Cowpea Transformation Experiment
Transgenic Cowpea Leaves Leaf painting assay on transgenic progeny (T2) demonstrate stable expression of the selectable marker gene. (nt: non transgenic; N: null segregant; T: transgenic segregant) Photo c/o Thomas J.V. Higgins.
Experiment designed by: J. C. Popelka, S. Gollasch & Thomas J.V. Higgins.
- Tissue culture phase:
A clean source of seeds, free of slow-growing microbial contamination in tissue culture seems to be critical. The most suitable explant we found is from immature or mature seed consisting of cotyledoary nodes, inducible to form multiple shoots. Several large seeded genotypes are screened for their ability to regenerate multiple shoots in tissue culture and most genotypes proved amenable. The optimal tissue culture medium consists of MS salts and benzylaminopurine.
- Transformation phase:
Several Agrobacterium tumefaciens strains were found to be effective vectors for gene transfer based on transient GUS expression. Both npt II and Bar genes are effective selectable marker genes in combination with the selective agents, geneticin and phosphinothricin, respectively.
Over twenty primary transgenic cowpea plants have been produced so far. These plants were established in the greenhouse and analysed for the bar gene by PCR as well as for phosphinothricin acetyl transferase (PAT) activity by enzyme assay. Seeds of all putative transgenic plants were collected and several seedlings (T1) were analysed for the bar gene by PCR and for PAT activity. Segregation in the T1 generation indicated a Mendelian pattern for most tranformation events. The T2 progeny of several events were confirmed as positive and homozygous plants have been identified after screening of T3 seedlings. The gene transfer system is now suitable for use in the genetic transformation of cowpea with recombinant genes including those for protection against insects. Share your results on cowpea transformation in the discussion forum.