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Muruganantham, M., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Institute for Plant Protection, Department of Plant Pathology and Weed Science, ARO Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
Amutha, S., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India
Selvaraj, N., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Department of Botany, E.V.R.College, Tiruchirappalli, Tamil Nadu, India
Vengadesan, G., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47906-2076, United States
Ganapathi, A., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India
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%). © 2007 The Society for In Vitro Biology.
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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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Muruganantham, M., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Institute for Plant Protection, Department of Plant Pathology and Weed Science, ARO Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel
Amutha, S., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India
Selvaraj, N., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Department of Botany, E.V.R.College, Tiruchirappalli, Tamil Nadu, India
Vengadesan, G., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India, Department of Forestry and Natural Resources, Purdue University, West Lafayette, IN 47906-2076, United States
Ganapathi, A., Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, Tamil Nadu 620 024, India
Efficient Agrobacterium-mediated transformation of Vigna mungo using immature cotyledonary-node explants and phosphinothricin as the selection agent
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%). © 2007 The Society for In Vitro Biology.
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