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  • Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.

Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.

Plant cell reports (2012-02-14)
Souvika Bakshi, Bedabrata Saha, Nand Kishor Roy, Sagarika Mishra, Sanjib Kumar Panda, Lingaraj Sahoo
ABSTRACT

A new method for obtaining transgenic cowpea was developed using positive selection based on the Escherichia coli 6-phosphomannose isomerase gene as the selectable marker and mannose as the selective agent. Only transformed cells were capable of utilizing mannose as a carbon source. Cotyledonary node explants from 4-day-old in vitro-germinated seedlings of cultivar Pusa Komal were inoculated with Agrobacterium tumefaciens strain EHA105 carrying the vector pNOV2819. Regenerating transformed shoots were selected on medium supplemented with a combination of 20 g/l mannose and 5 g/l sucrose as carbon source. The transformed shoots were rooted on medium devoid of mannose. Transformation efficiency based on PCR analysis of individual putative transformed shoots was 3.6%. Southern blot analysis on five randomly chosen PCR-positive plants confirmed the integration of the pmi transgene. Qualitative reverse transcription (qRT-PCR) analysis demonstrated the expression of pmi in T₀ transgenic plants. Chlorophenol red (CPR) assays confirmed the activity of PMI in transgenic plants, and the gene was transmitted to progeny in a Mendelian fashion. The transformation method presented here for cowpea using mannose selection is efficient and reproducible, and could be used to introduce a desirable gene(s) into cowpea for biotic and abiotic stress tolerance.

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Sigma-Aldrich
Phenol Red, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Phenol red solution, 0.5%, liquid, sterile-filtered, BioReagent, suitable for cell culture
Sigma-Aldrich
Phosphomannose Isomerase from Escherichia coli, recombinant, expressed in E. coli, ammonium sulfate suspension, ≥50 units/mg protein
Sigma-Aldrich
Phenol Red, ACS reagent