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Agrobacterium tumefaciens-mediated genetic transformation of sesame (Sesamum indicum L.)

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Abstract

Sesame (Sesamum indicum) is an important oil seed crop that has not yet been transformed genetically. We report herein for the first time successful recovery of fertile transgenic plants of sesame from cotyledon explants inoculated with Agrobacterium tumefaciens carrying a binary vector pCAMBIA2301 that contains a neomycin phosphotransferase gene (nptII) and a β-glucuronidase (GUS) gene (uidA) interrupted with an intron. Green shoots recovered from A. tumefaciens-infected explants on selection medium [Murashige and Skoog (MS) basal medium containing 25.0 μM benzyladenine (BA), 25.0 mg l−1 kanamycin and 400.0 mg l−1 cefotaxime] were rooted on MS basal medium containing 2.0 μM indole-3-butyric acid and 5.0 mg l−1 kanamycin. The rooted shoots were established in soil and grown to maturity to collect seeds. The presence, integration and expression of transgenes in putative T0 plants were confirmed by polymerase chain reaction (PCR), Southern blot hybridization and GUS histochemical assay, respectively. GUS activity was detected in vegetative and reproductive parts of T0 and T1 plants. Presence of thiol compounds in coculture medium and kanamycin selection at shoot regeneration and at rooting stages were found to be critical for transformation. The transgenes were inherited in Mendelian fashion in T1 progeny as detected by PCR. RT-PCR analysis of T1 plants confirmed the presence of transcripts of uidA gene. The transformation frequency was 1.01%, and 22–24 weeks were required from seed to seed generation time. This protocol can be used to transfer new traits in sesame for quantitative and qualitative improvement.

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Abbreviations

BA:

Benzyladenine

TDZ:

Thidiazuron

NPTII:

Neomycin phosphotransferase

GUS:

β-Glucuronidase

PCR:

Polymerase chain reaction

RT-PCR:

Reverse-transcriptase polymerase chain reaction

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Acknowledgments

We thank the Center for Application of Molecular Biology to International Agriculture (CAMBIA) for plasmid CAMBIA2301 and Dr. P. A. Kumar, NRC on Plant Biotechnology, IARI, New Delhi for providing laboratory facilities for the Southern blot hybridization. M.Y., D.C. and M.S. are grateful to MDU, Rohtak, CSIR and DBT, New Delhi, respectively, for the award of Research Fellowships.

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  1. Centre for Biotechnology, M.D. University, Rohtak, 124001, India

    Manju Yadav, Darshna Chaudhary, Manish Sainger & Pawan K. Jaiwal

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  1. Manju Yadav
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  2. Darshna Chaudhary
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  3. Manish Sainger
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  4. Pawan K. Jaiwal
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Correspondence to Pawan K. Jaiwal.

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Yadav, M., Chaudhary, D., Sainger, M. et al. Agrobacterium tumefaciens-mediated genetic transformation of sesame (Sesamum indicum L.). Plant Cell Tiss Organ Cult 103, 377–386 (2010). https://doi.org/10.1007/s11240-010-9791-8

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  • DOI: https://doi.org/10.1007/s11240-010-9791-8

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