2.8. GM SQUASH
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. For
example, when ZYMV or WMV2 infect a ZW-20 plant or another crossprotected squash plant, replication is blocked. Other modes of action of cross
protection have also been suggested.
2.8.1. Summary of Introduced Genetic Elements
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Code Name
Type Promoter,
other
CP
viral
coat
protein (Zucchini VR
Copies Form
Terminator
CaMV 35S
Native
CaMV 35S
Native
yellow mosaic potyvirus)
CP
viral coat protein (Watermelon VR
mosaic potyvirus 2)
2.8.2. Modification Method
The ZW-20 squash line was produced using an Agrobacteriummeditated 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.
2.8.3. Characteristics of the Modification
2.8.3.1. Expressed Material
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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.
2.8.4. Environmental Safety Considerations
2.8.4.1. 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.
2.8.4.2.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
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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.
Successful transmission of genetic material from CZW-3 squash via
pollen is possible to a limited number of squash relatives. 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.
2.8.4.3. Weediness Potential
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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. Squash volunteers are not uncommon in
areas next to production fields, but they do not readily establish feral or freeliving 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.
2.8.4.4. 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.
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2.8.4.5. 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.
2.8.5. Food Safety Considerations
2.8.5.1. Dietary Exposure
There was no reason to believe that the development of virusresistant 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.
2.8.5.2. 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.
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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.
2.8.5.3.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.
2.8.5.4. 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.
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