Novel Food Information - Reduced Lignin Alfalfa KK179

Health Canada has notified Monsanto Canada Inc. and Forage Genetics International (hereafter FGI) that it has no objection to the sale of food derived from Reduced Lignin Alfalfa KK179. The Department conducted a comprehensive assessment of this alfalfa event according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.


The following provides a summary of the notification from Monsanto Canada Inc. and FGI and the evaluation by Heath Canada. This document contains no confidential business information.

1. Introduction

Monsanto Canada Inc., in collaboration with FGI, developed Reduced Lignin Alfalfa KK179 (hereafter KK179 alfalfa) using recombinant DNA techniques to introduce a fragment of the Caffeoyl CoA 3-O-methyltransferase (CCOMT) coding sequence. This fragment sequence was introduced in an inverted repeat, so that expression will produce a double stranded RNA that will, via RNA interference (RNAi), suppress endogenous CCOMT RNA expression levels. CCOMT is an essential part of the G lignin monomer biosynthesis pathway and suppression of this enzyme results in lower levels of the G lignin and overall lignin content in KK179.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflects international guidance documents in this area (eg., Codex Alimentarius). The assessment considered: how KK179 alfalfa was developed; how the composition and nutritional quality of KK179 alfalfa compared to non-modified varieties; and what the potential is for KK179 alfalfa to be toxic or cause allergic reactions. Monsanto Canada and FGI have provided data which demonstrate that KK179 alfalfa is as safe and of the same nutritional quality as traditional alfalfa varieties in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). Foods derived from KK179 alfalfa are considered novel foods under the following part of the definition of novel foods: "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

  • (I) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.

2. Development of the Modified Plant

The petitioners have provided information describing the methods used to develop KK179 and molecular biology data that characterize the genetic change that results in reduced lignin production. Reduced Lignin Alfalfa KK179 was produced using Agrobacterium tumefaciens (A.  tumefaciens) mediated transformation of alfalfa variety R2336 with the transformation vector PV-MSPQ12633. This transformation vector was constructed to contain two T-DNA regions, one for the CCOMT fragment and one for the marker gene neomycin phosphotransferase II (nptII).

The first T-DNA (T-DNA I) contains the coding region that results in the reduced production of G lignin. This coding region consists of an inverted repeat of a 0.8 kb fragment of Caffeoyl CoA 3-O-methyltransferase (CCOMT). When transcribed this inverted repeat fragment will generate a double stranded RNA which, through endogenous RNA interference mechanisms, results in suppression of CCOMT protein expression.

The second T-DNA (T-DNA II) contains the coding regions for the marker gene neomycin phosphotransferase (npt II). The integration of this T-DNA in the alfalfa genome confers tolerance to the antibiotic neomycin. The integration of this gene cassette is not linked to the integration of T-DNA I and so it can be removed through selective breeding. The petitioners have stated that such a breeding program was undertaken and that the KK179 line does not contain any elements from T-DNA II, including the marker gene.

Alfalfa explants from R2336 were placed in tissue culture media and co-cultured with Agrobacterium tumefaciens carrying the plasmid PV-MSPQ12633. After three days of co-culturing, explants were placed on selection medium containing kanamycin and Timentin® to inhibit the growth of excess Agrobacterium and untransformed cells. The kanamycin resistant cells were developed via somatic embryogenesis and placed in media conductive to root and shoot development. Rooted plants (T0) with normal phenotypic characteristics were selected and transferred to soil for growth and further development. T0 plants were then crossed into Ms208, a conventional male sterile alfalfa, to produce F1 plants. The F1 plants were then segregated using PCR to identify plants positive for T-DNA I, but negative for T-DNA II. Segregated plants were then assessed for agronomic characteristics, resulting in a single plant, P0, being chosen for further breeding.

Due to self-incompatibility in alfalfa, and the negative impacts of breeding depression, it is not possible to breed pure isogenic lines through selfing. Therefore, the petitioners bred subsequent generations using traditional alfalfa breeding methods. After selecting P0, the plant was hand crossed with 10 elite alfalfa genotypes, each with fall dormancy 4 phenotypes. This produced the next generation of the KK179 line via a mechanism known as modified backcrossing. The generation produced from this crossing was known as MBC1. This generation was then crossed into the same 10 elite lines, resulting in MCB2. This generation was then crossed inter se (i.e., within itself), a polycross, to produce the first synthetic population (Syn1), which is the preferred generation for commercial development. In addition, the petitioner performed a second polycross, producing the advanced SYN1 generation (Syn1Adv), which is used in their forage feeding study.

Due to the complex nature of alfalfa breeding, and to ensure proper comparators for the testing of the KK179 lines, the petitioner also selected a single R2336 plant (C0) and performed an identical breeding program with this non-transgenic parental line resulting in the C0-Syn1 and C0-Syn1Adv generations. These generations were used as the conventional control generations in testing. Also included were both parental lines, R2336 and Ms208 as the conventional parental controls.

3. Characterization of the Modified Plant

The petitioners used Southern blots to confirm the number of copies of each functional element within the insert. To do this the petitioners used a variety of restriction enzymes and nine different probes, each specific for one or more functional elements the CCOMT cassette. The data presented by the petitioners confirm a single insertion of the CCOMT cassette at a single locus.

The petitioners undertook sequence analysis of the inserted CCOMT cassette to confirm the integrity of transferred DNA. The sequence analysis of the cassette demonstrated the integrity of the cassette and the functional elements sequences. This sequence analysis also noted a single base pair change 5’ of the insertion and a 120 bp deletion 3’ of the insertion. The single base pair change has been reported to be the result of a single nucleotide polymorphism in alfalfa. The deletion is considered likely to be the result of double strand break repair and is a common occurrence resulting from Agrobacterium-mediated transformations.

In addition to the Southern blots used to characterise the insert, the petitioners performed a series of Southern blots to demonstrate the absence of any T-DNA II sequences. Using a single probe, the petitioners probed KK179 genomic DNA after digestion with one of three restriction enzyme combinations. From the evidence presented by the petitioners, no genetic material from T-DNA II remains in KK179. The petitioners also performed a series of Southern blots to demonstrate the absence of any plasmid backbone sequences. Using four overlapping probes, the petitioners probed KK179 genomic DNA after digestion with one of three restriction enzyme combinations. From the evidence presented by the petitioners, no plasmid backbone has been transferred into KK179.

Generational stability of the single insert was determined across four generations of KK179 (P0, MBC1, MBC2 and Syn1). Southern blot analysis was presented for each generation confirming the presence of the T-DNA I cassette and its stability across generations. The petitioners also undertook testing on the MBC1, MBC2 and Syn1 generations to demonstrate that the insert is inherited in the expected manner for an insert at a single locus. Using PCR to analyze the generations, the petitioners tested for the presence of the T-DNA I. Based on this analysis, segregation ratios were determined and tested against the expected. These compared ratios were then analysed using Chi square to test the hypothesis of Mendelian inheritance. Based on the segregation ratios from the PCR, and confirmed by the Chi square analysis, the trait is inherited in the expected Mendelian manner. This indicates that the KK179 insert is integrated into the alfalfa genome at a single locus. This is also further evidence that the trait is stable across generations.

As recommended in the guidance published by Codex Alimentarius, the petitioners completed an analysis of any potential opening reading frames created by the insertion into the genomic DNA. The analysis was done use bioinformatics to assess the homology between any potential reading frames and databases of know toxins and allergens. Open Reading Frames spanning the 5' flanking sequence DNA-inserted DNA junctions, and 3' flanking sequence DNA-inserted DNA junctions were translated in all six reading frames. Unique putative peptides/polypeptides of at least 8 amino acids or greater in length from each reading frame were then compared to toxin, allergen and all protein databases. The petitioners have stated that there is no analytical data to indicate that any of these putative proteins is produced in KK179.

The results of the screen of all the putative polypeptides from the insert sequence ORFs showed no significant matches to the allergen or toxin databases. Two short, identical polypeptide matches with a sequence in the allergen database were reported. These sequences match the putative amino acid sequence that would be generated by the CCOMT sequences. As the mRNA produced from these sequences anneals to itself to produce dsRNA, which is then reduced to smaller segments, translation of the mRNA is not expected to occur and so this peptide will not be produced. Matches to sequences in the protein database were identified in all six frames. However, these matches were all either punctuated with several stop codons or were to elements contained in the insert that are not expressed. As a result, in the unlikely event that an unexpected translation product from the insert was produced, it is not expected that these polypeptides would be a toxin, allergen or a known protein that displays adverse biological activity.

The alignments of all the putative ORFs identified at the 5' and 3' insert junctions identified no biologically relevant structural similarity to known allergens, toxins or proteins. Furthermore, no contiguous 8 amino acid matches were found between the putative polypeptides and known allergens. As a result, in the unlikely event that a translation product spanning the 5' or 3' junctions was produced, it is not expected that these polypeptides would be a toxin, allergen or a known protein that display adverse biological activity.

As the intended effect of the introduced T-DNA I cassette is a reduction in lignin levels, via RNA interference, the petitioners have provided an analysis of CCOMT mRNA levels. Using total polyA+ RNA extracted from KK179 and the conventional control variety, the petitioners used Northern blots to demonstrate evidence of suppression in both forage and root tissue. The presented data demonstrated suppression of the CCOMT mRNA in both root and forage tissue confirming the suppression of this protein through the insertion of the CCOMT cassette in T-DNA I.

4. Product Information

Reduced Lignin alfalfa differs from conventional alfalfa by the insertion of a fragment of the Caffeoyl CoA 3-O-methyltransferase (CCOMT) coding sequence. This fragment sequence was introduced as an inverted repeat, so that expression will produce a double stranded RNA that will, via RNA interference (RNAi), suppress translation of endogenous CCOMT RNA  and levels of the CCOMT enzyme. CCOMT is an essential part of the G lignin monomer biosynthesis pathway and suppression of this enzyme results in lower levels of the G lignin and overall lignin content in KK179.

Lignin is a high molecular weight, polymeric molecule composed principally of three lignin monomeric subunits: guaiacyl (G), syringyl (S) and p-hydroxyphenyl (H) lignin. Lignin is a cell wall component, along with cellulose and hemicellulose that accumulates in the plant, particularly in the stem. Lignin is a natural constituent of all plants, but is indigestible and slows down the digestion of cellulose in the rumen of livestock. As a result, alfalfa with lower levels of lignin is desirable for forage producers.

5. Dietary Exposure

Monsanto Canada Inc. and FGI have indicated that KK179 alfalfa is not intended to be sold for food production purposes and is solely targeted to the forage market. They also noted that all growers buying KK179 seed would be required to sign a licensing agreement stating that the seed will not be used for food production. As such, Monsanto has indicated that dietary exposure through food would only be the result of the unintentional low level presence of this alfalfa. However, to ensure no alteration in their flow of commerce should such an event occur, Monsanto has requested a Letter of No Objection for food use of this product.

While this product is not intended to be sold for food production purposes, the assessment undertaken by the Food Directorate assumed that KK179 would be used in a similar manner to traditional food alfalfa varieties. The dietary exposure estimate was also based on the conservative estimate that all alfalfa consumed was KK179.

6. Nutrition

The nutrient data for this submission was obtained from KK179 and control alfalfa grown in 2011 at six locations in the United States, in randomized complete block designs with four plots for both KK179, control and reference varieties. All field trial experiments used to test the KK179 alfalfa were acceptable. All analyses of KK179, control and reference varieties were done using approved scientific and appropriate statistical methods.

KK179, control and reference varieties were analyzed for nutrients and anti-nutrients, in forage, as follows: Proximates (moisture, crude protein, crude fat, ash, carbohydrates (calculated); acid detergent fibre (ADF), neutral detergent fibre (NDF) and Acid Detergent Lignin (ADL)),  Minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), Amino Acids (essential and non-essential) Anti-Nutrients [daidzein, glycitein, genistein, coumestrol, formononetin, biochanin A, and saponins (total bayogenin, total hederagenin, total medicagenic acid, total soyasapogenol B, total soyasapogenol E, total zanhic acid and total saponins)], and Secondary Metabolite(s) (p-coumaric acid, ferulic acid, sinapic acid, total polyphenols, free phenylalanine and canavanine).

For combined locations, statistical differences (KK179 vs. control) were noted in 3 analytes as follows: ash, canavanine and ferulic acid.  Their mean values were within reference and/or literature ranges.  ADL was numerically lower (KK179 4.89% lower than control) although the difference was not statistically significant.

7. Toxicology

Bioinformatics analysis of the transgenic DNA insert and flanking regions was provided by the petitioners. Putative amino acid sequences from 6 reading frames (3 from sense and 3 from anti-sense strands) were compared to available FAARP protein (Genbank release 187.0) and toxin (TOX_2012) databases using the FASTA alignment tool. No statistically significant or toxicologically relevant alignments were detected based on the search criteria and, therefore, any protein made would not be expected to pose a toxic risk.  Furthermore, as RNAi mechanisms do not involve translation of novel proteins, the potential for generation of toxins is considered negligible.

Compositional analysis between KK179 and a conventional control and 14 alfalfa comparators showed no statistically significant or biologically meaningful differences in anti-nutrient or saponin content.

Exposure to exogenous RNA sequences including dsRNA (double stranded), siRNA (small interfering) and miRNA (microRNA), may present a potential health hazard as a result of interactions and/or interference with endogenous RNA mechanisms. The possibility of a toxicological concern was addressed through bioinformatics analyses as well as scientific rationale. Bioinformatics analyses were performed using the NCBI Basic Local Alignment Search Tool (BLAST; v.2.2.29) to compare the DNA insert to the human genome and transcriptome. The CCOMT insert was not found to have any matches with known human mRNA sequences based on the search criteria. Hits against the genomic database were noted, however, such hits were neither statistically significant nor considered biologically relevant. This suggests that dsRNA generated from the CCOMT insert would not be expected to directly interfere with human mRNA sequences.

The scientific rationale included a dietary intake estimate based on conservative assumptions for bioavailability and consumption. It was assumed that all alfalfa consumed is KK179, all small interfering RNA (siRNA) present in the plant is the sequence of interest expressed at the LOD (noting that total construct siRNA levels are below the LOD), and that all biological barriers do not hinder uptake. On this basis, a worst-case exposure estimate of 0.55 ng siRNA/kg bw/day was calculated based on Canadian consumption data.

This dietary intake estimate is several orders of magnitude lower than the level of exposure calculated in in vivo studies where no statistically or biologically significant increase in plasma or tissue levels of specific plant microRNAs (miRNAs) were observed after feeding mice and human subjects diets rich in such miRNAs. Compared to data provided in support of the submission, including data available in the scientific literature, the maximum potential siRNA dietary intake was considered negligible.

An assessment of allergenic potential of putative peptides associated with the transgenic DNA insert was performed by the petitioners using the FAARP allergen, gliadin and glutenin sequence database (AD_2012). Two alignments were found to be identical to a region in wheat (dehydrin), however, the petitioners stated that this sequence is derived from the partial CCOMT gene segment and is therefore an untranslated sequence. Specifically, it is unlikely that any novel proteins will be produced in KK179 as the RNA transcripts of the inverted repeat sequences have a double-stranded, hairpin secondary structure which should prevent translation and protein synthesis.

Alfalfa is not considered a priority allergen. Furthermore, as no new proteins are likely to be generated, the allergenic potential of KK179 is not expected to be different from that of conventional alfalfa.

On the basis of negligible homology with the human genome and transcriptome, a lack of significant putative amino acid alignment with known proteins or toxins, a low estimated dietary exposure and the lack of novel protein synthesis associated with the RNAi mechanism, KK179 alfalfa was not considered to pose an toxicological or allergenic risk to humans.


Health Canada’s review of the information presented in support of the food use of Reduced Lignin Alfalfa KK179 concluded that derived food products do not raise concerns related to safety. Health Canada is of the opinion that Reduced Lignin Alfalfa KK179 is similar to conventional alfalfa in terms of being an acceptable food source.

Health Canada's opinion deals only with the human food use of Reduced Lignin Alfalfa KK179. Issues related to the environmental safety of Reduced Lignin Alfalfa KK179 in Canada and its use as livestock feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada.  This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9

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