| Clubroot is a soil-borne disease caused by Plasmodiophora brassicae woronin,which seriously harms the production of cruciferous vegetables.Clubroot-resistant varieties are the first line of defense against clubroot.Free microspore culture technology can quickly create breeding resources,shorten breeding years and improve breeding efficiency.Clubroot-resistant Chinese Cabbage material is difficult to produce embryo or embryo induction rate due to genotype.At present,there are few studies on embryo induction and plant regeneration in microspore culture,in order to improve the regeneration efficiency of microspore cultured plants in Chinese cabbage.To quickly obtain the homozygous breeding resources of Clubroot-resistant Chinese Cabbage,and lay a rich material foundation for the breeding practice of Chinese cabbage heterosis,and improve the breeding efficiency.In this study,five kinds of Clubroot-resistant Chinese Cabbage were used as experimental materials to study the effects of heat shock time,genotype and medium additives on microspore induction rate.The whole pathway of microspore culture of Chinese cabbage and medium were observed.The effects of embryoid regeneration were studied to obtain regenerated plants.The main findings are as follows:1.By studying the effects of different heat shock time on the induction of microspore embryos in Chinese cabbage,the results showed that there were embryoid bodies in the microspores at 12 h,24h and 48 h after heat shock at 32 °C,and the embryo induction rate of heat shock was 2.1 h./ bud,both significantly higher than heat shock 12 h,24h,compared with heat shock 24 h increased by 35.2%,compared with heat shock 12 h increased by 26.2%,so the time for heat shock treatment of Chinese cabbage microspore is 48 h.2.The effects of different genotypes on microspore embryo induction were different.The embryoid bodies were obtained in all five materials,and the embryo induction rate was significantly different.The highest rate of CR3 embryo induction was 3.8 embryo/bud,followed by CR2,CR5,CR4,in turn,was 2.7 embryo/bud,2.5 embryo/bud,1.7 embryo/bud,and the CR1 induction rate was at least 1.2 embryo/bud.Therefore,genotype is the main factor affecting the induction rate of microspore embryos in Chinese cabbage.3.By studying the effect of adding exogenous substances on the induction of microspore embryos,it was found that there were significant differences in the induction rate of microspores in different sucrose concentrations.The average embryo induction rate of the five materials was the highest when adding 100g/L sucrose,which was 2.58 embryos./ bud,the embryo induction rate was increased by 89.7% compared with the addition of 130g/L sucrose,and the embryo induction rate was increased by 186% compared with the addition of 160g/L sucrose.Therefore,the sucrose concentration suitable for the formation of microspore embryos of Chinese cabbage is 100g/L;the ratio of different hormone combinations to the induction rate of microspores is different.The materials CR2,CR4 and CR5 are added with 0.1mg /L6-BA and 0.05 mg /LNAA.The embryo induction rate was the highest when combined,1.7 embryo/bud,1.4 embryo/bud,2.8 embryo/bud,which increased by 11.3%,36.7%,and 18.0%,respectively,compared with the control,while the materials CR1 and CR3 were added 0.1.The embryo induction rate was the highest whenmg/L 6-BA and 0.1 mg/L NAA combination were 1.0 embryo/bud and 3.5 embryo/bud,respectively,which increased by 40.0% and 16.9% compared with the control.Therefore,the hormone combination suitable for microspore embryo induction is 0.1mg/L 6-BA and 0.1mg/L NAA or 0.1mg/L 6-BA and 0.05mg/L NAA,and different proportions of hormones should be selected according to different varieties;4.The addition of a certain concentration of NaC1 in the medium increased the induction rate of microspore embryos.The induction rate of microspores in different concentrations was significantly different.The embryo induction rates of CR2 and CR5 were the highest at 200 mg/L,respectively.The embryo/bud,2.6 embryo/bud increased by 21.7% and 27.5% compared with the control,while the embryo induction rates of CR1,CR3 and CR4 were the highest when added with 150mg/L NaCl,1.1 embryo/bud,respectively.3.2 Embryo/bud,1.6 embryo/bud,increased by 17.5%,37.5%,and 43.3%,respectively,compared with the control.Therefore,the concentration of NaC1 induced by microspore embryos in the medium was 150 mg/L or 200 mg/L NaC1.5.By studying the effects of different solid media on the regeneration of microspores,it was found that the green transgenic rate was 75.99% when transferred to B5 medium,1.84 times higher than that of MS medium;the adventitious bud differentiation rate was 57.33%,1.39 times higher than MS;the callus induction rate was 52.67%,which was 1.63 times higher than MS.Therefore,the basic medium suitable for the regeneration of adventitious buds of Chinese cabbage embryos is B5 medium. |
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