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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.
2
Department of Applied Botany, Kuvempu University, Jnanasahyadri, Shankaraghatta - 577451, Shimoga District,
Karnataka, India.
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 TDZ - Thidiazuron (1-phenyl-3- [1,2,3-thiadiazol-5-yl]
(Bombyx mori L.), is exploited on a commercial scale for urea) S36, K-2, MR2 and S34 (Vijayan et al., 2004). V1 is
silk production (Wakhlu and Bhau 2000). In India, one of the most leading varieties in South India due to its
different mulberry varieties suitable for different agro- high yield potential and leaf quality.
climatic zones and agronomic practices have been Mulberry tree improvement through conventional
developed, amongst which a few promising clones are breeding is slow and also difficult due to its heterozygous
Victory1 (V1), nature (Ravindranan and Lakshmi Sita 1994; Song and
Sink 2006). Vegetative propagation of mulberry through
grafting is not economically viable (Bhau 1999). Although
*Corresponding author Email: nataraja_karaba@yahoo.com, propagation through stem cuttings is possible and being
Telephone: 91-8023636713; Fax: 91-8023636713 used, poor rooting ability of promising genotypes is a
major problem for large scale multiplication (Fotadar et
al., 1990). For targeted crop improvement through
Abbreviations: biotechnological approaches, attempts have been made
to standardize in vitro regeneration protocols in different
BAP - 6-benzylaminopurine mulberry varieties. Mulberry is a recalcitrant species in
GA3 - Gibberellic acid terms of tissue culture, and shoot regeneration is greatly
IBA - indole-3-butyric acid dependent on the genotype, type of explant and
MS - Murashige and Skoog combination of growth regulator used in the culture media
NAA - -Naphthalene acetic acid (Sahoo et al., 1997; Bhau and Wakhlu 2001; Feyissa et
PGR Plant growth regulators 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), light/dark using cool-white fluorescent lamps (photon flux
-2 -1
shoot tip and nodal segment (Yadav et al., 1990; Vijaya density, 55-75 mol m s ).
Chitra and Padmaja, 1999), leaf (Kapur et al., 2001) and
stem (Narayan et al., 1989), in vitro regeneration has Effect of growth regulators on multiple shoot
been attempted with various degrees of success. Since induction
there are variations in regeneration among mulberry
varieties (Tewary et al., 1996; Bhau and Wakhlu 2003; Based on the preliminary experiments, four plant growth
Rao et al., 2010), the main aim of the study was to regulators (PGR, Sigma-Aldrich, USA), 6-
develop and standardize a reproducible in vitro multiple benzylaminopurine (BAP), thidiazuron (TDZ), -
shoot induction protocol for quick regeneration of popular Naphthalene acetic acid (NAA) and Gibberellic acid (GA3)
variety Victory1 (V1). 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,
MATERIALS AND METHOD 1.25, 1.5, 1.75 and 2.0), either separately, or in
combination with NAA (0.25 mg/l). MS fortified with GA3
Plant material and explant preparation (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)
The plant material used in this study was obtained from 4 was used as secondary medium for proliferation and
year-old trees of the Morus (Morus alba L) variety, V1 elongation of the shoots. The number of shoots induced
grown in the mulberry garden of University of Agricultural per explant was recorded at 45 days after initiation of the
Sciences, Bangalore, India. Newly sprouted shoots were culture.
excised from field grown plants of mulberry. After
removing the leaves, the nodal sections were washed Rooting and plant acclimatization
thoroughly using running tap water and later sterilized
with detergent (Tween 20, Sigma-Aldrich, USA). Washed Healthy shoots having 5-7cm length (4-6 leaf stage)
explants were treated with 0.5 % (w/v) systemic fungicide were separated and transferred to rooting media
(Bavistin, BASF, Australia) for 30 min and rinsed two to containing full or half strength MS and Indole butyric acid,
three times with sterile distilled water. Explants were cut (IBA, 0.5 mg/l) with or without activated charcoal (1.0 %,
into 5 cm lengths and surface sterilized by immersing in w/v). Well rooted plantlets were removed from the culture
70% (v/v) ethyl alcohol (Changshu Yangyuan chemical, bottles, gently washed in sterile water to remove agar,
China) for 3 min followed by rinsing twice with sterilized transferred to plastic pots containing sterile soilrite and
distilled water and subsequently soaked in a solution of covered with transparent polyethylene sheet to maintain
0.1 % (w/v) mercuric chloride (HgCl2, Sigma-Aldrich, humidity. After 1525 days the hardened plants were
USA) for 12 min with gentle shaking. Traces of HgCl2 transplanted to pots filled with potting mixture 2:1:1
were completely removed by washing (five to six times) (garden soil, sand and farmyard manure), allowed to
with sterile double distilled water. grow in greenhouse and percentage of survival was
recorded 3 weeks after transplantation.

Medium preparation and establishment of aseptic Statistical analysis


culture
The data were analyzed by analysis of variance (ANOVA,
The MS (Murashige and Skoog, 1962) basal medium was SPSS software package for Windows, release 15.0;
prepared following standard protocol. The basal SPSS Inc., Chicago, IL, USA) to analyze the influence of
regeneration medium contained MS salts, vitamins, the basal media and the concentrations of plant growth
sucrose (3%, w/v) and bactoagar (0.8%, w/v). All growth regulators on mulberry shoot proliferation. Significant
regulators were added before the pH of the medium was difference between means were assessed by Duncan's
adjusted to 5.7. Twenty milliliters of medium were poured Multiple Range Test (DMRT) (P = 0.05) (Gomez and
into each culture tube (diameter, 2.5 cm; height, 15 cm) Gomez, 1976).
and sterilized by autoclaving at 121C 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 RESULTS
auxiliary bud were cultured in culture medium containing
MS salts and vitamins, supplemented with sucrose (3% Effect of growth regulators on multiple shoot
w/v) along with varied concentration/combination(s) of induction
cytokinin and auxin. Cultures after inoculation were
incubated at 25 2oC and 7080% relative humidity with Culture of nodal explants inoculated on MS medium
a photoperiod of 16/8 h supplemented with plant growth regulators increased
256 Int. Res. J. Plant Sci.

multiple shoot production as compared to hormone free Prolonged exposure to secondary medium resulted in the
medium (Table 1). Nodal explants inoculated on MS production of vitrified shoots with callus formation at the
medium supplemented with different base of micro shoots (Figure 1e).
concentration/combination(s) of growth regulators
initiated multiple shoot buds within 2 weeks of Rooting and plant acclimatization
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 Single healthy elongated shoots subcultured on rooting
regeneration of 85.67% (Table 1). Higher concentrations media (MS + IBA, 0.5 mg/l) with or without activated
of BAP (2.0 and 3.0 mg/l) along with TDZ (0.1 mg/l) and charcoal (1%, w/v) showed 100% initiations of roots
NAA (0.25 mg/l) resulted in reduction of regeneration as (Table 2; Figure 1f). Healthy, white colored and thick
compared to lower dose of BAP (1.0 mg/l) (Table 1). roots were noticed 2 weeks after subculture, which
Similarly, the shoot number was 7.33 per explant in MS subsequently turned brown after 4 weeks in the same
medium fortified with BAP (1.0 mg/l), TDZ (0.1 mg/l) and medium (Table 2; Figure 1g). The frequency and nature
NAA (0.25 mg/l), which was reduced to 5.67 and 4.33 of roots induced from regenerated shoots were similar in
when BAP concentrations were increased to 2.0 and 3.0 the presence or absence of activated charcoal (data not
mg/l, respectively (Table 1; Figure 1a). shown). Well rooted plantlets transferred to sterile soilrite
When nodal explants inoculated on MS medium in plastic pots were easily acclimatized to ex vitro
containing either BAP or TDZ, a maximum regeneration conditions and showed a survival rate of 100% (Figure
efficiency of 68.33 and 64.00% was observed at 1.0 and 1h). Fully grown plants were subsequently transplanted
0.1 mg/l BAP and TDZ, respectively (Table 1). The to large pots and maintained in the green house. The in
average number of shoots formed per explant varied vitro grown plants showed robust growth with 98%
significantly (P<0.05) between the different treatments, survival in the greenhouse.
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, DISCUSSION
there was significant reduction in shoot length (Table 1;
Figure 1b). Significant difference was noticed in the This work describes a robust multiple shoot induction
average number of leaves produced between the protocol in mulberry variety V1 using nodal explants. We
treatments. A maximum of 4.67 leaves per explant was noticed significant difference in regeneration frequency,
observed when MS medium was supplemented with BAP shoot number; shoot length among various PGRs which
(1.0 mg/l), TDZ (0.1 mg/l) and NAA (0.25 mg/l) (Table 1). 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
Effect of secondary medium in proliferation and efficiency of 80% in mulberry followed by Kim et al.
elongation (1985) (76%), Oka and Ohyama (1986) (53%) and
Narayan et al. (1989) (50%). In our study, a maximum
The effect of secondary medium in mulberry shoot regeneration efficiency of 85.67% were obtained with
proliferation and elongation was statistically significant in BAP (1.0 mg/l), TDZ (0.1 mg/l) and NAA (0.25 mg/l)
all treatments (P<0.05). After 45 days of culture in which is the highest reported with nodal explants in
initiation medium, shoots regenerated were sub-cultured in Morus species. Earlier reports by Kim et al. (1985) and
different concentration/combination(s) of GA3 containing Vijayan et al. (1998) suggest that combination of PGRs
secondary medium. Transfer of explants to secondary was found to be most effective in inducing higher
medium increased proliferation and elongated shoots percentage of multiple shoots than individual ones. The
(Table 2, Figure 1c). The secondary culture medium combination of BAP, TDZ and NAA was found to be
containing GA3, BAP and NAA was found to be suitable for better in inducing multiple shoots.
multiple shoot induction. The secondary medium, with 0.5, TDZ is known to be effective for woody plant tissue
1 and 0.25 mg/l GA3, BAP and NAA, respectively seems culture (Huetteman and Preece 1993), which can
to be the best combination for shoot proliferation and stimulate shoot proliferation in many recalcitrant species
elongation. Repeated subculture in the same secondary such as Cercis canadensis var. alba L. (Yusnita et al.
medium induced higher shoot proliferation with an 1990), muscadine grape (Gray and Benton 1991) and
average of 64.33 shoots per explant (Table 2, Figure 1d). Quercus robur L. (English oak; Chalupa, 1988). We
The maximum length of the shoots obtained was 5.20 cm noticed similar kind of response in mulberry (var. V1)
in the secondary medium supplemented with GA3 (0.5 where in, multiple shoots were induced from nodal
mg/l) with BAP (1.0 mg/l) and NAA (0.25 mg/l) (Table 2). explants at a high frequency on MS medium containing
There was no difference in the number of leaves TDZ. Higher concentrations of TDZ in culture media
produced per explant in different treatments in secondary favored the regeneration efficiency but affected the shoot
medium after 40 days of subculture (Table 2). number in
Sajeevan et al. 257

Table 1: Effect of various combinations of 6-benzylaminopurine (BAP), Thidiazuron (TDZ) and -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 Average length of Average no. of
shoots(cm) leaves/explant

BAP TDZ NAA

T1 - - - 45.33 3.53 gh 1.33 0.33 e 2.67 0.43 cd 3.33 0.33 bc


T2 0.5 - - 57.33 3.53 cdefg 2.67 0.33 cde 4.00 0.12 b 3.33 0.33 bc
T3 1.0 - - 68.33 4.00 bc 4.33 0.67 bc 4.93 0.18 a 4.00 0.58 abc
T4 1.5 - - 53.33 3.53 defgh 3.67 0.33 cd 4.13 0.35 b 4.00 0.58 abc
T5 2.0 - - 46.67 5.81 gh 4.00 0.58 cd 4.13 0.24 b 4.33 0.33 ab
T6 2.5 - - 64.00 7.06 bcde 3.00 0.90 cde 4.10 0.10 b 3.33 0.33 bc
T7 3.0 - - 65.33 2.31 bcd 3.33 0.33 cd 4.23 0.23 b 4.00 0.00 abc
T8 - 0.1 - 64.00 3.53 bcde 3.33 0.33 cd 1.87 0.18 fgh 4.33 0.33 ab
T9 - 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 -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 Average length of Average no. of Rooting (%)
/explant shoots (cm) leaves/explant

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. approach involving establishment of cultures on the
Reduced concentration of TDZ in the media greatly specific initiation medium (MS + BAP + TDZ + NAA) for
increased the regeneration efficiency and shoots 45 days and subsequent transfer to the secondary
induction without callus formation. Recent research on medium (MS + BAP + NAA + GA3) resulted in further
the propagation of Holarrhena antidysenterica proliferation and elongation of shoots. Repeated
(Mallikarjuna et al. 2007) also showed that TDZ is more subcultures in secondary medium produced 64.33 shoots
effective at lower concentrations. The inhibitory effects of per explant and well rooted, healthy plantlets were
TDZ on shoot elongation have been reported previously hardened and successfully established in the greenhouse
by Preece and Imel (1991), Feyissa et al. (2005), Raghu conditions. The micro-propagation system described in
et al. (2006) and Lyyra et al. (2006) and our results are in this study (Figure 2) provides an effective means for the
agreement with the findings above. A two-step large-scale propagation of high yielding mulberry
regeneration strategy, using TDZ to induce shoot varieties.
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 ACKNOWLEDGMENTS
blueberry (Vaccinium angustifolium Ait.) (Song and Sink
2004; Debnath 2005; 2009). Interestingly, in this study, We gratefully acknowledge the Department of
repeated subculture (2 cycles of 20 days interval) in Biotechnology (DBT), Government of India, New Delhi,
secondary medium containing GA3, BAP and NAA for providing financial support to carry out this work. We
significantly increased multiple shoots, with an average of wish to thank Professor Chandrashekar Reddy P for his
64.33 shoots per explant. Such modifications in media valuable suggestions and Ms. Pallavi BM for her help
compositions have produced useful results in a few species during the experiments.
such as apple (Fasolo et al,. 1989), pear (Singha and Bhatia
1988), and Populus (Russell and McCown 1986). Prolonged
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