Successful recovery of transgenic cowpea (Vigna unguiculata) using the 6-phosphomannose isomerase gene as the selectable marker.
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.
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati 781039, India.
Souvika
S
Bakshi
Bedabrata
B
Saha
Nand Kishor
NK
Roy
Sagarika
S
Mishra
Sanjib Kumar
SK
Panda
Lingaraj
L
Sahoo
D001681:Biological Assay; D020090:Chromosome Segregation / Q000187:drug effects / Q000235:genetics; D018548:Cotyledon / Q000187:drug effects / Q000254:growth & development; D003433:Crosses, Genetic; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007887:Fabaceae / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development*; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial / Q000235:genetics*; D005819:Genetic Markers; D008358:Mannose / Q000494:pharmacology; D008359:Mannose-6-Phosphate Isomerase / Q000235:genetics*; D010637:Phenolsulfonphthalein / Q000031:analogs & derivatives / Q000378:metabolism; D018520:Plant Shoots / Q000187:drug effects / Q000254:growth & development / Q000502:physiology; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D012038:Regeneration / Q000187:drug effects; D013395:Sucrose / Q000494:pharmacology; D014170:Transformation, Genetic / Q000187:drug effects
D005819:Genetic Markers; C007016:chlorophenol red; D013395:Sucrose; D008359:Mannose-6-Phosphate Isomerase; D010637:Phenolsulfonphthalein; D008358:Mannose