MICROPROPAGATION OF JATROPHA CURCAS (L.)

Indian J. Agric. Res., 43 (4) : 269-273, 2009 AGRICULTURAL RESEARCH COMMUNICATION CENTRE www.arccjournals.com / indianjournals.com MICROPROPAGATION OF JATROPHA CURCAS (L.) B.R. Ranwah, D.K. Gupta and M.A. Shah Department of Plant Breeding and Genetics, MPUAT, Udaipur ABSTRACT The leaf disc were enlarged in MS medium supplemented with 5 mg/l Kn, 0.5 mg/ l Kn and 5 mg/l BAP + 3 mg/l Kn, 3 mg/l BAP + 5 mg/l Kn and 5 mg/l BAP + 3 mg/ l Kn. However, green callus was observed in 10 mg/l BAP + 5 mg/l IBA. Quantity of callus was further increased in sub culturing on same medium. In sub culturing when medium was supplemented with charcoal primordial shoots were seen on enlarged leaf disc. On shoot tip explants leaves were seen in all the treatments. But, in sub culturing no further growth was observed. When epicotyl implanted on MS medium supplemented with 0.5 mg/l BAP + 5 mg/l Kn and charcoal one shoot was observed. On sub culturing two roots were initiated and enlarged upto 15.4 cm. In plumule explants one leaf and one leaf with one root was observed in 0.5 mg/l BAP + 3 mg/l Kn and 0.5 mg/l BAP + 5 mg/l Kn, respectively when medium was supplemented with charcoal. In hypocotyls no shooting was there however, best rooting i.e. early, maximum number and length was observed in treatment 5 mg/l IBA + 3 mg/l NAA shocked with 500 mg/l IBA. Key words: Jatropha, Physicnut, Tissue culture, Regeneration, Growth regulators. INTRODUCTION Jatropha curcas popularly known as ratanjot, Sobudam and physic nut is a large multipurpose oil yielding shrub, belonging to family Ephorbiaceae. This tropical and subtropical crop can thrive well in low rainfall regions and problematic soils. It is generally grown as a living fence around field to protect crops from live stock. The oil of this crop is used for burning, for making hard soap, candles, and as efficient substitute fuel for diesel engines (Stewart et al. 1981). The vegetative propagation of this plant is very easy but, looking to the large scale need of true to the type plants the micropropagation and tissue culture techniques becomes important. Looking to the importance, culture techniques have been developed for several Euphorbia species such as Euphorbia pulcherrima (Channaveeraiah et al., 1973), Euphorbia esula and Euphorbia cyparissias (Lee and Staratt, 1971); Euphorbia flanaganii (Silberstein et al., 1983); Euphorbia lathyris (Repley and Preece, 1986) and Jatropha integerrima (Sujatha and Dhingra, 1993). Inspite of lot of efforts made, the success in regeneration of plantlet is restricted to a limited number of crop species. The requirement of phytohormones and their concentrations varied from genotype to genotype of a species and organ to organ of a plant. The present paper reports the results of influence of plant growth regulators/ hormone on the regeneration of Jatropha curcas (L.). MATERIAL AND METHODS Explants leaf and shoot tip were taken from mature growing plants, and epicotyle, hypocotyle and plumule were taken from in

270 INDIAN JOURNAL OF AGRICULTURAL RESEARCH vitro seedlings. The explants were washed thoroughly with sterile double distilled water followed by surface sterilization with 0.1 per cent mercuric chloride (HgCl 2 ) solution for 5 minutes. The sterilized explants were again washed four times with auto sterile double distilled water. For culture media 42.24 g readymade powder of MS medium with agar and 500 mg/l calcium chloride were dissolved in double distilled water and raise the volume up to one liter. Activated charcoal (100 mg/l) was added in this medium wherever it was required. There after required quantity of fresh stock solution of phytohormones were added in the medium. The ph of the medium was adjusted to 5.8±0.2 before pouring of medium in conical flasks. The medium was autoclaved at 1.5 kg/cm 2 at 121 C temperature for 20 minutes. Shoot tips were introduced vertically into semisolid MS medium, leaf discs (5 mm in diameter) were placed in contact with medium and epicotyle, hypocotyle and plumule were cut transversely (0.5 cm diameter) and placed with the proximal cut end in contact with the medium. All culture were incubated at 25±2 C temperature in growth chamber and were exposed to 16/8 h light/dark period for shoot regeneration and 8/16 h light/ dark period for root regeneration. Observations on the days to shoot initiation, number of shoots at 15 DAI. Maximum length of shoot at 15 DAI (cm), days to root initiation, number of roots at 15DAI and maximum root length at 15DAI (cm) per responding treatment were recorded. Results were subjected to CRD. RESULTS AND DISCUSSION Results indicated that when the leaf disc implanted on without activated charcoal added MS medium were enlarged in treatment 5 mg/l Kn, 0.5 mg/l BAP + 3 mg/l Kn, 3 mg/l BAP + 3 mg/l Kn, 3 mg/l BAP + 5 mg/l Kn and 5 mg/l BAP + 3 mg/l Kn, but it was not uniform in all the replications. When the enlarged leaf disc sub cultured on activated charcoal supplemented MS medium further enlargement were observed in treatments 5 mg/ l Kn and 3 mg/l BAP + 3 mg/l Kn, but, again it was not uniform in all the replications. While in treatment 5 mg/l BAP + 3 mg/l Kn primordial shoots were observed on enlarged leaf discs, but, again it was not uniform in all the replications. In subsequent experiment these primordial shoots were sub cultured on activated charcoal added medium supplemented with various concentrations of BAP and Kn, but, no further growth was recorded. Sujatha and Mukta (1996) also reported that various concentrations of BAP and Kn are unable to support the growth of the explants. When leaf discs were inoculated on cytokinin along with the auxins green callusing was there. Maximum callus was observed on treatment 10 mg/l BAP + 5 mg/l IBA. The quantity of callus was further increased in sub culturing on same Table 1:Days to rooting, number of roots and length of root in hypocotyls without shock Phytohormone Concentration (mg/l) Days to rooting Number of Length of NAA 10 100 14.60c 1.40c 1.08b NAA 15 100 12.80b 9.00a 0.38c IBA + NAA 3 + 2 100 13.60bc 1.20c 0.45c IBA + NAA 10 + 5 100 10.20a 6.40b 1.33a Mean 12.80 4.50 0.73 SE 0.35 0.34 0.02 CD 5% 1.06 1.02 0.13 CV 6.18 16.85 13.09

Vol. 43, No. 4, 2009 271 Table 2: Days to rooting, number of roots and length of root in hypocotyls shocked with 500 mg/l IBA SN Phytohormone Concentration (mg/l) Days to rooting Number of Length of 1 IBA + NAA 2 + 2 100 16.60d 1.20f 0.28e 2 IBA + NAA 2 + 5 100 17.00d 3.40d 0.44d 3 IBA + NAA 3 + 2 100 14.00c 3.60d 0.42d 4 IBA + NAA 3 + 3 100 11.20b 4.40c 1.70b 5 IBA + NAA 3 + 5 100 12.40b 3.00d 1.42c 6 IBA + NAA 5 + 2 100 11.60b 8.40b 1.78b 7 IBA + NAA 5 + 3 100 9.20a 10.40a 3.00a 8 IBA + NAA 5 + 5 100 17.00d 2.20e 0.30e Mean 13.63 4.60 1.17 SE 0.52 0.28 0.002 CD 5% 1.26 0.77 0.10 CV 7.48 13.57 7.46 concentration. Sujatha and Mukta (1996) also observed callusing from leaves of Jatropha curcas in different concentration of BAP+IBA. Two leaves were sprouted on medium supplemented with 3 mg/l BAP + 3 mg/l Kn but, it was not uniform in all the replications. While in treatments viz. 0.5 mg/l Kn, 3 mg/l Kn, 0.5 mg/l BAP + 0.5 mg/l Kn and 3 mg/l BAP + 5 mg/l Kn only one leaf sprouted in 2 to 3 replications. When the responding explants were subcultured on same concentrations along with activated charcoal another leaves were sprouted in treatment 3 mg/l BAP + 5 mg/l Kn but it was not again uniform in all the replications. In further sub culturing neither much elongation nor multiple shooting was there, later on leaves became brown. One shoot was regenerated from epicotyle explants on activated added MS medium in treatment 0.5 mg/l BAP + 5 mg/l Kn in only one replication. Rest of the epicotyls became brown in all the replications in all the treatments. When this shoot was sub cultured two roots were initiated and enlarged up to 15.4 cm. Further, branching was also there on the longest root. Hypocotyls did not responded to any of the treatment. However, one leaf was initiated from plumule explant on charcoal added medium in treatment 0.5 mg/l BAP + 3 mg/l Kn and leaft along with root was initiated in treatment 0.5 mg/l BAP + 5 mg/l Kn but, it was only in one replication. Rest of the plumule explants became brown. The responding explants were subcultured on the same medium but, no further elongation was observed and leaves and roots became brown. In above shooting experiments it was observed that explants did not responded in most of the treatments on account of presence of latex. So use of antioxidants may help to avoid the toxicity of its latex. Thakur and Shukla (2004) also suggested the use of antioxidants on plants having latex to avoid toxicity of its latex. In above experiments no shooting was obtained therefore rooting on such shoots was not possible. However, to identify the protocol, rooting was studied on naturally available explants viz. hypocotyle and shoot tip were inoculated on activated charcoal added medium supplemented with different concentrations of auxins viz. IBA and NAA alone and in combinations with and without shocks. Shock was given by 500 mg/l IBA and NAA for 10 minutes separately wherever respective phytohormone was used in culture medium. Rooting was observed in some

272 INDIAN JOURNAL OF AGRICULTURAL RESEARCH Table 3: Days to rooting, number of roots and length of root in hypocotyls shocked with 500 mg/l NAA SN Phytohormone Concentration (mg/l) Days to rooting Number of Length of 1 NAA + IBA 2 + 3 100 14.00b 4.60bc 1.44a 2 NAA + IBA 2 + 5 100 12.00a 7.80a 1.58a 3 NAA + IBA 3 + 5 100 13.60b 4.80b 0.46b 4 NAA + IBA 5 + 2 100 16.40c 3.40c 0.42b 5 NAA + IBA 5 + 3 100 16.40c 1.20d 0.54b Mean 14.48 4.40 1.11 SE 4.78 21.07 11.88 CD 5% 0.91 1.22 0.17 CV 0.31 0.41 0.06 treatments both with and without shock on hypocotyle. In the three rooting experiments of hypocotyle with and without shock the earliest rooting was observed in the treatment 5 mg/l IBA + 3 mg/l NAA with shock of 500 mg/l IBA (9.2 DAI, Table 2). It was followed by 10 mg/l IBA + 5 NAA without shock (10.20 DAI) (Table 1) and 2 mg/l NAA + 5 mg/l IBA with the shock of 500 mg/l NAA (12.00 DAI) (Table 3). The maximum number of roots was observed in treatment 5 mg/l IBA + 3 mg/l NAA with shock of 500 mg/l IBA (10.4, Table 2). It was followed by 15 mg/l NAA without shock (9.00) and 2 mg/l NAA + 5 mg/l IBA with shock of 500 mg/l NAA (7.80). Similarly the longest root was in treatment 5 mg/l IBA + 3 mg/l NAA with shock of 500 mg/l IBA (3.00 cm, table 2).It was followed by 2 mg/l NAA + 5 mg/l IBA with the shock of 500 mg/l NAA (1.58 cm) and 10 mg/ l IBA + 5 mg/l NAA without shock (1.33 cm). Therefore, for rooting on hypocotyle best treatment was 5 mg/l IBA + 3 mg/l NAA, where rooting was earliest and number of roots and length of root were maximum. In the rooting experiments of shoot tip explants contamination rate was very high therefore no rooting could be recorded. Beniwal et al. (2004) also observed that the explants (shoot tip and nodal segments) from one year old un sprouted cuttings exhibited lower regeneration and a very high contamination rate (100 %) and reported that this may be due to systemic infection present in un sprouted cuttings. In all it can be concluded that all explants had some regeneration response whenever BAP was there except hypocotyle. In leaf disc response was at 5 mg/l BAP + 3 mg/l Kn, in shoot tip 3 mg/l BAP + 3 Kn and 3 mg/l BAP + 5 mg/l Kn, in epicotyle 0.5 mg/l BAP + 5 mg/l Kn and in plumule 0.5 mg/l BAP + 3 Kn or 0.5 mg/l BAP + 5 mg/l Kn. Though, frequency and growth was very poor. Therefore, on the basis of above results neither we can find out the explants nor hormonal combination and concentration for shooting. However, it is recommended that to reach on any conclusion large number of treatment combinations be tried in more number of replications. Callus can be generated and maintained by incubating leaf discs on MS medium supplemented with 10 mg/l BAP + 5 mg/l IBA as frequency and quantity of callus was very good on this combination. Similarly for rooting activated charcoal added MS medium supplemented with 5 mg/l IBA + 3 mg/l NAA with shock of 500 mg/l IBA was best treatment and can be used in future experiments.

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