Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335

Alkesh Hada; Veda Krishnan; Mansi Punjabi; Nabaneeta Basak; Vanita Pandey; Theboral Jeevaraj; Ashish Marathe; Amit K Gupta; Monica Jolly; Arun Kumar; Anil Dahuja; Markandan Manickavasagam; Andy Ganapathi; Archana Sachdev

doi:10.5511/plantbiotechnology.15.0901a

Original Papers

Refined glufosinate selection and its extent of exposure for improving the Agrobacterium-mediated transformation in Indian soybean (Glycine max) genotype JS-335

Alkesh Hada, Veda Krishnan, Mansi Punjabi, Nabaneeta Basak, Vanita Pandey, Theboral Jeevaraj, Ashish Marathe, Amit K Gupta, Monica Jolly, Arun Kumar, Anil Dahuja, Markandan Manickavasagam, Andy Ganapathi, Archana Sachdev

Author information

Alkesh Hada
Division of Biochemistry, Indian Agricultural Research Institute
Department of Biotechnology and Genetic Engineering, Bharathidasan University
Veda Krishnan
Division of Biochemistry, Indian Agricultural Research Institute
Department of Biotechnology and Genetic Engineering, Bharathidasan University
Mansi Punjabi
Division of Biochemistry, Indian Agricultural Research Institute
Nabaneeta Basak
Division of Biochemistry, Indian Agricultural Research Institute
Vanita Pandey
Division of Biochemistry, Indian Agricultural Research Institute
Theboral Jeevaraj
Department of Biotechnology and Genetic Engineering, Bharathidasan University
Ashish Marathe
Division of Biochemistry, Indian Agricultural Research Institute
Amit K Gupta
Division of Biochemistry, Indian Agricultural Research Institute
Monica Jolly
Division of Biochemistry, Indian Agricultural Research Institute
Arun Kumar
National Phytotron Facility, Indian Agricultural Research Institute
Anil Dahuja
Division of Biochemistry, Indian Agricultural Research Institute
Markandan Manickavasagam
Department of Biotechnology and Genetic Engineering, Bharathidasan University
Andy Ganapathi
Department of Biotechnology and Genetic Engineering, Bharathidasan University
Archana Sachdev
Division of Biochemistry, Indian Agricultural Research Institute

Keywords: Agrobacterium tumefaciens, ammonium glufosinate, cotyledonary-node, soybean, transformation

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Supplementary material

2016 Volume 33 Issue 5 Pages 341-350

DOI https://doi.org/10.5511/plantbiotechnology.15.0901a

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Abstract

Soybean like many other crops, in this genomic era, has well-established genomic database which provides a wide range of opportunities for improvement through genetic manipulation. But the growing demand for soybean transgenics with increased production and improved quality has been handicapped due to inefficient transformation strategies and hence an efficient, stable and reliable transformation system is of prime requisite. In the present study, Agrobacterium-mediated transformation was standardized by refining the glufosinate selection system in terms of dosage (0–6 mg l⁻¹) and degree of exposure. The cotyledonary node explants (with and without wounding) initially cultured on a non-selective shoot induction medium for 10 days before transferring them to the selective SIM with an optimized concentration of 5.0 mg l⁻¹ ammonium glufosinate, showed least selection escape frequency. Wounded cotyledonary node explants infected with Agrobacterium tumefaciens harboring pBIN-bar construct, showed an improved regeneration efficiency of 55.10% and transformation efficiency of 12.6% using Southern blotting in T₁ plants. Southern analysis of T₁ plants confirmed the integration of bar gene into the genomic DNA and the bar positive T₁ plants segregated in 3 : 1 ratio. This is the first report, to our knowledge, of a high transformation efficiency using Agrobacterium-mediated cot node-glufosinate system in an Indian soybean genotype.

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