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

<p style="text-align: justify;">Events J101 and J163 were field tested at several locations in the United States from1998 to 2004. These field trials were conducted to evaluate agronomic performance, disease and insect resistance, phenotypic equivalency, and for the multiplication of seed. Events J101 and J163 were compared to their non-transgenic counterpart which consisted of a population of null segregants obtained during the backcrossing of the transformed events into sets of nonmodified elite clones. No differences in disease and insect susceptibilities were observed between events J101 and J163 and the control population of null segregants. Phenotypic evaluations for events J101 and J163, including seed germination, seedling emergence, seedling vigour, vegetative vigour, dry matter yield, regrowth after cutting, and stand persistence were within the range of values observed for the control populations at each of the sites. Events J101 and J163 were generally equivalent to the control populations, indicating that the phenotype was not unintentionally altered by the genetic modification.</p>

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

<p style="text-align: justify;">The potential for introgression of the glyphosate-tolerance trait from events J101 and J163 into other alfalfa plants, or to wild or feral relatives of alfalfa, was investigated. Alfalfa is an outcrossing species and pollination is achieved by hymenopteran species, such as the leaf cutter bee (<i>Megachile rotundata</i>), honeybee (<i>Apis mellifera</i>), and bumble bee (<i>Bombus</i> spp.). Alfalfa outcrosses and hybridizes with members of the <i>M. sativa</i> complex (<i>M. sativa</i> subsp. <i>sativa</i> (cultivated alfalfa), subsp. <i>falcata</i>, subsp. <i>glutinosa</i>, subsp. <i>coerulea</i>, subsp. x <i>tunetana</i>, subsp. x <i>varia</i>, subsp. x <i>polychroa</i>, and subsp. x <i>hemicycla</i>). There have been reports of natural, but limited hybridization with <i>M. glomerata</i>; this species is indigenous to southern Europe and North Africa, and is not present in North America. <i>M. lupulina</i> (black medick) is an annual weed species indigenous to Europe and common in North America. Black medick does not hybridize with <i>M. sativa</i>.</p>

<p style="text-align: justify;">Alfalfa is primarily cultivated for its herbage. Ideally, alfalfa herbage is harvested (<i>i.e.</i>, cut) at the first onset of flowering to optimize the nutritional quality and dry matter yield. Gene outflow is therefore negligible under this type of management. However, gene outflow is significant when alfalfa is grown for the production of seed, or when plants are inadvertently allowed to flower and mature (<i>e.g.</i>, in old pastures, or unharvested hay fields). Alfalfa grown for seed production is physically isolated from other alfalfa fields to ensure varietal purity, thus limiting gene flow. Isolation distances ranging from 200 to 300 m are required by seed certification agencies in the United States and Canada for the production of pedigreed seed.</p>

<p style="text-align: justify;">The cultivation of glyphosate-tolerant alfalfa for seed production may therefore result in the development of glyphosate-tolerant hybrids with members of the <i>M. sativa</i> complex, and to a limited extent with <i>M. glomerata</i> in areas where this species is found. These hybrids will develop in areas where members of the<i>M. sativa</i> complex are indigenous (Europe, Asia, the Middle East and North Africa), as well as in North America where these species have become established and feral. Introgression of the trait into <i>M. glomerata</i> would occur only in areas where this species is indigenous. The glyphosate tolerance trait is not expected to provide a competitive advantage to hybrid plants unless these are growing in managed environments routinely subjected to glyphosate applications. Glyphosate-tolerant hybrids could be managed with conventional weed control practices, including herbicides other than glyphosate.</p>

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

<p style="text-align: justify;">Alfalfa does not possess characteristics typical of serious weeds, such as vigorous vegetative growth and abundant seed production. It is, however, considered an agricultural weed in Canada (Darbyshire, 2003), and an invasive species by the Southern Weed Science Society (Southern Weed Science Society, 1998). Free-living populations are widespread, and found in environments such as old hay fields, pastures, roadsides, and ditches. Alfalfa can also volunteer in a succeeding crop, and seed can lay dormant in the soil for many years. Seed dormancy in conventional alfalfa is characterized by the amount of hard seed, <i>i.e.</i>, seeds with a relatively hard and impermeable seed coat. Alfalfa seed normally contains variable amounts of hard seed, which requires scarification before germination will occur.</p>

<p style="text-align: justify;">Field trials of events J101 and J163 demonstrated that vegetative and reproductive growth, seed dormancy, germination and emergence and plant maturity were within the range observed for conventional alfalfa cultivars. The development of hard seed in events J101 and J163, and in the control population of null segregants was also investigated. The amount of hard seed in events J101 and J163 was significantly higher than in the conventional cultivars in one of the two years of the trials; however, these differences were attributed to the effect of environment rather than genotype. The results of these investigations led to the conclusion the alfalfa events J101 and J163 did not possess weediness characteristics, such as increased development of hard seed, compared to conventional alfalfa cultivars.</p>

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

<p style="text-align: justify;">The glyphosate-tolerant trait in events J101 and J163 is conferred by the novel protein CP4 EPSPS, an enzyme originally isolated from the soil bacterium <i>Agrobacterium</i> sp. strain CP4. The novel form of this enzyme is similar to that naturally found in plants, bacteria and fungi. The lack of toxicity of CP4 EPSPS has been substantiated in previous regulatory approvals of glyphosate-tolerant plants, and its high substrate specificity makes it unlikely to metabolize any other endogenous substances to produce toxic compounds. The cultivation of glyphosate-tolerant alfalfa cultivars derived from events J101 and J163 would therefore not be expected to result in deleterious effects on non-target and beneficial organisms attributed to the novel protein. The results of studies on CP4 EPSPS in previously approved glyphosate-tolerant crops were therefore used to evaluate the impact of this novel protein in J101 and J163. These studies evaluated the effect of CP4 EPSPS on pollinators (<i>e.g</i>. bees), soil organisms (<i>e.g.</i>, earthworms), and beneficial arthropods. No toxic effects on these beneficial and non-target species were reported in the studies. Results from field trials with J101 and J163 also demonstrated similar susceptibilities to diseases and insect pests of alfalfa, compared to conventional alfalfa cultivars. Alfalfa cultivars expressing the glyphosate-tolerance trait would therefore not be expected to become new hosts for plant-pathogens, or insects pests, any more than conventional alfalfa cultivars.</p>

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

<p style="text-align: justify;">Events J101 and J163 did not exhibit any novel phenotypic characteristics that would increase the survival of glyphosate-tolerant alfalfa in either unmanaged habitats, or in areas outside of the current geographical range of alfalfa production. However, the introgression of the glyphosate-tolerance trait into members of the M. sativa complex is possible when these are found growing in the vicinity of alfalfa populations containing either J101 or J163. This trait could also introgress into M. glomerata in areas where this species is endemic. The introgression of the glyphosate-tolerance trait into indigenous populations of the M. sativa complex, and M. glomerata, will not impart increased fitness, or weediness characteristics to these indigenous species, other than herbicide tolerance.</p>
