Study On Expanding Propagation And Identification Of F₁ In Vitro Culture Of Hybrid Embryo Of Caucasian Clover × White Clover

One of the key facters in Inner Mongolia was lack of nitrogen and supply of protein, which limited the development animal industry and land improvement. Caucasian clover had the characters of stronger resistance to the extreme climate, but the ability of nitrogen fixation was poorer, while white clover had the characters of stronger ability of nitrogen fixation, good quality and higher content of protein. In order to cultivate the new kinds of clover, which had the good characters of two kinds of clovers, 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. In the process of hybridization, the problem of hybrid embryo abortion was met. So this problem could be overcome efficiently by the means of hybrid embryo tissue culture. On the bases of older researches for hybrid of Caucasian clover and white clover, adventitious shoots were induced directly from the stems taken from the aseptic seedling of F1 in vitro culture which was crossed between the embryos of Caucasian clover and the embryos of white clover. The explants were cultured on proper media. After few generations of subculture, the callus from stems of F1 was transferred into differentiation medium. The study showed that somatic embryogenesis of F1 was affected by different concentrations of hormone. The means of cytology and isozyme maker were used to study the technology of tissue culture of hybrid F1 from Caucasian clover×white clover,which can be used as genetic marker to identify hybrid. The main results were as follows:1. MS+6-BA(2mg/L)+NAA(0.8mg/L)were the suitable medium induced the callus from stems of F1. MS +2,4-D(0.25mg/L)+6-BA(0.5mg/L)+NAA(0.25mg/L)+KT(0.5mg/L)+CH(250mg/L) was the suitable differentiation media for the callus from stems of F1.2. Histological observation was performed on embryogenic callus after differentiation. It was found that tubercular structure were formed on the surface of callus; they were embryogenic cell masses which can further developed and formed the somatic embryo.3. MS +2,4-D(0.25mg/L)+6-BA(2mg/L)and MS +2,4-D(0.1mg/L)+6-BA(2mg/L)were the suitable medium induced shoots from stems of F1.4. The chromosome number of hybrid F1 was 40, and hybrid F1 is pentaploid (2n=5x=40).5. The isoenzyme counts of the POD zymogram of hybrid F1 was the same as white clover, two basic bands of parents were inherited, the phenotype of zymogram was partial to female parent, that means F1 possessed two distinguished band and most of isoenzyme were stronger band.6. The GS of Caucasian clover and white clover was 0.3077, GS of hybrid F1 with female parent and male parent were 0.6154 and 0.4615 respectively. F1 was testified a true hybrid descendant on molecule level.