WO2011021211A2

WO2011021211A2 - A method for micropropagation of jatropha curcas

Info

Publication number: WO2011021211A2
Application number: PCT/IN2010/000469
Authority: WO
Inventors: Geetaa Singh; Sudheer Shetty
Original assignee: Labland Biotech Private Limited
Priority date: 2009-07-14
Filing date: 2010-07-14
Publication date: 2011-02-24
Also published as: WO2011021211A3

Abstract

Jatropha curcas is considered as a recalcitrant crop which does not easily respond to tissue culture. The instant disclosure provides a commercially viable tissue culture protocol that includes initiation, multiplication, shooting developments, root developments and greenhouse hardening (acclimatization). At each of the above stages, the instant disclosure provides innovative process protocols and media protocols that were able to successfully generate tissue culture plantlets of Jatropha curcas on a commercial scale.

Description

"A METHOD FOR MICROPROPAGATIQN OF

JATROPHA CURCAS"

FIELD OF THE DISCLOSURE

The present disclosure relates to plant biotechnology, particularly related to plant tissue culture. More particularly, the instant disclosure provides a protocol for micropropagation of Jatropha Curcas.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE

Cultivation of high yielding clones of J. curcas is vital to meet the large-scale demand and to ensure consistent supply of quality planting material for establishing sizable yielding plantations similar to other plantation crops. Attempts of micropropagation through tissue culture are going on in Indian labs using seeds or seedlings as the explants. The major limitation in large-scale cultivation as an energy crop is the low and inconsistent seed yield due to heterozygous nature of plants. Traditional propagation through stem cuttings is seasonal, prone to diseases and easy uprooting of established plants hamper the practical utility of this propagation method. Some reports on shoot bud induction through callus are available However, the presence of intermediary callus or callus-mediated regeneration is least desired for the production of true-to-type plants. Clonal propagation from seedling tissues, nodal segments of young seed raised plants, leaf, hypocotyl, stem and peduncle has been reported. However, the data on the number of plantlets produced, the rate of successful regeneration, the number of plants obtained is not clear from the reports. Besides, the commercial reproducibility of such protocols seems to have been not successful. Non-reproducibility and poor in vitro rooting leads to higher cost of production. These limitations have hampered the practical application of tissue culture methods. Research on reducing the production cost is also a priority.

Even though, attempts have been made by various institutions in the development of a successful protocol for the mass multiplication of elite Jatropha selections through tissue culture technique using nodal explants from adult, field grown plants appear to have been not yet complete. There is a need for a commercially viable tissue culture protocol for Jatropha to meet the huge demand for the quality planting material.

SUMMARY OF THE DISCLOSURE

Accordingly the present disclosure relates to a method for micropropagation of Jatropha curcas, comprising acts of a) identifying and treating mother plant to obtain explant, b) cleaning and pre-treating the explant to remove contaminants, c) inoculating the cleaned and the pre-treated explant into a medium and d) subculturing, multiplying, shooting, rooting and acclimatizing the inoculated explant to obtain the micropropagated Jatropha curcas; a method of acclimatization comprising acts of a) etiolating plantlet in a solidifying medium, b) washing the etiolated plantlet, c) blotting dry the washed plantlet, d) transferring the washed plantlet into hardening medium in net pots, e) transferring the net pots to greenhouse for hardening and f) transferring the net pot plants into poly- tunnels and nurturing in shade house for field transfer; a synergistic hardening medium composition comprising cocopeat in the range of about 50 % W/W to about 80% W/W, vermiculite in range of about 1% W/W to about 5% W/W, sand in range of about 10% W/W to about 25% W/W, granular charcoal in range of about -5% W/W to about 15% W/W, Carbendazim in range of about 0.1% W/W to about 0.5% W/W, Copper oxychloride in range of about 0.25% W/W to about 1% W/W, k-cyclin in range of about 0.01% W/W to about 0.1% W/W; a synergistic composition for inoculating and subculturing explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein said modification comprises a) addition of Benzyl Amino Purine ranging from about 0.01 mg/L to about 2.5 mg/L, preferably about 1.25 mg/L, Indole Acetic Acid in range of about 0.001 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L, and sodium phosphate in the range of about 150mg/L to about 200mg/L, preferably 170mg/L, b) omission of Glycine, Nicotinic acid and Pyridoxine, c) increase of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L, Thiamine in range of about 0.1 mg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 0.1 mg/L to about 5 mg/L, preferably about 0.5 mg/L and d) decrease of Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L, and Calcium chloride to range of about 310mg/L to about 350mg/L, preferably 330mg/L; a synergistic composition for multiplication of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises a) addition of Benzyl Amino Purine ranging from about 0.01 mg/L to about 2.5 mg/L, preferably about lmg/L to about 1.25 mg/L, Indole Acetic Acid in range of about 0.001 mg/L to about 0.5 mg/L, preferably about 0.05 mg/L, optionally Biotin in range of about 0.005 mg/L to about 2 mg/L, preferably about 0.05 mg/L, optionally Folic acid in range of about 0.005 mg/L to about 2 mg/L, preferably 0.5mg/L, sodium phosphate in the range of about 150mg/L to about 200mg/L preferably 170mg/L, b) omission of Glycine, Nicotinic acid and Pyridoxine, c) increasing of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L, thiamine in range of about O.lmg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L, and d) decreasing Magnesium sulphate to range of about 125mg/L to about 200mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L calcium chloride to range of about 250mg/L to about 350mg/L, preferably 330mg/L; a synergistic composition for shoot initiation of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises a) addition of Benzyl Amino Purine ranging from about 0.001 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L, Indole Butyric Acid in range of about 0.001 mg/L to about 2.0 mg/L, preferably about 0.1 mg/L, Biotin in range of about 0.005 mg/L to about 2 mg/L, preferably about 0.05 mg/L, Folic acid in range of about 0.005 mg/L to about 2 mg/L, preferably 0.05mg/L, sodium phosphate in the range of about 150mg/L to about 200mg/L preferably 170mg/L, b) omission of Glycine, Nicotinic acid and Pyridoxine, c) increasing of cupric sulphate in range of about 0.1 mg/L to about 2.5 mg/L, preferably about 0.25 mg/L, thiamine in range of about O. lmg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L and d) decreasing Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L, calcium chloride to range of about 310mg/L to about 350mg/L, preferably 330mg/L, sucrose to range of about 5000mg/L to about 25,000mg/L; a synergistic composition for shoot elongation of explant in micropropagation of Jatropha curcas, said composition comprising modified Economou and Read medium, wherein the said modification comprises a) addition of Indole Acetic Acid ranging from about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L, Indole Butyric Acid in range of about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L and iron sodiumethylenediaminotetraacetate in range of about 20mg/L to about 50mg/L, preferably 37mg/L, b) omission of Iron sodium Diethylene triamine pentaacetic acid, c) increasing of cupric sulphate in range of about 0.2 mg/L to about 2.5 mg/L, preferably about 0.25 mg/L, decreasing sucrose to range of about 5000mg/L to about 25,000mg/L preferably at 20000mg/L; a synergistic composition for rooting of shooted explant in micropropagation of Jatropha curcas, said composition comprising modified woody plant medium (WPM), wherein said modification comprises a) addition of Indole Acetic Acid in range of about 0.01 mg/L to about 2.5 mg/L, preferably about lmg/L, coconut water in range of about lOmL to about 150 mL, preferably about 50 mL, Nickel sulphate in range of about 0.0 lmg/L to about 0.1 mg/L, preferably about 0.045mg/L and Phloroglucinol in range of about 50mg/L to about 200mg/L, preferably about lOOmg/L, b) increase of calcium nitrate in range of about 540mg/L to about 570 mg/L, preferably about 556 mg/L, Magnesium sulphate to range of about 350 mg/L to about 390 mg/L, preferably 370 mg/L and Calcium chloride to range of about 85 mg/L to about 1 15 mg/L, preferably 96 mg/L, and c) decrease of Sodium Molybdate to range of about 0.00 lmg/L to about 0. lmg/L, preferably 0.025 mg/L and sucrose to range of about 5000mg/L to about 15,000mg/L preferably 15000 mg/L and ; a method of obtaining seeds of Jatropha curcas, said method comprising step of micropropagating Jatropha curcas according to claim 1, to obtain the seeds.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

Figure 1 shows a Flow chart for micropropagation of Jatropha curcas.

Figure 2 shows a Field grown elite adult plants of Jatropha curcas (6 Year old).

Figure 3 shows Endogenous fungal contamination without pretreatment.

Note the fungus arising from the cut ends of explants. Figure 4 shows Axenic cultures of Jatropha curcas positively responding in initiation medium after elaborate pretreatment.

Figure 5 (a) shows a 3-week-oId nodal explants consisting of 4-6 nodal buds along with shoot tip in initiation medium. Note the actively sprouting axillary buds.

Figure 5 (b) shows Initiation of single nodes where the bud is dormant even after 45 days of initiation.

Figure 6 shows subcultured explants after 20 days of initiation, each with 2 nodes and petiole intact.

Figure 7(a) shows browning and death of explants where internodes and petioles are dissected out.

Figure 7(b) shows healthy and vigorously growing explants where the petiole and internodes are intact in the initiation medium.

Figure 8 shows scooped out sprouts in 1^st multiplication medium at the end of 15 days of culture.

Figure 9 shows healthy, green, slightly elongated cultures in the 2^nd multiplication medium at the end of 15 days of culture.

Figure 10 shows individual shoots (1.5 cm) in 1^st shooting medium soon after transfer. Figure 11 shows individual shots (3 cm) in the 1^st shooting medium after 6 weeks of culture.

Figure 12 shows partly elongated shoots with severe leaf drop and culture drying in 2^nd shooting medium.

Figure 13 shows individual shoots (4-5 cm) in the improved 2^nd shooting medium after 6 weeks of culture.

Figure 14 shows rooted plantlets (5-6 cm) in the rooting medium ready for greenhouse transfer.

Figure 15 shows well rooted plantlets showing flexible, long roots with secondary roots. Figure 16 shows rooted plantlets transplanted to net pots and incubated in poly tunnels for primary hardening in the greenhouse.

Figure 17 shows semi hardened netpot plants with fresh leaves and profuse roots

Figure 18 shows hardened plants transferred to polybags and maintained in a warm walk- in poly tunnel. Figure 19 shows completely hardened tissue culture generated poly bag plants prior to field transfer.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a method for micropropagation of Jatropha curcas, comprising acts of:

(a) identifying and treating mother plant to obtain explant;

(b) cleaning and pre-treating the explant to remove contaminants;

(c) inoculating the cleaned and the pre-treated explant into a medium; and

(d) subculturing, multiplying, shooting, rooting and acclimatizing the inoculated explant to obtain the micropropagated Jatropha curcas.

In an embodiment of the present disclosure, the mother plant is 4-7 years old, preferably 6 years.

In another embodiment of the present disclosure, the mother plant yield at least 4Kg of seeds per plant per year.

In yet another embodiment of the present disclosure, the mother plant yield at least 40% oil per seed.

In still another embodiment of the present disclosure, the treating of the mother plant comprises act of spraying the mother plant with a mixture of antimicrobicide and insecticide.

In still another embodiment of the present disclosure, the mother plant is treated with the mixture of antimicrobicide and insecticide for about 5days to about 8 days, preferably 7days. (Please inform whether the treatment is done for one week continuously or only for one day and then after 8 days the explants are collected).

In still another embodiment of the present disclosure, the explant is obtained after 5days to 8 days, preferably 7 days of the treatment of the mother plant with the mixture of antimicrobicide and insecticide.

In still another embodiment of the present disclosure, the explant is selected from a group comprising node, stem, root, leaf and any other part, preferably node.

In still another embodiment of the present disclosure, the pre-treating the explant comprises acts of,

i) pre-treatment step-I, comprising acts of a. agitating the explant with an alcohol,

b. blotting dry the agitated explant;

c. treating the dried explant with a disinfectant;

d. washing the disinfected explant with sterile water;

e. soaking the washed explant in a solution of fungicides and antibiotics;

f. washing the soaked explant with sterile water; and

g. blotting dry the washed explant to obtain step I pre-treated explant.

ii) pre-treatment step-II, comprising acts of

a. sterilizing the step I pre-treated explant in airflow chambers; b. rinsing the sterilized explant in detergent solution;

c. treating the rinsed explant in mercuric chloride solution; and d. washing the treated explant with sterile water to obtain step II pre-treated explant .

In still another embodiment of the present disclosure, the explant is incubated after each step of inoculation, subculture, multiplication shooting and rooting.

In still another embodiment of the present disclosure, the inoculated, subcultured, multiplied, shooted and rooted explant develops into plantlets.

In still another embodiment of the present disclosure, the incubation is at temperature ranging from about 28°c to about 35°c, preferably 32 °c.

In still another embodiment of the present disclosure, the inoculation, subculture, multiplication, shooting and rooting are carried out at a pH in range of about 4.8 to about 5.4, preferably 5.2.

The present disclosure relates to a method of acclimatization comprising acts of ,

(a) etiolating plantlet in a solidifying medium;

(b) washing the etiolated plantlet ;

(c) blotting dry the washed plantlet;

(d) transferring the washed plantlet into hardening medium in net pots;

(e) transferring the net pots to greenhouse for hardening; and (f) transferring the net pot plants into poly-tunnels and nurturing in shade house for field transfer.

In an embodiment of the present disclosure, the solidifying medium is agar.

The present disclosure relates to a synergistic hardening medium composition comprising cocopeat in the range of about 50 % W/W to about 80% W/W, vermiculite in range of about 1% W/W to about 5% W/W, sand in range of about 10% W/W to about 25% W/W, granular charcoal in range of about -5% W/W to about 15% W/W, Carbendazim in range of about 0.1% W/W to about 0.5% W/W, Copper oxychloride in range of about 0.25% W/W to about 1% W/W, k-cyclin in range of about 0.01% W/W to about 0.1% W/W .

The present disclosure relates to a synergistic composition for inoculating and subculturing explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein said modification comprises:

(a) addition of Benzyl Amino Purine ranging from about 0.01 mg/L to about 2.5 mg/L, preferably about 1.25 mg/L, Indole Acetic Acid in range of about 0.001 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L, and sodium phosphate in the range of about 150mg/L to about 200mg/L, preferably 170mg/L;

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increase of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L, Thiamine in range of about 0.1 mg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 0.1 mg/L to about 5 mg/L, preferably about 0.5 mg/L ;and

(d) decrease of Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about

20mg/L, preferably 15mg/L, and Calcium chloride to range of about 310mg/L to about 350mg/L, preferably 330mg/L.

The present disclosure relates to a synergistic composition for multiplication of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises: (a) addition of Benzyl Amino Purine ranging from about 0.01 mg/L to about 2.5 mg/L, preferably about lmg/L to about 1.25 mg/L, Indole Acetic Acid in range of about 0.001 mg/L to about 0.5 mg/L, preferably about 0.05 mg/L, optionally Biotin in range of about 0.005 mg/L to about 2 mg/L, preferably about 0.05 mg/L, optionally Folic acid in range of about 0.005 mg/L to about 2 mg/L, preferably 0.5mg/L, sodium phosphate in the range of about 150mg/L to about 200mg/L preferably 170mg/L;

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increasing of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L; thiamine in range of about 0.1 mg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L; and

(d) decreasing Magnesium sulphate to range of about 125mg/L to about 200mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L; calcium chloride to range of about 250mg/L to about 350mg/L, preferably 330mg/L.

The present disclosure relates to a synergistic composition for shoot initiation of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises:

(a) addition of Benzyl Amino Purine ranging from about 0.001 mg/L to about 0.5 mg/L, preferably about 0.1 mg/L, Indole Butyric Acid in range of about 0.001 mg/L to about 2.0 mg/L, preferably about 0.1 mg/L, Biotin in range of about 0.005 mg/L to about 2 mg/L, preferably about 0.05 mg/L, Folic acid in range of about 0.005 mg/L to about 2 mg/L, preferably 0.05mg/L, sodium phosphate in the range of about 150mg/L to about 200mg/L preferably 170mg/L

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increasing of cupric sulphate in range of about 0.1 mg/L to about 2.5 mg/L, preferably about 0.25 mg/L; thiamine in range of about 0.1 mg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L; and (d) decreasing Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L' Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L; calcium chloride to range of about 310mg/L to about 350mg/L, preferably 330mg/L, sucrose to range of about 5000mg/L to about 25,000mg/L .

The present disclosure relates to a synergistic composition for shoot elongation of explant in micropropagation of Jatropha curcas, said composition comprising modified Economou and Read medium, wherein the said modification comprises:

(a) addition of Indole Acetic Acid ranging from about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L, Indole Butyric Acid in range of about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L and . iron sodiumethylenediaminotetraacetate in range of about 20mg/L to about 0mg/L, preferably 37mg/L.

(b) omission of Iron sodium Diethylene triamine pentaacetic acid;

(c) increasing of cupric sulphate in range of about 0.2 mg/L to about 2.5 mg/L, preferably about 0.25 mg/L; decreasing sucrose to range of about 5000mg/L to about 25,000mg/L preferably at 20000mg/L

The present disclosure relates to a synergistic composition for rooting of shooted explant in micropropagation of Jatropha curcas, said composition comprising modified woody plant medium (WPM), wherein said modification comprises:

(a) addition of Indole Acetic Acid in range of about 0.01 mg/L to about 2.5 mg/L, preferably about lmg/L, coconut water in range of about lOmL to about 150 mL, preferably about 50 mL, Nickel sulphate in range of about 0.0 lmg/L to about 0. lmg/L, preferably about 0.045mg/L and Phloroglucinol in range of about 50mg/L to about 200mg/L, preferably about lOOmg/L

(b) increase of calcium nitrate in range of about 540mg/L to about 570 mg/L, preferably about 556 mg/L; Magnesium sulphate to range of about 350 mg/L to about 390 mg/L, preferably 370 mg/L and Calcium chloride to range of about 85 mg/L to about 115 mg/L, preferably 96 mg L; and (c) decrease of Sodium Molybdate to range of about O.OOlmg/L to about O. lmg/L, preferably 0.025 mg/L and sucrose to range of about 5000mg/L to about 15,000mg/L preferably 15000 mg/L .

The present disclosure relates to a method of obtaining seeds of Jatropha curcas, said method comprising step of micropropagating Jatropha curcas according to claim 1, to obtain the seeds.

In an embodiment of the present disclosure, the seeds are used as source of oil.

In an embodiment of the present disclosure, the seeds and oil are used as insecticide and medicine.

Even though Jatropha plants are seen in many agroclimatic regions, sustainable high yield is not yet achieved. Jatropha is highly cross-pollinated plant. Variations among the same species are limited. It is well documented that some selections are rare and possess beneficial characteristics such as high yield, high oil content, drought resistance, photoperiod insensitivity, resistance/tolerance to major insect pests and diseases. Nonavailability of such quality planting materials in large numbers is the main constraint. Hence, it is desirable to develop commercially viable tissue culture protocol in order to multiply such superior selection to meet the huge demand for large-scale plantations. Here, the applicant report an effective regeneration protocol from stem explants of adult elite selections of Jatropha curcas collected from different agroclimatic regions of India whose agronomic performance (high seed yield and oil content) has been evaluated consistently since 6 years. These are appropriate base material to produce 'true-to-type' plantlets. The protocol will enable the supply of quality plantlets in requisite quantity as per the necessities of the global customers. The present investigation is very important for India and to the international community in the present situation of energy crisis and ever increasing petroleum oil prices. Long term benefit of the protocol is to develop superior clones of Jatropha curcas through tissue culture technique with reproducible higher yield of seeds and oil of superior quality. This will help to establish a sustainable economic production of Jatropha-based biodiesel, which will go long way to augment the dwindling, nonrenewable petroleum oil resources. The Jatropha plantations will help rehabilitation of vast marginal and eroded lands available and will generate large-scale employment opportunities to the rural poor.

Details of methods for explant selection, collection, specifications, pretreatments, composition of initiation medium and conditions to allow enhanced multiple shooting, rooting and acclimatization leading to increased production of Jatropha curcas plantlets are provided below.

The technology of the instant Application is further elaborated with the help of following examples. However, the examples should not be construed to limit the scope of the disclosure.

The process of tissue culture of Jatropha curcas involves two distinct parts. The laboratory based production called micropropagation consisting of a series of in vitro growth stages namely mother plant selection, pre-treatment and initiation, multiplication, shooting and rooting and the greenhouse hardening of the plantlets obtained in vitro to produce the finished product.

A. Mother plant selection and preparation

Several elite selections of Jatropha curcas were identified for the micropropagation process. These elite selections are with at least 40% seed oil and high seed yield of about 4 Kg/plant/year. Careful attention is given to these selections before micropropagation commences. The mother plants at the time of explant collection are free from any symptoms of pest infestation and diseases (Figure 2). The plants are sprayed with fungicides Bavistin (Carbendazim, BASF India limited), Dithane M45 (Mancozeb 75% WP wettable powder, Indofil Chemical Limited), Sulphur 80% WDG (Sulphex Gold, Excel Crop Care Limited) and Copper Oxychloride 50% WP, Blitox (Rallis India Limited) each at 0.2% (w/v) concentration. An insecticide, Rogor (ISAGRO, Asia) at 2 ml/L was also included to avoid any insect infestation.

After one week of treatment, the mother plants aged between 5 and 6 years are ready for harvesting the nodal explants. The following parameters are considered before harvesting of the explants.

Explants should always comprise of axiliary buds and meristem tip (shoot tip) intact and hence shoot tip consisting of 4-6 nodal buds are collected. Such branches are precisely 4-6 weeks old after they have sprouted from the main branches. Explants are collected in the morning hours of the day. The nodal explants, each measuring 4" in length, are dissected from the actively growing shoots. Explant collection is carried out during the months of February, March, April, September, October and November. The explants are collected during non-rainy days. Collection is restricted to warm early morning hours after a sunny day. Tissues are excised with a sharp plant cutter that is sterilized by dipping in 0.2% fungicide solution - Bavistin, Copper Oxychloride and -Cycline (Streptomycin sulphate 90% w/w, Tetracycline Hydrochloride 10% w/w, Karnataka Antibiotic and Pharmaceutical Limited).The excised explants are placed in a clean polypropylene box covered with polypropylene cap, and are carried to the production lab for further processing.

B. Explant cleaning and pretreatment

Pre-treatment is a process involving cleaning the explants in the laboratory. In the present investigation, two steps of pre-treatment are carried out.

I step of pre-treatment

a. The nodal explants (twigs) are grouped into 3 to 4 twigs and put into a 6" glass jars containing 100 ml of 70% ethanol.

b. The jars are manually agitated for 30 seconds.

c. The twigs are blotted dry, and then transferred to a fresh jar containing 250 ml sterile water + 10 ml Dettol liquid hand wash detergent. The process of manually swirling at an interval of 5 minutes for 45 minutes is repeated. The twigs are washed repeatedly with sterile distilled water 4 times to remove all the detergent.

All the twigs as treated above are soaked in a solution containing fungicides and antibiotics (Table 1) for 3 hours at ambient temperature (25±2 °C).

After incubation, the twigs are repeatedly washed 4 times in sterile distilled water and blot dried.

Table 1. Composition of pretreatment solution used for the surface sterilization of explants

Severe fungal and bacterial contamination, often endogenous fungal and bacterial contamination is a limiting factor for mass micropropagation. It has been found that endogenous fungal contamination (Figure 3) is a major problem in field grown plants. This reduces the number of explants available for mass micropropagation. In such situations, explant pre-treatment to recover maximum explants for commercial micropropagation becomes inevitable. Occurrence of endophytic bacteria in Jatropha curcas has been reported which expresses itself after 2-3 subcultures being the major limitation for multiplication and further growth. Explant pre-treatment using 10-15% Clorox and Tween20), 1% Bavistin for 30 min for 3 hours and 0.5% Bavistin for 5 min have been reported. However, these reports do not mention the severity of contamination and percent recovery of the explants. Further, these reports have not used explants harvested from field grown adult plants. 500 mg/L of Augmentin in the multiplication medium to effectively control endophytic bacteria has been reported. Further, it has also been reported to use it in the medium as an ingredient at reduced concentration. However it should be noted that use of antibiotics in the medium could lead to undesirable variations where-in the true-to-type quality could be affected.

Use of the fungicide and bactericide combination containing chemicals targeted to decontaminate both external and internal fungi as in the present disclosure. The antibiotics are also chosen to decontaminate surface-borne and tissue-borne Gram -ve and Gram +ve bacteria.

II step of pre-treatment

a. The twigs are shifted to sterilized Laminar air flow chambers.

b. The twigs are transferred to sterile glass jars and rinsed with sterile water containing 1 % (v/v) Tween-20.

c. The twigs are further treated with 0.2 % (w/v) mercuric chloride for 10 minutes, . followed by repeated sterile water wash for 5 times.

The elaborate pre-treatment procedure explained above has resulted to obtain 99 % axenic cultures essential for successful initiation process by controlling systemic and surface borne microbial contaminants (Figure 4). Such elaborate pre-treatment is to dislodge the water insoluble, residual pesticides and antibiotics.

C. Initiation

It is a critical step in plant tissue culture wherein the emergence of buds is activated by breaking the dormancy. This has impact on the mass production.

a. The explant is vertically anchored in modified MS basal medium (Murashige & Skoog, 1962), containing agar and essential micro/macro nutrients, vitamins, sugars and growth hormones and incubated for 2 weeks.

b. The pH of the medium is adjusted on the lower levels ranging between 4.8 and 5.4 preferably at 5.2 before autoclaving. c. The excised explants are inoculated in the culture bottles (Size: 250 ml, 109 mm x 70 mm) containing initiation medium and incubated at 30±2 °C with a 16 hours light /8 h dark cycle till the bud-break is observed.

d. The composition, concentration and incubation conditions are necessary for obtaining higher percentage of positively responding cultures.

A tender twig containing 4 nodes protected by sheath of the petiole and positioned by the internode which reduces the damage from pre-treatment chemicals to the initiating tissue (cambium) is used. The presence of 4 nodes in each explant comprising of axiliary buds and meristem tip (shoot tip) intact is essential (Figure 5a) in the successful initiation which increases the sites of emergence. Initiating single nodes almost always kills the explant or greatly delays the response (Figure 5b).

The concentration of mineral nutrients and phytohormones along with pH, temperature and photoperiod help in breaking the dormancy and accelerating the initiation process.

Most the reports published so far on the micropropagation of Jatropha curcas have employed either full strength MS or ½ strength MS medium without any modification.

These culture conditions employed in these reports consists of adopting pH levels of 5.7 or 5.8 and incubation temperatures of 25 °C. Whereas in the present disclosure it has been found that certain chemicals play an important role in the in vitro growth phase and their concentration need to be changed and hence certain modifications to the basal media composition were carried out.

In the present investigation, certain macro nutrients are added (NaH₂P0₄) preferably at 170 mg/L and certain amino acids (Glycine) and vitamins (Nicotinic acid, Pyridoxine) are omitted. The concentrations of a few nutrients (150 mg/L MgS0₄, 15 mg/L MnS0₄, 330 mg/L CaCl₂) have been reduced and concentrations of CuS0₄ (0.25 mg/L) and Thiamine (0.5 mg/L) are increased. The increased concentration of CuS0₄ (0.25 mg/L), concentration of Vitamin B6 (0.5 mg/L) and the use of minimum number of phytohormones (only BAP and IAA) are some of the important factors. Phytohormones BAP (0.01 to 2.5 mg/L) preferably at 1.25 mg/L and IAA (0.001 to 0.5 mg/L) preferably at 0.1 mg/L are important in the initiation medium.

The pH of the medium in the range of 4.8 to 5.4 preferably at 5.2 is adopted. The cultures in the medium with higher pH level (5.8), showed precocious leaf fall and culture browning.

Incubation temperatures ranging between 28 and 35°C preferably at 32 °C is maintained. The Jatropha cultures incubated at 25 to 28 °C were pale in colour and their growth response was found to be delayed. Jatropha curcas being a tropical plant, and greatly suffering during the lower climatic conditions in nature, prompted to increase the incubation temperatures up to 32 °C under which condition, cultures began to respond quickly with enhanced multiplication giving rise to healthy and green cultures.

Subculture 1: Subculture is a process which involves the transfer of cultures from medium after a certain period of growth to a fresh jar containing the same or different medium.

The twigs bearing 4 nodes with sprouted buds are transferred to new jars containing the same medium. The twigs are cut into two pieces each containing 2-3 nodes (Figure 6). Retaining the petiole and the internodes on either side of the node is critical. The incubation period is 2 weeks.

Subculture 2: After 2 weeks of initiation, the 4 node explants are cut into pieces each containing 2-3 nodes. Each, of the pieces must possess the petiole intact and transferred to fresh medium of the same composition. The incubation period is 2 weeks. At the end of the incubation period, the petiole detaches exposing the emerging sprout. This sprout is carefully scooped out and transferred to 1^st Multiplication medium.

It is important to note that as the sprout emerges out of the axils of the petiole, it drops off by itself, letting the sprout emerge in an active growth stage. Removal of the internodes and the petiole leads to browning of the culture and subsequent death of the sprouts (Figure 7a). Presence of the petiole and the internodes protects and promotes the healthy emergence of the sprout (Figure 7b) which is important for further growth and proliferation. Such sprout is physiologically more active as compared to those obtained from nodes without a petiole covering.

Supplementation of NaH₂P0₄ and enhanced concentrations of CuS0₄ (0.25 mg/L) and Thiamine (0.5 mg/L) and decreased concentration of MgS0₄ (150 mg/L), MnS0₄ (15 mg/L), CaCl₂ (330 mg/L) helped to obtain 100% establishment of initiated culture and subsequent culture multiplication, up to 10 shoots per explant. The synergistic effect of combination of chemicals, growth hormones and the use of multiple node initiation play a important role in obtaining increased axenic cultures that are healthy and vigorous for mass propagation as is evident from this study. Multiplication

Multiplication of explants involves two distinct stages - 1^st multiplication and 2^nd multiplication with slight changes in the composition of their growth medium. Both these media are prepared using modified MS basal salts as described previously.

1^st Multiplication: The scooped and rescued sprouts, are initially transferred to 1^st Multiplication medium, which quickly responded and produced multiple shoots consisting up to 10 shoots in a clump by 15 days thereby producing many healthy, physiologically active starting materials for mass production (Figure 8). Essentially, these shoots are to be transferred to 2^nd Multiplication medium. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °°C with a 16 hours iight/8 h dark cycle for 15 days as described previously.

1^st Multiplication medium: In the present investigation, certain vitamins like Biotin and Folic acid (0.005 to 2 mg/L) preferably at 0.05 mg/L and 0.5 mg/L respectively are supplementary. The addition of these vitamins are vital for the normal shoot growth and perpetual multiplication.

The compositions, concentration, period of culturing and incubation conditions are important for proper growth and proliferation of multiple shoots. The medium contains essential micro and macro nutrients, vitamins, sugars and growth hormones similar to that of initiation medium. However, the concentration of IAA is reduced to very low level (0.05 mg/L) in the 1^st Multiplication medium. Elimination of IAA in the medium does not promote quality cultures and perpetual multiplication. At higher concentration, the IAA promotes unwanted callus growth particularly when used for prolonged duration. Further, there is additional supplement of 2 vitamins viz. Biotin and Folic acid which are crucial for proper growth and proliferation of multiple shoots. Supplement of these vitamins leads to green colour and quick growth of cultures with enhanced multiplication. All these factors essentially produce quality multi-cultures (Figure 8).

In the present investigation, the use of minimum number of phytohormones like BAP at 1.25 mg/L and IAA at 0.05 mg/L for shoot multiplication of explants from adult plant is considered. In the present investigation, synergistic effect of BAP+IAA and addition of vitamins like Biotin and Folic acid rendered quality cultures with perpetual multiplication every 15 days with a multiplication ratio of 3 to 4 fold increase. In a commercial set up it is essential to maintain enormous numbers of quality multiplying cultures to cater to the production-dispatch chain.

T^d Multiplication: Bottles containing contamination-free cultures from the 1^st multiplication medium are transferred to 2^nd multiplication medium. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 15 days as described previously. The 2^nd multiplication medium is similar to the 1^st multiplication medium containing the essential micro and macro nutrients, vitamins, sugars and growth hormones. However, the concentration of BAP- is further reduced (lmg/L) than that used in 1^st multiplication medium and the two vitamins viz. Biotin and Folic acid are eliminated. This is helpful in proper growth and proliferation of multiple shoots with slight elongation of the individual shoots (Figure 9) which is crucial for shifting the shoots for the elongation medium. Retaining of vitamins in the 2^nd multiplication medium will lead to production of cultures with deep-green leaves that are brittle. The shoots obtained from such cultures fail to elongate properly.

The cultures in 2^nd multiplication medium grow to their full capacity again in 15 days. These need to be divided to smaller clumps of each consisting of 3-4 shoots and have to be transferred to fresh 2^nd multiplication medium to repeat the production process of multi cultures essential for mass production of plantlets. Eventually, the surplus shoots that have elongated to a height of 1.5 cm are channeled to the next stage for further growth.

In the present investigation it is found that for uninterrupted supply of shoots to rooting stage and then to the greenhouse it is essential to multiply and maintain large number of cultures in the 2^nd multiplication and subsequent stages. Shoot development (Shooting)

Shoot development involves two distinct stages - 1^st shooting and 2^nd shooting with changes in the composition of their growth medium. The shooting media are prepared using modified MS basal salts. 1^st Shooting (Shoot initiation)'. Individual shoots measuring about 1.5 cm from the 2^nd multiplication medium are transferred to the 1^st shooting medium (Figure 10) for appropriate elongation of shoots, an essential criteria prior to rooting of shoots. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to 1^st shooting medium consisting of proprietary basal salts specific to Jatropha curcas. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously.

At the end of 6 weeks, the cultures attain a height of about 3 cm (Figure 11). Shoot elongation is not only low, but is associated with challenges like leaf dropping and precocious shoot drying.

The 1^st shooting medium contains modified MS Medium as described earlier supplemented with IBA (0.1 mg/L), BAP (0.1 mg/L), Biotin (0.05)+Folic acid (0.05mg/L). Shoot elongation is highly dependent on several physical and chemical parameters. Partly elongated shoots suffer from severe leaf dropping and culture drying (Figure 12) and is assumed to be associated with toxic gas accumulation in the culture jars.

1^st Shooting medium: In the present investigation, vitamins like Biotin and Folic acid are additional. The addition of these vitamins in the shooting medium are vital for the normal shoot growth and appropriate elongation. Reverting to addition of Biotin and Folic acid was found to be extremely helpful in preventing precocious leaf fall and leaf yellowing. The use of minimum number of phytohormones (only BAP and IBA) at very low concentration (each 0.1 mg/L) completely inhibits multiplication and promotes vertical growth of individual cultures.

2^nd Shooting (Shoot elongation): The cultures measuring 3 cm from the 1^st shooting medium are transferred to the 2^nd shooting medium for further elongation. This stage encourages further elongation of plantlets to an appropriate length that are suitable for transferring to the rooting medium. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to 2^nd shooting medium consisting of modified basal salts of ER medium (Economou and Read, 1984). The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously. At the end of 6 weeks, the shoots attain a height of 4-5 cm (Figure 13) suitable to be transferred to the rooting medium.

Appropriate chemical formulation described in ER basal medium, period of culturing and incubation conditions are designed suitably in the 2^nd shooting medium to prevent leaf dropping and culture drying by manipulating macro/micro elements and additional chemicals and growth regulators in the medium. Besides, the shoot elongation is enhanced to 75% with appropriate formulation. The physical factors such as temperature, light and photoperiod are suitably modified which are totally different from that essential for multiplication stage. Further, the shooting stage is repeated twice as 1^st and 2^nd shooting to achieve appropriate shoot length prior to transfer to rooting stage. This consists of 8 - 10 weeks with 2 transfers at an interval of 4-6 weeks.

In the present investigation, the modified ER basal salts are used for 2^nd shooting medium. The ER medium is modified MS medium with low salt and is specifically used for shoot elongation of woody plants. However, this medium is also modified to suit shooting of Jatropha curcas cultures. The concentration of CuS0₄ (0.2 to 2.5 mg/L) preferably at 0.25 mg/L is increased and was found essential to enhance shoot elongation and prevent leaf dropping. Further, the concentration of sugar source (cane sugar) is reduced to a great extent (5 g to 25 g/L) preferably at 20 g/L. The chemical FeNaDTPA is replaced with FeNaEDTA at 0.037 g/L. The use of minimum number of phytohormones consisting of IBA and IAA (0. 1 to 2 mg/L) preferably at 1.5 mg/L each. The replacement of FeNaEDTA, consistent use of higher concentration of CuS0₄, reduction of sucrose content along with inclusion of auxins (IBA and IAA) synergistically resolved the leaf drying, precocious leaf falling and culture death in the present investigation. Shoots successfully elongated with almost 90% success. Presence of higher concentration CuS0₄ is added which has helped for obtaining commercial success of mass production of plantlets. Root development (Rooting)

This stage encourages the development of roots from sturdy and healthy shoots giving rise to plantlets of size 5-6 cm. Shoots of size 4-5 cm are transferred from the shooting medium to the rooting medium. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to rooting medium, consisting of modified WPM

(Lloyd & McCown, 1980) basal salts and plant growth hormones. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously. At the end of 6 weeks, the shoots will develop quality roots (Figure 14) suitable to be transferred to the greenhouse for hardening.

Rooting medium: In the present investigation, modified WPM basal salts are used in the rooting medium. The concentration of a few nutrients has been increased (CarN0₃]2.4H₂0 preferably at 556mg/L, MgS0₄ preferably at 370mg/L, CaCl₂ preferably at 96mg/L) and concentration of Sodium Molybdate preferably at 0.025mg/L is decreased. Further, NiS0 (0.01 to 0.1 mg/L) preferably at 0.045mg/L and phloroglucinol (50 to 200 mg/L) preferably at lOOmg/L are added as supplementary ingredients. Increased concentration of these compounds and addition of NiS0₄ and phloroglucinol in WPM basal medium is significant for the normal growth of roots and this additional combination of chemicals. The reduced concentration of Sodium Molybdate is necessary. Low concentrations of coconut water preferably at 50 ml/L in the rooting medium along with IBA and IAA (0.01 to 2.5 mg/L) of equal concentration preferably at 1.5 mg/L is used. Use of IBA alone or a combination of other growth hormones that have included Phloroglucinol, NAA, IAA, GA₃ etc has been reported . Further, reduction in the concentration of the carbohydrate source (Cane sugar) in the range of 5 g to 15 g/L preferably at 15 g/L for induction of healthy roots . The roots obtained in the present investigation are flexible, long, and sturdy and are with large number of secondary roots, essential criteria for successful greenhouse acclimatization (Figure 15).

In the present disclosure it is realized that root induction is low and is associated with other challenges like brittle roots, lack of secondary roots, precocious shoot drying and non-reproducibility of results. The roots obtained are delicate and highly vulnerable to physical damage. Often, the shoots shed the leaves and precociously dry up. The above mentioned chemical formulation in WPM medium along with manipulating the macro/micro elements and additional chemicals and growth regulators in the medium greatly helped to prevent leaf dropping and culture drying by. Besides, the root induction was enhanced to 85%. The physical factors such as temperature at 32 °C, 16 hour photoperiod, reduced sugar, addition of coconut water and phloroglucinol have all contributed to the success of obtaining large number of rooted plants regularly in the routine production schedule. Further, the rooting stage is repeated in case shoots do not root in the 1^st transfer to achieve rooting prior to transfer to greenhouse. This stage consists of prolonged growth period of 4-6 weeks with 1 or 2 transfers at an interval of 4- 6 weeks. Non-repeatability of results is attributed to loss of cell competence to the rooting hormones lost over repeated subcultures.

G.Greenhouse hardening (Acclimatization)

This stage involves the shift from the heterotrophic to autotrophic condition wherein plantlets are trained to grow in the outdoor environment and photosynthesize. Immediately upon transplanting, plantlets are kept in high humidity, gradually exposing them to outdoor conditions. Keeping the shoots actively growing is important because the acclimatization and development of autotrophic condition is dependent on the new growth after transfer from test tube environment. Rooted plants in tissue culture bottle are kept in greenhouse of 4,500 sft (100' X 45') fitted with fan and pad system for 1 week at a temperature of 28 ± 2 °C and Relative Humidity (RH) of 70-80 %, under diffused sunlight with 12 hour light and 12 hour dark cycle. The incubation of rooted plants in the tissue culture bottles along with agar in the greenhouse as specified above helps in etiolating the condensed nodes formed during the 1^st and 2^nd shooting and rooting stages. After one week, plants are taken out from the agar medium. The roots of the plants are washed in running tap water and excess water is blotted dry. Hardening media mix (HMM) is prepared by mixing coco peat, vermiculite, sand and granular charcoal in the ratio of 50:2:1 :2 (v/v/v/v). To this HMM, 5-10 g of Bavistin (Carbendazim), 10-20 g of Copper Oxychloride (CoC) and 1-2 g of K-cyclin (Streptomycin sulphate 90% w/w, Tetracycline hydrochloride 10 % w/w) are mixed and kept for one day. Fungicides and bactericides were added and HMM media was incubated for 24 hours. The next day, about 25 g of HMM is transferred to a net pot of size 45 mm height. The net pots are placed in 50 cavity pro tray (20" X 1 1 "). The washed plants are transferred individually to net pots containing the HMM in pro trays. Plants are kept in tunnels (Figure 16) on a movable bench in the greenhouse for hardening at a temperature range of 28±2 °°C and RH of 70-80 %. Plants are watered as and when required and are hardened for 45 days in the greenhouse poly tunnels under the said conditions by which time fresh leaves and profuse roots are developed (Figure 17). The net pot plants are then transferred to poly bags of appropriate size and continued to be incubated in walk-in-poly tunnels (Figure 18) to encourage profuse rooting, further etiolating of shoots and fresh leaf growth. After 10-15 days, the plants are shifted and nurtured in shade house at a temperature of 30±2 °C (Figure 19). At every shifting of plants to new location, leaf dropping and fresh leaf formation are common phenomena. The plants in the poly bags have to be nurtured for about 75 to 90 days prior to field transfer.

The acclimatization of rooted Jatropha curcas plantlets is a very sensitive technique. The plantlets are severely affected by neck rot by 8 to 10 days of planting. While the roots remain unaffected, the shoots turn yellow, with severe leaf drop and the entire shoot just melting away. Further, the plantlets need to be quickly planted with great skill. Highly trained staff with passion to handle the baby plants is teamed up for this activity. The process of acclimatization is carried out in two stages - primary hardening in the tunnels for about 45 days and a brief period in warm walk-in-poly tunnels in the net pots for additional 10 days.

Greenhouse acclimatization: The in , vitro rooted plantlets of Jatropha curcas are physiologically mature in the bottle conditions and posses highly condensed nodes. Repeated leaf shedding in the controlled conditions may drive the plants into some kind of dormant state. It is essential to etiolate the rooted plantlets in a precise manner under specific physical parameters for a brief period in order to break the in vitro induced dormancy. The plantlets are nevertheless sensitive to planting media, water content in the planting media, temperature and RH of the tunnels. These factors are clearly defined and a suitable planting mix consisting of coco peat, vermiculite, sand and granular charcoal is standardized to accomplish 60 to 85% hardening of plantlets. Addition of fungicides (Bavistin & CoC) and bactericides (K-cyclin) help in greatly reducing the fungal and bacterial damages caused during hardening process. The addition of granular charcoal not only gives porous texture to the HMM but may also adsorb toxic release of chemicals and plants if any, thereby enhancing the hardening efficiency. Use of warm walk-in-poly tunnels and hardening in two distinct stages of primary and secondary hardening is carried out.

The present disclosure is further elaborated by the following examples and figures, however the examples should not be construed to limit the scope of the disclosure. Example 1: Selection, Pre-treatment, initiation and sub-culturing of Jatropha curcas.

The following examples provide for the improved methods for explant selection, pretreatments and culturing of Jatropha curcas. The steps involved in the instant process of micropropagation are provided below in a stepwise format:

Mother plant selection and preparation Several elite selections of Jatropha curcas were identified for the micropropagation process. These elite selections are with at least 40% seed oil and high seed yield of about 4 Kg/plant/year. Such mother plants which are free from any symptoms of pest infestation and diseases are selected at the time of explant collection. These plants are sprayed with fungicides Bavistin, Dithane M45, Sulphur 80% WDG and Copper Oxychloride 50% WP, Blitox, each at 0.2% (w/v) concentration. Further, an insecticide, Rogor (ISAGRO, Asia) at 2 ml/L was also included to avoid any insect infestation.

After one week of treatment, the mother plants aged between 5 and 6 years are ready for harvesting the nodal explants. The following parameters are considered before harvesting of the explants. Explants should always comprise of axiliary buds and meristem tip (shoot tip) intact and hence shoot tip consisting of 4-6 nodal buds are collected. Such branches are precisely 4-6 weeks old after they have sprouted from the main branches. Explants are collected in the morning hours of the day. The explants are collected during non-rainy days. Tissues are excised with a sharp plant cutter that is sterilized by dipping in 0.2% fungicide solution - Bavistin, Copper Oxychloride and - Cycline. The excised explants are placed in a clean polypropylene box covered with polypropylene cap, and are carried to the production lab for further processing. Explant cleaning and pretreatment

Pre-treatment is a process involving cleaning the explants in the laboratory. In the present disclosure, two steps of pre-treatment are carried out.

I step of pre-treatment

The nodal explants (twigs) are grouped into 3 to 4 twigs and put into a 6" glass jars containing 100 ml of 70% ethanol and the jars are manually agitated for 30 seconds. Further, the twigs are blotted dry, and then transferred to a fresh jar containing 250 ml sterile water + 10 ml Dettol liquid hand wash detergent. The process of manually swirling at an interval of 5 minutes for 45 minutes is repeated. The twigs are washed repeatedly with sterile distilled water 4 times to remove all the detergent. All the twigs as treated above are soaked in a solution containing fungicides and antibiotics provided in the above Table 1, for 3 hours at ambient temperature (25±2 °C). After incubation, the twigs are repeatedly washed 4 times in sterile distilled water and blot dried.

II step of pre-treatment

The above treated twigs are shifted to sterilized Laminar air flow chambers, then the twigs are transferred to sterile glass jars and rinsed with sterile water containing 1 % (v/v) Tween-20.

The twigs are thereafter treated with 0.2 % (w/v) mercuric chloride for 10 minutes, followed by repeated sterile water wash for 5 times.

Initiation

The explant is vertically anchored in modified MS basal medium, containing agar and essential micro/macro nutrients, vitamins, sugars and growth hormones and incubated for 2 weeks. The pH of the medium is adjusted to 5.2 before autoclaving. The excised explants are inoculated in the culture bottles containing initiation medium and incubated at 30±2 °C with a 16 hours light /8 h dark cycle till the bud-break is observed. The composition, concentration and incubation conditions are necessary for obtaining higher percentage of positively responding cultures. A twig containing 4 nodes protected by sheath of the petiole and positioned by the internode is used as the explant.

In the present investigation, in the initiation media, certain macro nutrients are added (NaH₂P0₄) at 170 mg/L and certain amino acids (Glycine) and vitamins (Nicotinic acid, Pyridoxine) are omitted. The concentrations of nutrients 150 mg/L MgS0₄, 15 mg/L MnS0₄, 330 mg/L CaCl₂ have been reduced and concentrations of CuS0₄ (0.25 mg/L) and Thiamine (0.5 mg/L) are increased. The concentration of Vitamin B6 (0.5 mg/L) and the use of minimum number of phytohormones (only BAP and IAA) are some of the important factors. Phytohormones BAP at 1.25 mg/L and IAA at 0.1 mg/L are used in the initiation medium. The pH of the medium is set at 5.2 is adopted. The incubation temperature is maintained at 32 °C and the sub-culturing is done as follows: Subculture 1: The twigs bearing 4 nodes with sprouted buds are transferred to new jars containing the same medium. The twigs are cut into two pieces each containing 2-3 nodes. The incubation period is 2 weeks. Subculture 2: After 2 weeks of initiation, the 4 node explants are cut into pieces each containing 2-3 nodes. The incubation period is 2 weeks. At the end of the incubation period, the petiole detaches exposing the emerging sprout. This sprout is carefully scooped out and transferred to 1^st Multiplication medium. Supplementation of NaH₂P0₄ and enhanced concentrations of CuS0₄ (0.25 mg/L) and Thiamine (0.5 mg/L) and decreased concentration of MgS0₄ (150 mg/L), MnS0₄ (15 mg/L), CaCl₂ (330 mg/L) helped to obtain 100% establishment of initiated culture and subsequent culture multiplication, up to 10 shoots per explant. The synergistic effect of combination of chemicals, growth hormones and the use of multiple node initiation play a important role in obtaining increased axenic cultures that are healthy and vigorous for mass propagation as is evident from this study.

Example 2: Multiplication of sub-cultured explant.

The Jatropha carcus sub_^cultured is further subjected for multiplication which involves two distinct stages - 1^st multiplication and 2^nd multiplication. The multiplication step is carried out using multiplication medias, which are modified MS basal salts.

1^st Multiplication: The scooped and rescued sprouts obtained in the previous example, are initially transferred to 1^st Multiplication medium, which quickly responded and produced multiple shoots consisting up to 10 shoots in a clump by 15 days thereby producing many healthy, physiologically active starting materials for mass production. The pH of the medium was adjusted to 5.2 before autoclaving and the cultures were incubated at 32 °°C with a 16 hours light/8 h dark cycle for 15 days as described previously. The I^s multiplication medium comprised of certain vitamins like Biotin and Folic acid at 0.05 mg/L and 0.5 mg/L respectively are supplementary. The medium contains essential micro and macro nutrients, vitamins, sugars and growth hormones similar to that of initiation medium. However, the concentration of IAA is reduced to 0.05 mg/L in the 1^st Multiplication medium. Further, in the 1^st multiplication media the use of minimum number of phytohormones like BAP at 1.25 mg/L and IAA at 0.05 mg/L for shoot multiplication of explants from adult plant is done.

2^nd Multiplication: Bottles containing contamination-free cultures from the 1^st multiplication medium are transferred to 2^nd multiplication medium. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 15 days as described previously. The 2^nd multiplication medium is similar to the 1^st multiplication medium containing the essential micro and macro nutrients, vitamins, sugars and growth hormones. However, the concentration of BAP is further reduced to lmg/L and the two vitamins viz. Biotin and Folic acid are eliminated. The cultures in 2^nd multiplication medium grow to their full capacity again in 15 days. These need to be divided to smaller clumps of each consisting of 3-4 shoots and have to be transferred to fresh 2^nd multiplication medium to repeat the production process of multi cultures essential for mass production of plantlets. Eventually, the surplus shoots that have elongated to a height of 1.5 cm are channeled to the next stage for further growth. Example 3: Shooting or Shoot development of multiplied explant

Shoot development involves two distinct stages - 1^st shooting and 2^nd shooting with changes in the composition of their growth medium. The shooting media are prepared using modified MS basal salts. 1^st Shooting (Shoot initiation): Individual shoots measuring about 1.5 cm from the 2^nd multiplication medium are transferred to the 1^st shooting medium for appropriate elongation of shoots, an essential criteria prior to rooting of shoots. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to 1^st shooting medium consisting of proprietary basal salts specific to Jatropha curcas. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously. At the end of 6 weeks, the cultures attain a height of about 3 cm.

The 1^st shooting medium contains modified MS Medium as described earlier supplemented with IBA (0.1 mg/L), BAP (0.1 mg/L), Biotin (0.05)+Folic acid (0.05mg/L). Further, vitamins like Biotin and Folic acid are additional. The use of minimum number of phytohormones (only BAP and IBA) at very low concentration (each 0.1 mg/L) completely inhibits multiplication and promotes vertical growth of individual cultures. d Shooting (Shoot elongation): The cultures measuring 3 cm from the 1^st shooting medium are transferred to the 2^nd shooting medium for further elongation. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to 2^nd shooting medium consisting of modified basal salts of ER medium. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously. At the end of 6 weeks, the shoots attain a height of 4-5 cm suitable to be transferred to the rooting medium.

Further, the shooting stage is repeated twice as 1^st and 2^nd shooting to achieve appropriate shoot length prior to transfer to rooting stage. This consists of 8 - 10 weeks with 2 transfers at an interval of 4-6 weeks. In the present investigation, the modified ER basal salts are used for 2^nd shooting medium. The concentration of CuS0₄ at 0.25 mg/L is increased and was found essential to enhance shoot elongation and prevent leaf dropping. Further, the concentration of sugar source (cane sugar) is reduced to 20 g/L. The chemical FeNaDTPA is replaced with FeNaEDTA at 0.037 g/L. The use of minimum number of phytohormones consisting of IBA and IAA at 1.5 mg/L each. Example 4: Root development (Rooting) of the snooted explant

This stage encourages the development of roots from sturdy and healthy shoots giving rise to plantlets of size 5-6 cm. Shoots of size 4-5 cm are transferred from the shooting medium to the rooting medium. The cultures are trimmed at the base as found necessary; two shoots per bottle are transferred to rooting medium, consisting of modified WPM basal salts and plant growth hormones. The pH of the medium was adjusted on the lower levels at 5.2 before autoclaving and the cultures were incubated at 32 °C with a 16 hours light/8 h dark cycle for 4-6 weeks as described previously. At the end of 6 weeks, the shoots will develop quality roots suitable to be transferred to the greenhouse for hardening.

Rooting medium: In the present investigation, modified WPM basal salts are used in the rooting medium. The concentration of a few nutrients has been increased wherein Ca[N0₃]₂.4H₂0 at 556mg/L, MgS0₄ at 370mg/L, CaCl₂ at 96mg/L and concentration of Sodium Molybdate preferably at 0.025mg/L is decreased. Further, NiS0₄ is at 0.045mg/L and phloroglucinol at lOOmg/L are added as supplementary ingredients. Low concentrations of coconut water at 50 ml/L in the rooting medium along with IBA and IAA, each at 1.5 mg/L is used. Further, reduction in the concentration of the carbohydrate source (Cane sugar) is at 15 g/L for induction of healthy roots.

Further, the rooting stage is repeated in case shoots do not root in the 1^st transfer to achieve rooting prior to transfer to greenhouse. This stage consists of prolonged growth period of 4-6 weeks with 1 or 2 transfers at an interval of 4-6 weeks. Non-repeatability of results is attributed to loss of cell competence to the rooting hormones lost over repeated subcultures.

Example 5: Greenhouse hardening (Acclimatization) of the plantlet

This stage involves the shift from the heterotrophic to autotrophic condition wherein plantlets are trained to grow in the outdoor environment and photosynthesize. Rooted plants in tissue culture bottle are kept in greenhouse of 4,500 sft (100' X 45') fitted with fan and pad system for 1 week at a temperature of 28 ± 2 °C and Relative Humidity (RH) of 70-80 %, under diffused sunlight with 12 hour light and 12 hour dark cycle. The incubation of rooted plants in the tissue culture bottles along with agar in the greenhouse as specified above helps in etiolating the condensed nodes formed during the 1^st and 2^nd shooting and rooting stages. After one week, plants are taken out from the agar medium. The roots of the plants are washed in running tap water and excess water is blotted dry. Hardening media mix (HMM) is prepared by mixing coco peat, vermiculite, sand and granular charcoal in the ratio of 50:2:1 :2 (v/v/v/v). To this HMM, 5-10 g of Bavistin (Carbendazim), 10-20 g of Copper Oxychloride (CoC) and 1-2 g of K-cyclin (Streptomycin sulphate 90% w/w, Tetracycline hydrochloride 10 % w/w) are mixed and kept for one day. Fungicides and bactericides were added and HMM media was incubated for 24 hours. The next day, about 25 g of HMM is transferred to a net pot of size 45 mm height. The net pots are placed in 50 cavity pro tray (20" X 1 1 "). The washed plants are transferred individually to net pots containing the HMM in pro trays. Plants are kept in tunnels on a movable bench in the greenhouse for hardening at a temperature range of 28±2 °°C and RH of 70-80 %. Plants are watered as and when required and are hardened for 45 days in the greenhouse poly tunnels under the said conditions by which time fresh leaves and profuse roots are developed. The net pot plants are then transferred to poly bags of appropriate size and continued to be incubated in walk-in-poly tunnels to encourage profuse rooting, further etiolating of shoots and fresh leaf growth. After 10-15 days, the plants are shifted and nurtured in shade house at a temperature of 30±2 °C. At every shifting of plants to new location, leaf dropping and fresh leaf formation are common phenomena. The plants in the poly bags have to be nurtured for about 75 to 90 days prior to field transfer.

Claims

We claim:

1) A method for micropropagation of Jatropha curcas, comprising acts of:

(a) identifying and treating mother plant to obtain explant;

(b) cleaning and pre-treating the explant to remove contaminants;

(c) inoculating the cleaned and the pre-treated explant into a medium; and

2) The method as claimed in claim 1, wherein the mother plant is 4-7 years old, preferably 6 years.

3) The method as claimed in claim 2, wherein the mother plant yield at least 4Kg of seeds per plant per year.

4) The method as claimed in claim 2, wherein the mother plant yield at least 40% oil per seed.

5) The method as claimed in claim 1, wherein the treating of the mother plant comprises act of spraying the mother plant with a mixture of antimicrobicide and insecticide.

6) The method as claimed in claim 5, wherein the mother plant is treated with the mixture of antimicrobicide and insecticide for about 5days to about 8 days, preferably 7days. (Please inform whether the treatment is done for one week continuously or only for one day and then after 8 days the explants are collected).

7) The method as claimed in claim 1, wherein the explant is obtained after 5days to 8 days, preferably 7 days of the treatment of the mother plant with the mixture of antimicrobicide and insecticide.

8) The method as claimed in claim 1, wherein the explant is selected from a group comprising node, stem, root, leaf and any other part, preferably node.

9) The method as claimed in claim 1, wherein the pre-treating the explant comprises acts of:

i) pre-treatment step-I, comprising acts of:

(a) agitating the explant with an alcohol;

(b) blotting dry the agitated explant; (c) treating the dried explant with a disinfectant;

(d) washing the disinfected explant with sterile water;

(e) soaking the washed explant in a solution of fungicides and antibiotics;

(f) washing the soaked explant with sterile water; and

(g) blotting dry the washed explant to obtain step I pre-treated explant.

ii) pre-treatment step-II, comprising acts of:

(a) sterilizing the step I pre-treated explant in airflow chambers;

(b) rinsing the sterilized explant in detergent solution;

(c) treating the rinsed explant in mercuric chloride solution; and

(d) washing the treated explant with sterile water to obtain step II pre-treated explant .

10) The method as claimed in claim 1, wherein the explant is incubated after each step of inoculation, subculture, multiplication shooting and rooting.

1 1) The method as claimed in claim 1, wherein the inoculated, subcultured, multiplied, shooted and rooted explant develops into plantlets.

12) The method as claimed in claim 10, wherein the incubation is at temperature ranging from about 28°c to about 35°c, preferably 32 °c.

13) The method as claimed in claim 1, wherein the inoculation, subculture, multiplication, shooting and rooting are carried out at a pH in range of about 4.8 to about 5.4, preferably 5.2.

14) A method of acclimatization comprising acts of:

(a) etiolating plantlet in a solidifying medium;

(b) washing the etiolated plantlet ;

(c) blotting dry the washed plantlet;

(d) transferring the washed plantlet into hardening medium in net pots;

(e) transferring the net pots to greenhouse for hardening; and

(f) transferring the net pot plants into poly-tunnels and nurturing in shade house for field transfer.

15) The method as claimed in claim 14, wherein the solidifying medium is agar. 16) A synergistic hardening medium composition comprising cocopeat in the range of about 50 % W/W to about 80% W/W, vermiculite in range of about 1% W/W to about 5% W/W, sand in range of about 10% W/W to about 25% W/W, granular charcoal in range of about -5% W/W to about 15% W/W, Carbendazim in range of about 0.1% W/W to about 0.5% W/W, Copper oxychloride in range of about 0.25% W/W to about 1% W/W, k-cyclin in range of about 0.01% W/W to about 0.1% W/W .

17) A synergistic composition for inoculating and subculturing explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein said modification comprises:

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increase of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L, Thiamine in range of about 0.1 mg/L to about lmg L, preferably 0.5mg/L and vitamin B6 in range of about 0.1 mg/L to about 5 mg/L, preferably about 0.5 mg/L; and

(d) decrease of Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L, Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L, and Calcium chloride to range of about 310mg/L to

. about 350mg/L, preferably 330mg/L.

18) A synergistic composition for multiplication of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises:

(a) addition of Benzyl Amino Purine ranging from about 0.01 mg/L to about 2.5 mg/L, preferably about lmg/L to about 1.25 mg/L, Indole Acetic Acid in range of about 0.001 mg/L to about 0.5 mg/L, preferably about 0.05 mg/L, optionally Biotin in range of about 0.005 mg/L to about 2 mg/L, preferably about 0.05 mg/L, optionally Folic acid in range of about 0.005 mg/L to about 2 mg/L, preferably

0.5mg/L, sodium phosphate in the range of about 150mg/L to about 200mg/L preferably 170mg/L;

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increasing of Cupric sulphate in range of about 0.1 mg/L to about 1 mg/L, preferably about 0.25 mg/L; thiamine in range of about 0. lmg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L; and

19) A synergistic composition for shoot initiation of explant in micropropagation of Jatropha curcas, said composition comprising modified Murashige and Skoog medium, wherein the said modification comprises:

(b) omission of Glycine, Nicotinic acid and Pyridoxine;

(c) increasing of cupric sulphate in range of about 0.1 mg/L to about 2.5 mg/L, preferably about 0.25 mg/L; thiamine in range of about 0.1 mg/L to about lmg/L, preferably 0.5mg/L and vitamin B6 in range of about 1 mg/L to about 5 mg/L, preferably about 0.5 mg/L; and

(d) decreasing Magnesium sulphate to range of about 130mg/L to about 170mg/L, preferably 150mg/L' Manganese sulphate to range of about lOmg/L to about 20mg/L, preferably 15mg/L; calcium chloride to range of about 310mg/L to about 350mg/L, preferably 330mg/L, sucrose to range of about 5000mg/L to about 25, OOOmg/L . 20) A synergistic composition for shoot elongation of explant in micropropagation of Jatropha curcas, said composition comprising modified Economou and Read medium, wherein the said modification comprises:

(a) addition of Indole Acetic Acid ranging from about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L, Indole Butyric Acid in range of about 0.1 mg/L to about 2.0 mg/L, preferably about 1.5 mg/L and iron sodiumethylenediaminotetraacetate in range of about 20mg/L to about 50mg/L, preferably 37mg/L.

(b) omission of Iron sodium Diethylene triamine pentaacetic acid;

21) A synergistic composition for rooting of shooted explant in micropropagation of Jatropha curcas, said composition comprising modified woody plant medium (WPM), wherein said modification comprises:

(b) increase of calcium nitrate in range of about 540mg/L to about 570 mg/L, preferably about 556 mg/L; Magnesium sulphate to range of about 350 mg/L to about 390 mg/L, preferably 370 mg/L and Calcium chloride to range of about 85 mg/L to about 1 15 mg/L, preferably 96 mg/L; and

(c) decrease of Sodium Molybdate to range of about O.OOlmg/L to about 0. lmg/L, preferably 0.025 mg/L and sucrose to range of about 5000mg/L to about 15,000mg/L preferably 15000 mg/L .

22) A method of obtaining seeds of Jatropha curcas, said method comprising step of micropropagating Jatropha curcas according to claim 1, to obtain the seeds.

23) The method as claimed in claim 22, wherein the seeds are used as source of oil. 24) The method as claimed in claim 22 and 23, wherein the seeds and oil are used as insecticide and medicine.