1. Resistance to the Lepidopteran Insects<br>Bacillus thuringiensis subsp. kurstaki HD-73 (B.t.k.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces a CryIA(c) insecticidal, crystal protein that is non-toxic to humans, other vertebrates, and beneficial insects. This and similar proteins are used as environmentally acceptable foliar insecticides against lepidoptera. <br>The plant expressed toxin is similar to the Bacillus thuringiensis var. kurstaki HD-73, insecticidal, crystal protein. This protein is insecticidal to Lepidoptera larvae after cleavage to an approximately 600 amino acid, bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors on the insect, midgut epithelial cells, forming a pore which disrupts osmotic balance and eventually results in cell lysis then death. Current knowledge indicates that only sensitive insects possess such receptors. Specific Lepidopteran pests of cotton, sensitive to the protein are: cotton bollworm, tobacco budworm and pink bollworm. <br>The inserted gene codes for an insecticidal protein similar to a full length Cry IA(c) protein with some minor changes arising from the design of the gene. The complete sequence of the T-DNA with the genetic endpoints and the deduced amino acid sequence was included in the submission. The encoded Cry1A(c) plant produced protein is 99.4% homologous to the native protein from B.t.k.-HD73. <br>The cryIA gene is linked to a strong constitutive promoter. Protein production (was quantified for the three lines. Mean CryIA(c) expression in line 531 was 1.56 µg protein/g and 0.86 µg protein/g fresh weight in leaf and seed tissue respectively. In the whole plant the Cry1A(c) protein averaged 0.044 µg protein/g fresh weight, (25 µg/ plant) for a total of about 1.44 g/ acre. Expression varied approximately 3 fold over the growing season, with the levels peaking late in the season. Expression of the CryIA(c) protein was much higher in line 757. Average values were 12.6 µg protein/g fresh weight and 9.9 µg protein/g fresh weight in leaves and seeds respectively. Expression varied less than 3 fold over the season and peaked early in the season in leaf tissue. Whole plant amounts were 1.1 µg protein/g fresh weight (200 µg/plant) for a total of 12.2 g/ acre. Line 1076 produced the cry1A(c) protein at an average of 12.2 µg protein/g fresh weight and 12.7 µg protein/g fresh weight in leaves and seed respectively. Gene expression over the growing season varied less than 5 fold, with the highest concentration occurring early in the season. Whole plant totals were 1.1 µg protein/g fresh weight (389 µg/plant) for a total of 23.3 g/ acre. (Calculations per acre based on 60000 plants/acre). Expression in pollen and nectar was negligible in all 3 lines. <br>Biological activity of the plant expressed protein was compared to that of the native Btk-HD-73. The insecticidal protein was shown to be active on only lepidopteran species, even when fed at levels that were well above the concentrations that were effective on target species. Host range specificity was similar for the native and synthetic proteins Tobacco hornworm was the most sensitive species and tobacco budworm, corn earworm and European cornborer were relatively less sensitive. <br>The plant expressed protein degraded rapidly (less than 30 seconds) in simulated gastric fluid (pH 1.2) and in intestinal fluid (pH 7.5). A bioactive tryptic core remained after 21 hours in simulated intestinal fluid. Degradation of the protein was determined by western blot analysis and by insect bioactivity. Degradation of the B.t.k-HD-73 protein in soil proceeded with a half life of 41 days as determined by the insecticidal activity on tobacco budworm. <br>Protein allergens are normally resistant to digestion unlike the Cry1A(c) protein, which is rapidly metabolized. Unlike many known allergens the insecticidal protein is not glycosylated. Screening against a database of known allergens for both the full length protein and the tryptic fragment revealed no significant homology to any known allergenic protein. <br>2. Kanamycin Resistance<br>Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. <br>The kanamycin-resistance gene, isolated from the bacterium E. coli, codes for an enzyme that phosphorylates kanamycin, thereby preventing it from binding to ribosomes and rendering the cells resistant. <br>The gene is linked to a constitutive promoter. Expression levels for the NPT II protein in Line 531 were 3.14 µg protein /g fresh weight and 2.45 µg protein/g fresh weight in leaf and seed tissues respectively. Expression varied about 2 fold over the season. In line 757 NPT II expression was 6.9 µg protein/g fresh weight and 3.3 µg protein/g fresh weight in leaves and seeds respectively. For line 1076 the NPT II expression was 16.3 µg protein/g fresh weight and 7.9 µg protein/g fresh weight in leaves and seed respectively. Gene expression varied 2 to 3 fold over the growing season for line 757 and line 1076. <br>This protein is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids. <br>The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database. <br>The full nucleotide sequence and corresponding amino acid sequence were provided. <br>There was no detectable expression of the streptomycin/spectinomycin resistance marker used for selection of bacterial transformants during the development process. The gene is driven by a bacterial promoter and its lack of expression in the transformed plant was confirmed by specific ELISA.<br>3. Development Method<br>Coker line 312 cotton was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector. The vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and genes coding for lepidopteran and kanamycin resistance were inserted. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was demonstrated in the BollgardTM cotton.<br>4. Stable Integration into the Plant's Genome<br>The inserted T-DNA, including the partial genes, was well characterized by restriction enzyme analysis, for all 3 lines. The genes were stable across 4 generations, for lines 531 and 1076 as shown from data collected at 1 site. Line 757 has an intact T-DNA at one site and a partial T-DNA inserted at another site in the genome. The 2 insertions were inherited in a linked Mendelian manner. Stability for this line was tested and confirmed across 2 generations.