Composition and Stability of Leaf Condensed Tannins in White Clover ( Trifolium repens L) Expressing the Transcription Factor TaMYB14 ‐1 From Trifolium arvense

Condensed tannins (CTs) are phenolic metabolites that reduce rumen protein degradation, increase livestock productivity, prevent bloat and lower greenhouse gas emissions and nitrogen pollution of waterways. White clover ( Trifolium repens ), a protein‐rich forage legume, accumulates CTs in flowers, but not in leaves; however, foliar CTs can be induced through expression of TaMYB14‐1 , a MYB transcription factor from Trifolium arvense and a regulator of CT biosynthesis. This study examined the concentration, composition and in vitro protein binding capacity of white clover leaf CTs across successive breeding generations to establish the stability and inheritance of the trait. Total CT concentration was stable in heterozygous genotypes in the first two generations but increased significantly in later generations in response to selection for TaMYB14‐1 homozygosity. Importantly, the key CT structural attributes that determine the biological efficacy of CTs in the rumen, including the prodelphinidin:procyanidin ratio (~80:20) and mean degree of polymerisation (~9–12 units), remained stable across generations. Protein precipitation capacity (PPC) was numerically higher in advanced breeding lines compared to the primary transgenic plant, reflecting an increase in CT accumulation that coincides with moving from heterozygosity to homozygosity. Together these findings demonstrate that the white clover leaf CT trait is heritable and stable across generations. This supports its potential for incorporation into white clover breeding programmes aimed at improving forage quality and reducing the impact of livestock on greenhouse gas emissions.