International Research Journal of Plant Science (ISSN: 2141-5447) Vol. 2(8) pp. 254-261, August, 2011
Available online http://www.interesjournals.org/IRJPS
Copyright © 2011 International Research Journals
Full length Research Paper
An efficient in vitro protocol for multiple shoot
induction in mulberry, Morus alba L variety V1
R. S. Sajeevan1,2, S. Jeba Singh1, Karaba N Nataraja1* and M. B. Shivanna2
1
Department of Crop Physiology, University of Agricultural Sciences, GKVK, Bangalore, Karnataka, India.
Department of Applied Botany, Kuvempu University, Jnanasahyadri, Shankaraghatta - 577451, Shimoga District,
Karnataka, India.
2
Accepted 16 July, 2011
An efficient in vitro protocol for multiple shoot induction was standardized using nodal segment
explant in a most popular mulberry, Morus alba variety V1. Explants collected from the field grown
plants were cultured on Murashige and Skoog (MS) medium supplemented with different
concentration/combination(s) of phytohormones. Multiple shoots were induced from the nodal
segments after 45 days of incubation on shoot induction medium containing MS with BAP (1.0 mg/l),
TDZ (0.1 mg/l) and NAA (0.25 mg/l). Further proliferation and elongation of adventitious buds were
observed in secondary medium containing MS supplemented with BAP (1.0 mg/l), NAA (0.25 mg/l) and
Gibberellic acid (GA3, 0.5 mg/l). Rooting was induced on MS medium containing indole-3-butyric acid
(IBA, 0.5 mg/l) with or without charcoal (1 %, w/v) and well rooted plantlets were hardened in sterilized
soilrite. In vitro grown plantlets showed 98% survival under green house conditions. The protocol
developed would be of great use for mass propagation of mulberry and also support mulberry
transgenic programs.
Key words: In vitro-culture, multiple shoot induction, mulberry-var-V1, nodal explants.
INTRODUCTION
Mulberry (Morus species), a primary host of silkworms
(Bombyx mori L.), is exploited on a commercial scale for
silk production (Wakhlu and Bhau 2000). In India,
different mulberry varieties suitable for different agroclimatic zones and agronomic practices have been
developed, amongst which a few promising clones are
Victory1 (V1),
*Corresponding author Email: nataraja_karaba@yahoo.com,
Telephone: 91-8023636713; Fax: 91-8023636713
Abbreviations:
BAP - 6-benzylaminopurine
GA3 - Gibberellic acid
IBA - indole-3-butyric acid
MS - Murashige and Skoog
NAA - α-Naphthalene acetic acid
PGR – Plant growth regulators
TDZ - Thidiazuron (1-phenyl-3- [1,2,3-thiadiazol-5-yl]
urea) S36, K-2, MR2 and S34 (Vijayan et al., 2004). V1 is
one of the most leading varieties in South India due to its
high yield potential and leaf quality.
Mulberry tree improvement through conventional
breeding is slow and also difficult due to its heterozygous
nature (Ravindranan and Lakshmi Sita 1994; Song and
Sink 2006). Vegetative propagation of mulberry through
grafting is not economically viable (Bhau 1999). Although
propagation through stem cuttings is possible and being
used, poor rooting ability of promising genotypes is a
major problem for large scale multiplication (Fotadar et
al., 1990). For targeted crop improvement through
biotechnological approaches, attempts have been made
to standardize in vitro regeneration protocols in different
mulberry varieties. Mulberry is a recalcitrant species in
terms of tissue culture, and shoot regeneration is greatly
dependent on the genotype, type of explant and
combination of growth regulator used in the culture media
(Sahoo et al., 1997; Bhau and Wakhlu 2001; Feyissa et
al., 2005). Using different explants such as axillary bud
(Jain et al., 1990; Yamanouchi et al., 1999; Vijayan et al.,
Sajeevan et al. 255
2000), hypocotyl and cotyledon (Bhatnagar et al., 2001),
shoot tip and nodal segment (Yadav et al., 1990; Vijaya
Chitra and Padmaja, 1999), leaf (Kapur et al., 2001) and
stem (Narayan et al., 1989), in vitro regeneration has
been attempted with various degrees of success. Since
there are variations in regeneration among mulberry
varieties (Tewary et al., 1996; Bhau and Wakhlu 2003;
Rao et al., 2010), the main aim of the study was to
develop and standardize a reproducible in vitro multiple
shoot induction protocol for quick regeneration of popular
variety Victory1 (V1).
MATERIALS AND METHOD
Plant material and explant preparation
The plant material used in this study was obtained from 4
year-old trees of the Morus (Morus alba L) variety, V1
grown in the mulberry garden of University of Agricultural
Sciences, Bangalore, India. Newly sprouted shoots were
excised from field grown plants of mulberry. After
removing the leaves, the nodal sections were washed
thoroughly using running tap water and later sterilized
with detergent (Tween 20, Sigma-Aldrich, USA). Washed
explants were treated with 0.5 % (w/v) systemic fungicide
(Bavistin, BASF, Australia) for 30 min and rinsed two to
three times with sterile distilled water. Explants were cut
into 5 cm lengths and surface sterilized by immersing in
70% (v/v) ethyl alcohol (Changshu Yangyuan chemical,
China) for 3 min followed by rinsing twice with sterilized
distilled water and subsequently soaked in a solution of
0.1 % (w/v) mercuric chloride (HgCl2, Sigma-Aldrich,
USA) for 12 min with gentle shaking. Traces of HgCl2
were completely removed by washing (five to six times)
with sterile double distilled water.
Medium preparation and establishment of aseptic
culture
The MS (Murashige and Skoog, 1962) basal medium was
prepared following standard protocol. The basal
regeneration medium contained MS salts, vitamins,
sucrose (3%, w/v) and bactoagar (0.8%, w/v). All growth
regulators were added before the pH of the medium was
adjusted to 5.7. Twenty milliliters of medium were poured
into each culture tube (diameter, 2.5 cm; height, 15 cm)
and sterilized by autoclaving at 121°C for 15 min at 105
kPa. Ends of the surface sterilized nodal explants (3.0 cm
length) were trimmed off, and the explants with single
auxiliary bud were cultured in culture medium containing
MS salts and vitamins, supplemented with sucrose (3%
w/v) along with varied concentration/combination(s) of
cytokinin and auxin. Cultures after inoculation were
incubated at 25 ± 2oC and 70–80% relative humidity with
a photoperiod of 16/8 h
light/dark using cool-white fluorescent lamps (photon flux
-2 -1
density, 55-75 µmol m s ).
Effect of growth regulators on multiple shoot
induction
Based on the preliminary experiments, four plant growth
regulators
(PGR,
Sigma-Aldrich,
USA),
6benzylaminopurine (BAP), thidiazuron (TDZ), αNaphthalene acetic acid (NAA) and Gibberellic acid (GA3)
were tested. The sterile explants were inoculated on an
initiation medium supplemented with BAP (0.5, 1.0, 1.5,
2.0, 2.5, and 3.0 mg/l) and TDZ (0.1, 0.25, 0.5, 0.75, 1.0,
1.25, 1.5, 1.75 and 2.0), either separately, or in
combination with NAA (0.25 mg/l). MS fortified with GA3
(0.25, 0.5 and 1.0 mg/l), BAP (1.0, 2.0 and 3.0 mg/l) and
NAA (0.25 mg/l) of different concentration/combination(s)
was used as secondary medium for proliferation and
elongation of the shoots. The number of shoots induced
per explant was recorded at 45 days after initiation of the
culture.
Rooting and plant acclimatization
Healthy shoots having 5-7cm length (4-6 leaf stage)
were separated and transferred to rooting media
containing full or half strength MS and Indole butyric acid,
(IBA, 0.5 mg/l) with or without activated charcoal (1.0 %,
w/v). Well rooted plantlets were removed from the culture
bottles, gently washed in sterile water to remove agar,
transferred to plastic pots containing sterile soilrite and
covered with transparent polyethylene sheet to maintain
humidity. After 15–25 days the hardened plants were
transplanted to pots filled with potting mixture 2:1:1
(garden soil, sand and farmyard manure), allowed to
grow in greenhouse and percentage of survival was
recorded 3 weeks after transplantation.
Statistical analysis
The data were analyzed by analysis of variance (ANOVA,
SPSS software package for Windows, release 15.0;
SPSS Inc., Chicago, IL, USA) to analyze the influence of
the basal media and the concentrations of plant growth
regulators on mulberry shoot proliferation. Significant
difference between means were assessed by Duncan's
Multiple Range Test (DMRT) (P = 0.05) (Gomez and
Gomez, 1976).
RESULTS
Effect of growth regulators on multiple shoot
induction
Culture of nodal explants inoculated on MS medium
supplemented with plant growth regulators increased
256 Int. Res. J. Plant Sci.
multiple shoot production as compared to hormone free
medium (Table 1). Nodal explants inoculated on MS
medium
supplemented
with
different
concentration/combination(s) of
growth regulators
initiated multiple shoot buds within 2 weeks of
inoculation. The MS medium fortified with BAP (1.0 mg/l),
TDZ (0.1 mg/l) and NAA (0.25 mg/l) showed a maximum
regeneration of 85.67% (Table 1). Higher concentrations
of BAP (2.0 and 3.0 mg/l) along with TDZ (0.1 mg/l) and
NAA (0.25 mg/l) resulted in reduction of regeneration as
compared to lower dose of BAP (1.0 mg/l) (Table 1).
Similarly, the shoot number was 7.33 per explant in MS
medium fortified with BAP (1.0 mg/l), TDZ (0.1 mg/l) and
NAA (0.25 mg/l), which was reduced to 5.67 and 4.33
when BAP concentrations were increased to 2.0 and 3.0
mg/l, respectively (Table 1; Figure 1a).
When nodal explants inoculated on MS medium
containing either BAP or TDZ, a maximum regeneration
efficiency of 68.33 and 64.00% was observed at 1.0 and
0.1 mg/l BAP and TDZ, respectively (Table 1). The
average number of shoots formed per explant varied
significantly (P<0.05) between the different treatments,
and BAP at concentration of 1.0 mg/l induced 4.33
shoots per explant with an average shoot length of 4.93
cm. When TDZ was supplemented with MS medium,
there was significant reduction in shoot length (Table 1;
Figure 1b). Significant difference was noticed in the
average number of leaves produced between the
treatments. A maximum of 4.67 leaves per explant was
observed when MS medium was supplemented with BAP
(1.0 mg/l), TDZ (0.1 mg/l) and NAA (0.25 mg/l) (Table 1).
Effect of secondary medium in proliferation and
elongation
The effect of secondary medium in mulberry shoot
proliferation and elongation was statistically significant in
all treatments (P<0.05). After 45 days of culture in
initiation medium, shoots regenerated were sub-cultured in
different concentration/combination(s) of GA3 containing
secondary medium. Transfer of explants to secondary
medium increased proliferation and elongated shoots
(Table 2, Figure 1c). The secondary culture medium
containing GA3, BAP and NAA was found to be suitable for
multiple shoot induction. The secondary medium, with 0.5,
1 and 0.25 mg/l GA3, BAP and NAA, respectively seems
to be the best combination for shoot proliferation and
elongation. Repeated subculture in the same secondary
medium induced higher shoot proliferation with an
average of 64.33 shoots per explant (Table 2, Figure 1d).
The maximum length of the shoots obtained was 5.20 cm
in the secondary medium supplemented with GA3 (0.5
mg/l) with BAP (1.0 mg/l) and NAA (0.25 mg/l) (Table 2).
There was no difference in the number of leaves
produced per explant in different treatments in secondary
medium after 40 days of subculture (Table 2).
Prolonged exposure to secondary medium resulted in the
production of vitrified shoots with callus formation at the
base of micro shoots (Figure 1e).
Rooting and plant acclimatization
Single healthy elongated shoots subcultured on rooting
media (MS + IBA, 0.5 mg/l) with or without activated
charcoal (1%, w/v) showed 100% initiations of roots
(Table 2; Figure 1f). Healthy, white colored and thick
roots were noticed 2 weeks after subculture, which
subsequently turned brown after 4 weeks in the same
medium (Table 2; Figure 1g). The frequency and nature
of roots induced from regenerated shoots were similar in
the presence or absence of activated charcoal (data not
shown). Well rooted plantlets transferred to sterile soilrite
in plastic pots were easily acclimatized to ex vitro
conditions and showed a survival rate of 100% (Figure
1h). Fully grown plants were subsequently transplanted
to large pots and maintained in the green house. The in
vitro grown plants showed robust growth with 98%
survival in the greenhouse.
DISCUSSION
This work describes a robust multiple shoot induction
protocol in mulberry variety V1 using nodal explants. We
noticed significant difference in regeneration frequency,
shoot number; shoot length among various PGR’s which
was similar to earlier reports in Morus alba (Chitra and
Padmaja, 2005) and H. abyssinica (Feyissa et al. 2005).
Anis et al. (2003) reported the maximum regeneration
efficiency of 80% in mulberry followed by Kim et al.
(1985) (76%), Oka and Ohyama (1986) (53%) and
Narayan et al. (1989) (50%). In our study, a maximum
regeneration efficiency of 85.67% were obtained with
BAP (1.0 mg/l), TDZ (0.1 mg/l) and NAA (0.25 mg/l)
which is the highest reported with nodal explants in
Morus species. Earlier reports by Kim et al. (1985) and
Vijayan et al. (1998) suggest that combination of PGRs
was found to be most effective in inducing higher
percentage of multiple shoots than individual ones. The
combination of BAP, TDZ and NAA was found to be
better in inducing multiple shoots.
TDZ is known to be effective for woody plant tissue
culture (Huetteman and Preece 1993), which can
stimulate shoot proliferation in many recalcitrant species
such as Cercis canadensis var. alba L. (Yusnita et al.
1990), muscadine grape (Gray and Benton 1991) and
Quercus robur L. (English oak; Chalupa, 1988). We
noticed similar kind of response in mulberry (var. V1)
where in, multiple shoots were induced from nodal
explants at a high frequency on MS medium containing
TDZ. Higher concentrations of TDZ in culture media
favored the regeneration efficiency but affected the shoot
number in
Sajeevan et al. 257
Table 1: Effect of various combinations of 6-benzylaminopurine (BAP), Thidiazuron (TDZ) and α-naphthalene acetic acid (NAA) on regeneration (%), number of shoots, length
of shoots and number of leaves per explant of nodal cultures of Morus alba var. V1. Data were collected after 45 days of culture in initiation medium
Treatments
Plant Growth Regulators (mg/l)
Regeneration (%)
No of shoots /explant
BAP
TDZ
NAA
T1
-
-
-
45.33 ± 3.53
gh
1.33 ± 0.33
T2
0.5
-
-
57.33 ± 3.53
cdefg
2.67 ± 0.33 cde
T3
1.0
-
-
68.33 ± 4.00
bc
4.33 ± 0.67
bc
T4
1.5
-
-
53.33 ± 3.53
defgh
3.67 ± 0.33
T5
2.0
-
-
46.67 ± 5.81
T6
2.5
-
-
T7
3.0
-
T8
-
T9
e
Average length of
shoots(cm)
Average no. of
leaves/explant
2.67 ± 0.43
cd
3.33 ± 0.33 bc
4.00 ± 0.12
b
3.33 ± 0.33 bc
4.93 ± 0.18
a
4.00 ± 0.58 abc
cd
4.13 ± 0.35
b
4.00 ± 0.58 abc
gh
4.00 ± 0.58 cd
4.13 ± 0.24
b
4.33 ± 0.33 ab
64.00 ± 7.06
bcde
3.00 ± 0.90 cde
4.10 ± 0.10
b
3.33 ± 0.33 bc
-
65.33 ± 2.31
bcd
3.33 ± 0.33 cd
4.23 ± 0.23
b
4.00 ± 0.00 abc
0.1
-
64.00 ± 3.53
bcde
3.33 ± 0.33 cd
1.87 ± 0.18
fgh
4.33 ± 0.33 ab
-
0.25
-
46.67 ± 4.62
gh
3.33 ± 0.67 cd
1.53 ± 0.09
hi
3.33 ± 0.33 bc
T10
-
0.50
-
45.33 ± 3.53
gh
2.67 ± 0.88 cde
1.70 ± 0.17
ghi
3.67 ± 0.33 abc
T11
-
0.75
-
50.67 ± 3.53
fgh
3.67 ± 0.33 cd
1.90 ± 0.15
efgh
3.33 ± 0.33 bc
T12
-
1.0
-
54.67 ± 4.81
defgh
2.33 ± 0.33
de
2.43 ± 0.29
cdef
3.33 ± 0.33 bc
T13
-
1.25
-
52.00 ± 5.81
efgh
2.33 ± 0.33
de
1.67 ± 0.18
hi
3.67 ± 0.33 abc
T14
-
1.50
-
50.67 ± 2.31
fgh
2.67 ± 0.33 cde
1.50 ± 0.06
hi
3.33 ± 0.33 bc
T15
-
1.75
-
44.00 ± 3.53
h
2.67 ± 0.33 cde
1.13 ± 0.09
i
3.67 ± 0.33 abc
T16
-
2.0
-
42.67 ± 2.31
h
3.00 ± 0.58
cde
1.20 ± 0.12
i
3.00 ± 0.00 c
T17
1
0.1
0.25
85.67 ± 3.53
a
7.33 ± 0.33
a
3.00 ± 0.15
c
4.67 ± 0.33 a
T18
2
0.1
0.25
62.67 ± 1.20
bcdef
5.67 ± 0.33
b
2.53 ± 0.30
cde
4.00 ± 0.58 abc
T19
3
0,1
0.25
74.67 ± 5.81
ab
4.33 ± 0.88
bc
2.33 ± 0.20
defg
4.33 ± 0.33 ab
Means ± SE followed by different letters are significantly different at P=0.05 according to the Duncan's Multiple Range Test (DMRT).
Regeneration efficiency was calculated by (number of explants regenerated / total number to explants inoculated) x 100.
258 Int. Res. J. Plant Sci.
Figure 1: In vitro multiplication of Morus alba variety V1. (a) Multiple shoots emerging from the basal region, (b) Cluster of short
shoots in culture medium containing TDZ, (c) Elongated multiple shoots growing on culture medium, (d) Multiple shoots after 2
subcultures, (e) Vitrified shoots in culture medium, (f) Plantlets with healthy roots in root-induction medium, (g) Plantlet with well
developed roots after 4 weeks, (h) In vitro grown plant.
Figure 2: Schematic representation showing time frame for in vitro establishment of Morus alba variety V1.
Sajeevan et al. 259
Table 2: Effect of various combinations of 6-benzylaminopurine (BAP), gibberellic acid (GA3) and α-naphthalene acetic acid (NAA) on number of shoots
per explant and length of shoots of nodal cultures of Morus alba var. V1. Data were collected after 40 days of culture in secondary medium.
Treatments
Plant Growth Regulators (mg/l)
No of shoots
/explant
Average length of
shoots (cm)
Average no. of
leaves/explant
Rooting (%)
BAP
GA3
NAA
T1
-
0.25
-
30.33 ± 2.40 e
4.70 ± 0.35 ab
3.67 ± 0.33 a
95.33 ± 0.33 a
T2
-
0.5
-
42.33 ± 2.60 d
4.80 ± 0.12 ab
4.00 ± 0.57 a
98.66 ± 0.33 a
T3
-
1.0
-
42.33 ± 2.40 d
4.57 ± 0.15 bc
4.33 ± 0.33 a
98.00 ± 0.58 a
T4
1
0.25
0.25
49.33 ± 3.48 cd
5.03 ± 0.18 ab
4.00 ± 0.00 a
100.00 ± 0.00 a
T5
2
0.25
0.25
53.33 ± 2.33 bc
4.93 ± 0.20 ab
4.33 ± 0.33 a
97.66 ± 0.33 a
T6
3
0.25
0.25
61.00 ± 1.53 ab
4.90 ± 0.06 ab
3.67 ± 0.33 a
98.66 ± 0.33 a
T7
1
0.5
0.25
64.33 ± 3.18 a
5.20 ± 0.11 a
4.63 ± 0.33 a
100.00 ± 0.00 a
T8
2
0.5
0.25
62.67 ± 2.40 a
4.80 ± 0.15 ab
3.33 ± 0.33 a
98.66 ± 0.33 a
T9
3
0.5
0.25
58.67 ± 1.86 ab
4.10 ± 0.10 c
3.67 ± 0.33 a
97.33 ± 0.33 a
T10
1
1.0
0.25
57.67 ± 4.18 ab
4.87 ± 0.03 ab
3.67 ± 0.33 a
98.66 ± 0.33 a
T11
2
1.0
0.25
54.33 ± 2.33 bc
4.73 ± 0.18 ab
4.00 ± 0.58 a
95.33 ± 0.33 a
T12
3
1.0
0.25
49.33 ± 0.88 cd
4.17 ± 0.12 c
4.33 ± 0.33 a
97.66 ± 0.33 a
Means ± SE followed by different letters are significantly different at P=0.05 according to the Duncan's Multiple Range Test (DMRT).
260 Int. Res. J. Plant Sci.
subsequent subcultures due to callus formation.
Reduced concentration of TDZ in the media greatly
increased the regeneration efficiency and shoots
induction without callus formation. Recent research on
the
propagation
of
Holarrhena
antidysenterica
(Mallikarjuna et al. 2007) also showed that TDZ is more
effective at lower concentrations. The inhibitory effects of
TDZ on shoot elongation have been reported previously
by Preece and Imel (1991), Feyissa et al. (2005), Raghu
et al. (2006) and Lyyra et al. (2006) and our results are in
agreement with the findings above.
A two-step
regeneration strategy, using TDZ to induce shoot
formation followed by TDZ-free medium to promote shoot
proliferation and elongation, has been reported for
northern high bush blueberry cultivars and wild low bush
blueberry (Vaccinium angustifolium Ait.) (Song and Sink
2004; Debnath 2005; 2009). Interestingly, in this study,
repeated subculture (2 cycles of 20 days interval) in
secondary medium containing GA3, BAP and NAA
significantly increased multiple shoots, with an average of
64.33 shoots per explant. Such modifications in media
compositions have produced useful results in a few species
such as apple (Fasolo et al,. 1989), pear (Singha and Bhatia
1988), and Populus (Russell and McCown 1986). Prolonged
exposure to secondary medium resulted in the production of
abnormal shoots, with brittle leaves having a glassy
appearance called “vitrification”. This phenomenon has also
been observed in previous studies (Zhou et al., 2000;
Ozden-Tokatli et al., 2005; Chandra et al., 2006; Siddique
and Anis, 2007). Li et al. (2003) proposed that excess of
cytokinins along with the high water potential of the medium
were the major reasons for the vitrification of plantlets. The
vitrified shoots transferred to PGR free medium regained
normal growth and development.
Spontaneous rooting was observed in a few treatments
during the proliferation phase in secondary medium
containing NAA. The plantlets thus obtained could be
planted directly and acclimatized without passing through
the rooting phase in vitro. Those plantlets that did not
produce spontaneous roots in secondary medium were
cultured in rooting medium with IBA (0.5 mg/l) with or
without activated charcoal (1.0%, w/v). Root initiation of
cultured plantlets was observed within 15 days of
culturing and 100% rooting was noticed. High frequencies
of root induction were also reported for other plant
species such as Beta vulgaris (Gürel et al., 1995) and
Juniperus phoenicea (Loureiro et al. 2007). Well rooted
plantlets exhibited normal growth under green house
conditions with 98% survival rate. Plants derived from
multiple shoot induction protocol showed no apparent
difference in phenotype after 4 months of growing in the
greenhouse.
CONCLUSION
The present work describes an effective, high frequency
multiple shoot induction protocol for Indian mulberry
variety, V1 using nodal shoot segments. A two step
approach involving establishment of cultures on the
specific initiation medium (MS + BAP + TDZ + NAA) for
45 days and subsequent transfer to the secondary
medium (MS + BAP + NAA + GA3) resulted in further
proliferation and elongation of shoots. Repeated
subcultures in secondary medium produced 64.33 shoots
per explant and well rooted, healthy plantlets were
hardened and successfully established in the greenhouse
conditions. The micro-propagation system described in
this study (Figure 2) provides an effective means for the
large-scale propagation of high yielding mulberry
varieties.
ACKNOWLEDGMENTS
We gratefully acknowledge the Department of
Biotechnology (DBT), Government of India, New Delhi,
for providing financial support to carry out this work. We
wish to thank Professor Chandrashekar Reddy P for his
valuable suggestions and Ms. Pallavi BM for her help
during the experiments.
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