Database Product Description

MS3 (ACS-ZMØØ1-9)

Host Organism

Zea mays (Maize)

Trade Name

InVigor™

Trait

Glufosinate ammonium herbicide tolerance and male sterility

Trait Introduction

Electroporation of immature embryos.

Proposed Use

Production for human consumption and livestock feed.

Product Developer

Bayer CropScience (Aventis CropScience(AgrEvo))

Summary of Regulatory Approvals

Introduction Expand

Maize line MS3 (tradename InVigor®) was developed to provide a reliable hybridization system based on nuclear male sterility. The transgenic line was produced by genetically engineering plants to be male sterile and tolerant to the herbicide glufosinate ammonium, where herbicide tolerance was used as a selectable marker to identify the male sterile plants. These transformed maize lines contain the barnase gene for male sterility, isolated from Bacillus amyloliquefaciens, a common soil bacterium that is frequently used as a source for industrial enzymes. The barnase gene encodes for a ribonuclease enzyme (RNAse) that is expressed only in the tapetum cells of the pollen sac during anther development. The RNAse affects RNA production, disrupting normal cell functioning and arresting early anther development, thus leading to male sterility.

The male sterile maize line MS3 also contained the bar gene isolated from the common soil microorganism Streptomyces hygroscopicus, for use as a selectable marker. The bar gene encodes a phosphinothricin acetyl transferase (PAT) enzyme, which, when introduced into a plant cell, confers tolerance to the herbicide glufosinate ammonium. The herbicide tolerance trait was introduced into the maize line as a selectable marker to identify transformed plants during tissue culture regeneration, and as a field selection method to identify the male sterile lines at any growth stage. Under field conditions, plants that were not male-sterile could be eliminated by application of the herbicide glufosinate ammonium. The novel hybrid system provided an efficient and effective way to identify male-sterile plants for use in hybrid seed production.

Glufosinate ammonium is the active ingredient in phosphinothricin herbicides (Basta, Rely, Finale, and Liberty) and acts by inhibiting the plant enzyme glutamine synthetase, a key enzyme in plants that detoxifies ammonia by incorporating it into glutamine. Inhibition of this enzyme leads to an accumulation of ammonia in the plant tissues, which kills the plant within hours of application. PAT catalyses the acetylation of the herbicide phosphinothricin and thus detoxifies glufosinate ammonium into an inactive compound.

Summary of Introduced Genetic Elements Expand

Code	Name	Type	Promoter, other	Terminator	Copies	Form
barnase	barnase ribonuclease	MS	pTa 29 pollen specific promoter from Nicotiana tabacum		3 at 1 insertion site
bar	phosphinothricin N-acetyltransferase	HT	CaMV 35S	A. tumefaciens nopaline synthase (nos) 3'-untranslated region	3 at 1 insertion site	Modified
bla	beta lactamase	SM	bacterial promoter			Not expressed in plant tissues

Characteristics of Zea mays (Maize) Expand

Center of Origin	Reproduction	Toxins	Allergenicity
Mesoamerican region, now Mexico and Central America	Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.	No endogenous toxins or significant levels of antinutritional factors.	Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Donor Organism Characteristics Expand

Latin Name	Gene	Pathogenicity
Streptomyces hygroscopicus	bar	S. hygroscopicus is ubiquitous in the soil and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

Maize line MS3 was produced by transforming the inbred line H99 by electroporation of immature embryos in the presence of a preparation of linearized plasmid DNA. The plasmid mixture contained both genes of interest; the male sterility gene (barnase) and the herbicide resistance gene (bar) and additional genetic elements including barstar, bla and cat genes of which none were expressed in the modified maize plant.

The barnase gene encodes an RNAse enzyme that disrupts cell function during anther development and results in male sterility. The expression of the barnase gene was under control of an anther-specific promoter (pTa29), from Nicotiana tabacum, that turns on expression of the gene in the anthers where pollen is produced. The barnase gene was terminated by the 3' -polyadenylation signal of the nopaline synthase (nos) gene isolated from the Ti plasmid of Agrobacterium tumefaciens.

The bar gene encodes the PAT enzyme confering tolerance to glufosinate ammonium herbicide and was used as a selectable marker. Constitutive expression of the bar gene was under control of the cauliflower mosaic virus (CaMV) 35S promoter. Translocation of the introduced PAT enzyme to chloroplasts was accomplished by fusing sequences encoding the chloroplast transit peptide to the 5' terminus of the bar gene. Apart from the sequences encoding for RNAse and PAT, no other plant translatable DNA sequences were introduced into the maize plant genome.

The introduced DNA included the barstar gene from B. amyloliquefaciens that encodes a specific inhibitor of the barnase RNAse. The barstar gene was included to prevent the RNAse from disrupting the development of bacteria in which the introduced DNA was prepared. Also included were two antibiotic resistant genes the beta-lactamase gene (bla) and chloramphenicol acetyl transferase (cat) genes, encoding resistance to the antibiotics ampicillin and chloramphenicol, respectively. These were included as selectable marker genes to identify bacteria transformed with recombinant plasmids. The expression of these three genes (barstar, bla, and cat) was regulated by bacterial promoters that are not active in plants.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from MS3 demonstrated the integration of three copies of the DNA insert containing the barnase and bar genes at a single insertion site.

Genetic Stability of the Introduced Trait

The stability of the inserted DNA in the maize line MS3 was evaluated by segregation analyses over at least 6 generations demonstrating that the introduced trait segregated as a single dominant genetic locus and followed Mendelian rules of inheritance.hat showed over several generations. Comparisons between the original transgenic plant and plants from the fourth and sixth generations showed no differences in the gene integration pattern.

Expressed Material

The barnase RNAse was detected only in early stages of development of the tapetum cell layer of anthers. It was not detected in the other plant tissues tested: leaves, immature kernels, roots, dry and germinating seeds.
Expression levels of PAT were estimated through the quantification of RNA transcripts: 0.05 pg/µg of RNA was found in leaves and immature kernels, while none was detected in roots, dry and germinating seeds. A spectrophotometric assay showed no evidence for the presence of PAT in MS3 kernels.

Environmental Safety Considerations Expand

Field Testing

The male sterile maize line MS3 has been tested in the major maize growing regions of the United States since 1992 and has been extensively evaluated in the laboratory, greenhouse, and field experiments. Agronomic characteristics such as seed germination, vegetative vigour, time to maturity, time to tassel emergence, time to and process of silk extrusion, male and female fertility, yield parameters, disease and pest susceptibilities were compared to those of non-transgenic Zea mays counterparts and found to be within the normal range for commercial maize hybrids. Overall the field data reports and data on agronomic traits demonstrated that MS3 maize was as safe to grow as any other male sterile maize and had no potential to pose a plant pest risk.

Outcrossing

Multiple barriers, including sterility of the maize line MS3 ensured that gene introgression from these transformed lines into wild or cultivated sexually-compatible plants was extremely unlikely and such rare events would not increase the weediness potential of any resulting progeny or adversely impact biodiversity.

In the United States and Canada, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species is remote. Cultivated maize, Zea mays L. subsp. mays, is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum.

Weediness Potential

It was determined that the relevant introduced trait, male sterility, was unlikely to increase weediness of maize line MS3. There was no indication that the presence of the RNAse enzyme would convert maize into a weed. As well, resistance to glufosinate ammonium would not, in itself, render maize weedy or invasive of natural habitats.
Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. In agriculture, maize volunteers are not uncommon but are easily controlled by mechanical means or by using herbicides, other than glufosinate ammonium. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified maize line MS3 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and was not expected to impact on threatened or endangered species. The PAT enzyme responsible for glufosinate ammonium tolerance has no toxic or pathogenic properties; it has very specific enzymatic activity, does not possess proteolytic or heat stability typical of toxic compounds, and does not affect the metabolism of the plant.

Impact on Biodiversity

Maize line MS3 has no novel phenotypic characteristics that would extend the use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in the United States and Canada is remote, it was determined that the risk of transferring genetic traits from these maize lines to species in unmanaged environments was not a significant concern. It was determined that the relative impact on plant biodiversity was neutral, as was the impact on animal and microbe biodiversity since the introduced genes were not expected to alter the plant's metabolism and as such, novel compounds would not be produced.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Maize line MS3 was intended mainly for use in animal feed. The major human food uses for maize are extensively processed starch and oil fractions prepared by wet or dry milling procedures and include products such as corn syrup and corn oil, bran, grits, meal and flour. The human food uses of MS3 maize grain were not expected to be different from the uses of non-transgenic field maize varieties. As such, the dietary exposure of people to grain from MS3-derived maize hybrids was not prediced to differ from that for other commercially available field corn varieties. Furthermore, since neither of the introduced proteins were detected in the corn grain, there would be no dietary exposure to these introduced proteins.

Nutritional Data

The composition of grain from MS3-derived hybrid maize was compared to grain from non-transgenic maize. Parameters such as moisture, fibre, fat protein, ash, starch, oil, fatty acids and the amino acid composition were compared with no significant differences being observed. The use of grain from MS3-derived maize hybrids would therefore have no significant impact on the nutritional quality of the food supply.

Toxicity

The low potential for toxicity of transgenic maize line MS3 was demonstrated by examining the amino aid sequence homology and the characteristics of the proteins. An amino acid sequence comparison of the barnase enzyme with the sequences of known protein allergens and toxins in three public domain databases did not reveal any significant homologies, other than with ribonucleases from other bacilliform bacteria. Similar comparative analyses were completed for PAT enzyme. The amino acid sequence of PAT enzyme did not show significant homologies with any toxins or allergens, except with other phosphinothricin acetyltransferases originating from different organisms.
The full nucleotide sequence of the barnase gene was provided. The RNAse enzyme, encoded by the barnase gene, was a small single-domain protein, containing no disulfide bonds, metal-ion cofactors or other non-peptide components. When heated, it unfolded completely into an inactive form.

No toxic or allergic effects were expected from PAT as acetyltransferases are ubiquitous in nature, do not possess proteolytic or heat stability and are highly substrate specific. PAT has a extremely high substrate specificity for L-PPT and dimethylphosphinothricin (DMPT), and experimental data clearly showed that neither L-PPT's analog L-glutamic acid, D-PPT, nor any other amino acid can be acetylated by the PAT enzyme.

Allergenicity

The barnase RNAse and PAT enzymes are not likely to be allergenic. In addition to the amino acid sequence homology searches describe above, the potential for allergenicity of the PAT enzyme was assessed based upon the characteristics of known food allergens (stability to digestion, stability to processing). Unlike known protein allergens, these studies demonstrated that the PAT enzyme was rapidly inactivated when subjected to typical mammalian stomach conditions. In summary, the barnase RNAse and PAT enzyme were not derived from allergenic sources, do not share amino acid sequence homology with known allergens, and do not possess the physiochemical properties (heat and proteolytic digestion stability) normally associated with allergens.

Abstract Collapse

Maize (Zea mays L.), is grown primarily for its kernel, which is largely refined into products used in a wide range of food, medical, and industrial goods.

Only a small amount of whole maize kernel is consumed by humans. Maize oil is extracted from the germ of the maize kernel and maize is also a raw material in the manufacture of starch. A complex refining process converts the majority of this starch into sweeteners, syrups and fermentation products, including ethanol. Refined maize products, sweeteners, starch, and oil are abundant in processed foods such as breakfast cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as animal feed, with roughly70% of the crop fed to livestock, although an increasing amount is being used for the production of ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, pharmaceuticals and car parts.

The maize line MS3 was genetically engineered to express male sterility and tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme's usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death.

Glufosinate tolerance in this maize line is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces hygroscopicus. The PAT enzyme catalyzes the acetylation of phosphinothricin, detoxifying it into an inactive compound. The PAT enzyme is not known to have any toxic properties.

The male-sterile trait was introduced by inserting the barnase gene, isolated from Bacillus amyloliquefaciens, a common soil bacterium that is frequently used as a source for industrial enzymes. The barnase gene encodes for a ribonuclease enzyme (RNAse) that is expressed only in the tapetum cells of the pollen sac during anther development. The RNAse affects RNA production, disrupting normal cell functioning and arresting early anther development, thus leading to male sterility. The PAT enzyme was used as a selectable marker enabling identification of transformed plants during tissue culture regeneration, and as a field selection method to identify the male-sterile lines prior to flowering. Under field conditions, plants that were not male-sterile could be eliminated by application of the herbicide glufosinate ammonium. The novel hybrid system provided an efficient and effective way to identify male-sterile plants for use in hybrid seed production.

The male sterile maize line MS3 has been tested in the major maize growing regions of the United States since 1992 and has been extensively evaluated in laboratory, greenhouse, and field experiments. Agronomic characteristics such as seed germination, vegetative vigour, time to maturity, time to tassel emergence, time to and process of silk extrusion, male and female fertility, yield parameters, and disease and pest susceptibilities were compared to those of non-transgenic Zea mays counterparts and found to be within the normal range for commercial maize hybrids. Overall the field data reports and data on agronomic traits demonstrated that MS3 maize was as safe to grow as any other male sterile maize and had no potential to pose a plant pest risk. It was demonstrated that the transformed maize line did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Maize line MS3 was not expected to impact on threatened or endangered species.

Maize does not have any closely related species growing in the wild in the continental United States and Canada. Cultivated maize can naturally cross with annual teosinte (Zea mays ssp. mexicana) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in North America. Additionally, multiple barriers, including sterility of the maize line MS3, ensured that gene flow from this transformed line into wild or cultivated sexually-compatible plants was extremely unlikely. Gene exchange between maize line MS3 and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in Canada and the United States.

In an assessment of food and livestock feed safety the composition of grain from MS3-derived hybrid maize was compared to grain from non-transgenic maize. Parameters such as moisture, fibre, fat, protein, ash, starch, oil, and the fatty acid and amino acid composition were compared with no significant differences observed. It was therefore concluded that the use of grain from MS3-derived maize hybrids would have no significant impact on the nutritional quality of the food supply.

Potential toxicity and allergenicity of the PAT protein and the barnase RNase expressed in the transgenic maize line MS3 were investigated by searching for amino acid sequence homology with known toxins and allergens, and by examining their physiochemical properties. No significant homologies between the deduced amino acid sequence of the PAT protein or the barnase RNase and the sequences of known toxins or allergens were detected. Neither the PAT enzyme nor the RNase possess the proteolytic or heat stability characteristic of toxic compounds, and the PAT enzyme was readily digested under conditions simulating mammalian digestion. In summary, the barnase RNAse and PAT enzyme were not derived from allergenic sources, do not share amino acid sequence homology with known allergens, and do not possess the physiochemical properties (heat and proteolytic digestion stability) normally associated with allergens. Based on these properties, it was concluded that the RNase and the PAT protein, and thus MS3 maize, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office

• Decision Document 96-16: Determination of Environmental Safety of Plant Genetic Systems Inc.'s (PGS) Male Sterile Corn (Zea mays L.) Line MS3
  [PDF Size: 147.55K bytes]

Office of Food Biotechnology, Health Canada

• NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGYNOVEL HYBRIDIZATION SYSTEM FOR CORN (MS3)
  [PDF Size: 11.39K bytes]

U.S.Department of Agriculture, Animal and Plant Health Inspection Service

• Plant Genetic Systems (America) Inc. Petition for Determination of Nonregulated Status: Male Sterile, Glufosinate Tolerant Corn Transformation Even MS3
  [PDF Size: 12.68M bytes]

USDA-APHIS Environmental Assessment

• Response to Plant Genetic System's Petition for a Determination of Nonregulated Status for Male Sterile MS3 Corn
  [PDF Size: 54.07K bytes]

This record was last modified on Friday, March 26, 2010