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Acta Physiologiae Plantarum

, Volume 35, Issue 1, pp 41–54 | Cite as

Vacuum infiltration enhances the Agrobacterium-mediated genetic transformation in Indian soybean cultivars

  • Thankaraj Salammal Mariashibu
  • Kondeti Subramanyam
  • Muthukrishnan Arun
  • Subramanian Mayavan
  • Manoharan Rajesh
  • Jeevaraj Theboral
  • Markandan Manickavasagam
  • Andy GanapathiEmail author
Original Paper

Abstract

For the first time we have developed a reliable and efficient vacuum infiltration-assisted Agrobacterium-mediated genetic transformation (VIAAT) protocol for Indian soybean cultivars and recovered fertile transgenic soybean plants through somatic embryogenesis. Immature cotyledons were used as an explant and three Agrobacterium tumefaciens strains (EHA 101, EHA 105, and KYRT 1) harbouring the binary vector pCAMBIA1301 were experimented in the co-cultivation. The immature cotyledons were pre-cultured in liquid somatic embryo induction medium prior to vacuum infiltration with the Agrobacterium suspension and co-cultivated for 3 days on co-cultivation medium containing 50 mg l−1 citric acid, 100 µM acetosyringone, and 100 mg l−1 l-cysteine. The transformed somatic embryos were selected in liquid somatic embryo induction medium containing 10 mg l−1 hygromycin and the embryos were germinated in basal medium containing 20 mg l−1 hygromycin. The presence and integration of the hpt II and gus genes into the soybean genome were confirmed by GUS histochemical assay, polymerase chain reaction, and Southern hybridization. Among the different combinations tested, high transformation efficiency (9.45 %) was achieved when immature cotyledons of cv. Pusa 16 were pre-cultured for 18 h and vacuum infiltrated with Agrobacterium tumefaciens KYRT 1 for 2 min at 750 mm of Hg. Among six Indian soybean cultivars tested, Pusa 16 showed highest transformation efficiency of 9.45 %. The transformation efficiency of this method (VIAAT) was higher than previously reported sonication-assisted Agrobacterium-mediated transformation. These results suggest that an efficient Agrobacterium-mediated transformation protocol for stable integration of foreign genes into soybean has been developed.

Keywords

Immature cotyledon KYRT 1 Pre-culture Vacuum infiltration β-Glucuronidase 

Abbreviations

2,4-D

2,4-Dichlorophenoxy acetic acid

CaMV 35S

Cauliflower mosaic virus 35S promoter

FN macro

Finer and Nagasawa macro salts

FNL macro

Finer and Nagasawa lite macro salts

MS salts

Murashige and Skoogs salts

hpt II

Hygromycin phosphotransferase

gus

β-Glucuronidase gene

Notes

Acknowledgments

The authors are thankful to the Department of Biotechnology (DBT) of Ministry of Science and Technology, Government of India, for the financial support (BT/PR9622/AGR/02/464/2007) to carry out the present work.

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Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2012

Authors and Affiliations

  • Thankaraj Salammal Mariashibu
    • 1
    • 2
  • Kondeti Subramanyam
    • 1
  • Muthukrishnan Arun
    • 1
  • Subramanian Mayavan
    • 1
  • Manoharan Rajesh
    • 1
  • Jeevaraj Theboral
    • 1
  • Markandan Manickavasagam
    • 1
  • Andy Ganapathi
    • 1
    Email author
  1. 1.Department of Biotechnology, School of Life SciencesBharathidasan UniversityTiruchirappalliIndia
  2. 2.Temasek Life Sciences Laboratory Limited, 1 Research LinkNational University of SingaporeSingaporeSingapore

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