Study On Establishment Of The Plantlet Regeneration System For Red Clover And White Clover By Somatic Embryos

Triflium L.is the most important forage crops plant and turf grass. The tissue culture regeneration system will setαgood base for transform and the application of other technologies in the future. The experimental studied on the process of two clover embryogenic callus'inducing and embryoids'differentiating and regenerations'estalishing, different explant, different combinations and concentrations of hormones and others conditions effected to establish regeneration system, and got a complete set of tissue regeneration system.The materials were Trifolium pratense and four varieties of Trifolium repens, Koala,Persistent,Ladino and Palestine. The cotyledon, upside of hypocotyl, downside of hypocotyl and leaves were used as explants in vitro.Studied the effects of different explants and different treatments concentrated 2,4-D with light concentration 6-BA to the embryonic callus induction and embryoid differentiation in the modified SH basic media. And the suitable inducing time of trefoil different explants was searched. The results showed that the inductivity of embryogenic calli of the upside of hypocotyl and cotyledon were all higher. Nevertheless the downside of hypocotyl was lower. High concentrations of 2,4-D can promote the ratios of induction somatic embryos.The best medium inducing embryonic callus was modified SH+10 mg·L^-12,4-D+0.2 mg·L^-1 6-BA. In this combination ,the results of embryogenic callus induction of red clover and Palestine's upside of hypocotyl were best, the ratios of those two clovers embryogenic callus induction reached 88.5% and 57.9%. And red clover'embryogenic callus induction rates were higher than four varieties of White clover, because white clover'browning was more serious than the red clover significantly. but four varieties white clovers'embryoids differentiation rates were higher than the red clover in differentiation media MSO whitout hormones, and the embyogenic callus had a well-differentiated in differentiation media MSO. The ratios of embyoid differentiation of Persistent, Koala, Ladino ,Palestine and red clover were 45.3% 43.2% 45.7%, 33.7 %,respectively.The best subculture medium inducing callus was modified SH+5 mg·L^-12,4-D+0.5 mg·L^-1 6-BA. The best suitable inducing days of hypocotyl was 60⁶5d. That of cotyledon was 70⁷5d. During the induction of recultural embryogenic calluses reducing the concentration of the 2,4-D properly could promote the rate of embryogenic callus induction and reduce the white clover callus brown. The cycle of subculture should be 25～30d advisable. Using the leaves, stems , petioles from regeneration plant of red clover and white clover Persistent and koala to make for a second callus induction and the results proved: the regeneration of seedlings'different explants had been has the ability of inducing the somatic embryos high stabily and efficiently. Induction rate of Persistent and koala can reach 100%, red clover was lower comparatively, the highest reached 89%. The phenomenon of browning disappeared and the somatic embryogenesis synchrony improved during the second induction.The white clover'inducing time was shorten, especially Persistent ,its regeneration seedlings can differentiate from the callus after 40d.