GM Crop Database

Database Product Description

LLRICE06, LLRICE62 (ACS-OSØØ1-4, ACS-OSØØ2-5)
Host Organism
Oryza sativa (Rice)
Trade Name
Liberty-Link™
Trait
Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.
Trait Introduction
Direct DNA transfer system
Proposed Use

Production for human food, livestock feed and industrial uses.

Product Developer
Aventis CropScience

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2008 View
Canada 2006 2006 View
Colombia 2008 View
Mexico 2007 2007 View
Philippines 2012 2012 View
Russia 2007 View
South Africa 2011 2011 View
United States 2000 2000 1999

Introduction Expand

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.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
bar phosphinothricin N-acetyltransferase HT CaMV 35S CaMV 35S poly(A) signal

Characteristics of Oryza sativa (Rice) Expand

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 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

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 Expand

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 Expand

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 Expand

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.

Abstract Collapse

The major producers of rice were China, India, Indonesia, Bangladesh, Viet Nam, Thailand and Myanmar. Rice is grown primarily for its grain, which is the staple food for half of the world’s population, and has many uses in the food and industrial sectors, including use in livestock feed.

Harvested rice, or rough rice, is encased by an inedible hull or husk, which is removed before milling. The hulls are utilized as fuels, mulch, abrasives and animal feed products. Brown rice is what remains after the hulls are removed. The light brown colour of brown rice is due to the presence of bran layers and the rice germ surrounding the rice kernel. Brown rice can be milled into regular white or ‘polished rice’, where white rice is distinguished by the fact that the hulls, bran layers and rice germ are removed. The bran and rice germs are high in protein and nutrients and are used in specialty foods such as rice bran oil and also in livestock feed. Both brown and white rice kernels can be parboiled and/or milled into rice flour for use in breakfast cereals, baby foods and desserts and numerous other food products. Rice kernels are also used to produce beer and rice wine.

Weeds are a significant pest problem in rice production in the United States. The best approach to controlling weeds in rice involves a combination of good cultural (certified seed, variety selection), mechanical (crop rotations, preparation of seedbed), and chemical practices. In order to obtain high yield potential, high rates and often multiple applications of herbicides are required for weed control. Over the years, herbicide tolerant weeds have developed and resulted in the rapid decline in the effectiveness of several herbicides (e.g., Londax®; common name bensulfuron methyl) against these resistant weeds. The use of integrated weed management strategies will be the key to delaying the development of resistance in rice weeds.

Rice lines LLRICE06 and LLRICE62 were genetically 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 rice lines is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces hygroscopicus, the same organism from which glufosinate was originally isolated. 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 encoding gene (bar) was introduced into the rice genome by direct gene delivery transformation, and the resulting rice lines displayed field tolerance to phosphinothricin-containing herbicides.

LLRICE06 and LLRICE62 were tested in field trials in the United States (1997-1998). These tests demonstrated that the transformed lines did not exhibit weedy characteristics, or negatively affect beneficial or nontarget organisms, and were not expected to impact on threatened or endangered species.

Cultivated rice is primarily self-pollinating, but may cross-pollinate with other cultivated rice varieties although rates are less than one percent. Factors limiting cross-pollination in rice include flower morphology, inability of pollen to remain viable longer than a few minutes, and a lack of insect vectors for pollen spread. In the United States, the only wild species known to be compatible with 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. Due to the relative isolation of O. rufipogon, it is unlikely to hybridize with cultivated rice.

Cultivated rice may cross-pollinate with red rice, given suitable conditions, and form glufosinate-tolerant hybrids. However, the glufosinate-tolerance trait is not expected to provide a competitive advantage to hybrid plants unless grown in managed environments that are routinely subjected to glufosinate applications. In the event that a glufosinate-tolerant hybrid survived, the herbicide-tolerant individual would be easily controlled using mechanical and other available chemical means. The use of good cultural practices, crop and herbicide rotations, are also effective strategies for controlling the establishment of herbicide tolerant weeds.

The food and livestock feed safety of rice lines LLRICE06 and LLRICE62 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of rice was found to be equivalent to conventional varieties as shown by the analyses of grain, straw, and various processed fractions for key nutrients including proximates (moisture, ash, fat, protein, total dietary fibre, and carbohydrates), amino acid and fatty acid profiles and mineral content (calcium, phosphorus and iron). Assays were completed for anti-nutritional factors normally concentrated in the bran fraction of rice, such as phytic acid, trypsin inhibitor, and lectins. The levels of trypsin inhibitor and lectins in LLRICE06 and conventional rice were reported to be below the limit of detection, and the phytic acid content was the same for both transgenic and conventional varieties. The nutritional equivalence of LLRICE06 and LLRICE62 and conventional rice was confirmed in a 42-day feeding trial with male broiler chickens.

The potential for toxicity and allergenicity of the PAT protein was assessed by examining its physiochemical characteristics, amino acid sequence homology to known protein allergens, and susceptibility to in vitro digestion under conditions simulating the mammalian digestive tract. In each case, the PAT protein did not exhibit the characteristics commonly associated with food allergens. There were no amino acid sequence homologies with known allergens or toxins and the protein was readily digested in simulated gastric fluids. These studies were sufficient to provide a reasonable certainty that PAT protein has no allergenic potential.

There are reports of allergenic proteins within the rice endosperm. Laboratory studies determined there were no differences in the content of these allergenic proteins between rice lines LLRICE06 and LLRICE62 and conventional rice. These transgenic rice lines were also analyzed for the Osborne fraction, which contains prolamine proteins known to be related to Celiac disease. These proteins were investigated in brown rice samples and results showed there were no differences between rice lines LLRICE06 and LLRICE62 and conventional rice.

Links to Further Information Expand


This record was last modified on Wednesday, September 21, 2016