In this study, T medium (zigzag clover, 2n = 80 and T. pratense (red clover, 2n = 28) were hybridized and backcrossed four times to T. pratense (BC1, BC2, BC3 and BC4), and the seed production and vigor of the backcross progenies were assessed under field conditions. Both female and pollen fertility of the backcross progenies increased as the backcross generations advanced. Female and pollen fertility of BC4 were 21.3 and 65.3%, respectively. When honeybees were used as pollinators, almost all BC4 Plants produced mature seeds in the field. Most of the BC1, BC2, and BC3, plants died by the end of the fourth year in the field, whereas 61% of the BC4 plants survived for 4 yr. The number of chromosomes in 79% of the BC4 plants was more than 28, which is greater than that of T. pratense. Differences in leaflet size and shape between BC4 plants and T. pratense suggest that genes from T. medium were expressed in the BC4 plants.
Theses results indicate that BC4 plants may be useful for the improvement of the persistence of T. pratense.