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Title: A rapid method for optimization of Agrobacterium- mediated transformation of Indian soybean genotypes
Authors: Kumari, Sweta
Krishnan, Veda
Dahuja, Anil
Vinutha, T
Jolly, Monica
Sachdev, Archana
Keywords: Agrobacterium tumefaciens;Cotyledonary node;Glufosinate;Glycine max;β-Glucuronidase assay;Hypocotyl segment;Transformation
Issue Date: Oct-2016
Publisher: NISCAIR-CSIR, India
Abstract: An Agrobacterium-mediated transformation method optimized for two high yielding/popular Indian soybean genotypes JS335 and DS 9712, using the cotyledonary node (CN) and hypocotyl segment systems. The explants were co-cultivated with Agrobacterium tumefaciens harboring the binary vector with bar (phosphinothricin acetyltransferase gene, a selectable marker gene) and the gus reporter gene. The results revealed a higher regeneration frequency using CN as explants in the both cultivars JS335 (75%) and DS 9712 (50%), as compared to regeneration frequency of 44% (JS335) and 27% (DS 9712) using hypocotyl segments as explants. An addition of dithiothreitol (DTT) and sodium thiosulfatealong with L- cysteine during co-cultivation of JS-335 showed an increase (33%) in the transformation efficiency. Shoot induction on the non-selective shoot induction media (SIM) for 10 days before transferring the explants to SIM containing 5 mg/L of glufosinate greatly stimulated the regeneration of the transformed shoots. The transformed shoots elongated on selective shoot elongation media (SEM) (2×14 days) were successfully rooted and transplanted to pot mix and grown to maturity under greenhouse conditions. Stable integration confirmed by PCR and Southern analysis of the T1 progeny revealed a transformation efficiency of 3.1% and 2.4% in JS 335 and DS 9712, respectively. The protocol resulted in stable transformants of soybean, with T1 seeds developing to maturity within six months after the initiation of this protocol. The study indicated a better efficiency of the CN node transformation system over the hypocotyl segment system in cultivar JS335 and may be beneficial for versatile gene function analysis in popular Indian genotypes.
Page(s): 218-226
ISSN: 0975-0959 (Online); 0301-1208 (Print)
Appears in Collections:IJBB Vol.53(5&6) [October-December 2016]

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