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Improved Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merr.] following optimization of culture conditions and mechanical techniques

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Abstract

In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD600) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L−1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L−1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.

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Acknowledgements

The authors are very grateful to Dr. Andy Ganapathi (Vice Chancellor, Bharathiar University, Coimbatore, India) for his valuable guidance in improving soybean transformation.

Funding

This work was supported by National Agriculture Science Fund (NASF) program by the Indian Council of Agricultural Research (ICAR), India.

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  1. Alkesh Hada and Veda Krishnan contributed equally to this research.

Authors and Affiliations

  1. Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, 110012, India

    Alkesh Hada, Veda Krishnan, Monica Jolly, Shelly Praveen & Archana Sachdev

  2. Department of Biotechnology, National College (Autonomous), Tiruchirappalli, Tamil Nadu, 620001, India

    Alkesh Hada & M. S. Mohamed Jaabir

  3. Division of Pathology, Indian Agricultural Research Institute, New Delhi, 110012, India

    Archana Kumari

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Contributions

AS conceived and designed the experiments. AH and VK performed the experiments and compiled and analyzed the data. AK, MJ, and AH generated the pictures. AH, VK, and AS prepared the manuscript. SP and MSMJ helped in manuscript revision. All authors read and approved the final manuscript.

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Correspondence to Archana Sachdev.

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Hada, A., Krishnan, V., Mohamed Jaabir, M.S. et al. Improved Agrobacterium tumefaciens-mediated transformation of soybean [Glycine max (L.) Merr.] following optimization of culture conditions and mechanical techniques. In Vitro Cell.Dev.Biol.-Plant 54, 672–688 (2018). https://doi.org/10.1007/s11627-018-9944-8

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