and the improvement of rice and

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 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. 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 to create 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, 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. transfers Some toxins have been tested against the main cotton and rice pests and the results in 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 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, ) 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, 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. The gus Transgenic cotton plants should soon Photo CIRAD-UREA gene (encoding fê-glucuronidase), be available on the market. Calgene whose expression can be monitored and Monsanto, which have finalized agreements with Deltapine (a North in the presence of a suitable substrate by the formation of an indigo blue American seed company), have compound was used in these early applied for certification of transgenic stages. The transformation procedure cotton varieties with resistance to bromoxynyl or glyphosate is now fairly well controlled (herbicides) or certain lepidopterans and the results are reproducible. following insertion of B.t. genes. In Transgenic plants have been 1989, CIRAD, in collaboration with obtained using an A. tumefaciens strain carrying a containing a INRA, began transgenic research on cotton at the INRA Cell Biology selectable gene and the B.t. gene laboratory in Versailles (France). crylA(b), which is active against H. armigera, a major cotton pest in Genes of agronomic interest can be Africa and Asia. After selfing, these inserted in the cotton genome by an plants were able to set seed A. fume/ac/ens-mediated transforma­ (PANNETIER et ai, 1994). tion technique. Two B.t. genes are involved: the first, crylA(b), encodes B.t. genes inserted in the genome a toxin that is active against of the transgenic plant have to H. arm igera and Cryptophlebia be highly expressed in order to leucotreta; the second, crylC, which provide sufficient protection against was isolated and cloned in France at insect pests. However, as already the Institut Pasteur (SANCHIS et ai, 1989), encodes a protein that is very active against Spodoptera littoralis. 1. Somatic embryogenesis: organ fragments (here a piece of hypocotyl) are cultured on a suitable nutrient Cotton regeneration medium, resulting in formation of and expression disorganized cell clusters called "calIi". Some callus cells then give rise to Cotton plants are first regenerated in somatic embryos through a process that is Transformed cotton plant. vitro. Coker variety plants (C310, fairly similar to embryo formation from Photo C. Pannetier C312, C201) have been regenerated fertilized zygotes.

Agriculture et développement ■ Special issue - December 1 995 1 5 genetic engineering

The bacterium Plant cell Transformed plant cell Agrobacterium tumefaciens

A. tumefaciens transfers part of its genetic inform ation to the plant

Subsequent to injuries, A. tumefaciens induce tumors around the Figure 1. The bacterium Agrobacterium tumefaciens transfers some of its genetic crown of the plant. This disease is cal­ information to the plant. led crown gall. In 1974, a correlation was established between the disease and the presence of a high molecular weight plasmid in the bacterium, termed the Ti ("tumor inducing") plasmid. Tumoral tissue nuclear DNA

contains a Ti plasmid DNA fragment, RB i.e. T-DNA ("transferred DNA") (Figurei). Ti include genes that are responsible for bacterial virulence (Figure 2). T-DNA (for transferred DNA) carries oncogenes (O N C ), i.e. opine synthesis After transfer, genes carried by the genes (OPS). T-DNA are expressed in the plant. It is limited by "boundaries": right border These are oncogenes that control (RB) and left border (LB), composed synthesis of auxins and cytokinins, of a 25 nucleotide sequence. which induce continuous uncontrolled The region between these two boundaries proliferation of plant cells and tumor is transferred. formation. They also synthesize opines, On the plasmid, there are also VIR genes, used by bacteria as a growth substrate. which enable T-DNA transfer, with OPS genes controlling opine catabolism. ORI is the origin of replication. Figure 2. Diagram of the Ti plasmid.

A. tumefaciens features used to transfer interesting genes to cultivated plants

An interesting feature of A. tumefaciens is its ability to transfer genes. This RB prompted the idea of utilizing the feature to transfer interesting genes attached to T-DNA. Various vectors were created, particularly those involving a binary system (Figure 3). Two plasmids replace the Ti plasmid. The first one, often called the autonomous vector, carries a T-DNA limited by boundaries, within which genes to be transferred to the plant are contained. The second "disarmed Ti plasmid" (with T-DNA deleted), Both plasmids carry an origin of replication, carries the "trans-acting" virulence which controls their multiplication in function, i.e. enabling T-DNA transfer A. tumefaciens or E. coli. This bacterium from the autonomous vector. is used to construct the region to be Figure 3. Diagram of the binary system. inserted in the plant genome.

Agriculture et développement ■ Special issue - December 1 995 genetic engineering

mentioned, these genes are of was initially used to test the effects of bacterial origin and poorly expressed genetic modifications. Completely in plants. Two strategies were synthetic genes — reconstructed developed to improve B.t. gene from an ideal nucleotide sequence expression: modifications of the without any change in the amino nucleotide sequence and insertion acid sequence — have been inserted upstream of the gene of interest of in the cotton genome. sequences that provide more efficient translation (MAZIER et al., 1994). Circumventing insect resistance Potentially transformed cotton plants have been produced with T-DNA B.t. gene transfer and subsequent inserted from a vector containing the expression in the genome of Tobacco Mosaic Virus "omega commercial varieties are essential for leader" sequence, which potentially their future utilization. The aim of could enhance the translation research conducted at CIRAD and efficiency of genes placed INRA is to obtain sustained plant downstream. protection against pests. It is Studies were undertaken to modify important to avoid the development of insect resistance towards the toxin Anthonomus grandis the nucleotide sequence of the gene synthesized by the transgenic plant. on a cotton flower bud. encoding the toxin that is active Photo CIRAD-UREA against 5. littoral is. Tobacco — a Various strategies have been common model plant in cell and designed to reduce such risks. They laboratories — are generally based on lowering the

Construction of a plant expression vector

A chimeric gene had to be constructed techniques: or embryo so that the foreign gene (often called the , particle acceleration, transgene) could be expressed in the etc. plant (Figure 4). In the transformation vector, the gene of The promotor can be constitutive, i.e. interest is often associated with one or induce gene expression throughout the several selectable reporter genes (also in plant and at all stages of development, the form of chimeric genes). They or specific to an organ or tissue, or even enable relatively simple selection or be inducible by wounding, etc. detection of transformed cells or tissues. This chimeric gene is inserted in a The selectable genes are often antibiotic plasmid (autonomous circular DNA), (or herbicide) resistance genes. An which in turn is inserted in the E. coli gene encoding R-glucuronidase bacterium Escherichia coli. The (gus gene) is a common reporter gene. resulting transformation vector can be Its presence in the plant genome can be multiplied by culturing the bacterium. easily detected by histochemical It can then be transferred to techniques (in the presence of a suitable A. tumefaciens or used in plasmid substrate, Xgluc, an indigo blue form for so-called direct transformation precipitate is formed).

Chimeric genes are schematically divided into three parts:

(2) (1) (3) - a coding sequence corresponding Regulating Coding Terminal to the sequence of DNA, which is transcribed region sequence region into messenger RNA, which in turn is translated into a protein (1 ); - a promotor region, located upstream ■ ■■H of the coding sequence, is composed of several DNA sequences, one of which is recognized by the RNA polymerase, thus inducing Figure 4. Chimeric gene structure. the onset of DNA transcription (2); - a terminator sequence (3).

Agriculture et développement ■ Special issue - December 1 995 genetic engineering

selection pressure by limiting Institute (Japan), have invested in insect/toxin contacts: refuge plants2, genetic engineering of rice. The Agrobacterium transgenic/non-transgenic plant following results were obtained by rotations, plants expressing the public laboratories, with major tumefaciens- toxin only in certain organs, etc. funding from the larges-scale mediated (MACCAUGHEY & WHALON, 1992). Rockefeller Foundation programme that began in 1985. The strategy used involves associa­ transformation ting two genes that code for toxins The first genes of interest for insertion of plants with different modes of action. in rice are those that confer resis­ Hence, an insect might develop tance to insects (B.t. endotoxin resistance to one of the toxins, genes, plant protease inhibitors and but it will remain susceptible to lectins), viruses, bacteria, fungi and The transformation process the second. herbicides. Transgenic rice varieties have already been produced which Studies were thus launched to assess Transformation of cotton is described harbour a synthetic crylA(b) gene, a the association of genes encoding here to exemplify the general process. snowdrop lectin gene, potato and An organ fragment (hypocotyl fragment B.t. toxins and genes encoding cowpea protease inhibitor genes, the for cotton) is placed in contact with the protease inhibitors. These protease capsid protein of the stripe virus, and bacterium A. tumefaciens containing a inhibitors (HILDER et al., 1 987; that of the spherical and rod-shaped binary system — this is the inoculation RYAN, 1990) inhibit the insect's stage. The plant tissue is cultured for a tungro viruses and phosphinothricin digestive proteases and consequently specific period of time in vitro (under and sulfonylurea resistance genes. hinder its growth; their modes of aseptic conditions On modified nutrient However, very few of these trans­ action therefore differ from those of medium) — this is the coculture stage. genic varieties have been field tested. Antibiotics are then added to the B.t. toxins and they have a much culture medium, the first one prevents wider host range. The results of The gene transfer system is now bacterial growth and the second selects insect bioassays, i.e. blending operational for rice even though the transformed plant cells. protease inhibitors into a nutrient there are still problems concerning A selectable gene is inserted in the medium, highlighted their effects other cereals — nevertheless regular plasmid together with the gene of on a carpophagous lepidopteran, progress is being made in this area. interest; generally this gene provides H. armigera, and on a beetle, antibiotic resistance. Only cells Medium- and long-term research Anthonomus grandis (LE TAN- containing this antibiotic resistance studies aim at inserting genes that DUMANOIS, 1994). These tests were gene can undergo division in a cell could improve grain quality, increase medium containing the selective agent conducted in the CIRAD entomology tolerance to drought, salinity, anoxia (usually kanamycin). The gene of laboratory. and cold into the rice genome. interest is also inserted in these cells. More than 100 embryogenic cotton lines having integrated a protease Producing transgenic rice plants Embryogenesis in transformed inhibitor gene were obtained; dozens and regeneration problems cotton of transformed plant clones have been transferred to the greenhouse. The first transgenic rice plants were When regularly subcultured on Molecular and biochemical analysis obtained by direct gene transfer to medium containing plant hormones of these plants are under way, along and the selective agent, the transformed in temperate japónica cells give rise to a callus. In certain with prelim inary tests on insects. varieties (Taipei309, Nipponbare, culture medium and environmental The initial results indicate that Yamahoushi) in 1988 (ZHANG et ai, (lighting, temperature, etc.) conditions, high quantities of the protein are 1 988), 4 years after the first reports neoformation of plants from this callus synthesized by the plant, in the on obtaining transgenic dicots can be obtained by conventional leaves and bolls. through protoplast or A. tumefaciens micropropagation techniques. For mediated transformation. Indeed, it cotton, the callus gives rise to a was only in 1985 that regeneration of so-called embryogenic tissue within Transgenic rice protoplast-derived japónica rice which somatic embryos develop. In plants was fully mastered. The turn, these embryos give rise to young Few private plant plantlets which are subsequently companies, apart from Agracetus technique was then applied to indica transferred to the greenhouse (USA) and Japan Tobacco, Mitsui rice varieties (IR54, IR72) which are (see colour plates). Molecular and Toatsu and Plantech Research generally more difficult to regenerate biochemical analysis are then carried (PENG et al., 1992); however, the out to determine the copy number 2. Refuge plants: are untransformed technique was not very efficient with of inserted genes, and the level of plants that are grown with or close these genotypes. There are often expression of this gene in the plant. to transgenic plants (expressing the problems of male sterility and ploidy entomopathogenic toxin). levels in regenerated transgenic rice

Agriculture et développement ■ Special issue - December 1 995 genetic engineering

Embryogénie calli with developing cotton somatic embryos. Photo C. Pannetier

Transformed callus on a cotton hypocotyl fragment, developing on selection medium. Photo C. Pannetier

Young cotton plantlet developed from a somatic embryo. Photo C. Pannetier

Expression of the gus gene in young Chilo suppressalis damage in a rice field. regenerated rice plantlets. Photo M. Betbeder Photo E. Guiderdoni

Expression of the gus gene in immature rice embryo scutellum cells following microparticle bombardment. Photo F. Georget

Cross-section of the stem of a young transformed rice plant showing constitutive expression of the gus gene in cells from different rolled leaves. Photo E. Guiderdoni

Agriculture et développement ■ Special issue - December 1995 genetic engineering

plants. More variations (morpholo­ In 1993, a Taiwan research team gical modifications) occur in the (CHAN et al., 1993) and later a progeny as compared to protoplast- Japanese team (HIEI et al., 1994) derived plants that do not undergo obtained molecular evidence that any transformation treatment. These /4. tumefaciens can be used to plants already show differences transform rice. The efficiency is when compared to normally dependent on the regeneration germinated plants (DAVEY et al., potential of the genotype. 1991). Although there have been many publications on this process, Transfer of insect very few laboratories have used it to borer resistance develop efficient systems for routine mass production of true-to-type to Mediterranean rice transgenic rice plants. In 1990, CIRAD launched a gene transfer project for Mediterranean and rainfed rice varieties. The aim is Gene transfer techniques to produce rice resistant to the for rice Asian and European stemborer, Chilo suppressalis, a pyralid borer, In 1991, Agracetus obtained trans­ which causes crop losses of genic rice plants by microparticle 7-15 quintals/ha in Camargue acceleration of exogenous DNA into (France), and to the African Purified preparation of rice protoplasts, immature embryos of japónica and borers Maliarpha separatella and obtained through enzyme treatment indica varieties (CHRISTOU et al., Chilo zacconius. The studies are of cell suspensions, prior 1991). In other laboratories, being carried out in the BIOTROP to polyethylene glycol treatment. transformations by this technique research unit of CIRAD (Montpellier, Photo E. Guiderdoni were also carried out with cell France). suspensions and embryogenic calli. The statement that this technique is Rice varieties exist that are naturally universal is not quite correct, since tolerant to borers, but this trait is some immature embryos of various under polygenic control. This hinders rice varieties cannot regenerate conventional gene transfers and, in plants and, in any case, it would not addition, it is not certain that the be possible through a unique tolerance will remain stable after the process. rice variety is distributed widely. The project involves transfer of B.t. Overall, this is probably the most endotoxin genes, but initial work has promising transformation technique been specifically focused on for rice and cereals. Although the Mediterranean rice varieties and process is protected by many patents, C. suppressalis. The techniques it is used by an increasing number of developed (active toxin identifica­ laboratories. It seems better than the tion, transformation) could be protoplast transformation technique readily extended to tropical rice since it avoids many of the above varieties and pests. described variations, i.e. first genera­ Affinity and toxicity analysis tion plants are generally fertile and revealed that toxins encoded by morphologically normal. During in crylA(c), crylA(a) and crylB genes vitro culture, abnormalities in were the most active against Examples of molecular hybridisation regenerated plants are sometimes C. suppressalis larvae (FIUZA, 1995). with DNA of five rice plants. caused by the long regeneration Native genes of these three toxins Hybridisation is performed phase — which is shorter from were cloned and synthetic genes are with a radioactive probe composed embryos than protoplasts. It is being prepared, or have already been of a sequence from the transferred gene, difficult to determine which of these obtained, in collaboration with Prof. providing evidence that the transgene methods is superior in terms of their ALTOSAAR's laboratory (University has been inserted in the rice genome. efficiencies in producing true-to-type of Ottawa, Canada). This example involves a resistance transgenic plants since they have gene against ammonium glufosinate never been compared using the At the beginning of this project, a herbicides. same genotype or under the same technique involving direct gene Photo H. Chair laboratory conditions. transfer to protoplasts was mainly

Agriculture et développement ■ Special issue - December 1995 genetic engineering

used for genetic transformation of of the phosphinotricin acetyl elite Mediterranean rice varieties. transferase gene (or bar gene A system was developed for plant derived from the Streptomyces regeneration from embryogenic cell hygroscopicus bacterium). Cons­ suspension-derived protoplasts of titutive promotors were identified Ariete, Miara and Thaíbonnet rice that enable high expression in rice. varieties (GUIDERDONI & CHAIR, 1992). Variations observed in the An efficient system was created to field in the progeny of these plants produce transgenic cv Miara, Ariete were found to be genotype and Thaíbonnet plants at a rate of dependent — in terms of rate, 0.5-1 plants/100 000 protoplasts amplitude and direction — but they processed. However, there are could still be used in breeding still many problems In obtaining schemes (MEZENCEV et a i, 1995). true-to-type plants, e.g. albinism A protoplast transformation study associated with the use of protoplasts was also carried out, prompting the from mlcrospore-derlved calli, and development of an efficient gene obtention of higher than normal transfer technique. Genes could thus ploidy levels (2n). Promising results, be inserted in protoplasts after either which could solve some of these of the following treatments: chemical problems, have been obtained polyethylene glycol treatment through the use of protoplasts of or disruption with an electric somatic origin, and microparticle current (electroporation). Chemical acceleration of DNA into immature treatment was chosen for the project embryos (GEORGET, 1994). based on the results of comparative studies (CHAIR, 1995). Instead of The effects of constitutive expression using antibiotic resistance, selection of a native gene and a synthetic B.t. of transformed tissues was obtained gene on pyral borer resistance in a via resistance to phosphinotricin susceptible variety and a tolerant (Basta herbicide) through expression variety will soon be compared.

Direct gene transfer techniques

Electroporation millisecond range). The electric field thus disk or a screen) or in a drop created depolarizes phospholipid layers of water, depending on the type of Electroporation was first developed of the protoplast plasma membrane, particle gun used. With an aluminium for animal cells and bacteria. It inducing temporary holes through which foil device, pressure expansion after was subsequently applied to plant DNA molecules can enter the cells. the explosion of powder or neutral cells from which the cell walls had compressed, gas propulses the been enzymatically removed, i.e. Polyethylene glycol treatment protoplasts. It was considered essential macroprojectile, which is then Protoplasts, first suspended in a to remove the cell walls to allow DNA stopped by a stopping plate, while the saline buffer solution, are placed in to enter the cells. However, recent microprojectiles continue on towards the a polyethylene glycol solution (high success in obtaining transgenic plants target. With the plastic screen type, a molecular weight compound), with from zygotic embryos and callus flow of helium carries the microparticles bivalent cations (Mg2+ or Ca2+) and fragments, pretreated or not with an directly towards the target. With a drop plasmid DNA. The cations provide enzymatic solution, suggests that genes of water, an is created bridges between DNA and the plasma could be inserted through intact between electrodes, where the water membrane (both negatively charged). cell walls using high power voltage drop is deposited; the particle gun causes The polyethylene glycol fuses and and capacitance electroporation. its vaporization, thus propulsing the destabilizes the plasmalemma, allowing In practice, protoplasts are suspended DNA to enter the cells. microparticles. Microparticle propulsion in a saline buffer solution in the presence is performed in a chamber containing the of plasmid DNA. They are then placed in Microparticle acceleration target and in which a partial vacuum is a tank between electroporator terminals Plasmid DNA, precipitated on metal created to facilitate propulsion. For rice, which deliver a charge of capacitance (tungsten or gold) microparticles under the target can be a spread sample of (expressed in (jFarads) and voltage the action of alcohol or , is deposited embryogenic cell suspension, cal I i or (in volts) for a specific time (in the on a macroprojectile (an aluminum foil embryo scutellum, or microspores.

Agriculture et développement ■ Special issue - December 1995 genetic engineering

Thereafter, further combinations of available on the market. However, different synthetic genes or an for several transgenic varieties endotoxin gene and a protease especially with respect to herbicide inhibitor gene, controlled by resistance, there are current discus­ constitutive or regulated promotors, sions on some technical problems. could be constructed and inserted in In programmes under way at CIRAD, the rice genome. the first step in creating transgenic plants has been to develop reliable and reproducible gene transfer Prospects techniques. Insect resistance is the main goal of varietal Genetic transformation enables plant improvement. Close collaboration breeders to overcome limitations due between biologists, entomologists to barriers between species. After the and plant breeders is needed for discovery of this technique in the the effective use of transgenic early 1980s, there were great hopes plants containing genes encoding for an explosion of new varieties with entomopathogenic proteins. Various interesting traits in the 1990s. strategies, specifically aimed at Preliminary studies focused on limiting the development of introducing characters of high market resistance in plant pests, should be value (e.g. herbicide and insect designed for these transgenic Chilo suppressalis larvae in a rice sucker. resistance). It took quite some time varieties before their widespread Photo J. Escoute before genetic engineering had any distribution. marked impact in this field, but Continuous research on new sources transgenic varieties should soon be of resistance is essential for efficient long-term control. Studies on the efficiencies of various proteins Choices of transformation techniques (protease inhibitors, lectins, etc.) are presently under way. Insect genes that could be used to upset their Why is A. tumefaciens not used the possibility of large-scale development is a promising area of for rice? production of transgenic rice plants. study (STEWART, 1991). Rice, as is the case with other cereals, Direct gene transfer techniques resists A. tumefaciens infection. for rice The development of resistance to The fact that tumors do not form from insect pests remains a prime research In 1988, the first transgenic rice differentiated tissues of these plants is focus. Nevertheless, genetic plants were obtained by direct gene not related to the agrobacteria, but transfer techniques involving physical engineering offers considerable new rather to the inability of most cereal (electroporation) or chemical potential, and studies are, and will cells to proliferate. In 1986, Grimsley (polyethylene glycol) treatment of be, carried out to investigate all and collaborators demonstrated that protoplasts. The regeneration system cropping aspects, e.g. on resistance A. tumefaciens could insert its T-DNA first has to be fully mastered. into the nucleus of a cereal cell. The to abiotic stresses (salinity, tempera­ This approach was found to be lack of tumor formation from the inva­ ture, etc.), creation of F1 hybrids, and complicated to implement, only ded cereal cell is due to a difference in improvement of cotton fibre and rice the injury responses of monocot and applicable to a few genotypes, grain qualities. There are great gene showed a high risk of obtaining dicot cells: the former reacts by pools in wild-type varieties that have unwanted variations, because of the degenerating and the latter produces not yet been tapped. Wild species of long in vitro culture steps with total scar tissue. The inefficiency of gene cotton have many useful traits: cell isolation. Callus cell suspension transfers in grass species could be bacterial disease resistance, male explained by an absence of suitable and embryo scutellum transformation sterility, drought resistance, conditions for division in cells systems were developed to limit these technological features, insect resis­ that have been infected with an drawbacks. Transgenic rice plants agrobacterium. were thus obtained in 1991, and these tance, etc. However, there have been transformation techniques were very few successful insertions of Following initial studies by a Taiwan widely adopted for transgenic rice these traits into cultivated research team, Japanese scientists production. Interesting results were varieties to create new varieties. of the Japan Tobacco company were also recently obtained with other Biotechnology could extend current able to successfully coculture techniques, e.g. electroporation of horizons by enabling plant breeders proliferating embryogenic cells with embryo cells and treatment with a to utilize this high genetic diversity. agrobacteria, clearly demonstrating pulsed electric field. Access to these new techniques is an

Agriculture et développement ■ Special issue - December 1 995 genetic engineering

im portant challenge for less DEATON W.R., 1993. Growth and survival of developed countries. Many potential Bibliography Heliothis virescens (lepidoptera : noctuidae) on applications will be highly transgenic cotton containing a truncated form of beneficial, especially for developing CHAIR H., 1995. Transformation the delta endotoxin gene from Bacillus thuringiensis. J. Econ. Entomol. 86 :181-185. low-input cropping systems. Despite génétique de protoplastes de variétés méditerranéennes de riz (Oryza sativa L.). Thèse LE TAN-DUMANOIS V., 1994. Défense du delaying the expected benefits, de doctorat, biologie des populations et cotonnier contre les insectes ravageurs : étude analysis on the impacts of transgenic écologie, université des sciences et techniques d'une stratégie basée sur l'expression conjointe varieties (cropping practices, du Languedoc, Montpellier II, France, 78 p. d'inhibiteurs de protéases et de toxines de Bacillus thuringiensis dans la plante. Thèse de environmental impact, etc.) are CHAN M.T., CHANG H.H., HO S.I., TONC doctorat, génétique et amélioration des plantes, imperative before their agricultural W.F., YU S.M., 1993. Agrobacterium mediated université Paris-Sud, Orsay, France, 110 p. extension. Research should also production of transgenic rice plants expressing a chimeric amylase promoter/glucuronidase gene. MAZIER M., 1994. Création de plantes address the needs of less developed Plant. Mol. Biol. 22 : 491-506. transgéniques résistantes aux insectes. countries, and collaborative Utilisation du gène crylC. Thèse de doctorat, CHRISTOU P., FORD T.L., KOFRON M „ programmes could be set up to génétique et amélioration des plantes, 1991. Production of transgenic rice (Oryza université Paris-Sud, Orsay, France, 176 p. provide access to improved plant sativa L.) plants from agronomically MAC GAUGHEY W.H., W HALON M.E., material but also to the technology. important indica and japónica varieties via 1992. Managing insect resistance to Bacillus electric discharge particle acceleration of thuringiensis toxins. Science 258 :1451-1455. exogenous DNA into immature zygotic embryos. Bio/Technology 9 :957-962. MEZENCEV N., CLEMENT G., GUIDERDONI E., 1995. Variation among DAVEY M.R., KOTHARI S.L., ZHANG H., progenies of diploid plants regenerated from RECH E.L., COCKING E.C., LYNCH P.T., haploid cell suspension protoplasts of rice (Oriza 1991. Transgenic rice : Characterisation sativa L.). Plant Breeding 14 : 149-154. of protoplast-derived plants and their seed PANNETIER C., TOURNEUR )., progeny. J. Exp. Bot. 42 : 1159-1169. LE TAN V., MAZIER M „ COUZO P., 1994. FIROOZABADY E., DEBOER D.L., Genetic Engineering of cotton for Insect MERLO D.J., HALK E.L., AMERSON L.N., Pest Management in an INRA/ CIRAD RASHKA K.E., MURRAY E.E., 1987. research group. In Cotton Biotechnology Transformation of cotton (Gossypium REUR Technical series 92, C. Peeters (Ed.), FAO, hirsutum L.) by Agrobacterium tumefaciens Rome, Italy. and regeneration of transgenic plants. PENG J., K O N O N O W IC Z H., Plant Molecular Biology 10 : 105-116. HODGES T.K., 1992. Transgenic indica rice plants. Theor. Appl. Genet. 83 : 855-863. FIUZA L.M., 1995. Etude des sites récepteurs et de la toxicité des 3-endotoxines de PERLAK F.J., DEATON R.W., Bacillus Thuringiensis Berliner chez les larves de ARMSTRONG T.A., FUCHS R.L., SIMS S.R., la pyrale du riz, Chilo suppressalis Walker. GREENPLATE J.T., FISCHHOFF D.A., 1990. Thèse de doctorat en sciences agronomiques, Insect resistant cotton plants. Bio/Technology ENSAM, Montpellier, France, 1 79 p. 8 :939-943. RYAN C.A., 1990. Protease inhibitors in GEORGET F., 1994. Transformation plants : genes for improving defenses against de suspensions cellulaires et d'embryons insects and pathogens. Annu. Rev. Phytopathol. immatures de riz (Oriza sativa L.) par 28 :425-449. accélération de microparticules : étude des paramètres influençant l'expression transitoire et SANCHIS V., LERECLUS D „ MENOU G., stable du gène gus. M ém oire de DESS CHAUFAUX J„ GUO S., LECADET M.M., 1989. technologies du végétal, université d'Angers, Nucleotide sequence and analysis of the France, 33 p. N-terminal coding region of the Spodoptera-active 3-endotoxin gene of Bacillus GUIDERDONI E., CHAIR H., 1992. Plant thuringiensis aizawai 7.29. Mol. Microbiol. regeneration from haploid cell suspension 3 : 229-238. derived protoplasts of Mediterranean rice (Oriza SAVARY S., TENG P.S., 1994. Q uelle sativa cv Miara). Plant Cell Rep. 11:618-623. agriculture demain ? La Recherche 271 : HIEI Y., O HTA S., KOMARI T., 1320-1328. KUMASHIROT., 1994. Efficient transfor-mation STEWART J. MAC. D., 1991. Biotechnology of rice (Oryza sativa L.) mediated by of cotton. ICAC Review Articles on Cotton Agrobacterium and sequence analysis of the Production Research n° 3, CAB International, boundaries of the T-DNA. Plant ). 6 : 271-282. 50 p.

HILDER V.A., GATEHOUSE A.M.R., TROLINDER N.L., GOO DIN J.R., 1988. SHEERMAN S.E., BARKER R.F., BOULTER D „ Somatic embryogenesis in cotton (Cossypium). I. 1987. A novel mechanism of insect Effects of source of explant and hormone resistance engineered into tobacco. Nature regime. Plant Cell Tissue and Organ Culture 330 : 160-163. 12 : 31-42. ZH AN G H .M ., YANG H., RECH E.L., HOFTE H„ WHITELEY H.R., 1989. GOLDS T.L., DAVIS A.S., MULLIGAN B.J., Insecticidal crystal proteins of Bacillus COCKING E.C., DAVEY M.R., 1988. Transgenic thuringiensis. Microbiol. Rev. 53 : 242-255. Cotton boll damaged rice plants produced by electroporation JENKINS J.N., PARROT W.L., McCARTY mediated plasmid uptake into protoplasts. by Helicoverpa armigera. I.C., CALLAHAN F.E., BERBERICH S.A., Plant Cell Rep. 7 : 379-384. Photo CIRAD-UREA

Agriculture et développement ■ Special issue - December 1995 genetic engineering

Abstract... Resumen... Résumé...

C. PANNETIER, E. GUIDERDONI, B. HAU — Genetic C. PANNETIER, E. GUIDERDONI, B. HAU — Ingeniería C. PANNETIER, E. GUIDERDONI, B. HAU — Génie engineering and the improvement of rice genética y mejora del arroz y del algodón. génétique et amélioration du riz et du cotonnier. and cotton. La transferencia de genes que codifican proteinas Le tra n s fe rt de gènes codant pour des protéines The transfer of genes coding fo r entomopalhogenic entomopatógenas permite crear variedades de arroz y de entomopathogènes permet la création de variétés de riz et proteins makes it possible to create rice and cotton algodón que resisten a algunos insectos dañinos. El de cotonnier résistant à certains insectes ravageurs. Le varieties resistant to insect pests. The first focus is the use primer eje desarrollado es la utilización de genes que premier axe développé est l'utilisation de gènes codant of genes coding for "insecticide" toxins of the bacteria codifican toxinas "insecticidas" de la bacteria Bacillus pour des toxines « insecticides » de la bactérie Bacillus Bacillus thuringiensis. B.t. genes have thus been thuringiensis. De este modo, se han introducido genes de thuringiensis. Des gènes de B.t. ont été ainsi introduits introduced in various plants: cotton, poplar, potato, rice B.t. en diferentes especies: algodón, álamo, patata, arroz dans différentes espèces : cotonnier, peuplier, pomme de and maize. The first transformation of cotton was y maiz. Las primeras obtenciones de plantas de algodón terre, riz, maïs. Les premières obtentions de cotonniers achieved in 1987 via Agrobacterium tumefaciens and transformadas datan de 1987, a partir de la transformés datent de 1987, à partir de la transformation regeneration by somatic embryogenesis. Insecticide transformación mediante Agrobacterium tumefaciens y de via Agrobacterium tumefaciens et de la régénération par properties were confirmed in the field by several lines la regeneración por embriogénesis somática. Algunas embryogenèse somatique. Quelques lignées ont confirmé after substantial modification of the nucleotide sequence cepas confirmaron en el campo propiedades insecticidas au champ des propriétés insecticides, après d'importantes of the B.t. gene used, since B.t. genes of bacterial origin tras importantes modificaciones de la secuencia modifications de la séquence nudéotidique du gène de B.t. are very weakly expressed in plants. CIRAD, in nudeótida del gen B.t. utilizado, pues los genes de B.t., utilisé, car ces gènes d'origine bactérienne, s'expriment très collaboration with INRA, performs research on the de origen bacteriano, se expresan muy debilmente en un fa ible m en t dans un contexte végétal. Le CIRAD, transformation of cotton via Agrobacterium tumefaciens contexto vegetal. CIRAD, en colaboración con el Instituto en collaboration avec l'INRA, conduit des recherches sur la for resistance to Heliotis armigera (B.t. gene, crylA(b)) francés de Investigación Agrícola (INRA) está realizando transformation du cotonnier via A. tumefaciens pour la and to Spodoptera littoralis [B.t. gene, crylQ. However, investigaciones acerca de la transformación del algodón résistance à Heliotis arm igera— gène de B.t., crylA(bJ— use of plants synthesizing a single B.t. toxin may cause mediante Agrobacterium tumefaciens para la resistencia a et à Spodoptera littoralis— gène de B.t., crylC. Mais, l'uti­ the appearance of resistance in the target insects. Heliotis armigera (gen de B.t. crylA(b)) y a Spodoptera lisation de plantes synthétisant une seule toxine de Different strategies can be used to prevent the occurrence littoralis (gen de B.t. crylc). Sin embargo, la utilización de B .t. peut entraîner l'apparition de résistance chez of such resistance. That studied by the CIRAD/INRA team plantas que sintetizan una sola toxina de B.t. puede les insectes ciblés. Différentes stratégies peuvent être consists of combining two genes coding for provocar la aparición de resistencia en los insectos développées pour éviter l'apparition de cette résistance. entomopathogenic proteins with different modes of destinatarios. Pueden desarrollarse diferentes estrategias Celle qui est étudiée par l'équipe CIRAD/INRA consiste à action: B.t. toxins and protease inhibitors. Transgenic para evitar la aparición de esta resistencia. La estudiada associer deux gènes codant pour des protéines entomopa­ cotton plants incorporating a B.t. gene or expressing a por el equipo CIRAD/INRA consiste en asociar dos genes thogènes à modes d'action différents : les toxines de B.t. et protease inhibitor gene have been obtained and que codifican proteinas entomopatógenas de modos de les in hib iteu rs de protéases. Des cotonniers set seed. Rice tra n sfo rm a tio n was achieved only acción diferentes: las toxinas de B.t. y los inhibidores de transgéniques ayant intégré un gène de B.t. ou exprimant 4 years after the first dicot transgenic plants were proteasis. Se obtuvieron algodoneros transgénicos que un gène d'inhibiteur de protéase ont également obtained. However, regenerated transgenic rices have hablan integrado un gen de B.t. o que expresaban un gen été obtenus et ont produit des graines. Plus de more variations than true-to-type plants. Application of inhibidor de proteasis y que produjeron semillas. Más de quatre ans après les premières obtentions de dicotylédones resistance to borers ( Chilo suppressalis, Maliarpha cuatro años después de las primeras obtenciones de transgéniques, des riz transgéniques ont été produits. separatella and Chilo zacconius) in Mediterranean rice dicotiledóneas transgénicas, se produjeron arroces Cependant, les riz transgéniques régénérés présentent varieties has been undertaken by CIRAD. The transfer transgénicos. No obstante, los arroces transgénicos souvent des variations par rapport aux plantes conformes. method involves protoplast transformation. Overall, the regenerados presentan a menudo variaciones respecto a L'application au transfert de résistance aux foreurs des application of genetic engineering to cultivated plants has las plantas conformes. CIRAD ha comenzado la aplicación tiges IChilo suppressalis, Maliarpha separatella, Chilo been slower than expected, but transgenic varieties will a la transferencia de resistencia a los barreneros ( Chilo zacconius) pour les riz méditerranéens et tropicaux est soon be available on the market. Analysis of the impact suppressalis, Maliarpha separatella, Chilo zacconius) en engagée par le CIRAD. La méthode de transfert utilise for cropping techniques and the environment should los arroces mediterráneos. El método de transferencia la transformation des protoplostes. Dans l'ensemble, precede the release of transgenic varieties for cropping. utiliza la transformación de los protoplastos. En general, l'application du génie génétique aux plantes cultivées a été Keywords: cotton, rice, breeding, biotechnology, Bacillus la aplicación de la ingeniería genética a las plantas moins rapide que prévue, mais l'arrivée sur le marché de thuringiensis, insect pest control, toxin, protease inhibitor. cultivadas ha sido menos rápida que lo previsto, pero la variétés transgéniques est imminente. La vulgarisation des llegada en el mercado de variedades transgénicas es variétés transgéniques devra être précédée de l'analyse de inminente. La difusión en cultivo de las variedades l'im p a c t de cette intro du ctio n sur les techniques transgénicas deberá ir precedida del análisis de las culturales et sur l'environnement. consecuencias para las técnicas de cultivo y el medio Mots-clés : cotonnier, riz, génétique, biotechnologie, ambiente. Bacillus thuringiensis, lutte contre les insectes, toxine, Palabras clave: algodón, arroz, genética, biotecnologia, inhibiteur de protéase. Bacillus thuringiensis, lucha contra los insectos, toxina, inhibidor de proteasis.

Agriculture et développement ■ Special issue - December 1995