Study On The Tissue Culture Regeneration System Of Alfalfa(Medicago Sativa L.)

Medicago sativa L. is the most important fodder crop and water and soil conservation plant. The tissue culture regeneration system will set a good base for the application of other technologies and gene transform in the future. By comparing different explants, mediums and many disparate proportions of hormone, a full regeneration system of Medicago sativa L was achieved. At the same time, callus were used as materials to examine their endogenous hormone levels and histological studies .The results are followed:Hypocotyl is the most effective explants, the explants were cultured on SH with 2, 4-D 2.0 mg/L and KT0.025 mg/L to induce callus, after 3 weeks callus were put into the differentiation medium containing KT0.5~1.0mg/L on which the first embryo was visible as early as 5 days. Adding Peptone into the inducing medium can increase the embryo yield. Mature embryos were put into medium without any hormone to grow up, 74% callus produced embryos during the culture. The best medium for rooting is 1/2MS+ IBAl.Omg/L +15g/Lsucrose, on which 90% plants rooted. The full process went though about 100 days.IAA played an important role in callus induction. A lot of somatic embryos accured easily when IPA was in the highest level, IAA, ABA, GA was opposite, and with a high ratio of IPA/IAA. ABA was essential to embryos maturity with a suitable content .In the differentiation stage, adding high KT only increased the content of IPA, whereas reduced the content of IAA, GA and ABA rapidly.Histological studies indicated that there are much difference between embryonic callus and non-embryonic callus. Somatic embryos developed through multicellar proembryo, globular embryo, heart-shape embryo, Cotyledon-shape embryo, torpedo embryo. Somatic embryos can derive from both inside and outside of callus.