GM Crop Database

Database Product Description
 
ACS-OS1-4, ACS-OS2-5 (LLRICE06, LLRICE62)
Host Organism
Oryza sativa L. (Rice) Liberty-Link
 
Trait
Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.
 
Trait Introduction
Direct DNA transfer system.
 
Proposed Use
Production of rice for livestock feed, human food, and industrial uses.
 
Company Information
Aventis CropScience
P.O. Box 12014
2 T.W. Alexander Drive
Research Triangle Park
NC  US
 
 
Summary of Regulatory Approvals
 
Country Environment Food and/or Feed Food Feed Marketing
Australia 2008  
Canada 2006 2006  
Colombia 2008  
Mexico 2007  
United States 1999 2000  
Click on the country name for country-specific contact and regulatory information.
Notes
Canada Approval for Event LLrice62 only.
Mexico For LLRICE62 only
Australia For LLRICE62 only
Colombia For LLRICE62 only

Introduction
 
Rice lines LLRICE06 and LLRICE62 were developed using recombinant DNA techniques to allow the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta? Ignite? Rely? Liberty? Finale? RadicaleX?, as a weed control option in rice crops. Currently, rice growers in the United States control weeds through a combination of herbicides, crop rotation, and cultural practices such as flooding and tillage. The bar gene, which encodes the enzyme phosphinothricin N-acetyltransferase (PAT), was isolated from the common aerobic soil actinomycete, Streptomyces hygroscopicus strain HP632, and introduced by direct DNA transfer into the rice genome.

In addition to providing the trait of glufosinate herbicide tolerance, the PAT encoding gene was used as a selectable marker to identify transformed plants during regeneration in tissue culture. Glufosinate is a short name for the ammonium salt, glufosinate-ammonium. It is a broad-spectrum contact herbicide and is used to control a wide range of weeds after the crop emerges or for total vegetation control on land not used for cultivation. Glufosinate is a natural compound isolated from two species of Streptomyces fungi. It inhibits the activity of an enzyme, glutamine synthetase, which is necessary for the production of glutamine and for ammonia detoxification. The application of glufosinate leads to reduced glutamine and increased ammonia levels in the plant tissues. This causes photosynthesis to stop and the plant dies within a few days. Glufosinate also inhibits the same enzyme in animals. It is highly biodegradable, has no residual activity, and very low toxicity for humans and wild fauna. The PAT enzyme detoxifies phosphinothricin by acetylation into an inactive compound.

General Description
 
Transformation protocol. Transformation was achieved via direct gene transfer, therefore no vector agent was used . AgrEvo has requested that the details of the methodology be maintained by APHIS as confidential business information. Tissues were transformed with a fragment of DNA which contained the bar gene and from the plasmid, then used this fragment to transform the rice callus tissue.

Genes introduced to develop LLRICE06 and LLRICE62. Transformation events LLRICE06 and LLRICE62 were developed by introducing a single gene, the bar gene, into the varieties M202 and Bengal. The bar gene was derived from the soil-borne bacterium Streptomyces hygroscopicus, an organism which is not a plant pest. The bar gene encodes phosphinothricin-N-acetyltransferase (PAT), an enzyme which inactivates the herbicide glufosinate ammonium. The bar gene was engineered into the rice with noncoding DNA regulatory sequences attached to the coding region of bar. The regulatory sequences are the 35S promoter and terminator sequences derived from cauliflower mosaic virus. Neither the bar gene nor its associated noncoding DNA regulatory sequences cause plant disease or pose any plant pest risk.

Expression of genes introduced into lines LLRICE06 and LLRICE62. The rice transformation events LLRICE06 and LLRICE62 expressed the PAT protein encoded by the bar gene cassette. The PAT protein expressed in these plants was characterized and found to be equivalent to the previously characterized reference standards.
Reference: USDA-APHIS Reference No. 98-329-01p.

Summary of Introduced Genetic Elements
 
Code Name Type Promoter, other Terminator Copies Form
bar phosphinothricin N-acetyltransferase  (S. hygroscopicus) HT CaMV 35S
NULL
CaMV 35S poly(A) signal    

Characteristics of Oryza sativa (Rice)
 
Center of Origin Reproduction Toxins Allergenicity
Northern India and Southeast Asia, and southern China are believed to be the centre of origin of Asian rice (O. sativa). Wild progenitors of African cultivated rice (O. glaberrima) are grasses endemic to West Africa. Basically an autogamous plant propagating through seeds produced by self-pollination. Cross pollination between wild species and O. sativa cultivars has been reported to occur in natural habitats. Antinutrients including phytic acid, trypsin inhibitor, hemagglutinins (lectins) are present in the bran fraction. Allergenic proteins, 14-15 kDa range, present in the albumin plus globulin protein fraction from rice endosperm. Major 16 kDa allergenic protein is a member of the alpha-amylase/trypsin inhibitor family of proteins.

Donor Organism Characteristics
 
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
 
These transgenic rice lines were produced using a direct DNA transfer system in which DNA sequences encoding the PAT enzyme were introduced into callus tissue of the parental rice varieties M202, in the case of LLRICE06, and Bengal, in the case of LLRICE62. The nucleotide sequence of the native bar gene was modified to improve expression in plant cells, which was regulated by including promoter and transcription-termination and polyadenylation sequences derived from the 35S transcript of cauliflower mosaic virus (CaMV). The bar gene, including these regulatory sequences, was the only novel gene introduced into the parental rice varieties.

Characteristics of the Modification
 
The Introduced DNA
Southern blot analyses of genomic DNA from these transgenic rice lines revealed the integration of a single copy of the bar gene cassette into the LLRICE62 genome, and one complete plus one partial copy of the bar gene cassette into the LLRICE06 genome. No other plasmid encoded gene sequences were detected in the genomes of either of these transgenic lines.

Genetic Stability of the Introduced Trait
The herbicide tolerant trait from lines LLRICE06 and LLRICE62 was introduced into commercial rice varieties via traditional backcrossing, and segregated as a single genetic locus in progeny T1 and T2 generations. Southern blot analyses of genomic DNA from T2, T3, and T4 generations of LLRICE06, and from T2 and T3 generations of LLRICE62, yielded identical hybridization patterns, confirming the genetic stability of the original transformation events.

Expressed Material
The only newly expressed protein in the transformation events LLRICE06 and LLRICE62 was the PAT protein. The PAT protein expressed in these plants was characterized and found to be equivalent to the previously characterized reference standards.

The levels of PAT protein expressed in rice grain and various processed fractions derived from transgenic plants was quantitated by enzyme linked immunosorbent assay (ELISA) and determined to be very low. The assay detected both inactive and intact enzyme, such that positive detection did not necessarily indicate functional protein.

Environmental Safety Considerations
 
Field Testing
The rice lines LLRICE06 and LLRICE62 were field tested in the United States (1997 and 1998) and compared to their non-transgenic counterparts for agronomic characteristics, seed emergence, number of volunteers, disease susceptibility, and insect susceptibility and no obvious differences were detected. None of the field observations indicated that these transgenic rice lines had any increased potential to pose a plant pest risk.

Outcrossing
Cultivated rice (Oryza sativa L.) is primarily self-pollinating, although gene introgression into other cultivated rice is possible. Reported outcrossing rates are less than one percent and are limited by the biological characteristics of rice. Factors including flower morphology, inability of pollen to remain viable longer than a few minutes, and a lack of insect vectors for pollen spread contribute to the low propensity of rice to cross-pollinate.

In the United States, the only wild species known to be compatible to cultivated rice are O. rufipogon, which has been found in a single location in the Everglades of Florida, and red rice, a wild variant of cultivated O. sativa. It was considered very unlikely that cultivated rice would hybridize with O. rufipogon, since it occurs in a single isolated habitat that is not close to rice production areas. Consequently, red rice is considered to be the only wild species in which gene introgression from transgenic rice could occur.

Red rice is considered a weedy species in the cultivation of rice, as the reproduction of red rice favours specific environmental conditions (such as flooded fields) that are typical in the cultivation of commercial rice. Outside of rice production areas, red rice is not a weed species, however, it was assessed to determine whether it could become a weed pest if it hybridized with the glufosinate tolerant LLRICE06 and LLRICE62.

Gene flow from cultivated rice into red rice can occur, although the rate is likely to be very low with levels being dependent on the degree of overlapping of flowering periods. Thus, it was possible for the bar gene conferring tolerance to glufosinate to introgress into red rice and result in a glufosinate?tolerant red rice population. It was determined that the presence of glufosinate tolerance in hybrid populations would not change the fitness characteristics nor increase weediness (e.g., emergence vigor, final height, disease resistance, fecundity, shattering, and dormancy), except for a possible tolerance to the herbicide glufosinate ammonium. However, should this occurs, current weed control practices, such as tillage or other registered herbicides, would be an effective means of controlling the hybrid plants.

Weediness Potential
No competitive advantage was conferred to rice lines LLRICE06 and LLRICE62 other than that conferred by resistance to glufosinate ammonium herbicides. Resistance to glufosinate ammonium will not, in itself, render rice plants weedy or invasive of natural habitats since none of the reproductive or growth characteristics were altered by the transformation.

Cultivated rice is not listed as a common, serious or principal weed or a weed of current or potential importance in the United States. The transformed rice is an annual, does not shatter or disperse its seed, and has not acquired extended dormancy.

Secondary and Non-Target Adverse Effects
During field trials of LLRICE06 and LLRICE62, no toxicity or alteration of population levels were observed for beneficial insects, birds, or other species that frequent the fields. There were no qualitative differences between beneficial species populations present on transgenic and non-transgenic plants. Other than the production of the PAT enzyme, these plants were the same as the commercial rice varieties from which they were produced. The PAT enzyme has been extensively studied, and there have been no indications that it is toxic to organisms. Levels of the three known antinutrients found in rice (trypsin inhibitor, lectin, and phytic acid) were found to be in the expected range for transgenic seeds. Based on these findings, it was determined that these transgenic rice lines did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and were not expected to impact on threatened or endangered species.

Impact on Biodiversity
LLRICE06 and LLRICE62 have no novel phenotypic characteristics that would extend their use beyond the current geographic range of rice production. The cultivation of rice transformation events LLRICE06 and LLRICE62 would be equivalent to non-transgenic rice cultivars with respect to environmental impact.

Food and/or Feed Safety Considerations
 
Nutritional Data
The levels of proximates (moisture, ash, fat, protein, total dietary fibre, and carbohydrates) were determined in samples of grain, straw, and various processed fractions (e.g., rice hulls, brown rice, parboiled brown rice, polished rice, rice flour, rice bran, bran oil) from LLRICE62 and LLRICE06 (sprayed and unsprayed with glufosinate ammonium herbicide) and found to be within the normal range reported in the literature for commercial rice varieties. The amino acid and fatty acid profiles of grain from these transgenic varieties were similar to those of non-transgenic control varieties tested at the same time, and comparable to values reported in the scientific literature.

Rice contains a small number of antinutritional factors that are concentrated in the bran fraction and which, except for phytic acid, are subject to heat denaturation (inactivation). These antinutrients include: phytic acid, which is a storage form of phosphorus in plant seeds but also chelates calcium, zinc, iron, and magnesium in the digestive tract of animals thus interfering with absorption of these nutrients; trypsin inhibitor; and lectins, which are a class of proteins with specific binding affinities for particular carbohydrate moieties present on glycoproteins present in cell walls and cell plasma membranes, and have been associated with a range of antinutritive effects and some disease pathologies.

The levels of trypsin inhibitor and lectins in non-transgenic and transgenic LLRICE06 rough grain were reported to be below the limit of detection, and the phytic acid content did not vary significantly between transgenic and non-transgenic varieties.

The lack of any changes to nutritional composition and quality were also confirmed in a 42-day male broiler chicken feeding study.

Toxicity
The PAT enzyme does not have the characteristics of a toxin. It is specific for L?phosphinothricin and has no homology to proteins other than to similar PAT encoding genes from other organisms. In summary, no adverse effects were predicted given that the PAT enzyme is a minor constituent of human and animal food

Allergenicity
Searches of public amino acid sequence databases did not reveal any homologies between the deduced amino acid sequence of the introduced PAT protein and sequences of known toxins or allergens. The PAT enzyme does not exhibit the characteristics of an allergen. The PAT enzyme is both heat and acid labile, and looses 100% of its activity after cooking for 30 minutes at 75C. The enzyme is also inactivated after 30 minutes at a pH level of 4 or less. Should cooking not destroy the enzyme, it would be subject to oral ingestion by humans and subsequent rapid digestion. It was shown that PAT was degraded within minutes in simulated gastric juices.

Links to Further Information
 
Canadian Food Inspection Agency[PDF Size: 53480 bytes]
Decision Document DD2006-58; Determination of the Safety of Bayer CropScience?s Glufosinate Ammonium Tolerant Rice (Oryza sativa) Event LLrice62
Food Standards Australia New Zealand[PDF Size: 450877 bytes]
Final Assessment Report: Application A589 - Food derived from glufosinate ammonium-tolerant rice LLRICE62
Food Standards Australia New Zealand[PDF Size: 121154 bytes]
First Review Report: Application A589 - Food derived from glufosinate ammonium-tolerant rice line LLRICE62
Health Canada Novel Foods[PDF Size: 65147 bytes]
Novel Food Information; Glufosinate Tolerant Rice Event LLRICE62
U.S.Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 8781805 bytes]
AgrEvo USA Company Petition for Determination of Nonregulatory Status: LibertyLink Rice Transformation Events LLRICE06 and LLRICE62
US Food and Drug Administration[PDF Size: 509627 bytes]
Memorandum to file concerning glufosinate herbicide tolerant rice lines LLRICE06 and LLRICE62.
USDA-APHIS Environmental Assessment[PDF Size: 83754 bytes]
AgrEvo USA Company Petition 98-329-01p Determination of Nonregulated Status for Glufosinate Tolerant Rice Transformation Events LLRICE06 and LLRICE62

References
 
Cromwell, G.L., Henry, B.J., Scott, A.L., Gerngross, M.F., Dusek, D.L. and Fletcher, D.W. (2005). Glufosinate herbicide-tolerant (LibertyLink) rice vs. conventional rice in diets for growing-finishing swine. J. Anim. Sci. 83(5): 1068-1074.
Oberdoerfer, R.B., Shillito, R.D., de Beuckeleer, M. and Mitten, D.H. (2005). Rice (Oryza sativa L.) containing the bar gene is compositionally equivalent to the nontransgenic counterpart. J. Agric. Food Chem. 53(5): 1457-1465.


THIS RECORD WAS LAST MODIFIED ON WEDNESDAY, JANUARY 28, 2009
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