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Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant

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Abstract

This report describes Agrobacterium tumefaciens-mediated transformation of Withania somnifera—an important Indian medicinal plant. A. tumefaciens strain LBA4404, containing the binary vector pIG121Hm was used for transformation, along with the gusA reporter gene with intron under the transcriptional control of the Cauliflower Mosaic Virus (CaMV) 35S promoter. The leaf segments from two-and-a-half-month-old green house-grown seedlings were more efficient in transformation, as compared to those from the in vitro-grown shoots. Second expanded leaf from the shoot tip gave the highest transient transformation efficiency. Selection of transgenic shoots was done in the presence of 50 mg l−1 kanamycin. Polymerase chain reaction analysis of T0 transgenic plants showed the presence of gusA and nptII genes. The expression of these transgenes in T1 progeny was confirmed by RT-PCR. The integration of gusA gene was confirmed by Southern blot analysis. The transformation efficiency was found to be 1.67%.

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Abbreviations

AS:

Acetosyringone

BA:

Benzyladenine

CCM:

Cocultivation medium

GH-grown:

Green house-grown

gusA :

β-Glucuronidase

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

MIC:

Minimal inhibitory concentration

MS medium:

Murashige and Skoog (1962) medium

NAA:

α-Naphthaleneacetic acid

nptII :

Neomycin phosphotransferase

SIM:

Shoot induction medium

TTE:

Transient transformation efficiency

X-Gluc:

5-Bromo-4-chloro-3-indolyl-β-d-glucuronide

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Acknowledgements

The authors are thankful to the Council of Scientific and Industrial Research, New Delhi, Govt. of India, for providing the financial support to carry out this work under NMITLI scheme and Network Project (NWP-08). RT is thankful to the DST for J.C.Bose Fellowship.

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Authors and Affiliations

  1. National Botanical Research Institute, Council of Scientific and Industrial Research, Rana Pratap Marg, Lucknow, 226001, UP, India

    Vibha Pandey, Pratibha Misra, Pankaj Chaturvedi, Manoj K. Mishra, Prabodh K. Trivedi & Rakesh Tuli

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  1. Vibha Pandey
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  2. Pratibha Misra
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  3. Pankaj Chaturvedi
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  4. Manoj K. Mishra
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Correspondence to Rakesh Tuli.

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Communicated by P. Kumar.

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Pandey, V., Misra, P., Chaturvedi, P. et al. Agrobacterium tumefaciens-mediated transformation of Withania somnifera (L.) Dunal: an important medicinal plant. Plant Cell Rep 29, 133–141 (2010). https://doi.org/10.1007/s00299-009-0805-0

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