked aromatic chilli plantlet
MS medium
Day 0
4 weeks
Basal
40.0 ± 0.775c
50.0 ± 2.236cd
+5 mL/L YGE
41.16 ± 0.477b
65.0 ± 3.416a
+10 mL/L YGE
43.5 ± 1.204ab
60.0 ± 0ab
+20 mL/L YGE
41.0 ± 0.931b
52.5 ± 1.708cd
+5 mL/L OGE
45.16± 0.477a
63.33 ± 2.108a
+10 mL/L OGE
46.16 ± 1.014a
59.16 ± 0.833ab
+20 mL/L OGE
45.83 ±1.400a
55.0 ± 1.291bc
Values are means of 6 replications±SE and those sharing the same letter
are not significantly different. (YGE = young ginger juice, OGE = old
ginger juice)
that of control. Taken together, depending on concentrations and
age of ginger rhizome, juice of ginger rhizome added to basal MS
medium could generate more beneficial effects than basal MS
medium alone as far as formation and growth of roots in stem
explants of aromatic chilli was concerned.
Besides ginger juice, several complex natural substances have
also been shown to have beneficial effects on root formation and
elongation in vitro. It was hypothesized that extracellular extracts
of cyanbacterial culture might add some plant growth regulators
or other factors that act directly in promoting rooting response
(Manickavelu et al., 2006). Peptone together with NAA was found
better than NAA alone for root initiation and elongation in stem
explants of avocado (Nhut et al., 2008). It was thought that peptone
had an indirect rooting effect as it prevented deterioration of the
stem explants so that NAA could initiate root formation. In the
present study, ginger juice might have substances that augment the
action of endogenous auxin in the aromatic chilli stem explants.
Effect of culture treatments on ex vitro acclimatization:
There was virtually no significant difference in the survival
rates of all the exflasked plantlets of aromatic chilli under the ex
vitro acclimatization conditions in this study, regardless of the
previous in vitro culture treatments (Table 1A). This suggests
that the acclimatization conditions used in the present study were
adequate to ensure high survival rates of aromatic chilli plantlets
following exflasking.
Leaf production: At the time of exflasking, the aromatic
chilli plantlets developed on YGE-supplemented media had
fewer leaves per plantlet than control or those developed on
media supplemented with OGE (Table 1B). After 4 weeks of
acclimatization, exflasked plantlets previously cultured on media
supplemented with different concentrations of OGE formed 2030% more leaves per plantlet than control and those previously
cultured on media supplemented with YGE (Table 1B). Therefore,
it would seem more beneficial to add OGE rather than YGE to
MS basal medium as far as boosting leaf production in exflasked
plantlets during acclimatization was concerned.
Stem height: At the end of in vitro culture, the aromatic
chilli plantlets in control were shorter than those developed
on media supplemented with YGE or OGE (Table 1C). After
acclimatization, exflasked plantlets previously cultured on media
supplemented with 5 or 10 mL/L but not 20 mL/L YGE or OGE
were taller than control. Plantlets from the OGE treatments (5 or
10 mL/L) were about 15-30% taller than control. These results
were consistent with the notion that in vitro treatments could
have lasting or long-term effects on in vivo growth of exflasked
plants (Economou and Read, 1987; Nowak and Shulaev, 2003;
Iacona and Muleo, 2010).
In conclusion, this is the first report on utility of ginger juice
to influence growth and development of other plants. Aromatic
chilli plantlets exflasked from medium supplemented with OGE
(10 mL/L), generally grew better than those previously cultured
with YGE in terms of both increase in leaf number and stem
height during acclimatization. It would seem worthwhile in future
to identify the chemical basis of this effect of OGE. It has been
shown that ginger juice has antioxidants, carbohydrates and some
minerals (Shirin and Prakash, 2010). These are likely to vary with
different ages of the ginger rhizome from which the ginger juice
is extracted. It would be of interest to investigate more closely
if these substances present in ginger juice might promote root
initiation, root elongation and improved performance of exflasked
aromatic chilli plantlets after acclimatization. In addition, since it
is well-known that auxin and cytokinin are generally involved in
plant regeneration (Bodhipadma et al., 2011), it seems worthwhile
to investigate the occurrence and levels of these plant hormones
in ginger juice. Although it remains to be determined what factors
in ginger juice might be responsible for its effects on plant growth
and development, the practical benefit of ginger juice for improving
growth of exflasked aromatic chilli plantlets during acclimatization
has been clearly demonstrated which was the primary objective of
this study. It would be of further interest to determine if ginger
juice has any effect on other in vitro processes including callus
induction and growth, somatic embryogenesis etc.
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