High Efficiency Cultivation Of Refractory Material In Microspores And Polymorphism Analysis Of Doubled Haploid Plants (Brassica Oleracea L.)

This study makes further discussion and optimization of the microspore culture’s influence factors for embryo rate in the process,the establishment of regeneration system of Brassica DH root,the SSR analyzed by DH group,construction of DNA fingerprint database of DH system and provide reference for cabbage varieties such as specificity,authenticity,genetic relationship and hybridization.This experiment mainly studied the effect induceding microspore culture medium with the different concentrations of PEG4000,PEG6000,DMSO and four carbon sources on cabbage about embryo.The effects of different culture medium,the ratio of 6-BA and NAA and the root age on the regeneration of root segment of Brassica oleracea DH were studied and the difference of the regeneration ability of different genotypes was analyzed.1500 pairs of primers were used to screen primers with specificity and stability.The genetic stability and diversity of DH plants from the same mother plant were analyzed,and the coefficient of variation of cabbage DH was investigated in the field.For the refractory material,the single bud culture system of Brassica oleracea was established through the study of microspore culture process,and the feasibility and development prospect of single bud culture of cabbage were discussed.The main conclusions are as follows:1.Adding different concentrations of PEG4000,PEG6000,DMSO and different carbon sources in the NLN medium of the microspore culture of Brassica oleracea can significantly increase the expansion ratio and the embryo rate of the microspore in the cabbage,and the effect of induction is significant difference between the three kinds of materials.X17-4material in the processing of 0.0125M PEG4000 reached the highest induction rate of 12.9embryo/bud.Materials 438 added 0.2%DMSO can reach the maximum embryo rate of 13.5embryo/bud.Material X17-4 reached the maximum embryo rate of 15.2 embryo/bud in0.2M sucrose+0.2M sorbitol.2.The cabbage root sections were an effective propagation mode of DH explants propagation.The root explants of 20d root age can reach the best culture effect on MS+0.030mg/L NAA+4.5 mg/L6-BA+6mg/L AgNO₃ culture medium.The induction rate of callus and the induction rate of adventitious buds are up to 96%and 82%.The results showed that there was a significant difference in the induction of root differentiation between three DH genotypes of cabbage,and cabbage genotype was a major factor affecting root regeneration.3.26 primers for specific amplification were screened from 1500 primers of near species,and 106 bands were obtained by using these 26 pairs of primers to amplify 47 microspore plants’DNA from one mother plant,of which 43 bands were polymorphic,percentage of polymorphic bands was 41%.Each primer produced 4.07 bands and 1.72 polymorphic bands.The DH plants from one mother plant have good genetic stability and diversity,which are consistent with the results of field traits investigation.DH with the maternal source of microspore plant has a good genetic stability and genetic diversity,the findings and actual field characters.DH plants public exhibition’s coefficient of variation is 10.54%,the coefficient of variation of plant height is 11.49%,the coefficient of variation of external leaf number is 15.84%.4.It was observed that the single buds’microspore development can develop into embryos through intact embryonic development pathway in small petri dishes.Material 438 by single bud culture get the embryo rate of 10 embryo/bud,single bud of cabbage microspore culture is a new way to obtain embryoid more effective and feasible.