GM Crop Database

Database Product Description

CZW-3
Host Organism
Cucurbita pepo (Squash)
Trait
Resistance to viral infection, cucumber mosaic virus (CMV), watermelon mosaic virus (WMV) 2, zucchini yellow mosaic virus (ZYMV).
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption.

Product Developer
Asgrow (USA); Seminis Vegetable Inc. (Canada)

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1998 View
United States 1994 1994 1996

Introduction Expand

The yellow crookneck squash (Curcurbita pepo L.), line CZW-3 was developed using recombinant DNA techniques to be resistant to infection by three plant viruses that frequently infect commercial squash varieties, namely cucumber mosaic cucumovirus (CMV), zucchini yellow mosaic potyvirus (ZYMV), and watermelon mosaic potyvirus (WMV2). The novel variety was developed by insertion of the coat protein (CP) encoding sequences from these three single-stranded RNA viruses. The expression of viral CPs in this transgenic squash variety does not result in the formation of any infectious particles, or in the manifestation of any disease pathology, but rather enables the plant to resist infection by CMV, ZYMV and WMV2. These viruses are major pathogens of all cucurbit crops (squash, cucumbers, and their relatives) and are present in most regions where these crops are grown.

ZYMV and WMV2 are members of the potyvirus group, one of the largest groups of plant viruses, while CMV is the type member of the cucumovirus group. 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 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.
CZW-3 squash exhibits “pathogen derived resistance” to infection and subsequent disease caused by CMV, 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). Transgenic CZW-3 squash displayed a high level of resistance to systemic infection with CMV, ZYMV, and WMV 2, although 64% exhibited localized chlorotic dots that were mainly confined to older leaves. CZW-3 plants had a 50-fold increase in marketable yield compared to non-transgenic control plants.

An antibiotic resistance marker gene (neo) encoding the enzyme neomycin phosphotransferase II (NPTII), which inactivates aminoglycoside antibiotics such as kanamycin and neomycin, was also introduced into the genome of this variety. This gene was derived from a bacterial transposon (Tn5 transposable element from Escherichia coli) and was included as a selectable marker to identify transformed plants during tissue culture regeneration and multiplication. The expression of the neo gene in these plants has no agronomic significance and the safety of the NPTII enzyme as a food additive was evaluated by the United States Food and Drug Administration in 1994 (US FDA, 1994).

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP cucumber mosaic cucumovirus (CMV) VR

CaMV 35S

Native

CP zucchini yellow mosaic potyvirus (ZYMV) VR

CaMV 35S

Native

CP watermelon mosaic potyvirus (WMV2) VR

CaMV 35S

Native

nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens Native

Characteristics of Cucurbita pepo (Squash) Expand

Center of Origin Reproduction Toxins Allergenicity

The genus Cucurbita is indigenous to the Americas, with the center of distribution thought to be central or southern Mexico.

Squash pollination is by honeybees and wild bee species.

There are no known toxins in squash.

Allergies to squash are rare, but some people have exhibited rashes and mouth swelling after exposure to squash.

Modification Method Expand

The CZW-3 squash line was produced using an Agrobacterium-meditated transformation of the parental variety YS20 via a 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 CMV, 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, all three CP genes were fused to the 5' untranslated sequence from CMV to enhance translation of the transgene mRNA.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from CZW-3 indicated the presence of a single copy of the T-DNA containing intact sequences corresponding to the CP encoding genes form CMV, ZYMV, and WMV2, integrated at a single site. Additional analyses verified the presence of a single copy of the NPTII encoding neo antibiotic resistance marker gene, and no incorporation of plasmid backbone sequences outside of the T-DNA region.

Expressed Material

The level of expression of the CMV coat protein in transgenic squash was quantitated by enzyme linked immunosorbent assay (ELISA) and estimated to be 5.9-10.8 ng/g fruit tissue, which was of the same order of magnitude as the concentrations of ZYMV and WMV-2 coat proteins measured in the related squash line ZW-20. 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. The level of expression of NPTII (44 µg/g fruit tissue) was judged to be insignificant with respect to any human health risk due to exposure.

Environmental Safety Considerations Expand

Field Testing

The transgenic squash line CZW-3 was field tested in the United States (1993 – 1996). CZW-3 was extensively tested in the laboratory, greenhouse, and field experiments and based on these assessments it was determined that the plants exhibited the typical agronomic characteristics of the parent crookneck squash, with the addition of resistance to CMV, ZYMV and WMV2 infection. Field trial reports demonstrated that CZW-3 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 CZW-3 with FLCP plants indicate that FLCP x CZW-3 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 CMV, ZYMV and WMV2, suggesting that resistance to CMV, ZYMV and WMV2 would not provide any selective advantage. Should the virus resistance genes from CZW-3 transfer to FLCP plants, the selective pressure to maintain the virus resistance in natural populations of FLCP plants should be minimal, since all evidence supports the conclusion that FLCP populations are not under significant environmental stress from viral infection.

Weediness Potential

The transgenic squash line CZW-3 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 & Baumman, 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 CMV, ZYMV and WMV2 would result in CZW-3 squash becoming a weed. During field trials, the transgenic plants were examined for any characteristics that may increase their weediness potential, and none were observed. There were no major changes in seed germination, cucurbitin levels, seed set viability, susceptibility or resistance to pathogens or insects (except CMV, ZYMV and WMV2), and there were no differences in overwintering survivability between CZW-3 squash and nontransgenic squash. It was concluded that CZW-3 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 CZW-3 squash. The coat proteins expressed in CZW-3 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 CZW-3 squash. It was concluded that the genes inserted into the transgenic squash line CZW-3 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 CZW-3 had no novel phenotypic characteristics that would extend its use beyond the current geographic range of squash production. The impact of cultivation of CZW-3 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 for squash. Most yellow crookneck squash are consumed as a raw table vegetable or processed for the frozen food market. 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 to this product is anticipated to be the same as for other commercially available squash lines.

Nutritional Data

A compositional analysis conducted on transgenic CZW-3 squash and the non-transformed parental variety 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, dietary fibre, carbohydrates, calories, fructose, glucose, sucrose, lactose, maltose, vitamin C, beta-carotene, vitamin A, calcium, iron, and sodium.

Toxicity

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. The transgenic squash line CZW-3 and the parent variety were both non-bitter.

The CMV, 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 CMV, WMV2 and ZYMV coat proteins do not possess characteristics typical of known protein allergens. There were no regions of homology when the deduced amino acid sequences of these introduced proteins were compared to the amino acid sequences of known protein allergens. The CMV, 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, such as mosaic viruses, 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 CZW-3 squash line was developed using recombinant DNA techniques to resist infection by CMV, 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 CMV, 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).

CZW-3 squash was tested in field trials in the United States (1993–1996). These tests demonstrated that CZW-3 plants exhibited the typical agronomic characteristics of conventional crookneck squash, with the addition of resistance to CMV, ZYMV and WMV2 infection. CZW-3 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 CZW-3, 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 CZW-3 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 CZW-3 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, total fat, ash, dietary fibre, total 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 CZW-3 and the parent variety were both non-bitter, indicating that the cucurbitacin level in each line was less than 10 ppb.

The CMV, 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 CMV, ZYMV, and WMV2 CPs did not reveal any homology to known protein allergens and toxins. Furthermore, CMV-, 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 Tuesday, September 15, 2015