Establishment Of Regeneration System To Radiated Callus Of Trifolium Pratense Cv. Minshan

Via the cytological observation and establishment regeneration to radiated callus of Trifolium pratense cv. Minshan, the following results were gained.Sterilizing effect was different with different concentrations of sodium hypochlorite applied. The best sterilizing effect was obtained when the concentration of sodium hypochlorite was 2.0% and sterilizing time was 15 minutes. Under this condition, contamination rate was under 2.5% and had no effect on seed germination rate.The sucrose concentration was negative correlated with seed germination rate of Trifolium pratense cv. Minshan. While the sucrose concentration reduced from 20g/L to 0g/L, seed germination rate increased from 16% to 47% and seed germination time was uniform. This solved the problem of lower seed germination rate of Trifolium pratense cv. Minshan sown on the MS+20g/L sucrose medium.The establishment to regeneration system of Trifolium pratense cv. Minshan showed that, the optimal callus induction medium was MS+2 mg/L 2, 4-D+0.5 mg/L 6-BA+2 % sucrose+0.6 % agar,Using fast neutron to radiat the induced callus. Radiation dose inhibited the growth of callus significant. the half of callus died under 0.1032Gy absorbed dose of neutron + 0.1700Gy absorbed of photons that is the optimum radiation dose. Radiated callus appeared lots of mutation. Such as micronuclear, nuclear budding, small nuclear.double nuclear, and multinuclear. the best medium of bud induction medium after radiated callus was B5+0.06 mg/L NAA+2% sucrose+0.6% agar. In each bottle, 2~10 stems grown, and after transferred them into root induction medium, buds could continually grown. The optimal root induction medium was 1/2 MS+ 0.05mg/L NAA + 1.5% sucrose +0.8% agar.Inner structure of callus showed that, somatic embryos of Trifolium pratense cv. Minshan occurred in the callus and on the surface of it. Embryonic and non-embryonic callus had huge differences in histological and cytological. The development of somatic embryos experienced several stages such as multi-cellar pro-embryo, globular embryo, heart-shape embryo, cotyledon-shape embryo and torpedo embryo. Near the bottom of medium, angle-shaped cells which was 5～12 times longer than somatic cells were observed.