Cowpea is attacked by a range of damaging insect pests, both in the field and during grain storage. Conventional host-plant resistance has proved to be limited. Even after extensive screening of cowpea germplasm for resistance to pests such as the cowpea pod borer (Maruca vitrata) only weak sources of resistance were found, if any. Genetic transformation could be a helpful tool to develop insect-resistant germplasm, but no robust transformation system has been available for this staple crop. We have developed a transformation protocol for cowpea that we believe will facilitate the production of insect-resistant transgenic cowpeas. The following steps briefly describe our protocol:

In the tissue culture phase: A clean source of seeds that was free of microbial contamination of the slow-growing type in tissue culture was a critical parameter. The most suitable explant tissue was from immature or mature seed and consisted of cotyledoary nodes that could be induced to form multiple shoots. Several large-seeded genotypes were screened for their ability to regenerate multiple shoots in tissue culture and most genotypes proved amenable. The optimal tissue culture medium consisted of Murashigi and Skoog salts and benzyl-aminopurine.

In the transformation phase: Several Agrobacterium tumefaciens strains were shown to be effective vectors for gene transfer based on transient glucuronidase expression. Both Npt II and Bar genes were effective selectable markers in combination with the selective agents geneticin and phosphinothricin, respectively.

Over 20 primary transgenic cowpea plants have been produced so far. These plants were established in the greenhouse and analysed for the bar gene by polymerase chain reaction (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 of one (event T33) or several independent loci (event T42). The T2 progeny of event T33 were confirmed as positive and a homozygous line has been selected for seed multiplication.

The gene transfer system is now suitable for use in the genetic transformation of cowpea with recombinant genes including those for protection against insects.