ABSTRACT
Summary The objective of this research was to hybridize long-lived perennial Trifolium species with the agronomically desirable forage species red clover (Trifolium pratense L.). Numerous previous attempts to hybridize red clover with perennial Trifolium species using conventional crossing procedures have been unsuccessful. In the present research, in-vitro embryo culture methods were used to facilitate the recovery and growth of hybrid embryos prior to in-situ embryo abortion. The major effort was directed toward the cross of T. sarosiense Hazsl. with red clover. Preliminary efforts have been made to cross zigzag clover (T. medium L.) and T. alpestre L. with red clover. Crosses were made with the perennial species serving as female parents. Embryos were dissected aseptically from pollinated florets at 12-19 days after the crosses were made. The immature embryos, principally at heart stage, were cultured on agarsolidified nutrient media modified from that developed previously for tissue and cell cultures of red clover. The initial culture period was 8-14 days on a medium containing a high concentration of sucrose, a moderate level of auxin, and a low cytokinin activity. The high level of sucrose prevented precocious germination of radicles from the embryos, and the levels of growth regulators used allowed for the maturation of the embryos. Embryos that survived this culture treatment were transferred to a medium with low auxin and moderate cytokinin levels, which encouraged shoot germination. Hybrid shoot numbers were increased on a low-auxin, high-cytokinin medium and were subsequently rooted before transfer to soil in the greenhouse. Numerous hybrid plants have been obtained from the cross T. sarosiense x T. pratense. About 10% of the cultured embryos were rescued on the optimal culture sequence. Five F1 lines of the hybrid were grown successfully to maturity. Rescued plants were verified to be hybrid on the basis of cytological and genetical evidence. Preliminary efforts with the crosses of T. medium and T. alpestre with red clover have produced a few plants using the same methods, but verifications have not been performed. The embryo rescue procedures utilized in these studies have produced the first successful, verified cross of a perennial Trifolium species with red clover. These procedures should be useful for producing other interspecific hybrids of red clover. The transfer of genes for the perennial character to red clover from interspecific hybrids would be of great value in efficient production of red clover by improving field persistence.
