<p style="text-align: justify;">Alfalfa (<i>Medicago sativa</i> L.), also known as lucerne, is an herbaceous perennial legume (family Leguminosae) grown as a forage crop worldwide. In 2006, the world production of alfalfa was estimated at 436 million metric tonnes. The United States, Argentina and France are among the major alfalfa producing countries. The principal use of alfalfa is for ruminant livestock feed, mainly for the production of meat and milk. Alfalfa is also processed commercially as dehydrated pellets, and meal, and as compacted hay. Alfalfa is also an important source of nectar for honeybees.</p>

<p style="text-align: justify;">Alfalfa is grown mainly for hay and ensilage, either alone or in combination with temperate grass species such as bromegrass (<i>Bromus inermis</i> Leyss.) or timothy (<i>Phleum pratense</i> L.). It is valued as a forage crop due to its high nutrient content, digestibility and intake. Nutritional quality is maximized when the herbage is harvested at the first bloom stage; since regrowth occurs from buds at the base (<i>i.e.</i>, crown) of the plant, several cuts may be harvested per growing season. In pure stands, the protein content per hectare of alfalfa is higher than that of grain and oilseed crops. Alfalfa forage is also an excellent source of vitamins and minerals, especially calcium and vitamin A. The cultivation of alfalfa improves soil fertility and conservation. Alfalfa is a legume, and is thus to fix atmospheric nitrogen through a symbiotic association with the soil bacterium <i>Sinorhizobium (<i>Rhizobium</i>) meliloti</i>. Alfalfa adds significant amounts of nitrogen to the soil, thus reducing the nitrogen requirement for subsequent crops in a rotation. The cultivation of alfalfa reduces soil erosion since tillage is only required to prepare the seedbed, and stands of alfalfa can be maintained for several years. Soil organic matter and structure are improved from the cultivation of alfalfa due to its extensive and deep root system. Alfalfa is also grown in pastures and rangeland, and for erosion control.</p>

<p style="text-align: justify;">Weeds can reduce forage yield and quality in alfalfa production. The most critical period for the management of weeds is during crop establishment; alfalfa seedlings develop slowly and thus compete poorly with weeds. Weeds can be managed using a combination of cultural practices such preparation of a firm seed bed, sowing clean high quality seed, the use of pre-plant incorporated herbicides prior to seeding, and crop rotation to reduce the population of persistent weeds. Alfalfa can be sown along with a companion crop, usually a cereal such as oats (<i>Avena sativa</i>) or spring wheat (<i>Triticum aestivum</i>). The companion crop will germinate and establish sooner than the alfalfa, thus preventing weeds from becoming established. The use of a cover crop requires careful management to minimize competition for moisture, light and nutrients; poor stands can result if the species of cover crop is too competitive, if it is seeded too densely, harvested too late in the growing season, or if it lodges. Direct seeding of alfalfa, without a companion crop, usually results in a more vigorous stand in the year of establishment; however, proper weed control measures to minimize competition from weeds are required until the plants are well established. A combination of pre-emergence (<i>e.g.</i>, EPTC, benefin, trifluralin) and post-emergence (<i>e.g.</i>, 2,4-DB, sethoxydim, fluazifop-p-butyl) herbicides is usually recommended for the establishment of vigorous stands.</p>

<p style="text-align: justify;">Events J101 and J163 were developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup&reg;, as a weed control option in the production of pure stands of alfalfa, either as forage, or for seed. These genetically engineered lines each contain a novel form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This enzyme allows the plant to survive an otherwise lethal application of glyphosate. The EPSPS gene introduced into J101 and J163 was isolated from a strain of the common soil bacterium <i>Agrobacterium tumefaciens</i> strain CP4.</p>

<p style="text-align: justify;">The EPSPS enzyme is part of the shikimate pathway, an important biochemical pathway in plants involved in the production of aromatic amino acids and other aromatic compounds. When conventional plants are treated with glyphosate, the plants cannot produce the aromatic amino acids needed for growth and survival. EPSPS is present in all plants, bacteria, and fungi. It is not present in animals, since these organisms are unable to synthesize their own aromatic amino acids. Because the aromatic amino acid pathway is not present in mammals, birds, or aquatic life forms, glyphosate has little, if any, toxicity for these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources. Events J101 and J163 were developed by introducing the CP4 EPSPS gene into the proprietary <i>M. sativa</i> clone &lsquo;R2336&rsquo; using <i>Agrobacterium</i>-mediated transformation.</p>

<p style="text-align: justify;">Events J101 and J163 were field tested at several locations in the United States 1998 to 2004. Vegetative and reproductive growth, seed dormancy, emergence and germination, plant maturity, were found to be within the range of conventional alfalfa lines. Susceptibility to diseases and insects was not altered compared to conventional alfalfa. The cultivation of alfalfa populations containing either event J101 or J163 is therefore not expected to result in altered interactions with insects and diseases, including becoming new hosts for plant diseases and insects. Neither of these events demonstrated altered morphological, growth or seed dormancy characteristics such as those observed in aggressively weedy and invasive plant species.</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 relatives of alfalfa, was investigated. Alfalfa is an outcrossing species and pollination is achieved by bee 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 subspecies within 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 a related species found in Europe, Asia, North Africa and North America. Black medick is an annual and does not hybridize with <i>M. sativa</i>.</p>

<p style="text-align: justify;">The development of glyphosate-tolerant hybrids is therefore possible if alfalfa populations containing events J101 or J163 are grown in the vicinity of members of the <i>M. sativa</i> complex, and, to a limited extent, <i>M. glomerata</i>. These hybrids will develop where members of the <i>M. sativa</i> complex are indigenous (southern Europe, Asia, the Middle East and North Africa), as well as in North America where the <i>M. sativa</i> complex has become established. Introgression of the trait into <i>M. glomerata</i> would occur only in areas where this species is indigenous (southern Europe and North Africa). 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;">The livestock feed safety of events J101 and J163 was assessed from the following data and information: the history of safe consumption of the CP4 EPSPS enzyme in previously approved glyphosate-tolerant crops; the lack of toxicity or allergenicity of CP4 EPSPS, based on amino acid homology investigations; and the results of previous safety studies conducted with CP4 EPSPS. The nutritional equivalence and wholesomeness of J101 and J163, compared to a nontransgenic near-isogenic alfalfa populations and conventional alfalfa varieties, was demonstrated by the compositional analysis of the forage, including proximates (crude protein, fat, ash, and moisture), acid detergent fibre, neutral detergent fibre, amino acids, minerals, and levels of coumestrol.</p>
