<i>In Vitro</i> Plant Regeneration of <i>Morus indica</i> L. cv. V1 Using Leaf Explant

doi:10.4236/ajps.2013.410249

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American Journal of Plant Sciences, 2013, 4, 2001-2005

http://dx.doi.org/10.4236/ajps.2013.410249 Published Online October 2013 (http://www.scirp.org/journal/ajps)

2001

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: *[email protected]

Received March 26th, 2013; revised April 27th, 2013; accepted May 19th, 2013

tion License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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-

proaches.

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.

In Vitro Plant Regeneration of Morus indica L. cv. V1 Using Leaf Explant

2002

2. Materials and Methods

2.1. Explant Preparation and Surface

Sterilization

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

µmol·m−2·s−1.

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

treatment.

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.

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).

Medium

(mg/l) Sugar (%) Adventitious bud induction (%)

(±SE)

Regeneration

(%) (±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

transformation.

Table 2. Effect of different auxins on root induction.

Medium

(12MS + mg/l)

Percentage of

rooting

Mean root length (cm)

(±SD)

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

In Vitro Plant Regeneration of Morus indica L. cv. V1 Using Leaf Explant

2004

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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