<p style="text-align: justify;">1. Resistance to the European Corn Borer (ECB)</p>

<p style="text-align: justify;">Bacillus thuringiensis var. kurstaki strain HD-73 (B.t.k.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the delta&eth;-endotoxin Cry1A(c), which is active against certain lepidopteran insects, such as ECB. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.t.k. based foliar applied insecticides have been registered for over 30 years in Canada and have a long history of safe use.<br />
A synthetic gene coding the first 613 amino acids of cryIA(c) was developed to provide maximum expression in corn, and introduced into line DBT418. The gene codes for a protein similar to the Bacillus thuringiensis var. kurstaki HD-73 insecticidal crystal protein tryptic core fragment. The protein is insecticidal to lepidopteran larvae. Insecticidal delta&eth;-endotoxin activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually results in cell lysis. Specific Lepidopteran pests of corn sensitive to the protein are ECB and corn earworm.</p>

<p style="text-align: justify;">The cry1A(c) gene is linked to a strong constitutive promoter. Average protein level ranges in various tissues is as follows:</p>

<ul>
	<li style="text-align: justify;">harvest leaves: 459.6 to 1194.4 ng Cry1A(c) per gram tissue (dry weight)</li>
	<li style="text-align: justify;">harvest kernels: 36.0 to 42.8 ng Cry1A(c) per gram tissue (dry weight)</li>
	<li style="text-align: justify;">harvest root: 58.0 to 125.4 ng Cry1A(c) per gram tissue (dry weight)</li>
	<li style="text-align: justify;">harvest stalk: 40.9 to 123.6 ng Cry1A(c) per gram tissue (dry weight)</li>
</ul>

<p style="text-align: justify;">The Cry1A(c) protein was not detectable in pollen.</p>

<p style="text-align: justify;">The Cry1A(c) protein degrades readily in the environment. Monsanto studies with Cry1A(b) a similar protein indicated a half life of 1.6 days when the protein was a component of corn tissue. In Cotton tissue, Cry1A(c) has a half life of 41 days.</p>

<p style="text-align: justify;">Protein allergens are normally resistant to digestion unlike the Cry1A(c) protein which was shown to degrade readily in simulated gastric fluid. Unlike many known allergens the insecticidal protein is not glycosylated. A search for amino acid sequence similarity between the Cry1A(c) protein and known allergens, using the GENINFO- BLAST network server, revealed no significant amino acid sequence homologies. A search of a similarily constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(c) protein, with the exception of homologies to other B.t. insecticidal proteins.</p>

<p style="text-align: justify;">The full nucleotide sequence and corresponding amino acid sequence were provided.</p>

<p style="text-align: justify;">The 66 kDa protein expressed in plants was compared to the bacterial protein core fragment and shown to be of similar molecular weight and immunological reactivity. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein.</p>

<p style="text-align: justify;">2. Glufosinate Ammonium Herbicide Tolerance</p>

<p style="text-align: justify;">Phosphinothricin (L-PPT), the active ingredient of the herbicide glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application.<br />
The phosphinothricin tolerance gene (bar) engineered into DBT418 was isolated from a common aerobic soil streptomycete and codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity; experimental data clearly showed that neither L-PPT&#39;s analog L-glutamic acid, D-PPT, nor any protein amino acid can be acetylated by the PAT enzyme.</p>

<p style="text-align: justify;">The gene, modified for enhanced gene expression, is linked to a constitutive promoter. Data showed average protein level ranges in varoius tissues as follows:</p>

<ul>
	<li style="text-align: justify;">pollen stage leaves: 501.8 to 1099.4 ug PAT per g tissue (dry weight)</li>
	<li style="text-align: justify;">pollen stage stalk 66 to 77 ug PAT per g tissue (dry weight)</li>
	<li style="text-align: justify;">pollen stage root 27.5 to 69.5 ug PAT per g tissue (dry weight)</li>
	<li style="text-align: justify;">kernal 3.1 to 6.0 ug PAT per g tissue (dry weight)</li>
</ul>

<p style="text-align: justify;">The PAT protein was not detected in pollen.</p>

<p style="text-align: justify;">The expressed PAT enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post transcriptional modifications.The amino terminal sequence of DBT 418 derived PAT is the same as the native protein.<br />
Studies showed that the enzyme was completely degraded within five minutes when subjected to typical mammalian gastric conditions and is thus digested as a conventional dietary protein.</p>

<p style="text-align: justify;">The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENINFO-BLAST network server database.</p>

<p style="text-align: justify;">3. Development Method</p>

<p style="text-align: justify;">DBT418 was co-transformed with three vectors, one carrying a synthetic cry1A(c) gene and the second bearing the herbicide resistance gene. The third carried the potato proteinase inhibitor (pin II) gene. An ampicillin resistance gene, regulated by a bacterial promoter, also present in the vectors used to transform was not expressed. The three plasmid vectors were introduced by microprojectile bombardment into cultured plant cells. Glufosinate Ammonium tolerant transformed cells were selected, then cultured in tissue culture medium for plant regeneration.</p>

<p style="text-align: justify;">4. Stable Integration into the Plant&#39;s Genome</p>

<p style="text-align: justify;">Analysis indicated integration of two intact copies of the cry1A(c) gene, one intact copy of the bar gene and one rearranged copy of the bar gene. Partial copies of the pin II gene and alcohol dehydrogenase (adh) intron 1 were also integrated. There are four intact and one partial copy of the beta lactamase gene and four intact copies of the Col E1 origin of replication. The beta lactamase genes are not expressed and the pin II gene is non-functional and is not expressed.</p>
