<p style="text-align: justify;"><b>Field Testing:</b></p>

<p style="text-align: justify;">Field trials were established in 1996-1997 in Poland, Spain and Romania with C5 shoots grafted onto non-transgenic rootstocks. These trials were maintained for efficacy testing of the transgenic lines and deliberately inoculated with PPV using infected grafts or interspersed with infected trees to test for resistance to aphid transmitted virus. Further field trials in the United States were approved in 1995 and included trees which were rooted from C5 clones as well as grafted material. Pollen movement from this trial has been monitored between blocks of transgenic and non-transgenic trees.</p>

<p style="text-align: justify;"><b>Outcrossing:</b></p>

<p style="text-align: justify;">Outcrossing is expected in <i>P. domestica</i> due to the high levels of self incompatibility shown in this species. Seeds collected from open pollinated field trials have all shown a 1:1 segregation pattern of transgenic to non-transgenic, indicating that C5 is not self-pollinating nor being pollinated by other transgenic lines. Non-transgenic control trees of the same parental material in the experimental blocks have not produced any transgenic progeny, confirming that this line is self-incompatible and the transformation process has not altered the compatibility status of the material.<br />
Seeds collected from non-transgenic trees planted approximated 500 and 1000 meters from the transgenic plot were sampled over six years. Of 2,950 seeds tested, only two transgenic seeds were identified, from the 500 meter plot (outcrossing frequency was 2 in 850 seeds &ndash; 0.23%). It was considered that bees were the most likely vehicle of pollen movement. Further experiments with nine &lsquo;sentinel&rsquo; sites are being continuously monitored.</p>

<p style="text-align: justify;">Gene flow between <i>P. domestica</i> and other <i>Prunus</i> species is unlikely as these species are nearly all diploid while <i>P. domestica</i> is hexaploid. Successful crosses have been made between <i>P. domestica</i> and other species, but most hybrids were sterile and the literature contains many references to the difficulty of interspecific hybridisation within <i>Prunus</i>.<br />
Introgression between wild and cultivated species is not documented, but is physically possible as escapes of cultivated varieties are frequently found in woods, pastures and abandoned orchards. However, intercrosses with truly wild species is unlikely as these species are extremely different in morphology as well as in adaptation.</p>

<p style="text-align: justify;"><b>Weediness potential:</b></p>

<p style="text-align: justify;"><i>P. domestica</i> is not described as a weedy species and none of the <i>Prunus</i> species that may be sexually compatible with <i>P. domestica</i> are described as weedy species. In addition, plum is not listed as a noxious weed by the United States Federal authorities or on other weed lists.</p>

<p style="text-align: justify;"><b>Secondary and Non-Target Effects:</b></p>

<p style="text-align: justify;">The biology of the C5 plum trees with respect to their potential to affect non-target organisms such as beneficial insects (including pollinators such as bees), and biocontrol organisms was evaluated. The C5 plum does not express detectable coat protein from PPV, which eliminates concern of protein exposure to non-target organisms. Furthermore, even if C5 did express viral coat protein this would not increase the issue of potential impacts to non-target organisms as the PPV coat protein is not known to have any toxic properties. Plant viruses are ubiquitous in the environment and cause damage to fruits, leaves, seeds, flowers, stems, and roots of many important crop species. These viruses infect virtually every plant species and, under natural conditions, certain plant viruses are nearly always present on particular crop or weed hosts. Viral coat proteins are therefore routinely ingested by virtually all mammals when virus-infected fruits and vegetables are consumed. The small-interfering RNAs (siRNA) responsible for the PTGS resistance mechanism in C5 plum are also not of concern as nucleic acids are a normal part of every living organism and do not have toxic or allergenic properties.</p>

<p style="text-align: justify;">The <i>nptII</i> and &szlig;-glucuronidase genes are commonly used marker genes found in soil inhabiting <i>E. coli</i> bacteria. These bacteria are not plant or human pathogens, and do not cause disease symptoms or the production of infectious agents in plants. In addition, these marker genes are not known to cause adverse effects to non-target organisms and both have been granted exemption from the requirement of a tolerance by EPA for use in or on all raw agricultural commodities</p>

<p style="text-align: justify;"><b>Impact on Biodiversity:</b></p>

<p style="text-align: justify;">Analysis of available information indicates that C5 plum exhibits no traits that would cause increased weediness, that its unconfined cultivation should not lead to increased weediness of other cultivated plum or other sexually compatible relatives, and that it is unlikely to harm non-target organisms common to the agricultural ecosystem or threatened or endangered species. Based on this analysis, there is no apparent potential for significant impact to biodiversity.</p>

<p style="text-align: justify;">Analysis of data on agronomic performance, disease and insect susceptibility, and compositional profiles of the plums indicate no significant differences between C5 plum and non-transgenic counterparts that would be expected to cause either a direct or indirect plant pest effect on any raw or processed plant commodity. Data supplied by the applicant, including results of 10 years of field tests in various environments, suggest that the C5 plum trees have not had observable or measurable impacts on the ecosystems in which they have been allowed to grow. Based upon available information, it was determined that there are no past, present, or reasonably foreseeable actions that would aggregate with effects of the proposed action to create significant cumulative impacts or significantly reduce the long-term productivity or sustainability of any of the resources (soil, water, ecosystem quality, biodiversity, etc.) associated with the ecosystem in which C5 plum is planted.</p>
