Supporting Document 1

Supporting document 1 Safety assessment – Application A1085 Food derived from Reduced Lignin Lucerne Line KK179 Summary and conclusions Background A genetically modified (GM) lucerne line, KK179, has been developed that has reduced biosynthesis of guaiacyl lignin (G lignin), a major subunit of lignin. Lignin is a non- carbohydrate phenolic polymer deposited in plant cell walls, particularly in the vascular tissue, and is a contributor to the quality of forage eaten by grazing animals. The Applicants claim that growers will have the option of being able to harvest KK179 several days later than conventional lucerne without appreciable loss of forage quality typical in conventional lucerne at the same growth stage. The reduced level of lignin in lucerne KK179 has been achieved through the introduction of a partial caffeoyl CoA 3-O-methyltransferase (CCOMT) gene sequence derived from lucerne (Medicago sativa). The gene transcript acts, via suppression of the endogenous CCOMT gene, to reduce the lignin level. It is not intended that KK179 enter the food supply. However, a food approval is sought in case this inadvertently occurs. In conducting a safety assessment of food derived from lucerne line KK179, a number of criteria have been addressed including: a characterisation of the transferred genetic material and its origin, function and stability in the lucerne genome; compositional analyses; and evaluation of intended and unintended changes. This safety assessment report addresses only food safety and nutritional issues associated with the GM line. It therefore does not address: environmental risks related to the environmental release of GM plants used in food production the safety of animal feed or animals fed with feed derived from GM plants the safety per se of food derived from the non-GM (conventional) plant. i History of Use Lucerne is grown primarily for livestock feed and is grown throughout the world (approximately 30 million ha) as forage. It is often harvested for hay, but can also be made into silage and manufactured stock feed (meal and pellets). The main food products from M. sativa are alfalfa sprouts, comprising sprouted seeds packed into punnets that are used as a fresh vegetable in salads, sandwiches, soups and stir-fries. Other alfalfa products are widely available in specialised stores, for example alfalfa in the form of dried leaf, health drinks and teas. Molecular Characterisation Explants of the lucerne line ‘R2336’ were transformed via Agrobacterium-mediated transformation, the genes of interest being inserted via two separate T-DNAs. T-DNA I contains two CCOMT fragments, that when transcribed lead to the production of double- stranded RNAs (dsRNAs) that, via an RNA interference (RNAi) mechanism, suppress endogenous CCOMT RNA levels, leading to reduced biosynthesis of G-lignin. In order to select putative transformants, a T-DNA II was also inserted during the transformation procedure. This contained a neomycin phosphotransferase II (nptII) coding region that confers resistance to kanamycin. T-DNA II was removed from KK179 by selection. Comprehensive molecular analyses of lucerne line KK179 indicate there is a single insertion site at which there is a single copy of the T-DNA I. No DNA sequences from T-DNA II or from the backbone of the transformation vector, including antibiotic resistance marker genes, were transferred to the plant. The introduced genetic elements are stably inherited from one generation to the next. Northern blot analyses were used to compare the RNA levels associated with the endogenous CCOMT gene in forage and root tissue of KK179. The data show a clear reduction in the level of CCOMT mRNA in KK179 compared to the conventional control and hence that insertion of the CCOMT suppression cassette in T-DNA I has resulted in the intended modification. Compositional Analyses In order to establish the nutritional adequacy of forage from lucerne line KK179, samples were analysed for 50 analytes comprising nutrients; proximates (ash, fat, moisture, and protein), carbohydrates by calculation, acid detergent fibre, neutral detergent fibre, acid detergent lignin, minerals, amino acids and a number of anti-nutrients and secondary metabolites. In addition, p-coumaric acid, ferulic acid, sinapic acid, total polyphenols and free phenylalanine were also analysed to evaluate the effect of CCOMT suppression on the lignin pathway and cell wall-associated metabolites. As expected, the levels of lignin in general, and G lignin in particular, in KK179 were statistically significantly lower than in the control. The overall magnitude of the difference however was small, and the lignin levels were within the reference range obtained for non- GM reference varieties grown at the same time. While the difference in lignin levels between the GM line and the control is of agronomic significance, in that it enables the forage to be harvested at a later date without appreciable loss of forage quality, it is unlikely to have any nutritional significance to humans given the range of natural variation that exists in lucerne. For the remaining analytes, statistically significant differences were noted in only three analytes (ash, canavanine and ferulic acid). In all cases the differences were typically small ii and within the reference range obtained for non-GM reference varieties grown at the same time. Any observed differences are therefore considered to represent the natural variability that exists within lucerne. Conclusion No potential public health and safety concerns have been identified in the assessment of lucerne line KK179. On the basis of the data provided in the present Application, and other available information, food derived from lucerne line KK179 is considered to be as safe for human consumption as food derived from conventional lucerne cultivars. iii TABLE OF CONTENTS SUMMARY AND CONCLUSIONS ......................................................................................... I LIST OF TABLES ................................................................................................................. 2 LIST OF FIGURES ............................................................................................................... 2 LIST OF ABBREVIATIONS .................................................................................................. 3 1. INTRODUCTION ........................................................................................................... 4 2. HISTORY OF USE ........................................................................................................ 4 2.1 HOST AND DONOR ORGANISM ...................................................................................... 4 2.2 OTHER ORGANISMS ..................................................................................................... 6 3. MOLECULAR CHARACTERISATION .......................................................................... 6 3.1 METHOD USED IN THE GENETIC MODIFICATION .............................................................. 7 3.2 DESCRIPTION OF THE INTRODUCED GENETIC MATERIAL ................................................. 8 3.3 BREEDING TO OBTAIN LUCERNE LINE KK179 ............................................................... 11 3.4 CHARACTERISATION OF THE GENETIC MATERIAL IN THE PLANT ..................................... 13 3.5 STABILITY OF THE GENETIC CHANGE ........................................................................... 15 3.6 ANTIBIOTIC RESISTANCE MARKER GENES .................................................................... 16 3.7 CONCLUSION ............................................................................................................ 16 4. CHARACTERISATION OF NOVEL SUBSTANCES ................................................... 16 4.1 POTENTIAL ALLERGENICITY/TOXICITY OF ANY NOVEL ORFS CREATED BY THE TRANSFORMATION PROCEDURE .................................................................................. 17 4.2 THE EXPRESSION OF CCOMT IN KK179 ..................................................................... 15 5. COMPOSITIONAL ANALYSIS ................................................................................... 18 5.1 KEY COMPONENTS OF LUCERNE ................................................................................. 18 5.2 STUDY DESIGN, CONDUCT AND ANALYSIS .................................................................... 19 5.3 FORAGE COMPOSITION .............................................................................................. 20 5.4 CONCLUSION ............................................................................................................ 25 7. NUTRITIONAL IMPACT ............................................................................................. 26 REFERENCES ................................................................................................................... 26 1 LIST OF TABLES Table 1: Importation (kg) to A) Australia, between 2008 – 2012, and B) New Zealand in 2005 of lucerne seed for sowing/sprouting (by country of origin) ...................................... 5 Table 2: Description of the genetic elements contained in the two T-DNAs of PV- MSPQ12633 ............................................................................................................ 8 Table 3: KK179 generations used for various analyses ....................................................... 12 Table 4: Mean percentage ± S.E. of proximates and fibre in forage from C0 Syn1 and

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