Inc.EPA Docket control number **OPP-2004-0287**

FIFRA Scientific Advisory Panel

Federal Register, Vol. 69, No. 175, Pages 54780-54783, September 10,
2004

Comments by Seminis Vegetable Seeds

37437 State Highway 16

Woodland, California 95696

Dr. Keith Redenbaugh

Associate Director, Regulatory Affairs

<Keith.redenbaugh@seminis.com>

530 669-6170

October 8, 2004

Submitted via http://www.epa.gov/edocket/

To Whom It May Concern:

Seminis Vegetable Seeds, Inc. commercialized the first genetically
engineered (biotech) crop (ZW-20) containing viral coat protein genes in
1995, after receiving the following decisions from government agencies:

-   USDA Determination of Non-Regulated Status 12/07/94

-   FDA Completion of Consultation 4/5/95

-   EPA Exemption from the requirement of a tolerance 11/2/94

-   Canadian Food Inspection Agency for fruit importation 12/2/97

-   Health Canada approval 4/17/98

ZW-20 squash contains coat protein genes from Zucchini Yellows Mosaic
Virus and Watermelon Mosaic Virus 2.

Subsequently, Seminis commercialized a second genetically engineered
squash line (CZW-3) containing three viral coat protein genes in 2000,
after receiving the following decisions from government agencies:

-   USDA Determination of Non-Regulated Status 5/6/97

-   FDA Completion of Consultation 7/11/97

-   EPA Exemption from the requirement of a tolerance 8/14/97

-   Canadian Food Inspection Agency for fruit importation 12/2/97

-   Health Canada approval 4/17/98

CZW-3 squash contains coat protein genes from Cucumber Mosaic Virus,
Zucchini Yellows Mosaic Virus and Watermelon Mosaic Virus 2.

These products have been well received by squash farmers and provide
excellent resistance to the indicated plant viruses. Natural resistance
to these viruses is significantly inferior and does not provide the same
high level of virus resistance. In many cases, farmers have reported
success in producing a squash crop using the biotech varieties whereas
they had crop failure using other varieties due to these viruses.

### USDA APHIS oversight is sufficient and adequate

USDA APHIS conducted a rigorous 2-year review prior to its Determination
of Non-Regulated Status for the virus-resistant ZW-20 squash line (and
later, a similar review for CZW-3). The Determination was supported by
several years of data accumu­lation from initial contained tests in the
greenhouse, and small-scale field trials to large-scale field trials.
These tests included studies on gene flow and viral interactions such as
encapsidation. During the deregulation process USDA solicited public
comments on the petition and the comments received were carefully
considered. Furthermore, USDA consulted state extension agents in
relevant states and USDA requested additional studies from Asgrow (now
part of Seminis Vegetable Seeds), which were performed. All this was
done in addition to the usual independent scientific literature survey
and expert consultations USDA routinely does on top of the voluminous
information requested of petitioners.

The issues before this EPA Science Advisory Panel are important
questions, which USDA APHIS has considered when making its
Determinations of Non-Regulated Status for PVCP-PIPs, specifically in
the areas of gene flow and viral interactions. USDA APHIS has now a
12-year history of making PVCP-PIP Determinations. Under FFDCA and
FIFRA, EPA can finalize an exemption from regulation for PVCP-PIPs and
not create an unnecessary and duplicative registration process that USDA
APHIS adequately covers.

### Loss of product

Since 1994, Seminis has conducted research on other PVCP-PIP vegetables,
including tomato and melon. In most cases, the PVCP-PIPs resulted in
biotech plants that had excellent resistance to the target viruses. If
such resistance had come from wild relatives via wide hybridization
techniques, as has been done in plant breeding for decades, these virus
resistant varieties would have been commercialized and farmers today
would be benefiting from improved virus resistance. However, because of
the regulatory costs to obtain approvals of PVCP-PIPs, Seminis
determined that these other PVCP-PIPs would not be able to justify
sufficient value-added to pay for the regulatory approvals. Therefore,
the vegetable PVCP-PIP projects were discontinued, which is unfortunate
for farmers who still must battle virus infestations.

It should be noted, that under today's regulatory burden, Seminis would
not be able to justify the investment to create our two biotech squash
products, ZW-20 and CZW-3, which are currently providing significant
benefit to American farmers. Squash is not a high-value crop. The
premium that farmers are willing to pay for virus resistant squash is
simply not enough to cover today's regulatory expenses. In fact, because
of PIP registration costs, it is likely that these virus resistant
squash varieties would have to be removed from the market should they no
longer be exempt from the requirement of a tolerance. This would place a
significant burden on those farmers who have come to depend on these
varieties in order to produce marketable crops.

### Regulatory burden

Development of biotech horticultural crops (minor crops) has slowed
significantly over the past five years, as seen by a decrease in field
trials of horticultural crops (see figure below) and a cessation or
severe reduction in research activity and product development in
industry. Even academic scientists are wondering if it will ever be
possible for them to release the biotech varieties they are developing
in the horticultural field. There are several reasons for this,
including the European Union's moratorium on biotech approvals, lack of
tolerance levels for adventitious presence in seed, and, of importance
to this Science Advisory Panel and the EPA, significantly increased
regulatory costs. While progress in the US has slowed and all approvals
in the EU have stopped until just recently, some countries, such as
China, continue to develop biotech products for their internal markets.
It is predicted that within a few years, China will emerge as the leader
in biotech horticultural crops.

US field trials from 1987 to February 2004 (Redenbaugh, K. and A.
McHughen. 2004. Regulatory challenges reduce opportunities for
horticultural biotechnology. California Agriculture 58:106-115),
compiled from http://www.nbiap.vt.edu/cfdocs/fieldtests1.cfm

![](media/image1.wmf){width="5.134722222222222in"
height="2.951388888888889in"}

The regulatory requirements to develop improved biotech varieties
(over-and-above the costs to develop traditionally bred varieties) is
now at least \$1 million per allele (if limited strictly to the US) and
more likely at \$5 million or more per allele, depending on the number
of countries in which approvals are required (an allele or genetic trait
is a transformation event). Due to the international trade in
horticultural commodities, there are few examples of products under
development in which both the seed and the product could be contained
solely in the US. More likely, a biotech variety will need approvals in
a number of countries to which the product might be exported. For
example, biotech processing tomatoes grown in California would end up
being exported as tomato paste or other products to many countries
around the world, each of which must give food approval prior to
commercialization. And, if the processed product contains seeds that
might be viable, environmental studies and approvals will also be
required in the importing country, even if the importation is intended
only for food consumption. Importing countries may also impose
additional and unique requirements, such as labeling or the ability to
trace the product back to the producing farm, as in the EU.

Any additional regulatory burden, such as registration of PVCP-PIPs,
would further reduce the opportunities to develop biotech horticultural
crops.

## Seminis request and recommendation

Seminis strongly recommends that the EPA continue to provide exemption
from regulation for PVCP-PIPs. USDA adequately addresses the issues
raised by EPA, including gene flow, transencapsidation and recombination
and has held numerous public fora and workshops to gather information
and obtain public input on PVCP-PIPs. EPA registration is unnecessary
and duplicative of the USDA oversight it has provided for 12 years.

**Seminis responses to FIFRA SAP questions**

1.  What scientific evidence supports or refutes the idea that plant
    viruses have significant effects on reproduction, survival, and
    growth of plant populations in natural settings? Is there scientific
    evidence that plant populations freed from viral pressure could have
    increased competitive ability leading to changes in plant population
    dynamics?

> **Research by Fuchs and Gonsalves with commercial biotech squash
> provided evidence that viruses are not limiting the growth, and fruit
> and seed production of a wild population of squash. They concluded
> that "there is little, if any, evidence that the introduction of virus
> resistance could provide wild squash species with a tremendous
> selective advantage." See attached report from the 8^th^ International
> Symposium on the Biosafety of Genetically Modified Organisms.**

2.  Please comment on the validity of the Agency list of crops that have
    no wild or weedy relatives in the United States with which they can
    produce viable hybrids in nature (i.e., tomato, potato, soybean, and
    corn)?

Seminis has extensive experience with tomato and sweet corn, neither of
which have wild or weedy relatives in the United States.

3.  Please identify other crops that have no wild or weedy relatives in
    the United States with which they can produce viable hybrid in
    nature, e.g., papaya, peanut, and/or chickpea.

> **There are no wild or weedy relatives of beans (*Phaseolus
> vulgaris*), cucumber, eggplant, onion, pea, pepper, and spinach in the
> United States. *Brassica oleracea* (cauliflower, broccoli, cabbage,
> etc.) has wild relatives, but embryo rescue is usually required for
> such crosses.**
>
> **There are no cucurbit crops (squash, melon, pumpkin) with
> conspecific weedy relatives in the United States. All of the
> conspecific wild cucurbits are insignificant with respect to
> weediness.**

4.  What laboratory techniques used to achieve genetic exchange between
    species (e.g., embryo rescue, use of intermediate bridging crosses,
    protoplast fusion) are not indicative of possible genetic exchange
    between these species in the field? Conversely, what techniques, if
    any, used in laboratory or greenhouse experiments provide the most
    reliable indication of ability to hybridize in the field?

Species, which can only be crossed using embryo rescue, use of
intermediate bridging crosses, hand crossing, protoplast fusion, etc.
are of little concern for gene flow in natural settings. Such species
should not be considered as potential recipients of transgenes in the
natural environment. For laboratory or greenhouse experiments,
appropriate techniques for ability to hybridize are wind and insect
pollination. Wind pollination can be simulated by providing air flow
within a greenhouse or via mechanical means. For insect pollination, the
appropriate pollinator for the species being studied must be chosen.

It is possible to use embryo rescue and/or bridging crosses to transfer
genes between several members of the genus *Cucurbita*, but the
intermediate individuals are sterile and/or remarkably weak, such that
they would not survive in a natural environment.

5.  Given that current bioconfinement techniques are not 100% effective,
    what would the environmental implications be of extremely low
    transfer rates of virus resistance genes over time?

The movement of genes between sexually compatible species has occurred
for centuries using common breeding practices. A major breeding strategy
in traditional plant improvement programs is to significantly increase
the level of natural PIPs to combat disease, insects or other pests.
Seminis is unaware of any traditional breeding efforts for virus
resistance that have led to the transfer of the PIP with resultant
negative impact on the environment.

> **Since EPA has determined that PIPs in traditionally bred plants are
> exempt from all FIFRA requirements (except for an adverse effects
> reporting requirement), it follows that these same PIPs moved to other
> sexually compatible plants, regardless of method, should also be
> exempt. Furthermore, should there be an adverse effect, the developer
> would be required by FIFRA to report the effect, at which time the
> Agency would respond accordingly. The potential of increasing a PIP is
> equally great in sexually compatible species, regardless of the method
> for transferring the PIP.**

See also response to Question 6.

6.  Please comment on the prevalence of tolerance and/or resistance to
    viruses in wild relatives of crops.

The majority of traditional viral resistance (if not all) in commercial
vegetable crops such as tomato, pepper and squash originated in wild
species. Examples include resistance to tobamovirus, geminivirus,
potyvirus and tospovirus in tomato and pepper and resistance to CMV,
potyvirus and geminivirus in squash. Wild relatives of *Cucurbita* spp.
crops include buffalo gourd and swamp gourd. The swamp gourd (*C.
okeechobeensis*) is the original source for resistance to some viruses
that is used by traditional breeding techniques.

> **Biotech virus-resistant squash has been commercially planted for 9
> years in the United States. During this time, there have been no
> reports of squash or wild relatives becoming a pest problem. There are
> no reports of wild or weedy squash relatives gaining a selective
> advantage due to proximity to virus-resistant squash fields. Farmers
> are often quick to report back to seed companies on any problems, such
> as those just described, but Seminis has received no farmer complaints
> on these issues. The observations over the past 9 years confirm the
> validity and completeness of the USDA Determinations of Non-Regulated
> Status as well as supporting EPA's 1994 proposed rule exempting from
> tolerance viral coat protein genes.**

The USDA acknowledged that pollen flow will likely occur between biotech
and wild squash and that movement of virus resistance genes into the
wild populations were likely to be retained in a population if the genes
confer an advantage to the plant containing the new genes. The evidence
from Seminis' virus survey of wild populations indicated that there is
not continuous virus pressure on these wild populations and under those
circumstances, wild population with the virus resistance gene will not
have any selective advantage over wild populations without the virus
genes. Therefore the selective pressure to maintain the virus-resistant
trait in the wild population will be minimal.

Natural populations of free living *Cucurbita* populations (FLCP) appear
to be largely free of infection by CMV, ZYMV and WMV2, strongly
suggesting that resistance to CMV, ZYMV and WMV2 would not provide any
selective advantage. Should the virus resistance genes from Seminis
biotech squash transfer to FLCP, the selective pressure to maintain the
virus resistance in natural populations of FLCP would be minimal, since
all evidence supports the conclusion that FLCP are not under significant
environmental stress from viral infection.

While these types of studies are useful for determining the
environmental safety of each new virus-resistant, biotech crop, the
oversight provided by the USDA ensures that all these issues are
considered and evaluated.

> **EPA's suggestion that the Agency could require PVCP-PIP registration
> would directly duplicate what USDA requires. Therefore, a duplicate
> submission to EPA would not be necessary. It is Seminis' opinion that,
> based on USDA regulatory oversight, all PVCP-PIPs should be exempted
> from tolerance.**

7.  Please specify techniques that do or do not provide measures of
    tolerance and/or resistance that are relevant to field conditions.

Mechanical inoculation and subsequent incubation in greenhouse
conditions, in general, provide measures of resistance that are relevant
to field conditions.

Visual ratings of symptoms are the preferred method in squash for
evaluating resistance to ZYMV, WMV, CMV, PRSV (Papaya Ringspot Virus),
SqLCV (Squash Leaf Curl Virus), and other viruses in the field. Symptoms
of infection are generally severe and obvious, resulting in plant
distortion, discoloration, and possibly death. ELISA techniques can be
used to identify which viruses are present, but this technique does not
necessarily confirm which virus is causing the symptoms observed. Plants
which are resistant to symptom development may serve as a host for a
particular virus, without displaying severe symptoms, so ELISA alone
cannot be used to determine resistance.

8.  How do environmental or other factors (e.g., temporal variations)
    affect tolerance and/or resistance? Given the expected variability,
    what measures of tolerance and/or resistance would be reliable?

Environmental factors strongly affect epiphytotics of disease, primarily
by affecting the population of vectors. Heavy rain, for example, can
decrease vector populations of aphids or whiteflies, decreasing disease
transmission. Presence or absence of wind can carry disease vectors.
Temperature has a similarly strong affect on viral vectors.

Both temperature and light intensity affect the expression of
resistance. Temperature higher than 30 C, for example, breaks down the
resistance to tomato spot wilt virus in pepper. Conversely, CMV is more
severe at lower temperatures. High light intensity generally favors the
expression of resistance. Evaluation of resistance in a greenhouse with
adequate temperature and light control or in an incubator with more
precise environmental control would provide reliable measures of
resistance.

Given that these environmental factors have such strong affects,
measures of resistance are challenging to standardize. Visual ratings of
disease symptoms are the only "reliable" method for evaluating
resistance, as these relate directly to the health of the individual
plant, without regard to viral presence.

9.  What would be the ecological significance if a plant population
    acquired a small increase in viral tolerance and/or resistance above
    a naturally-occurring level?

    There would be little ecological impact on most wild plant
    populations because naturally occurring resistance is already
    relatively high. This is the case for wild relatives of *Cucurbita*
    spp., which typically grow in marginal areas or disturbed
    environments (e.g. roadsides, tree-fall clearings). Even a large
    increase in viral tolerance is not likely to have a large
    consequence for plant populations.

10. Please comment on how necessary and/or sufficient these conditions
    are to minimize the potential for the PVCP-PIP to harm the
    environment through gene flow from the plant containing the PVCP-PIP
    to wild or weedy relatives. Would any other conditions work as well
    or better?

11. To what extent are *novel* viral interactions (e.g., recombination,
    heterologous encapsidation) involving a viral transgene an
    environmental concern?

    Crop plants in commercial field are often infected with multiple
    viruses. For example, some pepper plants have been shown to be
    infected with up to 7 viruses. Recombination and possibly
    heterologous encapsidation are likely occurring naturally,
    irrespective of the presence of PVCP-PIPs. The risk of novel viral
    interactions involving a viral transgene in biotech plants would not
    be higher than that already existing in traditional crop plants.
    Because traditional sources of resistance to plant viruses are less
    effective and do not confer complete resistance, the plants still
    support viral populations. As a result, the overall number and
    volume of viral particles consumed is higher with the traditional
    sources of resistance. The higher viral populations in the
    non-resistant or partially resistant plant populations will, if
    anything, lead to a higher probability of transencapsidation.

> **Research by Fuchs and Gonsalves with biotech squash indicated that
> heterologous encapsidation, but not recombination, may occur at a very
> low rate (2%). In another experiment, the rate was 0%. Based on their
> experiments, they concluded that such encapsidation was "not durable
> and had restricted impact on the environment." See attached report
> from the 8^th^ International Symposium on the Biosafety of Genetically
> Modified Organisms.**

# **The NRC Committee in its 2000 report concluded that regarding recombination between transgenes and viral pathogens, in all cases examined, only homologous sequences were exchanged. No experimental data indicate that recombination can occur between virus genomes and transgene sequences that are derived from distantly related or unrelated viruses. Since trans­encapsi­dation does not involve exchange of genetic material, any unique insect vectoring properties of a transencapsidated virus genome will not be inherited.** 

The USDA as well as Seminis' petitions also addressed this issue.
Cucurbits are readily infected with multiple viruses including CMV, ZYMV
and WMV2. The virus titer within multiple infected plants can be several
hundred times greater than the level of the biotech RNA or coat protein
in ZW-20 or CZW-3 plants. The elements contained in ZW-20 and CZW-3 do
not pose an exposure of viral components since these components have had
the potential to interact with one another in nature in mixed
infections. The biotech lines may actually reduce the probability of
recombina­tion merely by the fact that the virus titer of CMV, ZYMV and
WMV2 is significantly reduced in the biotech lines ZW-20 and CZW-3.

12. What conclusions can be drawn as to whether the likelihood of
    recombination and/or heterologous encapsidation would be increased
    or decreased in a transgenic plant compared to its non-bioengineered
    counterpart?

Seminis knows of no evidence showing that the frequency of viral
recombination and/or heterologous encapsidation in a biotech plant is
higher or lower than that in traditionally-bred plants.

13. How effective is deleting the 3' untranslated region of the PVCP
    gene as a method for reducing the frequency of recombination in the
    region of the PVCP gene? Is this method universally applicable to
    all potential PVCP-PIP constructs? Would any other methods work as
    well or better? Which methods are sufficiently effective and
    reproducible such that actual measurement of rates to verify rate
    reduction would be unnecessary?

14. Are any methods for inhibiting heterologous encapsidation or
    transmission by insect vectors universally applicable to al
    PVCP-PIPs? Which methods are sufficiently effective and reproducible
    such that actual measurement of rates to verify rate reduction would
    be unnecessary?

15. How technically feasible would it be to measure rates of
    recombination, heterologous encapsidation, and vector transmission
    in PVCP-PIP transgenic plants in order to show that rates are
    reduced?

16. Please comment on how necessary and/or sufficient each of these
    conditions is to minimize the potential for novel viral
    interactions. Please address specifically what combination would be
    most effective or what conditions could be modified, added, or
    deleted to ensure that potential consequences of novel viral
    interactions in PVCP-PIP transgenic plants are minimized.

> **The PVCP-PIP biotech plants do not introduce any significantly new
> genetic material to the environment that was not already present.**

17. To what degree and in what ways might a PVCP gene be modified (e.g.,
    through truncations, deletions, insertions, or point mutations)
    while still retaining scientific support for the idea that humans
    have consumed the products of such genes for generations and that
    such products therefore present no new dietary exposures?

18. What are the potential adverse effects, if any, of such
    modifications on nontarget species (e.g., wildlife and insects that
    consume the PVCP-PIP)?

19. To what degree and in what ways might a PVCP gene be modified (e.g.,
    through truncations, deletions, insertions, or point mutations)
    before it would be a concern that novel viral interactions due to
    the modifications could occur because the PVCP gene would be
    significantly different from any existing in nature?

20. Would any additional requirements related to PVCP-PIP identity and
    composition (e.g., demonstration that the transgene has been stably
    inserted) be needed for significant reduction of risks associated
    with PVCP-PIPs?

21. Are there any consideration beyond gene flow, recombination, and
    heterologous encapsidation as posed in the preceding questions that
    the Agency should consider in evaluating the risk potential of
    PVCP-PIPs (e.g., synergy)?

 

No. Even though there was no evidence to suggest that potyviral coat
proteins are involved in synergy, Asgrow inoculated both ZW-20 and CZW-3
squash plants with several common squash-infecting viruses. Using other
common cucurbit viruses, like CMV and PRV, Asgrow demonstrated that
there was no synergy between the ZYMV and WMV2 transgenes and other
viruses that commonly infect squash. This demonstrated that the
transgenes would not result in other viruses causing severe symptoms.
Although highly unlikely, if synergistic symptoms occurred in ZW-20 or
CZW-3 plants, this would only be an agronomic phenomenon and have no
long-term environmental impact (American Institute of Biological
Sciences (AIBS). 1995. Biotech virus-resistant plants and new plant
viruses. Meeting report from AIBS workshop sponsored by U.S. Department
of Agriculture. 47pp).

**2000 National Research Council (NRC) Report**

**"Genetically Modified Pest-Protected Plants: Science and Regulation"**

**Seminis' Response**

Following are Seminis' comments on the information, analyses, and
conclusions on viral coat proteins, with specific focus on Seminis'
biotech virus-resistant **squash lines ZW-20 and CZW-3**

### General

The NRC report presented the biotech virus-resistant squash as a case
study to illustrate potential risks posed by virus-derived transgenes.
In the report, the Committee concludes that USDA's assessment about how
the spread of virus-protective transgenes will affect free-living
*Cucurbita pepo* populations is not well supported by scientific
studies, suggesting that the review by the USDA was not rigorous enough.

It should be noted that the deregulation of the biotech virus-resistant
ZW-20 squash line by USDA was the result of a rigorous 2-year review by
USDA, and information was gathered from an array of sources. It was
supported by several years of data accumu­lation from initial contained
tests in the greenhouse, and small-scale field trials to large-scale
field trials. During the deregulation process USDA solicited public
comments on the petition and the comments received were carefully
considered. Furthermore, USDA consulted state extension agents in
relevant states and USDA requested additional studies from Asgrow (now
part of Seminis Vegetable Seeds), which were performed. All this was
done in addition to the usual independent scientific literature survey
and expert consultations USDA routinely does on top of the voluminous
information requested of petitioners.

### Health issues

The health concerns about biotech virus protected squash have been
related to both viral and bacterial genes that are expressed in all the
plant's cells. As mentioned in the NRC report, human and animal
consumption of plants with viral coat proteins is widely considered to
be safe, on the basis of common exposure to these proteins in
non-biotech squash. Asgrow performed a quantitative ELISA analysis of
the viral coat protein and for CZW-3 also of the linked NPT II protein.
The average coat protein levels in greenhouse grown fruits of both
biotech ZW-20 and CZW-3 were found to be significantly lower that viral
coat proteins found in virus infected non-biotech cucurbit fruits
collected from the local grocery store. In the fruit from biotech CZW-3
there were very low levels of NPT II protein. The NPT II protein is
ubiquitous in the environ­ment (Flavell *et al.*1992). The safety of NPT
II was detailed in publications of Fuchs *et al.* (1993a, b), Flavell
*et al.* (1992) and Nap *et al.* (1992). All data available indicate
that the kanamycin resistance gene can be safely used. The FDA has
approved many crops with the kanamycin resistance gene. Furthermore, FDA
has given food additive status to the NPT II protein.

### Environmental issues

As the NRC report indicates the major environmentally adverse effects
that have been discussed in connection with virus-resistant crops
pertain to effects of viral coat protein genes on the pathogenicity of
other viruses and consequences of crop-to-wild gene flow that could
allow beneficial transgenes to move into feral crop plants or closely
related weeds.

# [Putative effects of viral coat protein genes on the pathogenicity of other viruses.]{.underline}

The first issue was studied experimentally and it was concluded that the
risks that other viruses would become transmissible (from
heteroencapsidation) or that the non-pathogenic viruses would become
more virulent (from recombination) were exceedingly small (Fuchs *et
al.* 1998).

# The Committee concludes that regarding recombination between transgenes and viral pathogens, in all cases examined, only homologous sequences were exchanged. No experimental data indicate that recombination can occur between virus genomes and transgene sequences that are derived from distantly related or unrelated viruses.

Regarding transencapsidation and gain-of-transmission characters, it is
highly unlikely that functional coat proteins expressed in biotech
plants pose a significant risk of expansion of host range to new crop or
non-crop hosts. Trans­encapsi­dation does not involve exchange of genetic
material, meaning that any unique insect vectoring properties of a
transencapsidated virus genome will not be inherited. No data indicate
that expression of viral coat proteins enhance the virulence of
heterologous viruses. Studies by Fuchs & Gonsalves show that
heteroencapsidation will not cause epidemics and this is not likely to
have significant consequences to the environment.

Asgrow addressed the concerns about the squash also posing the risk that
its virus genes or the coat proteins they produced might interact with
other viruses to produce new viruses by conducting a study on mixed
infections of ZW-20. Using other common cucurbit viruses, like CMV and
PRV, Asgrow demonstrated that there was no synergy between the ZYMV and
WMV2 transgenes and other viruses that commonly infect squash. This
demonstrated that the transgenes would not result in other viruses
causing severe symptoms.

USDA and the petition also addressed this issue. Cucurbits are readily
infected with multiple viruses including ZYMV and WMV2. The virus titer
within multiple infected plants can be several hundred times greater
than the level of the biotech RNA or coat protein in ZW-20. The elements
contained in ZW-20 do not pose an exposure of viral components since
these components have had the potential to interact with one another in
nature in mixed infections. The biotech line may actually reduce the
probability of recombina­tion merely by the fact that the virus titer of
ZYMV and WMV2 is significantly reduced in the biotech line ZW-20.

*The second issue, whether (wild) relatives could benefit from viral
coat protein genes was according to the NRC report more controversial.*

*Crop-to-crop gene flow*

The genus *Cucurbita* includes five domesticated species and 22 wild
species (Decker 1988). The common ancestor of all cucurbits is probably
an annual gourd-producing plant that was first used in the New World
agriculture about 10,000 years ago. The *C. pepo* lineage appears to be
composed of two subsets, formally identified as two subspecies *ovifera*
and *pepo*. Subspecies *pepo* includes domesticated types, pumpkins,
zucchini, marrow varieties, and some ornamental gourds, whereas
subspecies *ovifera* var. *ovifera* includes the remaining ornamental
gourds varieties and acorn, crookneck, straightneck, scallop and yellow
squash (Wilson 1993). The five cultivated cucurbit species include *C.
pepo*, *C. maxima*, *C. mixta*, and *C. moschata*. The fifth species is
*C. ficifolia*, which is mostly cultivated in South America.
Inter-specific hybridization has been extensively investigated and is
well understood in the four first-mentioned cultivated species. F~1~
hybrids can be obtained in breeding programs, but only with difficulty
and such hybrids usually are sterile. There is no evidence of
spontaneous hybridization among these four species despite the fact that
they have been grown side by side under cultivation for many generations
(Whitaker & Robinson 1986).

A weed pest is a plant that grows persistently in locations where it is
unwanted. There are several definitions of weediness, but they all have
the undesirable nature of weeds from the point of view of humans in
common (de Wet & Harlan 1975). Significant differences were found in the
distribution of weedy characteristics among weeds, 'normal plants' and
crops (Baker 1965; Keeler 1989). Baker (1965) described 12 common weed
attributes, which include rapid growth to germination and flowering in
many environ­ments, internally controlled discontinuous germination,
long-lived seeds, continuous seed production, use of wind or
unspecialized insects for pollination if outcrossing occurs, high seed
production and good competitive­ness. Baker's list of weed attributes can
be used as an imperfect guide to the likelihood that a plant will behave
as a weed. Keeler (1989) analyzed *C. maxima*, a close relative to *C.
pepo*, and stated that *C. maxima* possess 3 out of 15 characteristics
of plants that are notably successful weeds. Those characteristics are:
continuous production of seeds as long as growing conditions permits,
use of unspecialized insects as pollinators, and strong competitiveness
with other plants. However, *C. pepo* that has been bred for
agricultural use, e.g. yellow crookneck squash, has few traits that are
associated with weediness. However, despite the extensive cultivation of
*C. pepo* in the US and Mexico since antiquity, there is no body of
scientific reports of significant weediness of *C. pepo* in those
countries. *C. pepo*, yellow crookneck squash is not listed as weed in
the Federal Noxious Weed Act (7 U.S.C. 2801-2813) (see Westbrooks 1998)
and is not reported by the Weed Society of America to be a common or
troublesome weed anywhere in the US (Holm *et al.* 1979; Muenscher
1980). Also in the NRC report it is mentioned that volunteer squash
plants are not known to spread and become weeds. Over-wintering
volunteers are always killed after germination by spring frosts, as
squash is very sensitive to cold. The Committee stated that it knows of
no scientific evidence that crop-to-crop gene flow has caused health or
environmental risks to date. Contamination with pollen from other farms
is likely to be very low in most cases. Based on traditional breeding
practices, isolation distances has been established for a range of
(outcrossing) species, which can also be used as reference for the
isolation of biotech (squash) crops.

# Crop-to-wild gene flow

Interspecific hybridization has been extensively investigated. In the
United States, there exist three free-living subspecies of *C. pepo*
(FLCP) that can cross with cultivated varieties of *C. pepo* without
loss of fertility (Whitaker & Bemis 1964; Nee 1990). These include the
free-living gourds in Texas designated *C. pepo* ssp*. ovifera* var*.
texana* and free-living gourds in Illinois, Arkansas and Oklahoma
designated *C. pepo* ssp*. ovifera* var*.* *ozarkana* (Wilson 1993) and
a putative relative of *C. pepo* that was rediscovered at several sites
in northeastern Mexico during the early 1980's, designated *C. pepo*
ssp. *fraterna* (Nee 1990). Both ssp. *ovifera* can cross with
cultivated *C. pepo* varieties through natural pollination mediated by
honey bees without loss of fertility (Kirkpatrick *et al.* 1985). Field
experiments done by Fuchs & Gonsalves showed that the coat protein genes
of CMV, ZYMV and WMV2 could readily move from biotech squash into *C.
texana* upon hybridization and introgression. Movement of the coat
protein genes provided resistance against these three viruses to *C.
texana* and a selective advantage under conditions of high virus
pressure in contrast to under low virus pressure where there is no
selective advantage. However, under high virus pressure, transfer of
transgenes to *C. texana* virtually did not occur because the *C.
texana* plants severely affected by the virus and produced very few
flowers.

*C. texana* is not a noxious weed. The question is, would it become a
noxious weed if it became virus-resistant? Free living squash
populations (FLCP) are not reported to be a serious problem in unmanaged
or agriculture ecosystems. Squash is not listed as a weed in the Federal
Noxious Weed Act (7 U.S.C. 2801-2813) and is not reported by the Weed
Society of America to be a common or troublesome weed in the U.S.
(Bridges & Baumman 1992). FLCP have previously been listed as a
significant weed in soybean and cotton fields in Arkansas. The Arkansas
representative to the Weed Society of America, Dr. Baldwin stated that
FLCP are not currently as significant a problem in this region as they
were in the past. Dr. Weidemann from the Univ. of Arkansas, who
conducted research to identify biological control agents to eliminate
FLCP from soybean fields, confirmed that FLCP are only a minor problem
in Arkansas in recent years because FLCP have been controlled through
the use of new herbicides. As mentioned in the NRC report, in 1977 FLCP
were listed as one of the top-10 most important weeds in Arkansas.

The critical question was whether viruses kept the population of wild
squash, which produces inedible gourds, in check. In other words, if
gene flow occurs between VR biotech and wild relatives, will the VR gene
be retained? Asgrow conducted a survey to test this. The evidence from
the virus survey of wild populations indicated that there is not
continuous virus pressure on these wild relatives and under those
circumstances the virus resistance gene will not have any selective
advantage over plants that lack this gene.

Some experts in biosafety research said the study of 14 plants from 9
locations was too limited to draw valuable conclusions. However, the
survey did not represent a mere 14 plants, but 9 populations of FLCP,
each population consisting of many individuals. It was based on
extension agents visits to 9 sites in Mississippi, Louisiana, and
Arkansas with instructions to survey the populations for the presence of
virus symptoms, which would readily be visible on FLCP. If the extension
agents saw symptoms, they were to take leaf samples for Asgrow to
analyze. However, no visible symptoms were observed in any of the
locations. As an extra precaution, our collectors were asked to collect
a representative vine to double check through ELISA, double diffusion
serology and inoculations onto indicator plants in order to confirm and
validate visual evaluation.

Field experiments done by Fuchs & Gonsalves indeed showed that under
conditions of low virus pressure, hybrids that expressed the three CP
genes did not appear to have any selective advantage over *C. texana*
and their non-biotech counterparts. Furthermore, preliminary survey by
Fuchs & Gonsalves indicated that *C. texana* are not readily infected by
viruses in their natural ecosystems. Therefore, the selective pressure
to maintain the virus resistance trait in the wild population will be
minimal. In conclusion, based on field experiments, free-living *C.
texana* resistant to ZYMV, CMV and WMV2 are unlikely to become a
significant threat to the environment as invasive and eventually even
more noxious weed pests.

The concern regarding the risk of biotech virus resistance causing wild
relatives to become more weedy seems to ignore the fact that resistance
genes to these viruses already exist in *C. pepo* and can possibly cross
into wild relatives. Thus, the biotech virus-resistant squash does not
contain traits that are not already present in the germplasm pool.
Zucchini squash with resistance to CMV have been available from seed
companies for many years. Traditionally bred varieties with resistance
to ZYMV and WMV are also commercially available. There are also other
species of wild squash, which are present in the US that are resistant
to PRV, CMV and WMV. Despite the fact that they have these resistances,
these species are not known to be significant weed problems in the US.

In the report, commissioned by APHIS, Wilson, a squash expert at Texas
A&M Univ. was asked "if crop/weed interaction has occurred within the
*C. pepo* complex of domesticated and free-living forms throughout the
3,000 year history of human agricultural activity in the Eastern US,
then why be concerned about possible involvement of transgenic strains?"
All available evidence, both archaeological and botanical, indicates
that new, domesticated elements of *C. pepo* complex have been
sequentially introduced into the agricultural systems of eastern North
America over the past 3,000 to 7,000 years. Wilson claims that the
source of transgenes, and unknown interactions between these unique
genetic elements, which are not part of the *C. pepo* complex, and the
*C. pepo* genome represent an unknown and untested factor. The process
of injecting a foreign genetic element, a functional gene that has no
precedent within the phylogenetic history of the crop/weed system,
constitutes a biological risk. This reasoning seems to overlook the fact
that resistance genes to these viruses already exist in *C. pepo*.
Traditionally bred varieties with resistance to ZYMV, CMV and WMV are
commercially available and there are species of wild squash carrying
resistance to PRV, CMV and WMV. As said earlier the biotech
virus-resistant squash does not contain traits that are not already
present in the germplasm pool. The introduced CMV, WMV2 and ZYMV coat
protein genes encode viral coat proteins substantially similar to viral
coat proteins found in abundance in virus infected traditionally bred
squash fruits as was found when viral coat protein levels were
determined in non-biotech squash fruits collected from local grocery
stores. No convincing data were generated that backed Dr. Wilson's
statement that data presented in his report point towards the clear
presence of risk regarding increased weediness and loss of crop plant
biodiversity. The 'africanized honey bee' which Dr. Wilson mentions as
example of the impact on natural populations due to both intentional and
accidental human manipulations is not relevant for the squash case. Most
if not all cases in which human intervention has caused adverse
ecological effects refer to introduction of exotics, like the rabbits
and Opuntia cactus in Australia. Placing virus-resistant squash in its
natural habitat will not have such drastic effects. Dr. Wilson's
concerns about the introduction of biotech virus-resistant squash are
not backed by other prominent scientists in the field including Drs.
Provvidenti and Robinson of Cornell University, who are both world
renown in the area of breeding and pathology in Cucurbits. They
supported the release of the virus-resistant biotech squash.

# Exemption of viral coat proteins

In addition to exempting plant-pesticides derived from sexually
compatible plants, the 1994 and 1997 EPA documents propose a number of
more specific exemptions. EPA generally provides more reasonable
scientific justification for these exemptions. One specific class of
plant products that was proposed for categorical exemption was viral
coat proteins. Viral coat proteins are already present in foods because
of natural virus infec­tions of crops and have not caused obvious medical
problems, so health concerns are considered minimal. The EPA exemption
of viral coat proteins is also based on considerations that \"include
the low potential for adverse effects to non-target organisms and the
potential benefits (environmental and economic) of utilizing virus coat
protein mediated resistance.\" The NRC committee, in general, agrees
with this assessment of the minimal health and non-target effects posed
by viral coat protein expression in crop plants and concludes that
"Viral coat proteins in biotech pest-protected plants are not expected
to jeopardize human health because consumers already ingest these
com­pounds in non-biotech food. However, the committee questions the
categorical exemption of all viral coat proteins under FIFRA due to
concerns about outcrossing with weedy relatives".

In the report, it is stated that although ecological concerns are
discussed and a more restrictive exemption that considers outcrossing is
presented, the proposed rule favors complete exemption of viral coat
proteins. It is not completely clear what is meant by the proposed rule
because the Committee advises that "EPA should not categorically exempt
viral coat proteins from regulation under FIFRA. Rather, EPA should
adopt an approach, such as the Agency's alternative proposal, in which
the Agency can limit its exemption considering the gene transfer risks
associated with the introduction of viral coat proteins to plants".
Following the decision trees for viral coat proteins in virus-resistant
squash for health and weedy-relative concerns as given in para­graphs
3.3.1 and 3.3.2 of the NRC report, in both cases lead to exemption as
indicated hereunder.

[Health concern:]{.underline}

1\) Is the substance found in plant parts that consumers, including
human and non-human consumers, such as food animals or pets eat or
workers come into contact with?

**a) Yes or Unknown---go to 2**

b\) No---exempt from health concerns

2\) Is the substance known to have general chemical and physical
properties common to many allergens?

a\) Yes or Unknown---subject to safety assessment

**b) No---go to 3**

3\) Is the substance similar to substances that people now eat or come
into contact with, and can confident predictions of safety based on the
similarities be made?

**a) Yes---go to 4**

b\) No or Unknown---subject to safety assessment

4\) Is the expected exposure to the substance substantially greater than
current exposures?

a\) Yes or Unknown---subject to safety assessment

**b) No---go to 5**

5\) Is there a reasonable chance, based on known properties of the
substances, that its production will lead to harmful concentrations of
toxicants or allergens that consumers eat or workers come into contact
with?

a\) Yes or Unknown---subject to safety assessment

**b) No---exempt from health concerns**

[Ecological concern:]{.underline}

1\) Does the cultivated plant occur in feral populations or hybridize
with related species in the United States?

**a) Yes or More data needed---go to 2**

b\) No---exempt from weedy-relative considerations

2\) Have feral populations or wild relatives been reported as weedy or
invasive in the United States or have a reasonable potential to become
weedy?

**a) Yes or More data needed---go to 3**

b\) No---exempt from weedy-relative considerations

3\) Does the gene for resistance confers a specific type of resistance
or a greatly enhanced degree of resistance that is not found in feral
populations or sexually compatible wild relatives in the United States?

**a) Yes or More data needed---go to 4**

b\) No---exempt from weedy-relative considerations

4\) Is it reasonable to expect that this trait could have a substantial
impact on the population dynamics of feral plants or wild relatives and
will lead to increased abundance?

a\) Yes or More data needed---subject to weedy-relative considerations

**b) No---Exempt from weedy-relative considerations**

### Conclusion

In a New York Times article of Nov. 3 1999, a squash grower was
interviewed. He indicated that viruses devastating his squash plants,
giving no yield and, besides breeding virus-resistant varieties either
through biotech or traditional breeding, there is no cure to this
disease. Therefore, the biotech virus-resistant squash plants, which
show very high protection, are especially valuable in light of the
difficulties to obtain similar multiple resistance by traditional
breeding strategies or to control vector populations by cultural
practices and the use of chemicals.

In summary, the NRC report provides no compelling reason for the
withdrawal of the decision to consider Seminis' ZW-20 and CZW-3 biotech
virus-resistant squash non-regulated products. The report suggest
further areas of study regarding the putative outcrossing from
cultivated *C. pepo* into free living *C. pepo* subspecies. Such further
research has been done or is taking place in public research centers,
(partly) funded by USDA grants, like the 3-year study taking place at
the USDA Agricultural Research Service, Madison (WI) about the gene flow
from biotech *C. pepo* into free living population by Staub, Quemada &
Walters and the 3-year study of Falk (UC, Davis) on the incidence and
origin of new viruses in multiple virus-resistant Cucurbits. Additional
work on the area of outcrossing or recombination continues to support
the finding of the USDA determination. Additionally, these studies
reflect a desire to maintain a stewardship component for this
technology. Seminis have provided samples to university researchers
funded by USDA to assist in such stewardship research.

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