Genetic Transformation of Cowpea

Contributers: J. C. Popelka, S. Gollasch & Thomas J.V. Higgins.

Cowpea infected by pod borer Maruca vitrata. Photo c/o Thomas J.V. Higgins.

The aim of this open source project is to develop a method of transforming cowpea with TransBacter. We hope that the genetic transformation of cowpea will lead to insect resistant crops.

Cowpea is attacked by a range of insect pests, in the field and during grain storage. Conventional host plant resistance has been limited. Even after extensive screening of cowpea germplasm to pests such as the cowpea pod borer (Maruca vitrata) and the cowpea weevil (Callosobruchus maculatus) 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 is available for this staple crop.

The recently developed Agrobacterium transformation protocol for cowpea may facilitate the production of insect resistant transgenic cowpeas. TransBacter could now be used to transform cowpea.

What to do in this project?

• Read about Agrobacterium transformation of cowpea.
• Let us know your thoughts in the cowpea discussion forum.

Your ideas will help form this project!

Cowpea Transformation Experiment

Experiment designed by: J. C. Popelka, S. Gollasch & Thomas J.V. Higgins.

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.

Methods

1. 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.
2. 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.

Expression of the reporter gene GUS in cowpea callus (left) and leaves (right) of regenerated plants. Photo c/o Thomas J.V. Higgins.

Current Results

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.

The information contained in this page was believed to be correct at the time it was collated. New patents and patent applications, altered status of patents, and case law may have resulted in changes in the landscape. CAMBIA makes no warranty that it is correct or up to date at this time and accepts no liability for any use that might be made of it. Corrections or updates to the information are welcome, please send an email to info@bios.net.