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Agrobacterium-mediated genetic transformation of Withania somnifera using nodal explants

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Abstract

Withania somnifera is an important medicinal plant and used to cure many diseases. Direct regeneration method was standardized for the nodal explants of W. somnifera. In this method, the maximum of 42.4 ± 2.68 shoots produced per explant was achieved at 1.5 mg l−1 BAP with 0.3 mg l−1 IAA in the second subculture. Transformation was performed in the nodal explants of W. somnifera via direct regeneration using Agrobacterium tumefaciens strain EHA105 that harbored a binary vector pGA492, which carrying kanamycin resistant (nptII), phosphinothricin resistant (bar) and an intron containing β-glucuronidase (gus-intron) genes. The sensitivity of nodal explants to kanamycin (300 mg l−1) was determined for the selection of transformed plants. Transformation was confirmed by histochemical β-glucuronidase (GUS) assay and amplification of the nptII gene by polymerase chain reaction (PCR). PCR and southern blot analyses confirmed the integration of nptII gene in the genome of W. somnifera and the transformation frequency of 3.16 % has been achieved. This is the first report on the genetic transformation of W. somnifera using nodal explants, which may aid in the transformation of any other character gene for improving therapeutic value.

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Abbreviations

AMGT:

Agrobacterium-mediated genetic transformation

BAP:

6-Benzyl amino purine

IAA:

Indole-3-acetic acid

NAA:

Naphthalene acetic acid

IBA:

Indole-3-butyric acid

GA3 :

Gibberellic acid

CaMV:

Cauliflower mosaic virus

GUS :

β-Glucuronidase

nptII:

Neomycin phosphotransferase II

PCR:

Polymerase chain reaction

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Acknowledgments

This work was supported by the facility from Bharathidasan University, (CSIR-Govt. of India) India and Pai Chai University, Korea and by the grant (111074-3) from Bio-industry Technology Development Program of Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea through the IPET.

Conflict of interest

Authors have declared that no competing interests exist.

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Authors and Affiliations

  1. Department of Biochemistry, Government Arts College (Autonomous), Kumbakonam, 612 001, Tamil Nadu, India

    Rajangam Udayakumar

  2. Department of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024, Tamilnadu, India

    Rajangam Udayakumar, Sampath Kasthurirengan, Thankaraj Salammal Mariashibu, Jesudass Joseph Sahaya Rayan & Andy Ganapathi

  3. Department of Biology and Medicinal Science, Pai Chai University, Daejeon, 302-735, Korea

    Rajangam Udayakumar, Sei Chang Kim, Jae Jin Kim & Chang Won Choi

Authors
  1. Rajangam Udayakumar
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  2. Sampath Kasthurirengan
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  3. Thankaraj Salammal Mariashibu
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  4. Jesudass Joseph Sahaya Rayan
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  5. Andy Ganapathi
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  6. Sei Chang Kim
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  7. Jae Jin Kim
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  8. Chang Won Choi
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Corresponding authors

Correspondence to Andy Ganapathi or Chang Won Choi.

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Communicated by J.-H. Liu.

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Udayakumar, R., Kasthurirengan, S., Mariashibu, T.S. et al. Agrobacterium-mediated genetic transformation of Withania somnifera using nodal explants. Acta Physiol Plant 36, 1969–1980 (2014). https://doi.org/10.1007/s11738-014-1572-2

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  • DOI: https://doi.org/10.1007/s11738-014-1572-2

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