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Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in Indian Soybean Cultivars

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Abstract

Soybean is a recalcitrant crop to Agrobacterium-mediated genetic transformation. Development of highly efficient, reproducible, and genotype-independent transformation protocol is highly desirable for soybean genetic improvement. Hence, an improved Agrobacterium-mediated genetic transformation protocol has been developed for cultivar PK 416 by evaluating various parameters including Agrobacterium tumefaciens strains (LBA4404, EHA101, and EHA105 harboring pCAMBIA1304 plasmid), sonication duration, vacuum infiltration pressure, and vacuum duration using cotyledonary node explants of soybean prepared from 7-day-old seedlings. The transformed plants were successfully developed through direct organogenesis system. Transgene expression was assessed by GUS histochemical and gfp visual assays, and integration was analyzed by PCR and Southern blot hybridization. Among the different combinations and durations evaluated, a maximum transformation efficiency of 18.6 % was achieved when the cotyledonary node explants of cv. PK 416 were sonicated for 20 s and vacuum infiltered for 2 min at 250 mmHg in A. tumefaciens EHA105 suspension. The amenability of the standardized protocol was tested on four more soybean cultivars JS 90-41, Hara Soy, Co 1, and Co 2 in which all the cultivars responded favorably with transformation efficiency ranging from 13.3 to 16.6 %. The transformation protocol developed in the present study would be useful to transform diverse soybean cultivars with desirable traits.

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Abbreviations

MS salts:

Murashige and Skoogs salts

B5 vitamins:

Gamborg vitamins

BA:

N 6-Benzylaminopurine

GA3 :

Gibberellic acid

MES:

2-(N-Morpholino) ethanesulfonic acid

IBA:

Indole-3-butyric acid

CaMV 35S:

Cauliflower mosaic virus 35S promoter

hpt II:

Hygromycin phosphotransferase II gene

npt II:

Neomycin phosphotransferase II gene

gfp-gus :

Green fluorescent protein-β-glucuronidase fusion gene

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Acknowledgments

The authors are thankful to the Department of Biotechnology (DBT) of Ministry of Science and Technology, New Delhi, Government of India for the financial support (BT/PR9622/AGR/02/464/2007). The corresponding author is thankful to University Grants Commission (UGC), Govt. of. India for providing fellowship under UGC–BSR scheme. Subramanyam and Sivanandhan are thankful to Council of Scientific and Industrial Research (CSIR), Govt. of India for the award of Senior Research Fellowship (SRF) to carry out their doctoral work.

Conflict of interest

The authors declare that they have no competing interests.

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Authors and Affiliations

  1. Department of Biotechnology & Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620024,, Tamil Nadu, India

    Muthukrishnan Arun, Kondeti Subramanyam, Thankaraj Salammal Mariashibu, Jeevaraj Theboral, Ganeshan Shivanandhan, Markandan Manickavasagam & Andy Ganapathi

  2. Department of Horticultural Sciences, Kyungpook National University, Daegu, 702-701, South Korea

    Muthukrishnan Arun

  3. Department of Forest Resources, College of Forest and Environmental Sciences, Kangwon National University, Chunchon, 200-701, Republic of Korea

    Kondeti Subramanyam

  4. Temasek Life Sciences Laboratory Limited, 1 Research Link, National University of Singapore, Singapore, 117604, Singapore

    Thankaraj Salammal Mariashibu

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  1. Muthukrishnan Arun
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  2. Kondeti Subramanyam
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  3. Thankaraj Salammal Mariashibu
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Correspondence to Andy Ganapathi.

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Arun, M., Subramanyam, K., Mariashibu, T.S. et al. Application of Sonication in Combination with Vacuum Infiltration Enhances the Agrobacterium-Mediated Genetic Transformation in Indian Soybean Cultivars. Appl Biochem Biotechnol 175, 2266–2287 (2015). https://doi.org/10.1007/s12010-014-1360-x

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