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Micropropagation of Jatropha Curcas L. with Different Hormonal Treatments Article · January 2015
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Indian Res. J. Genet. & Biotech 7(1) : 35 – 40 (2015)
Micropropagation of Jatropha Curcas L. with Different Hormonal Treatments 1
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Sadhana Jadon , Vishwajeet Singh , Ankita Shrivastava , Nitin Wahi , Seema bhadauria 1
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Department of Botany, R.B.S. College, Agra University, Agra-282 002 (U.P.), India New Era Research 3 Foundation, Agra-282 007 (U.P.), India Department of Biotechnology, I.A.H, G.L.A. University, Mathura281 406 (U.P.), India (Received: November 2014; Revised: December 2015; Accepted: January 2015) Abstract An efficient regeneration method via axillary nodes has been developed for Jatropha curcas, which is a medicinal as well as a promising plant for biofuel production. This was achieved on MS medium with BA (3.0 mg/lit) + IBA (1.0 mg/lit) + Adenine sulphate (25 mg/lit) + Glutamine (50 mg/lit) + L-arginine (15 mg/lit) + Citric acid (25 mg/lit) within three to four weeks of inoculation. On an average, within a period of three subcultures, a single initial axillary node explants generated 100 shoots respectively there by making the procedure economic and time effective. Plants were rooted on half strength MS medium supplemented with IBA (1.0-4.0 mg/L) and NAA (1.0-4.0 mg/L). The highest frequency of root induction was on the medium with 3.0 mg/lit IBA. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the nursery. Key words: Jatropha curcas, Axillary buds, Micropropagation, MS medium, IBA (Indole-3-butyric acid). Introduction Jatropha curcas L., a soft wood perennial plant belongs to family Euphorbiaceae, commonly known as Jamalghota, Physic nut, Ratanjot or Purgative nut. Jatropha curcas is one of the most valuable crude drugs of primitive times and is still widely used in modern medicine. It has natural distribution covering the Neo tropic from Mexico to Brazil including the Caribbean Island (Grim, 1996). It is now distributed throughout the entire tropics of Africa and Asia as well (Grim, 1996). In recent years this plant has received extensive attention of many scientists in view of its great economic importance, medicinal significant and for its seed oil as commercial source of fuel (Datta & Pandy, 1993). The superior quality oil can be extracted from the seeds. The oil can be used as a mixed fuel for diesel/gasoline engines (Yoshifumi, 1982). The oil is not edible due to the presence of toxic substance “Curcascine” (Gandhi et al., 1995). It is conventionally used in making soaps, candles, paints, lubricants and medicinally as a purgative (Dastur, 1951; Sujatha & Mukta, 1996). It is also recommended as a drought resistant plant suitable for erosion control and is non palatable for grazing animals due to the toxicity (Munch
& Kiefer, 1989). Plant cell cultures are generally more desirable than a solid medium because of higher growth rates resulting from a high medium to tissue contact. Plant cell and tissue cultures provide an alternative approach to plants cultivars which are difficult to cultivate, has a long seed dormancy period, or has a low product yield due to its slow growth rate. By cell culture or tissue culture plant growth may be significantly increased thus making the procedure highly economical in nature resolving the above problems encountered in conventional farming (Hippolyte et al., 1992; Zhong et al., 1994). The advantages of plant cell culture have for enhancing shoot proliferation and growth is reported in several species by different researchers (Ilan et al., 1995; Escalona et al., 1999). Further studies have been carried out on the effect of environmental condition on culture induction, maintenance, somatic embryogenesis, transformation and plant regeneration (Kim et al., 2003, 2004, 2005; Rao et al., 2006, Nakamura & Ishikawa, 2006). However despite their economic importance, drought resistance characteristic and medicinal value only a limited number of in vitro culture studies have been reported, including plant regeneration from callus (Sujatha & Mukta, 1996). The present study was framed with the following sets of objectives:
Corresponding authors- e-mail:
[email protected] Published by the Indian Society of Genetics, Biotechnology Research and Development Biotech Bhawan 5 E Nikhil Estate, DPS Road, Shastripuram, Agra 282007 Online management by www.isgbrd.co.in
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To test different growth regulator formulations, to establish callus and cell suspension cultures and to develop a long term maintenance on large scale.
medium comprising with NAA (1.0-4.0 mg/L) and IBA (1.0- 4.0 mg/L).
To develop a cost effective, rapid, economic strategy, for the effective micro propagation of Jatropha curcas.
Culture were maintained in a culture room under cool -2 1 white fluorescent light (80-100 µ mole photon m s- ) of 16 hr photoperiod and 8 hr dark periods at a temperature of 24±2°C and relative humidity of 5055%. The cultures were observed after one week. Each experiment had 3 replicates with 10 cultures per replicate. The experiments were repeated thrice and observations represented within the tables as mean of the three repeated experiments.
Materials and Methods Procurement of Ex-Plant The materials and methods of plant tissue culture were the standard ones as propagated within tissue and organ culture fundamental methods (Gamborg and Phillips, 2004). The explants used for the in vitro propagation of Jatropha curcas were axillary nodes of 1-1.5 cm collected from 3-months old plants (before flowering) from the agricultural farms of R.B.S. College, Agra. The explants were first washed with tap water for about half an hour, followed by 1 to 4 drops of liquid soap for 10 to 20 minutes. After washing with tap water properly the explants were surface sterilized with 0.1% mercuric chloride solution for 2 to 3 minutes. This was followed by washing with sterile distilled water for 3-4 times to remove the traces of mercuric chloride solution.
Culture Conditions
Acclimatization and transfer of plantlets to Soil Rooted micro shoots were placed on filter paper supports in sterile distilled water for a week after washing off all traces of agar. The plantlets were then transfers to pots containing garden soil mixed with vermiculite and sand (1:1:1) under controlled growth chamber conditions (24±2°C and 50-55% relative humidity). After 30 days, the plants were kept under shade for 2 weeks and then placed outdoors under full sun. Statistical Analysis
Culture media and Incubation Culture media consisted of MS media (Murashige and Skoog, 1962) supplemented with 3% w/v sucrose and 0.8% w/v agar (Himedia, India) for induction and multiplication of shoots. Explants were cultured on MS medium supplemented with 6-benzylaminopurine (BA) (1.0-4.0 mg/lit), 1-naphthalene acetic acid (NAA) (0.252.0 mg/lit) and indole-3-butyric acid (IBA) (0.25-2.0 mg/lit) either individually or in combination. Along with growth hormones additives like Adenine sulphate (25 mg/lit), Glutamine (50 mg/lit), L-arginine (15 mg/L) and Citric acid (50 mg/L) were also used for induction and multiplication of shoots. The pH of the medium was adjusted to 5.8 before gelling with agar and autoclave at 121°C at 15 lbs pressure for 20 minutes. The surface sterilized explants were inoculated on the above media under aseptic conditions. Rooting of Shoots The elongated shoots 5-6 cm long regenerated from mature in vitro explants were excised and rooted on
Statistical analysis was carried out by calculating the mean values and standard errors, that are represented within the figures and tables. Mean were compared using Students t-test. Results & Discussion Effect of Growth Regulators on Shoot induction Explants cultures on MS medium containing growth regulators (BA/ IBA/ NAA) singly as well as in combination showed varied response with respect to shoot length, number of shoot buds obtained per explants (Table-1). Explants remain green and fresh but failed to develop shoots & buds in lacuna of growth regulators. Maximum growth was found within treatment having MS medium + 6-benzyl aminopurine (BA) (3.0 mg/lit) along with indole-3-butyric acid (IBA) (1.0 mg/lit) which showed 6.99 shoot buds with 4.42 fold increase in shoot length over control (p
