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A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant

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Abstract

In spite of the economic importance of sesame (Sesamum indicum L.) and the recent availability of its genome sequence, a high-frequency transformation protocol is still not available. The only two existing Agrobacterium-mediated transformation protocols that are available have poor transformation efficiencies of less than 2 %. In the present study, we report a high-frequency, simple, and reproducible transformation protocol for sesame. Transformation was done using de-embryonated cotyledons via somatic embryogenic stages. All the critical parameters of transformation, like incubation period of explants in pre-regeneration medium prior to infection by Agrobacterium tumefaciens, cocultivation period, concentrations of acetosyringone in cocultivation medium, kanamycin concentration, and concentration of plant hormones, including 6-benzylaminopurine, have been optimized. This protocol is superior to the two existing protocols in its high regeneration and transformation efficiencies. The transformed sesame lines have been tested by PCR, RT-PCR for neomycin phosphotransferase II gene expression, and β-glucuronidase (GUS) assay. The regeneration frequency and transformation efficiency are 57.33 and 42.66 %, respectively. T0 and T1 generation transgenic plants were analyzed, and several T1 plants homozygous for the transgenes were obtained.

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Abbreviations

BAP:

6-Benzylaminopurine

IAA:

Indole-3-acetic acid

ABA:

Abscisic acid

Kan:

Kanamycin

GUS:

β-Glucuronidase

MS:

Murashige and Skoog

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Acknowledgments

This work was partially supported by the Department of Biotechnology, Ministry of Science and Technology, Government of India (DBT). S. Chowdhury is thankful to the University Grants Commission (UGC), New Delhi and A. Basu is thankful to the Council for Scientific and Industrial research (CSIR), New Delhi for providing junior research fellowship. We are grateful to Prof. Tapas Dasgupta, Institute of Agricultural Science, University of Calcutta, for providing us the S. indicum L. cultivar VRI-1 seeds. We are also grateful to Prof. Sampa Das, Bose Institute, Kolkata for providing the A. tumefaciens strain LBA4404 and the vector pCAMBIA 2301.

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The authors declare they have no conflict of interest.

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Authors and Affiliations

  1. Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, 35, Ballygunge Circular Road, Kolkata, 700019, India

    Supriyo Chowdhury, Arpita Basu & Surekha Kundu

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  1. Supriyo Chowdhury
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  2. Arpita Basu
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  3. Surekha Kundu
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Correspondence to Surekha Kundu.

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Chowdhury, S., Basu, A. & Kundu, S. A new high-frequency Agrobacterium-mediated transformation technique for Sesamum indicum L. using de-embryonated cotyledon as explant. Protoplasma 251, 1175–1190 (2014). https://doi.org/10.1007/s00709-014-0625-0

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