| Clovers(Trifolium L. )are some annual or perennial legumes that can improve soil fertility,increase ground mulch, contral weed growing, keep soil humidity and reduce soil erosion. Caucasian clover had the characters of high yield and stronger resistance to the extreme climate, but the ability of nitrogen fixation was poorer; while white clover had the characters of high yield and stronger ability of nitrogen fixation, but its root systems were shallow and need wet and warm environment. In order to cultivate the new variety of clover, sexploid Caucasian clover introduced from New Zealand and tetraploid white clover grew in Hill Da xinganling in Inner Mongolia were used as parents to conduct interspecific distant hybridization. Caucasian clover and white clover were two species which had great genetic differentiation.In the process of hybridization, the problems of incompatibility, hybrid embryo abortion, and hybrid sterility were met. These problems could be overcome efficiently by the means of hybrid embryo tissue culture then backcross. On the bases of older generations′researches for the characters of Caucasian clover and white clover, the means of tissue culture, cytology and isozyme maker were used to study the technology of tissue culture of hybrid embryo from Caucasian clover×white clover. The main results were as follows:1. The karyotype formula of Caucasian clover was 2n=6x=48=44m+4sm, belonged to"2B"type; the karyotype formula of white clover was 2n=4x=32=22m+10sm(2Sat); belonged to"2A"type.2. The regeneration ability of stems was higher than that of leaves. MS+2,4-D0.25㎎/L +6BA0.5㎎/L+3% sucrose, MS+2,4-D0.2㎎/L+6BA1.0㎎/L+2% sucrose, MS+2,4-D 0.25㎎/L+6BA 1.0㎎/L were better for callus induction, proliferation and regeneration of Caucasian clover, and the rate of regeneration reached 75%; the completed white clover plantlet was obtained on the MB medium with 2,4-D2.0㎎/L+6BA 0.75㎎/L.3. When the Caucasian clover was female parent, white clove was male parent, pollinated at 8:00~9:00, and the embryo was picked after 12~13d from pollination, the hybrid embryo was sterilized by NaClO(10%)for 6.5~7min,then cultured on eight improved MS media, the highest germination rate(8%)could be obtained. The higher frequency propagation was got on the media added 2,4-D0.25㎎/L and 6BA0.50㎎/L .4. The stems of hybrid F1 were transplanted on media with 6BA2mg/L and 2,4-D0.25mg/L in the first step, then they were transplanted on media with 6BA2mg/L and NAA 0.5mg/L after 10 days. Their callus grew faster and the quality was better on the later media. The best medium for roots and leaves was added with 6BA2mg/L and NAA 0.5mg/L.5. The isoenzyme counts of the POD zymogram of callus of hybrid F1 was between Caucasian clover and white clover, two basic bands of parents were inherited, the phenotype of zymogram was partial to female parent, that means F1 possessed a distinguished band and most of isoenzyme were stronger. The study on effection of hormone combinations for the zymogram showed the variation of hybrid F1 POD zynogram was effected strongly by NAA.6. The GS of Caucasian clover and white clover was 0.3333, GS of F1 with female parent and male parent were 0.6667 and 0.5455 respectively. F1 was testified a true hybrid descendant on molecule level. |
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