| Brassica oleracea var.Acephala is rich in vitamins,mineral elements and some bioactive ingredients,strong cold resistance,which in the cold winter can be used as vegetables,but also can be assembled colorful winter flower beds.The breeding methods of kale are mainly seed breeding,which is one of the most significant crops with heterosis,so it is more important to get pure lines.Free microspore technology is one of the fast ways to obtain pure line inbred lines.Therefore,increasing the embryo rate of free microspores and improving the direct regeneration rate of embryoid bodies can shorten the time of homozygous inbred lines and improve breeding effectiveness.Clones can maintain the excellent traits of the varieties.The construction of the regeneration system of the cabbage in vitro is an effective method to expand the propagation of free microspore plants.In this study,homozygous breeding varieties obtained from free microspore culture were used as test materials.Efficient and stable regeneration system,access to a large number of homozygous clones.In this study,19 factors affecting embryogenesis were analyzed in 19 subspecies cabbage,including genotype,developmental period,sucrose concentration,low temperature pretreatment and high temperature heat shock.The effects of embryos on the direct regeneration of shoots were discussed,how to obtain robust plants,and explored the factors of bud induction,and established a highly efficient system for the regeneration of microspore plants.1.In the process of microspore induction of kale,12 of the 19 materials were able to obtain embryoid bodies.Among them,the highest rate of emission is "red peacock",the lowest is "6BZ",which "42 red","42 white","edible","DHZ","DFZ","black wrinkle-powder"," D10×6BZ " 7 genotypes were not obtained by repeated experiments.Therefore,this study suggests that genotype is the main factor affecting the embryogenesis of microspore in kale.2.The size of buds can reflect the development process of microspores,but there are great differences between different genotype materials and microspore development period.When the length of the cabbage cabbage was 2.51～4.00mm,the free microspores were in the early stage of mononuclear and single nucleus late and double nuclei.At this time,the free microspores had higher embryo induction rate.3.The free microspores of different genotypes had different requirements on the treatment of low temperature.On the whole,the genotypes were raised at 4℃ for 24-48h before microspore culture,Over time will inhibit the induction rate of microspores.High temperature heat shock at 33℃ heat shock 24-48h on the cabbage microspore embryoid embryo embryo formation rate has a certain role in promoting.The sucrose concentration of different genotypes was about 130g·L-1 and 160 g·L-1 for the microspore culture.4.Pretreatment of cotyledon embryos at 4℃for 4 days,then transferred to solid medium,more conducive to direct reproduction of kaleidae embryoid plants.Compared with embryos of other developmental stages,the success rate of cotyledon embryoid induction was higher,MS was the most suitable medium for direct regeneration of cotyledon progeny,and it was easier to reproduce directly.The optimal concentration of hormone in the regeneration of cabbage was in the range of 0.2 mg·L-1 6-BA + 0.05 mg·L-1 NAA.The addition of appropriate concentration of NAA in 1/2MS medium had a significant effect on the growth of microspore plants and their rooting.NAA concentration of 0.05 mg·L-1 and 0.1 mg·L-1 is conducive to rapid and efficient cultivation of robust microspore plants.5.In the process of bud induction regeneration,axillary buds are the ideal regenerated explants.Adding a certain concentration of 6-BA to the medium is helpful for the growth of regenerated shoots.The hormone ratio is MS + 1.0 mg·L-1 6-BA + 0.05 mg·L-1 NAA is most suitable for inducing axillary bud regeneration.6.The addition of 6-BA in the culture medium was helpful to the subculture of regenerated shoots.When the concentration of 6-BA was 0.2 mg·L-1,the subculture was the best.When the concentration of NAA was 0.2 mg L-1,the bud growth potential was the best,the leaf color was green,the leaf area was the largest,the stems were stout,and the growth was strong.7.The root length of 0.5～1.5cm was the most suitable for the transplanting of regenerated plants.The mortality rate of later seedlings was lower and the plant growth status was the best. |
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