Effects Of Antioxidants On Isolated Microspore Culture Embryogenesis And Ploidy Identifications Of Regeneration Plants In Non-heading Chinese Cabbage

The technique of isolated microspore culture utilizes the pluripotency of cells.Under sterile environmental conditions,the original gametophytic pathway is changed by extrusion and grinding,temperature,laser and other stress treatments,so that it turns to the sporophytic development pathway and develops into a whole plant by induction and culture.In the non-heading Chinese cabbage breeding,the traditional breeding method takes 6～8 years to obtain homozygous plants,and the microspore culture technology only takes 1 to 2 years to obtain a large number of double haploid plants,greatly reducing the breeding time and cost.In this study,the factors influencing the isolated microspore culture of non-heading Chinese cabbage and the identification of microspores regenerated plant culture and ploidy level were investigated,and the aim is to improve the incidence of free microspore embryos,simplify microspore culture techniques,improve the efficiency of homozygous plants of non-heading Chinese cabbage,obtain homozygous materials,and promote the breeding of new varieties.The main findings are as follows:(1)The effects of genotypes on embryogenesis of non-heading Chinese cabbage were studied by using 14 non-heading Chinese cabbages with different genotypes.The results showed that all 14 cultivars obtained embryoids,and the rate of embryogenesis reached 100%,The highest rate of embryogenesis was ’Kuaicai’,with an average of 26.8 embryos/mL,followed by ’NHCC-224’,with an average of 12.9 embryos/mL;and ’NHCC-063’ has the lowest rate of embryogenesis,only 0.2 embryos/mL.(2)According to the embryogenesis,three varieties with different rates of embryogenesis,’NHCC-224’,’Kuaicai’ and ’NHCC-068’,were selected to explore the effects of ascorbic acid,different ratios of ascorbic acid and 6-BA,sodium ascorbate on the rate of microspores embryogenesis.Ascorbic acid with a concentration of 0-20 mg/L can increase the embryogenic rate of microspores,and inhibit embryogenesis when the concentration is higher than 20 mg/L.Adding 6-BA and ascorbic acid at the same time is better than using either one alone,but there are differences in different genotypes;0-0.2 μmol/L of sodium ascorbate can increase the rate of embryogenesis,and 0.2 μmol/L inhibits embryogenesis.(3)The embryoids obtained from different microspore cultures were transferred on solid medium to obtain regenerated plants.The regenerated plants of ’Suzhouqing × Aijiaohuang’had the highest survival rate,up to 87.27%.(4)The ploidy level of the regenerated plants was identified by morphological identification and flow cytometry analysis.It was found that with the increase of ploidy level,the plant growth was enhanced,the leaves became larger,and the inflorescence became longer.Haploid flower buds are small and not full,without pollen.The ploidy identification of all regenerated plants revealed that there were haploid,diploid,tetraploid and chimera,and the highest rate of diploid was 92%for ’Suzhouqing × Aijiaohuang’,while the doubling rate of ’Wuyueman’ is the lowest,only 40%,chimeras appear in ’Suzhouqing × Aijiaohuang’,’Wuyueman’ and ’Dafengcai’.The overall spontaneous doubling rate is high.