METHOD OF INTRODUCTION OF NOVEL GENES INTO J. CURCAS USING AGROBACTERIUM MEDIATED TRANSFORMATION

Field of Invention:

The present invention relates to the method of introduction of a novel wild Agrobacterium strain, validation of the trait (female flowers), perpetuation of the character, advantages for vegetatively propagated plants like J. curcas. The developed J. curcas plant is named as NANDAN-18.

Background of the Invention:

J. curcas (physic nut) is one of the important species that 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 produces non- edible oil and has no competition with food crops for resources as it can be grown in marginal and waste lands with minimum inputs. The full potential of J. curcas has not been realized due to several technological and economic reasons. One of the major reasons is the lack of high yielding varieties with high oil content. Improving seed and oil yield and other agronomically important traits is the major challenge in J. curcas improvement. The presence of high yielding varieties of Jatropha can generate interest among the growers for sustainable cultivation leading to higher production of biodiesel.

The inflorescence in J. curcas is axillary paniculate polychasial cyme formed terminally on branches and is complex, possessing main and co-florescences with paracladia. The average male to female (M/F) flower ratio is 20:1, which changes drastically (108:1) with the fall in temperature (Dhillon et al., Indian J. Agroforest, 8:18, 2006). Flowers are unisexual, monoecious, greenish yellow colored in terminal long peduncled paniculate cymes. Male flowers: calyx segments 5, nearly equal, elliptic or obviate; corolla is campanulate, lobes 5, connate, hairy inside, exceeding the calyx, each lobe bears inside a gland at the base, stamens 10 in two series, outer five filaments free, inner five filaments connate, anthers dithecous erect, opening by longitudinal slit. Female flowers: sepals up to 18 mm long, persistent; calyx as in male, corolla 4 scarcely exceeding the calyx lobes united, villous inside; ovary 3-locular, ellipsoid, 1.5-2 mm in diameter, style bifid, ovules solitary in each cell (Tiwari, In: Jatropha and biodieselAst ed. New Delhi, Ocean books Ltd., 2007).

The objectives for genetic up gradation of J. curcas should aim at more number of female flowers or pistillate plants, high seed yield with high oil content, early maturity, resistance to pests and diseases, drought tolerance, reduced plant height and high natural ramification of branches. Seed yield of J. curcas is determined by number of inflorescences per plant, number of fertile pistillate flowers per inflorescence, seeds per capsule and 100-seed weight. 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.

Sujatha (Sujatha, Indian J. Agroforest. 8:58, 2006) and Sujatha et. al.(Sujatha et. al., Biotechnol. Adv. 26:424, 208) reported that genetic improvement and domestication of J. curcas should follow the same course as that of castor (Ricinus communis L.) as both belongs to the Euphorbiaceae family. Castor has been improved from a perennial wild to annual domesticate, having short internodes with varying flower sexuality ratios from completely pistillate to predominantly male types (Singh, Castor Ricinus communis (Euphorbiaceae). In: Simmonds NW, editor. Evolution of crop plants. Longman, London, p.84, 1976). The success is due to the use of mutation techniques, selection of germplasm and identification of pistillate variants (Hegde et. al., In: Castor in India. DOR, Hyderabad, India, 2003). J. curcas can be improved through assessment of variation in wild source and selection of superior/elite genotypes and application of mutation, alien gene transfer through inter-specific transformation and biotechnological interventions to bring the change in the desired traits. Enhancement of productivity can be achieved through development of pistillate plants and/or to identify divergent parents, which can later be exploited by heterosis. Development of pistillate plants through mutation and inter-specific transformation techniques is time consuming. Meanwhile, physiological manipulation of sexuality, applying gametocides to enhance the M/F ratio may be considered to increase yield. Breeding system plays a critical role in deciding the route of plant evolution and the results of breeding system indicated 32.9% fruit setting under self pollination and 89.7% under natural pollination in Jatropha, which indicated that J. curcas is self-compatible and tended to cross-pollinate (Qing et. al., J. South China Agricult. Univ., 28: 62, 2007).

Foreign DNA is usually delivered into plant nucleus via bombardment-mediated or Agrobacterium-mediated transformation. Bombardment-mediated transformation, or biolistics, is a process by which DNA can be delivered into cells in association with high- velocity microprojectiles (Sanford, Plant Physiol. 79: 206 (1990); Klein et. al., Proc. Natl. Acad. Sci. USA 85: 8502 (1988); Finer and McMullen, Plant. Cell Rep. 8: 586 (1990). Although several plant species have been transformed by biolistic methods, the frequency of stable transformation can be quite low due to the absence of a mechanism to mediate the integration of the foreign DNA into the plant genome. In addition, bombardment of plant cells with DNA results in the delivery of more than one copy or the partial integration of the gene of interest into the plant cell genome (Hansen and Chilton, Proc. Natl. Acad. Sci. USA 93: 14978 (1996), which causes deleterious changes to other traits of the targeted cell.

Unlike biolistic methods, Agrobacterium tumefaciens mediated transformation has become a method of choice for basic plant research, as well as a principal technology for generating transformed plants for the agricultural biotechnology industry (Stafford, Bot. Rev., 66: 99, 2000; Gelvin, Micro. Mol. Bio. Rev. 67:16, 2003). This procedure has the advantage to result in stable integration of defined DNA sequences into the plant genome and it often results in a lower transgene copy number, fewer DNA rearrangements and higher long-term stability of expression as compared to direct DNA delivery methods (Dai et. al., Mol. Breed., 7:25, 2001; Travella et. al., Plant Cell Rep., 23:780, 2005). Agrobacterium is a soil born phytopathogen that integrates a piece of DNA (T-DNA) into the genome of a large number of dicotyledonous and few monocotyledonous plants (Chilton et. al., Cell 11:263, 1977; Hoekema, et. al. al., Nature 303: 179, 1985; Bevan, Nucleic Acids Res. 12: 8711, 1984; Sheng and Citovsky, The Plant Cell 8: 1699, 1996). The T-DNA is flanked by specific sequences, called the right and left borders (Wang et. al. al., Science 235: 587, 1987). The expression of this transferred DNA results in neoplastic growths (tumors) on the host plant. However, because the T-DNA element is defined by its borders, any gene of interest can replace the coding region of the wild type T-DNA. As a consequence, Agrobacterium can be used to produce transformed plants expressing genes of interest.

The Ti plasmid of A. tumefaciens is a mega plasmid (~ 200kb) containing two major regions, a T-DNA and a vir (virulence) region. The T-DNA carries necessary oncogenes for expression of crown gall (tumor) phenotype in plants. There are two major types of T- DNA, nopaline type and octopine type. In a typical nopaline type plasmid, the T-DNA carries genes iaaH, iaaM and ipt. Both iaaH and iaaM are involved in auxin biosynthesis, while ipt is essential for cytokinin biosynthesis and the whole T-DNA is expressed as a single transcriptional unit (Pratik Satya, Plant genetic engineering - applications and achievements, In: Genomics and Genetic Engineering, NIP A, New Delhi, 2007). Upon integration and expression of the T-DNA-located genes iaaH and iaaM there is an increased synthesis of auxin (Kado, In Genes Involved in Microbe-Plant Interactions. Eds. Verma & Hohn, pp. 311-336. Springer-Verlag, Wien, New York, 1984) that enhances ethylene synthesis in the tumor by stimulating the transcription of ACC synthase genes (Yang & Hoffman, Ann. Rev. Plant Physiol. 35: 155, 1984; Yoon et. ah, Plant Cell Physiol. 38: 217, 1997).

Ethylene production in A. tumefaciens induced tumors may depend on different signals. The auxin content was up to 13 times higher in tumors than in the uninfected stem in Ricinus. Time-dependent auxin measurements showed kinetics similar to ACC accumulation. It is known that auxin stimulates the rate of ethylene biosynthesis in a tissue-dependent manner (Visser et. al., Plant Physiol. 112: 1687, 1996) by inducing the gene expression of ACC synthase and increasing its activity (Nakagawa et. al., Plant Cell Physiol. 32: 1 153 (1990); Olson et. al., Proc. Natl. Acad. Sci., USA 88: 5340 (1991); Yoon et. al., Plant Cell Physiol. 38: 217, 1997). Thus, auxin is most likely to be the signal for ethylene production in A. tumefaciens-mduced tumors.

Sex expression in plant species is influenced by genetic, environmental and hormonal factors. Monoecious strains of cucumber (Cucumis sativus L.) and musk melon (C. melo L.) bear staminate (male) and pistillate (female) flowers. Gynoecious strains normally produce only pistillate flowers. Other cucumber and muskmelon strains produce staminate or pistillate and in addition, perfect (hermaphrodite) flowers in various combinations (Byers et. al., Proc. Nat. Acad. Sci. USA 69: 717 (1972). Exogenous application of auxin (Galun, Phyton 13: 1, 1959) and inhibitors of gibberellin biosynthesis (Halevy and Rudich, Physiol. Plant 20: 1052, 1967) promote monoecious strains to form pistillate flowers. Determination of endogenous growth substances indicate that strains with genetically strong female sex expression contain more auxin (Galun et. al., Plant Physiol. 40: 321, 1965). Ethylene and 2-chloroethylphosphonic acid (Ethephon), an ethylene releasing compound, found to promote femaleness in cucurbits (Iwahori et. al., Plant Physiol. 46: 412, 1970). Exogenous application of auxin increases ethylene production by cucumber plants (Shanon and De LaGuardia, Nature 223:183, 1969) and some response to auxin are now attributed to auxin-induced ethylene synthesis (Burg and Burg, In Biochemistry and Physiology of Plant Growth Substances, eds. Wightman and Setterfield, The Range Press Ltd. Canada, pp.1275, 1967).

Several scientists have reported the transformation method of Jatropha curcas (Nitish Kumar et al., Industrial Crops & Products 32:41, 2010; Meiru Li et al., Plant Cell Tiss Organ Cult. 2007; Johnson and Deore, US 2010/0251421, 2010; Mao et al., WO 2010/071608, 2010; Jingli Pan et al, African J Biotech, 9: 6477, 2010) but nobody has used transformation as a tool for transformation of monoecious Jatropha curcas plant into unisexual pistillate plant mediated by Agrobacterium tumefaciens.

Object of the Invention:

The object of the present invention is to develop a new, unique and superior transformed J. curcas plant with more number of pistillate flowers by Agrobacterium-mediated transformation.

Summary of the Invention:

The present invention discloses the method of transformation of monoecious J. curcas plant to a unisexual pistillate plant and the transformed J. curcas plant named as NANDAN-18.

In another aspect, the present invention relates to method for producing a transformed J. curcas plant by a novel wild Agrobacterium strain isolated and used for transformation, wherein the A. tumefaciens is a non-disarmed strain containing the oncogenes (iaaH, iaaM and ipt) in its T-DNA region. Thus, any process using the transformed J. curcas plant NANDAN-18 in further transgenic production including the gene expression of ACC synthase and increasing its activity; backcross, crosses to population, clonal propagation including grafting using NANDAN-18 as scion and/or stock and micropropagation are part of this invention. All plants which are a progeny of or descend from NANDAN-18 are within the scope of this invention. It is an aspect of this invention for J. curcas transformed NANDAN-18 to be used in crosses with other, different, J. curcas plants to produce first generation (Fi) J. curcas hybrid seeds and plants with superior characteristics.

Another object of the present invention relates to use of J. curcas transformed NANDAN- 18 in crosses with other, different, J. curcas plants to produce first filial generation J. curcas hybrid (Fi) seeds and plants with superior characteristics and to provide a method of regeneration of J. curcas hybrid from tissue culture.

In further aspect, the present invention discloses regenerable cells for use in tissue culture (micropropagation) of NANDAN-18. The tissue culture is preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing transformed J. curcas plant, and of regenerating plants having substantially the same genotype as the foregoing transformed J. 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 transformed J. curcas, NANDAN-18 plants regenerated from the tissue cultures of the invention.

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 methods 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 Inflorescence of NANDAN-18 Figure 2 Fruit bunch of NANDAN-18 Detailed Description of the Invention

The present invention describes J. curcas transformed NANDAN-18 as a unisexual pistillate plant with high seed yielding capacity, more number of inflorescences per plant, inflorescence producing only pistillate flowers, less number of abortive female flowers / unfilled capsules with three seeds per capsule and higher 100-seed weight with altered morphological characters.

Explant source

The selected J. curcas clones were grown in a greenhouse under controlled and disease free condition. The leaves were collected from greenhouse and washed under running tap water for 10 minutes and immersed in 1% (w/v) Carbendazim solution for 20 minutes. The leaves were than surface sterilized 0.1% (w/v) HgCl₂ for 1 minute followed by repeated washing with sterile water for five times. After blot-dry on sterile filter paper, the leaves were carefully dissected keeping the midrib and cut into disc of 1cm² size and used as explants for co cultivation with A. tumefaciens, callus induction and shoot regeneration.

Transformation procedure

A native non-disarmed A. tumefaciens strain isolated from the soil harboring the oncogenes was used for transformation. Single colony of the bacterial strain was inoculated in 25 ml of liquid YEB medium (lg yeast extract, 5g beef extract, 5g peptone, 5g sucrose per L and 2 mM MgS04, pH 7-8). Bacterial cultures were grown overnight at 28 °C until OD reached to 0.8. The cells were collected by centrifuging at 5000 rpm for 5 min at 25°C and the pellet was resuspended in liquid co cultivation medium (MS medium (Murashige and Skoog, Physiol. Plantarum 15: 473, 1962) containing 2.22 μΜ of BA, 2.27 μΜ of TDZ and 0.49 μΜ of IB A, pH adjusted to 5.8) supplemented with 100 μΜ acetosyringone. The leaf disc explants were inoculated in bacterial suspension for 30 min with occasional shaking. The explants were then blotted on sterile filter paper and co cultivated in petridishes lined with filter paper, moistened with liquid co cultivation medium supplemented with 100 μΜ acetosyringone for 3 days at 25 °C in dark. After 3 days of co cultivation, the explants were washed five times with sterile distilled water containing 500 mg/1 of cefotaxime to remove Agrobacteria and blot-dried on sterile filter paper. Callus induction and shoot multiplication

Blot dried leaf disc explants were placed on the MS medium supplemented with 2.22 μΜ of BA, 2.27 μΜ of TDZ and 0.24 μΜ of IBA for induction of callus. The explants formed white friable callus after 2 weeks of culture. The callus were transferred to MS medium supplemented with 2.22 μΜ of BA and 0.24 μΜ of IBA for shoot bud induction. Within 4 weeks of transfer cluster of multiple shoots were developed from the callus. After 4 weeks, the regenerated shoots were transferred onto MS medium supplemented with 2.22 μΜ of BA, 2.32 μΜ of Kinetin and 0.72 μΜ of GA₃ for shoot elongation and bud multiplication. The elongated shoots were rooted on ½MS medium supplemented with 2.69 μΜ of NAA within 3 weeks.

Characterization of the transformed J. curcas plant NANDAN-18

Morphological and Phenological characters of NANDAN-18

The transformed J. curcas plant (NANDAN-18) is identified at pre-flowering stage based on the following morphological characteristics as mentioned in Table 1. At flowering it is identified by their production of only unisexual pistillate flowers (Figure 1). The observations at post-flowering and yield attributes are mentioned in Table 2.

Determination of ACC biosynthesis in NANDAN-18

The T-DNA located genes iaaHmd iaaM upon integration and expression in NANDAN- 18 plant genome, increased synthesis of auxin takes place that enhances endogenous ethylene synthesis in cells by stimulating the transcription of 1-Ammino-cyclopropane-l- carboxylic acid (ACC) synthase genes that produces ACC, the close precursor of ethylene. The quantity of ACC was determined following Lizada & Yang (Lizada and Yang, Analytical Biochemistry, 100: 140, 1979). The ACC content was 74 times higher in the stem of NANDAN-18 (7200 pmol g 'FW) than in the normal stem (97 pmol g^_1FW) at six month old stage.

Endogenous ethylene biosynthesis in NANDAN-18

1-Ammino-cyclopropane-l-carboxylic acid (ACC) is then converted to ethylene by ACC oxidase enzyme. The quantity of ethylene was determined by gas-chromatograph and the quantity was 135 times higher in the stem of NANDAN-18 (810 pmol g^"*FW) than in the normal stem (6 pmol g^_1FW) at six month old stage.

Further production and multiplication of transformed plants can occur by tissue culture and regeneration. Tissue culture of various tissues of J. 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. Mol. Bio. 30: 475 (2004). Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce J. curcas plants having the physiological and morphological characteristics of J. curcas transformed NANDAN-18.

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 J. 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-18.

The present invention contemplates a J. curcas plant regenerated from tissue culture of the transformed J. curcas plant of the present invention. As is well known in the art, tissue culture of J. curcas can be used for the in vitro regeneration of a J. curcas plant. Tissue culture of various tissues of J. 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. Jlant 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 J. curcas plants having the physiological and morphological characteristics of NANDAN-18.

Another process involves producing a population of NANDAN-18 progeny J. curcas plants, comprising crossing NANDAN-18 with another J. curcas plant, thereby producing a population of J. curcas plants, which on average, derive 50% of their alleles from NANDAN-18. A plant of this population may be selected and repeatedly selfed or sibbed with a J. curcas plant resulting from these successive filial generations. One embodiments of this invention is the J. curcas cultivar produced by this process and that has obtained at least 50 % of its alleles from transformed NANDAN-18.

Progeny of J. curcas transformed NANDAN-18 may also be characterized through their filial relationship with NANDAN-18, as for example, being within a certain number of breeding crosses of NANDAN-18. 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-18 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-18.

TABLES:

Morphological characteristics of transformed J. curcas plant (NANDAN-18) at pre- flowering stage as compared normal J. curcas are presented in Table 1.

The phonological characteristics at post-flowering and yield attributes of transformed J. curcas plant (NANDAN-18) as compared normal J. curcas are mentioned in Table 2.

The results in Table 2 compare the yield, number of inflorescences per plant, number of pistillate flowers per plant, male to female flower ratio, seeds per capsule, 100-seed weight of the transformed, NANDAN-18 of the recent invention. As shown in Table-2, bearing only unisexual pistillate flowers (Figure 1) on the plant, male to female flower ratio being zero, more seeds per capsule, and higher 100-seed weight than normal J. curcas plant.

TABLE 1: MORPHOLOGICAL CHARACTERISTICS AT PRE-FLOWERING STAGE

Plant Morphological Characters NANDAN-18 Normal Jatropha curcas

Plant height 220 cm 240 cm

Basal girth 52 cm 48 cm

Canopy diameter, across 232 cm 223 cm

No. of primary branches 3 4

No. of secondary branches 10 12

No. of tertiary branches 46 52

Internodal length 2.5 cm 2.6 cm

Branching pattern Divergent Divergent

Location of branches Basal Basal

Leaf Characters

Size Medium Medium to large

Shape Standard, lobes 5, bent Standard, lobes 5

upward

Color Light green to pale yellow Green

Pubescence Absent Absent

Petiole length 14.6 cm 15.3 cm

Petiole color Light green Green

Leaf aestivation Spiral Spiral

TABLE 2: THE PHONOLOGICAL CHARACTERISTICS AT POST-FLOWERING AND YIELD ATTRIBUTES

Yield and Yield Components NANDAN-18 Normal Jatropha curcas

Yield (plant^"1 year^"') 8.56 kg 1.01 kg

Average number of inflorescences 158 42

(plant year^"1)

Average number of pistillate flowers 32 16

per inflorescences

Average number of seeds per fruit 2.8 2.6

100-seed weight 62 g 58

Inflorescence and Capsule Characters

Inflorescence type Loose Loose to Compact

Male to female flower ratio 0 25

Flower color Light green Green

Capsule size (diameter) Medium (2.0 - 2.5 cm) Medium (2.5 - 2.8

Pedicel length 1.32 cm 1.24 cm

Seed Characters

Seed coat color Black Black

Shape class (Length / width ratio) Long Long

Size (measurements) Length 16 mm 17 mm

Width 10.2 mm 10.4 mm

L / W 1.57 1.63

Seed weight 0.62 g 0..58 g

Oil content 31.5 % 30.2 %

Disease Resistance

Powdery mildew (Erysiphe Moderately tolerant Moderately tolerant euphorbiae)

Insect Pest Resistance

Inflorescence & capsule borer Moderately tolerant Moderately tolerant (Pempelia morosalis Salam Uller)

Mealy bug (Ferrisia vergata Ckll.) Moderately tolerant Moderately tolerant

Bugs (Scutellera nobilis Fabr.) Moderately Moderately

susceptible susceptible