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

• Published in: Applied biochemistry and biotechnology 2015-01, Vol.175 (1), p.436-453
• Main Authors: Tiwari, Vivekanand, Chaturvedi, Amit Kumar, Mishra, Avinash, Jha, Bhavanath
• Format: Article
• Language: English
• Subjects: Acetic acid; Agricultural biotechnology; Agrobacterium - genetics; Arachis - genetics; Arachis - growth & development; Arachis hypogaea; Bacteria; Biochemistry; Biotechnology; Chemistry; Chemistry and Materials Science; Cotyledon - genetics; Cotyledon - growth & development; Crops, Agricultural; Cultivars; Cultivation; Edible oils; Industrial crops; Legumes; Plants, Genetically Modified; Selective breeding; Shoots; Transformation, Genetic; Transgenic plants
• ISSN: 0273-2289; 1559-0291
• DOI: 10.1007/s12010-014-1286-3

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.