Title of Invention


Abstract This invention provides an efficient process for in vitro propagation of Jatropha through direct regeneration of leaf disc without any intermediary callus phase. This process provides a commercially viable process for producing true-to-type plants of Jatropha on a large scale.
Full Text FORM 2
(39 of 1970)
PROVSIONAL SPECIFICATION (See Section 10; rule 13)
An Indian Company having its registered office at
Chitrakoot, 2nd Floor,
Ganpatrao Kadam Marg,
Shree Ram Mills Compound,
Lower Parel, Mumbai 400 013,
Maharashtra, India
The following specification describes and ascertains the nature of this invention:

The present invention relates to a method for highly efficient direct regeneration of plantlets through leaf disc of Jatropha for producing a commercially viable Jatropha plant, mainly for extracting biodiesel from the plant, without any intermediary callus phase.
Jatropha Curcas is a plant of Latin American origin, widely spread throughout the arid and semi-arid tropical regions of the world. Jatropha is a large genus comprising over 170 species. The commonly occurring species in India are J. curcas, J. glandulifera, J. gossypifolia, J. multifida, J. nana, J. panduraefolia, J. villosa and J. podagrica. Jatropha is a small tree or shrub with smooth gray bark, which exudes a whitish colored, watery, latex when cut. Normally, it grows between three and five meters in height, but can attain a height of up to eight or ten meters under favorable conditions. It is a drought resistant plant, living up to 50 years and growing on marginal lands. It has large green to pale green leaves, which are aligned alternate to sub-opposite. The leaves are three-five lobed with a spiral phyllotaxis. The petiole of the flowers ranges between 6-23 mm in length. Flowers are formed in hot seasons. In conditions where continuous growth occurs, an imbalance of pistillate or staminate flower production results in a higher number of female flowers. Fruits are produced in winter when the shrub is leafless. Each inflorescence yields a bunch of approximately 10 or more ovoid fruits.
A three, bi-valved cocci is formed after the seeds mature and the fleshy exocarp dries. The seeds become mature when the capsule changes from green to yellow, two to four months after fertilization. The blackish, thin-shelled seeds are oblong and resemble small castor seeds.
This plant has various medicinal uses especially in nutraceuticals, pharmaceutical, dermatological, and personal care products. The latex of Jatropha curcas has anticancer properties due to the presence of an alkaloid known as "jatrophine". The tender twigs are

used for cleaning teeth. The juice of the leaf is used for external application to treat piles. The roots are used as an antidote for snake-bites. The bark yields a dark blue dye used for coloring cloth, fish net and lines.
The seeds are used for anthelmithic purposes. The oil of the seeds has special properties of commercial importance in that it has a very high saponification value and is extensively used for making soaps. Further, it also burns without emitting smoke. Most of these species are ornamental, except for J. curcas and J. glandulifera which are oil yielding species (Renu Swarup, 2004. Biotechnological interventions to improve Jatropha seeds and oil quality. SAARC Oils & Fats Today, August, pp. 39-41.). The seeds contain semi-dry oil that has been found useful for medicinal and veterinary purposes (Gubitz, G. M; Mittelbach, M and Trabi, M (1999). Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresource Technol. Vol. 67, pp. 73-82).
The oil content is 25-30% in the seeds and 50-60% in the kernel. The oil contains 21% saturated fatty acids and 79% unsaturated fatty acids. The Jatropha oils are linolenic acid (C18:2) and oleic acid (C18:l) which together account for. up to 80% of the oil composition. Palmitic acid (C16:0) and stearic acid (C18:0) are other fatty acids present in this oil. The oil is non-edible, however it has the potential to provide a promising and commercially viable alternative to diesel oil as it has similiar desirable physicochemical and performance characteristics as diesel. Of late, the plant J. curcas has attracted particular attention as a tropical energy plant. The seed oil can be used as a diesel engine fuel for it has characteristics close to those of the fossil fuel, diesel. Moreover, biodiesel extracted from Jatropha meets the European EN 14214 standards for a pure and blended automotive fuel for diesel engines due to its non-toxic and biodegradable nature. Jatropha curcas seed yields approach 6-8 MT/ha with ca 37% oil. Such yield can produce the equivalent of 2100-2800 liters of fuel oil/ha, whose energy is equivalent to 19,800-26,400 kwh/ha (Gaydou, A. M; Menet, L; Ravelojaona G and Geneste, P. (1982). Vegetable energy sources in Madagascar: ethyl alcohol and its oil seeds. Oleagineux, Vol .37 (3), pp. 135-141.).

Micropropagation can be defined as in vitro regeneration of plants from organs, tissues, cells or protoplasts using techniques like tissue culture for developing true-to type resultant plants of a selected genotype. In general, tissue from a plant, commonly known as explants, is isolated from a plant whose multiplication is desired to create a sterile tissue culture of that species in vitro. A culture is initiated from the explant. Once a culture is stabilized and growing well in vitro, multiplication of the tissue or regeneration of entire plant can be carried out. Shoots (tips, nodes or internodes) and leaf pieces are commonly used but cultures can be generated from many different tissues. Juvenile tissues generally respond best. Besides the type of explant, the chemical composition of the culture medium and the physical environment of cultures have been found to have great influence on the regeneration capacity, multiplication ratio, and growth and development of new plants in the culture system. Therefore, one needs to optimize these factors for individual plant species.
Earlier studies have shown, either callus mediated regeneration or direct shoot bud with interspersed callus from hypocotyls, leaves, and petioles. However, plant tissue regeneration through a callus stage is vulnerable to somaclonal variations and hence will not ensure true-to-type plants from elite mother plants. Thus, there remains a need for methods that allow propagation of true-to-type plants.
There is a need to develop a protocol by which Jatropha species with desirable traits can be produced. Some of the desirable traits that a Jatropha species essentially need producing bio fuel is seed yield and oil content in the Jatropha species. Other desirable traits are dwarf stem, fatty acid content, early flowering, and synchronous maturity.. Therefore, in view of the above, there is a need of the hour to develop tissue culture protocols for the rapid propagation of selected elite variety genotypes of Jatropha and for further genetic improvement of Jatropha. The present invention is directed towards the rapid propagation of selected elite variety genotypes of Jatropha and genetically

improved quality of the plant, without any intermediary callus phase and also aims at producing Jatropha species of desirable traits.
The present invention for the first time provides a process, for in vitro propagation of Jatropha through direct regeneration of leaf discs without any intermediary callus stage.
Plant tissue culture is rapidly becoming a commercial method for large-scale propagation of the elite varieties and for plants difficult to propagate rapidly by conventional methods. Tissue culture is particularly useful for multiplication of plants, which are slow growing (turmeric, ginger, cardamom); cross-pollinated (coconut, teak, eucalyptus, cashew, mango and those which show wide variation in the progeny), male sterile lines (cotton, sorghum, pearl millet); and newly virus free plants by meristem culture (sugarcane, potatoes, tapioca, etc).
Known methods of plant tissue culture have involved passage through the callus stage, which means that there is a high possibility of somaclonal variations.
The present invention provides a process for in vitro propagation of Jatropha through direct regeneration of leaf discs without any intermediary callus stage, thus avoiding associated somaclonal variations arising during the callus stage. This process can be used for:
a) Large-scale propagation of true-to-type Jatropha plants
b) Improvement of economic traits
c) Regeneration of leaf discs transformed by particle gun and Agrobacterium for pathway engineering of Jatropha oil
d) an alternative to exchange and conservation of high-yielding germplasm of Jatropha.

Figures 1 and 2 show the initiation of minute shoot buds from Jatropha leaf discs.
Figures 3 and 4 show multiple shoot buds occurring on the surface of leaf disc.
Figures 5 and 6 show the further growth of multiple shoot buds (Note that shoot bud
induction is without any callus growth.)
Figure 7 shows the various stages of shoot bud induction.
Figure 8: Shows the rooting of the plantlets.
"Micropropagation" refers to the in vitro regeneration of plants from organs, tissues, cells or protoplasts and the true to type propagation of a selected genotype using in vitro culture techniques.
"Callus" refers to an unorganized or undifferentiated mass of proliferative cells produced either in culture or in nature.
"Gene transfer" refers to incorporation of new DNA into an organism's cells, usually by a vector.
As used herein, the term "transformed" refers to a cell, tissue, organ, or organism into which has been introduced a foreign polynucleotide molecule, such as a construct. Preferably, the introduced polynucleotide molecule is integrated into the genomic DNA of the recipient cell, tissue, organ, or organism such that subsequent progeny inherits the introduced polynucleotide molecule. A "transgenic" or "transformed" cell or organism also includes progeny of the cell or organism and progeny produced from a breeding program employing such a transgenic plant as a parent in a cross and exhibiting an altered phenotype resulting from the presence of a foreign polynucleotide molecule.

"True-to type propagation" means that all characteristics present in mother plant will also be present in next generation, i.e., the plantlets will be the true type of mother plant.
"Genotype" refers to the combination of alleles located on homologous chromosomes that determines a specific characteristic or trait.
"Agrobacterium mediated transformation" is the use of Agrobacterium to transfer DNA to plant cells harnessed for the purposes of plant genetic engineering.
"Leaf -disc regeneration" refers to direct regeneration of plantlets through leaf disc without passing through the callus stage.
"High yielding germplasm" refers to a process, which conserves genetic material, especially its specific molecular and chemical constitution, which is the physical basis of the inherited qualities of an organism.
"Somaclonal variations" refers to the genetic variability, which can arise during tissue culture.
A "high-yield" Jatropha curcas plant is one having an increased number and/or weight of seeds per plant as compared to the average plant. For example, Jatropha curcas with yield of approximately 3 kg of seeds per plant is considered to be a high yield plant.
A "high oil content" Jatropha curcas plant is one having seeds with increased oil content. For example, Jatropha curcas having seeds with 38% oil content is considered to be a "high oil content" plant.
The term "treatment to reduce microbial contamination" refers to common methods known in the art for reducing the level of bacteria and fungi that can interfere with the micropropagation process. These methods can include, but are not limited to, cleaning

with detergents or spraying with fungicides, insecticides, pesticides, and other surface-sterilizing agents.
The term "elite variety" refers to any type of plant having characteristics that are desirable. For example, a high yield plant having seeds with high oil content is desirable for the use of Jatropha for oil production.
The present invention provides a process for in vitro propagation from leaf discs, an easily obtainable explant of Jatropha. This method is useful for obtaining plantlets without intermediary callus pase.. The present invention also provides true-to-type Jatropha and transgenic Jatropha made using methods of this invention.
The process for the producing a true-to-type Jatropha curcas has steps including, but not limited to:
1) Selecting a mother plant; involves collecting dry seeds from known source, which is high yielding variety and their germination in vitro using tissue culture protocols and isolation of embryo and cotyledons from the dry seeds under aseptic conditions by treating the mother plant to reduce microbial contamination like removing any contaminants such as fungus, bacteria, microbes etc., from the surface of seeds after dehusking and their pre incubation. The culturing of embryo and cotyledons to induce complete plantlet, in a medium comprising of:
a) salts of conventional medium
b) vitamins of conventional medium
c) carbon source
d) phytohormones
e) gelling agent, and
at a pH range of 5.4 to 6.2 and sterilizing medium. The cultures were incubated at 20-30°C, under cool white fluorescent light and continuing the culture of the explants until complete plantlets are formed

2) isolating a leaf from the mother plant; s from both in vitro germinated seedlings and field grown plants
3) isolating a leaf disc from the leaf;
4) treating the leaf disc to reduce microbial contamination; by incorporation of antibiotics or combination of antibiotics into the culture media to control bacterial contamination associated with seed home, inherent contamination and
4) micropropagating the leaf disc by culruring them in a medium capable of inducing shoot buds directly. The medium comprising of
a) salts of conventional medium
b) vitamins of conventional medium
c) carbon source
d) phytohormones
e) and gelling agents
at a pH range of 5.4 to 6.2 and sterilizing the medium by autoclaving. The cultures were incubated at 20-3 5°C under cool white fluorescent lamps for a minimum period of 2-3 months to induce shoots with sub culruring every 30 days. Subsequently cultured in a rooting medium and subjected to hardening.
The growth regulators employed in the culture medium or selected from cytokinins such
as BAP (6 benzyl amino purine), zeatin; auxinsa such as IAA (Indole-3-acetic acid), NAA
(Naphthelene acetic acid), and cytokinin active urea such as TDZ (l-phenyl-3, 1,2,3
thidiazola-5-yl urea). Gelling agents such as agar (0.6 to 1.2% w/v) or phytagel (0.2 to
0.5% w/v) or any gelling agent at appropriate concentration suitable for shoot
In some emabodiments, the mother plant is a field grown plant. In other embodiments, the
plant is germinated in vitro.
In other embodiements the mother plant can be invitro propagated.
The process for producing a Jatropha curcas has the following steps
Selection of the Mother Plant

The mother plant is either a field-grown plant or a plant micropropagated from another plant.
The Jatropha curcas mother plants to be used in methods of this invention can be selected using methods known to those of skill in the art. In preferred embodiments, healthy plants are selected. Health can be determined by assessing the plants for their size, weight, general growth, appearance, and absence of infection or contamination.
When the mother plant is to be used in a method for production of true-to-type Jatropha curcas, elite variety plants are preferred. Elite variety plants are those having desirable traits, such as dwarf stem, early flowering, and synchronous maturity. In preferred embodiments, the elite variety plants are those with high yield and high oil content.
Micropropagation of the Mother Plant
In some embodiments, a micropropagated mother plant instead of a field-grown plant is used to reduce the likelihood of contamination during the methods of the invention. The micropropagated plant may be generated from another micropropagated plant or a field-grown plant.
A micropropagated mother plant can be generated from a field-grown plant by methods including, but not limited to, the following steps: isolating seeds from a field-grown plant; reducing the microbial contamination of the seeds isolated from the field-grown plant; isolating the embryo and cotyledons from the seeds; and culturing the embryo and cotyledons to produce plantlets.
A typical procedure includes the following steps. Dry seeds are collected from a known source using methods known to those of skill in the art. If to be used in a method for production of true-to-type plants, the source is preferably an elite variety. The seeds are dehusked and then microbial contamination of the seed surface is reduced using standard methods known in the art, including those described herein for reducing the microbial contamination of mother plants and explants. Embryo and cotyledons are then isolated

from the dry seeds under aseptic conditions and cultured in media capable of inducing complete plantlets from the embryo and cotyledons. The media typically includes a standard concentration of salts and vitamins, a carbon source, phytohormones, gelling agent, antibiotics to control bacterial contamination associated with seed borne, inherent contamination, is at a pH ranging from 5.4 to 6.2, and has been sterilized by autoclaving. Common phytohormones used for micropropagation are described herein. The cultures are typically incubated at temperatures between 20 and 30°C under cool, white fluorescent light. Once complete plantlets are formed, very young leaves can be isolated from the in vitro germinated seedlings and leaf discs are isolated using methods known in the art.
Preparation of the Mother Plants
Microbial contamination of mother plants can be reduced by spraying the plants with agents such as fungicides, insecticides, pesticides or the like. Preferred fungicides for the pretreatment of the mother plant include Bavistin™, Captan™, Dithane™, Thiram™, Thiovit™, or combinations thereof at a concentration of about 0.05% to 0.2%. Preferred insecticides for the pretreatment of the mother plant include, but are not limited to, Rogor™, Nuvacron, Fastac™, Ultracid™ 40-WP, Thiodane™ at a concentration of about 0.005% to 0.02%.
Preparation of Leaf Explants
The present invention provides methods for efficient in vitro mass culture of Jatropha curcas using leaf discs.
Isolation of Leaves
The leaf discs are isolated from leaves of the selected mother plants. In preferred embodiments, leaf discs are isolated from young leaves, such as the second, third, or fourth leaf of the selected mother plant. In particularly preferred embodiments, the leaf disc is isolated from the [????] leaf.

Preferably, the leaf discs used in the present invention are selected from healthy, fresh, disease-free plants. The leaf discs may be isolated from either field grown or micropropagated mother plants.
Reduction of Microbial Contamination
Microbial contamination of the explants can be reduced by standard methods such as
cleaning and sterilization. Cleaning can be performed using methods known to those of
skill in the art, for example, by shaking explants in dilutions of a mild detergent, such as
Sterilization can be performed using any method known to those of skill in the art, for example, by treatment with fungicide, a surface sterilizing agent, or combinations thereof. The explant may be subjected to a single round of sterilization or multiple rounds of sterilization. For example, the explant may go through a primary sterilization step with the fungicide Bavastitin and then go through a secondary sterilization with a surface sterilizing agent like sodium hypochlorite or mercuric chloride.
Micropropagation of Leaf Explants
The present invention provides a method for efficient in vitro mass culture of Jatropha curcas using leaf discs and culture in media without passage through a callus stage.
Other aspects of the micropropagation process can be performed using methods known to those of skill in the art in plant tissue culture. Micropropagation typically involves the following steps:
1) culturing explants in initiation media to generate multiple shoots
2) transferring shoots to proliferation and elongation media
3) transferring the elongated shoots to rooting media
4) hardening the plantlets, and
5) transferring the hardened plantlets to the field.

The media for shoot development typically includes a standard concentration of salts and vitamins, a carbon- source, phytohormones, gelling agent, antibiotics to control bacterial contamination associated with seed borne, inherent contamination, is at a pH range of 5.4 to 6.2, and has been sterilized by emobidments. Common phytohormones used for micropropagation are described herein. The cultures are incubated at 20-35°C under cool, white fluorescent lamps for a minimum period of 2-3 months to induce shoots with sub-culturing every 30 days.
General Media Composition
The basal media used to culture Jatropha can be any of those already known in the field of the art for plant tissue culture, such as Murashige & Skoog, Gamborg' s, Vacin & Went, White's, Schenk & Hildebrandt or the like.
In some embodiments, the proliferation and shoot elongation medium and rooting medium have the same level of nutrients and hormones as the initiation medium. In other embodiments, the compositions are similar but not precisely the same.
The basal media can be supplemented with various carbon sources. The carbon source may be sucrose or glucose, typically, at a concentration of about 2-5%. The carbon source may also be sugar alcohol like myo-inositol, typically, at a concentration of about 50-500 mg per liter.
In some embodiments, the basal media will include gelling agents such as agar, alginic acid, carrageenan, gellangum. Typical concentrations are 0.5-1%.
Hormones in Media
The phytohormone can be any hormone that will affect growth in the desired manner
during different stages of tissue culture. Examples of suitable hormones include natural
or synthetic auxin, cytokinin, gibberellin, or cytokinin-active urea derivatives.
The cytokinins used can include, but are not limited to, 6-aminopurine(adenine), 6-
aminopurine hydrochloride, 6-aminopurine hemisulfatea, 6-benzyI aminopurine (BAP),

kinetin, zeatin, ^-substituted derivatives, or derivatives of these compounds. Preferred
cytokinin-active urea derivatives include, but are not limited to, thiadiziron, diphenylurea,
N-phenyl-N,-(4-pyridyl) urea or their derivatives.
The auxins used can include, but are not limited to, naphthalene acetic acid,
naphthaleneacetamide, naphthoxyacetic acid, indole acetic acid, indole butyric acid
(IBA), 4-chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic Acid (2,4-D), 2,4,5-
trichlorophenoxyacetic acid, or the like and their derivatives.
The phytohormone may be used singly or in combination with two or more other
The concentration of the phytohormone present in the media will depend on the stage of the method of the invention. The present invention provides that concentration is between 0.01 mg per liter to 10 mg per liter; preferably the concentration is between 0.01 mg/L and 0.05 mg/L. For auxins, the preferred range is 0-2mg/l or more preferably lmg/1. In one embodiment of this invention, a cytokinin is used in the initiation and proliferation/elongation media while an auxin is used in the rooting media.
Culture Conditions
In certain embodiments, the culture conditions (i.e., light cycle, light intensity, media, temperature, relative humidity) are the same throughout the initiation, proliferation and elongation, and rooting stages. Subculturing is performed as necessary; preferably, every 3 to 4 weeks.
Once well-formed roots are obtained, plantlets can be hardened on soil, sand, moss, charcoal or other media either alone or in combination in defined ratio. The plantlets can then be transferred to the fields by direct sowing or transplanting of the cuttings.
Micropropagated true-to-type Jatropha curcas plants and Jatropha curcas can be produced using the above-described methods. True-to-type plants are generated by selecting a plant and micro propagating a plant from the leaf disc.

Tsugawa, H; Kagami, T and Suzuki, M (2004). High-frequency transformation of Lobelia erinus L by Agrobacterium mediated gene transfer. Plant Cell Rep. Vol. 22 (10), pp. 759-764.
Snvastava, P. S (1974). In vitro induction of triploid roots and shoots from mature endosperm of Jatrophapanduraefolia. Z. Pflanzenphysiol. Vol. 66, pp. 93-96.
Snvastava, P. S and John, B. M (1974). Morphogenesis in mature endosperm cultures of Jatropha panduraefolia, Beitr. Biol. Pflanz. Vol. 50, pp. 255-268.
Sujatha, M and Dhingra, M (1993). Rapid plant regeneration from various explants of Jatropha integerrima. Plant Cell Tiss. Org. Cult. Vol. 35, pp. 293-296.
Sujatha, M and Mukta, N (1996). Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas. Plant Cell Tiss. Org. Cult. Vol 44, pp. 135-141.
Weida, L; Qim, W; Lin, Tang; Fang, Y and Fang, C (2003). Induction of callus from Jatropha curcas and its rapid propagation.. Yingyong Yu Huanjing Shengwu Xuebao, Vol. 9 (2), pp. 127-130.
Johri, B. M and Srivastava, P. S (1973). Morphogenesis in endosperm cultures. Z. Pflanzenphysiol Vol. 70, pp. 285-304
Chandran, K and Sujatha M (1996). Isolation and purification of protoplasts of castor (Ricinus communis L) and Jatropha spp. GAU Res. J. Vol. 21, pp. 110-111.
All of the references cited herein are hereby incorporated by reference.

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, are within the scope of the invention.

Dated this 31st day of March 2006

K. V. Subramaniam
For Reliance Life Sciences Pvt. Ltd.

This invention provides an efficient process for in vitro propagation of Jatropha through direct regeneration of leaf disc without any intermediary callus phase. This process provides a commercially viable process for producing true-to-type plants of Jatropha on a large scale.






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Patent Number 246173
Indian Patent Application Number 490/MUM/2006
PG Journal Number 08/2011
Publication Date 25-Feb-2011
Grant Date 18-Feb-2011
Date of Filing 31-Mar-2006
# Inventor's Name Inventor's Address
1 JOHNSON TANGIRALA SUDHAKAR Reliance Life Sciences Pvt. Ltd. DALSC, Plot No R-282 TTC Area of MIDC, Rabale, Navi Mumbai - 400701
PCT International Classification Number A01H4/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA