The lentil line RH44 was developed to allow the use of imidazolinone herbicides, as a weed control option in lentil production. This trait was developed using chemically induced seed mutagenesis and whole plant selection procedures. This rice line expresses a mutated form of the acetohydroxyacid synthase (AHAS) enzyme, which renders the plant tolerant to levels of imazethapyr used in weed control.

AHAS catalyses the first step in the biosynthesis of the branched-chain amino acids isoleucine, leucine and valine, and is active in the glycolytic pathway of plant metabolism. When conventional lentil plants are treated imazethapyr, the herbicide binds to a specific site on the enzyme, thereby inhibiting its activity. The result of this enzyme inhibition is a decrease in the synthesis of these amino acids, and an accumulation of toxic levels of alpha-ketoglutarate, all of which results in the eventual death of the plant.

RH44 lentil was isolated from a population derived from the seed of several lentil cultivars treated with ethyl methanesulfonate (EMS), a substance known to induce point mutations within the genome of organisms. The selection of herbicide tolerant plants was made from whole plants treated with an imidazolinone herbicide. One imidazolinone tolerant plant was selected from this population. Conventional breeding and seed increase techniques were then used to develop the imidazolinone-tolerant line RH44.

The Introduced DNA
Since RH44 is a product of mutagenesis and conventional seed increase techniques there was no introduction or incorporation of heterologous DNA into the plant genome. The tolerance to imidazolinone is due to a single point mutation in the AHAS encoding gene such that the amino acid sequence of the mutated enzyme differs by one amino acid from that of the unmodified enzyme. This single change in the amino acid sequence alters the binding site on AHAS to imidazolinone, such that the herbicide cannot inhibit the enzyme’s activity. This mutation of the AHAS gene was identified by sequencing the gene, which showed the single nucleotide change in the coding sequence for AHAS.

Genetic Stability of the Trait
The RH44 line is several generations removed from the original mutant herbicide tolerant plant. The imidazolinone trait in line RH44 segregates according to the expected Mendelian ratio for a single dominant allele.

Expressed Material
The modified AHAS gene, conferring tolerance to imazethapyr, is under control of the native AHAS promoter and is believed to be constitutively expressed. RH44 lentil therefore demonstrates whole plant tolerance to imidazolinone herbicides.

In the unmodified plant, the levels of valine, leucine and isoleucine are regulated by feedback inhibition. A mutation in the AHAS enzyme could affect the regulation of the biosynthesis of these amino acids. Data were submitted to show that the activity of the modified AHAS in RH44 lentil was not altered: the mutation did not affect feedback inhibition and therefore, the regulation and levels of these amino acids. The amino acid composition of RH44 was compared to that of commercial cultivars and found to be equivalent in terms of the levels of valine, leucine and isoleucine.

Outcrossing
Lentil (L. culinaris) is predominantly self-pollinating, with less than one percent cross-pollination (CFIA, 2003). As such, an isolation distance of only 3 metres is required for pedigreed seed production in Canada (Canadian Seed Growers Association, 2000). The potential for introgression of the herbicide tolerance trait into unmodified lentil plants is therefore negligible. Lentil can successfully hybridize with its wild progenitor Lens orientalis. This wild relative of lentil grows in the Near East, and from the western Mediterranean area to Ethiopia. Introgression of the herbicide tolerance trait from RH44 plants into L. orientalis is therefore possible in these regions. Neither Lens orientalis, nor any other wild relative of lentil grow in Canada.

Weediness Potential
Lentil does not exhibit characteristics that would render it either weedy or invasive of unmanaged habitats. While some lentil pods shatter at harvest, these seeds do not normally volunteer in any subsequent crop. In Canada, lentil seeds that germinate in the fall do not survive the winter. Those seeds that do germinate the following spring compete poorly with other crops and weeds. Unlike legumes species such as alfalfa or red clover, lentil seed does not exhibit dormancy characteristics such as hard seed. Since lentil seedlings grow slowly are not competitive, lentil could not establish itself successfully in unmanaged habitats. Lentil does not establish successfully in unmanaged habitats; the seedlings grow slowly and thus compete poorly with other plants.

Field trials were conducted to evaluate the agronomic performance of RH44 compared to conventional varieties. There were no significant differences in either seed yield, germination or days to maturity between RH44 and its unmodified comparators. These results indicated that since none of the growth and reproductive characteristics were unintentionally altered, RH44 would not be expected to become weedy or invasive.

Secondary and Non-Target Effects
The characterization of the modified AHAS gene containing a single base pair change, and the resulting modified enzyme led to the conclusion that the expression of the modified AHAS does not result in altered toxic or allergenic properties. The AHAS enzyme is not a known toxin, does not confer resistance to agricultural pests and is present in plants and micro-organisms with a history of safe use. Based on this information, it was determined that the RH44 lentil line will not result in altered impacts on non-target organisms, compared to conventional lentil cultivars.

The agronomic performance and disease and insect susceptibilities of RH44 were found to be in the range observed by conventional commercial lentil cultivars. RH44 lentil therefore does not display altered morphological or physiological characteristics that would alter its interaction with non-target organisms. Also, since RH44 did not exhibit any altered susceptibilities to either insect pests or plant diseases, it is not expected to display an altered plant pest potential.

Impact on Biodiversity
RH44 does not exhibit novel phenotypic characteristics that would extend its use beyond the current geographic range of lentil production. The insect and disease susceptibilities of RH44 were similar to those of conventional lentil cultivars. Furthermore, there does not exist the potential for outcrossing to wild lentil relatives in Canada. The impact of the cultivation of line RH44 in Canada would therefore be equivalent to that of conventional lentil cultivars.

The potential for outcrossing exists in areas of the world where wild relatives of lentil are indigenous. The cultivation of RH44 lentil in these areas, such as the Near East, the western Mediterrenean area, and Ethiopia, could result in the introgression of the imidazolinone-tolerance trait into these related species. The impact on biodiversity of these herbicide tolerant hybrids would be minimal outside of selection pressure from imidazolinone herbicides.

Dietary Exposure
The modification to the AHAS enzyme in RH44 will not result in any change in the consumption pattern of lentil. The availability of many lentil varieties for cultivation, the diversity of lentil varieties in phenotypic traits, and the normal variation in lentil composition due to differences in growing conditions, all result in a wide variation in the composition of conventional lentil. Thus, the cultivation of RH44 would not be expected to change the dietary exposure to humans, any more than do commercially available lentil cultivars.

Nutritional and Compositional Data
Nutritional components of line RH44 were measured analytically and compared to those of unmodified commercial lentil cultivars. These components included proximates (crude protein, crude fat, crude fibre, ash, moisture), amino acids, fatty acids, vitamins (thiamine, niacin, pantothenic acid, B6), and minerals (magnesium, zinc, iron, phosphorus). The levels of these nutrients in RH44 lentil were within the range observed for currently registered commercial cultivars in Canada. The levels of the antinutritional factors phytic acid and trypsin inhibitor for RH44 were also within the range observed in these commercial cultivars.

Certain components were not included in the compositional analysis of RH44 lentil. These were folacin, riboflavin, vitamin C, copper, potassium and manganese, and lectin. The developer of RH44 provided a rationale to the reviewing regulatory agencies stating that since the overall composition, physiology and phenological characteristics of RH44 were not significantly changed, there was no indication that the levels of these other nutritional components and antinutrients would be outside of the acceptable range for conventional lentil cultivars. The lentil line RH44 is also not intended as a stand-alone cultivar, but rather as genetic stock (and source of the imidazolinone-tolerance trait) for lentil cultivar improvement.

The results of these compositional analyses, and the submitted rationale, led to the conclusion that the lentil line RH44, and any progeny containing its imidazolinone-tolerance trait, would not be nutritionally or compositionally different than conventional commercial lentil cultivars.

Toxicity and Allergenicity
The potential for toxicity and allergenicity of RH44 was investigated by examining the amino acid sequence homology and the characteristics of the altered AHAS protein. The unmodified form of AHAS is heat sensitive and susceptible to trypsin degradation. The AHAS from RH44 demonstrated no enzyme activity after 1 min of heating at 100ºC. The modified AHAS was also completely degraded after 30 minutes of treatment with trypsin, to simulate gastric digestibilitiy. The unmodified form of AHAS demonstrated no amino acid similarity to known toxins or allergens. The amino acid sequence of the mutated AHAS differs by only one amino acid compared to unmodified rice.

The potential for increased allergenicity of the seed from RH44 was investigated, specifically with regard to endogenous lentil allergens that induce an IgE reaction in susceptible humans. The data submitted showed that protein banding patterns and IgE activity from sera obtained from lentil allergic persons were similar among RH44 lentil and the conventional lentil cultivars.

The results of the amino acid sequence homology investigations, heat sensitivity and digestibility studies, and immunological assays led to the conclusion that the lentil line RH44 did not demonstrate any potential for toxicity and novel allergenicity, or any altered endogenous allergenicity, compared to conventional lentil culivars.

Decision Document DD2004-46 Determination of the Safety of the BASF Canada Imidazolinone-Tolerant Lentil Line RH44

Novel Food Information - Food Biotechnology: Imidazolinone Tolerant Clearfield™ Lentil (RH44)

Canadian Food Inspection Agency. (2003). Biology Document BIO2003-12: The Biology of Lens culinaris Medikus (Lentil). Canadian Food Inspection Agency, Plant Products Directorate.

Canadian Seed Growers Association. (2000). Regulations and procedures for pedigreed seed production. Circ. 6-94. Canadian Seed Growers Association, Ottawa ON.