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Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile

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Abstract

Agrobacterium tumefaciens (EHA-105 harboring pCAMBIA 1304)-mediated transgenic plant production via direct regeneration from leaf and elite somaclones generation through indirect regeneration in Stevia rebaudiana is reported. Optimum direct regeneration frequency along with highest transformation frequency was found on MS + 1 mg/l BAP + 1 mg/l NAA, while indirect regeneration from callus was obtained on MS + 1 mg/l BAP + 2 mg/l NAA. Successful transfer of GUS-positive (GUS assay and PCR-based confirmation) transgenic as well as four somaclones up to glasshouse acclimatization has been achieved. Inter-simple sequence repeat (ISSR) profiling of transgenic and somaclonal plants showed a total of 113 bands, out of which 49 were monomorphic (43.36 %) and 64 were polymorphic (56.64 %). Transgenic plant was found to be closer to mother plant, while on the basis of steviol, stevioside, and rebaudioside A profile, somaclone S2 was found to be the best and showed maximum variability in ISSR profiling.

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Acknowledgments

We gratefully acknowledge the financial support received from the Council of Scientific & Industrial Research as CSIR-SRF, Government of India, and New Delhi to carry out this work and Director CIMAP for providing all the necessary facilities. We are highly thankful to Dr. Shakti Mehrotra for providing ISSR markers. Help rendered by Mr. Anupam Maurya during HPLC analysis is highly acknowledged.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Plant Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow, 226015, India

    Shamshad Ahmad Khan & Laiq Ur Rahman

  2. Analytical Chemistry Division, Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow, 226015, India

    Karuna Shanker & Manju Singh

Authors
  1. Shamshad Ahmad Khan
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  2. Laiq Ur Rahman
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  3. Karuna Shanker
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  4. Manju Singh
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Correspondence to Shamshad Ahmad Khan.

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Khan, S.A., Ur Rahman, L., Shanker, K. et al. Agrobacterium tumefaciens-mediated transgenic plant and somaclone production through direct and indirect regeneration from leaves in Stevia rebaudiana with their glycoside profile. Protoplasma 251, 661–670 (2014). https://doi.org/10.1007/s00709-013-0568-x

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  • DOI: https://doi.org/10.1007/s00709-013-0568-x

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