In vitro clonal propagation of Jatropha curcas (L.) using nodal explants and assessment of genetic...

Current Biotica 5(1): 1-16

1

In vitro clonal propagation of Jatropha curcas (L.) using nodal explants and

assessment of genetic fidelity through RAPD markers

Subramanyam Koona1* Subramanyam Kondeti2, Muralidhararao Doulathabadh

3 and

Rajasekhar Pinnamaneni4

1,4,*

Department of Biotechnology, Sreenidhi Institute of Science and Technology, JNTU,

Yamnampet, Ghatkesar, R.R. District, Hyderabad – 501 301, India 2Plant Molecular Biology Unit, Department of Biotechnology, Bharathidasan University,

Tiruchirappalli – 620 024, Tamilnadu, India 3Department of Biotechnology, Sri Krishnadevaraya University, Anantapur – 515 003, A. P., India

*E-mail: [email protected]

ABSTRACT

An efficient micropropagation protocol was developed for Jatropha curcas (L.) using

nodal explants, and its genetic fidelity and was assessed using RAPD markers. Auxillary shoot

buds were induced from seven–month–old nodal explants on MS medium supplemented with

Kn, BAP, and TDZ along with AdS. A maximum number of shoot buds (6.2 ± 0.56) were

induced on MS medium fortified with 21.6 µM BAP and 54.2 µM AdS. Shoot multiplication

from the auxiliary buds was significantly enhanced (about 32.0 ± 0.01) on MS medium

containing 0.4 µM IBA, 4.4 µM Kn, 21.6 µM BAP and 27.1 µM AdS. About 54% rooting

was achieved on MS medium supplemented with 1.0 µM IBA. The well–rooted plantlets were

established in green house with 91% survival frequency. A total of 15 RAPD primers

produced in 925 distinct and reproducible bands and confirmed the genetic fidelity of the

regenerated Jatropha curcas plants.

KEYWORDS : Auxillary shoot buds, Clonal propagation, Genetic fidelity, Jatropha curcas,

RAPD

ABBREVIATIONS

RAPD Randomly amplified polymorphism of DNA

MS Murashige and Skoog

Kn Kinetin

BAP Benzylamino purine

TDZ Thidiazuron

AdS Adenine sulphate

IBA Indole-3-butyric acid


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INTRODUCTION

Jatropha curcas (L.) species is a

perennial, monoecious shrub or a small tree,

native to America, widely distributed in

tropics. J. curcas, a member of

Euphorbiaceae family, can grow well under

any unfavorable agro climatic conditions,

because of its low moisture demands,

fertilizer requirements and tolerance to high

temperature (Tiwari et al. 1994). It is found

throughout the tropics and known by nearly

200 names, which indicate its significance

(Koushik et al. 2007). Recently, the

economic importance of J. curcas has been

increasing because of its oil used as a fuel,

biofuel and is being looked at as an

important alternative fuel in the overall

energy security worldwide. The oil is also a

rich source of hydrocarbon (27.0–48.5% of

seed oil) (Martin and Mayeux 1985). It

promises for its use as an oil crop for

biodiesel (Henning 1998). The main

advantages of using biodiesel are its

renewability, better quality exhaust gas

emission, and biodegradability. It does not

contribute to a rise in the level of carbon

dioxide in the atmosphere (Korbitz 1999;

Beet et al. 2002; Sims 2001). Extracts from

the plant are known for their medicinal

properties and their effects on a wide array

of organisms including insects, pests, and

nematodes (Jain and Trivedi 1997). It is a

multipurpose tree species, which fits for

agro forestry and other afforestation

programmes (Wood and Boorely 1991).

Conventional agriculture uses seeds

and cuttings for its propagation. But the

seeds are heterozygous in nature, and the

cuttings are seasonal. Moreover, it is

reported that vegetative cuttings are not

deep-rooted and are easily uprooted as they

do not form a taproot system. Seed set has

been reported to be low in vegetatively

propagated plants (Sujatha et al. 2006).

Tissue culture studies were

undertaken in different species of Jatropha.

Morphogenesis from an endosperm tissue

has been reported in J. panduraefolia

(Srivastava 1971; Srivastava and Johri

1974). High frequency regeneration from

various explants of J. integerrima has been

reported (Sujatha and Dhingra 1993).

Using different explants, plant

regeneration protocols have also been

described in J. curcas (Sujatha et al. 2006;

Qin et al. 2004; Rajore and Batra 2005;

Sujatha and Mukta 1996), but multiplication

rate was low for field applications. Nodal

meristems are an important source tissue for

micropropagation (Mukul Manjari et al.

2007) and plants raised from these are

comparatively more resistant to genetic

variation (Pierik 1991). Keeping in mind the

economical importance of Jatropha curcas,

critical analysis of the earlier protocols

necessitated formulating a well-documented,

reproducible, in vitro micropropagation

protocol.

The genetic integrity of

micropropagated plants can be determined

with the use of various techniques. Various

authors have found RAPD technique useful

in examining genetic fidelity of tissue

culture clones. With the use of RAPD

markers, clonal fidelity of micropropagated

plants has been determined in Pinus

thunbergii (Goto et al. 1998), Lilium

(Varshney et al. 2001), and Tylophora

indica (Jayanthi and Mandal 2001). Anna

and Ewa (2004) have confirmed the

suitability of RAPD technique for

determining the genetic fidelity in two

micro-propagated Drosera species, D.

anglica and D. binata. The present study has

been taken up to develop an efficient micro

propagation protocol using nodal explants

and assess its genetic fidelity using RAPD

markers.


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MATERIALS AND METHODS

Plant material

Jatropha curcas (L.) seeds were

procured from the National Bureau of Plant

Genetic Resources (NBPGR), New Delhi,

India. The seeds were germinated in sterile

vermiculate mixture at 25–30°C in light and

later transferred to field. Nodal explants

were collected from these plants to

standardize the micropropagation protocol.

Nodal explants of 2–3 cm long, collected

from the seven–month–old plants were

immersed in 1% systemic fungicide

“Bavistin” (w/v) (BASF India Ltd) for 3 h,

they were surface sterilized by soaking in

0.1% mercuric chloride for 5 min, and

finally rinsed three times with sterile double

distilled water to remove remnants of

mercuric chloride.

Induction of shoots

For auxillary shoot bud induction,

about 1 cm long nodal explants were

inoculated onto Murashige and Skoog’s

(MS) medium (Murashige and Skoog 1962)

supplemented with 54.2 µM AdS in

combination with cytokinins viz., 2.2–

36.6µM Kn, 2.1–34.6 µM BAP, and 2.2–

36.4µM TDZ (shoot induction medium)

separately. The pH of the medium was

adjusted to 5.7 prior to autoclaving at 121°C

for 15 min. Cultures were incubated at

25±1°C under 16 h photo period provided

by cool white fluorescent lamps (Philips,

New Delhi) for 4 weeks. Shoot induction

was recorded after 4 weeks of culture in

each treatment which was replicated thrice

that consisted of 10 nodal explants each.

Shoot multiplication and elongation

After 4 weeks of culturing, the

induced auxillary shoot buds were separated

from nodal explants, transferred onto MS

medium fortified with 27.1 µM AdS in

combination with 2.1–21.6 µM BAP alone

or in combination with 0.2–1.6 µM IBA and

2.2–22.6 µM Kn (shoot multiplication

medium) for efficient shoot multiplication.

After 3 weeks of shoot multiplication, the

shoot clusters were sub cultured into the

same medium for elongation. The cultures

were maintained at 25 ±1°C under 16 h

photoperiod for another 3 weeks.

Root initiation and elongation

The elongated shoots were separated

from the shoot clusters and placed in rooting

medium consisting of MS medium

supplemented with 0.5–10 µM IBA for 3

weeks under 16 h photo period at 25 ±1ºC

temperature. The root elongation was

achieved on only MS basal medium in 2

weeks.

Hardening and field transfer

The plantlets with well–developed

roots were gently removed from the culture

flasks and thoroughly washed in sterile

double distilled water. The plantlets were

then transferred into pots containing a

mixture of soil and vermiculate in the ratio

of 1:1 and covered with clear polythene bags

to maintain high humidity. After 3 weeks of

hardening, the plantlets were transferred to

big earthen pots.

Statistical analysis

The experiments were carried out in

a completely randomized design. Data were

analyzed by analysis of variance (ANOVA)

to detect significant differences between

means at P ≤ 0.05. Means differing

significantly were compared using Duncan’s

Multiple Range Test (DMRT).


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Assessment of genetic fidelity

The total genomic DNA was isolated

from the leaves of mother plant and ten

randomly selected tissue culture-raised J.

curcas plants by following modified Cetyl

Tri Methyl Ammonium Bromide (CTAB)

method (Doyle and Doyle 1990). The

isolated genomic DNA concentration and

quality were cheecked by using the

spectrophotometer (BL198 ELICO, INDIA)

at 260 nm. Fifteen RAPD primers were used

for the analysis of genetic fidelity. The

RAPD amplification were performed using

20 µl PCR containing 50 ng of template

DNA, 1 x PCR Buffer, 2.5 mM MgCl2

(Bangalore Genei Pvt. Ltd., Bangalore), 200

µM dNTP mix (Genetix, New Delhi, India),

0.5 µM of RAPD primer (OPERON

Technologies, Inc., USA), 0.2 U of Taq

DNA polymerase (Fermentas Pvt. Ltd).

The PCR was performed at an initial

denaturation at 94ºC for 5 min followed by

35 cycles of 1 min denaturation at 94ºC, 1

min annealing at 36ºC, and 2 minutes

extension at 72ºC with final extension at

72ºC for 5 min using thermal cycler (UVI

Gene technologies Pvt. Ltd. INDIA). The

PCR products along with the λ DNA ECoR I

– Hind III double digest were separated on

1.4% agarose gel in 1X TBE buffer

containing ethidium bromide at 150 V and

photographed under ultraviolet light using

Gel Doc 2000 (Bio - RAD).

Data analysis

RAPD markers for parent plant and

regenerates were visually scored for the

presence (1) and absence (0) for each

primer. By comparing the banding patterns

of parent and regenerates for a specific

primer, specific bands were identified, and

faint or unclear bands were not considered.

Similarity matrices were generated by

percent similarity, and dendrogram was

constructed by using the Unweighed Pair

Group method with arithmetic Average

(UPGMA) with the MVSP 3.0 (Multi

Variate Statistical Analysis; [Kovach

Computing Services, 1987-1998]) to show

genetic relationships as revealed by the

percent similarity.

RESULTS

The nodal explants cultured on MS

medium supplemented with 54.2 µM AdS

with different cytokinins (Kn, TDZ, and

BAP) showed varied response (Table. 1).

The better response (6.2 ± 0.56

shoots per nodal explant) was obtained in

the presence of 54.2 µM AdS, 21.6 µM BAP

and was found to be significantly (P ≤ 0.05

level) higher than shoots induced per nodal

explant in other concentrations of cytokinins

(Kn, TDZ) used in the present study (Table

1, Fig. 1 C).

Four-week-old induced shoot buds

on MS medium supplemented with 54.2 µM

AdS and 21.6 µM BAP were transferred

onto MS basal medium fortified with 27.1

µM AdS with different cytokinins (BAP,

IBA, and Kn) singly or in combination

showed varied response (Table 2). In the

present study, transfer of shoot buds onto

MS medium with lower concentration of

AdS (27.1 µM) and with same concentration

of BAP (21.6 µM) produced 10.2 ± 0.05

shoots per nodal explant with average shoot

length of 1.5 ± 0.08 cm. Below or above the

21.6 µM BAP, shoot number and shoot

length gradually reduced (Table. 2). In

order to increase the shoot number, we

analyzed the effect of IBA and Kn in

combination with 21.6 µM BAP. A 0.4 µM

IBA and 4.4 µM Kn along with 21.6 µM

BAP produced maximum number of shoots

(32.0 ± 0.01 shoots per nodal explant) with

avarage shoot length of 2.1 ± 0.01 cm

(Table 2, Fig. 1 D and E ).


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When approximately 2.0 cm length

elongated shoots were inoculated onto MS

medium with lower concentrations of IBA

(0.5–1.0 µM), 54% of the inoculated shoots

were induced roots with average number of

5.6 ± 0.04 roots per shoot at 1.0 µM

concentration within three weeks (Table 3,

Fig. 1 F). Below or above 1.0 µM IBA,

percentage of response to root formation

drastically reduced. A distinct tap root

system developed with slender and white

secondary roots; this was considered

important for hardening and field transfer

(Fig. 1 G). Significant increase in root

length occurred on transfer to MS basal

medium for another 2 weeks. The well–

rooted plantlets were separated from the

medium, thoroughly washed with distilled

water, dipped for 1 h in 0.1% (w/v) bavistin

(systemic fungicide), transplanted to plastic

pots containing a mixture of soil and

vermiculate in equal ratio and covered with

polythene bags to maintain high humidity.

The plantlets were irrigated with tap water

as and when required. Polythene covers

were completely withdrawn after 3 weeks of

hardening. Plants were then transferred to

potted soil for further growth (Fig. 1 H). The

plants ranged from 86.0 ± 0.12 to 102.0 ±

0.21 cm in height after 10 months (Fig. 1 I).

RAPD pattern of 10 randomly

selected micropropagules obtained from

nodal explants were compared with mother

plant. Of the fifteen primers (Table 4)

screened, 14 primers yielded clear,

reproducible bands. The number of bands

for each primer varied from three in OPA-06

to nine in OPB-01. Each primer produced

amplification products in the size range 0.2

kb in OPB- 04 to 2.0 kb in OPA-01. The 14

tested primers yielded totally 925 scorable

bands (number of propagules x number of

screened markers) with an average of six

bands per primer. The entire tested primers

produced monomorphic pattern across all

the shoots, as examples, the patterns

obtained for primers OPA-01 and OPA-06

and OPA-07 are shown in (Fig. 2 A–C).

UPGMA Dendrogram revealed that

regenerates and mother plant grouped in

single cluster with 100% similarity. Genetic

Similarity was also found to be 100%

between the mother plant and regenerates,

and between the regenerates (Table 5;

Fig. 3), confirming the genetic uniformity of

the micro propagated plant material.

DISCUSSION

The best shoot induction response

(6.2 ± 0.56 shoots per nodal explant) was

obtained in the presence of 54.2 µM adenine

sulphate, 21.6 µM BAP. At the same

concentration of 21.6 µM BA, a lower

initiation (1.5 ± 0.2 shoot buds per explant;

2.2 ± 0.6 shoots per nodal explant) and

shoot bud proliferation were reported

(Rajore and Batra 2005). This differential

response may be attributed to the specific

age and physiological condition of the donor

plant from which the nodal explants were

excised. Nodes cultured on medium with

different concentrations of Kn showed lower

induction and proliferation of axillary shoot-

buds. Though TDZ is known to induce

cytokinin–like effects in a number of plant

species, particularly woody (Barrueto et al.

1999) as well as herbaceous crop species,

the present study showed inefficient effect

of TDZ on shoot induction, similar findings

were also reported in Jatropha curcas

(Sujatha et al. 2006).

The present study revealed that

shoots cultured on medium with

combinations of BA and Kn did not

proliferate further or showed slow response.

On the other hand, transferring the cultures

from a set having higher concentration of

BAP (21.6 µM) and 54.2 µM adenine


6

sulphate to a set with lower concentration of

AdS (27.1 µM), in combination with 21.6

µM BAP, 0.4 µM IBA, and 4.4 µM Kn, led

to a significantly higher nodal shoot

multiplication (32.0 ± 0.01 shoots per nodal

explant) within the next 3 weeks, which was

not recorded previously with this

concentrations. This was possibly due to a

combined effect of different growth

regulators along with other additives.

Lowering of growth regulators in

micropropagation studies to achieve higher

rate of multiplication has been reported in

Holarrhena antidysenterica (Kumar and

Sharma 2005). However, it has been

observed in Jatropha that it requires higher

concentration of only one type of cytokinin

(BAP) for induction phase and favors lower

concentration of another type of cytokinins

(IBA and Kn) along with other additives for

escalation and proliferation of shoot

cultures. In a recent report, Sujatha et al.

(2006) indicated similar effect in J. curcas

with BA and TDZ, but the authors obtained

lesser number of shoots (2.0 ± 0.8 to 12.3 ±

1.7) with comparatively higher amount of

cytokinin.

The genetic integrity of

micropropagated plants can be determined

with the RAPD as it has the advantage of

ease, quick performance, relatively low cost

and requires very little plant material

(Rafalski et al. 1993). The results presented

in this study demonstrate that the RAPD

technique proved to be effective in

generating reproducible results useful in

assessment of genetic fidelity of

micropropagated propagules in Jatropha

cultivars.

CONCLUSION

A simple, efficient and high fidelity

protocol for mass propagation of Jatropha

curcas from nodal explants has been

established. Using this protocol, it is

possible to produce viable, uniform and

healthy plants with maximum survival rate

for the proposed in vitro germplasm. The

protocol should also provide an efficient

means for large-scale cultivation and in vitro

multiplication of J. curcas. It is evident that

the RAPD technique proved to be effective

in generating reproducible results that are

useful in assessment of genetic stability of in

vitro propagated propagules in J. curcas.

ACKNOWLEDGMENTS

Authors are thankful to the National Bureau

of Plant Genetic Resources (NBPGR), New

Delhi, India, Jawaharlal Nehru

Technological University, Anantapur, Sri

Krishnadevaraya University, Anantapur, and

Gandhi Krishi Vignana Kendra (GKVK),

Sreedher Bhat’s Research laboratory (SBL),

Bangalore, for facilitating the research work.

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Table 1: Effect of different types of medium and concentrations of cytokinins on shoot

induction of J. curcas

MS basal medium

+ 54.2 µM AdS +

Conc. of

cytokinin (µM)

Average number

of shoots (9> 0.5 cm)

developed per nodal

Average length of

shoots developed

(cm ± SE)

Kn

TDZ

BAP

2.2

4.4

11.3

22.6

36.6

2.2

4.4

11.4

22.7

36.4

2.1

4.2

10.8

21.6

34.6

2.2 ± 0.17b

2.0 ± 0.10b

2.4 ± 0.12b

3.5 ± 0.35c

1.0 ± 0.01a

3.0 ± 0.02c

1.2 ± 0.04a

1.0 ± 0.05a

1.0 ± 0.02a

1.0 ± 0.01a

1.0 ± 0.01a

2.0 ± 0.13b

2.7 ± 0.23bc

6.2 ± 0.56f

5.3 ± 0.40e

0.8 ± 0.02a

0.8 ± 0.04a

0.8 ± 0.06a

0.9 ± 0.07a

0.8 ± 0.02a

0.8 ± 0.03a

0.9 ± 0.06a

0.9 ± 0.02a

0.8 ± 0.03a

0.9 ± 0.02a

0.8 ± 0.02a

0.8 ± 0.05a

0.9 ± 0.03a

2.0 ± 0.18b

1.4 ± 0.12ab

Data were recorded after 4 weeks of culture. Each treatment was repeated thrice and each

replicate consisted of 10 nodal explants. Means having different letters as superscripts are

significantly different from each other (P ≤ 0.05) according to DMRT.


10

Table 2: Effect of growth regulators on shoot multiplication and elongation from nodal

explants of J. curcas

Shoot multiplication and elongation

medium* with growth regulators (µM)

BAP IBA Kn

Number of

shoots per explants

Length of shoots

(cm)

2.1

4.2

10.8

21.6

34.6

21.6

21.6

21.6

21.6

21.6

21.6

21.6

21.6

-

-

-

-

-

0.2

0.4

0.8

1.6

0.4

0.4

0.4

0.4

-

-

-

-

-

-

-

-

-

2.2

4.4

11.3

22.6

2.0 ± 0.01a

4.0 ± 0.03b

8.0 ± 0.03a

10.2 ± 0.05c

7.0 ± 0.05e

18.2 ± 0.01a

22.1 ± 0.09a

12.0 ± 0.03b

8.5 ± 0.02c

26.3 ± 0.03g

32.0 ± 0.01f

21.2 ± 0.02e

15.1 ± 0.01d

0.8 ± 0.02a

0.8 ± 0.05a

1.0 ± 0.05a

1.5 ± 0.08b

1.2 ± 0.04ab

1.8. ± 0.01a

2.0 ± 0.03a

1.7 ± 0.01a

1.1 ± 0.04b

1.3. ± 0.01a

2.1 ± 0.01a

1.7 ± 0.02a

1.1 ± 0.03b

*MS basal medium + 27.1 µM AdS along with different plant growth regulators. Data were

recorded after 3 weeks of culture. Each treatment was repeated thrice and each replicate

consisted of 10 nodal explants. Means having different letters as superscripts are significantly

different from each other (P ≤ 0.05) according to DMRT.


11

Table 3: Effect of IBA on initiation of roots in excised shoots of J. curcas

MS + IBA

(µM)

Percentage of

root induction

Number of roots

per shoot

Length of roots

(cm ± SE)

0.5

1.0

2.5

5.0

10.0

15

54

20

16

10

1.0 ± 0.06a

5.6 ± 0.04b

4.1 ± 0.02a

2.4 ± 0.06a

2.1 ± 0.01a

2.8 ± 0.09a

5.9 ± 0.10b

3.8 ± 0.06a

2.9 ± 0.04a

2.4 ± 0.02a

Data were recorded after 3 weeks of culture. Each treatment was repeated thrice and each

replicate consisted of 20 shoots. Means having different letters as superscripts are significantly

different from each other (P ≤ 0.05) according to DMRT.


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Table 4: Number of amplification products generated from each RAPD primers in the

analysis of genetic fidelity of in vitro propagated J. curcas plants.

S.No Primer Sequence (5’-3’) Number of

generated bands

1

2

3

4

5

6

7

8

9

10

11

12

13

14

OPA-01

OPA-02

OPA-03

OPA-04

OPA-05

OPA-06

OPA-07

OPA-08

OPA-09

OPA-10

OPB-01

OPB-02

OPB-04

OPB-05

CAGGCCCTTC

TGCCGAGCTG

AGTCAGCCAC

AATCGGGCTG

AGGGGTCTTG

GGTCCCTGAC

GAAACGGGTG

GTGACGTAGG

GGGTAACGCC

GTGATCGCAG

GTTTCGCTCC

TGATCCCTGG

GGACTGGAGT

TGCGCCCTTC

07

05

05

06

08

03

06

05

05

08

09

08

06

04


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Table 5: Genetic Similarity among J. curcas parent (P) and ten randomly selected nodal

regenerates (R1-R10) developed by percent Similarity based on the RAPD data from each

primer.

Parent R1 R2 R3 R4 R5 R6 R7 R8 R9 R10

Parent 100

R1 100 100

R2 100 100 100

R3 100 100 100 100

R4 100 100 100 100 100

R5 100 100 100 100 100 100

R6 100 100 100 100 100 100 100

R7 100 100 100 100 100 100 100 100

R8 100 100 100 100 100 100 100 100 100

R9 100 100 100 100 100 100 100 100 100 100

R10 100 100 100 100 100 100 100 100 100 100 100


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Fig. 1: In vitro clonal propagation of Jatropha curcas (L.) nodal explants. A. Nodal explant from

the seven-month old mother plant. B. Nodal explant after the first week of culture on shoot

induction medium (MS medium containing 54.2 µM AdS and 21.6 µM BAP). C. Auxillary shoot

bud induction from nodal explant after 2 weeks of culture on shoot induction medium (MS

medium containing 54.2 µM AdS and 21.6 µM BAP). D. Shoot multiplication after 3 weeks of

culturing on shoot multiplication medium (MS medium containing 27.1 µM AdS, 21.6 µM BAP,

0.4 µM IBA, and 4.4 µM Kn). E. Elongated shoots on shoot multiplication medium (MS

medium containing 27.1 µM AdS, 21.6 µM BAP, 0.4 µM IBA, and 4.4 µM Kn) after 6 weeks of

culturing. F. Root initiation from the elongated shoots on rooting medium (MS medium

containing 1.0 µm IBA) after 3 weeks of culturing. G. In vitro roots with slender and white tap

root system. H. Jatropha curcas (L.) plants after 10 weeks of hardening. I. Ten month-old

micropropagated Jatropha curcas (L.) plant in big earthen pot under natural sun light


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Fig.2: Agarose gel electrophoresis and RAPD banding pattern of Jatropha curcas (L.) mother

and regenerated lines generated with primer OPA-01 (A), OPA-06 (B), and OPA-07 (C). Lane

P: Mother plant; Lanes 1 – 10: Regenerated plant lines; Lane M: λ DNA EcoR I and Hind III

double digest


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Fig.3: Dendrogram generated from the RAPD data obtained from the parent (mother

plant) and nodal explant derived regenerated plant lines (R1-R10) based on percent

similarity

[MS received: 20-4-2011;

MS accepted: 03-6-2011]

In vitro clonal propagation of Jatropha curcas (L.) using nodal explants and assessment of genetic...

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