The Research On Breeding Of Ogura CMS Fertility Restoration Materials For Brassica Oleracea L. Through Embryo Rescue Technique

Brassica oleracea L.var.capitata L.,known as Cabbage,is an important vegetable crop in China.It is a kind of Cruciferous Brassica species,which has obvious heterosis and its annual cultivation area is reached to 9×10⁵ hm².The ogura cytoplasmic male sterility line is abbreviated as Ogura CMS,whose material manifests as maternal inheritance,pollen abortion,and normal pistil.It is easily recovered by the dominant nuclear restorer gene of restorer materials and facilitates the control of seed sources by breeders.However,it cannot perform normal self-segregation and further innovation of germplasm resources.Since the restorer lines of Ogura CMS have not been found in cole crops,there is an urgent need to carry out transgenesis from other crops containing the Ogura CMS restorer gene.In order to obtain the cabbage material containing the Ogura CMS restorer gene quickly and efficiently,the interspecific crossbreeding of Brassica oleracea L.and Brassica napus L.restorer material was performed in this study.The ovaries and ovules were separately performed embryo rescue through B5 and MS solid media to obtain F₁ hybrid plants.The restorer genes of Brassica napus were transferred to cabbage.Simultaneously,the optimum extraction period,medium addition components,and hybridization combinations for this type of distant hybridization embryo rescue were preliminary selected.The ploidy identification of flow cytometry,SSR molecular markers and morphological identification were used to identify the authenticity of the F₁ hybrid plants.The main results are as follows:1.The 71 strains of interspecific hybrids between Brassica oleracea L.and Brassica napus L.were obtained,of which 67 strains of F₁ hybrids obtained by ovule culture were far more than 4 strains of F₁ hybrids obtained by ovary culture.The best time of ovary culture was 11 days after pollination and 16 days after ovule pollination.The embryo rate was too low for early culture,while the ovule abortion was common for late culture.When the ovary culture was（B5 minimal medium+6-BA 0.5 mg/L+NAA 0.1 mg/L+0.5%CH+0.5%CA）,the seedling emergence rate of ovary culture was best.When the ovule culture was（MS minimal medium+GA 0.1 mg/L+NAA 0.1 mg/L+0.5%CH+0.5%CA）,the seedling emergence rate of ovule culture was best which reached to 5.871%.The addition of appropriate growth regulators,casein and activated charcoal to the culture medium was more conducive to ovule growth and development.4 hybrid strains were obtained from the ovary culture using the cross combination of M14CMS×RFO-46,while 21 hybrid strains were obtained from the ovule culture using the cross combination of M09CMS×RFO-46.The experimental results showed that ovule culture was better than ovary culture in terms of comprehensive performance.2.The ploidy of the 71 hybrids obtained were identified by flow cytometry.The location of horizontal axis corresponding to the peak of fluorescence intensity of 67 strains was between the parents,and the preliminary judgement was that they were true hybrid triploid plants.At the same time,a pair of primer BoE-134 was screened out from 10 pairs of core recovery primers in combination with SSR molecular markers.6%Polyacrylamide gel electrophoresis analysis was performed on parental and hybrid F₁ plants.The result showed that 67 of them amplified parental specific bands and other bands.This indicates that true distant hybrid has the characteristics of back-to-family heritability and intense isolation;The morphological characteristics of the F₁ plants showed super-parental advantages as a whole,with 0.2%colchicine doubled the F₁ plants,the pollen viability was between 11%and 60%,and the fertility recovered.Distant hybridization combined with embryo rescue technology can promote the exchange of excellent genes among species and provide theoretical basis for the creation of new germplasm resources.3.The hybrid breeding technology of Brassica oleracea L.and Brassica napus L.was obtained by combining field distant hybridization technology,embryo rescued in vitro culture technology and identification of hybrid strains.