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Efficient Agrobacterium-mediated transformation of Vigna mungo using immature cotyledonary-node explants and phosphinothricin as the selection agent

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Abstract

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T0) and in the seedlings of the T1 generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).

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Acknowledgments

The authors thank the Department of Biotechnology (DBT), Government of India for the financial support (Grant no. BT/AGR/PR1446/07/68/99). The senior author is grateful to Prof. D. Girija and Mrs. Beena, Center for Plant Biotechnology and Molecular Biology, Kerala Agricultural University, Trissur, Kerala, for providing assistance and the opportunity to perform the radioactive labeling and Southern hybridization. The authors are grateful to Dr. Victor Gaba, Department of Plant Pathology, ARO Volcani Center, Bet Dagan, Israel for the critical comments on the manuscript.

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Author notes

  1. M. Muruganantham

    Present address: Institute for Plant Protection, Department of Plant Pathology and Weed Science, ARO Volcani Center, P.O. Box 6, Bet Dagan, 50250, Israel

Authors and Affiliations

  1. Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620 024, India

    M. Muruganantham, S. Amutha, N. Selvaraj, G. Vengadesan & A. Ganapathi

  2. Department of Botany, E.V.R.College, Tiruchirappalli, Tamil Nadu, India

    N. Selvaraj

  3. Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN, 47906-2076, USA

    G. Vengadesan

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  1. M. Muruganantham
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  2. S. Amutha
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  3. N. Selvaraj
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Correspondence to M. Muruganantham.

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Editor: T.J. Jones

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Muruganantham, M., Amutha, S., Selvaraj, N. et al. Efficient Agrobacterium-mediated transformation of Vigna mungo using immature cotyledonary-node explants and phosphinothricin as the selection agent. In Vitro Cell.Dev.Biol.-Plant 43, 550–557 (2007). https://doi.org/10.1007/s11627-007-9060-7

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