FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENT RULES, 2003
PROVISIONAL SPECIFICATION
(See Section 10; rule 13)
Plant regeneration from immature embryos of Jatropha curcas L.


RELIANCE LIFE SCIENCES PVT.LTD an Indian Company having its Registered Office at Dhirubhai Ambani Life Sciences Centre, R-282, TTC Area of MIDC, Thane Belapur Road, Rabale, Navi Mumbai - 400 701

Maharashtra India.

The following specification particularly describes and ascertains the nature of this invention and the manner in which it is performed:-


TECHNICAL FIELD
The present invention relates to the efficient regeneration method from immature embryos of Jatropha curcas L. The present invention in particular relates to plant regeneration Jatropha curcas L from immature embryos that is independent of genotypes.
BACKGROUND ART
Jatropha curcas L. (Euphorbiaceae) is an important biofuel crop commonly found in tropical and partially subtropical areas. Its seeds are source of non-edible semi-dry oil. There is a growing interest in the use of Jatropha curcas seed oil to alleviate the energy crisis. J. curcas oil is relatively simple to convert to biodiesel by chemical (Berchmans and Hirata, 2008) or biological trans-esterification (Modi et al, 2007). In addition to the low production cost, J. curcas biofuel has been reported to be non-toxic, clean and eco-friendly (Datta et al., 2007, Deore and Johnson 2008b). Moreover, the fuel properties of Jatropha biodiesel are comparable to those of diesel and confirming to the American and European standards (Tiwari et al, 2007). Besides its use as a biofuel crop, it is also desired due to its drought hardiness, rapid growth and easy propagation, low cost of seeds, oil content and small gestation period, wide adoption, production on good and degraded soils (Jones and Miller, 1991). In addition to its usage as an energy crop, Jatropha seeds and other parts of plant are a source of pesticides, cosmetics and anti¬cancer medicines (Gubitz et al., 1999).
This drought resistant plant which can be easily cultivated by low income farmers on marginal and degraded lands could benefit as an energy provision to remote areas. In India, work on biodiesel from Jatropha plantation to seed crushing to trans-esterification is being pursued actively by both governmental and non-governmental organizations. However, a major bottleneck in the widespread adoption of the technology is the non¬availability of superior clones in large numbers. Some of traits such as higher seed yield, oil content, synchronous maturity and early flowering can be introduced to make this technology sustainable and viable.
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Micropropagation and other tissue culture techniques could be usefully employed for the multiplication of desired clones (Deore and Johnson, 2008a). The earlier investigations on tissue culture of the genus Jatropha was confined to endosperm culture of J. panduraefolia (Srivastava 1971, Srivastava and Johri, 1974). Later, tissue culture protocols have been extended to both direct and callus mediated shoot regeneration and somatic embryogenesis of J. curcas using epicotyl, hypocotyl, peduncle, axillary buds, nodal segments, leaf segments and shoot tips as an explant source (Sujatha and Dingra, 1993; Sujatha 1996; Sujatha and Mukta 1996; Qin et al, 2004; Jha et al, 2007; Datta et al, 2007 and Deore and Johnson, 2008a).
Of all explants,, leaf segments have been found to be most responding tissue. Earlier it was reported that high frequency plant regeneration directly from leaf disc cultures without any intermediary callus stage is essential to obtain txue-to-type plants (Deore and Johnson, 2008). Although extensive work has been carried out using different explants, however till date the morphogenic potential of embryo cultures in J. curcas has not been reported..
The inventors of the present invention has been successfully evaluated the embryo's morphogenic potential in J. curcas and has further studied the effects of various factors such as age of explant, shoot regeneration and morphogenesis in J. curcas.
The present invention has developed an efficient and reproducible method having high multiplication rate of shoot regeneration from immature embryo cultures. The method developed can also be extended to Agrobacterium mediated genetic transformation
OBJECTIVES OF THE INVENTION:
It is the aim of the present invention to develop a efficient method for regeneration from immature embryo cultures in J. curcas.
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It is the aim of the present invention to develop an efficient and reproducible process having high multiplication rate of shoot regeneration from immature embryo cultures of Jatropha Curcas.
It is the aim of the present study to identify stage and physiological age of immature embryo to obtain high frequency callus induction to obtain subsequent plantlet regeneration
It is the aim of the present study to identify type and concentration of growth additives such as, but not limited to, copper sulphate, 1-glutamine, casein hydrolysate, 1-proline, silver nitrate alone or in combination to improve morphogenic callus induction and subsequent plant regeneration.
It is the aim of the present invention to extend the process of regeneration from immature embryo cultures to Agrobacterium mediated genetic transformation.
It is the aim of the present invention to provide the best suitable nutrient media with optimum growth regulators and other components required for different modes and phases of regeneration.
It is an important aspect of the present invention to provide the optimum growth conditions with respect to physical parameters like temperature, relative humidity, photoperiod and light intensity for all the stages of culture.
It is an important aspect of the present invention to provide the optimum sub-culture interval during in vitro culture.
It is also an additional aspect of the present invention to provide a hardening protocol that proves the efficient hardening of the micropropagated plants with as much as about 100% rate of survival in the field.
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SUMMARY OF INVENTION
The present invention provides the the morphogenic potential of immature embryo cultures of Jatropha curcas. The present invention in particular provides a reproducible, genotype independent protocol having high multiplication rate of shoot regeneration from immature embryos of Jatropha curcas
In one embodiment, the present invention provides the study of the morphogenic potential of Jatropha curcas and the effect of the various factors on efficient regeneration from immature embryos.
In one embodiment, the present invention provides the study on the various developmental stages of the explant- immature embryos. In one preferred aspect the present invention has studied the morphogenic potential of embryos collected from fruits 2- 6 weeks after pollination (WAP).
In one embodiment, the present invention has studied the critical parameters of the embryo for morphogenic callus development. In one preferred embodiment, the size and physiological age of the embryo was studied for establishment of morphogenic callus.
In one embodiment, the present invention provides the effect of various combinations and concentrations of auxins and cytokinins on morphogenic callus induction.
In one embodiment the present invention provides the effect of various growth additives for improvement in the morphogenic callus induction.
In one embodiment, the present invention provides the effect of various combinations and concentrations of auxins and cytokinins on subsequent plant regeneration
In one embodiment the present invention provides the study of the various media components on the root generation.
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In one embodiment the present invention provides a method for regeneration from immature embryos which can be extended to Agrobacterium transformations.
In one embodiment the present invention provides an efficient and reproducible method for regeneration for Jatropha curcas of various varieties.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present specification and are included to further
demonstrate certain aspects of the present disclosure, the inventions of which can be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. la: Induction of callus from immature embryo cultures of Jatropha curcas FIG. Ib-d: Stages in subsequent plant regeneration from morphogenic callus FIG. le-f: Tissue cultured plant during acclimatization FIG. lg: Field transferred plant
DESCRIPTION OF EMBODIMENTS
DEFINITIONS:
The term "immature embryos" as used herein refers to the embryos which are not ripen (means not mature for germination in soil) is known as immature embryos approximately 4-6 weeks after WAP, when the fruits are still green in color.
The term "morphogenic potential" as used herein refers to the cultures which are going to differentiate and give rise to roots or shoots or plantlets.
The term "plant regeneration" as used herein refers to the production of shoots or plantlets from cultures.
The term "callus induction" as used herein refers to the production of an undifferentiated mass of cells is known as callus induction.
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The term " Jatropha sp" as used herein refers to the plant belongs to family Euphorbiaceae and has a characteristic to give Biofuel from the mature seeds and includes all varieties.
The term "explant" as used herein refers to the part of the plant which is used for the culture initiation is known as explant- immature embryo / cotyledons.
The term "growth additives" as used herein refers to the chemical components in addition to growth regulators which are going to help the growth of cultures for the callus induction or plant regeneration
The term "growth regulators" as used herein refers to the chemical compound which used to regulate the growth of the plants in vitro or in vivo e.g. auxins or cytokinins.
The following are the expanded terms for the abbreviations used herein the specification
2,4-D: 2,4- Dichlorophenoxyacetic acid, BAP: 6-Benzylaminopurine, CH: Casein hydrolysate, GA3: Gibberellic acid, IAA: Indole-3-acetic acid, IBA: Indole-3-butyric acid, Kn: Kinetin,
MS: Murashige and Skoog basal medium, NAA: a-Naphthaleneacetic acid, PIC: 4-Amino-3,5,6-tri-chloropicolinic acid, PVP: Polyvinyl pyrrolidone, WAP: Weeks after pollination CIM: callus induction media
In preferred embodiments of the present invention the process for shoot regeneration from immature embryos of Jatropha sp broadly comprises of:
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1) Selection of fruits from different Jatropha sp.
2) Isolation of immature embryos
3) Induction of morphogenic callus on callus induction media (CIM) media - 1
4) Improvement of morphogenic callus induction on CIM media- 2
5) Regeneration of morphogenic callus on regeneration medium
6) Improvement of regeneration capacity adding growth additives
7) Transfer of plantlets in rooting media
8) Subjecting the plantlets to acclimatization
9) Transfer of the plantlets to the fields
Immature embryos (1.1-1.5cm) obtained from fruits 6 \veeks after pollination (WAP) showed high ability to produce morphogenic callus and subsequent plant regeneration. In this study, immature embryos of J. curcas from various developmental stages have been tested for their morphogenic potential. Developmental stage of immature embryo has been reported as an important factor for regeneration (He et al., 1988). Abdullah et al., (2005) demonstrated potential role of immature embryos in genetic transformation and direct organogenesis. In present study, the percent of morphogenic callus induction (100%) and subsequent plant regeneration (70%) was highest in immature embryos (1.1-1.5 cm) obtained from size class-4. In rest of the size classes either morphogenic callus induction was very poor or subsequent plant regeneration ^as not observed (Table 1).
Induction of morphogenic callus was critical stage in tissue culture where the quality and type of callus influences subsequent plant regeneration. So far. to our knowledge, no comprehensive study was undertaken to study the relationship between morphogenic potential and the developmental stage of embryo in J. curcas. Results from present study found that the optimum developmental stage of immature Embryo for morphogenic callus induction and subsequent plant regeneration in J. curcas was from immature fruits obtained 6 WAP.
Plant growth regulators, especially cytokinins and auxins alone and in combination are known to play a very important role in the process of callus induction and its proliferation. However, the optimum concentrations for inducing morphogenic callus differ according to the type of auxin, genotype and explant source. In the present study.
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IBA in combination with Kn induced lowest percentage of callus while, in combination with BAP promoted highest percentage of morphogenic callus induction (85.7%), maximum subsequent plant regeneration (70%) and highest number of shoots per explant (4.7) (Table 3). BAP has been reported to be more beneficial than other cytokinins for micropropagation of various members of Euphorbiaceae (Tideman and Hawker, 1982; Ripley and Preece, 1986 and Sujatha and Reddy, 1998). In Jatropha integerrima, a high frequency callus induction was reported in media containing IBA+BAP in hypocotyls, stem, peduncle and leaf explants (Sujatha and Reddy, 2000). Similarly in J. curcas BAP promoted higher regeneration from hypocotyls and petiole explants than kinetin (Sujatha and Mukta, 1996). The favourable influence of BAP on the morphogenic capacity of the explants was also reported in other species; castor (Sarvesh et a!., 1992, Sujatha and Reddy, 1998), Euphorbia peplus (Tideman and Hawker, 1982) and E. hirta (Baburaj et al., 1987). But all above studies were on explants other than immature embryo. No comparative data on response of immature embryo to cytokinins is available.
The present results indicate the efficiency of IBA-BA combination on the induction of morphogenic callus (Table 3). The differential response of cytokinins, i.e., in this case BAP and Kn may be attributed to difference in uptake, levels of endogenous growth regulators and recognition by cells. Induction of morphogenic callus was a critical stage where subsequent plant regeneration is highly dependent and governed by growth regulators used during callus induction stage. The best response of morphogenic callus induction (85.7%), subsequent plant regeneration (70%) and maximum number of plantlets (4.7) were obtained on Murashige and Skoog's (MS) with 3% sucrose in combination of low levels of IBA (0.5mg/l) and BAP (1.0 mg/I).
After determining the optimum combination of IBA+BA (CIM-1), the incorporation of growth additives such as 1-proline, 1-glutamine, casein hydrolysate, CUS04 or AgNo3 to further been reported that the rate of plant regeneration increases with the addition of casein hydrolysate, and amino acids such as 1-glutamine and 1-proline (Minhas et al. 1999). The concept of addition of amino acids has been very well studied. Rao et al.. (1995), O'Kennedy et al, (2004) obtained enhanced regeneration with the addition of
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amino acids in the culture medium. The present study showed the supplementation of 1-glutamine (200 mg/1) alone to CIM-1 had induced morphogenic callus in 85.7% explants but subsequent plant regeneration was less. Casein hydrolysate supplementation individually induced callus induction in 42.9 to 57.1% of explants depending on concentration used, number of shoots produced per explant varied from 2.7 to 4.7 (Table 4). But when casein hydrolysate and glutamine were used in combination, there was morphogenic callus induction in 100% explants, subsequent plant regeneration in 90% explants and 9.1 shoots produced per explant, indicating synergistic effect of both additives.
Many reports have shown positive effect of AgNo3 in plant tissue cultures, e.g., cassava (Zhang et al., 2001), rape seed (Akasaka-Kennedy, 2005). AgNo3 is a potent inhibitor of ethylene action, and ethylene is considered to suppress shoot organogenesis in vitro. Zhang et al., (1998) considered that the increased shoot regeneration frequency by AgNo3 is caused by un-interruption of an ethylene signal transduction pathway. In the present study, supplementing AgNo3 to CIM-1 resulted in increase in shoot regeneration (80%) and increase in shoots produced per explant (up to 6) from immature embryo cultures of J. curcas. Increase in copper concentration was also found to enhance the regeneration frequency (Nirwan and Kothari, 2003). Dahleen (1995) determined that increasing the copper concentration by as much as 50-fold that in MS medium improved plant regeneration of two barley cultivars. In the present investigation, addition of copper in from of CuSo4 to CIM-1, although increased morphogenic callus induction and subsequent plant regeneration but number of shoots produced per explant were very low (Table 4).
Supplementation of CIM-I with casein hydrolysate, glutamine and CuSo4 together had best results, attaining 100% morphogenic callus induction, 90% subsequent plant regeneration and up to 10 shoots per explant. The incorporation of combination of growth additives to immature embryos of J. curcas appear to be critical in obtaining increased morphogenic callus induction and subsequent plantlet regeneration as evident from present report.
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The highest frequency of morphogenic callus induction (100%), subsequent plant
regeneration (90%) and maximum number of plantlets (10) were observed on MS medium with 3% sucrose in supplementation of 0.5 mg/1 IBA + 1.0 mg/1 BAP + 100 mg/1 casein hydrolysate + 200 mg/11-glutamine + 50uM CuS04.
The incorporation of combination of growth additives to immature embryo cultures appear to be critical in obtaining maximum morphogenic callus induction.
The in vitro regenerated plants (2-4 cm in length) rooted on 14 strength MS medium with 2.0% (w/v) sucrose supplemented with 0.5 mg/1 IBA and 342 mg/1 trehalose, after 4-8 weeks.
The rooted plants after acclimatization successfully transferred to the field in different agro-climatic zones of India. The regenerated plants showed no phenotypic abnormalities.
The developed protocol was evaluated on five elite lines of J. curcas and results have been reproduced. Many times, morphogenesis in callus cultures is genotype dependant. When optimized protocol using immature embryo as an explant was extended to five other lines of J. curcas collected from various geographical locations of India; S003 (Jagdalpur district in Chhattisgarh); S002 (Sarguja area of Chattisgadh); S006 (Churu district of Rajastan); S007 (Hanumangad district of Rajasthan) and S009 (Sirohi area of Rajasthan), the results were successfully reproduced indicating that protocol is genotype independent. To date 1000 plants have been regenerated with this protocol, while several of them are planted in different agro climatic zones, we found no apparent phenotypic variations in regenerated plants.
EXAMPLES
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor
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to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
EXAMPLE 1:
Plant material
Fruits from five different lines of Jatropha curcas L. were collected from various geographical locations of India; S003 (Jagdalpur district in Chhattisgarh); S002 (Sarguja area of Chattisgadh); S006 (Churu district of Rajastan); S007 (Hanumangad district of Rajasthan) and S009 (Sirohi area of Rajasthan)". Fruits were collected after 3 w, 4 w, 5w or 6 weeks after pollination (WAP) and were divided into four groups, viz., immature fruit size class-l, 2, 3 and 4. The seed size, texture and color were the criteria to select the right stage of the explant. The immature fruit size class-1 and 2 were (3 and 4 WAP: respectively), characterized by green colour fruit, whitish seeds with watery endosperm. The immature fruit size class-3 (5 WAP) were characterized by green colour fruit, light brown seeds with watery and few celled thick endosperm while the immature fruit size class-4 (6 WAP) characterized by dark green colour fruit, dark brown to black seeds with white, developed endosperm. Fruit sizes and their respective immature embryo sizes were recorded (Table 1).
Embryo isolation
Fresh fruits were dissected and outer fleshy layer was removed by surgical scalpel to remove seeds. The seeds were surface sterilized with 0.1 % (w/v) Bavistin (BASF, India) for 30 minutes followed by four rinses with sterile distilled water. Then the immature seeds were surface sterilized with 70% Ethanol (v/v) for 3 minutes and rinsed by sterile distilled water followed by 2.5 % Sodium Hypochlorite (v/v) for 30 minutes and rinsed five times with sterile distilled water. Afterwards, the seed coat was removed with the help of sterile mortar and pestle under laminar air flow cabinet. Removed seed
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coat was discarded. Now from these de-coated seeds immature embryos (immature embryonal axis + cotyledons) were isolated carefully by dissecting endosperm. able 1: Effect of the developmental stage of immature embryo on morphogenic callus induction and subsequent plant regeneration capacity of Jatropha curcas L. in vitro

Age of the
explant (WAP)* Stages of physiological maturity of fruit Size class of the fruits Size of the fruits (cm) Embryo '
size
(cm) Percent of
callus induction (after 4 weeks of culture) Percent of
plant
«** regeneration
3 Immature, green 1 2.4 0.2-0.3 No response -
4 Immature, green 2 2.6 0.4-0.5 No response -
5 Immature, green 3 2.8 0.7-0.9 30 No
6 Immature, dark green 4 3.2 1.1-1.5 100 70
* Weeks after pollination
**Data obtained 4 weeks after culture on callus induction medium (CIM-2) consisting of
MS medium with 3% sucrose supplemented with 100 mg/1 casein hydrolysate + 200 mg/1
glutamine + 0.5 mg/1 IBA + 1.0 mg/1 BAP + 50uM CuS04
***Data obtained 4 weeks after culture on regeneration medium (RM) consisting of MS
medium + 3% Sucrose + 500 mg/1 polyvinyl pyrrolidone + 30mg/l citric acid + 1.0 mg/1
BAP+ 0.5 mg/1 Kn + 0.25 mg/1 IBA
The effect of the age of the immature embryo on callus induction and subsequent plant regeneration was studied (Table 1). To determine, the green immature fruits were collected at 3, 4. 5 and 6 weeks after pollination (WAP) whose sizes were 2.4, 2.6. 2.8 and 3.2 cm, respectively (Table 1). Four size classes of immature embryos (0.2-0.3 cm; 0.4-0.5 cm; 0.7-0.9 cm and 1.1-1.5 cm) were cultured on MS (Murashige and Skoog, 1962) basal medium supplemented with 0, 0.5, 1.0, 2.5 and 5.0 mg/1 of 2,4-D, PIC, IBA, IAA, Kn or BAP (data not shown). There were significant differences in the callus formation among size classes of immature embryos. The immature embryo size class-4 (1.1-1.5 cm) obtained from immature fruits 6 WAP produced highest callus formation (100%) on MS medium supplemented with lower quantity of IBA (0.5 or 1.0 mg/1). The
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subsequent plant regeneration was also highest (70%) in above size class (Table 1). The Immature embryos size classes 1-3 (0.2-0.3 cm; 0.4-0.5 cm and 0.7-0.9 cm) obtained from fruits 3, 4 and 5 WAP, respectively usually did not produce morphogenic callus and large embryos (i.e., beyond 1.5 cm) obtained from fruits beyond 7 WAP germinated but failed to produce morphogenic callus (Data not shown). In each experiment 7 explants with two replications were taken for the callus induction. Each experiment was repeated thrice.
Developmental stage of the explant is reported to be crucial factor for in vitro regeneration in cereal crops (Castillo et al., 1998). The optimal size and age of the embryo varies with plant genotype. In this study, the optimum developmental stage and size of immature embryos of J! curcas for morphogenic callus induction and subsequent plant regeneration was found to be size class-4, 1.1-1.5 cm in length obtained from immature fruit 6WAP.
Immature embryos are most responsive source to regenerate plantlets among other explants in culture. In this study, immature embryos of J. curcas from various developmental stages have been tested for their morphogenic potential. Developmental stage of immature embryo has been reported as an important factor for regeneration (He et al., 1988). Abdullah et al., (2005) demonstrated potential role of immature embryos in genetic transformation and direct organogenesis. In present study, the percent of morphogenic callus induction (100%) and subsequent plant regeneration (70%) was highest in immature embryos (1.1-1.5 cm) obtained from size class-4. In rest of the size classes either morphogenic callus induction was very poor or subsequent plant regeneration was not observed (Table 1). Induction of morphogenic callus was critical stage in tissue culture where the quality and type of callus influences subsequent plant regeneration. So far, to our knowledge, no comprehensive study was undertaken to study the relationship between morphogenic potential and the developmental stage of embryo in J. curcas. Results from present study found that the optimum developmental stage of immature embryo for morphogenic callus induction and subsequent plant regeneration in J. curcas was from immature fruits obtained 6 WAP.
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EXAMPLE 2: Callus induction medium
The immature embryos were longitudinally dissected into 6-7 small pieces and placed horizontally on 90 mm dia. plastic Petri plates (Greiner, Germany) containing 25ml medium. They were cultured on 36 different combinations of the callus induction medium (Table 2). The media contained MS (Murashige and Skoog, 1962) basal salts with 3% sucrose, pH 5.8 solidified with 0.75% agar (Hi-media, Mumbai) and supplemented with different concentrations of auxins (2.4-D, IAA, IBA, NAA, PIC) or cytokinins (BAP or Kn) alone or in combinations. The data on callus induction response were collected after 4 weeks of culture on callus induction medium.. Among auxins, IBA showed best response of callus induction (data not shown). Subsequently, further experiments were carried out with IBA in combination with cytokinins BAP and Kn (Table 2).
Table 2: Effect of various growth regulators on induction of morphogenic callus and subsequent plant regeneration from immature embryo cultures of Jatropha curcas L

Growth regulators (mg/1) * Callus
formation
(%) Subsequent plant regeneration** (%) Shoots per
explant
(Mean)
Auxin Cytokinin
0.2 IBA 0.2 Kn 28.6 2.0

0.5 Kn 28.6 1.0

1.0 Kn 14.3 - -
0.5 IBA 0.2 Kn 14.3 - -

0.5 Kn 14.3 - -

I.OKn 28.5 - -
1.0 IBA 0.2 Kn 28.5 - -

0.5 Kn 28.5 - -

l.OKn 14.3 - -
0.2 IBA 0.2 BAP 14.3 - -

0.5 BAP 28.6 0 0.0

1.0 BAP 42.9 20 3.6
0.5 IBA 0.2 BAP 42.9 25 3.9

0.5 BAP 57.1 40 2.0

1.0 BAP 85.7 70 4.7
1.0 IBA 0.2 BAP 71.4 0 0.0

0.5 BAP 42.9 0 0.0

1.0 BAP 57.1 40 1.3
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Data scored after 4 -weeks of culture
*Growth regulators were supplemented to MS medium containing 3% sucrose. **Only green, compact and regenerating calli were transferred to regeneration medium containing MS medium+ 3% sucrose+ 500 mg/1 polyvinyl pyrrolidone + 30mg/l citric acid+ 1.0 mg/1 BAP, 0.5 mg/1 Kn+ 0.25 mg/1 IBA+ 50uM CuS04
Immature embryos grown on MS basal medium supplemented with 3% sucrose in combination with IBA (0.2, 0.5 and 1.0 mg/1) and cytokinins- BAP or Kn (0.2, 0.5 and 1.0 mg/1) exhibited a wide range of callus induction percentage (14.3 - 85.7%). In media with 0.5 mg/1 IBA in combination with any concentration of BAP or Kn (Table 2), cream and friable non regenerable callus and green, or whitish green very compact, regenerable callus was found (Fig. la). IBA in combination with Kn induced lowest percentage of callus whereas, in combination with BAP induced highest percentage of morphogenic callus. But the best result was observed on MS medium supplemented with 0.5 mg/1 IBA + 1.0 mg/1 BAP (i.e. 85.7%). This medium was termed as CIM-1.
After identifying the most suitable medium for the callus induction (termed as CIM-1) CIM-1- was supplemented with different growth additives (casein hydrolysate, 1-glutamine, 1-proline, silver nitrate, copper sulphate either alone or in combination) in different concentration (Table 3) in order to improve morphogenic potential of the callus. This medium is termed as CIM-2. Cultures grown on above media showed the callus formation percentage with the range of 42.9% (with 100 mg/1 casein hydrolysate, alone) till 100% (with 100 mg/1 casein hydrolysate + 200 mg/1 glutamine). However, the green, compact, morphogenic calli (Fig 2A) were found on CIM-1 medium supplemented with 100 mg/1 casein hydrolysate + 200 mg/1 1-glutamine and CIM-1 with 50|aM CuS04. Subsequent plant regeneration was also found to be highest (90%) on above media. But. when CIM-1 was supplemented with combination of casein hydrolysate (100 mg/1) + glutamine (200 mg/1) + CuS04 (50uM), percentage of callus induction and subsequent plant regeneration as well as the number of shoots per explant were increased tremendously (Table 3).
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Table 3: Supplementation of different growth additives to the CIM-1 and their influence on further improvement of the morphogenic callus induction and subsequent plant regeneration from immature embryo cultures of Jatropha curcas L.

Treatments* Callus formation (%) Subsequent
plant
regeneration**
(%) Shoots per explant (Mean)
lOOmg/lCH 42.9 30 4.7
200mg/lCH. 57.1 50 4.4
500 mg/1 CH 57.1 60 2.7
100 mg/11-glutamine 71.4 50 2.0
200 mg/11-glutamine 85.7 45 2.0
500 mg/11-glutamine 71.4 70 4.0
100 mg/1 CH+100 mg/1 1-glutamine 85.7 70 3.8
100 mg/1 CH +200 mg/11-glutamine 100.0 90 9.1
100 mg/1 CH +500 mg/11-glutamine 85.7 80 0.0
10 mg/11-proline 57.1 40 1.0
50 mg/11-proline 57.1 50 1.0
100 mg/1 -proline 42.9 40 0.0
10 uMAgN03 71.4 50 1.0
50 nM AgN03 71.4 70 6.0
100uMAgNO3 85.7 80 2.0
5 uM CuS04 85.7 60 1.3
50 uM CuS04 85.7. 90 1.0
100uMCuSO4 71.4 70 0.0
100 mg/1 CH +200
mg/1 l-glutamine+ 50
uM CuS04 100.0 90 10.0
Data scored after 4 -weeks of culture
*MS medium supplemented with 3% sucrose+ 0.5mg/l IBA + 1.0 mg/1 BAP was taken as
a basal medium for the callus induction
* * Only green, compact and regenerating calli were transferred to regeneration medium
containing MS medium with 3% sucrose+ 500 mg/I polyvinyl pyrrolidone (PVP)+
30mg/l citric acid+ 1.0 mg/1 BAP, 0.5 mg/1 Kn+ 0.25 m^l IBA+ 50uM CuS04.

When casein hydrolysate and 1-glutamine used alone, lower percent of (30-70%) subsequent plant regeneration was observed in comparison to a combination of both additives (70-90%). L-proline when used alone, subsequent plant regeneration was low (40-50%). AgN03 at 100 uM showed 80% subsequent plant regeneration but in comparison to C11SO4 at 50 uM it was low (90%) although both are ethylene inhibitors. Finally, the callus induction medium arrived at consisted of MS medium with 3% sucrose supplemented with 0.5 mg/1 IBA+ 1.0 mg/1 BAP + 100 mg/1 casein hydrolysate + 200 mg/11-glutamine + 50 uM Q1SO4. This medium was termed as CIM-2.
Rest of the growth additives such as l-proline, AgNCb did not have marked influence on callus induction compared to rest of the growth additives. It was found from present study that there was remarkable increase in callus induction percentage and subsequent plant regeneration when CIM-1 was supplemented with combination of growth additives (Fig. lb). Our efforts in defining optimum medium to enhance morphogenic callus induction percentage and subsequent plant regeneration in immature embryo cultures of J. curcas by supplementing CIM-1 with combination of growth additives proved rewarding. CIM-2 was tried, at least, 20 times for morphogenic callus induction and provided reproducible results morphogenic calli and subsequent plant regeneration consistently.
The data were recorded after 4 weeks of culture on CIM-2 to evaluate induction of morphogenic calli.
EXAMPLE 3: Maintenance medium
The morphogenic callus after 4 weeks of culture on CIM-2 was subcultured further on to maintenance medium for the further proliferation of morphogenetic calli. To define optimum maintenance medium we used 36 different combinations of growth regulators, in combination with growth additives (data not shown). Finally the maintenance medium consisted of MS medium with 3% sucrose supplemented with 100 mg/1 casein hydrolysate + 200 mg/1 glutamine + 50uM CUSO4 with reduced concentrations of IBA (0.25 mg/1).
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EXAMPLE 4: Regeneration medium
For. plant regeneration our group has developed a medium for proliferation and elongation of shoot buds (Deore and Johnson 2008). This medium consisted MS medium with 3% sucrose supplemented with 1.0 mg/1 BAP+ 0.50 mg/1 Kn + 0.125 mg/1 IAA+ 0.125 mg/1 IBA + 0.5 mg/1 GA3 and was taken as a reference medium for the present investigation. In order to improve regeneration capacity of morphogenic calli eight different media were designed supplemented with growth additives, viz., copper sulphate at 50uM, casein hydrolysate at 100mg/l, 1-glutamine at 200mg/l, silver nitrate at 50, 100 uM and trehalose at 34.2, 171, 342, 682 mg/1 alone and in combination (Table 4). To study the influence of growth additives on regeneration capacity of morphogenic calli eight different media were designed for optimizing above regeneration medium (Table 4).
Table 4: Optimization of regeneration medium for plantlet regeneration from morphogenic callus obtained from immature embryo cultures of Jatropha curcas L

Treatment Responded explant (%) Regenerated shoots per explant
Base medium (BM)* 60 S^
BM+34.2 mg/1 trehalose 30 s+
BM+171mg/I trehalose 40 s*
BM+342 mg/1 trehalose 30 s+
BM+682 mg/1 trehalose 40 S"1"
BM+50mM CuSo4 90 s+++
BM+100 mg/1 CH+ 200 mg/11-glutamine 10 s+
MS basal salt+3%sucrose+0.125 mg/1 IBA+ 0.125 mg/1 IAA+0.5 mg/1 GA3 50 s^
MS basal salt+3%sucrose+0.125 mg/1 IBA+ 0.125 mg/1 IAA+0.5 mg/1 GA3+ 50 uM AgN03 60 s~
Data recorded after 4-weeks of culture.
*Base medium contained MS medium + 3% sucrose + 500 mg/1 polyvinyl pyrrolidone
(PVP) + 30mg/l citric acid + 1.0 mg/1 BAP + 0.5 mg/1 Kn + 0.25 mg/I IBA
1 shoot per callus ^ 2-4 Shoots per callus
more than 4 shoots per callus
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Only green, compact and morphogenic calli were used for optimization. Twenty callus pieces were transferred to each medium composition and the regeneration capacity was evaluated after 30 d of culture. While addition of trehalose did not have significant influence on number of shoots regenerated, highest shoot regeneration was observed on medium supplemented with CUS04 (50 urn) with 4 shoots per callus piece (Table 2 and Fig. I c&d). Finally the best regeneration medium arrived at consisted of MS medium with 3% sucrose supplemented with 500 mg/1 PVP+ 30mg/l citric acid+ 1.0 mg/1 BAP+ 0.5 mg/1 Kn+ 0.25 mg/1 IBA + 0.5 mg/1 GA3+ 50uM CuS04. The regeneration capacity was evaluated after 30 d.
EXAMPLE 5: Root induction
Regenerated shoots (2-4 cm in length) were transferred to 8 different media combinations for root induction and supplemented with different concentrations of zeatin or NAA or IAA or IBA with or without trehalose (Table 5). Data on root induction was scored after 30 days of culture. In all of these experiments the pH was adjusted to 5.8 before autoclaving and the medium was solidified with 0.7% agar (Hi-media, Mumbai) and sterilized for 20 min at 121°C temperature. All the cultures were incubated in a culture room at 16-h (day) / 8 h (dark) photoperiod cycle illuminated with cool fluorescent lamps at an intensity of 30 umol m'2 s"'at 24+2°C.
Table 5: Effect of different growth regulators on root induction in regenerated shoots of Jatropha curcas L.

Growth regulators* Root induction response No. of roots per plant

type percent
0.1 mg/1 IBA PR 20-30 1-2
0.1 mg/1 IBA+ 0.1 mg/1 Zeatin+ 0.5 mg/1 GA3 - - -
0.5 mg/1 GA3 - - -
0.1 mg/1 Zeatin - - -
0.1 mg/1 IAA+ 0.5 mg/1 GA3 - - -
0.5 mg/1 NAA - - -
0.1 mg/1 IBA+ 342 mg/1 trehalose PR and SR 60-80 >2
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Data scored after 4-weks of culture
* Vz MS medium with 2.0% of sucrose has taken as basal medium for rooting experiments
PR= Primary roots
SR= Secondary roots
Root formation was observed on medium supplemented either with IBA (0.1 mg/I) alone in 20-30% culture or IBA (0.1 mg/1) in combination with 342 mg/1 trehalose in 60-80% cultures. The best result was found on I/2 MS medium with 2% sucrose and supplemented with 0.1 mg/1 IBA and 342 mg/1 trehalose (Table 5). On this medium, profuse rooting with secondary roots was also observed. There was no root formation on V2 MS medium with 2% sucrose supplemented with only zeatin or NAA or GA3 or any combination thereof
EXAMPLE 6: Field transfer of regenerated plants
The well developed regenerated plantlets with 4-5 cm long shoots and 5-9 cm long primary roots with secondary branches were transferred to the soil for the first acclimatization (Fig. le). For the 1st acclimatization they were grown successfully in incubator at 24 °C and 12 h photoperiod with 80% relative humidity in the growth chamber, these plantlets were covered with polythene bags to retain the humidity. . If these plantlets were not covered with the polythene bags, then the plants tend to die after couple of days
The well rooted regenerated plants were transferred to the soil in an environmental chamber (Sanyo, Japan) at 80% relative humidity, 24 ± 2°C temperature and 12 hour photoperiod with 30 (jmol m"2 s"'at light intensity. The soil mixture contained garden soil: coco peat: sand: vermicompost in 1:1:1:1, respectively. These plantlets were covered for a week with the polythene bags to retain moisture and to avoid any unwanted desiccation. After 1 week the polythene bags were removed and the plants were incubated in the same chamber for 2-3 weeks. Then these plants were transferred to the poly house at 28-30° C. When the plants became approximately 30cm tall then these were transferred to the field in open environment at natural condition.
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Afterwards these plantlets were transferred to the poly house (Fig. If). When the plants were totally acclimatized and reached to 24-30 cm. height, they were transferred to the field in natural field conditions (Fig.lg). The plants were grown very well under field conditions. The regenerated plantlets developed by this protocol were delivered to different fields and locations in India. The plant were survived happily in all different locations.
When the same protocol was followed for further experiments, the reproducibility was proven (data not shown). This protocol would be very helpful for the further transformation experiments. When the plants were transferred for the acclimatization, 50-70% plantlets could be survived from in vitro to in vivo conditions. For further acclimatization, these plants were transferred to poly house at 30° C. When plants were totally acclimatized in polyhouse could be transferred to the field successfully under different environmental conditions.
Example 7: Evaluation of developed protocol on other genotypes
When the developed protocol was used for the tissue culture experiments of other elite lines (S 002, S 006, S 007, S 009) ofJatropha curcas, it also worked reproducibly. The plant regeneration was also observed in all the lines (Table 6). The fruits were taken with the range of 3.1 to 3.4 cm. big and the size of embryos were 1.0 to 1.5 cm which gave the best response for the immature embryo culture experiments of different elite lines. This experiment has proven that the developed protocol is very reproducible not only with line R 044 but also other elite lines of Jatropha curcas.
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Table 6: In vitro culture response of immature embryo cultures of five elite lines of Jatropha curcas L. - Reproducibility of optimized protocol for morphogenic callus induction and subsequent plant regeneration.

Name of the line Size of the fruits (cm) Age of the
explant
(WAP) Embryo size including cotyledon (cm) Response for morphogenic callus induction Subsequent
plant
regeneration
(%r .
R044 3.4 6 1.1-1.5 100 90
S002 3.1 6 1.2-1.4 70 70
S006 3.2 6 1.2-1.5 70 65
S007 3.2 6 1.1-1.5 95 90
S009 3.1 6 1.0-1.4 90 90
*CIM-2 consisting of MS medium with 3% sucrose supplemented with 100 mg/1 casein hydrolysate + 200 mg/1 glutamine + 0.5 mg/1 IBA + 1.0 mg/1 BAP + 50uM CuS04 was used for morphogenic callus induction. Data scored after 4 weeks of culture. ** Regeneration medium consisting of MS medium + 3% Sucrose + 500 mg/1 polyvinyl pyrrolidone + 30mg/l citric acid + 1.0 mg/1 BAP+ 0.5 mg/1 Kn + 0.25 mg/1 IBA+ 50 uM CUS04 was used for plant regeneration. Data scored after 4 weeks of culture
REFERENCES
1. Abdullah R, Zainal A, Heng YW, Li CL, Beng YC, Phing LM, Sirajuddin SA: Ping WYS, Joseph JL, Jusoh SA, Muad MR, Huey YL (2005) Immature embryo: A useful tool for oil palm (Elaeis guineensis Jacq.) genetic transformation studies. E. J. Biotechnol. 8: 25-34
2. Akasaka-Kennedy Y, Yoshida H, Takahata Y (2005) Efficient plant regeneration from leaves of rapeseed (Brassica napus L): the influence of AgNo3 and genotype. Plant Cell Rep. 24:649-654
23

3. Baburaj S, Dhamotharan R, Santhaguru K (1987) Regeneration in leaf callus cultures of Euphorbia hirta Linn. Curr Sci. 56:194
4. Berchmans HJ, Hirata S (2008) Biodiesel production from crude Jatropha curcas L. seed oil with a high content of free fatty acids. Bioresour. Technol. 99:1716-1721.
5. Binott JJ, Songa JM, Ininda J, Njagi EM, Machuka J (2008) Plant regeneration from immature embryos of Kenyan maize inbred lines and their respective single cross hybrids through somatic embryogenesis. African J. Biotechnol. 7: 981-987
6. Castillo AM, Egana B, Sanz JM, Cistue L (1998) Somatic embryogenesis and plant regeneration from barley cultivars grown in Spain. Plant Cell Rep. 17: 941-945
7. Choi P.-S, Cho D.-Y, Soh W.-Y (2003) Plant regeneration from immature embryo cultures of Vigna unguiculata. Biol. Plant. 47:305-308
8. Dahleen LS (1995) Improved plant regeneration from barley callus cultures by increased copper levels. Plant Cell Tiss. Org. Cult. 43:267-269
9. Datta MM, Mukherjee P, Ghosh B, Jha TB (2007) In vitro clonal propagation of biodiesel plant (Jatropha curcas L.) Curr. Sci. 93: 1438-1442.
10. Deore A, Johnson TS (2008a) High-frequency plant regeneration from leaf-disc cultures of Jatropha curcas L.: an important biodiesel. Plant. Biotechnol. Rep. 2:7-11.
11. Deore A, Johnson TS (2008b) Occurrence of vivipary in Jatropha curcas L. Curr. Sci. 95:321-322.
12. Gubitz GM, Mittelbach M, Trabi M (1999) Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresour. Technol. 67: 73-82.
24

13. He DG, Yang YM, Scott KJ (1988) A comparison of scutellum callus and epiblast callus induction in wheat: the effect of genotype, embryo age and medium. Plant Sci. 57:225
14. Jha TB, Mukherjee P, Datta MM (2007) Somatic embryogenesis in Jatropha curcas Linn.., an important biofuel plant. Plant Biotech Rep. 1:135-140.
15. Jones N, Miller JH (1991) Jatropha curcas- a multipurpose species for problematic sites. Land Resources Series 1:1-12.
16. Minhas D, Rajam MV, Grover A (1999) Maintenance of callus growth during sub-culturing is a genotype dependant response in rice: Mature seed-derived callus from IR-54 rice cultivar lacks culture ability. Curr. Sci. 77:1410-1413
17. Modi MK, Reddy JR, Rao BV, Prasad RB (2007) Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor. Bioresour. Technol. 98:1260-1264.
18. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15: 473-497
19. Nirwan RS, Kothari SL (2003) High copper levels improve callus induction and plant regeneration in Sorghum bicolor (L.) Moench. In Vitro (Plant) 39:161-164
20. O'kennedy MA. Smith G, Botha FC (2004) Improved regeneration efficiency of a pearl millet {Pennisetum glaucum) breeding line. South Africa J. Bot. 70:502-508
21. Qin W, Wei-Da Lu, Yi L, Shu-lin P, Ying X, Lin T, Fang C (2004) Plant regeneration from epicotyl explant of Jatropha curcas. J Plant physiol Mol Biol 30: 475-478
22.Rajore S, Batra A (2005) Efficient plant regeneration via shoot tip explant in Jatropha curcas L. J Plant Biochem Biotech 14: 73-75
25

23. Rao AM. Padmasree K, Kishor, PBK (1995) Enhanced plant regeneration in grain and sweet sorghum by asparagines, proline and cefotaxime. Plant Cell Rep. 15:72-75
24. Ripley KP, Preece JE (1986) Micropropagation of Euphorbia lathyris L. Plant Cell Tiss. Org. Cult. 5:213-218
25. Roy AT, De UN (1990) Tissue culture and plant regeneration from immature embryo explants of Calotropis gigantea (Linn.) R. Br. Plant Cell Tiss. Org. Cult. 20: 229-233
26. Sarvesh A, Rao DM, Reddy TP (1992) Callus initiation and plantlet regeneration from epicotyl and cotyledonary explants of castor ((Ricinus communis L.). Adv. Plant Sci. 5:124-128
27. Srivastava PS (1971) In vitro induction of triploid roots and shoots from mature endosperm of Jatropha panduraefolia. Z. Pflanzenphysiol. 66:93-96
28. Srivastava PS, John BM (1974) Morphogenesis in mature endosperm cultures of Jatropha panduraefolia. Beitr. Biol. Pflanz. 50:255-268
29. Sujatha M, Dhingra M (1993) Rapid plant regeneration from various explants of Jatropha mtegerrima. Plant cell tiss. Org. cult. 35:293-296
30. Sujatha M, Mukta N (1996) Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas L. Plant cell tiss. Org. cult 44:135-141
31. Sujatha M, Reddy TP (1998) Differential cytokinin effects on the stimulation of in vitro, shoot proliferation from meristematic explants of castor (Ricinus communis L.). Plant Cell Rep. 17:561-566
32. Sujatha M, Reddy TP (2000) Morphogenic responses of Jatropha integerimma explants to cytokinins. Biol Bratislava 55: 99-104
26

33. Thepsamran N, Thepsithar C, Thongpukdee A In vitro multiple shoot induction of physic nut {Jatropha curcas)
34. Tideman J, Hawker JS (1982) In vitro propagation of late* producing plants. Ann Bot 49: 273-279
35. Tiwari AK, Kumar A, Raheman, H (2007) Biodiesel production from Jatropha (Jatropha
. curcas) with high free fatty acids: an optimized process. Biomass Bioenergy 31: 569-575.
36. Zhang FL, Takahata Y, Xu JB (1998) Medium and genotype factors influencing shoot regeneration from cotyledonary explants of Chinese cabbage {Brassica campestris L). Plant Cell Rep. 17:780-786
37. Zhang P, Phansiri S, Puonti-Kaerlas J (2001) Improvement of cassava shoot organogenesis by the use of silver nitrate in vitro. Plant Cell Tiss. Org. Cult. 67:45-54
Thus, while we have described fundamental novel features of the invention, it will be understood that various omissions and substitutions and changes in the form and details may be possible without departing from the spirit of the invention. For example, it is expressly intended that all combinations of those elements and/or method steps, which perform substantially the same function in substantially the same way to achieve the same results, be within the scope of the invention
Dated this 13th day of februry , 2009
For Reliance Life Sciences Pvt. Ltd
K. V. Subramaniam President