ORIGINAL ARTICLE

Somatic embryogenesis in Jatropha curcas Linn., an importantbiofuel plant

Timir baran Jha Æ Priyanka Mukherjee ÆMukul Manjari Datta

Received: 28 March 2007 / Accepted: 2 May 2007 / Published online: 26 July 2007

� Korean Society for Plant Biotechnology and Springer 2007

Abstract Jatropha curcas L. is one potential source of

non-edible biofuel-producing energy crop. Its importance

also lies in its medicinal properties. The species is pri-

marily propagated through heterozygous seeds, and thus

the seed oil content varies from 4 to 40%. Moreover, due to

its perennial nature, seed setting requires 2 to 3 years time.

The seed viability and rate of germination are low, and

quality seed screening is another laborious task; thus, seed

propagation alone cannot provide quality planting material

for sustainable use. Somatic embryogenesis, a powerful

tool of plant biotechnology for faster and quality plant

production has been successfully applied to regenerate

plants in Jatropha curcas for the first time. Embryogenic

calli were obtained from leaf explants on MS basal medium

supplemented with only 9.3 lM Kn. Induction of globular

somatic embryos from 58% of the cultures was achieved on

MS medium with different concentrations of 2.3–4.6 lM

Kn and 0.5–4.9 lM IBA; 2.3 lM Kn and 1.0 lM IBA

proved to be the most effective combination for somatic

embryo induction in Jatropha curcas. Addition of 13.6 lM

adenine sulphate stimulated the process of development of

somatic embryos. Mature somatic embryos were converted

to plantlets on half strength MS basal medium with 90%

survival rate in the field condition. The whole process re-

quired 12–16 weeks of culture for completion of all steps

of plant regeneration. This protocol of somatic embryo-

genesis in Jatropha curcas may be an ideal system for

future transgenic research.

Keywords Jatropha curcas � Somatic embryogenesis �Biofuel � Adenine sulphate � Secondary somatic

embryogenesis

Introduction

Jatropha curcas L., a member of the family Euphorbia-

ceae, is considered one potential source of a non-edible

biofuel-producing energy crop throughout the world.

Jatropha biofuel contains more oxygen, with a higher

cetane value increasing the combustion quality, is clean,

non-toxic, eco-friendly and economic due to its low

production cost. It can be a good plantation material for

eco-restoration in all types of wasteland and also serves as

an important medicinal plant. Seeds, constituting the pri-

mary source of non-edible oil productions, are genetically

heterozygous as Jatropha sp. forms artificial and natural

hybrid complexes readily and poses a problem to genetic

fidelity (Prabakaran and Sujatha 1999). Thus, the percent-

age of oil varies from 4 to 40% within the species. Con-

ventional propagation of J.curcas is beset with problems of

poor seed viability, low germination, scanty and delayed

rooting of seedlings and vegetative cuttings (Heller 1996

and Openshaw 2000). Plants propagated by cuttings show a

lower longevity and possess a lower drought and disease

resistance than those propagated by seeds (Heller 1996).

Plants produced from cuttings do not produce true taproots

(hence are less drought tolerant); rather, they produce

pseudo-taproots that may penetrate only one-half to two-

thirds the depth of the soil compared to taproots produced

on seed grown plants (Heller 1996). Considering its enor-

mous potential, a large amount of quality planting material

is required for future use. Thus, improvement of the crop

through the application of plant biotechnological methods

T. b. Jha � P. Mukherjee (&) � M. M. Datta

Department of Botany, Presidency College,

Kolkata 700073, India

e-mail: presibot@vsnl.net

123

Plant Biotechnol Rep (2007) 1:135–140

DOI 10.1007/s11816-007-0027-2

is felt. Micropropagation of J.curcas has been reported by

various authors (Sujatha and Mukta 1996; Sardana et al.

2000; Qin et al. 2004; Rajore and Batra 2005; Sujatha et al.

2006), using different tissues except for nodal explants

from the field grown plants, but in all of the cases the

multiplication rate was low for application. An efficient

micropropagation protocol using nodal explants from field

grown plants has been achieved in our laboratory (publi-

cation is under consideration). However, no report of

complete plant regeneration through somatic embryogen-

esis is available in this species. To date, all applied re-

search focuses on somatic embryogenesis, and it is now

considered as the gateway to many more technologies.

Plant propagation by somatic embryogenesis not only helps

to obtain a large number of plants year round, but also can

act as a powerful tool for genetic improvement of any plant

species because of its single cell origin (Bhansali et al.

1991). The present study focuses on the process of plant

regeneration from leaf tissues of J. curcas through somatic

embryogenesis and successful field establishment of the

acclimatized plants.

Materials and methods

Plant materials

Leaves were excised from 7-month-old Jatropha curcas L.

plants grown in the experimental garden of Presidency

College, Kolkata. Leaves of sizes 2.0 · 1.0 cm to

2.5 · 2.0 cm near to the apical meristem were selected for

culture. All the explants were collected from this donor

plant for the present investigation.

Sterilization of leaf explants

The leaves were first thoroughly washed with tap water,

then dipped in 0.5% Bavistin (BASF India Ltd.) solution

for 5 min and washed two to three times with sterile dis-

tilled water. The leaves were then surface sterilized in 0.1%

HgCl2 (w/v) solution for 5–8 min and washed four to five

times with sterile distilled water to remove any traces of

the HgCl2.

Induction of embryogenic callus

The leaves were cut into small pieces and cultured with the

lower surface in contact with MS (Murashige and Skoog

1962) basal medium with 3% sucrose (w/v) (Merck, India)

containing 2.3–37.2 lM Kn and 2.2–35.5 lM BA indi-

vidually as well as in combination (Table 1) for 4 weeks.

The pH of all media was adjusted to 5.7 ± 0.1 before

adding 0.7% agar (Qualigens, India) and prior to auto-

claving. The medium was autoclaved at 121�C and

15 lbs in. m–2 for 15 min.

The leaf segments placed in culture tubes (150 · 25 mm)

plugged with non-absorbent cotton plugs contained 20 ml of

medium each. All the cultures were incubated under 16/8 h

light–dark cycles (artificial light 80 lmol–2 s–1) at 22 ± 2�C.

All the experiments were repeated three times.

Table 1 The influences of

cytokinins on embryogenic

callus induction frequency from

leaf explants of Jatropha curcas

Data recorded after 4 weeks of

culture. Each treatment was

replicated three times, and each

replicate consisted of 10–15

explants. Values represent the

means ± SE

Means followed by the same

letter are not significantly

different at the 0.05 level of

confidence

Concentration of cytokinin (lM) Callus induction (%) Morphology of callus

Kinetin BA

2.3 0 32.4 ± 5.8c Soft friable, light yellowish

4.6 0 44.7 ± 8.2d Soft friable, light yellowish

9.3 0 56.0 ± 9.6e Nodular, creamish ,embryogenic

13.9 0 37.9 ± 7.5c Compact, greenish brown

23.2 0 35.7 ± 6.1c Compact, greenish brown

37.2 0 19.2 ± 3.8b Compact, dark brown

0 2.2 35.0 ± 6.7c Soft friable, light green

0 4.4 41.5 ± 8.4cd Soft friable, light yellowish

0 8.9 47.7 ± 8.9d Compact, light yellow

0 13.3 38.1 ± 7.2c Compact, light brown

0 22.2 42.3 ± 8.0d Compact, light yellow

0 35.5 34.8 ± 6.4c Soft friable, brown

2.3 2.2 21.3 ± 4.3b Soft friable, light brown

4.6 4.4 11.2 ± 2.2a Compact, white

11.6 11.1 13.5 ± 2.9a Soft friable, light green

23.2 22.2 10.4 ± 2.5a Soft friable, white

136 Plant Biotechnol Rep (2007) 1:135–140

123

Embryo formation, development and germination

The 4-week-old embryogenic callus cultures obtained at a

concentration of 9.3 lM Kn were transferred from initia-

tion medium [MS basal medium +3% sucrose (w/v) +

9.3 lM Kn] to MS basal medium supplemented with

different concentrations of 2.3–4.6 lM Kn and 0.5–4.9 lM

IBA (Table 2). After another 4 weeks of culture, the

embryogenic calli induced globular somatic embryos in

embryo tissue proliferation medium [MS basal medium +

3% sucrose (w/v) + 2.3 lM Kn + 1.0 lM IBA]. The 8-

week-old embryogenic calli with globular somatic embryos

were subcultured onto the same media for 2 subsequent

weeks. Light and temperature conditions were the same as

mentioned earlier. After 10 weeks of culture, the globular

somatic embryos that had developed were transferred to

somatic embryo maturation medium [MS basal medium +

3% sucrose (w/v) 2.3 lM Kn + 1.0 lM IBA + 13.6 lM

adenine sulphate] for 4 weeks. Different concentrations of

5.4–54.3 lM adenine sulphate were tested on somatic

embryo maturation. For germination of mature somatic

embryos, the 14-week-old embryos were transferred to

conversion medium (MS half-strength basal medium sup-

plemented with 3% sucrose) for 2 weeks.

Hardening and field transfer

The embryo-derived plantlets were taken out of the culture

vessels, thoroughly washed with tap water, dipped for 1 h

in 0.1% (w/v) bavistin (systemic fungicide) and transferred

to plastic pots containing a mixture of (1:1) sand and

vermicompost and covered with polythene bags. The

plantlets were irrigated with tap water as and when re-

quired. After 4 weeks these plantlets were transferred to

bigger pots containing garden soil mixed with organic

manure. When the plantlets showed signs of establishment

in pots with the appearance of new leaves, the polythene

bags were removed gradually for acclimatization to field

conditions.

Chromosomal analysis

Cytological studies from randomly selected somatic em-

bryos were carried out using saturated PDB solution as

pretreating chemical for 4 h followed by overnight fixation

in Carnoy’s fluid. Materials were stained with 2% aceto-

orcein, HCl (9:1) solution (Sharma and Sharma 1990). The

stained materials were squashed for microscopic observa-

tion, and photographs were taken under Zeiss photomi-

croscope.

Statistical analysis

The experiments were set up in a randomized design. Data

were analyzed by analysis of variance (ANOVA) to detect

significant differences between means (Sokal and Rohlf

1987). Means differing significantly were compared using

Duncan’s multiple range test (DMRT) at a 5% probability

level. Variability of data has also been expressed as the

mean ± standard error (SE).

Table 2 Somatic embryogenesis from embryogenic callus, germination and plantlet conversion in Jatropha curcas

Concentration of growth regulator

with 13.6 lM adenine sulphate

Percentage of

embryogenesis (%)

Number of

somatic embryos

Number of somatic

embryos germinated

Number of somatic

plantlets recovered

Kinetin (lM) IBA (lM)

2.32 0.5 72 40.0 ± 10.6cd 18.0 ± 4.5b 2.0 ± 0.56b

1.0 80 58.5 ± 12.7d 24.0 ± 6.8c 5.0 ± 0.87c

2.5 67 32.6 ± 8.9c 15.5 ± 4.6b 2.0 ± 0.22b

4.9 58 37.8 ± 9.2c 10.0 ± 3.0a 2.0 ± 0.35b

3.5 0.5 67 31.7 ± 8.3c 12.5 ± 3.2ab 1.5 ± 0.24ab

1.0 53 25.5 ± 7.4b 10.0 ± 3.4a 1.0 ± 0.05a

2.5 58 22.8 ± 7.2b 16.0 ± 4.9b 1.0 ± 0.08a

4.9 50 23.0 ± 7.9b 8.5 ± 2.3a 1.0 ± 0.04a

4.6 0.5 60 16.0 ± 4.3ab 0 0

1.0 57 11.5 ± 3.8a 0 0

2.5 48 8.0 ± 2.7a 0 0

4.9 42 5.6 ± 1.8a 0 0

Data was recorded after 6–12 weeks. Each treatment was replicated three times, and each replicate consisted of 20 explants. Values represent the

means ± SE

Means having different letters in superscript are significantly different from each other (P < 0.05) according to Duncan’s multiple range test

Plant Biotechnol Rep (2007) 1:135–140 137

123

Results and discussions

Somatic embryogenesis has been documented in some of the

species of the family Euphorbiaceae as in Hevea sp

(Michaux-Ferriere et al. 1992) and Cassava sp (Raemakers

et al. 2000), but not so prominent in any of the species of

Jatropha. The present investigation is a well-documented

study of somatic embryogenesis in Jatropha curcas. The

type and concentrations of the plant growth regulators were

the strong determining factors for somatic embryogenesis in

J. curcas. Leaf pieces were used as the primary explant. Leaf

sections cultured on MS basal medium supplemented with

various concentrations of cytokinin started swelling after 5–

7 days. Initiation of callus was noted on the cut surfaces of

the leaf sections after 2 weeks of culture. Leaf explants

cultured on MS medium supplemented with 9.3 lM Kn

showed significantly (P < 0.05) higher induction of callus

(56.0%) in comparison to other concentrations of cytokinins

studied (Table 1). The initiation medium showed the

development of nodular, creamish, embryogenic calli within

4 weeks of culture (Fig. 1a). Subsequent transfer of the

embryogenic calli in MS medium with different lowered

concentrations of Kn with IBA showed varied results (Ta-

ble 2). The highest frequency (80%) of globular somatic

embryos (58.5 ± 12.7) of callus was recorded in the com-

bination of 2.3 lM Kn and 1.0 lM IBA after 4–6 weeks of

culture (Fig. 1b). The role of cytokinins and auxins in the

different stages of somatic embryogenesis is well estab-

lished (Fujimara and Komamine 1980; Lo Schiavo et al.

1989; Litz and Gray 1995), but what is important is finding

out the triggering combination and concentrations of plant

growth regulators besides other factors that vary from cell to

cell even within a particular type of tissue of a plant species.

Earlier studies have reported that the continued presence of

an auxin promotes the completion of the globular stage

during embryogenesis (Lo Schiavo et al. 1989; Litz and

Gray 1995). In our study, IBA promoted the completion of

the globular stage of the embryos. The combined favorable

influence of auxin and cytokinins observed in the present

system is in accordance with the culture response of somatic

embryogenesis in Coffea arabica (Neuenschwander and

Baumann 1992). Clusters of globular somatic embryos were

visible during the first 2–3 weeks in embryo tissue prolif-

eration medium. Highly organized, round, creamish globu-

lar somatic embryos differentiated on the edges of the callus

by the fourth week of culture. It was also found to be

embedded in the embryogenic callus tissue, while other

areas of the calli remained white and translucent. Globular

somatic embryos on subculture to the same embryo tissue

proliferation medium were found to gradually convert into

heart-shaped, torpedo and cotyledonary stages embryos

with distinct bipolarity with globular stage embryos domi-

nating in culture (Fig. 1c). Asynchronous development of

different stages of somatic embryos within a culture indi-

cates the complexity of the mechanism of somatic

embryogenesis. Our study required a minimum time of 4–

6 weeks for conversion of globular somatic embryos to

germinated plantlets. Addition of 13.6 lM adenine sulphate

along with 2.32 lM Kn and 1.0 lM IBA led to the signif-

icantly (P < 0.05) highest mean number of mature somatic

embryos (24.0 ± 6.8) after 4 weeks of culture, which served

the best concentration and combination of PGRs (Table 2,

Fig. 1d). However, addition of 5.4 lM, 27.0 lM and

54.3 lM adenine sulphate to maturation medium did not

show any further development. Adenine sulphate is known

to enhance the efficiency of maturation of somatic embryos,

which is a critical step in somatic embryogenesis. Earlier

reports by many authors (Das et al. 1993; Martin 2003) also

support the role of adenine sulphate in somatic embryo

maturation.

An interesting observation was the induction of a low

frequency of secondary embryogenesis along the shoot and

root poles of well-developed primary embryos grown in the

maturation medium (Fig. 1e). The synergistic combination

of reduced Kn, IBA and adenine sulphate not only pro-

moted the growth of shoot and root, but also secondary

embryo formation. In this study, both proliferation and

germination of secondary embryos are successfully re-

ported for the first time. Somatic embryogenic cells can act

independently from neighboring cells and undergo somatic

embryogenesis, or they can continue to differentiate into

secondary embryogenesis (Raemakers et al. 1995). This

continuous proliferation of somatic embryos via secondary

somatic embryogenesis is both cost and time effective, and

is independent of the explant source.

The critical step of conversion of mature somatic em-

bryos into somatic plantlets was obtained on transfer of 14-

weeks-old mature somatic embryos cultured on maturation

medium to conversion medium for 2 weeks (Table 2). The

formation of distinct bipolar somatic embryos with root

and shoot poles may be attributed to the presence of cyt-

okinins, as cytokinins stimulate shoot and tap root forma-

tion (Chang 1991). After a certain developmental stage,

transfer of the embryos to growth regulator free solid 0.5

MS medium resulted in improved germination forming

complete plantlets with well-developed shoot and tap root

systems attaining an average length of 4.2 cm. The plant-

lets grew well with green leaves and produced three to five

roots/plantlets initially. The well-formed tap root system

allows the acclimatization of about 90% of the plants.

Somatic embryo-derived plants are now growing luxuri-

antly in field conditions (Fig. 1g).

Chromosomal analysis revealed a diploid parental

chromosome number of 2n = 22 very small chromosomes

(Fig. 1f). No cytological anomaly was observed in our

studies, indicating a genotypic stability.

138 Plant Biotechnol Rep (2007) 1:135–140

123

The protocol of plant regeneration through somatic

embryogenesis in the biofuel plant Jatropha curcas

developed in our laboratory is the first well-documented

report. The protocol can be used for production of quality

planting material at a faster rate than the available micro-

propagation protocols in Jatropha curcas mentioned earlier

(Sujatha and Mukta 1996; Sardana et al. 2000; Qin et al.

2004; Rajore and Batra 2005; Sujatha et al. 2006). Somatic

embryogenesis in Jatropha curcas may be a system for any

future transformation and metabolic engineering studies.

Acknowledgments Financial assistance from the Department of

Botany, Presidency College, Kolkata is gratefully acknowledged.

References

Bhansali R (1990) Somatic embryogenesis and regeneration of

plantlets in pomegranate. Ann Bot 66:249–254

Chang WC (1991) Bamboos. In: Bajaj YPS (ed) Biotechnology in

agriculture and forestry, vol. 16. Springer, Berlin, pp 211–237

Das P, Rout GR, Das AB (1993) Somatic embryogenesis in callus

cultures of Mussaenda erythrophylla cvs. Plant Cell Tissue

Organ Cult 35:199–201

Fujimura T, Komamine A (1980) Mode of action of 2,4-D and

zeatin on somatic embryogenesis in carrot suspension culture.

Z Pflazenphysiol 99:1–8

Heller J (1996) Physic nut, Jatropha curcas L. Promoting the

conservation and use of underutilized and neglected crops. No 1.

International Plant Genetic Resource Institute, Rome

Litz RE, Gray DJ (1995) Somatic embryogenesis for agricultural

improvement. World J Microbiol Biotechnol 11:416–425

Lo Schiavo F (1989) DNA methylation of embryogenic carrot cell

cultures and its variations as caused by mutation, differentiation,

hormones and hypomethylating drugs. Theor Appl Genet

77:325–331

Martin KP (2003) Plant regeneration through direct somatic embryo-

genesis on seed coat explants of cashew (Anacardium occiden-tale L.). Sci Hortic 98:299–304

Michaux-Ferriere N, Grout H, Carron M P (1992) Origin and

ontogenesis of somatic embryos in Hevea brasiliensis (Euphor-

biaceae). Am J Bot 79:174–180

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassays with tobacco tissue cultures. Physiol Plant 15:473–479

Neuenschwander B, Baumann TW (1992) A novel type of somatic

embryogenesis in Coffea arabica. Plant Cell Rep 10:608–612

Openshaw K (2000) A review of Jatropha curcas: an oil plant of

unfulfilled promise. Biomass Bioenerg 19:1–15

Prabakaran AJ, Sujatha M (1999) Jatropha tanjorensis Ellis and

Saroja, a natural interspecific hybrid occurring in Tamil Nadu,

India. Genet Resour Crop Evol 46:23–218

Fig. 1 Somatic embryogenesis

and plant regeneration in

Jatropha curcas. aEmbryogenic callus after

4 weeks of culture (bar2.0 mm). b Development of

globular somatic embryos after

4-6 weeks (bar 1.0 mm). cDevelopment of cotyledonary

stage of somatic embryo

showing distinct bipolarity (bar2.0 mm). d Different

developmental stages showing

globular to cotyledonary stage

of somatic embryogenesis (bar1.0 cm). e Secondary embryo

formation on the shoot pole of

the bipolar somatic embryo (bar5.0 mm). f A mitotic plate

showing 2n = 22 very small

chromosomes. g A complete

hardened plant of Jatrophacurcas (bar 10.0 cm)

Plant Biotechnol Rep (2007) 1:135–140 139

123

Raemakers CJJM, Jacobsen E, Visser RGF (1995) Secondary somatic

embryogenesis and applications in plant breeding. Euphytica

81:93–107

Raemakers K, Jacobsen E, Visser R (2000) The use of somatic

embryogenesis for plant propagation in Cassava. Mol Biotech

14:215–221

Rajore S, Batra A (2005) Efficient plant regeneration via shoot tip

explant in Jatropha curcas. J Plant Biochem Biotech 14:73–75

Sardana J, Batra A, Ali DJ (2000) An expeditious method for

regeneration of somatic embryos in Jatropha curcas L. Phyto-

morphology 50:239–242

Sharma AK, Sharma A (1990) Study of plant chromosomes from

tissue culture. Chromosomal techniques theory and practice, 3rd

edn. Aditya Books, pp 317–338

Sokal R, Rohlf FJ (1987) Introduction to Biostatistics, 2nd edn.

Freeman WH, New York

Sujatha M, Mukta N (1996) Morphogenesis and plant regeneration

from tissue cultures of Jatropha curcas. Plant Cell Tissue Organ

Cult 44:135–141

Sujatha M, Makkar HPS, Becker K (2006) Shoot bud proliferation

from axillary nodes and leaf sections of non-toxic Jatrophacurcas L. Plant Growth Reg 47:83–90

Wei Q, Lu WD, Loao Y, Pan SL, Xu Y, Tang L, Chen Fang (2004)

Plant regeneration from epicotyl explants of Jatropha curcas.

J Plant Physiol Mol Biol 30:475–478

140 Plant Biotechnol Rep (2007) 1:135–140

123