An Efficient Method of Agrobacterium-Mediated Genetic Transformation and Regeneration in Local Indian Cultivar of Groundnut (Arachis hypogaea) Using Grafting

作者：Tiwari Vivekanand; Chaturvedi Amit Kumar; Mishra Avinash^*; Jha Bhavanath

来源：Applied Biochemistry and Biotechnology, 2015, 175(1): 436-453.

DOI：10.1007/s12010-014-1286-3

• Agrobacterium
• Grafting
• Groundnut
• Regeneration
• Transformation
• Tissue culture

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 mu M 6-benzylaminopurine (BAP), while the highest number of shoot buds per explant, 17.67 +/- 3.51, was found with 20 mu M BAP and 10 mu M 2,4-dichlorophenoxyacetic acid (2,4-D). The bacterial culture OD, acetosyringone and l-cysteine concentration were optimized as 1.8, 200 mu M 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 beta-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.

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