WO2009072141A2

WO2009072141A2 - Jatropha curcas hybrid nandan-1 for high seed yield and construction of molecular markers specific to it

Info

Publication number: WO2009072141A2
Application number: PCT/IN2008/000471
Authority: WO
Inventors: Koteswara Rao Karanam; Jayakumar Bhavanasi
Original assignee: Nandan Biomatrix Limited
Priority date: 2007-12-06
Filing date: 2008-07-25
Publication date: 2009-06-11
Also published as: AP2010005319A0; WO2009072141A4; WO2009072141A3

Abstract

A Jatropha curcas hybrid designated as Nandan-1, responsible for high seed yield is disclosed. The process of invention relates to a new and distinctive Jatropha curcas hybrid designated as Nandan-1 for high seed yield and construction of molecular marker specific to hybrid Nandan-1. We would like to patent our hybrid Nandan -1 in the process of invention and for the construction of molecular marker responsible for high seed yield and is specific to hybrid Nandan -1 and hence we are here by enclosing the same.

Description

JATROPHA CURCAS HYBRID NANDAN l FOR HIGH SEED YIELD AND CONSTRUCTION OF MOLECULAR MARKERS SPECIFIC TO IT

Field Of Invention:

The present investigation relates to a new and distinctive Jatropha curcas hybrid designated as Nandan-1 and construction of molecular marker specific to hybrid Nandan-1. All publications cited in this application are herein incorporated by reference.

Background Of The Invention:

In India, the tree species considered for biodiesel production are those that are not used as edible oil, which do not complete with food crops for resources and not used in traditional medicine. Jatropha curcas is one of the important species and has gained attention in tropical and sub-tropical countries for the use of its seed oil as a commercially viable and potential alternative source of fuel. It is a drought-resistant small tree that can reach a height of up to 5 m.

Breeding objectives depend on use of the specific crop; increasing yield is a primary objective in all programs. Seed yield of Jatropha curcas is determined by number of inflorescences per plant, number of fertile pistillate flowers per inflorescence, seeds per capsule and 100-seed weight. As the maximum number of seeds per capsule is limited and the agronomic factor of planting density^ does not offer much flexibility for increasing yields, selection should focus on the other yield components to obtain higher yield. Heritable variation exists for all of these components except number of seeds per capsule, and breeders may directly or indirectly select for increases in any of them.

Among the oil borne tree species, Jatropha curcas is considered to be the most prospective plant due to its hardiness, rapid growth, easy propagation, drought endurance, high oil content, low gestation period and ability to grow on degraded soils and waste lands with low to high rainfall. There are highly significant correlations in different seed samples between the 100-seed weight and percent of crude fat content. This is interesting from the breeders' point of view, as simple selection for high 100-seed weight could imply increased crude fat contents. But the shrubs which produce seeds of a high 100- seed weight, and consequently a higher crude fat content, may not yield more oil per hectare. Hence to solve this, the development of a high yielding Jatropha curcas hybrid is very necessary

Numerous steps are involved in the development of any novel, desirable cultivar. Plant breeding begins with the analysis and definition of problems and weakness of the current cultivars, followed by the fixation of program goals, and the definition of specific breeding objectives. The next step is selection of parental lines that possesses the traits, required to meet the program goals. The goal is to combine in a single cultivar an improved combination of desirable traits from the parental sources. These important traits may include higher yield, resistance to disease and insect pests, better canopy structure, tolerance to environmental stresses, better agronomic characteristics and higher oil content in oil bearing plants.

US7,005,298 relates to in vitro micropropagation and phytofortification of a phytopharmaceutical plant comprising: a) culturing a sterile explant of said phytopharmaceutical plant on an induction medium comprising at least one plant growth regulator having cytokinin activity, to form regenerated tissue; b) transferring said regenerated tissue to a first basal medium lacking said plant growth regulator having cytokinin activity, and culturing to form plantlets; and c) sub culturing said plantlets onto a second basal medium supplemented with one, or more than one additive of interest at an amount from about 50 to about 200 mg/L.

US 20070218175 discloses and claims a method of producing a triglyceride solution, comprising dissolving triglyceride in a liquid fatty acid alkyl ester to form a triglyceride solution from an oil-bearing plant. US20030028927 discloses method of detecting genetic variation in a plant genome, comprising inhibiting Hsp90 activity in at least one plant cell and method of identifying a plant polymorphism.

US20020194646 discloses a method of creating a transfected or transgenic plant chosen from the group consisting of ornamental, horticultural, forestry, medicinal or Nicotiana sp. plants, exhibiting a dwarf phenotype comprising: expressing in the plant the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122 or the mRNA encoded by the DNA identified by a polynucleotide sequence chosen from the group consisting of SEQ. ID NO: 1-122.

WO2006043281 discloses an improved process for the preparation of biodiesel from triglyceride oils through transesterification. More specifically, the invention relates to preparation of fatty acid methyl ester of oil mechanically expelled from whole seeds of Jatropha curcas. The utilization of the co products for preparing value added products and the integrated approach to utilize and manage the effluent streams make the process environmentally friendly.

CNl 799340 discloses a method of mass production grease tung seed sprout through tissue culture which provides a high effective way for the grease tung artificial breeding, germ-free sprout and species of high quality breeding.

Thus the present invention relates to new and distinctive Jatropha curcas hybrid which is responsible for high seed yield and construction of molecular marker specific to hybrid Jatropha curcas.

The goal of Jatropha curcas hybridization is to develop new, unique and superior Jatropha curcas hybrids. The breeder initially selects and crosses two or more parental lines, followed by selection among the many new genetic combinations. The breeder can theoretically generate billions of new and different genetic combinations via crossing. The breeder has no direct control at the cellular level; therefore, two breeders will never develop the same line, or even very similar line, having the same traits of Jatropha curcas. Choice of breeding process to select for the improved combination of traits depends on the mode of plant reproduction, the heritability of the traits being improved, and the type of cultivar used commercially. For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants.

There are possibilities for the breeder to modify the process. In addition, hybrid cultivars could be bred to use the heterosis effect. The existence of male sterile cultivars would facilitate crossings. Emasculation was not necessary for hybridization in the insect-free greenhouse due to absence of insect vectors and the time lag of anthesis of staminate flowers. The standard routine of bagging should be sufficient in the field. However, to avoid self-pollination if staminate and pistillate flowers were to open simultaneously, emasculation in this unisexual species could be required as Jatropha curcas is self- compatible and this can be achieved very easily as staminate and pistillate flowers look very distinct species.

Jatropha curcas is an important and valuable oil seed crop. Thus, a continuing goal of Jatropha curcas breeders is to develop stable, high yielding hybrid that are agronomically sound. The reasons for this goal are to maximize the amount of seed oil produced on the land used and to supply biofuel. To accomplish this goal, the Jatropha curcas breeder must select and develop Jatropha curcas plants that have the traits that result in superior hybrid.

The two important features of cross-pollinated species are inbreeding depression and heterosis. Population improvement schemes generally aim at keeping inbreeding at a low level to avoid its ill effects, but an effort to exploit heterosis is rarely made. Heterosis is the basis of hybrid varieties and hybrid varieties are the best means for exploiting heterosis. Object Of The Invention:

The object of the present invention is to develop a new, unique and superior Jatropha curcas hybrid, Nandan-1 and method of construction of a hybrid specific molecular marker responsible for high seed yield and its progeny.

Summary Of The Invention:

The present invention discloses to a hybrid Jatropha curcas plant designated as Nandan- 1, responsible for higher seed yield and construction of hybrid specific molecular marker.

In another aspect, the present invention relates to processes for producing a Jatropha curcas plant by crossing (JCZ-P34 x JCZ-I84) followed by biparental crossing adopting North Carolina Design II. Thus, any process using the hybrid Jatropha curcas plant Nandan-1 in backcross, hybrid production, crosses to population, clonal propagation and micro propagation are part of this invention. All plants which are a progeny of or descend from Nandan-1 are within the scope of this invention. It is an aspect of this invention for Jatropha curcas hybrid Nandan-1 to be used in crosses with other, different, Jatropha curcas plants to produce first generation (Fi) Jatropha curcas hybrid seeds and plants with superior characteristics.

Another object of the present invention relates to use of Jatropha curcas hybrid Nandan- 1 in crosses with other, different, Jatropha curcas plants to produce first generation (F₁) Jatropha curcas hybrid Nandan-1 seeds and plants with superior characteristics and to provide a method of regeneration of Jatropha curcas hybrid from tissue culture.

In further aspect, the present invention discloses regenerable cells for use in tissue culture (micro propagation) of Nandan-1. The tissue culture is preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Jatropha curcas plant, and of regenerating plants having substantially the same genotype as the foregoing Jatropha curcas plant. Preferably, the regeneration cells in such tissue cultures are embryos, protoplasts, meristematic cells, callus, pollen, leaves, anthers, pistils, root tips, seeds or stems. Still further, the present invention provides Jatropha curcas, Nandan-1 plants regenerated from the tissue cultures of the invention.

A plant having essentially all the physiological and morphological characteristics mean a plant having the physiological and morphological characteristics of the cultivar, except for the characteristics derived from the hybridization.

• 100-Seed weight. The weight of 100 Jatropha curcas seeds as measured in grams.

• Regeneration. Regeneration refers to the development of a plant from tissue culture.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.

The following embodiments and aspects thereof are described in conjunction with procedures, tools and processes which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

Description of the Drawings:

Figure 1 Gel electrophoresis of RAPD and PCR analysis for detecting Polymorphism

Figure 2 DNA Sequence of Nandan 1 (939 bp)

Detailed Description Of The Invention

The present invention describes Jatropha curcas hybrid Nandan-1 as a high seed yielding plant with more number of inflorescences per plant, more number of pistillate flowers per inflorescence, high female to male flower ratio, less number of abortive female flowers / unfilled capsules with three seeds per capsule and higher 100-seed weight.

Germplasm accessions have been collected from different agro climatic conditions of the country covering the parts of South, North, West, and East & Central India. The performances of these germplasm accessions in respect to yield and yield components and other morphological characters have been studied for two consecutive years. From these accessions two germplasm accessions were selected for the hybridization program. The female parent (JCZ-P34) was selected for having the character of producing cymose inflorescences with more number of pistillate flowers per inflorescence with highly fertile ovule and very less number of abortive flowers. The pollen parent (JCZ-I84) was selected due to its potentiality for producing more number of inflorescences per plant. Stability of the characters identified for selection of both the female and male parents has been established for two consecutive years.

The selected plants were then crossed (JCZ-P34 x JCZ-I84) for the production of Nandan-1. Jatropha curcas hybrid Nandan-1 has the following morphological and other characteristics as mentioned in Table 1, recorded at the age of three years.

This invention also is directed to methods for producing a Jatropha curcas hybrid by crossing a first parent Jatropha curcas plant with a second parent Jatropha curcas plant, wherein the first or second Jatropha curcas plant is a Jatropha curcas from the hybrid Nandan-1. Further, both first and second parent Jatropha curcas plants may be from the hybrid Nandan-1. Therefore, any methods using the hybrid Nandan-1 are part of this invention: selfing, backcross, hybrid breeding and crosses to populations. Any plants produced using Jatropha curcas hybrid Nandan-1 as a parent are within the scope of this invention.

This invention also is directed for producing Jatropha curcas hybrid Nandan-1 with a second Jatropha curcas plant and growing the progeny seed, and repeating the crossing and growing steps with the Jatropha curcas hybrid Nandan-1-derived plant from 0 to 7 times. Thus, any such processes using the Jatropha curcas hybrid Nandan-1 are part of this invention: selling, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using Jatropha curcas hybrid Nandan-1 as a parent are within the scope of this invention, including plants derived from Jatropha curcas hybrid Nandan-1.

It should be understood that the parents of hybrid Nandan-1, through routine manipulation of cytoplasmic and other factors, are produced in a male-sterile form. Such embodiments are also contemplated within the scope of the present claims.

Identification Of The Marker Molecular Characterization Of Nandan-1

Plant Material

Attempts were made to develop diagnostic molecular markers for the high yielding hybrid Nandan-1. For this purpose, molecular analysis of the hybrid Nandan-1 was carried out with 9 more diverse Jatropha curcas lines that exhibited variability in seed characters (large versus small), maturity pattern (extended flowering versus synchronous maturity), leaf size (small versus medium) and yield (high vs low).

DNA Extraction

Total genomic DNA was extracted from younger leaves of the hybrid (Nandan-1) following the standard CTAB process with minor modifications (Doyle and Doyle 1987). Five grams of leaves were ground in liquid nitrogen, then homogenized in 20 ml of extraction buffer (2% CTAB, 20 niM EDTA, 2% PVP, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0 and 1% β-mercaptoethanol) and incubated at 65 ⁰C for Ih. The supernatant was treated with RNase A (100 μg/ml), incubated at 37 ⁰C for 30 min and twice extracted with chloroform: isoamylalcohol (24:1 v/v). The DNA was precipitated with isopropanol and washed twice with 70% ethanol. The pelleted DNA was air dried and resuspended in 500 μl of sterile Millipore water and stored overnight at -20 ⁰C.

RAPD And PCR Analysis

A total of 200 decamer primers from Operon kits - OPB to OPK (Operon technologies, Alameda, USA) were used for DNA amplification according to the process of Williams et al. (1990). The PCR amplification reaction (10 μl) consisted of 2.5 ng of DNA, Ix PCR buffer (10 mM Tris pH 9.0, 50 mM KCl, 1.5 mM MgCl₂), 100 μM of each of the four dNTPs, 0.4 μM of RAPD primer and 0.3 U of Taq DNA polymerase (Bangalore Genei, India). PCR amplifications were performed in an GeneAmp 9700 Thermal Cycler (Eppendorf) with an initial denaturation at 94 ⁰C for 3 min followed by 45 cycles at 94 ⁰C iυ

for 45 s, 36 ⁰C for 30 s and 72 ⁰C for 2 min with a final extension at 72 ⁰C for 7 min. The PCR products were separated on 1.5% agarose gel in Ix TAE buffer by electrophoresis at 100 V for 3 h and visualized with ethidium bromide staining under gel documentation system. In general, RAPD markers suffer from a lack of reproducibility, but to check the consistency of the electrophoretic patterns and the polymorphism detected, every PCR reaction was repeated twice. All the PCR amplifications included a negative control (no DNA) to avoid erroneous interpretations.

The 200 tested primers gave robust amplification profiles. The polymorphism detected and these Polymorphic bands were checked for accession specific bands. Only one marker was found specific to Nandan-1.

Further, the distinction of the hybrid Nandan-1 has been accomplished through development of a molecular marker specific to the hybrid. The molecular marker gives a specific band of 939bp (M 13 primer) in Nandan-1. The inheritance of the marker was validated by checking it on progeny (20) resulting from this promising hybrid

Further reproduction of the hybrid can occur by tissue culture and regeneration. Tissue culture of various tissues of Jatropha curcas and regeneration of plants there from is well known and widely published. For example, reference may be had to Lin et. al., Plant Physiol Commun 38: 252 (2002); Lu et. al., Environ. Biol. 9: 127 (2003); Sujatha et. al., Plant Cell Tiss. Org. Cult.44: 135(1996); and Wei Qin, J. Plant Physiol. MoI Bio. 30: 475 (2004).Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce Jatropha curcas plants having the physiological and morphological characteristics of Jatropha curcas hybrid Nandan-1.

As used herein, the term 'tissue culture' indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, plant meristems, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as embryos, pollen flowers seeds, inflorescences, leaves, stems, roots, root tips, anthers, and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants.

As used herein, the term 'plant' includes plant cells, plant protoplasts, plant cells of tissue culture from which Jatropha curcas plants can be regenerated, plant calli, plant meristems, and plant cells that are intact in plants or parts of plants, such as pollen, flowers, embryos, ovules, seeds, inflorescence, leaves, stems, pistils, anthers and the like. Thus another aspect of this invention is to provide for cells which upon growth and differentiation produce a cultivar having essentially all of the physiological and morphological characteristics of Nandan-1.

The present invention contemplates a Jatropha curcas plant regenerated from tissue culture of the hybrid Jatropha curcas plant of the present invention. As is well known in the art, tissue culture of Jatropha curcas can be used for the in vitro regeneration of a Jatropha curcas plant. Tissue culture of various tissues of Jatropha curcas and regeneration of plants there from is well known and widely published. For example, reference may be had to Lin et. al., Plant Physiol Commun 38: 252 (2002); Lu et. al., Environ. Biol. 9: 127 (2003); Sujatha et. a\.,Plant Cell Tiss. Org. Cult. 44:135 (1996); and Wei Qin, J. Plant Physiol. MoI. Bio. 30:475 (2004). Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce Jatropha curcas plants having the physiological and morphological characteristics of Nandan-1.

Another process involves producing a population of Nandan-1 progeny Jatropha curcas plants, comprising crossing Nandan-1 with another Jatropha curcas plant, thereby producing a population of Jatropha curcas plants, which on average, derive 50% of their alleles from Nandan-1. A plant of this population may be selected and repeatedly selfed or sibbed with a Jatropha curcas plant resulting from these successive filial generations. One embodiments of this invention is the Jatropha curcas cultivar produced by this process and that has obtained at least 50 % of its alleles from hybrid Nandan-1. The same process may be used with one or all four of the parents of Nandan-1. Progeny of Jatropha curcas hybrid Nandan-1 may also be characterized through their filial relationship with Nandan-1, as for example, being within a certain number of breeding crosses of Nandan-1. A breeding cross is a cross made to introduce new genetics into the progeny, and is distinguished from a cross, such as a self or a sib cross, made to select among existing genetic alleles. The lower the number of breeding crosses in the pedigree, the closer the relationship between Nandan-1 and its progeny. For example, progeny produced by the processes describes herein may be within 1, 2, 3, 4, or 5 breeding crosses of Nandan-1.

TABLES:

The following tables present the data on the traits and characteristics of Jatropha curcas hybrid Nandan-1 as compare to its parental lines JCZ-P34 and JCZ-I84.

The results in Table 2 compare the yield, number of inflorescences per plant, number of pistillate flowers per plant, female to male flower ratio, seeds per capsule, 100-seed weight of Nandan-1, the hybrid of the recent invention produced by crossing JCZ-P34 and JCZ-I84. As shown in Table-2, hybrid Nandan-1 has significant higher yield, more number of inflorescences per plant, more number of pistillate flowers per plant, low female to male flower ratio, more seeds per capsule, and higher 100-seed weight than both of the parents of Nandan-1.

In Table 2 below, column 2 shows the yield, column 3 shows number of inflorescences per plant, column 4 shows number of pistillate flowers per plant, column 5 shows female to male flower ratio, column 6 shows seeds per capsule, and column 7 shows 100-seed weight. TABLE 1: HYBRID DESCRIPTION INFORMATION

Seed Characters

Seed coat color Black

Shape class (Length / width ratio) Long

Size (measurements) Len eth Width L / W

18 mm 11.3 mm 1.6

100-seed weight 65 g

Oil content 29.5 %

Disease Resistance

Powdery mildew {Erysiphe euphorbiae) Moderately resistant

Insect Pest Resistance

Inflorescence & capsule borer (Pempelia Moderately resistant morosalis) Moderately resistant

Leaf miner {Neurobathra curcassi Busck.) Moderately susceptible

Bugs (Scutellera nobilis Fabr.)

TABLE 2:

Claims

We Claim:

1. An intra specific hybrid of Jatropha curcas plant designated as Nandan-1 produced by crossing JCZ-P34 and JCZ-I84 and applying biparental crossing process adopting North Carolina Design at F₂ generation following selection.

2. A Jatropha curcas hybrid plant (Nandan-1) or a part thereof, produced by asexual propagation or tissue culture process or growing the seed of the claim 1.

3. Pollen of the hybrid plant (Nandan-1) of claim 2.

4. An ovule of the hybrid plant (Nandan-1) of claim 2.

5. A Jatropha curcas hybrid (Nandan-1) plant or a part thereof; is having all of the physiological and morphological characteristics of the Jatropha curcas hybrid (Nandan-1) plant of claim 2.

6. A tissue culture cells produced from the Jatropha curcas hybrid (Nandan-1) plant of claim 2, wherein said cells . of the tissue culture are produced from a plant part selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, inflorescence, and stems.

7. A protoplast produced from the hybrid (Nandan-1) plant of claim 2.

8. A Jatropha curcas hybrid (Nandan-1) plant regenerated from the tissue culture of claim 6, wherein the plant has all the morphological and physiological characteristics of hybrid Nandan-1.

9. A process of producing the Jatropha curcas intra specific hybrid Nandan-1 by crossing JCZ-P34 and JCZ-I84 and then applying biparental crossing process adopting North Carolina Design at F₂ generation following selection.

10. An intraspecific hybrid Nandan-1 plant or part thereof prepared by the process of claim 9.

11. A method of producing an intra specific hybrid Nandan-1 of Jatropha curcas plant with the morphological and physiological characteristics of hybrid Nandan-1 according to claim 9 or claim 10, comprising regeneration of a tissue culture.

12. A method of producing an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 according to any one of claims 9 to 11 wherein the Nandan-1 exhibits a high yield, resistance to disease and insect pests, improved canopy structure, tolerance to environmental stresses, improved agronomic characteristics and/or higher seed yield.

13. A method of producing an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 according to any one of claims 9 to 12, wherein the female parent used in the method comprises the characteristics of producing cymase inflorescence, a highly fertile ovule and a small number of abortive flowers.

14. A method of producing an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 according to any one of claims 9 to 13, wherein the male parent used in the method comprises the characteristics of producing a greater number of inflorescences per plant.

15. A method of producing an intra specific hybrid Nandan-1 of Jatropha curcas plant according to any one of claims 9 to 14, wherein the intra specific hybrid Nandan-1 of Jatropha curcas plant is then crossed with a Jatropha curcas or part thereof, to produce a population of Nandan-1 progeny comprising 20 to 70% of alleles from the Nandan-1 parent.

16. A method of producing an intra specific hybrid of Jatropha curcas plant designated Nandan-1 according to any one of claims 9 to 15, wherein the Nandan-1 product comprises the sequence SEQ .ID.l (M 13 Primer)

17. A method of producing an intra specific hybrid of Jatropha curcas plant designated Nandan-1 according to any one of claims 9 to 16, wherein part of the Nandan - 1 is a product comprising the sequence SEQ .ID.l (M 13 Primer)

18. A method of producing an intra specific hybrid Nandan-1 of Jatropha curcas plant according to any one of claims 9 to 17 wherein the Nandan-1 product comprises a sterile male form.

19. An intra specific hybrid Nandan-1 plant or part thereof prepared by the method of any one of claims 9 to 18.

20. A method of producing pollen from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1, comprising forming the plant by the method of any one of claims 9 to 18 and extracting pollen there from.

21. A method of producing an ovule from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1, comprising forming the plant by the method of any one of claims 9 to 18 and extracting an ovule there from.

22. A method of producing a protoplast from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1, comprising forming the plant by the method of any one of claims 9 to 18 and extracting a protoplast there from.

23. A method of producing a microbiological product from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 comprising forming the plant by the method of any one of claims 9 to 18 and extracting a microbiological product.

24. A method of producing a microbiological product from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 comprising forming the plant by the method of any one of claims 9 to 18 and extracting a microbiological product selected from either a pollen cell or an ovule cell.

25. A method of producing a microbiological product from an intra specific hybrid of Jatropha curcas plant designated as Nandan-1 comprising forming the plant by the method of any one of claims 9 to 18 and extracting a microbiological product comprising one or more of embryos, protoplasts, meristematic cells, callus, leaves, anthers, pistils, root tips, seeds or stems.

26. A method of culturing tissue cells from an intra specific hybrid of Jatropha curcas plant according to any one of claims 9 to 18, comprising culturing tissue cells produced from the Jatropha curcas plant, where cells are selected from the group consisting of leaves, pollen, embryos, cotyledons, hypocotyls, meristematic cells, roots, root tips, pistils, anthers, flowers, inflorescence, callus, seeds and stems.

27. Use of the intra specific hybrid Nandan-1 plant or part thereof, according to claim 19 in a process for the production of a biofuel.

28. Use according to claim 27 wherein the biofuel is biodiesel.

29. A biofuel generated by the use according to claim 27 or claim 28.