Agrobacterium Tumefaciens-mediated Transformation System Research Articles

Emerging factors that influence efficiency of T-DNA gene transfer into Phalaenopsis violacea orchid via Agrobacterium tumefaciens–mediated transformation system

An early step in the Agrobacterium-mediated transformation of Phalaenopsis violacea orchid was investigated in the plant-bacterium interaction. Directed movement in response to chemical attractants is the crucial importance to Agrobacterium tumefaciens strains. Chemotaxis of Agrobacterium tumefaciens strains (EHA 101 and 105) towards wounded orchid tissues has been studied by using swarm agar plates. The results obtained indicate a minor role of chemotaxis in determining host specificity and suggest that it could not be responsible for the absence of tumorigenesis in Phalaenopsis violacea orchid under natural conditions. Various aspects of transformation were examined using transient gusA gene expression in efforts to improve the efficiency of producing transformants. Hypervirulent Agrobacterium tumefaciens strains, EHA 101 and 105, harboring the pCAMBIA 1304 plasmid containing gusA gene as a reporter marker, were used for transformation study. The effects of different concentrations of L-cysteine, silver nitrate, and various temperatures (0C) during co-cultivation in half-strength MS medium supplemented with 5% of banana Mas extract on transformation efficiency were evaluated. Strain EHA 105 proved significantly better than EHA 101 based on higher percentage of gusA gene expression and even spread of GUS staining on PLBs. In addition, 200mg.L-1 L-cysteine, 60µM silver nitrate, and under the temperature at 240C gave the highest percentage of transient GUS expression. The results from transient transformation of PLBs suggested that Agrobacterium-mediated transfer of T-DNA is highly efficient. Therefore, by combining the best treatments, an efficient and reproducible of Agrobacterium-mediated transformation procedure could be continued for the production of transgenic Phalaenopsis violacea orchid.

A high-throughput Agrobacterium tumefaciens-mediated transformation system for molecular breeding and functional genomics of rice (Oryza sativa L.)

Agrobacterium tumefaciens-mediated transformation is the preferred method for genetic transformation of rice. Here we provide a comprehensive and high-through-put protocol for Agrobacterium-mediated transformation of rice for generating the large numbers of transgenic plants that are required for functional analysis and for evaluating traits of agronomic importance. In a project designed to produce nitrogen use efficient rice plants, we refined/improved several factors including optimization of conditions for inducing vir genes prior to transformation, infection and co-cultivation medium for better interaction of target tissues with Agrobacterium. These optimizations supported not only the survival of co-cultured calli in high frequency, but also the production of multiple resistant cell lines per co-cultured embryogenic and nodular callus. In addition, improvements in plant tissue culture, selection and regeneration media has enabled us to produce large numbers of transgenic rice plants containing genes of agronomical importance. Partial desiccation and ABA treatment to hygromycin-resistant callus tissue significantly (P<0.05) enhanced both regeneration frequency and regeneration of transgenic plantlets per callus tissue. Regeneration frequency was further improved by optimizing the concentration of copper sulphate in the regeneration medium. The majority of the transgenic plants obtained using this improved protocol displayed a normal phenotype and the gene of interest was inherited by the progeny in a Mendelian fashion. Homozygous plants of selected lines showed stable phenotypes both in soil and hydroponic conditions even after five generations. Availability of such a high-throughput Agrobacterium-mediated transformation system will improve future opportunities for rice genetics and functional genomics study.

Early GFP gene assessments influencing Agrobacterium tumefaciens-mediated transformation system in Phalaenopsis violacea orchid

An initial step in the genetic transformation of Phalaenopsis violacea orchid species was investigated in the plant-Agrobacterium interaction. Agrobacterium tumefaciens strains EHA 101 and 105, harboring the pCAMBIA 1304 plasmid contains gfp gene as the reporter gene marker, were used in this transformation study. The spectrophotometric GFP assay provides information on the amount of inoculated Agrobacterium tumefaciens that effectively bound to various orchid tissues. Different temperatures during co-cultivation period, the concentration of Lcysteine, calcium (CaCl2) and silver nitrate (AgNO3) in co-cultivation medium during cocultivation period were identified to be major and important factors in enhancing the increase percentage of transient gfp gene expression in PLBs. Agrobacterium tumefaciens EHA 105 was proved to be better bacterial strain in transforming the targeted PLBs than EHA 101, based on the notably higher transient expression of gfp gene in all the optimization parameters that were tested. Highest T-DNA delivery efficiencies were obtained when P. violacea PLBs were co-cultivated with Agrobacterium tumefaciens strain EHA 105 in half-strength MS medium supplemented with 5% of banana cultivar, Mas extract containing 200 mgL-1 L-cysteine, 60µM silver nitrate, without calcium in the medium during co-cultivation in the dark condition at 24°C. The results from the transient gfp gene expression of PLBs suggested that Agrobacterium-mediated transfer of T-DNA to the naturally recalcitrant P. violacea is feasible and is highly efficient. Hence, the use of the gfp marker gene during in vitro screening of the transgenic cells has enabled the visual selection of orchid transformed by Agrobacterium tumefaciens at higher frequency rates.

Agrobacterium-mediated transformation as a useful tool for the molecular genetic study of the phytopathogen Curvularia lunata

In order to explore the molecular mechanisms of virulence and genetic variance of Curvularia lunata in maize, an ATMT (Agrobacterium tumefaciens-mediated transformation) system was established in order to create a wide range of insertional transformants of C. lunata. Our results showed that the germinating conidia were the ideal starting material for transformation. Based on our optimised transformation condition, the transformation efficiency of C. lunata with T-DNA was improved greatly, and the average transformation frequency was as high as 84 ± 5 transformants per 1 × 106 germlings. Southern blotting results of 39 randomly-selected transformants showed a unique hybridisation pattern and predominant single-copy insertions. An ATMT library containing approximate 3000 transformants was generated, and four types of transformants were screened in terms of growth rate, sporulation, mycelial pigmentation, and toxin production in vitro. This library will be used to identify genes involved in the virulence of the pathogen.

Establishment of Agrobacterium tumefaciens -Mediated Transformation of an Oleaginous Fungus, Mortierella alpina 1S-4, and Its Application for Eicosapentaenoic Acid Producer Breeding

Gene manipulation tools for an arachidonic-producing filamentous fungus, Mortierella alpina 1S-4, have not been sufficiently developed. In this study, Agrobacterium tumefaciens-mediated transformation (ATMT) was investigated for M. alpina 1S-4 transformation, using the uracil-auxotrophic mutant (ura5(-) strain) of M. alpina 1S-4 as a host strain and the homologous ura5 gene as a selectable marker gene. Furthermore, the gene for omega3-desaturase, catalyzing the conversion of n-6 fatty acid to n-3 fatty acid, was overexpressed in M. alpina 1S-4 by employing the ATMT system. As a result, we revealed that the frequency of transformation surpassed 400 transformants/10(8) spores, most of the integrated T-DNA appeared as a single copy at a random position in chromosomal DNA, and most of the transformants (60 to 80%) showed mitotic stability. Moreover, the accumulation of n-3 fatty acid in transformants was observed under the conditions of optimal omega3-desaturase gene expression. In particular, eicosapentaenoic acid (20:5n-3), an end product of n-3 fatty acids synthesized in M. alpina 1S-4, reached a maximum of 40% of total fatty acids. In conclusion, the ATMT system was found to be effective and suitable for the industrial strain Mortierella alpina 1S-4 and will be a useful tool for basic mutagenesis research and for industrial breeding of this strain.

Expression of a gfp gene in Penicillium nordicum under control of the promoter of the ochratoxin A polyketide synthase gene

A gfp reporter gene strain of Penicillium nordicum was constructed in order to be able to study the influence of environmental parameters on ochratoxin A biosynthesis. To introduce the gfp gene an Agrobacterium tumefaciens mediated transformation system (ATMT) for P. nordicum was established resulting in a transformation efficiency of about 60 transformants per µg of DNA. The selection principle was based on the hygromycin B resistance gene located on the TI-DNA fragment of the binary vector system. PCR and Southern blot hybridization revealed that the TI-DNA was integrated into the chromosome of P. nordicum. To show that the GFP protein can be used as a reporter gene in P. nordicum, this species was subsequently transformed with a vector, carrying a gfp gene under the control of the strong constitutive gpd promoter of A. nidulans. Moreover in this vector construction the gfp gene contained a stuA nuclear localization domain. Successful transformed strains showed a strong GFP activity located in the nuclei after light stimulation in contrast to the wild type which showed only very weak unspecific auto fluorescence under these conditions. Based on this proof of principle a vector was constructed in which the promoter of the otapksPN gene, the gene of the ochratoxin A polyketide synthase of P. nordicum was placed in front of the gfp gene. This construct was transformed into P. nordicum by ATMT and the resulting transformants were analysed by fluorescence microscopy. In these transformants the whole mycelial cells showed GFP activity after light stimulation, whereas the wild type strain did not. When the transformed strains were grown on medium which suppressed ochratoxin A biosynthesis, a very low level of fluorescence could be detected, whereas a high level of fluorescence was seen after growth on medium supportive for ochratoxin A biosynthesis.

Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum.

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 10(6) conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alternatively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

Additional virulence genes in conjunction with efficient selection scheme, and compatible culture regime enhance recovery of stable transgenic plants in cowpea via Agrobacterium tumefaciens-mediated transformation

A critical step in the development of robust Agrobacterium tumefaciens-mediated transformation system in recalcitrant grain legume, cowpea is the establishment of optimal conditions for efficient T-DNA delivery into target tissue and recovery of transgenic plants. A dramatic increase in efficiency of T-DNA delivery was achieved by constitutive expression of additional vir genes in resident pSB1 vector in Agrobacterium strain LBA4404. A geneticin based selection system permitted rapid and efficient identification of transgenic shoots without interfering with their regeneration, and eliminated the bulk of escapes. Supplementation of 0.5 μM kinetin to medium containing 5.0 μM benzyl aminopurine after 1 week of culture followed by 3 weeks of culture were found critical for optimal multiplication and elongation of transformed shoots from cotyledonary node explants. Combining these three developments, we recovered fertile transgenic plants at a frequency of 1.64%, significantly higher than previous reports. The presence, integration, expression and inheritance of transgenes were confirmed by molecular analysis. The protocol developed for cultivar Pusa Komal will facilitate the transfer of desirable traits into cowpea.

Earlier Flowering Induced by Over‐Expression of CO Gene Does Not Accompany Increase of Artemisinin Biosynthesis in Artemisia annua

The early flowering gene CONSTANS (CO) from Arabidopsis thaliana was transferred into Artemisia annua using the Agrobacterium tumefaciens-mediated transformation system. The plant expression vector pBI CO was constructed by inserting the CO gene into the binary vector pBI121 under the control of CaMV 35S promoter. Analyses of PCR, PCR Southern blot, and Southern blot revealed that the transgenic plants contained the foreign CO gene. The results of RT-PCT and RT-PCR Southern blot suggested that the foreign CO gene had expressed at the transcriptional level. Although the flowering time of the CO transgenic plant was about 2 weeks earlier than that of the non-transgenic plant under short-day conditions, no significant difference in artemisinin content was found between the flowering transgenic plant and the non-flowering non-transgenic plant. These results show that the usually observed increase of artemisinin content before plant flowering under natural conditions is not a direct consequence of flowering itself, perhaps there is even no direct linkage between flowering and artemisinin biosynthesis.

Agrobacterium tumefaciens-mediated high frequency genetic transformation of an Indian cowpea ( Vigna unguiculata L. Walp.) cultivar and transmission of transgenes into progeny

A reproducible Agrobacterium tumefaciens-mediated genetic transformation system for the production of fertile transgenic plants of cowpea ( Vigna unguiculata) that transmits transgenes into progeny in Mendelian fashion has been developed. The cotyledonary node explants excised from 4-d-old seedlings, raised in vitro on medium containing salts of Murashige and Skoog's and vitamins of Gamborg's media (MBM) and BAP (10 μM), were co-cultured with Agrobacterium strain EHA105 harboring a binary vector pCAMBIA2301 that carried β-glucuronidase ( uidA) interrupted with an intron and neomycin phosphotransferase ( nptII) genes as a reporter and a selectable marker, respectively. The green shoots recovered from Agro-infected explants on selection medium (MBM containing BAP 5 μM, kanamycin 85 mg l −1 and cefotaxime 500 mg l −1) were rooted on MS supplemented with IBA (2.5 μM) and kanamycin (10 mg l −1). The rooted shoots that were found positive for the nptII gene by polymerase chain reaction (PCR) were established in soil and grown to maturity to collect the seeds. The integration of nptII gene into the cowpea genome was confirmed by Southern analysis. GUS activity was detected in vegetative and reproductive organs of T 0 plants and T 1 seedlings. The transgenes inherited in Mendelian fashion in T 1 progeny as detected by PCR. The transformation efficiency was 0.76% and 20–23 weeks were required from seed to seed generation time. This protocol can be used to introduce agronomically desired genes in cowpea for its genetic improvement.

Agrobacterium tumefaciens-mediated genetic transformation of barley ( Hordeum vulgare L.)

A critical step in the development of a robust Agrobacterium tumefaciens-mediated transformation system for a recalcitrant species, like barley, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. We used Agrobacterium strain LBA4404 harbouring either binary vector pUGAB7, which contains a T-DNA incorporating the bar gene and uidA gene, or pYF133, which contains hpt gene and gfp gene. These were used to investigate and optimize major T-DNA delivery and tissue culture variables in the immature embryos of barley (cv. Golden Promise). A number of factors produced significant difference in T-DNA delivery. These included pre-culture of immature embryos, co-cultivation, presence of acetosyringone and sonication and vacuum filtration assisted inoculation of 1-day precultured immature embryos. Plant regeneration was achieved via somatic embryogenesis. Regeneration media showed significant difference in their capacity to support regeneration of transgenic plants. Optimizing factors for T-DNA delivery resulted in bialaphos and hygromycin resistant barley transgenic plants with transformation efficiencies ranging from 2.6 to 5.6% and 3.3 to 6.7%, respectively. Molecular analysis of transgenic plants demonstrated that transgenes were integrated into the barley genome and subsequently transmitted into progeny at Mendelian ratios. The developed protocol will facilitate the insertion of desirable traits into barley via Agrobacterium-mediated transformation.

Transformation of the entomopathogenic fungus Paecilomyces fumosoroseus with Agrobacterium tumefaciens

To test the suitability of the Agrobacterium tumefaciens-mediated transformation (AMT) method with Paecilomyces fumosoroseus, a fungal pathogen that causes diseases in a wide range of insects including whiteflies. Conidia of P. fumosoroseus were successfully transformed to hygromycin B resistance using the hph gene of Escherichia coli as the selectable marker. Transformation frequencies were 58.3 +/- 18.5, 98.3 +/- 24.8 and 169.7 +/- 35.5 (+/-SEM) transformants per 10(5), 10(6) and 10(7) target conidia respectively. After confirmation by PCR, transformants were subjected to Southern analysis, and the results revealed that 45% (four of nine) of the transformants contained single-copy integration of the T-DNA. In our AMT system, we efficiently transformed conidia of P. fumosoroseus. The employment of this method circumvents time-consuming protoplast preparation and allows the isolation of transformants containing single-copy integration of the T-DNA. Considering the efficiency of Ag. tumefaciens-mediated transformation, this method represents a useful tool for insertional mutagenesis to characterize genes that are important for the pathogenicity of P. fumosoroseus.

Agrobacterium-mediated transformation of Perilla (Perilla frutescens)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l−1 phosphinothricin (PPT) and 150 mg l−1 kanamycin, respectively, for shoot induction and 1 mg l−1 PPT and 125 mg l−1 kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T0 and T1 plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T0 and T1 transgenic plants.

Disruption of the subtilase gene, albin1, in Ophiostoma piliferum.

Wood sapstaining fungi produce multiple proteases that break down wood protein. Three groups of subtilases have been identified in sapstaining fungi; however, it is not known if these groups have distinct physiological roles (B. Hoffman and C. Breuil, Curr. Genet. 41:168-175, 2002). In this work we examined the role of the subtilase Albin1 from Ophiostoma piliferum. Reamplification of cDNA ends PCR was used to obtain the albin1 gene sequence. The encoded subtilase is probably extracellular and involved in nutrient acquisition. This gene was disrupted with an Agrobacterium tumefaciens-mediated transformation system. Two of the disruptants obtained had significantly lower levels of proteolytic activity, slower growth in bovine serum albumin, and significantly reduced growth on wood. Thus, albin1 plays an important role in O. piliferum's ability to acquire nitrogen from wood proteins.

Moxalactam as a counter-selection antibiotic for Agrobacterium-mediated transformation and its positive effects on Theobroma cacao somatic embryogenesis

Previous work in a number of laboratories has led to the development of a highly efficient cacao somatic embryogenesis (SE) protocol, establishing the basis for an Agrobacterium tumefaciens-mediated genetic transformation system. Such a system will be valuable as a basic research tool, and possibly in the future as a means to introduce specific disease resistance or other genes into cacao. However, during our initial attempts at transformation, cacao tissue was frequently destroyed due to Agrobacterium overgrowth following co-cultivation. Furthermore, the addition of the antibiotic cefotaxime, commonly applied to tissue culture media to eliminate Agrobacterium post infection, decreased cacao somatic embryo production by 86%. To define antibiotic conditions sufficient to suppress Agrobacterium while not interfering with cacao SE, we evaluated the efficacy of four antibiotics. Two cephalosporins, cefotaxime and moxalactam; and two penicillins, amoxicillin and carbenicillin. Moxalactam, a β-Lactam antibiotic, was proven to effectively suppress Agrobacterium growth. Furthermore, at certain concentrations, it also significantly enhanced the efficiency of cacao SE. A possible mechanism for this enhancement is proposed.

Agrobacterium tumefaciens -mediated transformation of Ocimum basilicum and O. citriodorum

Transgenic basil (Ocimum spp.) was produced for the first time using an Agrobacterium tumefaciens-mediated transformation system. β-Glucuronidase (GUS) transient expression experiments showed a 96% or higher infection rate using Agrobacterium strains GV3101 and EHA105. Inoculated leaf explants were regenerated on Murashige and Skoog medium supplemented with thidiazuron, naphthaleneacetic acid and kanamycin. Two sweet basil (cvs SBA28 and Purple Ruffles, O. basilicum) and two lemon basil (cv Sweet Dani, O. citriodorum) plants were regenerated after in vitro cultivation. Stable transformation of both species was verified by polymerase chain reaction amplification (PCR) of both the gus (697 bp) and nptII (320 bp) gene fragments and by Southern blot analysis for the gus gene. Inheritance of the transgenes was verified by the GUS colorimetric assay and PCR analysis of the T1 generation of Ocimum transgenic plants, and the observed segregation ratio (3:1) indicated a single gene for GUS. Transgenic T1 plants exhibited identical phenotypic characteristics and accumulated high concentrations of linalool (118.98 µg g–1), 1.8-cineole (20.86 µg g–1), eugenol (69.3 µg g–1), neral (468.61 µg g–1) and geraniol (649.1 µg g–1) in volatile oil profiles similar to those of the non-transgenic controls.

Expression of a chimeric farnesyl diphosphate synthase gene in Artemisia annua L. transgenic plants via Agrobacterium tumefaciens-mediated transformation

An Agrobacterium tumefaciens-mediated transformation system was developed for Artemisia annua L. Using this system a cDNA encoding farnesyl diphosphate synthase (FDS placed under a CaMV 35S promoter) was transferred into A. annua via A. tumefaciens strain LB4404. Leaf or leaf discs were used as explants to be infected with A. tumefaciens and an optimal concentration of 20 mg/l kanamycin was applied to select kanamycin resistant shoots. Forty-five lines of resistance kanamycin shoots transformed with FDS were established. Analysis of PCR showed that at least 20 shoots transformed with the FDS gene were PCR positive. Southern blot analysis suggested the foreign FDS gene had been integrated into the A. annua genome, and Northern blot analysis revealed that the foreign FDS gene expressed at the transcriptional level in five shoot lines (F-1, F-4, F-61, F-62 and F-73 shoot lines). Analysis of artemisinin demonstrated that about 8∼10 mg/g DW of artemisinin were then detected in transgenic plants regenerated from five shoot lines, this is about 2–3 times higher than that in the control.

The Effect of Elevated Concentrations of Fructose 2,6-Bisphosphate on Carbon Metabolism during Deacidification in the Crassulacean Acid Metabolism Plant Kalanchöe daigremontiana.

In C(3) plants, the metabolite fructose 2,6-bisphosphate (Fru 2,6-P(2)) has an important role in the regulation of carbon partitioning during photosynthesis. To investigate the impact of Fru 2,6-P(2) on carbon metabolism during Crassulacean acid metabolism (CAM), we have developed an Agrobacterium tumefaciens-mediated transformation system in order to alter genetically the obligate CAM plant Kalanchöe daigremontiana. To our knowledge, this is the first report to use genetic manipulation of a CAM species to increase our understanding of this important form of plant metabolism. Transgenic plants were generated containing a modified rat liver 6-phosphofructo-2-kinase gene. In the plants analyzed the activity of 6-phosphofructo-2-kinase ranged from 175% to 198% of that observed in wild-type plants, resulting in Fru 2,6-P(2) concentrations that were 228% to 350% of wild-type plants after 2 h of illumination. A range of metabolic measurements were made on these transgenic plants to investigate the possible roles of Fru 2,6-P(2) during Suc, starch, and malic acid metabolism across the deacidification period of CAM. The results suggest that Fru 2,6-P(2) plays a major role in regulating partitioning between Suc and starch synthesis during photosynthesis. However, alterations in Fru 2,6-P(2) levels had little effect on malate mobilization during CAM fluxes.

Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens.

A rapid Agrobacterium tumefaciens-mediated transformation system for wheat was developed using freshly isolated immature embryos, precultured immature embryos, and embryogenic calli as explants. The explants were inoculated with a disarmed A. tumefaciens strain C58 (ABI) harboring the binary vector pMON18365 containing the [beta]-glucuronidase gene with an intron, and a selectable marker, the neomycin phosphotransferase II gene. Various factors were found to influence the transfer-DNA delivery efficiency, such as explant tissue and surfactants present in the inoculation medium. The inoculated immature embryos or embryogenic calli were selected on G418-containing media. Transgenic plants were regenerated from all three types of explants. The total time required from inoculation to the establishment of plants in soil was 2.5 to 3 months. So far, more than 100 transgenic events have been produced. Almost all transformants were morphologically normal. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. One to five copies of the transgene were integrated into the wheat genome without rearrangement. Approximately 35% of the transgenic plants received a single copy of the transgenes based on Southern analysis of 26 events. Transgenes in T1 progeny segregated in a Mendelian fashion in most of the transgenic plants.

Introduction of new traits into cotton through genetic engineering: insect resistance as example.

The main goal of gene transfer into cotton is the development of insect-resistant varieties. The stakes are important since cotton protection against insects uses almost 24% of the world's chemical insecticides market, which is not without consequences on the environment. The first approach was to introduce and express in the cotton genome, genes from the bacterium Bacillus thuringiensis (B.t.) which produces entomopathogenic toxins. The development of an efficient Agrobacterium tumefaciens mediated transformation system was the first step. The expression of B.t. genes was studied and synthetic genes more adapted to a plant genome have been constructed. Studies on their expression in cotton is underway. The second focus was to develop strategies that would minimize the risks of inducing insect resistance. The main approach is to associate several genes coding for entomopathogenic proteins with different modes of action. Genes encoding protease inhibitors were chosen. One possibility is to associate a B.t. gene and a gene encoding a protease inhibitor. Several protease inhibitors were tested in artificial diets on major pests of cotton. The corresponding genes have been introduced into the cotton genome. These various orientations of the research program will be presented.