TECHNICAL FIELD OF THE INVENTION:

The present invention relates to a new and distinctive Jatropha interspecific hybrid designated as Nandan-5. More particularly, it concerns to interspecific hybrid plants derived from the cross of Jatropha curcas with Jatropha integerrima species.

BACKGROUND AND PRIOR ART:

Jatropha curcas L. (Family Euphorbiaceae) occurs naturally in Central America, but it is cultivated in many tropical and subtropical areas of America, Africa and Asia (Lin et al, 2004). 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 crude fat content. This is interesting from the breeders point of view, as to create genetic diversity in the plants through different hybridization programme with simple selection for bushy type plants, resistant to insect-pests, high yield, and high oil content, long productive life span of around 40 years, easy adaptation in marginal/poor soil, suitability of oil as fuel without being refined. The plant parts are also used as a food source of organic fertilizer, insecticide and medicine for cancer, piles, snakebite, paralysis, dropsy, etc. However the soft stem character makes the plant highly susceptible to lodging and root-rot disease, wherever ample moisture is available. Also, the plant is highly sensitive to frost and temperature below zero degree. Jatropha integerrima, commonly called spicy Jatropha or peregrina, is an evergreen shrub or small tree native to the West Indies, especially Cuba. It has been introduced in southern parts of the United States, where it is rapidly becoming popular as a spectacular plant that blooms most of the year with clusters of bright red flowers and handsome dark green leaves. Jatropha integerrima is frost tolerant, root-rot resistant with sturdy stem and high degree of resistance to leaf eating caterpillars (Lakshminarayan and Sujatha, 2008) has assumed importance owing to its seed oil which is rich in linoleic acid.

Breeding objectives depend on use of the specific crop; increasing yield is a primary objective in all programs. Numerous steps are involved in the development of any novel, desirable cultivar. Hybridization program 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 those possess the traits, required to meet the program goals. The goal is to combine an improved combination of desirable traits in a single cultivar from the parental sources. These important traits may include higher yield, resistance to disease and insect-pest, better canopy structure, production of dwarf plants, tolerance to environmental stress, better agronomic characteristics and higher oil content in oil bearing plants.

As Jatropha curcas is a cross-pollinated crop, the existence of male sterile cultivars would facilitate crossings. The interspecific hybridization in Jatropha species plays a significant role in crop improvement by transferring useful traits such as high oil content, maximum number of seeds, more female flowers and hard stem character for promotion of Jatropha as a biofuel crop. An understanding of the biological nature of this crossability barrier will provide a way to successful production of new hybrids.

The following are the patents either issued or in the process of issuing related to Jatropha, but none of them are citing about plant material improvement for interspecific hybridization using the presently used crosses till F2BC3 generation.

US/2007/0218175 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.

US/2003/0028927 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.

WO/2006/061256 disclose the methods relates to interspecific hybrids of the genus Allium, specifically plants of the Allium cepa or Allium fistulosum species, which are resistant to the downy mildew of onion caused by the fungus Peronospora destructor, due to a Pd resistance locus, wherein any fragment of a chromosome, comprising the Pd resistance locus, can be present homozygously in progeny without causing lethality.

WO/2006/043281 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.

WO/2009/081416 discloses the method of the interspecific hybridization of the uncultivated Jatropha gossypifolia with the cultivated Jatropha curcas species. The new interspecific hybrid plant thus invented is found to have unique traits that differed from either parent.

An article by Rupert E. A. et al. of 1970 relates to the experimental studies of relationships in the genus Jatropha I. J. curcas x integerrima.

Dhillon et al, 2009, studied the wide range of variation in vegetative and reproductive stages of interspecific hybrids (Fl) between J. curcas and J. integerrima.

The goal of interspecific hybridization between J. curcas and J. integerrima are combining the desirable traits, such as high seed yield, high oil content, resistance to root-rot and frost tolerance. The development of improved cultivars through hybridization has brought a major contribution for increased productivity and quality of plants in different parents and is been followed for back cross (F2BC3) development and simultaneously molecular marker techniques are often used for fostering plant improvement. The traditional method of hybrid identification based on morphological characters and is influenced by environmental factors, thus frequently lacks the resolving power to identify hybrids at the juvenile stage. Whereas now the status of the hybrid plants are been assessed at the DNA level using molecular markers i.e. SSR (Simple Sequence Repeats) and SNP (Single Nucleotide Polymorphism).

A similar interspecific hybrid was developed by Parthiban et al, (2009) entitled " Hybrid progenies in Jatropha - a new development", but that does not resemble the present invention, Nandan-5, which is been developed for profuse flowering, better canopy, hard stem character, high seed yield, high oil content, frost tolerant and root-rot resistant through the method of hybridization programme (F2BC3).

SUMMARY OF THE INVENTION:

The invention provides an interspecific hybrid Jatropha plant or part thereof derived from the cross of Jatropha curcas as female parent and Jatropha integerrima as pollen parent. Further provided by the invention are any parts of the interspecific hybrid plant, including a flower, branch cutting, pollen, ovule or any other plant part, including a single cell or collection of cells of such a plant. Still further, provided by the invention is an interspecific hybrid Jatropha plant or part thereof clonally propagated from such a plant i.e. any generation of a Jatropha interspecific hybrid plant clonally derived from a plant provided by the present invention. The invention also provides a plant or any part thereof produced by crossing a Jatropha integerrima plant to a Jatropha curcas plant, including any clonal propagation thereof.

The invention further provides an interspecific hybrid Jatropha plant or part thereof produced by tissue culture procedure or growing the seed.

Definition: In the context of the present application, the following terms are defined in the following manner:

Introgression: Natural introduction of genes of one species into another through the process of interspecific hybridization followed by successive backcrosses to the recurrent parents. Each species may thereby become more variable and show certain characters of the other species.

In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

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.

Tissue culture: It 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.

Regeneration: Regeneration refers to the development of a plant via tissue culture.

Clonal propagation: It means any generation of a plant clonally propagated or derived from a plant. Such clonal propagation may be carried out, for example, by taking a cutting of a plant and cultivating the saple cutting such that it produces roots. The rooted cutting may then be grown as a new plant which is a clone of the starting plant. Clonal propagation may be carried out with an essentially unlimited number of times to produce great numbers of clonally propagated plants, each of which is exactly identical to the starting plant from which the cutting is taken.

BRIEF DESCRIPTION OF FIGURES:

Figure 1: Gel picture of Nandan-5 interspecific hybrid showing the band separation between parents and hybrids using Sc75 marker.

Figure 2: Backcrossing between Jatropha curcas and Jatropha integerrima up to F2BC3

DETAILED DESCRIPTION OF THE INVENTION:

The invention provides a novel interspecific Jatropha hybrid Nandan-5 plant with unique characteristics like profuse flowering, hardiness of stem, leaves with pigmented petiole, high seed yield and high oil content. Nandan-5 is resistant to root-rot disease and more tolerant to environmental stresses especially drought.

The invention involves procedures for creating plants with novel characteristics. It is produced from interspecific hybridization between two species of Jatropha where Jatropha curcas is the female parent and Jatropha integerrima is the pollen parent. The crossed seeds were grown as Fi population. Five plants were selected on the basis of physiological and morphological character of the Fi progeny {Jc x Ji) are grown and pooled to allow selfing to generate F2 Population. Seeds are harvested at maturity and grown as described previously. The F2 plant JBC-24-57 was selected on the basis of desirable characters of the F2 population for the backcross (BC) with J, curcas up to F2BC3 population for transferring the desirable characters. The JHZ-13-23 plant among F2BC3 plants was selected and designated as Nandan-5 interspecific hybrid. This interspecific hybrid showed its uniqueness in characters than both the parents, as described in the following hybrid description information.

This invention also directs to the procedures for producing a Jatropha inter or intraspecific hybrid by crossing a first parent Jatropha curcas plant with a second parent Jatropha integerrima plant, or vice-versa wherein the plant used for back cross is a Jatropha plant from the hybrid Nandan-5. Therefore, any procedures using the interspecific hybrid Nandan-5 are part of this invention: selfing, backcross, hybrid breeding and crossing to populations. Any plants produced using Jatropha interspecific hybrid Nandan-5 as a parent are within the scope of this invention. (Refer Figure 2)

In addition, the invention provides a process of preparing an interspecific hybrid Jatropha plant or part thereof comprising the steps of, obtaining a cutting of interspecific hybrid Jatropha Nandan-5. Accordingly, any derivatives of a plant derived from the Nandan-5 are specifically within the scope of the invention. As such, those of skill in the art will recognize that once a Jatropha interspecific hybrid plant is prepared from Nandan-5, it may be propagated for a potentially unlimited number of generations. Each plant produced by such a process for the propagation additionally from a part of the present invention.

The invention also relates to the hybrid plants and any further progeny of Nandan-5 as a pollen donor. The invention is directed to procedures for producing a Jatropha plant by crossing J. curcas or J. integerrima as female parent and Nandan-5 as male parent or vice-versa. The procedures using the interspecific hybrid Jatropha plant Nandan-5 in backcross, hybrid production, crosses to population, clonal propagation, micropropagation and the like are part of this invention. All plants which are a progeny of or descend from Nandan-5 are within the scope of this invention. This is an invention for interspecific Jatropha hybrid Nandan-5 to be used in crosses with other, different, Jatropha plants to produce first filial generation Jatropha hybrid seeds and plants.

Accordingly, the female parent used for producing Nandan-5 having characteristics of high seed yield, high oil content and root-rot resistant. Also, the male parent used for producing Nandan-5 having characteristics of greater number of inflorescences per plant, hardiness of stem, profuse flowering and flower pigmentation.

The present invention also provides regenerable cells for use in tissue culture of Nandan-5. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing Jatropha interspecific hybrid plant, and of regenerating plants having substantially the same genotype as the foregoing Jatropha curcas plant. Preferably, the regeneration cells in such tissue cultures will be root, root-tips, stem, stem slips, leaves, flowers, inflorescence, seeds, pollen, anthers, pistils, embryos, protoplast, callus, meristematic cells, cotyledons or hypocotyls

This invention also is directed to procedures for producing Jatropha interspecific hybrid Nandan-5 with a second Jatropha plant and growing the progeny seed, and repeating the crossing and growing steps with the Jatropha interspecific hybrid Nandan-5-derived plant from 0 to 7 times. Thus, any such procedures using the Jatropha interspecific hybrid Nandan-5 are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using Jatropha interspecific hybrid Nandan-5 as a parent are within the scope of this invention, including plants derived from Jatropha interspecific hybrid Nandan-5.

In addition, the invention also provides the use of interspecific hybrid Nandan-5 plant or part thereof, in a process for the production of a biofuel such as biodiesel.

MOLECULAR CHARACTERIZATION OF NANDAN-5 Plant Material

Attempts were made to develop diagnostic molecular markers for the high yielding hybrid Nandan-5. For this purpose, molecular analysis of the hybrid Nandan-5 was carried out with 9 more diverse Jatropha curccts lines that exhibited variability in seed characters (large v/s small), maturity pattern (extended flowering v/s synchronous maturity), leaf size (small v/s medium) and yield (high v/s low).

DNA Extraction

Total genomic DNA was extracted from younger leaves of the hybrid (Nandan-5) following the standard CTAB method 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 raM EDTA, 2% PVP, 1.4 M NaCl, 100 mM Tris-HCl pH 8.0 and 1 % |3-mercaptoethanol) and incubated at 65°C for 1 h. The supernatant was treated with RNaseA (lOOug/ml), incubated at 37°C 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 (j.1 of sterile Millipore water and stored overnight at -20 °C for use in PCR.

PCR Analysis

A total of 45 SSR primers were used for DNA amplification according to the process of Williams et al. (1990). The PCR amplification reaction (10 ul) consisted of 2.5 ng of DNA, lx PCR buffer (10 mM Tris pH 9.0, 50 mM KC1, 1.5 mM MgCl2), 100 uM of each of the four dNTPs, 0.4 uM 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 C for 5 min for initial denaturation, 94 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min repeating for 40 cycles with a final extension at 72 °C for 7 min. The PCR products were separated on 3.0 % agarose gel in lx TAE buffer by electrophoresis at 100 V for 3 h and visualized with ethidium bromide staining under gel documentation system. The polymorphism detected was observed and the polymorphic band was checked for accession specificity. Only one marker- Sc75 was found specific to Nandan-5.

Characterization using SSR markers confirmed the hybridity. The gel picture (Fig. 1) confirms the similarity of the material with J. integerrima and also the hybridity between J. curcas and J. integerrima as parental lines. The banding pattern of the material is exactly the same as that of J. integerrima. Further, the distinction of the hybrid Nandan-5 has been accomplished through development of a molecular marker specific to the hybrid. The molecular marker Sc75 primer is confirming Nandan-5. The inheritance of the markers was validated by checking its progeny resulting from this promising hybrid.

Further reproduction of the hybrid can occur by tissue culture and regeneration. Tissue culture of various tissues of Jatropha species 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. Mol. Bio. 30: 475 (2004). Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce Jatropha plants having the physiological and morphological characteristics of Jatropha interspecific hybrid Nandan-5.

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-5.

The utility of Jatropha interspecific hybrid Nandan-5 also extends to crosses with other species. Commonly, suitable species will be of the family Euphorbiaceae, and especially of the genera Jatropha.

This invention also is directed to procedures for producing a Jatropha inter or intraspecific hybrid by crossing a first parent Jatropha integerrima plant with a second parent Jatropha curcas plant, or vice-versa wherein the first or second parent plant is a Jatropha plant from the hybrid Nandan-5. Further, both first and second parent Jatropha plant may be from the hybrid Nandan-5. Therefore, any processes using the interspecific hybrid Nandan-5 are part of this invention: selfmg, backcross, hybrid breeding and crosses to populations. Any plants produced using Jatropha interspecific hybrid Nandan-5 as a parent are within the scope of this invention: selfing, backcross, hybrid production, crosses to populations and the like. All plants produced using Jatropha interspecific hybrid Nandan-5 as a parent are within the scope of this invention, including those developed from varieties derived from Jatropha interspecific hybrid Nandan-5. Any Jatropha plant produced by crossing Nandan-5 and comprising 20 to 70% of alleles from the Nandan-5 parent are within the scope of this invention. Advantageously, the Jatropha interspecific hybrid of the present invention could be used in crosses with other, different, Jatropha species to produce the first filial generation Jatropha hybrid seeds and plants with superior characteristics. Nandan-5 can also be used for transformation where exogenous genes are introduced and expressed by any of a number protocol known to these of skill in the art are intended to be within the scope of this invention.

The following describes breeding procedures that may be used with interspecific hybrid Nandan-5 in the development of further Jatropha plants. One such embodiments is a procedure for developing an Nandan-5-derived progeny Jatropha plant in a breeding program through back cross comprising: obtaining the Jatropha plant, or a part thereof, of Jatropha interspecific hybrid plant Nandan-5, utilizing said plant or plant part as a source of breeding material and selecting an Nandan-5 progeny plant with molecular markers in common with Nandan-5 and/or with morphological and/or physiological characteristics selected from the characteristics listed in Table-1. Breeding steps that may be used in the Jatropha breeding program include pedigree breeding, mutation breeding, and recurrent selection. In conjugation with these steps, techniques such as RFLP-enhanced selection, genetic marker enhanced selection like SSR markers and the making of double haploids may be utilized.

One of the ordinary skills in the art of plant breeding would know how to evaluate the traits of two plant types to determine if there is no significant difference between the two traits expressed by those plant types. Thus, the invention includes Jatropha interspecific hybrid.

Nandan-5 progeny plants comprising a combination of at least two Nandan-5 traits. Using techniques described herein, molecular markers may be used to identify said progeny plant as a Nandan-5 progeny plant. Mean trait values may be used to determine whether trait difference are significant, and preferably the traits are measured on plants grown under the same environmental conditions. Once such a variety is developed its value is substantial since it is important to advance the germplasm base as a whole in order to maintain or improve traits such as yield, disease resistance, insect pest resistance, and plant performance in extreme environmental conditions.

Progeny of Jatropha interspecific hybrid Nandan-5 may also be characterized through their filial relationship with Nandan-5, as for example, being within a certain number of breeding crosses of Nandan-5. 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-5 and its progeny.

Tables

The following tables present data on the traits and characteristics of Jatropha interspecific hybrid Nandan-5 as compare to its parental lines Jatropha curcas and Jatropha integerrima as pollen parent. The results in Table 2 compare the distinctive morphological characters of Nandan-5 with its parents.

TABLE 1: INTERSPECIFIC HYBRID DESCRIPTION INFORMATION

Table 2: Distinctive morphological characters of Nandan-5 with its parents

We Claim:'-

1. An interspecific hybrid of Jatropha curcas plant designated as Nandan-5 or a part thereof, produced by backcross breeding between Jatropha curcas as female parent and Jatropha integerrima as pollen parent up to F2BC3.

2. An interspecific hybrid of Jatropha curcas plant designated as Nandan-5 or a part thereof, produced by tissue culture procedure or growing the seed of the claim 1.

3. Pollen of an interspecific hybrid Nandan-5 plant of claim 2.

4. A root-rot resistant Jatropha plant of the hybrid Nandan-5 plant of claim 2.

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

6. A protoplast produced from an interspecific hybrid Nandan -5 plant of claim 1 or claim 2.

7. Tissue culture cells produced from the interspecific Jatropha curcas hybrid Nandan-5 plant of claim 2, wherein said cells of the tissue culture are produced from a plant part selected from the group consisting of root, root-tips, stem, stem slips, leaves, flowers, inflorescence, seeds, pollen, anthers, pistils, embryos, protoplast, callus, meristematic cells, cotyledons or hypocotyls.

8. An interspecific Jatropha curcas hybrid Nandan-5 plant regenerated from the tissue culture of claim 7, wherein the plant has all the morphological and physiological characteristics of interspecific hybrid Nandan-5.

9. A process of producing the Jatropha interspecific hybrid Nandan-5 plant or parts thereof by crossing Jatropha curcas as female parent with Jatropha integerrima as pollen parent and its Fi, F2, F2BC1, F2BC2 and F2BC3 plants of claim 1.

10. An interspecific hybrid Nandan-5 plant or part thereof prepared by the process of claim 9.

11. A process of producing an interspecific hybrid of Jatropha plant with the morphological and physiological characteristics of hybrid Nandan-S according to claim 9 or claim 10, comprising regeneration from tissue culture.

12. A process of producing an interspecific hybrid of Jatropha plant designated as Nandan-5 according to claims 9 to 11 wherein the Nandan-5 exhibits a high yield; resistance to root rot disease and insect pests, improved canopy structure, tolerance to environmental stresses, improved agronomic characteristics and/or higher seed yield.

13. A process of producing an interspecific hybrid of Jatropha plant designated as Nandan-5 according to claims 9 to 12, wherein the female parent used in the method comprises the characteristics of producing high seed yield, high oil content and root-rot resistant.

14. A process of producing an interspecific hybrid of Jatropha plant designated as Nandan-5 according to claims 9 to 12, wherein the male parent used in the method comprises the characteristics of producing a greater number of inflorescences per plant, hardiness of stem, profuse flowering and flower pigmentation.

15. A process of producing an interspecific hybrid of Jatropha plant according to claims 9 to 14, wherein the interspecific hybrid Nandan-5 of Jatropha plant is then crossed with a Jatropha curcas or part thereof, to produce a population of Nandan-5 progeny comprising 20 to 70% of alleles from the Nandan-5 parent.

16. A process of producing an interspecific hybrid of Jatropha plant designated Nandan-5 according to any one of claims 9 to 15, wherein the Nandan-5 product comprises the Sc75 SSR marker.

17. A process of producing an interspecific hybrid Nandan-5 of Jatropha plant according to claims 9 to 16 wherein the Nandan-5 product comprises a root-rot resistant form.

18. An interspecific hybrid Nandan-5 plant or part thereof prepared by the method of claims 9 to 17.

19. A process of producing profuse flowering from interspecific hybrid of Jatropha plant designated as Nandan-5, comprising forming the plant by the method of claims 9 to 18.

20. A process of producing a high seed yield from inter specific hybrid of Jatropha plant designated as Nandan-5, comprising forming the plant by the method of claims 9 to 18.

21. A process of producing a root-rot resistant from interspecific hybrid of Jatropha plant designated as Nandan-5, comprising forming the plant by the method of claims 9 to 18.

22. A process of culturing tissue cells from an inter specific hybrid of Jatropha plant according to claims 9 to 18, comprising culturing tissue cells produced from the Jatropha plant, where cells are selected from the group consisting of root, root-tips, stem, stem slips, leaves, flowers, inflorescence, seeds, pollen, anthers, pistils, embryos, protoplast, callus, meristematic cells, cotyledons or hypocotyls

23. Use of the interspecific hybrid Nandan-5 plant or part thereof, according to claim 18 in a process for the production of a biofuel.

24. Use according to claim 23 wherein the biofuel is biodiesel.

25. A biofuel generated by the use according to claim 23 or claim 24.

26. An interspecific hybrid of Jatropha curcas Nandan-5 plant or a part thereof according to any one of the proceeding claims having unique characteristics like profuse flowering, hardiness of stem, leaves with pigmented petiole, high seed yield and high oil content.