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Genetic Engineering and the Improvement of Rice and Cotton

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Genetic Engineering and the Improvement of Rice and Cotton Genetic engineering and the improvement of rice and cotton C. PANNETIER Insect pests cause considerable damage to certain CIRAD-CA/INRA, Centre de Versailles, 7 8 0 26 Versailles Cedex, France crops — forcing farmers to conduct massive chemical E. GUIDERDONI pesticide treatments. Radical changes in plant protection CIRAD-CA/BIOTROP, BP 5035, 34032 Montpellier Cedex 1, France concepts are thus necessary to reduce negative impacts B. HAU QRAD-CA, BP 5035, on the environment and ecological balances. Genes encoding entomopathogenic proteins could be Research on genetic transformation of cotton for insect resistance was carried out in the Cell inserted in rice and cotton plants to create Biology laboratory of the Versailles (France) station of INRA, directed by Y. Chupeau. transgenic pest-resistant varieties. The following scientists were or are involved in the programme: J. Tourneur (CNRS research scientist) P. Couzi (INRA technician) ice and cotton crops have Chemical pesticide treatment was M. Mazier (Ph.D. student 1991-94) been substantially improved long considered to be the only way to V. Dumanois-Le Tan (Ph.D. student 1991-94) control insect pests. However, they M. Giband (CIRAD research scientist) R (i.e. yields and quality) P. Montoro (CIRAD postdoctoral student) by conventional plant breeding upset the natural biological balance, In the BIOTROP (CIRAD) laboratory techniques. However, some pro­ are generally unselective and often in Montpellier, the following scientists blems concerning these crops such kill both the target pests and their were or are involved in the rice genetic as resistance to pests (especially natural enemies. Intensive use of engineering programme: T. Legavre (CIRAD-GERDAT research scientist) insects) are more difficult to solve these compounds can lead to the H. Chair (Ph.D. student 1991-94) using these techniques. Without development of resistance in target N. Mezencev (postgraduate DEA student, control, 40% of the world's cotton insects, which necessitates an Rennes, 1992) crops would be destroyed. increase in treatment dosages. As a D. Ferrand (postgraduate DESS student, Lepidopterans, mainly Helicoverpa result, almost 30% of all chemical Angers, 1993) pesticides sold worldwide are used F. Georget (postgraduate DESS student, armigera, Helicoverpa zea, Heliothis Angers, 1994) virescens and Pectinophora for cotton crop protection. S. Plchot (postgraduate DEA student, ENSAIA, gossypiella, are responsible for 70% Nancy, 1994). of these losses. In rice, stemborers An integrated pest management CIRAD: Centre de coopération internationale en recherche agronomique (also lepidopterans) cause 10-30% strategy has been implemented in pour le développement, France of the crop losses, depending on the recent years to reduce the effects of CNRS: Centre national de la recherche extent of crop intensification. abusive chemical treatments. It scientifique, France Moreover, yield-boosting techniques combines all methods with crop CSIRO: Commonwealth Scientific often involve massive use of protection potential: cropping and Industrial Organization, Australia INRA: Institut national de la recherche polluting chemical compounds, techniques, biological control, use agronomique, France which is not in line with sustainable of genetic plant traits (studies on USDA: United States Department of Agriculture, agriculture objectives (SAVARY resistant varieties) and chemical USA & TENG, 1994). pesticide treatments. Agriculture et développement ■ Special issue - December 1995 genetic engineering Integrated control programmes are The bacterium Bacillus thuringiensis very complicated to set up, requiring excellent technical skills and an and creation of transgenic overall understanding of the pests and agroecological situation. By insect resistant plants genetic engineering techniques, genes coding for entomopathogenic proteins can be inserted and The B.t. bacterium was first discovered in Japan in 1902 in a silkworm breeding unit. expressed in plants; they are now In 1911, it was again isolated in a flour moth population and characterized by Berliner in Thuringen (Germany). being used to create insect resistant varieties. B.t. synthesis of entomopathogenic toxins Research scientists are able to "directly" modify the genome B.t. is a gram-positive bacterium that synthesizes entomopathogenic crystalline inclusions during sporulation. The crystalline structure of the inclusion is an assembly through genetic transformation of protoxin subunits, called 5-endotoxins. Most B.t. strains produce several different techniques. Researchers were crystalline proteins. Different strains can contain the same proteins. Cry 8-endotoxins previously only able to make use of are highly specific. the variability within the plant At least 40 genes encoding protoxins from a wide range of B.t. isolates have been species or, if highly sophisticated isolated and sequenced. These genes were classified by HOFTE & WHITELEY (1989), techniques were available, within based on protein toxin structural homologies and specificities. The genes are the genus of the plant under study. categorized in four main classes: cryl, cryll, crylll and cryIV, each divided into W ith access to this new tool, they subclasses. New toxins are constantly being identified. now have a wide range of different The 5-endotoxins are solubilized in the insect's gut (due to the alcaline pH). They are possibilities. The first focus was activated by gut proteases that cleave the protoxin into a smaller polypeptide, to use the entomopathogenic the toxin. This toxin then binds to the surfaces of epithelial cells in the midgut, properties of the bacterium Bacillus inducing lesions that destroy the cells and lead to death of the insect. thuringiensis Berliner (commonly called B.t.) to obtain insect Use of B.t. to create insect resistant plants resistance. The chief advantage of this application is that it does not threaten humans, mammals or non-target fauna. The use of B.t. strains in biopesticide spray treatments — for more than 30 years with different B.t. strain formulations — has been relatively limited, mainly because of the low field persistence. Transgenic plants The transgenic plant strategy is of considerable interest because it enables toxin production by the plant. The entire plant is therefore protected, especially against for the tropics and insects such as borers that infest plant parts that spraying treatments often cannot subtropics reach. The toxin molecule affects early instar phases of the insect. The system is environmentally safe since the product is retained within the plant tissues. The first results on B.t. gene transfers Some toxins have been tested against the main cotton and rice pests and the results in tobacco and tomato were publi­ highlighted interesting genes that could be inserted in the genomes of these two crops shed in 1987. Since then, B.t. genes (Table 1 ). have been transferred to various other crop species, e.g. cotton, poplar, potato, rice, maize. Transgenic cotton plants Table 1. Toxins active against the main rice and cotton pests. The first transgenic cotton Insect Type of pest Active toxin plants were obtained in 1987 (FIROOZABADY et al., 1987; Cotton UMBECK et a l.,1 987) through Spodoptera littoralis phyllophagous lepidopteran CrylC transformation by Agrobacterium Helicoverpa armigera carpophagous lepidopteran CrylA(b), CrylA(c) tumefaciens and regeneration Pectinophora gossypiella carpophagous lepidopteran CrylA(b), CrylA(c) of transformed cells by somatic Cryptophlebia leucotreta carpophagous lepidopteran CrylA(b), CrylA(c) embryogenesis. Private North Rice American companies (Agracetus, Chilo suppressalis lepidopteran borer CrylA(a), CrylA(c), CrylB Calgene, Monsanto) then invested heavily to produce cotton plants that are resistant to certain lepidopterans. Agriculture et développement ■ Special issue - December 1995 genetic engineering Monsanto reported obtaining plants by a somatic embryogenesis with high resistance to these pests technique1, based on the results of (PERLAK et ai, 1990), but they were TROLINDER & GOODIN (1988). not tested in the field. A few lines The main focus has been on transfor­ then showed some "insecticide" mation of varieties showing good features in the field (JENKINS et al., regeneration potential, rather than on 1993). These plants were obtained developing somatic embryogenesis through significant modifications in techniques and transformation of the nucleotide sequence of the B.t. varieties created by CIRAD. gene used, i.e. crylA(b). Indeed, B.t. According to published results, very genes of bacterial origin are not few varieties other than cvs Coker highly expressed in plants. have been successfully regenerated. Very interesting results were M oreover, transgenes can be obtained by Dr. N. TROLINDER's transferred from transformed cv team (USDA, USA), who transferred Coker plants to commercial varieties a gene from the soil bacterium by standard backcrossing techniques. Alcaligenes eutrophus to produce The main parameters involved in transgenic plants resistant to the A. tumefaciens-mediated transforma­ herbicide 2,4-D (2,4 dichlorophe- tion using hypocotyl fragments have noxyacetic acid). This research was been defined. continued by CSIRO (Australia), which is also investigating the A reporter gene whose expression creation of insect resistant varieties in can be readily detected by collaboration with Monsanto. histochemical techniques is used at Spodoptera littoralis on a cotton leaf. this development phase.
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