Two Agrobacterium-mediated Transformation Protocols of White Clover (Trifolium Repens) Through the Callus System

Abstract White clover ( Trifolium repens ) is one of the most widely cultivated livestock forage legumes co-cultivated worldwide with pasture grass in a mixed-sward setting, however, its persistence and aesthetic quality are severely affected by abiotic stresses. In this study, regeneration of white clover plants was conducted through a callus system for 4-5 months with a regeneration frequency of 36-41%. Inoculating 4-day-old cotyledons into MS media fortified with 0.4 mg·L -1 6-BA and 2 mg·L -1 2,4-D significantly increased the callus formation rate. Roots and cotyledons were better induced, followed by hypocotyls, leaves, and petioles. The development of differentiated structures performed effectively on MS supplemented with 1 mg·L -1 6-BA and 0.1 mg·L -1 NAA. Further, we determined factors affecting the Agrobacterium tumefaciens -mediated transient transformation for root-derived callus and 4-day-old cotyledons. The parameters that facilitated transient transformation were: Agrobacterium suspension density of 0.5 (OD600), 20 mg·L -1 AS, and 4-days co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus formation in root segments (Protocol A) and transformation before callus initiation in 4-day-old cotyledons (Protocol B). The transformation frequencies varied from 1.92% to 3.17% in Protocol A and from 2.76% to 3.47% in Protocol B. We offer the possibility to regenerate multiple transgenic white clover from a single genetic background. In addition to assistance in identification of functional genes associated with yield, resistance and aesthetic quality, our research will also contribute to successful genetic manipulation and genome editing in white clover.