GM Crop Database

Database Product Description

J101, J163 (MON-ØØ1Ø1-8, MON-ØØ163-7)
Host Organism
Medicago sativa (Alfalfa)
Trait
Herbicide tolerant, glyphosate.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for livestock feed.

Product Developer
Monsanto Company and Forage Genetics International

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2006
Canada 2005 2005 2005 View
Japan 2005 2006 2006
Korea 2007 2007
Mexico 2005 2005
Philippines 2006 2006
United States 2005 2005 2011 View

Introduction Expand

Events J101 and J163 were developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in the production of pure stands of alfalfa, either as forage or for seed. These genetically engineered alfalfa populations contain a glyphosate-tolerant form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the soil bacterium Agrobacterium tumefaciens strain CP4. The novel form of this enzyme is termed hereafter CP4 EPSPS.

The EPSPS enzyme is part of the shikimate pathway that is involved in the production of aromatic amino acids and other aromatic compounds in plants. When conventional plants are treated with glyphosate, the herbicide binds to EPSPS, thereby preventing the synthesis of aromatic amino acids needed for plant growth. The CP4 EPSPS enzyme in events J101 and J1638 has a reduced affinity for glyphosate; its enzymatic activity is therefore not hindered by the herbicide.

EPSPS is present in all plants, bacteria, fungi, but not in animals, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic pathway is not present in mammalian, avian or aquatic life forms, glyphosate has little if any toxicity for these organisms. The EPSPS enzyme is normally present in food derived from plant and microbial sources.

Events J101 and J163 were each developed by introducing the CP4 EPSPS coding sequences into the alfalfa clone ‘R2336’ using Agrobacterium-mediated transformation.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT

enhanced Figwort Mosaic Virus (FMV) 35S; petunia HSP 70 5' untranslated leader sequence

chloroplast transit peptide from A. thaliana EPSPS gene (CTP2)

Pisum sativum (pea) ribulose-1,5-bisphosphate carboxylase small subunit E9 gene

1 functional

Native

Characteristics of Medicago sativa (Alfalfa) Expand

Center of Origin Reproduction Toxins Allergenicity
Asia Minor, Transcaucasia, Turkmenistan, and Iran Outcrossing, insect-pollinated (bees). Hybridizes with members of the M. sativa complex; limited hybridization with M. glomerata. Seed dormancy variable, depending on amount of hard seed. Saponins, such as medicagenic acid, which cause bloat in ruminants; coumestrol and coumestan, phytoestrogens, which can cause reproductive problems in livestock. No known endogenous allergens

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Agrobacterium tumefaciens strain CP4 CP4 epsps

Agrobacterium tumefaciens is a common soil bacterium that is responsible for causing crown gall disease in susceptible plants. There have been no reports of adverse effects on humans or animals.

Modification Method Expand

Events J101 and J163 were developed by Agrobacterium-mediated transformation of plant cells from the proprietary alfalfa clone ‘R2336.’ Plant transformation was achieved with the plasmid vector PV-MSHT4. The T-DNA portion of this plasmid vector contained one cp4 epsps gene expression cassette coding for glyphosate tolerance. The cp4 epsps gene was linked to a chloroplast transit peptide (cpt2) sequence, derived from the Arabidopsis thaliana epsps gene. The expression of the cp4 epsps gene was regulated by an enhanced 35S promoter from the Figwort Mosaic Virus, and the petunia heat shock protein 70 5’ untranslated leader sequence. The transcriptional termination sequences were derived from the pea (Pisum sativum) ribulose-1,5-biphosphate carboxylase (Rubisco) small subunit E9 gene.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of the genomic DNA of events J101 and J163 confirmed the insertion of a single intact copy of the cp4 epsps gene cassette. For each of the transformation events, the cp4 epsps gene cassette was integrated at a different locus. All of the elements of the gene cassette were integrated, including the promoter, enhancer, terminator and chlorophyll transit peptide sequences. None of the vector backbone sequences from the PV-MSHT4 vector were integrated into the genome of either J101 or J163.

The cp4 epsps gene was isolated from Agrobacterium tumefaciens strain CP4. In plants, EPSPS is a chloroplast-localized enzyme that is transported from the cytosol to the chloroplast by a chlorophyll transit peptide. In events J101 and J163, the inserted ctp2 gene codes for a chlorophyll transit peptide that binds to the CP4 EPSPS enzyme to enable transportation to the chloroplast.

Genetic Stability of the Introduced Trait

Mendelian segregation studies were conducted to investigate the stability of inheritance of the glyphosate-tolerance trait in J101 and J163 alfalfa populations. Segregation data were obtained from five generations of introgression of the trait from events J101 and J163 into unrelated alfalfa germplasm. This was accomplished by initially outcrossing the transformed plants to unrelated elite alfalfa clones. The F1 progeny were subsequently outcrossed to a second set of unrelated elite clones to produce a modified backcross generation. Three additional modified backcross generations were developed with three other sets of unrelated clones. Southern blot analysis and chi-square analysis of the resulting segregation data demonstrated that the glyphosate-tolerance trait in events J101 and J163 was inherited as a single-locus dominant trait, and was stable across five generations.

Expressed material

The quantification of the expression levels of CP4 EPSPS in alfalfa populations containing each of J101 and J163 was achieved using enzyme-linked immunosorbent assay (ELISA) analysis. Samples for analysis were obtained from field trials of these varieties grown at six sites in 2001. The mean expression level of CP4 EPSPS in plants containing events J101 was 257 µg/g fresh weight, and 270 µg/g fresh weight in plants containing event J163.

Environmental Safety Considerations Expand

Field Testing

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.

Outcrossing

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 (Megachile rotundata), honeybee (Apis mellifera), and bumble bee (Bombus spp.). Alfalfa outcrosses and hybridizes with members of the M. sativa complex (M. sativa subsp. sativa (cultivated alfalfa), subsp. falcata, subsp. glutinosa, subsp. coerulea, subsp. x tunetana, subsp. x varia, subsp. x polychroa, and subsp. x hemicycla). There have been reports of natural, but limited hybridization with M. glomerata; this species is indigenous to southern Europe and North Africa, and is not present in North America. M. lupulina (black medick) is an annual weed species indigenous to Europe and common in North America. Black medick does not hybridize with M. sativa.

Alfalfa is primarily cultivated for its herbage. Ideally, alfalfa herbage is harvested (i.e., 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 (e.g., 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.

The cultivation of glyphosate-tolerant alfalfa for seed production may therefore result in the development of glyphosate-tolerant hybrids with members of the M. sativa complex, and to a limited extent with M. glomerata in areas where this species is found. These hybrids will develop in areas where members of the M. sativa 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 M. glomerata 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.

Weediness Potential

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.e., 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.

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.

Secondary and Non-Target Adverse Effects

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 Agrobacterium 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 (e.g. bees), soil organisms (e.g., 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.

Impact on Biodiversity:

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.

Food and/or Feed Safety Considerations Expand

Dietary exposure

The genetic modification in J101 and J163 will not result in changes in the use of alfalfa as an animal feed. Any cultivars resulting from crosses with either of events J101 or J163 are expected to be used in similar applications as conventional alfalfa cultivars, with the sole exception that these cultivars can only be cultivated as pure stands. In mixed grass-alfalfa stands, the grass portion would be eliminated by the use of glyphosate. The nutritional quality analysis of these alfalfa lines did not reveal any novel compositional characteristics compared to conventional cultivars, and the nutritional composition was within the normal range of variability observed in conventional alfalfa. Furthermore, due to the availability of many alfalfa cultivars adapted to different environmental and management conditions, the dietary exposure of livestock animals to forage from either J101 or J163 alfalfa cultivars is therefore expected to be similar to that of conventional alfalfa.

Nutritional and Compositional Data

The nutritional composition of lines J101 and J163 was determined analytically and compared to that of a null segregant control population (from backcrosses with parental populations), and to twelve conventional alfalfa cultivars. Samples were obtained from second cut forage, harvested at early to late bloom, from five locations in the United States. Forage samples were analyzed for 35 nutritional components including proximates (crude protein, fat, ash, and moisture), acid detergent fibre, neutral detergent fibre, amino acids, and nine minerals, including calcium, magnesium, phosphorus and potassium. A 99% tolerance interval for the compositional constituents was developed using the twelve conventional alfalfa cultivars.

Statistically significant differences were observed in the levels of cystine, histidine, lysine, tyrosine, acid detergent fibre, neutral detergent fibre, and lignin in either J101 or J163, compared to the control population. While some of these differences were observed within and among locations, all of the compositional values of J101 and J163 were within the tolerance interval established for each constituent, and were also comparable to published literature values.
Coumestrol is an antinutritional estrogenic compound found naturally in alfalfa. Forage samples from trials conducted at four locations were analyzed for coumestrol. No significant differences in the levels of coumestrol were observed among lines J101, J163, the control line, and the commercial cultivars.

Results from the nutritional and compositional analyses supported the conclusion that forage from events J101 and J163 is nutritionally comparable to that from currently available commercial cultivars of alfalfa.

Toxicity and Allergenicity

The potential for toxicity and allergenicity of the alfalfa events J101 and J163 was assessed from data and information obtained from previous studies and investigations conducted in support of the safety of CP4 EPSPS, and the gene donor A. tumefaciens strain CP4. Results from these studies and investigations have demonstrated that CP4 EPSPS is functionally equivalent to native EPSPS, except for a reduced affinity for glyphosate; the cp4 epsps gene donor has a history of safe use; CP4 EPSPS is not homologous to known toxins and allergens; the protein is rapidly digested in simulated gastric fluids; and that no treatment-related adverse effects were observed in acute toxicity tests where mice were fed up to 572 mg of CP4 EPSPS per kg of body weight. The no-observed adverse effects level of 572 mg/kg is approximately 20 times the amount of CP4 EPSPS to which a ruminant animal would be exposed when consuming a typical daily ration of alfalfa forage. The results of these studies and investigations, and data demonstrating the relatively low expression levels of CP4 EPSPS, led to the conclusion that the alfalfa events J101 and J163 are no more toxic or allergenic than conventional alfalfa cultivars.

Abstract Collapse

Alfalfa (Medicago sativa 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.

Alfalfa is grown mainly for hay and ensilage, either alone or in combination with temperate grass species such as bromegrass (Bromus inermis Leyss.) or timothy (Phleum pratense 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.e., 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 Sinorhizobium (Rhizobium) meliloti. 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.

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 (Avena sativa) or spring wheat (Triticum aestivum). 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 (e.g., EPTC, benefin, trifluralin) and post-emergence (e.g., 2,4-DB, sethoxydim, fluazifop-p-butyl) herbicides is usually recommended for the establishment of vigorous stands.

Events J101 and J163 were developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, 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 Agrobacterium tumefaciens strain CP4.

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 M. sativa clone ‘R2336’ using Agrobacterium-mediated transformation.

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.

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 (Megachile rotundata), honeybee (Apis mellifera), and bumble bee (Bombus spp.). Alfalfa outcrosses and hybridizes with subspecies within the M. sativa complex (M. sativa subsp. sativa (cultivated alfalfa), subsp. falcata , subsp. glutinosa, subsp. coerulea, subsp. x tunetana, subsp. x varia, subsp. x polychroa, and subsp. x hemicycla. There have been reports of natural, but limited hybridization with M. glomerata; this species is indigenous to southern Europe and North Africa, and is not present in North America. M. lupulina (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 M. sativa.

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 M. sativa complex, and, to a limited extent, M. glomerata. These hybrids will develop where members of the M. sativa complex are indigenous (southern Europe, Asia, the Middle East and North Africa), as well as in North America where the M. sativa complex has become established. Introgression of the trait into M. glomerata 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.

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.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biosafety Office Food Standards Australia New Zealand Health Canada Novel Foods Japanese Biosafety Clearing House, Ministry of Environment Philippines Department of Agriculture, Bureau of Plant Industry United States (USDA) United States Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Friday, May 5, 2017