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Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants |
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| Authors: | Perumal Venkatachalam Natesan Geetha Narayanasamipillai Jayabalan Saravanababu Lakshmi Sita |
| Institution: | (1) Department of Plant Science, School of Life Sciences, Bharathidasan University, 620 024 Tiruchirappalli, India;(2) Biotechnology Division, The Rubber Research Institute of India, Kottayam, 686 009 Kerala, India;(3) Department of Microbiology and Cell Biology, Indian Institute of Science, 560 012 Bangalore, India;(4) Department of Botany, C.N. College, Erode, India |
| Abstract: | Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed. |
| Keywords: | Agrobacterium tumefaciens Arachis hypogaea Gene expression Polymerase chain reaction Southern hybridization Transformation |
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