Bacillus Thuringiensis Toxin Gene Research Articles

Optimization of Agrobacterium-mediated genetic transformation of Fraxinus nigra and development of black ash for possible emerald ash borer resistance

Emerald ash borer (EAB; Agrilus planipennis Fairmaire) is the most devastating insect pest of North American ash species, including black ash (Fraxinus nigra Marsh.). As a first step in an effort to develop transgenic black ash plants resistant to EAB, we successfully established an efficient Agrobacterium-mediated transformation system for black ash hypocotyls. Kanamycin and timentin at 40 and 300 mg L−1, respectively, were most effective to select transformed explants and control excess Agrobacterium growth. Using a plant transformation vector harboring the enhanced green fluorescent protein (eGFP) gene, the effects of Agrobacterium strain, bacterial density, and the concentration of Silwet L-77 on transformation efficiency were evaluated. The best result was obtained when Agrobacterium strain EHA105 was used at a density of OD600 = 1.0. Silwet L-77 failed to promote transformation frequency and showed an adverse effect at higher concentrations (> 0.015%). Using this optimized transformation system, transgenic black ash shoots expressing a synthetic Bacillus thuringiensis toxin gene (cry8D2) were regenerated. Although no morphological abnormality was observed, transgenic shoots showed severe growth restriction. Three independent transgenic lines were selected for further assessment. All selected lines contained two copies of the cry8D2 gene, and the expression of the transgene was verified in transcript and protein levels. These transgenic shoots can be used for future bioassay to evaluate its efficacy against EAB.

Tri-Trophic Impacts of Bt-Transgenic Maize on Parasitoid Size and Fluctuating Asymmetry in Native vs. Novel Host-Parasitoid Interactions in East Africa.

Environmental stress can affect trait size and cause an increase in the fluctuating asymmetry (FA) of bilateral morphological traits in many animals. For insect parasitoids, feeding of hosts on transgenic maize, expressing a Bacillus thuringiensis toxin gene is a potential environmental stressor. We compared the size of antennae, forewings, and tibia, as well as their FA values, in two parasitoids developed on two East African host species feeding on non-transgenic vs. transgenic maize. The two lepidopteran stem-borer hosts were the native Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and a recent invader, Chilo partellus Swinhoe (Lepidoptera: Crambidae). The two braconid parasitoids were the native, gregarious larval endoparasitoid Cotesia sesamiae and the recently introduced Cotesia flavipes. Both parasitoids attacked both hosts, creating evolutionarily old vs. novel interactions. Transient feeding of hosts on transgenic maize had various effects on FA, depending on trait as well as the host and parasitoid species. These effects were usually stronger in evolutionarily novel host–parasitoid associations than in the older, native ones. These parameters have capacity to more sensitively indicate the effects of potential stressors and merit further consideration.

An Empirical Assessment of Transgene Flow from a Bt Transgenic Poplar Plantation.

To assess the possible impact of transgenic poplar plantations on the ecosystem, we analyzed the frequency and distance of gene flow from a mature male transgenic Populus nigra plantation carrying the Bacillus thuringiensis toxin gene (Bt poplar) and the survival of Bt poplar seeds. The resultant Bt poplar seeds occurred at a frequency of ~0.15% at 0 m to ~0.02% at 500 m from the Bt poplar plantation. The germination of Bt poplar seeds diminished within three weeks in the field (germination rate from 68% to 0%) compared to 48% after three weeks of storage at 4°C. The survival rate of seedlings in the field was 0% without any treatment but increased to 1.7% under the addition of four treatments (cleaning and trimming, watering, weeding, and covering with plastic film to maintain moisture) after being seeded in the field for eight weeks. The results of this study indicate that gene flow originating from the Bt poplar plantation occurred at an extremely low level through pollen or seeds under natural conditions. This study provides first-hand field data on the extent of transgene flow in poplar plantations and offers guidance for the risk assessment of transgenic poplar plantations.

Variant cry1Ab entomocidal Bacillus thuringiensis toxin gene facilitates the recovery of an increased number of lepidopteran insect resistant independent rice transformants against yellow stem borer (Scirpophaga incertulus) inflicted damage

The primary technical constraint plant scientists face in generating insect resistant transgenic crops with insecticidal Bacillus thuringiensis (Bt) crystal protein (Cry) genes remains failing to generate sufficiently large numbers of effective resistant transgenic plant lines. One possible means to overcome this challenge is through deployment of a Cry toxin gene that contains high levels of insecticidal specific activity for target insect pests. In the present study, we tested this hypothesis using a natural variant of the Cry1Ab toxin under laboratory conditions that possessed increased insecticidal potency against the yellow stem borer (YSB, Scirpophaga incertulus), one of the most damaging rice insect pests. Following adoption of a stringent selection strategy for YSB resistant transgenic rice lines under field conditions, results showed recovery of a significantly higher number of YSB resistant independent transgenic plant lines with the variant cry1Ab gene relative to transgenic plant lines harbouring cry1Ab berliner gene. Structural homology modelling of the variant toxin peptide with the Cry1Aa toxin molecule, circular dichroism spectral analysis, and hydropathy plot analysis indicated that serine substitution by phenylalanine at amino acid position 223 of the Cry1Ab toxin molecule resulted in a changed role for α-helix 7 in domain I of Cry1Ab for enhanced toxicity.

The Feasibility of Producing Oil Palm with Altered Lignin Content to Control Ganoderma Disease

AbstractOil palm is a major crop which is grown for the production of vegetable oil used in foods, cosmetics and biodiesel. The palm is of major economic importance in southeast Asia where it is grown extensively in Malaysia and Indonesia. There is concern about Ganoderma rots of oil palm which need to be controlled to prevent major infection. However, the basic mechanism of white‐rot infection has been ignored. White rot implies that fungi attack the lignin component of woody tissue leaving the white cellulose exposed. The fungus grows within palms by utilizing cellulose in the tree. By altering the lignin fraction of oil palm losses may be reduced. Methods for altering lignin in plants are reviewed here to indicate how similar transformations could be attempted for oil palm. In addition, progress in transforming oil palm is described. Lignin is extremely complex and this may explain why it has not been studied in oil palm. Some crops transformed with Bacillus thuringiensis toxin genes have increased lignin and modified oil palm have been produced using B. thuringiensis genes. These require to be tested for lignin concentration and structure. The nomenclature of the disease organism is discussed. The prospects for altered lignin oil palm are considered herein.

GM plants for your health

When will agricultural biotechnologies, such as genetically modified (GM) crops, reach Europe? This was the main question at the Agricultural Biotechnology International Conference (ABIC)—the largest of its kind—that took place in September this year in Cologne, Germany. Given that the ABIC was accompanied by a parallel conference organized by critics of GM crops and foods, this is an appropriate question. Most of the European Union (EU) member states have not yet approved the GM crops that are used widely and safely elsewhere in the world. Moreover, although the EU has finally lifted its moratorium on GM crops, and has passed new regulations for growing and marketing GM foods, national politics, legislation and ideological views about consumer and environmental protection have further hampered their use. European consumers remain wary of agricultural biotechnology and its products, as they do not see any direct benefits from GM crops and are, therefore, understandably reluctant to accept them. But it is only a matter of time before GM foods arrive on supermarket shelves across Europe, predicts Ashley O'Sullivan, President and CEO of Ag‐West Bio Inc. (Saskatoon, Saskatchewan, Canada). “The reality for legislation to regulate agricultural biotechnology is that the train has left the station and there is no way of going back,” he added. > …to convince the cautious European public, agricultural biotechnology still has to […] offer products that directly benefit consumers But to convince the cautious European public, agricultural biotechnology still has to show that it can do more than increase the returns to farmers, and offer products that directly benefit consumers. The next wave of GM plants, which are currently being developed and tested in academic and industry laboratories around the world, including Europe, may soon do this. A range of new GM crops in the research pipeline will offer direct benefits to …

Mating and dispersal behaviour of Scirpophaga incertulas and Chilo suppressalis (Lepidoptera; Pyralidae) in relation to resistance management for rice transformed with Bacillus thuringiensis toxin genes

Information on the mating and dispersal behaviour of the rice stem borers Scirpophaga incertulas and Chilo suppressalis is necessary for the design of resistance management strategies for rice varieties genetically engineered with toxin genes from Bacillus thuringiensis (Bt). We investigated whether S. incertulas and C. suppressalis move from the site of adult eclosion before mating to determine the likelihood that random mating will occur between adults eclosing in fields of Bt rice and refuge fields of non-Bt rice. Observations were made under laboratory, screenhouse and field conditions. Almost all S. incertulas males and females flew out of sight after cuticle expansion and hardening, as did most C. suppressalis. Approximately 15% of C. suppressalis females called and mated within 3 m of the site of eclosion, and approximately 5% of C. suppressalis males mated within 5 m of the site of eclosion. These results suggest that random mating is likely to occur between S. incertulas eclosing in Bt rice fields and nearby refuge fields. Although a small proportion of C. suppressalis did not move far before mating, random mating of moths from Bt and nearby refuge fields is also likely to occur in this species if Bt-resistant moths are rare.

Field survey and greenhouse evaluation of non-rice host plants of the striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), as refuges for resistance management of rice transformed with Bacillus thuringiensis toxin genes.

Field survey and greenhouse evaluation of non-rice host plants of the striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae), as refuges for resistance management of rice transformed with Bacillus thuringiensis toxin genes - Volume 92 Issue 3

Effect of rice lines transformed with Bacillus thuringiensis toxin genes on the brown planthopper and its predator Cyrtorhinus lividipennis

AbstractFive transgenic rice lines, each containing an insecticidal toxin gene from Bacillus thuringiensis (Bt) under control of a different promoter, were tested for effects on two non‐target insects: the brown planthopper, Nilaparvata lugens (Stål) (Homoptera: Delphacidae), and its predator Cyrtorhinus lividipennis (Hemiptera: Miridae). Bt toxin was detected by ELISA in the honeydew of N. lugens that fed on rice lines with the CaMV 35S and actin promoters. Nilaparvata lugens produced greater volumes of acidic honeydew (derived from xylem feeding) on all five Bt rice lines than on non‐transgenic control lines. The amount of honeydew derived from phloem feeding did not differ between Bt and control lines. There were no differences between N. lugens reared on Bt and control lines in any of the five fitness parameters measured (survival to the adult stage, male and female weight, and male and female developmental time). There were no differences between C. lividipennis reared on N. lugens nymphs from Bt and control lines, in any of the three fitness parameters examined (survival to the adult stage and male and female developmental time). Our results indicate that N. lugens and its natural enemies will be exposed to Bt toxins from rice lines transformed with some Bt gene constructs, but that this exposure might not affect N. lugens and C. lividipennis fitness.

Transformation of Liquidambar styraciflua using Agrobacterium tumefaciens

We describe the molecular transformation of Liquidambar styraciflua using Agrobacterium tumefaciens. A binary TI-plasmid vector containing a chimeric neomycin phosphotransferagene which confers resistance to kanamycin and either a chimeric Bacillus thuringiensis toxin gene, a chimeric E. coli β-glucuronida(GUS), or a chimeric tobacco anionic peroxidase gene was introduced into sweetgum by co-cultivation with Agrobacterium tumefaciens. Sweetgum shoots regenerated in the presence of kanamycin were confirmed to be transformed by genomic DNA blots or the presence of GUS activity. The optimization of the transformation protocol and the incorporation of molecular transformation into a rapid germplasm improvement protocol are discussed.

Prospects for genetic engineering of insect resistance in forest trees

Gene transfer and recombinant DNA methods provide opportunities for enhancing insect resistance of forest trees by importing genes from other species and by manipulating native genes to create novel forms of resistance. Current opportunities for enhancing insect resistance include insertion of the toxin gene from the bacterium Bacillus thuringiensis and transfer of proteinase inhibitor genes from other plant species. Work is under way in a number of laboratories throughout the world to insert Bacillus thuringiensis toxin genes into forest tree species. Other strategies, such as the manipulation and transfer of chitinase genes, lectin genes, baculovirus genes, and genes encoding enzymes involved in the production of novel secondary compounds, also hold promise but require more information before their likelihood of success can be judged. Use of genetically engineered, resistant trees should be environmentally safer than controlling insect pests with insecticides. This is primarily because engineered trees affect only species that feed on them, and even then will generally be harmful to only a limited number of insect taxa. The main environmental risk associated with the use of engineered trees is that insects may counter-evolve to overcome their resistance. This would be particularly significant, for example, if counter-evolution to the Bacillus thuringiensis toxin gene in trees also precluded the use of insecticides containing Bacillus thuringiensis. We argue, however, that the risks of serious counter-evolution can be reduced to an acceptable level by maintaining genetic diversity in the forest, using multiple genes for resistance, and employing forest management practices that mitigate the potential for counter-evolution. Genetically engineered resistance should be more effective than the spraying of insecticides because the toxins are delivered as soon as insects begin feeding, can be produced continuously, and are delivered within tissues — thereby contacting insects that are difficult to reach with exterior sprays. We hypothesize that the improved nutrition of coniferous trees provided by intensive forest management should allow them to make heavier investments in novel nitrogen-based defensive compounds such as proteins and alkaloids, and that genetic engineering can help to take advantage of this opportunity. The greatest limitations to the current use of genetic engineering to improve insect resistance of trees are: insufficient knowledge of the molecular biology of insect development, insect pathogenesis, and tree defenses against insects; inefficient systems for insertion of genes into large numbers of tree genotypes; inability to produce sterile trees, necessary to prevent the release of engineered genes into natural or feral populations; concerns about insect counter-evolution to overcome the effects of engineered resistance genes; a lack of public understanding of the true benefits and risks of genetic engineering. In the short term, the greatest benefits from recombinant DNA and genetic engineering technology will be to provide new avenues for understanding tree-insect interactions, and thus new options for combating insect pests.

Peptide mapping of different Bacillus thuringiensis toxin gene products by CNBr cleavage in SDS-PAGE gels

A cyanogen bromide fragmentation reaction of 3 hr in sodium dodecyl sulfate gel slices was used for the degradation of the toxins coded by the three cryIA genes from Bacillus thuringiensis kurstaki. Peptide patterns diagnostic for each toxin gene product were observed. Treatment longer than 3 hr led to the weakening and disappearance of protein bands. In 9 out of 16 wild B. thuringiensis strains tested, it was determined that only one of the cryIA genes was being expressed; in 6 strains, one to two genes were identified, with the presence of an additional gene possible and masked because of the overlap of peptide bands; in one strain none of the genes was expressed.