Potential role of transgenic approaches in the control of cowpea insect pests

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1 Potential role of transgenic approaches in the control of cowpea insect pests 3.4 Potential role of transgenic approaches in the control of cowpea insect pests J. Machuka 1 Abstract Crops incompatibility makes conventional breeding approaches untenable in transferring available insect resistance from wild Vigna sp. into cowpea. The alternative recourse is to isolate and transfer alien resistance genes using genetic transformation. This has the added advantage of using useful genes from distantly related organisms to control cowpea pests. Artificial diet bioassays carried out on the Maruca pod borer, pod sucking bugs, and cowpea weevils indicate that these insects can be controlled by Bacillus thuringiensis crystal proteins, plant lectins, protease, α-amylase inhibitors, chitinases, and/or ribosome-inactivating proteins. The challenge now is to express the genes encoding these proteins in transgenic cowpea and hope that what happens in artificial diets will, at least in some cases, be replicated in transgenics. Other candidate genes include enzymes encoding biochemical pathways in secondary metabolism. It can be anticipated that useful information emerging from current global genomics efforts in crop species, including model legumes, will have a bearing on cowpea improvement through genetic engineering. What cowpea researchers need to do now is develop a comprehensive pest resistance management strategy. Such a strategy must take into account criteria such as transformation of elite cowpea lines that are adapted to each of the major agroecological zones, gene flow between cultivated and wild cowpea, and strategies for dissemination and adoption of biotechnologically improved cowpea lines. This paper reviews previous work on candidate genes, presents some recent results, and makes projections on how research on cowpea breeding through genetic modification for insect resistance may move from the laboratory into farmers fields, especially in sub-saharan Africa. Introduction Grain yield losses in cowpea (Vigna unguiculata) are mainly due to biotic stresses, especially insect pests, including aphids, thrips, Maruca pod-borer (MPB [Maruca vitrata]), bruchids, and pod-sucking bugs (PSB). Although modest levels of host plant resistance are available in cowpea germplasm, there is nearly none to MPB. Insect resistance genes are present in wild cowpea relatives (Vigna spp.) as well as other non-vigna legumes that are infested by MBP such as African yam bean (AYB [Sphenostylis stenocarpa]). However, breeding barriers make conventional breeding approaches untenable in transferring resistance from wild Vigna and other legumes into cowpea. The alternative recourse is to isolate and transfer alien resistance genes using genetic transformation. This has the added advantage of using useful genes from distantly related organisms to control cowpea pests. Artificial diet bioassays carried out on MPB, PSB, and cowpea weevils indicate that these insects can be controlled by Bacillus thuringiensis (Bt) crystal proteins, plant 1. PO Box 347, Kilifi, Kenya. 213

2 Biotechnology for cowpea lectins, protease and α-amylase inhibitors, chitinases and/or ribosome-inactivating proteins. The challenge now is to express the genes encoding these proteins in transgenic cowpeas and hope that what happens in artificial diets will, at least in some cases, be replicated in transgenics. Other candidate genes include enzymes encoding biochemical pathways in secondary metabolism. This paper reviews research related to identification of candidate insect resistance genes and makes projections on how cowpea genetic modification breeding for insect resistance may move from the laboratory into farmers fields, especially in sub-saharan Africa. Methods for isolation of insect resistance factors The first step in generating insect resistant transgenic crops is to identify insecticidal proteins or compounds that are active against the target pest. The most common way of doing this involves the use of artificial diets or seeds that contain proteins, secondary metabolites, or other compounds that are suspected or known to have anti-insect properties (Duck and Evola 1997). Bacillus thuringiensis crystal proteins were the first to be used to generate transgenic insect resistant crops (reviewed by Krattiger 1997). Proteins from other microorganisms as well as plants have also been used for direct screening for insecticidal activities (Schulera et al. 1998). The common higher plant defense proteins tested to date include lectins, protease, and α-amylase inhibitors (Duck and Evola 1997). In addition to screening known factors, random screening without bias regarding origin or source of protein, chemical, or extract may be performed. The compounds or proteins may even be purchased from commercial sources. For example, Streptomyces cholesterol oxidase, a potent insecticidal enzyme against the cotton boll weevil, was isolated by screening culture filtrates from over microbial fermentations (Purcell 1997). Callus-based insect bioassays from susceptible and resistant crop lines have also been used to investigate insect resistance (Williams et al. 1987). Map-based cloning using techniques such as chromosome walking which utilize molecular probes that map near resistance loci is another approach to isolate genes for deployment in genetic engineering for insect resistance (Gibson and Somerville 1993). Although some work has been done to transfer insect resistance genes from mammals and insects into crops (Schulera et al. 1998), the following discussion focuses mainly on microbial and plant genes that have potential for deployment in transgenic insect resistance in cowpea. Resistance genes from microorganisms Bacillus thuringiensis is a spore-forming soil bacterium that produces insecticidal protein crystals, also called Bt toxins, endotoxins, or crystal (Cry) proteins, within its cells during sporulation. Spores and purified protein crystals of several Bt strains have been used as microbial insecticides since the 1950s and now have an established role in some integrated pest management systems (Fietelson et al. 1992). Different strains of Bt produce different crystal proteins, coded for by Cry genes that are highly toxic to specific insects, nematodes, and other invertebrates. Bt toxins tend to be specific in their activities either to Lepidoptera, Coleoptera or other insects. Their mechanism of action is not quite clear, but it is believed that the proteins damage the membrane of the insect s midgut epithelial cells, causing massive water uptake (Gatehouse et al. 1992). This may in turn lead to the disruption of the electrical K+ and ph gradients by creating pores, resulting in irreversible damage to the midgut wall. 214

3 Potential role of transgenic approaches in the control of cowpea insect pests To date, several genes encoding different Bt toxins have been engineered into crop plants (Schulera et al. 1998). Research at IITA has shown that Cry1Ab, Cry1C, and CryIIA proteins are toxic to MPB (Jackai, unpublished). For control of cowpea pests, it is imperative that other different Bt toxins be tested in artificial diets or seeds for their efficacy against MPB, bruchids, and PSB for which assay systems are available (Jakai and Raulston 1988; Shade et al. 1986). Moreover, artificial insect resistance assays need to be developed for other problematic pests, particulary thrips, to allow screening of Cry proteins against these pests. Bacillus thuringiensis also produces vegetative insecticidal proteins (ViPs) when it is not sporulating (Estruch et al. 1996; Warren 1997). The ViPs are unrelated to crystal proteins and appear to be active against lepidopteran pests such as fall armyworm, beet armyworm, corn rootworm, and tobacco budworm (Estruch et al. 1997). Other candidate genes for insect protection include Streptomyces cholesterol oxidase (Purcell 1997), fungal chitinases (Kramer et al. 1997), the isopentenyl-transferase gene (ipt) from Agrobacterium tumefaciens (Smigocki et al. 1997) and genes encoding insect viral RNAs (Hanzlik and Gordon1997). Additionally, the bacterium Photorrhabdus luminescens, which lives in entomophagous nematodes has recently been shown to produce insecticidal toxins that may be useful for transgenic insect control (Bowen et al. 1998). Insect resistance genes from higher plants It is important to discover new genes that can be pyramided with Bt genes to enhance resistance levels. Other limitations of Bt genes include possibilities of resistance breakdown, limited scope of pests covered by Cry proteins, and public perception issues (Stewart 1999). To overcome some of these limitations, plant-derived genes have been cloned and transferred into several crop species (Schulera et al. 1997, Snow and Palma 1997). Genes for bruchid resistance Coleopteran insects in the family Bruchidae cause serious cowpea grain losses in storage. Callosobruchus maculatus is key among these pests. Through conventional breeding efforts at IITA and elsewhere, modest levels of resistance to C. maculatus have been attained (Singh and Jackai 1985). To enhance these modest resistance levels, efforts have also been underway to identify plant genes that affect C. maculatus development. The majority of artificial seed bioassays have involved the use of plant lectins (Gatehouse et al. 1984, 1991; Heusing et al. 1991a; Machuka et al. 1999a, 1999b, 2000; Murdock et al. 1990; Omitogun et al. 1999; Pratt et al. 1990). Vicilins (7S seed storage proteins) and protease and α-amylase inhibitors and α-amylase inhibitor-like proteins (AIL), are also insecticidal to bruchids (Hilder et al. 1987; Ishimoto et al. 1999; Pittendrigh et al. 1997; Sales et al. 1996; Yunes et al. 1998; Huesing et al. 1991c). Table 1 summarizes the toxicity mechanism of these proteins. Transgenic pea and azuki seeds containing the bean α amylase inhibitor are resistant to bruchid beetles (Ishimoto et al. 1996, Shade et al. 1994). Plans are underway to introduce this gene into modestly bruchid resistant IITA cowpea lines once the transformation system becomes routine. Various compounds are toxic to cowpea beetles. For example, leaf, fruit, seed, and oil extracts from some African shrubs possess larvicidal and ovicidal activities against C. maculatus (Leonard et al. 1993, Seck et al. 1993). However, these toxins are more applicable in biocontrol than transgenic insect control strategies, at least in the short term. 215

4 Biotechnology for cowpea Table 1. Candidate genes for transgenic resistance to bruchids. Protein Possible mechanism(s) of action Cowpea vicilins α-amylase inhibitors Cowpea protease inhibitors e.g. cystein, Bowman-Birk, trypsin, and chymotrypsin inhibitors Lectins Bind insect chitin Inhibition in insect α-amylases Depletion of essential amino acids resulting from hypersecretion of digestive enzymes Inhibition of insect digestive proteases Carbohydrate binding to insect midgut epithelium or peritrophic matrix/ membrane Resistance to proteolysis Genes for resistance to the Maruca pod borer Unlike studies focusing on cowpea weevils, only two studies have been reported that pertain to the biological effects of plant lectins on growth, development, and fecundity of MPB in artificial diet bioassays (Machuka et al. 1999b, 2000). Table 2 shows the list of plant lectins so far tested for their effects against MPB. At least 26 lectins from 15 plant families and representing seven carbohydrate-binding specificity groups have been tested. Results from this screening work indicated that mannose-specific lectins from twayblade (Listera ovata) and snowdrop (Galanthus nivalis) have detrimental effects on MPB larval development at all stages of development. Others, such as wheat germ and jackfruit agglutinins possess latent effects that only manifest at (a) subsequent unique stage(s). A type 1 ribosome-inactivating protein (RIP) from Iris and bean (Phaseolus vulgaris) α-mylase inhibitor are not toxic to MPB larvae although the latter mildly affects pupal development and adult emergence (Machuka et al. 1999b). The galactose-specific seed lectin from Nigerian-grown African yam bean (Sphenostylis stenocarpa) does not affect MPB larval development, although it inhibits C. maculatus development (Machuka et al. 2000). Generally, relatively few lectins are toxic to lepidopteran insects, even when they have been found stable to proteolysis by enzymes in the insect gut (Czapla and Lang 1990; Czapla 1997; Gatehouse et al. 1995). Apart from lectins, plant proteinaceous inhibitors (PIs) of insect proteinases (serine, cysteine, aspartic, and metallo proteinases) are considered potential candidates for gene transfer for insect resistance (Ryan 1990). Serine proteases are the dominant class in lepidopteran insects larvae such as MPB, whereas coleopteran species have a wider range of dominant gut proteinases (Gerald et al. 1997). Since serine and cysteine PIs mainly inhibit the growth and development of lepidopteran (and coleopteran) species, it would be useful to screen a wide range of these PIs against MPB in artificial diets. To date, more than 14 different plant PI genes have been introduced into crop plants, with efforts concentrated on serine PIs from the plant families Fabaceae, Solanaceae, and Poaceae (Koiwa et al. 1997, Schulera et al. 1998). So far, the most active PI identified is the cowpea trypsin inhibitor (CpTI), isolated from an IITA bruchid resistant line, TVnu 2027 (Hilder et al. 1987). Serine PI-like proteins have been identified from seeds of Nigerian-grown velvetbeans (Mucuna spp.) (Machuka 2000a). These proteins, as well as affinity purified trypsin and chymotrypsin inhibitors from two wild Vigna species (V. vexillata and V. oblongifolia) and AYB, are not toxic to MPB (Machuka unpublished). The advantage of 216

5 Potential role of transgenic approaches in the control of cowpea insect pests Table 2. Plant lectins tested against the Maruca pod borer in artificial diets. Lectin specificity Lectin Plant family group ASA, Allium sativum (garlic) agglutinin Alliaceae AUA, Allium ursinum (ramson) lectin Alliaceae *GNA, Galanthus nivalis (snowdrop) agglutinin Amaryllidaceae Mannose *LOA, Listera ovata (twayblade) agglutinin *NPA, Narcissus pseudonarcissus (daffodil) agglutinin Orchidaceaee *CSA, Calystegia sepium (hedge bindweed) agglutinin Mannose/maltose PSL, Pisum sativum (garden pea) lectin Convolvulacea Mannose/glucose SSA, Sphenostylis stenocarpa (African yam bean) Fabaceae agglutin Apiaceae APA, Aegopodium podagraria (ground elder) lectin Curcurbitaceae BDA, Bryonia dioica agglutinin (white bryony) Galactose/ *BPA, Bauhinia purpurea agglutinin Moraceae N-acetyl- (carmel s foot tree) galactosamine DBA, Dolichos biflorus agglutinin (horse gram) Fabaceae *IRA, Iris hybrid agglutinin (Dutch iris) Iridaceae JCA, Artocarpus integrifolia lectin (jackfruit) Caesalpiniaceae SBA, Glycine max agglutinin (soybean) *SNA-II, Sambucus nigra agglutinin (elderberry) Fabaceae Sambucaseae DSL, Datura stramonium lectin (jimson weed) Solanaceae *UDA, Urtica dioica agglutinin (stinging nettle) Urticacaceae N-acetylglucosamine WGA, Triticum aestivum (wheat germ) (Wheat) Gramineae agglutinin MAA, Maackia amurensis (Maackia) agglutinin Fabaceae Sialic acid SNA-I, Sambucus nigra (elderberry) agglutinin Sambucaceae CAA, Colchicum autumnale (meadow saffron) Liliaceae agglutinin PHA-E, Phaseolus vulgaris (red kidney bean) Fabaceae Complex glycan phytohemagglutinin isoform E *PHA-L, Phaseolus vulgaris (red kidney bean) Fabaceae phytohemagglutinin isoform L Liliaceae TLC-I, Tulipa hybrid (tulip) agglutinin Fabaceae RPA, Robinia pseudoacacia (false/black acacia) agglutinin Candidate lectins for transgenic resistance to Maruca pod borer. Detailed references of names and classification of lectins and pod borer bioassays can be found in Van Damme et al. (1998a, b) and Machuka et al. (1999b, 2000). using PIs and other genes from plants, especially edible ones, for enhanced insect resistance is that the nutritional penalty after gene transfer is absent or minimal and there are fewer public perception problems. This has been demonstrated through mammalian toxicity tests, for example, in the case of the cowpea trypsin inhibitor gene (Pusztai et al. 1992). Recently, it has been shown that expression of plant proteases rather than protease inhibitors may be a novel insect defence mechanism in plants (Pechan et al. 2000). Based on the use of Arginine Sepharose B chromatography for isolation of animal serine proteases, novel insecticidal proteins against MPB larvae have been isolated from Mucuna seeds (Machuka 2000b). Although protein database searches revealed that the N-terminus of these proteins is similar to a novel human synovial membrane fluid protein, it is not clear exactly what these proteins are and what their role is in plants. Other candidate genes 217

6 Biotechnology for cowpea that may be implicated in MPB resistance may include chitinases and lectin-like proteins (Colucci et al. 1999, Machuka and Okeola 2000). Genes for resistance to pod-sucking bugs PSBs are probably the next most serious pests of cowpea for which conventional breeding approaches have been inadequate. Omitogun et al. (1999) were the first to demonstrate that crude lectin-enriched extracts from AYB affect development of the cowpea coreid bug (Clavigralla tomentosicollis [Stal]) (Hemiptera: Coreidae). Subsequently, the purified seed lectin (SSA) from AYB has been shown to be toxic to C. tomentosicollis in an artificial cowpea seed system (Machuka et al. 1999a, Okeola et al. 2000). Wheat germ agglutinin, the nonprotein amino acid (para-aminophenylalanine, PAPA) from V. vexillata, and a cysteine protease inhibitor (E-64) also inhibit development of C. tomentosicollis nymphs (Jackai, Shade, and Murdock, unpublished). More studies are needed to identify other candidate proteins for resistance to PSB. Some ecological issues related to projected transgenic cowpea release It is clear from the above survey that candidate genes for transgenic insect control in cowpea are available. In order to realise the potential of this approach it is imperative to establish a stable genetic transformation system for this crop. At the same time, it is also crucial for cowpea scientists to begin to discuss the ecological issues associated with release of transgenic cowpeas, particularly in Africa. Although it is true that pest resistance genes identified in wild and cultivated Vigna germplasm have been incorporated into cultivated varieties by farmers and breeders for several years (Singh et al. 1990; Fatokun 1991; Jackai et al. 1996) the use of genetic engineering raises questions related to the transfer of transgenes to compatible wild or weedy Vigna species related to cowpea (Krattiger 1997; Snow and Palma 1997; Stewart 1999). Some of the issues to consider at this point include the possibility that introduced pest resistance may confer added fitness to cowpea, resulting in enhancement of weedy characteristics due to its increased ability to survive and spread outside of cultivation. Secondly, would transgenic cowpeas transfer pest resistance (or other traits) by natural hybridization to produce hybrid progeny that are more aggressive or more difficult to control? Although gene flow to related species is likely to be limited to V. unguiculata subspecies such as V. unguiculata ssp. dekindtiana, it is important to carry out field trials to determine rates of gene flow. Such a study is underway at IITA (Fatokun, personal communication). Obviously, information will be required from many disciplines such as weed science, agronomy, population biology and genetics, entomology, plant breeding, ecology, plant pathology, molecular biology, and from farmers. Conclusion Reliable and efficient bioassay systems need to be continuously developed and refined to aid the discovery of insecticidal proteins for control of key cowpea pests. It can be anticipated that useful information emerging from current global genomics efforts in crop species, including model legumes, will have a bearing on cowpea improvement through genetic engineering. What cowpea researchers need to do now is develop a comprehensive pest resistance management strategy that incorporates transgenic approaches. Such 218

7 Potential role of transgenic approaches in the control of cowpea insect pests a strategy must take into account criteria such as transformation of elite cowpea lines that are adapted to each of the major agroecological zones, gene flow between cultivated and wild cowpeas, and strategies for dissemination and adoption of biotechnologicallyimproved cowpea lines. Aknowledgements Thanks to all the technicians in the Cellular and Molecular Technology Laboratory at IITA for work related to characterization of insecticidal proteins from African legumes. References Bowen, D., T.A. Rocheleau, M. Blackburn, O. Andreev, E. Golubeva, R. Bhartia, and R.H. ffrench- Constant Insecticidal toxins from bacterium Photorhabdus luminescens. Science 280: Colucci, G., J. Machuka, and M.J. Chrispeels cdna cloning of a class III acid chitinase from the African yam bean (Sphenostylis stenocarpa) (Accession no. AF137070). Plant Physiology 120: 633. Czapla, T.H Plant lectins as insect control agents in transgenic plant. Pages in Advances in insect control: the role of transgenic plants, edited by N. Carozzi and M. Koziel. Taylor and Francis, London, UK. Czapla, T.H. and B.A. Lang Effect of plant lectins on the larval development of European corn borer (Lepidoptera: Pyralidae) and southern corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology 83: D Silva, I., G.G. Poirier, and M.C. Heath Activation of cysteine proteases in cowpea plants during the hypersensitive response a form of programmed cell death. Experimental and Cell Research 245: Duck, N.B. and S. Evola Use of transgenes to increase host plant resistance to insects: opportunities and challenges. Pages 1 18 in Advances in insect control: the role of transgenic plants, edited by N. Carozzi and M.G. Koziel. Taylor and Francis, London, UK. Estruch, J.J., N.B. Carozzi, N.B. Duck, G.W. Warren, and M.G. Koziel Transgenic plants: an emerging approach to pest control. Nature Biotechnology 15: Estruch, J.J., G.W. Warren, M.A. Mullins, G.J. Nye, J.A. Craig, and M.G. Koziel ViP3A, a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects. Proceedings National Academy of Sciences of the USA 93: Fatokun, C.A Wide hybridization in cowpea: problems and prospects. Euphytica 54: Fietelson J., J. Payne, and L. Kim Bacillus thuringiensis: insects and beyond. Bio/Technology 10: Gatehouse, A.M.R., F.M. Dewey, J. Dove, and K.A. Fenton Effect of seed lectins from Phaseolus vulgaris on the development of larvae of Callosobruchus maculatus, mechanism of toxicity. Journal of the Science of Food and Agriculture 35: Gatehouse, A.M.R., D.S. Howe, J.E. Flemming, V.A. Hilder, and J.A. Gatehouse Biochemical basis of insect resistance in winged bean (Psophocarpus tetragonolobus) seeds. Journal of Science Food and Agriculture 55: Gatehouse A.M.R., V.A. Hilder, and D. Boulter Plant genetic manipulation for crop protection. CAB International, Oxford, UK. Gatehouse, A.M.R., K.S. Powell, W.J. Peumans, E.J.M. Van Damme, and J.A. Gatehouse Insecticidal properties of lectins; their potential in plant protection. Pages in Lectins: biomedical perspectives, edited by A.J. Pusztai and S. Bardocz. Taylor and Francis, London, UK. 219

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9 Potential role of transgenic approaches in the control of cowpea insect pests Machuka, J.S., O.G. Okeola, M.J. Chrispeels, and L.E.N. Jackai African yam bean seed lectins affect the development of the cowpea weevil but do not affect the development of larvae of legume pod borer. Phytochemistry 53: Murdock, L.L., J.E. Huesing, S.S. Nielsen, R.C. Pratt, and R.E. Shade Biological effects of plant lectins on the cowpea weevil. Phytochemistry 29: Okeola, O.G., J. Machuka, and I.O. Fasidi Insecticidal activities of the African yam bean seed lectin on the development of bruchid beetle and pod sucking bugs, in Abstracts, Proceedings of World Cowpea Conference III, September IITA, Ibadan, Nigeria. Omitogun, O.G., L.E.N. Jackai, and G. Thottappilly Isolation of insecticidal lectin-enriched extracts from African yam bean, Sphenostylis stenocarpa Harms, and other legume species. Entomologia Experimentalis et Applicata 90: Pechan, T., L. Ye, Y. Chang, A. Mitra, L. Lin, F.M. Davis, W.P. Williams, and Dawn S. Luthe A unique 33-kd cysteine proteinase accumulates in response to larval feeding in maize genotypes resistant to fall armyworm and other Lepidoptera. Plant Cell 12: Pittendrigh, B.R., J.E. Huesing, R.E. Shade, and L.L. Murdock Effects of Cry1A/Cry1B Bt endotoxins, PAPA, protease and α-amylase inhibitors, on the development of the rice weevil, Sitophilus oryzae, using an artificial seed bioassay. Entomologia Experimentalis et Applicata 82: Pratt, R.C., N.K. Singh, R.E. Shade, L.L. Murdock, and R.A. Bressan Isolation and partial characterization of a seed lectin from Tepary Bean that delays bruchid beetle development. Plant Physiology 93: Purcell, L Cholesterol oxidase for the control of boll weevil. Pages in Advances in insect control: the role of transgenic plants, edited by N. Carozzi and M. Koziel. Taylor and Francis, London, UK. Pusztai A, G. Grant, D.J. Brown, J.C. Stewart, S. Bardocz, S.W. Ewen, A.M.R. Gatehouse, and V. Hilder Nutritional evaluation of the trypsin (EC ) inhibitor from cowpea (Vigna unguiculata Walp.). British Journal of Nutrition 68: Ryan, C.A Protease inhibitors in plants: genes for improving defenses against insects and pathogens. Annual Review of Phytopathology 28: Shade R.E., H.E. Schroeder, J.J. Pueyo, L.M. Tabe, L.L. Murdock, T.J.V. Higgins, and M.J. Chrispeels Transgenic pea seeds expressing the alpha-amylase inhibitor of the common bean are resistant to bruchid beetles. Bio/Technology 12: Sales M.P., V.M. Gomes, K.V. Fernandes, and J. Xavier-Filho Chitin-binding proteins from cowpea (Vigna unguiculata) seeds. Brazillian Journal of Medical and Biological Research 29: Shade, R.E., L.L. Murdock, D.E. Foard, and M.A. Pomeroy Artificial seed system for bioassay of cowpea weevil (Coleoptera: Bruchidae) growth and development. Environmental Entomology 15: Shade, R.E., H.E. Schroeder, J.J. Pueyo, L.M. Tabe, L.L. Murdock, T.J.V. Higgins, and M.J. Chrispeels Transgenic pea seeds expressing the α-amylase inhibitor of the common bean are resistant to bruchid beetles. Biotechnology 12: Schulera, T.H., G. Poppya, B.R. Kerrya, and I. Denholmb Insect-resistant transgenic plants. Trends in Biotechnology 16: Seck, D., G. Lognay, E. Haubruge, J-P. Wathelet, M. Marler, C. Gaspqr, and M. Severin Biological activity of the shrub Boscia senegalensis (Pers.) Lam. Ex Poir. (Capparaceae) on stored grain pests. Journal of Chemical Ecology 19: Singh, S.R. and L.E.N. Jackai Insect pests of cowpea in Africa: their life cycle, economic importance, and potential for control. Pages in Cowpea research, production, and utilization, edited by S.R. Singh and K.O. Rachie. John Wiley and Sons, Chichester, UK. 221

10 Biotechnology for cowpea Singh S.R., L.E.N. Jackai, J.H.R. Dos Santos, and C.B. Adalla Insect pests of cowpea. Pages in Insect pests of tropical food legumes. John Wiley and Sons, Chichester, UK. Smigocki, A., S. Heu, I. Mccanna, C. Wozniak, and G. Buta Insecticidal compounds induced by regulated overproduction of cytokinins in transgenic plants. Pages in Advances in insect control: the role of transgenic plants, edited by N. Carozzi and M. Koziel. Taylor and Francis, London, UK. Snow, A.A. and P.M. Palma Commercialization of transgenic plants: potential ecological risks. BioScience 47: Stewart, C.N Insecticidal transgenes into nature: gene flow, ecological effects, relevancy, and monitoring. Pages in Gene flow and agriculture, relevance for transgenic crops. Proceedings of a symposium held at the University of Keele, April 1999, edited by P.J.W. Lutman. British Crop Protection Council, Surrey, UK. Van Damme, E.J.M., W.J. Peumans, A. Pusztai, and S. Bardocz. 1998a. A handbook of plant lectins: properties and biomedical applications. John Wiley and Sons, Chichester, UK. Van Damme, E.J.M., W.J. Peumans, A. Barre, and P. Rougé. 1998b. Plant lectins: a composite of several distinct families of structurally and evolutionary related proteins with diverse biological roles. Critical Reviews in Plant Sciences 17: Warren, G.W Vegetative insecticidal proteins: novel proteins for control of corn pests. Pages in Advances in insect control: the role of transgenic plants, edited by N. Carozzi and M. Koziel. Taylor and Francis, London, UK. Williams, W.P., P.M. Buckley, and F. Davis Tissue culture and its use in investigations of resistance of maize. Agriculture, Ecosystems and Environment 18: Yunes, A.N., T.M. de Andrade, P.M. Sales, R.A. Morais, V.S. Fernandez, V.M. Gomes, and J. Xavier- Filho Legume seed vicilins interfere with the development of the cowpea weevil (Callosobruchus maculatus). Journal of Agricultural and Food Chemistry 76: