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

Plant Cell Rep. 2012 Jun;31(6):1093-103. doi: 10.1007/s00299-012-1230-3. Epub 2012 Feb 11.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay
  • Chromosome Segregation / drug effects
  • Chromosome Segregation / genetics
  • Cotyledon / drug effects
  • Cotyledon / growth & development
  • Crosses, Genetic
  • Escherichia coli / enzymology*
  • Escherichia coli / genetics*
  • Fabaceae / drug effects
  • Fabaceae / genetics*
  • Fabaceae / growth & development*
  • Gene Transfer Techniques*
  • Genes, Bacterial / genetics*
  • Genetic Markers
  • Mannose / pharmacology
  • Mannose-6-Phosphate Isomerase / genetics*
  • Phenolsulfonphthalein / analogs & derivatives
  • Phenolsulfonphthalein / metabolism
  • Plant Shoots / drug effects
  • Plant Shoots / growth & development
  • Plant Shoots / physiology
  • Plants, Genetically Modified
  • Plasmids / genetics
  • Regeneration / drug effects
  • Sucrose / pharmacology
  • Transformation, Genetic / drug effects

Substances

  • Genetic Markers
  • chlorophenol red
  • Sucrose
  • Mannose-6-Phosphate Isomerase
  • Phenolsulfonphthalein
  • Mannose