| Cucumber（Cucumis sativus L.,2n=14）is a melon crop of Cucurbitaceae.It is a worldwide vegetable with important economic value.Cucumber is a cross-pollinating crop,which has obvious hybrid vigor.The varieties used in production are almost all F1 generation.The process of hybrid breeding is seriously restricted,due to the extensive purification time of homozygous parents.Obtaining genotype homozygous double haploid（DH）by in vitro gynogenesis culture can accelerate the breeding process and improve the breeding efficiency.In vitro culture of non-pollinated ovary is one of the way to obtain double haploid of cucumber,but there are few systematic and comprehensive study.To optimize the system of high frequency embryo induction for unfertilized ovary culture in cucumber,different genotypes were used as the experimental materials to study the factors that affecting the embryogenesis of unfertilized ovary in cucumber and plant regeneration.The ploidy of the regenerated plants was identified by flow cytometry and morphologic observation,and the homozygosity of the callus and embryoid regenerated plants were identified by SSR markers.In order to explore the application of slow growth conservation in vitro of cucumber,3533 was cultured on the base medium(MS+0.2 mg·L-16-BA+0.2 mg·L-1NAA)containing with different concentractions of sucrose and CCC,the height,withering rate and survival rate of regenerated plantlets were investigated.Root striked,domestication,transplanting and agronomic characters for the embryo sac regeneration plants which were studied.SSR marker was used to determine the homozygosity of the next generation of diploid from ovary culture.An efficient and stable ovary culture technology system was set up.The results showed that:1.Study on in vitro culture system of unpollinated ovary of cucumberUsing unfertilized ovaries of cucumber were used as explants,to study the effects of genotype,sowing seasons,cultivation methods,sampling flowering period,ovary development period,concentration of TDZ,time and temperature of heat shock pretreatment on ovary culture were investigated.In the same condition,the frequency of embryogenesis was significantly different among these genotypes.The highest embryo induction rate was SG033,reaching 95.93%,the lowest embryo induction rate was SG035,reaching 8.15%.The embryogenesis ability of hybrids was significantly higher than the weak parents.The effect of different ovary development period on ovary culture is significant.In the treatment of different factor combinations,the embryo induction rate was the highest in treatment number 11(Autumn sowing,planting in plastic greenhouse,sample collected in the period of flowering,unfertilized ovary 1 day before flowering,4℃for 4 days,inoculated on induction medium MS+0.08 mg·L-1TDZ+30 g·L-1sucrose+7 g·L-1agar,heat shock and dark treatment at 35℃for 2 days).The embryogenesis capacity of the weak responsive genotype could be significantly ameliorated with treatment 11 after crossed with the genotype with strong embryogenesis ability.2.Identification and subculture of regenerated plants in ovary cultureThe identification of regenerated plants includes ploidy identification and homozygosity testing.In this paper,ploidy and homozygosity of regenerated plant lines obtained were identified by DNA flow cytometry and SSR.The results demonstrated that the group of regenerated plants was diverse,which included haploid,diploid,triploid and chimera plant types.SSR markers were used to identify the source of diploid regenerated plants.The frequency of DH plant regeneration from embryoid was 100%,and from callus was 20%.The deformed seedlings of cucumber were rescued and prevented.When the GA concentration in MS medium reached 0.9mg·L-1the‘blunt with blossom’phenomenon of regenerated plant could be effectively vanished.97.78%of in vitro shoots conserved on MS supplemented with 0.2 mg·L-16-BA,0.02 mg·L-1NAA,30 g·L-1sucrose and 7 g·L-1agar,added with 200 mg·L-1CCC for 50 d.The lowest rate of etiolation was 15.56%,and the growth of cucumber tissue culture seedlings was good.3.Domestication,transplantation and character observation of regenerated plants in ovary cultureRoots grew faster and stronger when 0.02 mg·L-1NAA was added in the 1/2 MS medium.The plant survival rate of the canned bottle upside down for moisturizing,nutrient solution spraying the next day is higher（98.33%）,and the new leaves grow vigorously.There were significant differences in the performance of individual regenerated plants.Among them,SSR21563 and CS24 primers have good specificity,clear bands and obvious band spacing,which are the primers for the purity identification of cucumber.The next generation of diploid from 2649 ovary culture was identified as a homozygous DH line by SSR markers. |
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