American Journal of Plant Sciences, 2013, 4, 2001-2005 Published Online October 2013 (
In Vitro Plant Regeneration of Morus indica L. cv. V1
Using Leaf Explant
Melur Kodandaram Raghunath1, Karaba N. Nataraja2*, Jyothi Sainath Meghana1,
Radha Sivarajan Sajeevan2, Mala V. Rajan1, Syed Mashayak Hussaine Qadri1
1Central Sericultural Research and Training Institute, Srirampura, Mysore, India; 2Department of Crop Physiology, University of
Agricultural Sciences, GKVK, Bangalore, India.
Email: *
Received March 26th, 2013; revised April 27th, 2013; accepted May 19th, 2013
Copyright © 2013 Melur Kodandaram Raghunath et al. This is an open access article distributed under the Creative Commons Attribu-
tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Adventitious bud induction and plantlet regeneration were studied in a popular mulberry variety, V1 using leaf as an
explant. Fully expanded leaf explants were cultured on Murashige and Skoog’s (MS) medium supplemented with thidi-
azuron (TDZ) (0.5 - 4.0 mg/l), 6-benzylaminopurine (BAP) (0.5 - 2.0 mg/l), indole acetic acid (IAA) (2.0 mg/l), gibber-
lic acid (GA3) (1.0 - 2.0 mg/l) silver nitrate (AgNO3) (2.0 mg/l) and different carbon sources such as sucrose, fructose
and glucose (10% - 30%) either individually or in combination to induce adventitious buds and regeneration. The high-
est percentage (63%) of adventitious bud formation and regeneration (68%) was achieved in the medium containing MS
with TDZ (1.0 mg/l), IAA (2.0 mg/l) and AgNO3 (2.0 mg/l). For subsequent regeneration and shoot elongation the MS
medium having BAP (1.0 mg/l), GA3 (2.0 mg/l) and AgNO3 (2.0 mg/l) was found to be suitable. Amongst the carbon
sources tested, the most suitable carbon source was found to be sucrose (3%) followed by fructose (2%) for adventitious
bud formation. Excised in vitro shoots were rooted (60% - 80%) in half strength MS medium supplemented with in-
dole-3-butyric acid (1.0 mg/l). The well rooted plantlets were hardened in soil + sand + farm yard manure (FYM) mix-
ture with a success rate of 70% - 90%. Since in vitro regeneration is highly genotype-dependent in mulberry, the stan-
dardized protocol can be effectively used for further improvement of this leading genotype using biotechnological ap-
Keywords: In Vitro Regeneration; Adventitious Bud; Thidiazuron; Mulberry cv. V1; Carbon Source
1. Introduction
Mulberry (Mor u s spp.) is a woody perennial tree of im-
portance to the sericulture industry as mulberry leaf is the
sole food for the silkworm (Bombyx mori L.) larvae. Ow-
ing to its long juvenile period and heterozygosity [1], im-
provement of specific characters through conventional
breeding is cumbersome and time consuming. Therefore
targeted manipulation of elite genotypes through incor-
poration of specific genes encoding desired traits using
modern biotechnological methods offers a new opportu-
nity for crop improvement. An efficient in vitro regenera-
tion procedure is pre-requisite for transgenic approach in
any crops. Information on development and standardiza-
tion of in vitro regeneration protocols in promising mul-
berry genotypes is limited, although there are reports
in a few genotypes. Studies have been made in mulberry
to examine the impact of various growth regulators on in
vitro organogenesis and plant regeneration by using dif-
ferent explants viz. leaf, internodal segment, hypocotyls
and cotyledons [2-12]. However, the shoot differentiation
from callus is confined only to a few genotypes and re-
peatability of protocols developed was not assured due to
the recalcitrant nature of the plant. In this study, we made
an attempt to develop and standardize in vitro regenera-
tion protocol in a widely cultivated mulberry variety, V1
using leaf explants. The variety is highly popular due to
its economic characters under irrigated conditions and
the foliage is suitable for both young and late age bivol-
tine silkworm rearing. The major emphasis was given to
investigate the effect of Thidiazuron (TDZ), a substituted
phenyl urea and different carbon sources in inducing ad-
ventitious buds and efficient regeneration in V1.
*Corresponding author.
Copyright © 2013 SciRes. AJPS
In Vitro Plant Regeneration of Morus indica L. cv. V1 Using Leaf Explant
2. Materials and Methods
2.1. Explant Preparation and Surface
Fully expanded leaf from the top 2nd - 5th position was
collected from actively growing shoots of (six months
old) mulberry variety, V1. The explants were thoroughly
washed in running tap water for 1 hour, followed by im-
mersion in a 1% (v/v) liquid detergent (Labolene, Quali-
genes, India) for 4 - 5 minutes and again washed thor-
oughly in running tap water to remove the traces of the
detergent. Surface sterilization was done by treating the
explants with 0.1% (v/v) HgCl2 (Qualigens, India) for 3 -
4 minutes and rinsing in sterile distilled water to remove
traces of HgCl2.
2.2. Induction of Adventitious Shoot Buds and
Plant Regeneration
The surface sterilized leaf explants were cultured on Mu-
rashige and Skoog’s (1962) [13] supplemented with thi-
diazuron (TDZ) (0.5 - 4.0 mg/l), indole acetic acid (IAA)
(2.0 mg/l), silver nitrate (AgNO3) (2.0 mg/l) either added
singly or in combination. As a carbon source, sucrose
(1% - 3%) was added singly or a mixture of sucrose,
fructose and glucose in the proportion of 1:1. The pH of
the medium was adjusted to 5.8 before gelling with agar-
agar (0.8%, Himedia, India). The induced shoot buds were
transferred to various shoot induction media supplement-
ed with 6-benzylaminopurine (BAP) (0.5 - 2.0 mg/l), gib-
berlic acid (GA3) (1.0 - 2.0 mg/l) and silver nitrate
(AgNO3) (1.0 - 2.0 mg/l). All the cultures were main-
tained at 25˚C ± 2˚C under 16/8 h (day/night) photope-
riod with light provided by cool, white fluorescent tubes
(Phillips, TL 40 W/54) at a light intensity of 150 - 200
2.3. Rooting and Hardening of Plantlets
Regenerated in vitro shoots of 3 - 4 cm long were clipped
off and transferred to half strength MS media fortified
with NAA or IBA (1.0 mg/l) to induce roots. The well
rooted plantlets were transplanted to small earthen pots
filled with potting mixture containing garden soil, sand
and FYM (2:1:1 ratio) for hardening and establishment
under controlled condition. The transparent plastic cups
were inverted over the potted plantlets to maintain high
humidity and three weeks after hardening, the plantlets
were transplanted to field.
2.4. Data Recording
The data on adventitious bud formation and subsequent
regeneration were recorded after 4 and 8 weeks of cul-
tures respectively. The percentage of rooting and root
length were recorded after 20 days and 30 days of sub-
culture respectively on rooting medium. Each value of
data represented the mean (±SE or SD) of 24 cultures per
3. Results and Discussion
In vitro regeneration in some perennial plants like mul-
berry is regulated by several factors and regeneration ef-
ficiency is highly genotype-dependent. Similar to other
species, the morphogenic response of mulberry leaf ex-
plants was influenced by concentrations and combina-
tions of the phytohormones and carbon source supple-
mented in the medium. First sign of leaf expansion,
swelling at the midrib region and basal cut ends with
slight callusing were noticed after 10 - 15 days in all the
media tested. After 30 days, nodule-like structures were
formed on the midrib region and basal cut ends. These
structures later turned into the shoot buds (Figures 1(a)
and (b)) and subsequently into the shoots, after 45 - 55
days (Figure 1(c)). The adventitious bud formation was
maximum at the basal cut ends compared to the midrib
region. Of the different adventitious bud induction media
tested, TDZ (1.0 mg/l), IAA (2.0 mg/l), AgNO3 (2.0 mg/l)
and sucrose (3%) resulted in maximum response of ad-
ventitious bud formation (63%, Table 1). The substituted
phenyl urea, TDZ, has been shown to be an effective re-
gulator of in vitro morphogenesis of many dicot plants
especially in woody perennials, influencing callusing, ad-
ventitious bud formation, shoot regeneration, somatic em-
bryogenesis and protoplast division [10,14,15]. Similar to
other studies, in this study, addition of TDZ had benefi-
cial effects in inducing bud regeneration. The regenera-
tion ability of the adventitious buds transferred to differ-
ent regeneration medium was significantly higher (68%,
Table 1) in BAP (1.0 mg/l), GA3 (2.0 mg/l) and AgNO3
(2.0 mg/l) containing medium (Figure 1(c)). Similar
types of findings were also reported in earlier studies in
mulberry [10,15,16].
Carbon source is one of the very important compo-
nents of the nutrient media. In the present investigation,
different concentrations of sucrose (2% and 3%) and
those in combination with fructose and glucose were test-
ed for adventitious bud formation. Among the carbon
sources tested, the most suitable carbon source was found
to be sucrose (3%) followed by fructose (2%, Table 1).
As reported earlier, sucrose seems to be the best source
of carbon for in vitro regeneration [2,3,10,15,16] of mul-
berry variety, V1.
In vitro regenerated shoots were rooted successfully
with rooting per cent between 60 - 80 and the mean root
length ranged from 3.58 - 5.20 cm on 12 MS supple-
mented with indole butyric acid (1.0 mg/l) medium after
30 days (Figure 1(d), Table 2). Well-rooted plantlets were
hardened with a success rate of 70% - 90% (Figure 1(e)
and (f)). Similar observations were reported in M.
Copyright © 2013 SciRes. AJPS
In Vitro Plant Regeneration of Morus indica L. cv. V1 Using Leaf Explant 2003
Table 1. Response of leaf explants cultures of V1 mulberry genotype. Sugar (%): A. Sucro se (3%), B. Sucrose (2%), C. Fruc-
tose (3%), D. Fructose (2%); E. Sucrose (3%), F. Glucose (2%), G. Sucrose + Fructose (1:1), H. Sucrose + Glucose (1:1).
(mg/l) Sugar (%) Adventitious bud induction (%)
(%) (±SE)
A 10.0 ± 5.77(16.35) 1.67 ± 1.67
B 23.3 ± 10.93(27.78) 13.33 ± 1.68
C 13.3 ± 1.68(21.34) 3.33 ± 1.67
D 36.67 ± 6.67(37.14) 23.33 ± 3.33
E 13.33 ± 1.68(21.34) 21.67 ± 3.33
F 18.33 ± 3.33(25.19) 5.00 ± 1.67
G 41.67 ± 3.33(40.18) 0.00 ± 0.00
1. MS + TDZ (0.5) + IAA (2.0) +
AgNO3 (2.0)
H 38.33 ± 3.33(38.22) 0.00 ± 0.00
A 63.33 ± 1.67(52.74) 68.33 ± 4.41
B 51.67 ± 3.33((45.96) 60.00 ± 2.89
C 26.67 ± 4.41(30.95) 18.33 ± 1.67
D 23.33 ± 1.67(28.86) 13.33 ± 1.67
E 28.33 ± 8.82(31.64) 51.67 ± 3.33
F 28.33 ± 3.33(32.09) 21.67 ± 1.67
G 15.00 ± 1.33(22.77) 3.33 ± 1.67
2. MS + TDZ (1.0) + IAA (2.0) +
AgNO3 (2.0)
H 23.33 ± 1.68(28.86) 30.00 ± 2.89
A 16.67 ± 4.47(23.74) 5.00 ± 2.89
B 11.67 ± 1.67(19.86) 0.00 ± 0.00
C 8.33 ± 4.11(15.09) 5.00 ± 5.00
D 61.67 ± 1.67(51.76) 31.67 ± 6.67
E 48.33 ± 4.41(44.03) 18.33 ± 3.33
F 31.67 ± 6.67(34.04) 10.00 ± 1.67
G 16.67 ± 4.41(23.74) 5.00 ± 2.89
3. MS + TDZ (2.0) + IAA (2.0) +
AgNO3 (2.0)
H 41.67 ± 6.00(40.11) 28.33 ± 3.33
A 28.33 ± 8.82(31.64) 5.00 ± 2.89
B 51.67 ± 1.67(45.96) 11.67 ± 1.67
C 20.00 ± 2.89(26.45) 16.67 ± 8.33
D 20.00 ± 5.00(26.15) 3.33 ± 3.33
E 33.33 ± 1.67(35.25) 13.33 ± 1.67
F 31.68 ± 6.67(34.04) 8.33 ± 1.67
G 25.00 ± 10.00(29.23) 10.00 ± 5.00
4. MS + TDZ (4.0) + IAA (2.0) +
AgNO3 (2.0)
H 41.68 ± 3.33(40.17) 20.00 ± 3.33
CD at 5% Medium 4.99 3.15
Sugar 7.06 4.46
Medium × Sugar 9.85 8.98
laeviagata [17]; M. indica var. C176 and C776 [18] and
other mulberry varieties [15,16].
The results generated in this study provided a reliable
and high frequency regeneration protocol with high re-
producibility. The standardized protocol could suitably
be used for large scale in vitro propagation and genetic
Table 2. Effect of different auxins on root induction.
(12MS + mg/l)
Percentage of
Mean root length (cm)
IBA (0.5) 60.00 3.58 ± 0.83
IBA (1.0) 80.00 5.20 ± 0.90
NAA (0.5) 55.00 1.16 ± 0.30
NAA (1.0) 35.00 1.20 ± 0.47
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In Vitro Plant Regeneration of Morus indica L. cv. V1 Using Leaf Explant
Figure 1. Protocol for plantlet regeneration from leaf explants in V1 mulberry variety.
4. Acknowledgements
We gratefully acknowledge the Department of Biotech-
nology (DBT), Government of India, New Delhi, for pro-
viding financial support to carry out this work.
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