GM Crop Database

Database Product Description

ZW20
Host Organism
Cucurbita pepo L. (Squash)
Trait
Resistance to viral infection, watermelon mosaic virus (WMV) 2, zucchini yellow mosaic virus (ZYMV).
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use
Production of squash for human consumption, either fresh or processed.
Product Developer
Upjohn (USA); Seminis Vegetable Inc. (Canada)

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1998 View
United States 1997 1997 1994

Introduction Expand

The modified yellow crookneck squash (Curcurbita pepo L.), line ZW-20 was developed using recombinant DNA techniques to resist infection by two plant viruses that frequently infect commercial squash varieties, namely zucchini yellow mosaic potyvirus (ZYMV) and watermelon mosaic potyvirus (WMV2). This novel variety was developed by insertion of the coat protein (CP) encoding sequences from ZYMV and WMV2 into the plant genome. The expression of these introduced gene sequences does not result in the formation of infectious virus particles, nor does it result in any disease pathology, but rather enables ZW-20 squash plants to resist infection by these two viruses.

ZYMV and WMV2 are members of the potyvirus group, which is one of the largest groups of plant viruses. ZYMV produces a severe disease consisting of mosaic (patchwork of yellow chlorotic tissues and green uninfected tissues), yellowing, shoestringing, stunting, and fruit and seed deformations on cucurbits, such as zucchini squash, muskmelon, cucumber, and watermelon. WMV2 causes mosaic and mottle diseases of cantaloupe, pumpkin, squash, and watermelon. On a given cucurbit host, ZYMV usually causes more severe symptoms than WMV2.

The squash line ZW-20 exhibits resistance to infection and subsequent disease caused by ZYMV and WMV2 through a process that is related to viral cross-protection. Although the exact mechanism by which the viral protection occurs is unknown, most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication, uncoating (removal of CP) from the incoming virus (Register & Nelson, 1992). For example, when ZYMV or WMV2 infect a ZW-20 plant or another cross-protected squash plant, replication is blocked. Other modes of action of cross protection have also been suggested (Matthews, 1991).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP viral coat protein VR

CaMV 35S

Native

CP viral coat protein VR CaMV 35S Native

Characteristics of Cucurbita pepo L. (Squash) Expand

Center of Origin Reproduction Toxins Allergenicity

Modification Method Expand

The ZW-20 squash line was produced using an Agrobacterium-meditated transformation protocol in which the transfer-DNA (T-DNA) region of the bacterial tumour inducing (Ti) plasmid was modified using recombinant DNA techniques to contain the coat protein (CP) encoding sequences from ZYMV and WMV2. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome, and there was no incorporation of backbone plasmid sequences outside of the T-DNA region.

The constitutive expression of the CP genes was regulated by the 35S promoter and termination sequences from cauliflower mosaic virus (CaMV). In addition, WMV2 CP gene was fused to the 5' intergenic region and the first 48 nucleotides (N-terminus) of the CMV coat protein gene to enhance translation of the transgene mRNA.

The plasmid vector used for transformation also contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII), which conferred the trait of resistance to aminoglycoside antibiotics that was used as a selection mechanism during plant tissue culture regeneration and propagation. During varietal development, only plants lacking this gene were selected for commercialization and the NPTII encoding sequences were, therefore, not present in the ZW-20 genome.

Characteristics of the Modification Expand

Expressed Material

The levels of expression of ZYMV and WMV2 coat proteins were quantitated by enzyme linked immunosorbent assay (ELISA) and found to range between 73 – 1218 ng/g fresh weight and 47-112 ng/g fresh weight, respectively. These concentrations were significantly lower those measured in naturally virus infected samples of zucchini squash, yellow crookneck squash, cantaloupe and honeydew melons obtained from supermarket shelves. These latter levels have been estimated to be up to 268 and 421 fold greater than the corresponding amounts of ZYMV and WMV2 CP produced in transgenic squash.

Environmental Safety Considerations Expand

Field Testing

The transgenic squash line ZW-20 was field tested at 46 sites in the United States between 1990 and 1993. Based on testing performed in the laboratory, greenhouse, and in research test plots, it was determined that the plants exhibited the typical agronomic characteristics of the parent crookneck squash, with the addition of resistance to ZYMV and WMV2 infection. The transgenic plants did not exhibit weedy characteristics and had no effect on nontarget organisms or the general environment.

Outcrossing

Squash (C. pepo) and all other species of Cucurbita are monoecious (having both male and female flowers on the same plant) and produce heavy sticky pollen grains. Pollination requires an arthropod vector, usually a bee, to transmit the pollen from the staminate to the pistillate flower. Interspecific hybridization has been extensively investigated and is well understood in the four cultivated species of C. pepo: C. mixta, C. moschata, and C. maxima. It is reported that F1 hybrids can be obtained in breeding programs, but only with difficulty and such hybrids are usually sterile. There is no evidence of spontaneous hybridization among these four species despite that fact that they have been grown side by side under cultivation for many generations (Whitaker & Robinson, 1986).

Successful transmission of genetic material from CZW-3 squash via pollen is possible to a limited number of squash relatives (Wilson, 1993). In the United States, there exists two free-living subspecies of C. pepo that can cross with cultivated squash varieties without loss of fertility (designated as free-living Cucuribita pepo or FLCP). These include the free living gourds in many states including Texas (C. pepo ssp. ovifera var. texana) and free-living gourds in Illinois, Arkansas, and Oklahoma (C. pepo ssp. ovifera var. ozarkana.). FLCP plants have grown in proximity to new, improved cultivars of squash, and there have been no reports to suggest that disease resistance traits have introgressed into FLCP plants to produce hybrid populations that pose increased problems as weeds. Furthermore, field tests with hybrid plants derived from controlled crosses of ZW-20 with FLCP plants indicate that FLCP x ZW-20 hybrids do not appear to be strong competitors when growing in fields that have not been tilled to remove competing wild plants.

Natural populations of FLCP appear to be largely free of infection by ZYMV and WMV2, suggesting that resistance to ZYMV and WMV2 would not provide any selective advantage. Should the virus resistance genes from ZW-20 transfer to FLCP plants, the selective pressure to maintain the virus resistance in natural populations of FLCP plants should be minimal, as all evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection.

Weediness Potential

The transgenic squash line ZW-20 was not likely to present a plant pest risk as a weed. The parent plant, yellow crookneck squash, is an agricultural crop plant that exhibits no appreciable weedy characteristics. In general there are no reports of squash to be a common or troublesome weed anywhere in the United States (Bridges & Baumann, 1992). Squash volunteers are not uncommon in areas next to production fields, but they do not readily establish feral or free-living populations. Volunteers are controlled by mechanical means or herbicides.

There are no indications that resistance to ZYMV and WMV2 would result in ZW-20 squash becoming a weed. Plant characteristics that may increase their weediness potential were observed and reports indicated that ZW-20 squash retained the agronomic characteristics of the parental line. There were no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except ZYMV and WMV2), and there were no differences in overwintering survivability between ZW-20 squash and nontransgenic squash. It was concluded that ZW-20 was unlikely to increase the weediness of yellow crookneck squash and was no more likely to become a weed than virus-resistant plants.

Secondary and Non-Target Adverse Effects

No direct pathogenic properties, nor any hypothetical mechanisms for pathogenesis toward beneficial organisms, such as bees and earthworms, were identified for ZW-20 squash. The coat proteins expressed in ZW-20 squash are not known to have any toxic properties. In fact, these viral coat proteins are routinely ingested by virtually all animals, including humans, when squash is consumed. Naturally occurring infections of susceptible squash varieties result in concentrations of coat proteins far higher than those that occur in the tissues of the ZW-20 squash. It was concluded that the genes inserted into the transgenic squash line ZW-20 would not result in any deleterious effects or significant impacts on nontarget organisms, including threatened and endangered species or beneficial organisms.

Impact on Biodiversity

The squash line ZW-20 had no novel phenotypic characteristics that would extend its use beyond the current geographic range of squash production. The impact of cultivation of ZW-20 squash on biodiversity was likely to be comparable to that from non-transgenic varieties.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

There was no reason to believe that the development of virus-resistant squash plants would result in a change in fresh marketing or processing procedures. Most yellow crookneck squash are consumed as a raw table vegetable or processed for the frozen food market. The fruit of ZW-20 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 to this product is anticipated to be the same as for other commercially available squash lines.

Nutritional Data

A compositional analysis was conducted on transgenic ZW-20 squash and the non-transformed parental variety in three different locations. The analysis of nutrients from transgenic ZW-20 squash and non-transgenic squash revealed only small differences that were within the range of variability normally reported for squash. It was concluded that there were no significant differences in the content of protein, moisture, fat, ash, total dietary fibre, carbohydrates, calories, fructose, glucose, sucrose, lactose, maltose, vitamin C, beta-carotene, vitamin A, calcium, iron, and sodium. The consumption of products from modified squash would have no significant impact on the nutritional quality of the food supply.

Cucurbitis plants, such as squash, produce alkaloids classified as cucurbitacins, bitter-tasting compounds that discourages feeding by herbivores. It was reported that cucurbitacin B and cucurbitacin E are detectable by taste at sensitivity levels as low as 1 –10 ppb. A standard test in plant breeding for the presence of cucurbitacins involves tasting the product to determine its bitterness. Both the transgenic squash line ZW-20 and its parent variety were non-bitter.

Toxicity

The WMV2 and ZYMV 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.

Allergenicity

The WMV2 and ZYMV coat proteins do not possess characteristics typical of known protein allergens. Comparisons of the deduced amino acid sequence for each of these proteins with the sequences of known protein allergens did not reveal any significant homologies. The WMV2 and ZYMV coat proteins are extremely unlikely to be allergens.

Abstract Collapse

Yellow crookneck squash (Cucurbita pepo L.), together with pumpkins, gourds, and other squash, were grown in 87 countries in 2004, with a combined harvest of 19 million metric tonnes. The major producers of these vegetables were China, India, Ukraine, the United States, Egypt, Mexico, the Islamic Republic of Iran, and Cuba. Yellow crookneck squash is grown primarily for the fresh market and the processed squash industry, which includes frozen foods, dehydrated and canned products.

Squashes, pumpkins and gourds are mostly annual, warm season species in the genus Cucurbita. They are closely related to cucumber, muskmelon and watermelon. There are four major species in the Cucurbita genus: pepo, mixta, moschata, and maxima, and one minor species, ficifolia, which is a perennial plant that grows in the Andes. The terms pumpkin and squash have no precise botanical meaning and may refer to any of the above New World species. Squashes are also called vine crops because, originally, the cultivars in this family all grew on vines. While most of these crops still grow on vines, some of the newest cultivars have a bush growth habit, making them easier to grow in smaller spaces. The fruits come in an astonishing assortment of shapes and sizes, from tiny, marble-sized pumpkins of the Caribbean islands to giant gourds more than seven feet long. All of the New World species, except ficifolia, are annuals.

Viral diseases are a limiting factor to squash production, particularly during summer and fall months. Mosaic viruses include the cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic potyvirus (WMV2). It is often hard to differentiate virus infections from one another based on visual symptoms, which include mosaic patterns (light green, dark green) in leaves, puckering, leaf distortion, stunting, shortened internodes and misshapen fruit. Aphids vector each of the viruses mentioned above and CMV is also seed-transmitted. Often two or more viruses are detected in a single plant, and the viruses generally overwinter in weed hosts. Preventative control measures include avoiding late season planting and removing weeds around fields. Insect control has not proven effective. Other than removal of virus-infected plants, there are no other effective control measures for these crops once infected.

The ZW20 squash line was developed using recombinant DNA techniques to resist infection by ZYMV and WMV2 by inserting virus-derived sequences that encode the coat proteins (CPs) from each of these viruses. The introduced viral sequences do not result in the formation of any infectious particles, nor does their expression result in any disease pathology.

This transgenic squash exhibits “pathogen-derived resistance” to infection and subsequent disease caused by ZYMV and WMV2 through a process that is related to viral cross-protection. Although the exact mechanism by which the viral protection occurs is unknown, most evidence suggests that expression of viral CP by a plant interferes with one of the first steps in viral replication, uncoating (removal of CP) from the incoming virus (Register & Nelson, 1992). Other modes of action of cross-protection have also been suggested (Matthews, 1991).

Squash line ZW20 was tested in field trials in the United States (1990–1993). These tests demonstrated that ZW20 plants exhibited the typical agronomic characteristics of conventional crookneck squash, with the addition of resistance to ZYMV and WMV2 infection. ZW20 was comparable to conventional squash varieties and did not exhibit weedy characteristics, and had no effect on nontarget organisms or the general environment.

Squash (C. pepo) and all other species of Cucurbita are monoecious, such that the male and female flowers are separate structures but still on the same plant. Pollination requires an insect vector, usually a bee, to transmit the pollen from the male flower to the female flower. Squash varieties, including ZW20, will cross-pollinate with other varieties within the same species, such as zucchini squash and acorn squash.

There are several related species to cultivated squash, which include C. mixta, C. moschata, and C. maxima and wild populations of C. pepo referred to as free living Cucurbita pepo (FLCP). There is no evidence that squash can naturally cross-pollinate with relatives from C. mixta, C. moschata, and C. maxima, despite the fact that they have been grown side by side under cultivation for many generations. Cultivated squash can cross-pollinate with FLCP and form fertile hybrids. Observations of FCLP plants indicate that disease resistance traits have not be transferred to FCLP. If virus resistance genes from ZW20 were transferred to FLCP plants, the selective pressure in natural populations of FLCP plants to maintain the virus resistance gene would be minimal. Evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection.

The food and livestock feed safety of squash line ZW20 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of squash fruit was found to be equivalent to conventional varieties by the analyses of protein, moisture, fat, ash, total dietary fibre, carbohydrates, calories, sugar profile (fructose, glucose, sucrose, lactose, maltose), vitamin C, beta-carotene, vitamin A, calcium, iron, and sodium. Cucurbit plants, such as squash, produce alkaloids classified as cucurbitacins, which are bitter-tasting compounds that discourage feeding by herbivores. A standard test in plant breeding for the presence of cucurbitacins involves tasting the product to determine its bitterness and is able to detect levels between 1–10 ppb. The transgenic squash line ZW20 and the parent variety were both non-bitter, indicating that the cucurbitacin level in each was less than 10 ppb.

The ZYMV and WMV2 coat proteins do not possess characteristics typical of known protein allergens or toxins such as heat stability and resistance to digestion by simulated gastric fluids. Comparisons of the deduced amino acid sequence of the plant-expressed ZYMV and WMV2 CPs did not reveal any homology to known protein allergens and toxins. Furthermore, ZYMV- and WMV2-infected squash naturally contains higher levels of viral CP than expressed in this transgenic squash, and there has been no evidence of adverse effects linked to the consumption of virus-infected squash.

Links to Further Information Expand

International Committee on Taxonomy of Viruses (ICTV) Universal Virus Database Office of Food Biotechnology, Health Canada U.S. Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Environmental Assessment

This record was last modified on Friday, March 26, 2010