title: An efficient method of agrobacterium-mediated genetic transformation and regeneration in local Indian cultivar of groundnut (Arachis hypogaea) using grafting.
abstract: Groundnut (Arachis hypogaea L.) is an industrial crop used as a source of edible oil and nutrients. In this study, an efficient method of regeneration and Agrobacterium-mediated genetic transformation is reported for a local cultivar GG-20 using de-embryonated cotyledon explant. A high regeneration 52.69 ± 2.32 % was achieved by this method with 66.6 μM 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 ± 3.51, was found with 20 μM BAP and 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and L-cysteine concentration were optimized as 1.8, 200 μM and 50 mg L(-1), respectively, in co-cultivation media. It was observed that the addition of 2,4-D in co-cultivation media induced accumulation of endogenous indole-3-acetic acid (IAA). The optimized protocol exhibited 85 % transformation efficiency followed by 14.65 ± 1.06 % regeneration, of which 3.82 ± 0.6 % explants were survived on hygromycin after selection. Finally, 14.58 ± 2.95 % shoots (regenerated on survived explants) were rooted on rooting media (RM3). In grafting method, regenerated shoots (after hygromycin selection) were grafted on the non-transformed stocks with 100 % survival and new leaves emerged in 3 weeks. The putative transgenic plants were then confirmed by PCR, Southern hybridization, reverse transcriptase PCR (RT-PCR) and β-glucuronidase (GUS) histochemical assay. The reported method is efficient and rapid and can also be applied to other crops which are recalcitrant and difficult in rooting.
authors.affiliation: Discipline of Marine Biotechnology and Ecology, CSIR-Central Salt and Marine Chemicals Research Institute, G.B. Road, Bhavnagar, 364002, Gujarat, India.
authors.forename: Vivekanand
authors.initials: V
authors.lastname: Tiwari
authors.forename: Amit Kumar
authors.initials: AK
authors.lastname: Chaturvedi
authors.forename: Avinash
authors.initials: A
authors.lastname: Mishra
authors.forename: Bhavanath
authors.initials: B
authors.lastname: Jha
mesh_terms: D060054:Agrobacterium / Q000235:genetics*; D010367:Arachis / Q000235:genetics* / Q000254:growth & development; D018548:Cotyledon / Q000235:genetics / Q000254:growth & development; D018556:Crops, Agricultural; D030821:Plants, Genetically Modified; D014170:Transformation, Genetic