*4.2. Plant Material*

Monocultures of selected pasture legumes listed in Table 4 were established in 2016 and 2017 (Experiment 1) at the Charles Sturt University research farm in Wagga Wagga, NSW, Australia (35.04◦ S, 147.36◦ E) on a red sodosol soil [75]. Each planting was arranged as a randomized complete block design with five replications. Individual pasture legumes were established in plots by direct-seeding with a drill adapted for small-seeded legumes in late May to early June of each year, with plots measuring approximately 4 × 20 m. Following on from experimentation to assess legume establishment and performance [75], above-ground plant tissues (leaf, stem, and flower) were collected from established plots in the third week of October in 2016 and 2017 after >50% flowering was achieved in each species. This corresponded to the growth stage in which peak concentrations of phytoestrogen have been reported in vegetative tissue [76]. An additional trial was also established at Charles Sturt University research farm in 2016 and 2017 which focused on chemical composition in two newly released biserrula cultivars (cv. Casbah and Mauro) using four replicates per cultivar (Experiment 2). In this case, composite fresh plant samples were collected from each plot (two sub-samples per cultivar) at pre-bud, pre-bloom, 50% bloom, full bloom, and senescence between mid-July to mid-November in 2016 and 2017, approximately every three weeks. All plant material was placed on ice at collection and subsequently stored at −20 ◦C until extraction.
