During the past decade the pesticidal bacterium Bacillus thuringiensis has been the subject of intensive research. These efforts have yielded considerable data about the complex relationships between the structure, mechanism of action, and genetics of the organism’s pesticidal crystal proteins, and a coherent picture of these relationships is beginning to emerge. Other studies have focused on the ecological role of the B. thuringiensis crystal proteins, their performance in agricultural and other natural settings, and the evolution of resistance mechanisms in target pests. Armed with this knowledge base and with the tools of modern biotechnology, researchers are now reporting promising results in engineering more-useful toxins and formulations, in creating transgenic plants that express pesticidal activity, and in constructing integrated management strategies to insure that these products are utilized with maximum efficiency and benefit.

Bacillus thuringiensis and Its Pesticidal Crystal Proteins

▪ Abstract This review examines potential impacts of transgenic cultivars on insect population dynamics and evolution. Experience with classically bred, insecticidal cultivars has demonstrated that a solid understanding of both the target insect's ecology and the cultivar's performance under varied field conditions will be essential for predicting area-wide effects of transgenic cultivars on pest and natural enemy dynamics. This experience has also demonstrated the evolutionary capacity of pests for adaptive response to insecticidal traits in crops. Biochemical and genetic studies of insect adaptation to the Bacillus thuringiensis (Bt) toxins expressed by currently marketed transgenic cultivars indicate a high risk for rapid adaptation if these cultivars are misused. Theoretical and practical issues involved in implementing strategies to delay pest adaptation to insecticidal cultivars are reviewed. Emphasis is placed on examining the “high dose”/refuge strategy that has become the goal of industry and reg...

Sustainability of Transgenic Insecticidal Cultivars: Integrating Pest Genetics and Ecology

The fundamental discoveries of Darwin and Mendel established the scientific basis for plant breeding and genetics at the turn of the 20th century. Similarly, the recent integration of advances in biotechnology, genomic research, and molecular marker applications with conventional plant breeding

/pdf/molecular-plant-breeding-as-the-foundation-for-21st-century-4wu0zq6rzj.pdf

Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement

Transformants of maize inbred A188 were efficiently produced from immature embryos cocultivated with Agrobacterium tumefaciens that carried "super-binary" vectors. Frequencies of transformation (independent transgenic plants/embryos) were between 5% and 30%. Almost all transformants were normal in morphology, and more than 70% were fertile. Stable integration, expression, and inheritance of the transgenes were confirmed by molecular and genetic analysis. Between one and three copies of the transgenes were integrated with little rearrangement, and the boundaries of T-DNA were similar to those in transgenic dicotyledons and rice. F1 hybrids between A188 and five other inbreds were transformed at low frequencies.

High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens

The origin of agriculture led to the domestication of many plant species and to the exploitation of natural resources. It took almost 10,000 years for food grain production to reach 1 billion tons, in 1960, and only 40 years to reach 2 billion tons, in 2000. This unprecedented increase, which has been named the 'green revolution', resulted from the creation of genetically improved crop varieties, combined with the application of improved agronomic practices.

Green revolution: the way forward

We introduced a synthetic gene encoding a truncated version of the CryIA(b) protein derived from Bacillus thuringiensis into immature embryos of an elite line of maize using microprojectile bombardment. This gene was expressed using either the CaMV 35S promoter or a combination of two tissue specific promoters derived from maize. High levels of CryIA(b) protein were obtained using both promoter configurations. Hybrid maize plants resulting from crosses of transgenic elite inbred plants with commercial inbred lines were evaluated for resistance to European corn borer under field conditions. Plants expressing high levels of the insecticidal protein exhibited excellent resistance to repeated heavy infestations of this pest.

Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis

A novel transgenic corn event designated VIP1034, is disclosed. The invention relates to seeds of corn plants comprising the transgenic genotype of the transgenic corn event VIP1034, to corn plants comprising the transgenic genotype of VIP1034 and to methods for producing a corn plant by crossing a corn plant comprising the transgenic genotype with itself or another corn variety. The invention further relates to assays for detecting the presence of the VIP1034 event based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the VIP1034 event and of genomic sequences flanking the insertion sites.

Novel corn event

A selectable marker system for plant transformation that does not require the use of antibiotics or herbicides was developed. The selectable marker consists of the manA gene from Escherichia coli under the control of a plant promoter that encodes for phosphomannose isomerase, pmi. Only transgenic plants were able to metabolize the selection agent, mannose, into a usable source of carbon, fructose. Transgenic plants were produced efficiently after delivery by Biolistics™ of the pmi gene into maize and wheat tissues, with mean transformation frequencies of 45% for maize and 20% for wheat. Adjustment of the sucrose and mannose levels in the selection medium essentially eliminated escapes. Transgenic events can be identified as early as 2 months for wheat and 4 months for maize. A simple test, a modified chlorophenol red assay, was used for early identification of transgenic events expressing the pmi gene. Transformation frequencies for both crops exceeded those obtained with the bar and pat genes with selection on either Basta® or bialaphos.

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker.

Phosphomannose isomerase (PMI) catalyzes the reversible interconversion of mannose 6-phosphate and fructose 6-phosphate. Plant cells lacking this enzyme are incapable of surviving on synthetic medium containing mannose as a carbon source. Maize, wheat and barley plants, genetically modified to express the Escherichia coli manA gene (pmi) under the control of a plant promoter, were able to survive selection on mannose-containing medium. Transformation frequencies averaged 45% for maize transformation via Biolistics™ 35% for maize Agrobacterium-mediated transformation, 20% for wheat, 3% for barley, and 2% for watermelon transformation. Moreover, the frequencies exceeded those obtained for maize and wheat using the pat or bar gene with Basa® selection. A preliminary safety assessment has been conducted for PMI. Purified PMI protein demonstrates no adverse, effects in an acute mouse toxicity test. Purified PMI protein was readily digested in simulated mammalian gastric and intestinal fluids. Plants derived from surgar beet and corn cells that had been genetically modified to express the E. coli manA gene were evaluated for biochemical changes in mannose-associated pathways. No detectable changes in glycoprotein profiles were detected in PMI-transformed plants as compared to nontransgenic controls. The yield and nutritional composition of grain from PMI-transformed corn plants compared to their non-transformed isogenic counterparts were also determined and no statistically significant differences were found. The inherent safety of a system based on simple sugar metabolism coupled with high transformation frequencies for monocots make pmi and ideal selectable marker for plant transformation.

John Dawson

Papers

Field Performance of Elite Transgenic Maize Plants Expressing an Insecticidal Protein Derived from Bacillus thuringiensis

Novel corn event

Efficient biolistic transformation of maize (Zea mays L.) and wheat (Triticum aestivum L.) using the phosphomannose isomerase gene, pmi, as the selectable marker.

Phosphomannose isomerase: An efficient selectable marker for plant transformation

Transgenic Maize for the Control of European Corn Borer and Other Maize Insect Pests