Towards better insect management strategy: restriction of insecticidal gene expression to biting sites in transgenic cotton

Most of the commercialized Bt crops express cry genes under 35S promoter that induces strong gene expression in all plant parts. However, targeted foreign gene expression in plants is esteemed more important as public may be likely to accept ‘less intrusive’ expression of transgene. We developed plant expression constructs harboring cry1Ac gene under control of wound-inducible promoter (AoPR1) to confine Bt gene expression in insect wounding parts of the plants in comparison with cry1Ac gene under the control of 35S promoter. The constructs were used to transform four Turkish cotton cultivars (GSN-12, STN-468, Ozbek-100 and Ayhan-107) through Agrobacterium tumefaciens strains GV2260 containing binary vectors p35SAcBAR.101 and AoPR1AcBAR.101 harboring cry1Ac gene under control of 35S and AoPR1, respectively. Phosphinothricin (PPT) was used at concentration of 5 mg L^?1 for selection of primary transformants. The primary transformants were analyzed for transgene presence and expression standard molecular techniques. The transformants exhibited appreciable mortality rates against larvae of Spodoptera exigua and S. littoralis. It was found that mechanical wounding of T ₁ transgenic plants was effective in inducing expression of cry1Ac protein as accumulated levels of cry1Ac protein increased during post-wounding period. We conclude that use of wound-inducible promoter to drive insecticidal gene(s) can be regarded as a valuable insect-resistant management strategy since the promoter activity is limited to insect biting sites of plant. There is no Bt toxin accumulation in unwounded plant organs, seed and crop residues, cotton products and by-products, thus minimizing food and environmental concerns.     

Sequential transformation to pyramid two Bt genes in vegetable Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> L.) and its potential for control of diamondback moth larvae

Vegetable Indian mustard (Brassica juncea cv. “Green Wave”) plants that control Plutella xylostella (diamondback moth) (DBM) were produced by introduction of one or two Bacillus thuringiensis (Bt) genes. A cry1Ac Bt gene associated with the nptII gene for kanamycin selection or a cry1C Bt gene with the hpt gene for hygromycin selection was introduced individually through Agrobacterium-mediated transformation of seedling explants. A cry1C line was then transformed with the cry1Ac gene to produce pyramided cry1Ac + cry1C plants. Sixteen cry1C, five cry1Ac, and six cry1Ac + cry1C plants were produced. PCR and Southern analyses confirmed the presence of the cry1C, cry1Ac or pyramided cry1Ac + cry1C genes in the Indian mustard genome. ELISA analysis showed that production of Bt proteins varied greatly among individual transgenic plants, ranging from undetectable to over 1,000 ng Bt/mg total soluble protein. The levels of the Bt proteins were correlated with the effectiveness of control of diamondback moth (DBM) larvae. Insect bioassays indicated that both the cry1C and cry1Ac plants were toxic to susceptible DBM. The cry1C plants also controlled Cry1A-resistant DBM while cry1Ac plants controlled Cry1C-resistant DBM, and the pyramided cry1Ac + cry1C plants effectively controlled all three types of DBM. These Bt-transgenic plants could be used either for direct control of DBM and other lepidopteran insect pests or for tests of “dead-end” trap crops as protection of high value non-transgenic crucifer vegetables such as cabbage.     

Expression of Cry1Ac in Transgenic Tobacco Plants Under the Control of a Wound-Inducible Promoter (AoPR1) Isolated from Asparagus officinalis to Control Heliothis virescens and Manduca sexta

Expression of cry1Ac gene from Bacillus thuringiensis (Bt) was evaluated under the control of a wound-inducible AoPR1 promoter from Asparagus officinalis in transgenic tobacco plants. The leaves of transgenic plants were mechanically wounded to evaluate the activity of the AoPR1 promoter in driving the expression of Cry1Ac protein at the wound site. Our results indicate that mechanical wounding of transgenic plants was effective in inducing the expression of Cry1Ac protein. As a result of this induction, the accumulated levels of Cry1Ac protein increased during 6–72 h post-wounding period. The leaves of transgenic tobacco plants were evaluated for resistance against Heliothis virescens and Manduca sexta in insect bioassays in two different ways. The detached tobacco leaves were either fed directly to the insect larvae or they were first mechanically wounded followed by a 72 h post-wounding feeding period. Complete protection of mechanically wounded leaves of transgenic plants was observed within 24 h of the bioassay. The leaves of transgenic plants fed directly (without pre-wounding) to the larvae achieved the same level of protection between 24 and 72 h of the bioassay.     

Elite Indica Transgenic Rice Plants Expressing Modified Cry1Ac Endotoxin of Bacillus Thuringiensis Show Enhanced Resistance to Yellow Stem Borer (Scirpophaga Incertulas)

Bt-transgenics of elite indica rice breeding lines (IR-64, Pusa Basmati-1 and Karnal Local) were generated through biolistic of Agrobacterium-mediated approaches. A synthetic cry1Ac gene, codon optimised for rice and driven by the maize ubiquitin-1 promoter, was used. Over 200 putative transformants of IR-64 and Pusa Basmati-1 and 26 of the Karnal Local were regenerated following use of the hpt (hygromycin phosphotransferase) selection system. Initial transformation frequency was in the range of 1 to 2% for particle bombardment while it was comparatively higher ( 9%) for Agrobacterium. An improved selection procedure, involving longer selection on the antibiotic-supplemented medium, enhanced the frequency of Bt-transformants and reduced the number of escapes. Molecular evaluation revealed multiple transgene insertions in transformants, whether generated through biolistic or Agrobacterium. In the latter case, it was also observed that all genes on the T-DNA do not necessarily get transferred as an intact insert. Selected Bt-lines of IR-64 and Pusa Basmati-1, having Bt-titers of 0.1% (of total soluble protein) and above were evaluated for resistance against manual infestation of freshly hatched neonate larvae of yellow stem borers collected from a hot spot stem borer infested area in northern India. Several Bt-lines were identified showing 100% mortality of larvae, within 4-days of infestation, in cut-stem as well as vegetative stage whole plant assays. However, there was an occasional white head even among such plants when assayed at the reproductive stage. Results are discussed in the light of resistance management strategies for deployment of Bt-rice.     

Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene

Transposon-mediated repositioning of transgenes is an attractive strategy to generate plants that are free of selectable markers and T-DNA inserts. By using a minimal number of transformation events a large number of transgene insertions in the genome can be obtained so as to benefit from position effects in the genome that can contribute to higher levels of expression. We constructed a Bacillus thuringiensis synthetic cry1B gene expressed under control of the maize ubiquitin promoter between minimal terminal inverted repeats of the maize Ac-Ds transposon system, which was cloned in the 5' untranslated sequence of a gfp gene used as an excision marker. The T-DNA also harboured the Ac transposase gene driven by the CaMV 35S promoter and the hph gene conferring resistance to the antibiotic hygromycin. Sixty-eight independent rice (Oryza sativa L.) transformants were regenerated and molecularly analysed revealing excision and reinsertion of the Ds-cry1B element in 37% and 25% respectively of the transformation events. Five independent transformants harbouring 2–4 reinserted Ds-Cry1B copies were analysed in the T1 progeny, revealing 0.2 to 1.4 new transpositions per plant. Out segregation of the cry1B gene from the T-DNA insertion site was observed in 17 T1 plants, representing 10 independent repositioning events without selection. Western analysis of leaf protein extracts of these plants revealed detectable Cry1B in all the plants indicating efficient expression of the transgene reinsertions. Stability of position and expression of the cry1B transgene was further confirmed in T2 progeny of T-DNA-free T1 plants. New T-DNA-free repositioning events were also identified in T2 progenies of T1 plants heterozygous for the T-DNA. Furthermore, preliminary whole plant bioassay of T-DNA-free lines challenged with striped stem borer larvae suggested that they are protected against SSB attacks. These results indicate that transposon mediated relocation of the gene of interest is a powerful method for generating T-DNA integration site-free transgenic plants and exploiting favourable position effects in the plant genome.     

The −689/+197 region of the maize protease inhibitor gene directs high level,wound-inducible expression of the cry1B gene which protects transgenic rice plants from stemborer attack

To investigate the activity of the regulatory region of the maize (Zea mays L.) proteinase inhibitor (mpi) gene, we transferred into rice (Oryza sativa L.) plants the –689/+197 (C1) fragment of the mpi genomic clone fused to either theuidA gene or a synthetic Bacillus thuringiensis cry1B gene. Although uidA and cry1B encode very different proteins consistent results were obtained from their respective histochemical and fluorometric and immunoblot detections in T₃ transgenic rice lines. In response to mechanical wounding, a 4–5 fold increase in GUS activity and a Cry1B accumulation reaching 0.1–0.2% of total soluble proteins were observed from basal and undetectable levels respectively in leaf tissue. The establishment of the time-course of wound response in both systems revealed a maximum induction level 12–16 h after treatment. From both systems we also deduced that the C1 region is not active in pollen and seed endosperm. Three independent transformation events expressing cry1B under the control of the C1 region exhibited protection against striped stem borer damage and showed 100% mortality of second instar larvae 8 days after release. These results illustrate the first evidence that wound-inducible expression of a Bacillus thuringiensis endotoxin gene affords full protection to transgenic rice plants.     

Expression of multiple insecticidal genes confers broad resistance against a range of different rice pests

We report the simultaneous introduction of three insecticidal genes (the Bt genes cry1Ac and cry2A, and the snowdrop lectin gene gna) into commercially important indica rice varieties M7 and Basmati 370, by particle bombardment. Transgenic plants expressed Cry1Ac, Cry2A and GNA at different levels, either singly or in combination at 0.03–1%, 0.01–0.5% and 0.01–2.5% of total soluble protein, respectively. The transgenes showed stable transmission and expression, and R₁ transgenic plants provided significant (p<0.01) protection against three of the most important insect pests of rice: rice leaf folder (Cnaphalocrocis medinalis), yellow stemborer (Scirpophaga incertulas) and brown planthopper (Nilaparvata lugens). The triple transformants showed significantly (p<0.05) higher resistance to these insects than plants expressing single transgenes. Bioassays using the triple-transgenic plants showed 100% eradication of the rice leaf folder and yellow stem borer, and 25% reduction in the survival of the brown planthopper. The greatest reduction in insect survival, and the greatest reduction in plant damage, occurred in plants expressing all three transgenes. This approach maximises the utility of gene transfer technology to introduce combinations of genes whose products disrupt different biochemical or physiological processes in the same insect, providing a multi-mechanism defence.     

Tolerance of Bt corn (MON 810) to maize stem borer, Chilo partellus (Lepidoptera: Pyralidae)

Transgenic corn (MON 810), expressing the Bacillus thuringiensis (Bt) protein, Cry1Ab, was evaluated under greenhouse conditions for its tolerance to the maize stem borer, Chilo partellus. Bt corn (MON 810) provided effective protection against the stem borer even under a high level of larval infestation in the greenhouse. The observed tolerance is examined and discussed in the light of the susceptibility of C. partellus to the Cry1Ab protein in laboratory bioassays. The implications of the tissue concentrations of Cry1Ab in MON 810, and baseline susceptibility recorded in the current study, for insect-resistance management are discussed.     

Efficient genetic transformation of okra (Abelmoschus esculentus (L.) Moench) and generation of insect-resistant transgenic plants expressing the cry1Ac gene

Key message

Agrobacterium -mediated transformation system for okra using embryos was devised and the transgenic Bt plants showed resistance to the target pest, okra shoot, and fruit borer ( Earias vittella ).

Abstract

Okra is an important vegetable crop and progress in genetic improvement via genetic transformation has been impeded by its recalcitrant nature. In this paper, we describe a procedure using embryo explants for Agrobacterium-mediated transformation and tissue culture-based plant regeneration for efficient genetic transformation of okra. Twenty-one transgenic okra lines expressing the Bacillus thuringiensis gene cry1Ac were generated from five transformation experiments. Molecular analysis (PCR and Southern) confirmed the presence of the transgene and double-antibody sandwich ELISA analysis revealed Cry1Ac protein expression in the transgenic plants. All 21 transgenic plants were phenotypically normal and fertile. T₁ generation plants from these lines were used in segregation analysis of the transgene. Ten transgenic lines were selected randomly for Southern hybridization and the results confirmed the presence of transgene integration into the genome. Normal Mendelian inheritance (3:1) of cry1Ac gene was observed in 12 lines out of the 21 T₀ lines. We selected 11 transgenic lines segregating in a 3:1 ratio for the presence of one transgene for insect bioassays using larvae of fruit and shoot borer (Earias vittella). Fruit from seven transgenic lines caused 100 % larval mortality. We demonstrate an efficient transformation system for okra which will accelerate the development of transgenic okra with novel agronomically useful traits.     

Linear transgene constructs lacking vector backbone sequences generate transgenic rice plants which accumulate higher levels of proteins conferring insect resistance

Biolistic transformation was used to introduce genes encoding the insecticidal proteins snowdrop lectin (Galanthus nivalis agglutinin; GNA) and cry1Ac Bt toxin (-endotoxin from Bacillus thuringiensis) into elite rice (Oryza sativa) cultivars. Plant transformation was carried out in parallel experiments simultaneously by using either whole plasmids containing suitable gene constructs, or the corresponding minimal gene cassettes, which were linear DNA fragments lacking vector sequences excised from the plasmids. Both transformation methods generated similar numbers of independent transformation events. Selected R₀ clonal plant lines were further characterised for presence and expression of transgenes. Co-transformation of the unselected genes (cry1Ac and gna) with the selectable marker (hpt) was at least as efficient for transformation with minimal gene cassettes as with whole plasmid DNA, and higher levels of accumulation of the insecticidal gene products GNA and cry1Ac were observed in plants resulting from minimal gene cassette transformation. Insect bioassays with major pests of rice showed that transgenic plants expressing gna showed enhanced resistance to brown planthopper (Nilaparvata lugens), and plants expressing cry1Ac were protected against attack by striped stem borer (Chilo suppressalis). Expression of both transgenes gave protection against both pests, but did not increase protection against either pest significantly over the levels observed in plants containing a single insecticidal transgene.     

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2^nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.     

Expression of <Emphasis Type="Italic">GNA</Emphasis> and biting site-restricted <Emphasis Type="Italic">cry1Ac</Emphasis> in cotton; an efficient attribution to insect pest management strategies

Insect-resistant transgenic cotton has been commercialized for two decades. Most of the introduced cultivars express Bt gene(s) constitutively under the control of 35S promoter in whole-plant tissues. However, there have been other promoters considered by researchers to confine the toxin expression to targeted organ and tissues. We developed a triple-gene construct including GNA, cry1Ac and cp4 epsps genes. We attempted to confine cry1Ac expression to insect biting sites by cloning it to downstream of a wound-inducible promoter isolated from Asparagus officinalis (AoPR1). Moreover, to broaden the range of resistance, GNA was driven by the 35S promoter to target the sap-sucking insects like aphids which impose large losses in cotton production. To select the transformants in selection medium and for glyphosate tolerance, GNA and cry1Ac genes were accompanied with cp4 epsps gene. Two binary vectors harboring desired genes were constructed and utilized in the study (pGTGNAoC1AC and pGTGN35C1AC). Transformation of cultivar GSN-12 was carried out by employing Agrobacterium tumefaciens strain EHA105. Plantlets were primarily screened under glyphosate (N-phosphonomethyl glycine) selection pressure and subsequently subjected to molecular and biotoxicity assays. Introduction of cry1Ac and GNA to cotton plant conferred resistance to Spodoptera littoralis and Aphis gossypii Glover. Restriction of cry1Ac toxin protein to insect biting sites along with a plant lectin attributes significantly to insect pest management strategies.     

Influence of transgenic cottons with <Emphasis Type="Italic">Bacillus thuringiensis cry1Ac</Emphasis> gene on the natural enemies of <Emphasis Type="Italic">Helicoverpa armigera</Emphasis>

Transgenic cotton has been released for cultivation in several parts of the world to increase crop productivity. However, concerns have been raised regarding the possible undesirable effects of genetically modified crops on non-target organisms in the eco-system. Therefore, we studied the effects of transgenic cottons with cry1Ac gene from Bacillus thuringiensis Berliner (Bt) on the natural enemies of cotton bollworm/legume pod borer, Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) under field and laboratory conditions. There was no apparent effect of transgenic cotton on the relative abundance of predatory spiders (Clubiona sp. and Neoscona sp.), coccinellid (Cheilomenes sexmaculatus Fab.), and the chrysopid (Chrysoperla carnea Stephens). However, the abundance of spiders, coccinellids, and chrysopids was quite low in insecticide protected plots towards end of the cropping season. There was a significant reduction in cocoon formation and adult emergence of the ichneumonid parasitoid, Campoletis chlorideae Uchnida reared on H. armigera larvae fed on the leaves of transgenic cottons before and after parasitization. However, no Bt toxins were detected in H. armigera larvae and the parasitoid cocoons with enzyme linked immunosorbent assay. Reduction in cocoon formation was because of early mortality of the H. armigera larvae due to Bt toxins in the leaves of transgenic cotton. There was a slight reduction in adult weight and fecundity, and prolongation of the larval period when the parasitoid was raised on H. armigera larvae fed on the leaves of transgenic cotton before and after parasitization. Survival and development of C. chlorideae was also poor when H. armigera larvae were fed on the leaves of cotton hybrid Mech 184. The adverse effects of transgenic cotton on survival and development of C. chlorideae were largely due to early mortality, and possibly poor nutritional quality of H. armigera larvae due to toxic effects of the transgene.     

Combining cry1Ac with QTL alleles from PI 229358 to improve soybean resistance to lepidopteran pests

A QTL conditioning corn earworm resistance in soybean PI 229358 and asynthetic Bacillus thuringiensis cry1Ac transgene from therecurrent parent Jack-Bt were pyramided intoBC₂F₃ plants by marker-assisted selection. Segregatingindividuals were genotyped at SSR markers linked to an anitbiosis/antixenosisQTL on linkage group M, and were tested for the presence ofcry1Ac. Marker-assisted selection was used during andafter the two backcrosses to develop a series of BC₂F₃plants with or without the crylAc transgene and the QTLconditioning for resistance BC₂F₃ plants that werehomozygous for parental alleles at markers on LG M, and whicheither had or lacked cry1Ac, were assigned to one of fourpossible genotype classes. These plants were used in no-choice, detached leaffeeding bioassays with corn earworm and soybean looper larvae (Lepidoptera:Noctuidae) to evaluate the relative antibiosis in the different genotypeclasses. Resistance was measured as larval weight gain and degree of foliageconsumption. Few larvae of either species survived on leaves expressing theCry1Ac protein. Though not as great as the effect of Cry1Ac, the PI229358-derived LG M QTL also had a detrimental effect on larval weights of bothpest species, and on defoliation by corn earworm, but did not reduce defoliation bysoybean looper. Weights of soybean looper larvae fed foliage from transgenicplants with the PI-derived QTL were lower than those of larvae fed transgenictissue with the corresponding Jack chromosomal segment. This work demonstratesthe usefulness of SSRs for marker-assisted selection in soybean, and shows thatcombining transgene-and QTL-mediated resistance to lepidopteran pests may be aviable strategy for insect control.     

The Bt gene <Emphasis Type="Italic">cry2Aa2</Emphasis> driven by a tissue specific ST-LS1 promoter from potato effectively controls <Emphasis Type="Italic">Heliothis virescens</Emphasis>

Expression of the Cry2Aa2 protein was targeted specifically to the green tissues of transgenic tobacco Nicotiana tabacum cv. Xanthi plants. This deployment was achieved by using the promoter region of the gene encoding the Solanum tuberosum leaf and stem specific (ST-LS1) protein. The accumulated levels of toxin in the leaves were found to be effective in achieving 100 mortality of Heliothis virescens larvae. The levels of Cry2Aa2 expression in the leaves of these transgenic plants were up to 0.21 of the total soluble proteins. Bioassays with R₁ transgenic plants indicated the inheritance of cry2Aa2 in the progeny plants. Tissue-specific expression of the Bt toxin in transgenic plants may help in controlling the potential occurrence of insect resistance by limiting the amount of toxin to only predated tissues. The results reported here validate the use of the ST-LS1 gene promoter for a targeted expression of Bt toxins in green tissues of plants.     

Transgenic rice plants with a synthetic cry1Ab gene from Bacillus thuringiensis were highly resistant to eight lepidopteran rice pest species

To fully explore the resistance potential of transgenic rice produced by Agrobacterium-mediated transformation, an elite line KMD1 was assessed for its resistance to eight lepidopteran rice pest species. KMD1 contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter. It was derived from a commercial japonica Chinese rice variety Xiushui 11, and bred true for both agronomic traits and a cry1Ab gene when the bioassays were done in 1998 in the R₅ generation. The eight lepidopteran pest species were: four Pyralidae species: Chilo suppressalis (striped stem borer, SSB), Scirpophaga incertulas (yellow stem borer, YSB), Cnaphalocrocis medinalis (leaf folder), Herpitogramma licarisalis; two Noctuidae: Sesamia inferens (pink stem borer, PSB) and Naranga anescens; one Stayridae: Mycalesis gotama; and one Hesperiidae, Parnara guttata. In laboratory bioassays, 100% mortality was observed in all insect species when their newly hatched or third-instar larvae were fed KMD1 leaf tissues, whereas only 9.65% of the neonates and none of the third-instar larvae died when fed the leaf tissues of non-transgenic control. Moreover, the leaf area of control tissues consumed in four days by stem borers was 20 to 40 times higher than that of KMD1 tissues, and the area of control tissues eaten by leaf-feeding species was 120 to 180 times greater than that of the transgenic tissues. Under natural infestation, no KMD1 plant was visibly damaged by the SSB, YSB and leaf folder in field evaluation. On the other hand, 80, 9.3 and 88.7% of control plants were injured by SSB, YSB, and leaf folder, respectively. These data disclosed that the transgenic line was highly resistant to a broad spectrum of lepidopteran insect species and could be useful in insect resistance breeding programs.     

β-Chemokines Are Produced in Lungs of Mice with Mycoplasma Respiratory Disease

The abilities of Bacillus polymyxa and Bacillus thuringiensis to survive on the rice phyllospere were compared; it was found that B. polymyxa colonizes the crop better. This study also showed that B. polymyxa inoculation to rice plants increased the shoot and the root growth of the crop. Efforts were made to introduce the cry1Ac gene of B. thuringiensis subsp. kurstaki into B. polymyxa so that the application of such transgenic B. polymyxa strains would prove to be dually beneficial to rice crops both as a biopesticide and as a biofertilizer. Immunoblot analysis of the recombinant organism containing the cry1Ac gene, strain BP113, indicated efficient expression of this gene in the heterologous host. Bioassays with the first instar larvae of the yellow stem borer of rice (Scirpophaga incertulas) revealed that the protein preparations from BP113 were toxic. Received: 12 August 1998 / Accepted: 25 September 1998