Development of two protocols for Agrobacterium-mediated transformation of white clover (Trifolium repens) via the...

White clover (Trifolium repens) is one of the most widely cultivated livestock forage plants whose persistence is severely affected by abiotic stresses. For the white clover, efficient regeneration systems is still a great necessity. In this study, 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 induction rate. Roots and cotyledons proved to be better explants, followed by hypocotyls, leaves, and petioles for callus induction. The development of differentiated structures occurred effectively on MS supplemented with 1 mg·L^-1 6-BA and 0.1 mg·L^-1 NAA. To increase transformation, we investigated various factors affecting the Agrobacterium tumefaciens transformation in white clover. The optimal conditions for root-derived callus and 4-day-old cotyledons were as follows: Agrobacterium suspension density with OD600 of 0.5, 20 mg·L^-1 AS, and 4 days of co-cultivation duration. Subsequently, we developed two transformation protocols: transformation after callus induction from 4-day-old roots (Protocol A) and transformation before initiation of callus from 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 report the possibility to regenerate multiple transgenic white clover plants from a single genetic background. Our research may also contribute to successful genetic manipulation and genome editing in white clover.

The online version contains supplementary material available at 10.1007/s13205-023-03591-2.