Transgenics in groundnut (Arachis hypogaea L.) expressing cry1AcF gene for resistance to Spodoptera litura (F.)

Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T₂ generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 μg/g FW. Further, Southern blot analysis of ten T₂ transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T₁ and T₂ generations, respectively. Segregation analysis of the selected lines in the T₃ generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T₂ generation, the cry1AcF gene was effective against S. litura only to some extent.     

Transformation and inheritance of Bt genes inGossypium hirsutum

Transgenic plants offer many unique opportunities for managing pest populations. However, the inheritance, integration, and expression of multiple transgenes are prerequisite for maintaining sustainable resistance against insects in crops. We took a gene-pyramiding approach to produce Bt cotton expressing two Bt genes,cry1Ac andcry2A. Using sonication-assistedAgrobacterium-mediated transformation (SAAT), we achieved an efficiency of 6.26%. Putative transgenic plants were confirmed via PCR, Southern hybridization, and western-blotting. Those showing mortality of 75 to 100% for the second instar ofHeliothis armigera (compared with 0% for the control) were considered Bt-positive. Transgenes were segregated according to a 3:1 Mendelian inheritance pattern in the T1 generation forHeliothis resistance. In our insect bioassay, the control plants showed >95% leaf damage, and insects reached the 4^th instar stage of larval growth. In contrast, leaf damage on transgenic plants was limited to only a few bites, and insect mortality was 75 to 100%. ELISA confirmed transgene expression, and Bt protein was detected in leaf tissue. This performance was consistent with that of the parent transgenics. PCR and Southern blots verified integration of thecry1Ac andcry2A genes into the progeny. Therefore, this strategy provides a pathway toward cotton improvement and the development of durable resistance against insect damage.     

cry Genes profiling and the toxicity of isolates of Bacillus thuringiensis from soil samples against American bollworm,Helicoverpa armigera

Aims: The aim of this study was to search for Bacillus thuringiensis (Bt) harbouring cry1A gene which could effectively control cotton pest, American bollworm, Helicoverpa armigera. Methods and Results: cry gene profiling of 50 Bt isolates showed the presence of cry1, cry2, cry3, cry4, cry7, cry8 and cry9 genes. None of the isolates harboured cry1 gene alone. It was always found in combination with cry3. There was no isolate positive for cry10 gene. Considering isolates with single cry genes, the frequency of cry4 was predominant (22%) followed cry2 (6%), cry3 (4%) and cry8 (2%). Isolates having two cry genes in combination had 14% incidence for cry2 + cry4, 12% for cry3 + cry4 and 10% for cry1 + cry3. The most dominant three gene linkage was cry1 + cry3 + cry4. Further profiling of cry1 gene showed that cry1K gene was abundantly present in all combinations such as cry1A, cry1D, cry1F and cry1I. However, cry1C existed independent of other subtypes. Finally, the Bt isolates with cry1A were analyzed for 16S rRNA gene, which showed two distinct groups of isolates on the basis of sequence homology. Bioassays of spore–crystal mixtures of SBS‐Bt4, 8, 17, 21 and 26 harbouring cry1 against neonate larvae of H. armigera showed LC₅₀ 1288, 1202, 467·7, 524·8 and 108·5 μg ml^?1. The SBS‐Bt26 showed fourfold higher toxicity than the cry 1Ac harbouring positive control, HD‐73. Conclusions: None of the isolates harboured single cry 1 gene. They were always in combination of two or three genes. A Bt isolate (Bt26) had fourfold higher toxicity against H. armigera larvae compared with the positive control HD 73 and hence can be commercially exploited to control insect pest. Significance and Impact of the Study: The inter relationship between the cry genes content and the toxicity may allow better understanding of Bt ecology.     

Novel synthetic Bacillus thuringiensiscry1B gene and the cry1B-cry1Ab translational fusion confer resistance to southwestern corn borer, sugarcane borer and fall armyworm in transgenic tropical maize

In order to develop a resistance management strategy to control tropical pests based on the co-expression of different toxins, a fully modified Bacillus thuringiensis cry1B gene and the translational fusion cry1B-cry1Ab gene have been developed. Both constructs were cloned under the control of a maize ubiquitin-1 or a rice actin-1 promoter and linked to the bar gene driven by the CaMV 35S promoter. Immature embryos from the tropical lines CML72, CML216, and their hybrids, were used as the target for transformation by microprojectile bombardment. Twenty five percent of the transformed maize plants with cry1B expressed a protein that is active against southwestern corn borer and sugarcane borer. Ten percent of the transgenic maize expressed single fusion proteins from the translational fusion gene cry1B-1Ab and showed resistance to these two pests as well as to the fall armyworm. Transgenic maize plants that carried the cry1B gene in T1 to T3 progenies transmitted trangenes with expected Mendelian segregation and conferred resistance to the two target insects. Molecular analyses confirmed the cry genes integration, the copy number, the size of protein(s) expressed in maize plants, the transmission, and the inheritance of the introduced cry gene. These new transgenic products will provide another recourse for reducing the build-up of resistance in pest populations. Received: 25 September 2000 / Accepted: 15 December 2000     

Survival of Helicoverpa armigera larvae on and Bt toxin expression in various parts of transgenic Bt cotton (Bollgard II) plants

There is no conclusive evidence that Helicoverpa spp. (Lepidoptera: Noctuidae) in Australia have evolved significant levels of resistance to Bollgard II^® cotton (which expresses two Bt toxin genes, cry1Ac and cry2Ab). However, there is evidence of surviving larvae on Bollgard II cotton in the field. The distribution and survival of early‐instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae were examined on whole Bollgard II and non‐Bt cotton plants in greenhouse bioassays. The expression of Cry toxins in various parts of Bollgard II plants was compared to the survival of larvae in those locations. Only 1% of larvae survived after 6 days on greenhouse‐grown Bollgard II plants compared to 31% on non‐Bt cotton plants. Overall, and across all time intervals, more larvae survived on reproductive parts (squares, flowers, and bolls) than on vegetative parts (leaves, stems, and petioles) on Bollgard II plants. The concentration of Cry1Ac toxin did not differ between plant structures, whereas Cry2Ab toxin differed significantly, but there was no relationship between the level of expression and the location of larvae. This study provides no evidence that lower expression of Cry toxins in the reproductive parts of plants explains the survival of H. armigera larvae on Bollgard II cotton.     

Diversity of Bacillus thuringiensis strains from soil in China and their pesticidal activities

A large number of Bacillus thuringiensis (Bt) isolates have been obtained from soil samples in China. The flagellar antigen serotypes, cry genes and crystal proteins of 570 Bt isolates were determined, and the pesticidal activity was assayed against the insects, Plutella xylostella, Heliothis armigera, Phaedon brassicae and Locusta migratoria manilensis, and the snail, Oncomelania hupensis. The results indicated that the Bt isolates were distributed within 35 H-serotypes, in which isolates of H3 were the most abundant (20%) followed by H5 (13%), H7 (9%) and H4 (8.7%), whereas isolates of other H-serotypes were less than 6%. The percentage of isolates containing the genes cry1Ac, cry1Aa/cry1Ac, cry1Aa/cry1Ac/cry1Ab, and cry1Aa/cry1Ac/cry1C was 14.7%, 6.6%, 5.6%, and 7.0%, respectively, while 265 isolates, representing 46.5% of the 570 Bt isolates, did not show any amplification product for the genes cry1Aa, cry1Ac, cry1C, cry2, cry3, cry4, cry7Aa. Some of the 570 Bt isolates caused high mortality of the assayed pests with 14.9%, 6%, 1.6%, 1.1%, and 0.2% of the isolates killing more than 90% of P. xylostella, H. armigera, P. brassicae, O. hupensis, and L. migratoria manilensis, respectively. The remaining 76.2% of the 570 isolates caused no mortality or less than 90% mortality against the tested insect and snail species.     

Characterisation and expression of a novel holotype crystal protein gene, <Emphasis Type="Italic">cry56Aa1</Emphasis>, from <Emphasis Type="Italic">Bacillus</Emphasis> <Emphasis Type="Italic">thuringiensis</Emphasis> strain Ywc2-8

Bacillus thuringiensis isolate Ywc2-8, from soil in Sichuan Basin in western China, contains a spherical crystal harbouring two insecticidal crystal proteins with masses of 70 kDa and 130 kDa. A novel cry-type gene, encoding a 664 amino acid protein with 34% homology to cry29Aa1, was found and cloned from this strain. This gene belongs to a novel holotype cry and is designated as cry56Aa1. It was expressed in E. coli. Insecticidal activity assays showed that recombinant Cry56Aa1 was toxic to both Dipteran (Aedes aegypti) and Lepidopteran (Plutella xylostella and Helicoverpa armigera) pests. Cloning of this gene may help to overcome the increasing resistance of pests to currently used insecticides.     

Natural occurrence of Bacillus thuringiensis in leguminous phylloplanes in the New Delhi region of India

Bacillus thuringiensis (Bt) isolates were present on the phylloplanes of chickpea (Cicer arietinum), pigeon pea (Cajanus cajan), pea (Pisum sativum) and mung bean (Vigna radiata). Bt index (ratio of the number of Bt colonies to the total number of spore-forming colonies per g of leaves) differed significantly among these plants, with the highest (0.20) in the chickpea phylloplane, followed by pigeon pea (0.17). Bt population of the chickpea phylloplane varied with plant age, being maximal in 45-day-old plants. Diversity was observed among Bt isolates for growth (up to 10-fold difference), antibiotic resistance, PCR product profile and toxicity to Helicoverpa armigera. Two isolates with high activity towards H. armigera were found. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetically modified coffee plants expressing the Bacillus thuringiensis cry1Ac gene for resistance to leaf miner

 A synthetic version of the cry1Ac gene of Bacillus thuringiensis has been used for the transformation of coffee species (Coffea canephora and C. arabica) to confer resistance to an important pest, the coffee leaf miner (Perileucoptera coffeella and other Leucoptera spp). Somatic embryos were co-cultivated with the LBA4404 strain of Agrobacterium tumefaciens containing the cry1Ac gene. More than 100 transformed plants from independent transformation events were obtained for each coffee genotype. The integration and expression of the cry1Ac gene was studied, and effective resistance of transgenic plants against leaf miner was verified in bioassays with the insects. These plants could represent a good opportunity to analyse the impact of genetic engineering of perennial crops for sustainable resistance to an obligate endocarpic pest using a B. thuringiensis insecticidal protein. Received: 7April 1999 / Revision received: 20 July 1999 / Accepted: 22 July 1999     

Codon optimization of <Emphasis Type="Italic">cry1Ab</Emphasis> gene for hyper expression in plant organelles

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.     

Cloning and characterization of truncated <Emphasis Type="Italic">cry1Ab</Emphasis> gene from a new indigenous isolate of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

The insecticidal crystal protein(s) encoded by cry gene(s) of Bacillus thuringiensis (Bt) have been used for insect control both as biopesticides and in transgenic plants. A new 3′-truncated cry1Ab gene was cloned from an indigenous isolate of Bt, A19-31. Nucleotide sequencing and homology search revealed that the deduced amino acid sequence of Cry1Ab toxin of Bt strain A19-31 had a variation of two amino acid residues with the holotype sequence, Cry1Ab1. Expression of the 3′-truncated cry1Ab gene was studied in an acrystalliferous strain of Bt (4Q7). SDS-PAGE and immunostrip analysis of spore-crystal mixture revealed a low level expression of the 3′-truncated cry1Ab gene. Insecticidal activity assay showed that the recombinant 3′-truncated cry1Ab gene product was toxic to larvae of both Helicoverpa armigera and Spodoptera litura.     

Detection and characterisation of a Bacillus thuringiensis crystal protein with nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus

Nematicidal Bacillus thuringiensis (Bt) strains were isolated from forests in Zhejiang, China for further characterisation. PCR analysis was performed with nine pairs of primers specific for cry1, cry2, cry3, cry4, cry5, cry6, cry9, cry11 and cry13 to characterise and classify cry gene groups from Bt isolates. The isolates from individual cry groups were tested for nematicidal activity against the pinewood nematode Bursaphelenchus xylophilus, which is implicated in pine wilt disease. PCR identified 14 different categories of cry gene combinations, indicating a large diversity of cry genes. The cry1 gene was by far the most abundant in Bt isolates and was found in 68% of samples. The Bt isolates zjfc85 and zjfc392 were from two distinct classes, but shared the same cry5 amplification profile and the same ~130 kDa protein; they had the highest nematicidal activity against pinewood nematode during the 48 h exposure tests, resulting in 90 and 59% mortality (9% of mortality under control conditions), respectively. The ~130 kDa Cry protein from isolate zjfc85 was purified and named as Cry5Ba3. Bioassay results indicated pinewood nematode was highly susceptible to Cry5Ba3 and exhibited profound growth abnormalities after exposure to Cry5Ba3. Our results are a novel finding and provide a potential strategy to manage pine wilt disease caused by B. xylophilus based on a nematicidal Bt.     

Two different Bacillus thuringiensis toxin genes confer resistance to beet armyworm (Spodoptera exigua Hübner) in transgenic Bt-shallots (Allium cepa L.)

Agrobacterium-mediated genetic transformation was applied to produce beet armyworm (Spodoptera exigua Hübner) resistant tropical shallots (Allium cepa L. group Aggregatum). A cry1Ca or a H04 hybrid gene from Bacillus thuringiensis, driven by the chrysanthemum ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (Rubisco SSU) promoter, along with the hygromycin phosphotransferase gene (hpt) driven by the CaMV 35S promoter, was employed for genetic transformation. An average transformation frequency of 3.68% was obtained from two shallot cultivars, Tropix and Kuning. After transfer of the in vitro plants to the greenhouse 69% of the cry1Ca and 39% of the H04 transgenic shallots survived the first half year. After one year of cultivation in the greenhouse the remaining cry1Ca and H04 transgenic plants grew vigorously and had a normal bulb formation, although the cry1Ca transgenic plants (and controls) had darker green leaves compared to their H04 counterparts. Standard PCR, adaptor ligation PCR and Southern analyses confirmed the integration of T-DNA into the shallot genome. Northern blot and ELISA analyses revealed expression of the cry1Ca or H04 gene in the transgenic plants. The amount of Cry1Ca expressed in transgenic plants was higher than the expression levels of H04 (0.39 vs. 0.16% of the total soluble leaf proteins, respectively). There was a good correlation between protein expression and beet armyworm resistance. Cry1Ca or H04 gene expression of at least 0.22 or 0.08% of the total soluble protein in shallot leaves was sufficient to give a complete resistance against beet armyworm. This confirms earlier observations that the H04 toxin is more toxic to S. exigua than the Cry1Ca toxin. The results from this study suggest that the cry1Ca and H04 transgenic shallots developed could be used for introducing resistance to beet armyworm in (sub) tropical shallot.     

Development of cotton transgenics with antisense <Emphasis Type="Italic">AV2</Emphasis> gene for resistance against cotton leaf curl virus (CLCuD) via <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Cotton transgenics for resistance against cotton leaf curl disease using antisense movement protein gene (AV2) were developed in an Indian variety (F846) via Agrobacterium-mediated transformation using the protocol developed previously. A binary vector pPZP carrying the antisense AV2 (350 bp) gene along with the nptII gene was used. Transgenic nature of the putative transgenics was confirmed by molecular analysis. Shoots were induced on selection medium and subcultured on rooting medium containing IBA and 75 mg l^–1 kanamycin. Transgenic plants were recovered in 12–16 weeks from the time of gene transfer to establishment in pots. Preliminary analysis of the field-established plantlets was conducted by PCR. T₁ plants were obtained from T₀ seeds, the presence of the AV2 and nptIIgenes in the transgenic plants was verified by PCR and integration of T-DNA with AV2 into the plant genome of putative transgenics was further confirmed by Southern blot analysis. Several T₁ lines were maintained in the greenhouse. Progeny analysis of these plants by PCR analysis showed a classical Mendelian pattern of inheritance.     

Isolation and characterization of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains from different grain habitats in Turkey

Bacillus thuringiensis (Bt) is a gram-positive, spore-forming bacterium and it produces insecticidal crystal (cry) proteins during sporulation. Because the genetic diversity and toxic potential of Bt strains differ from region to region, strains have been collected and characterized all over the world. The aim of this study is to isolate Bt strains in grain-related habitats in Turkey and to characterize them on the basis of crystal morphology, cry gene content, and chromosomal and plasmid DNA profiles. Four approaches were taken analysis with phase contrast (PC) microscopy, polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE) and plasmid isolation. Ninety-six samples were collected from Central Anatolia and the Aegean region. Bt was isolated from 61 of 96 samples (63.5) and 500 Bt-like colonies were obtained. One hundred and sixty three of the colonies were identified as Bt based on cry protein formation using PC microscopy. Among the examined colonies, the overall proportion identified (as Bt index) was 0.33. We found that 103 isolates were positive for the five different cry genes (cry1, cry2, cry3, cry4 and cry9) examined with PCR. In addition, plasmid profiling of 37 cry gene-positive isolates indicated that the 15 kb plasmid band was present in all isolates; however, 11 of 37 isolates had more than one plasmid band at different sizes. Finally, chromosomal DNA profiling by PFGE gave rise to different DNA patterns for isolates containing the same cry gene which suggests a high level of diversity among the Bt strains isolated.