Fundamental Studies On Somatic Fusion Of Alfalfa And Sweet Clover

There are abundant and various vita min as well as inorganic salt in the Medicago, besides the ratio of protein weight to dry matter weigh is about 17%-20%. The Melilotus suaveolen are strongly resistant to the drought, cold, salt-alkali, and leanness. Thus, the basic researching reference for expecting a new forage cultivar characterized by both Medicago, high nutritional value, and Melilotus suaveolen, strong stress resistance, could be accumulated by researching tissue culture either of Medicago and Melilotus suaveolen, also which provided the prerequisite to their somatic fusion each other. This presentation has studied the founding of regeneration system of four Medicago cultivar (Medicago varia cv. Gannong No.1, M. sativa cv. Ameristand, M. sativa cv. WL324 and M. sativa cv. Sanditi), through somatic embryo, and observation of somatic embryo cytological structure in Gannong No.1, in addition, also the effects of basic medium, culture cycle and exogenous plant hormone on inducing callus of Melilotus albus have been investigated individually, using the cotyledon of Gannong No.1 and Melilotus albus as the material for isolating protoplasts, and then cultured, all have been conducted in our work. The research details and results as below:1. Throughout screening the best appropriate conditions of inducing Medicago callus from culture cycle, explants, hormones and light intensity etc., we had attained a whole regeneration system by vitro tissue culture, additionally the embryo cytological observation had been carried out. The results showed: (1) For the Gangnong No.1, the most optimum is 14d of culture cycle, hypocotyl as explant, 2,4-D (2.0 mg/L) + KT (0.2 mg/L) as the combination and concentration of the hormones; although the light practically was thought of the unavailability for callus inducing ratio, all with 100%, under alternate lighting and dark condition the callus will increasely thrive and grow rapidly, while the color is green or green and yellow stripes, but the browning ratio a little bit higher. (2) After transport the callus onto the somatic embryogenesis medium, it was found that, 4 Medicago cultivars callus induced under alternate lighting and dark are much easier to form somatic embryos, the highest ratio, among 20.83%～56.25%, appeared in Gangnong No. 1, the next was Ameristand, Sanditi and WL324 in order of ratio; the best somatic embryogenesis medium is UM + NAA (0.1mg/L) + KT (0.5mg/L), with 56.25% of embryogenetic ratio; adding appropriate PEG6000 can facilitate embryogenesis, and the optimal concentration is 6% PEG6000. (3) The cytological observation indicated that before embryogenesis, there had to go through four stages, globular embryo, heart-shape embryo, torpedo embryo and cotyledon-shape embryo stage.2. This experiment had worked over the effect of basic media, explants, culture cycle and plant growth regulators on inducing callus of Melilotus albus Results showed that there was the highest inducing callus ratio, when cultured in UM basic medium, on usage of hypocotyl as the explant from which callus could thrived most in three explants, and after 21 days, furthermore, as the concentration of 2,4-D and KT increased, the number of formed calli became more and more firm and dry, callus ratios of cotyledon and hypocotyl declined gradually, when that of radicle was increased along with the increase of KT concentration and then reduced.3. The cotyledons of Medicago varia Gannong No.1 and Melilotus albus were selected as the material for isolation protoplasts, and then cultured. The best enzyme solution for Gannong No.1 protoplasts isolation was comprised of 2.5% (Cellulase Onozuka R-10) + 0.5% (Macerozyme R-10), and the highest yield, 46.75×106/g, was obtained after 12h treated. As far as Melilotus albus, the enzyme solution contained 2.0% (Cellulase Onozuka R-10) + 0.5% (Macerozyme R-10) + 0.5% (Pectinase), and after 24h-treated the highest yield reached 5.00×106/g. Both of Medicago varia and Melilotus albus protoplasts were cultured in KM8P medium supplied with 2,4-D (1.5mg/L) + KT (0.2 mg/L), and some tiny callus saw with naked eyes appeared after about 2 month culture.