ARCHIVED - irus Resistant Squash Line CZW-3

October 1999

Health Canada has notified Seminis Vegetable Seeds Inc. that it has no objection to the food use of the transgenic squash line CZW-3, which has been developed to resist infection by three plant viruses that commonly infect squashes, watermelon mosaic virus 2 (WMV2), zucchini yellows mosaic virus (ZYMV) and cucumber mosaic virus (CMV). The Department conducted a comprehensive assessment of CZW-3 according to its Guidelines for the Safety Assessment of Novel Foods (September, 1994). These guidelines are based upon internationally accepted principles for establishing the safety of foods derived from genetically modified organisms.


The following provides a summary regarding the Seminis Vegetable Seeds Inc. notification to Health Canada and contains no confidential business information.

1. Introduction

The yellow crookneck squash (Curcurbita pepo L.), line CZW-3, was developed through a specific genetic modification to be resistant to infection by three plant viruses WMV2, ZYMV and CMV. WMV2 and ZYMV are members of the potyvirus group and CMV is the type member of the cucumovirus group. The novel variety was developed by insertion of the coat protein (CP) encoding sequences from these three single-stranded RNA viruses. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology. The genetically modified squash exhibits the trait of resistance to infection and subsequent disease caused by these viruses through a process that is related to viral cross-protection.

2. Development of the Modified Plant

The CZW-3 squash line was created by Agrobacterium-mediated transformation in which the transfer- DNA (T-DNA) contained the coat protein genes from each of the three viruses. The constitutive expression of these genes was regulated by the 35S promoter from cauliflower mosaic virus. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII) from the Tn5 transposon of Echerichia coli, strain K12. The expression of NPTII activity was used as a selectable trait to screen transformed plants for the presence of the viral CP genes. There was no incorporation of plasmid DNA sequences outside of the T-DNA region as verified by Southern blot analysis.

3. Product Information

Constitutive expression of the CMV coat protein in transgenic squash was estimated to be 5.9-10.8 mg/kg fruit tissue. This level of expression was significantly lower than measured levels of ZYMV, WMV2 and CMV coat protein in zucchini squash, yellow crookneck squash, cantaloupe and honeydew melons from supermarket shelves. Human consumption of viral coat proteins occurs whenever virus-infected plant products are consumed, or where crops have been protected from virus infection by classical crossprotection using a mild or symptomless strain of the virus. The level of expression of NPTII (44 mg/kg fruit tissue) was judged to be insignificant with respect to any human health risk due to exposure. Other than susceptibility to virus infection, the disease, pest and other agronomic characteristics of line CZW-3 were comparable to non-transgenic counterparts. Additionally, the levels of curcurbitacins, based on reference to the standard taste evaluation for curcurbitacins B and E, are non-bitter.

4. Dietary Exposure

The fruit of CZW-3 is intended primarily for human consumption and the genetic modification will not result in any change in the consumption pattern for this product. Consequently, the dietary exposure of Canadians to this product is anticipated to be the same as for other commercially available squash lines.

5. Nutrition

The analysis of nutrients from transgenic CZW-3 squash and non-transgenic squash revealed only small differences that were within the range of variability normally reported for squash. The consumption of this product would, therefore, have no significant impact on the nutritional quality of the Canadian food supply.

6. Safety

a) Potential Toxicity:

The WMV2, ZYMV and CMV coat protein sequences were compared to databases of known protein toxins and did not show homologies with known mammalian protein toxins. The history of known safe consumption of these proteins from virus-infected plant products provides additional evidence of lack of toxicity.

b) Potential Allergenicity:

The WMV2, ZYMV and CMV coat proteins do not possess characteristics typical of known protein allergens. There were no regions of homology when the sequences of these introduced proteins were compared to the amino acid sequences of known protein allergens. The WMV2, ZYMV and CMV coat proteins are extremely unlikely to be allergens.


Health Canada's review of the information presented in support of the food use of virus resistant squash CZW-3 concluded that this squash does not raise concerns related to human food safety. Health Canada is of the opinion that CZW-3 is as safe and nutritious as current commercial squash varieties. Health Canada's opinion deals only with the food use of this virus resistant squash. Issues related to growing virus resistant squash in Canada and its use as animal feed are addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Protection Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

For further information, please contact:

Office of Food Biotechnology
Food Directorate
Health Protection Branch
Health Canada
Tunney's Pasture
Ottawa, Ontario K1A 0L2
Telephone: (613) 941-5535
Facsimile: (613) 952-6400

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