Increased expression of the insect control protein genes ofBacilus thunrngiensis in plants has been critical to the development of genetically improved plants with agronomically acceptable levels of insect resistance. The expression of the cryIA(b) gene was compared to partially modIfied (3% nucleotide difference) and to fully modified (21% nucleotide difference) crylA(b) and crylA(c) genes in tobacco and tomato. The modified genes increased the frequency of plants that produced the proteins at quantities sufficient to control insects and dramatically increased the levels of these proteins. Among the most highly expressing transformed plants for each gene, the plants with the partially modified crylA(b) gene had a 10-fold higher level ofinsect control protein and plants with the fully modified crylA(b) had a 100-fold higher level of CryIA Insect control proteins from a prokaryotic source, Bacillus thuringiensis var. kurstaki (B.t.k.; ref. 1) are specific for lepidopteran insects and exhibit no activity against humans, other vertebrates, and beneficial insects (2). These properties have made the genes of these insect-specific proteins attractive candidates for genetic modification of crops for protection against lepidopteran pests. Genes encoding lepidopteran-specific insect control proteins have been cloned and sequenced. Truncated genes, which produce insecticidally active protein, have been expressed in tomato (3), tobacco (4), and cotton (5). Field tests of these plants revealed that higher levels of insect control protein in the plant tissue would be required to obtain commercially useful plants (6).The insect control proteins are highly expressed in their natural host, B. thuringiensis. Up to 50% of the total protein in sporulated cultures ofB.t.k. are the insect control proteins deposited as crystals within the cell. Insect control protein genes are expressed well in Escherichia coli (7) or Pseudomonas (8). Poor expression in plants is a well-reported characteristic of the B.t.k. insect control proteins. Truncating the gene, keeping essentially the N-terminal half of the protein intact, results in improved expression of the gene in plants to barely detectable levels (3, 4). The use ofdifferent promoters, fusion proteins, and leader sequences has not significantly increased insect control protein gene expression (4, 9).We hypothesized that a gene with a sequence adapted for a Gram-positive prokaryote may not be the appropriate coding sequence for efficient plant expression. Examination of the insect control protein gene coding sequence indicated that it differs significantly from plant genes in G+C content. Multiple sequences motifs that are not common in the coding region of plant genes were found to be common in the wild-type (WT) crylA(b) sequence. These included localized regions of A+T richness resembling plant introns (10), potential plant polyadenylylation signal sequences (11), ATTTA sequences, which have been shown to destabilize mRNA in other systems (12), and codons rarely used in plants...

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.

Russet Burbank potato plants have been genetically improved to resist insect attack and damage by Colorado potato beetles (Leptinotarsa decemlineata (Say)) by the insertion of a cryIIIA gene encoding the insect control protein of Bacillus thuringiensis var. tenebrionis. A modified gene that dramatically improved plant expression of this protein was utilized. Its expression in Russet Burbank potato plants resulted in protection from damage by all insect stages in the laboratory and in dramatic levels of protection at multiple field locations. Analysis of these genetically modified potatoes indicated that they conform to the standards for Russet Burbank potatoes in terms of agronomic and quality characteristics including taste.

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Bollgard cotton is the trademark given to a number of varieties of cotton bio-engineered to produce an insecticidal protein from Bacillus thuringiensis (Bt). When produced by the modified cotton plants, this protein controls certain lepidopterous cotton insect pests. Commercially available since 1996, these cotton varieties are purchased under a license agreement in which the growers pay a fee and agree to abide by the terms, which include a 1-year license to use the technology and agreement to participate in an insect resistance management program. Today, Bollgard cotton is grown on more than one-third of all cotton acreage in the USA. This product has reduced cotton production costs and insecticide use by providing an effective alternative to chemical insecticides for the control of tobacco budworm, Heliothis virescens; cotton bollworm, Helicoverpa zea; and pink bollworm, Pectinophora gossypiella. The specificity and safety profile of the Bt protein produced in planta in cotton was maintained. It has retained its selectivity for lepidopterous insects and lacks the characteristics found in potential allergenic proteins. Fiber quality, the agronomic characteristics of the plant and seed composition remain unchanged. New cotton technology is being developed to provide improved insect control and a wider spectrum of activity. These future products could further reduce insecticide use in the production of cotton, while maintaining the high level of safety and reliability that has been demonstrated by five seasons of Bollgard cotton use.

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