Cowpeas are nutritious grains that provide the main source of protein, highly digestible energy and vitamins to some of the world's poorest people. The demand for cowpeas is high but yields remain critically low, largely because of insect pests. Cowpea germplasm contains little or no resistance to major insect pests and a gene technology approach to adding insect protection traits is now a high priority. We have adapted features of several legume and other transformation systems and reproducibly obtained transgenic cowpeas that obey Mendelian rules in transmitting the transgene to their progeny. Critical parameters in this transformation system include the choice of cotyledonary nodes from developing or mature seeds as explants and a tissue culture medium devoid of auxins in the early stages, but including the cytokinin BAP at low levels during shoot initiation and elongation. Addition of thiol-compounds during infection and co-culture with Agrobacterium and the choice of the bar gene for selection with phosphinothricin were also important. Transgenic cowpeas that transmit the transgenes to their progeny can be recovered at a rate of one fertile plant per thousand explants. These results pave the way for the introduction of new traits into cowpea and the first genes to be trialled will include those with potential to protect against insect pests.

Bruchid larvae cause major losses of grain legume crops throughout the world. Some bruchid species, such as the cowpea weevil and the azuki bean weevil, are pests that damage stored seeds. Others, such as the pea weevil (Bruchus pisorum), attack the crop growing i n the field. We transferred the cDNA encoding the a-amylase inhibitor (a-AI) found in the seeds of the common bean (Phaseolus vulgaris) into pea (Pisum sativum) using Agrobacferium-mediated transformation. Expression was driven by the promoter of phytohemagglutinin, another bean seed protein. The a-amylase inhibitor gene was stably expressed in the transgenic pea seeds at least to the T, seed generation, and a-AI accumulated i n the seeds up to 3% of soluble protein. This level is somewhat higher than that normally found in beans, which contain 1 to 2 % a-AI. In the 1 , seed generation the development of pea weevil larvae was blocked at an early stage. Seed damage was minimal and seed yield was not significantly reduced in the transgenic plants. These results confirm the feasibility of protecting other grain legumes such as lentils, mungbean, groundnuts, and chickpeas against a variety of bruchids using the same approach. Although a-AI also inhibits human a-amylase, cooked peas should not have a negative impact on human energy metabolism.The common bean (Phaseolus vulgaris L.) contains a family of structurally related seed proteins: PHA-E and -L, arcelin, and a-AI. PHA-E and PHA-L are strong agglutinins, i.e. classical lectins that bind carbohydrate, and arcelin, which is found only in certain wild accessions of the common bean, may be a weak agglutinin (Hartweck et al., 1991). The bean a-AI has 65 to 70% amino acid sequence identity with the other three but lacks at least one of the conserved residues needed for lectin activity. Its biochemical mode of action is to form a one-to-one complex with certain amylases (for reviews, see Chrispeels and Raikhel, 1991;Rouge et al., 1993).

A vip3Ba gene reconstructed for plant expression was used in transforming cowpea via Agrobacterium tumefaciens. Transgenic lines expressing Vip3Ba protein were used in insect feeding trials to assess protection against Maruca and were found to be completely protected from this pest. We propose that the vip-cowpea lines could be combined with existing cry-transgenic cowpea to introgress this additional resistance trait and thus avoid or greatly delay the development of resistance in Maruca.

An improved cowpea transformation method utilizing Agrobacterium -mediated gene delivery to explants derived from the cotyledonary nodes of imbibed cowpea seed is described. The explants were regenerated following a sonication procedure and a stringent selection comprising alternating regimes of kanamycin and geneticin. The method was reproducible and led to the recovery of independent fertile transgenic plants in the greenhouse at a level of about one per cent of starting explants. A transgene encoding an insecticidal protein from Bacillus thuringiensis was used to demonstrate the efficacy of the system.

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