In Vitro Cellular & Developmental Biology - Plant

, Volume 54, Issue 6, pp 672–688 | Cite as

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

  • Alkesh Hada
  • Veda Krishnan
  • M. S. Mohamed Jaabir
  • Archana Kumari
  • Monica Jolly
  • Shelly Praveen
  • Archana SachdevEmail author
Plant Tissue Culture


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.


Agrobacterium tumefaciens Half-seed explants Soybean transformation Regeneration 



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

Author 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.


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

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

11627_2018_9944_MOESM1_ESM.docx (233 kb)
ESM 1 (DOCX 232 kb)


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Copyright information

© The Society for In Vitro Biology 2018

Authors and Affiliations

  • Alkesh Hada
    • 1
    • 2
  • Veda Krishnan
    • 1
  • M. S. Mohamed Jaabir
    • 2
  • Archana Kumari
    • 3
  • Monica Jolly
    • 1
  • Shelly Praveen
    • 1
  • Archana Sachdev
    • 1
    Email author
  1. 1.Division of BiochemistryIndian Agricultural Research InstituteNew DelhiIndia
  2. 2.Department of BiotechnologyNational College (Autonomous)TiruchirappalliIndia
  3. 3.Division of PathologyIndian Agricultural Research InstituteNew DelhiIndia

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