GM Crop Database

Database Product Description

MS1, RF1 =PGS1 (ACS-BNØØ4-7 x ACS-BNØØ1-4)
Host Organism
Brassica napus (Argentine Canola)
Trait
Glufosinate ammonium herbicide tolerance and fertility restored.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Aventis CropScience (formerly Plant Genetic Systems)

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2002 2002 2003
Canada 1994 1995 1995
China 2004 2004
European Union 2005 2005 View
Japan 1996 1996 1996
Korea 2005 2008
South Africa 2001 2001
United States 1996 1996 2002 View

Introduction Expand

The MS1 and RF1 canola lines (Brassica napus) were developed using genetic engineering techniques to provide a pollination control system for production of hybrid oilseed rape (MS1 x RF1). The novel hybridization system involves the use of two parental lines, a male sterile line MS1 and a fertility restorer line RF1. The transgenic MS1 plants do not produce viable pollen grains and cannot self-pollinate. In order to completely restore fertility in the hybrid progeny, line MS1 must be pollinated by a modified plant containing a fertility restorer gene, such as line RF1. The resultant F1 hybrid seed derived from cross between MS1 x RF1, produces hybrid plants that produce pollen and are completely fertile.

The transgenic line MS1 (B91-4) was produced by genetically engineering plants to be male sterile and tolerant to the herbicide glufosinate ammonium (as a selectable marker). The parental line MS1 contains the barnase gene for male sterility, isolated from Bacillus amyloliquefaciens, a common soil bacterium that occurs naturally in the soil and in various organisms including bacteria and plants, and frequently used as a source for industrial enzymes. The barnase gene encodes for a ribonuclease enzyme (RNAse) 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 transgenic line RF1 (B93-101) was produced by genetically engineering plants to restore fertility in the hybrid line and to be tolerant to the herbicide glufosinate ammonium (as a selectable marker). Transgenic RF1 plants contain the barstar gene isolated from Bacillus amyloliquefaciens. The barstar gene codes for a ribonuclease inhibitor (barstar enzyme) expressed only in the tapetum cells of the pollen sac during anther development. The ribonuclease inhibitor (barstar enzyme) specifically inhibits barnase RNAse expressed by the MS1 line. Together, the RNAse and the ribonuclease inhibitor form a very stable one-to-one complex, in which the RNAse is inactivated. As a result, when pollen from the restorer line RF1 is crossed to the male sterile line MS1, the resultant progeny express the RNAse inhibitor in the tapetum cells of the anthers allowing hybrid plants to develop normal anthers and restore fertility.
Both trangenic canola lines MS1 and RF1 contain the bar gene that confers tolerance to the post-emergence, broad-spectrum phosphinothricin herbicides (Liberty®). The bar gene, isolated from the common soil microorganism Streptomyces hygroscopicus, encodes a phosphinothricin acetyl transferase (PAT) enzyme. The active ingredient in phosphinothricin herbicides is glufosinate ammounium which acts by inhibiting the plant enzyme glutamine synthetase, leading to the accumulation of phytotoxic levels of ammonia killing the plant within hours of application. PAT detoxifies glufosinate ammonium by acetylation into an inactive compound, eliminating its herbicidal activity. The herbicide tolerance trait was introduced into the canola lines as a selectable marker to identify transformed plants cells in the laboratory, and as a field selection method to obtain 100% hybrid seed.

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 1 Introduced into MS1
barstar barnase ribonuclease inhibitor RF anther-specific promoter 1 Introduced into RF1
bar phosphinothricin N-acetyltransferase HT PSsuAra from Arabidopsis thaliana chloroplast transit peptide from A. thaliana 1 Introduced into both MS1 and RF1
nptII neomycin phosphotransferase II SM nopaline synthase (nos) from A. tumefaciens octopine synthase Native; both MS1 and RF1

Characteristics of Brassica napus (Argentine Canola) Expand

Center of Origin Reproduction Toxins Allergenicity

The species is native to India.

Canola flowers can self-pollinate, and they can also be cross-pollinated by insects and by wind.­

Brassica species can contain erucic acid and various glucosinolates, which can be toxic. However, commercial canola varieties have been bred to reduce the levels of these substances. Canola may contain elevated levels of tannins, which reduce the digestibility of seed protein, and sinapine, which is a bitter substance that can reduce the palatability of feeds made from canola meal.

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Occupational exposure to pollen and seed flour have been associated with allergic reactions in humans. There are no known allergic reactions to canola oil.

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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

The MS1 and RF1 canola lines were both produced using Agrobacterium-mediated transformation of the Brassica napus cultivar 'Drakkar'. The T-DNA region of the Ti plasmid was "disarmed" by removal of virA genes, normally associated with the pathogenicity and disease-causing properties of A. tumefaciens, and replaced with the genes of interest for each transgenic line MS1 and RF1. During transformation, the T-DNA portion was transferred into the plant cells and stably integrated into the plant's genome.

Line MS1 was produced from the insertion of T-DNA containing the barnase gene together with an anther specific promoter, pTa29, from Nicotiana tabacum, terminated by part of the 3´non-coding region (3´nos) of the nopaline synthase gene from Agrobacterium tumefaciens. Similarly, line RF1 was produced from T-DNA containing the barstar gene under the control of the pTa29 anther-specific promoter from Nicotiana tabacum and 3'nos terminator.

In addition, each T-DNA contained a copy of the bar gene from S. hygroscopicus, which encodes the PAT enzyme, and the neomycin phosphotransferase II (NPTII) encoding neo gene from the Tn5 transposon of Escherichia coli, strain K12. Expression of the bar gene was regulated by the PSsuAra promoter from Arabidopsis thaliana and post-translational targeting of the gene product to the chloroplast organelles was accomplished by fusion of the 5’-terminal coding sequence with the chloroplast transit peptide DNA sequence from A. thaliana. The expression of NPTII activity, under control of the nopaline synthase promoter from A. tumefaciens, was used as a selectable trait for screening transformed plants for the presence of the barnase and barstar genes, respectively.

Characteristics of the Modification Expand

The Introduced DNA

Southern Blot and segregation analyses demonstrated the stable integration of the inserted DNA at a single genetic locus in both MS1 and RF1. The barnase, bar and neo genes were integrated into MS1 and similarly, the barstar, bar and neo genes were integrated in RF1. Additional Souther blot analyses demonstrated that there was no incorporation of any plasmid sequences outside of the T-DNA region.

The insertion site was very well characterized. Deletion studies showed no evidence that a native plant gene was interrupted by the transformation event. Brassica napus is an amphidiploid composed of two genomes B. rapa/B. oleracea. The insertion site was found to be located in the B. rapa portion of the genome.

Genetic Stability of the Introduced Trait

MS1 and RF1 are several generations removed from the original transformants. Comparisons to the original transformants, in addition to data from several generations of backcrossing demonstrated the stable inheritance of the novel traits in lines MS1 and RF1. Segregation analysis confirmed the data from Southern blots and indicated that the novel traits in both transformation events segregated as a single locus according to the rules of Mendelian inheritance.

Expressed Material

Transcription of the bar gene was detected in both leaf and flower bud tissue by Northern blot analysis. Similar analyses demonstrated that the barnase and barstar genes were only transcribed in the flower bud tissue of MS1 and RF1 plants, respectively. Both genes were expressed only during the early stages of anther development in the tapetum cell layer of the anther.

The NPTII encoding gene was linked to a weak constitutive promoter and expression of this enzyme was not detected in pollen samples or unprocessed honey.

Environmental Safety Considerations Expand

Field Testing

The canola lines MS1 and RF1, and the hybrid MS1 x RF1 were field tested in Canada from 1991 to 1994. Agronomic and adaptation characteristics such as vegetative vigour, overwintering capacity, flowering period, seed production, seed dissemination, germination and establishment, and seed dormancy were within the normal range of expression of characteristics in unmodified counterparts. As expected, seed production of the hybrid line was greater than that of unmodified counterparts. Flowers of the MS1 line had undeveloped anthers, slightly smaller petals and did not produce fertile pollen, but nectar production remained unchanged and normal insect pollination was observed. Stress adaptation was evaluated, including its resistance or susceptibility to major B. napus pests and pathogens (e.g., blackleg, sclerotinia, flea beetles, diamondback moth larvae) and determined to fall within the ranges currently displayed by commercial varieties. The lines were tested under various environmental conditions, and showed no differences in agronomic performance when compared to unmodified counterparts under the same conditions. Overall the field data reports demonstrated that MS1 and RF1, and the hybrid MS1 x RF1 had no potential to pose a plant pest risk.

Outcrossing

The sterility of the B. napus line MS1 ensured that gene introgression from MS1 into wild or cultivated sexually-compatible plants was extremely unlikely. The MS1 plants can act as pollen recipients, but the progeny would be partially male sterile.

Transgenic line RF1 and hybrid plants displayed normal reproductive characteristics. Brassica napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that gene flow is most likely to occur with B. rapa.

The genes coding for male sterility or fertility restoration do not confer any ecological advantage to potential hybrid offspring of MS1 or RF1 plants. If glufosinate ammonium tolerant individuals arose through interspecific or intergeneric hybridization, the novel traits would confer no competitive advantage to these plants unless these populations were routinely subject to herbicide treatments. In the event that a glufosinate ammonium tolerant B. napus survived, these herbicide-tolerant individuals would be easily controlled using mechanical and other available chemical means. It was concluded that gene flow from the transgenic lines MS1 and RF1 or their hybrids to canola relatives was possible, but would not result in increased weediness or invasiveness of these relatives.

Weediness Potential

Field studies on invasiveness and survival characteristics comparing the MS1, RF1 and hybrid MS1xRF1 to unmodified plants determined that the transgenic lines were not different from their counterparts in this respect. Published data showed that seed survival of transgenic seeds expressing kanamycin resistance and glufosinate ammonium tolerance was significantly lower than seed survival of unmodified counterparts, when seeded under a variety of wild conditions. Competition studies, between transgenic lines and barley sown together at various ratios, demonstrated that the transgenic lines did not confer any novel competitive advantage.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified lines MS1, RF1, and hybrid MS1 x RF1 did not have a significant adverse impact on nontarget organisms or organisms beneficial to plants or agriculture, and were not expected to impact on threatened or endangered species. The barnase and barstar proteins did not result in altered toxicity or allergenicity properties and are only produced in the tapetum cell layer of anthers at a specific developmental stage. In addition, detailed studies of pollination behavior in the field and in greenhouses showed no effects on honeybees. An avian dietary test was performed with the seed eating canary bird (Serinus canaria domestica) and a feeding study was performed with the domesticated rabbit (Oryctolagus cuniculus). Both studies determined no differences in food consumption, behavior and body weight between birds or rabbits fed with the transgenics or unmodified counterparts. Microorganisms living in the rhizosphere surrounding the roots were compared quantitatively and qualitatively and there were no differences observed between plots of transgenic plants versus plots containing unmodified B. napus.

Impact on Biodiversity

The transgenic lines MS1, RF1 and their hybrids have no novel phenotypic characteristics which would extend their use beyond the current geographic range of canola/rapeseed production. Since outcross species are only found in disturbed habitats, transfer of novel traits would not have an impact on unmanaged environments. Studies have shown that these lines are not invasive of natural habitats and that they are no more competitive than the unmodified counterparts, both in natural and managed ecosystems. It was determined that the relative impact on biodiversity of MS1, RF1 and MS1xRF1 was equivalent to that of currently commercialized canola lines.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of canola products is limited to the refined oil. Refined edible canola oil does not contain any detectable protein and consists of purified triglycerides (96-97%). As the introduced gene products are not detectable in the refined oil produced from transgenic canola, there will be no human exposure to these proteins based on normal consumption patterns.

Nutritional Data

The analysis of nutrients from transgenic canola lines MS1 and RF1 and non-transgenic canola did not reveal any significant differences in the levels crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. The fatty acid composition of oils extracted from both transgenic and non-transgenic canola was statistically the same and within the normal range for canola oil. The transgenic lines meet the standards for canola oil in Canada of containing less than 2% erucic acid and less than 30 micromoles/g glucosinolates in the oil-free meal. It was determined that the consumption of refined oil from MS1, RF1 or hybrids derived therefrom would have no significant impact on the nutritional quality of the food supply in Canada.

Toxicity and Allergenicity

Since only the processed oil from transgenic MS1, RF1, or hybrids derived therefrom (MS1xRF1), will be available for human consumption and the processing removes proteinaceous material, there are no additional toxicity or allergenicity concerns with this product. This was further demonstrated by examining the amino aid sequence homology and the characteristics of the novel proteins RNAse (barnase), RNAse inhibitor (barstar),PAT (bar) and neomycin phosphotransferase (NPTII).

Comparative analyses with amino acid sequences of known protein toxins and allergens in public domain databases did not reveal any homologies. The only sequence homologies were with other related proteins. For example, barnase encoding RNAse was similar to ribonucleases from other bacilli, PAT was similar to other phosphinothricin acetyltransferases originating from different organisms, and NPTII was found to be similar to other peptides that have a similar function (aminoglycoside 3'-phosphotransferases, kanamycin kinases, streptomycin 3'-kinases. No resemblance with potential toxins or allergens was observed.

Abstract Collapse

Argentine or oilseed rape (Brassica napus) was grown as a commercial crop in over 50 countries, with a combined harvest of 48.9 million metric tonnes in 2006. The major producers of rapeseed are China, Canada, India, Germany, France, the United Kingdom and Australia. Canola is a genetic variation of B. napus that was developed through conventional breeding to contain low levels of the natural rapeseed toxins, glucosinolate and erucic acid. Canola is grown for its seed, which represents a major source of edible vegetable oil and is also used in livestock feeds.

The only food use of canola is as a refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortenings, cooking and salad oils. Canola meal, a byproduct of the oil production process, is added to livestock feed rations. An increasing amount of oil is being used for biodiesel production, especially in Europe.

The canola lines MS1 and RF1 were developed using genetic engineering techniques to provide a pollination control system for the production of hybrid oilseed rape (MS1xRF1) expressing male sterility and tolerance to glufosinate ammonium. The novel hybridization system involves the use of two parental lines, a male sterile line MS1 and a fertility restorer line RF1. The transgenic MS1 plants do not produce viable pollen grains and cannot self-pollinate. In order to completely restore fertility in the hybrid progeny, line MS1 must be pollinated by a modified plant containing a fertility restorer gene, such as line RF1. The resultant F1 hybrid seed, derived from the cross between MS1 x RF1, generates hybrid plants that produce pollen and are completely fertile.

The male-sterile trait was introduced in MS1 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 transgenic line RF1 was produced by genetically engineering plants to restore fertility in the hybrid line. Transgenic RF1 plants contain the barstar gene, also isolated from Bacillus amyloliquefaciens. The barstar gene codes for a ribonuclease inhibitor (barstar enzyme) expressed only in the tapetum cells of the pollen sac during anther development. The ribonuclease inhibitor (barstar enzyme) specifically inhibits barnase RNAse expressed by the MS1 line. Together, the RNAse and the ribonuclease inhibitor form a very stable one-to-one complex, in which the RNAse is inactivated. As a result, when pollen from the restorer line RF1 is crossed to the male sterile line MS1, the resultant progeny express the RNAse inhibitor in the tapetum cells of the anthers, allowing hybrid plants to develop normal anthers and restoring fertility.

Both transgenic lines MS1 and RF1 were also engineered to express 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 these canola lines was 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 PAT enzyme was used as a selectable marker enabling identification of transformed plants during tissue culture regeneration, and as a field selection method to obtain 100% hybrid seed.

The canola lines MS1 and RF1, and the hybrid MS1xRF1, were field tested in Canada from 1991 to 1994. Agronomic characteristics such as vegetative vigour, overwintering capacity, flowering period, and seed germination and dormancy were within the normal range of expression found in unmodified canola varieties. As expected, seed production of the hybrid line was greater than that of the unmodified counterparts. Flowers of the MS1 line had undeveloped anthers, slightly smaller petals and did not produce fertile pollen, but nectar production remained unchanged and normal insect pollination was observed. Stress adaptation was evaluated, including susceptibilities to major insect pests and diseases, and was determined to fall within the ranges currently displayed by commercial varieties. It was demonstrated that the transformed canola lines did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Canola lines MS1, RF1 and their progeny were not expected to impact on threatened or endangered species.

Brassica napus may outcross (up to 30% of the time) with plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that cross breeding is most likely to occur with B. rapa. Due to the ability of canola to outcross with related plants, the formation of glufosinate-tolerant hybrids is possible. However, the glufosinate-tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environments routinely subjected to glufosinate applications. Additionally, multiple barriers, including the male-sterility of the canola line MS1, ensured that gene flow from this transformed line into wild or cultivated sexually compatible plants was extremely unlikely. In the event that a glufosinate-tolerant hybrid survived, the herbicide-tolerant individual could be easily controlled using mechanical and/or other available chemical means.

The human consumption of canola products is limited to the refined oil. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of the introduced proteins were detected in the refined oil of MS1 or RF1 canola. Furthermore, processing would destroy the enzymatic activity of the introduced gene products. As such, there will be no human exposure to these proteins as a result of the consumption of refined oil from these transgenic lines.

The analysis of nutrients from transgenic canola lines MS1 and RF1 and non-transgenic canola did not reveal any significant differences in the levels crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. The fatty acid composition of oils extracted from both transgenic and non-transgenic canola was statistically the same, and within the normal range for canola oil. It was determined that the consumption of refined oil from MS1, RF1 or hybrids derived from these lines would have no significant impact on the nutritional quality of the food supply.

Potential toxicity and allergenicity of the transformed canola lines was further investigated through examination of the amino acid sequences and physiochemical characteristics of the novel proteins RNAse (barnase), RNAse inhibitor (barstar), and PAT (bar). No significant homologies between the amino acid sequences of these novel proteins and those of known toxins or allergens were detected. It was concluded that these proteins, and thus MS1, RF1 and MS1xRF1 hybrid canola lines, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office European Commission European Commission: Community Register of GM Food and Feed Food Standards Australia New Zealand Japanese Biosafety Clearing House, Ministry of Environment Office of Food Biotechnology, Health Canada Office of the Gene Technology Regulator THE COMMISSION OF THE EUROPEAN COMMUNITIES U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S. Food and Drug Administration U.S.Department of Agriculture, Animal and Plant Health Inspection Service

This record was last modified on Wednesday, June 24, 2015