Efficient in vitro plant regeneration from shoot apices and gene transfer by particle bombardment in Jatropha curcas
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An efficient and reproducible in vitro plant regeneration system from shoot apices was developed in Jatropha curcas. Benzylaminopurine (BAP; 2.5 μM) was most effective in inducing an average of 6.2 shoots per shoot apex. Incorporation of gibberellic acid (GA3; 0.5 μM) to basal medium was found essential for elongation of shoots. The BAP-habituated mother explants continuously produced shoots during successive subculture without any loss of morphogenic potential. The shoots rooted efficiently on half-strength MS medium. The rooted plantlets were acclimatized with more than 98 % success and the plants transferred to soil:compost in nursery showed no sign of variation compared to the seed-grown plants. The whole process of culture initiation to plant establishment was accomplished within 5–6 weeks. A genetic transformation system in J. curcas was established for the first time, using bombardment of particles coated with plasmid pBI426 with a GUS-NPT II fusion protein under the control of a double 35S cauliflower mosaic virus (CaMV) promoter. The β-glucuronidase (GUS) activity in J. curcas shoot apices was significantly affected by the gold particle size, bombardment pressure, target distance, macrocarrier travel distance, number of bombardments, and type and duration of osmotic pre-treatment. The proliferating bombarded shoot apices were screened on medium supplemented with 25 mg dm−3 kanamycin and surviving shoots were rooted on medium devoid of kanamycin. The integration of the transgene into genomic DNA of transgenic plants was confirmed by PCR and Southern blot hybridization. The transgenic plants showed insertion of single to multiple copies of the transgene.
Additional key wordsbenzylaminopurine β-glucuronidase gibberellic acid micropropagation PCR Southern blot
α- naphthaleneacetic acid
Murashige and Skoog’s (1962) medium
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The authors thank NRC (PBI), Canada for pBI426 plasmid, and NEDFi Guwahati and DARL Pithoragarh India for financial assistance.
- Choi, H.W., Lemaux, P.G., Cho, M.J.: Increased chromosomal variation in transgenic versus nontransgenic barley (Hordeum vulgare L.) plants. — Crop Sci. 40: 524–533, 2000.Google Scholar
- Figueiredo, S.F.L., Albarello, N., Viana, V.R.C.: Micropropagation of Rollinia mucosa (Jacq.) Baill. — In Vitro cell. Dev. Biol. Plant. 37: 471–475, 2001.Google Scholar
- Fu, X., Duc, L.T., Fontana, S., Bong, B.B., Tinjuangjun, P., Sudhakar, D., Twyman, R.M., Christou, P., Kohli, A.: Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. — Transgenic Res. 9: 11–19, 2000.CrossRefPubMedGoogle Scholar
- Geetha, N., Venkatachalam, P., Prakash, V., Sita Lakshmi, G.: High frequency induction of multiple shoots and plant regeneration from seedling explants of pigeonpea (Cajanus cajan). — Curr Sci. 75: 1036–1040, 1998.Google Scholar
- Gopi, C., Vatsala, T.M., Ponmurugan, P.: In vitro multiple shoot proliferation and plant regeneration of Vanilla planifolia Andr. — a commercial spicy orchid. — J. Plant Biotechnol. 8: 37–41, 2006.Google Scholar
- Heller, J.: Physic Nut, Jatropha curcas L. Promoting the Conservation and Use of Underutilized and Neglected Crops. — International Plant Genetic Resource Institute, Rome 1996.Google Scholar
- Iglesias, V.A., Gisel, A., Bilang, R., Leduc, N., Potrykus, I., Sautter, C.: Transient expression of visible marker genes in meristem cells of wheat embryos after ballistic microtargetting. — Planta 192: 84–91, 1994.Google Scholar
- Johri, B.M., Srivastava, P.S.: Morphogenesis in endoserm cultures. — Z. Pflanzenphysiol. 70: 285–304, 1973.Google Scholar
- Jordan, M., Oyanedel, E.: Regeneration of Pouteria lucuma (Sapotaceae) plants in vitro. — Plant Cell Tissue Organ Cult. 31: 249–252, 1992.Google Scholar
- Lee, C.W., Yeckes, J., Thomas, J.C.: Tissue culture propagation of Euphorbia lathyris and Asclepias erosa. — Hort. Sci. 17: 533, 1982.Google Scholar
- Nair, N.G., Kartha, K.K., Gamborg, O.L.: Effect of growth regulators on plant regeneration from shoot apical meristems of cassava (Manihot esculenta Crantz) and on the culture of internodes in vitro. — Z. Pflanzenphysiol. 95: 51–56, 1979.Google Scholar
- Ramesh, M., Gupta, A.K.: Transient expression of β-glucuronidase gene in indica and japonica rice (Oryza sativa L.) callus cultures after different stages of co-bombardment. — Afr. J. Biotechnol. 4: 596–600, 2005.Google Scholar
- Rogers, S.O., Benedich, A.J.: Extraction of DNA from plant tissues. In: Gelvin, S.B., Schilperoot, R.A. (ed.): Plant Molecular Biology Manual. Pp. A6/1–10. Kluwer Academic Publishers, Dordrecht 1988.Google Scholar
- Srivastava, P.S.: In vitro induction of triploid roots and shoots from mature endosperm of Jatropha panduraefolia. — Z. Pflanzenphysiol. 66: 93–96, 1971.Google Scholar
- Srivastava, P.S., Johri, B.M.: Morphogensis in mature endosperm cultures of Jatropha panduraefolia Beitr. — Biol. Pflanz. 50: 255–268, 1974.Google Scholar
- Tideman, J., Hawker, J.S.: In vitro propagation of latex producing plants. — Ann. Bot. 49: 273–279, 1982.Google Scholar