How Relevant is Recalcitrance for the Recovery of Transgenic Cowpea: Implications of Selection Strategies
Identifieur interne : 002365 ( Main/Exploration ); précédent : 002364; suivant : 002366How Relevant is Recalcitrance for the Recovery of Transgenic Cowpea: Implications of Selection Strategies
Auteurs : Souvika Bakshi [Inde] ; Lingaraj Sahoo [Inde]Source :
- Journal of Plant Growth Regulation [ 0721-7595 ] ; 2013-03-01.
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Abstract
Abstract: A highly efficient cowpea stable transformation system was developed with a 44 % increase in efficiency compared to the existing transformation methods by employing an improved kanamycin selection regime, and fertile transgenic plants expressing Btcry1Ac were generated. Agrobacterium-cocultivated 4-day-old cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days. Surviving explants were shifted to kanamycin-free media supplemented with a reduced dosage (2.5 μM) of 6-benzyl aminopurine resulting in profuse proliferation of kanamycin-resistant shoots with a 63.6 % increase in shoot length within 15 days of culture. The elongated shoots readily developed roots on kanamycin-free rooting media and fertile plants were obtained following transfer to soil. β-glucuronidase histochemical assay, polymerase chain reaction, and reverse-transcriptase PCR analyses of T0 and T1 generations confirmed the transgenic nature of the selected plants. Of the four selection regimes tested, the established regime exhibited maximum selection efficiency (41.6 %). Analysis of T1 progeny of all the T0 transformants recovered through established selection regime showed that three-fourths had a segregation ratio of 3:1 and the remaining had a typical segregation ratio of 15:1 for segregation of Btcry1Ac. The expression of Btcry1Ac was demonstrated in T1 transgenic lines through a rapid qualitative immunodiagnostic assay and Western blot analysis. The established transformation method should provide new opportunities for the genetic improvement of cowpea for biotic and abiotic stress tolerance and nutritional quality.
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DOI: 10.1007/s00344-012-9284-6
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Agrobacterium-cocultivated 4-day-old cotyledonary node explants were selected on medium containing 150 mg/l kanamycin for 20 days. Surviving explants were shifted to kanamycin-free media supplemented with a reduced dosage (2.5 μM) of 6-benzyl aminopurine resulting in profuse proliferation of kanamycin-resistant shoots with a 63.6 % increase in shoot length within 15 days of culture. The elongated shoots readily developed roots on kanamycin-free rooting media and fertile plants were obtained following transfer to soil. β-glucuronidase histochemical assay, polymerase chain reaction, and reverse-transcriptase PCR analyses of T0 and T1 generations confirmed the transgenic nature of the selected plants. Of the four selection regimes tested, the established regime exhibited maximum selection efficiency (41.6 %). Analysis of T1 progeny of all the T0 transformants recovered through established selection regime showed that three-fourths had a segregation ratio of 3:1 and the remaining had a typical segregation ratio of 15:1 for segregation of Btcry1Ac. The expression of Btcry1Ac was demonstrated in T1 transgenic lines through a rapid qualitative immunodiagnostic assay and Western blot analysis. 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