Research On Haploid Induce Via In Vitro Unpollinated Ovary Culture Of Cucumber

Cucumber(Cucumis sativus L.)is an important annual Cucurbitaceae vegetable crop,widely cultivated in the world.Cucumber has a narrow genetic basis and lack of germplasm resources.The F1 generation is widely used in production due to its significant heterosis,but the process of obtaining homozygous parents using conventional breeding methods is long.Obtaining genotype homozygous double haploid(DH)by doubling the haploid chromosome can shorten the breeding time.In vitro culture of unpollinated ovary is the main way to obtain double haploid of cucumber.To establish an efficient and stable culture system,in this paper,the unpollinated ovary of F1 generation cucumbers with different genotypes was used as the donor material to conduct in vitro culture of unpollinated ovary.In vitro culture,the effects of various factors on the in vitro culture of unpollinated ovary of cucumber were discussed,and regenerated plants were obtained.The ploidy of the obtained plants was analyzed by flow cytometry and chromosome count,and the homozygosity of the plants was identified by SSR markers.The seedling rate and traits of sac regeneration plants were counted.To explore the process and mechanism of embryo sac regenerating plants,paraffin sections were used to observe the development status of embryo sac at different development stages and different parts,and different media were used in the culture.Observation of paraffin sections and determination of hormone content in embryo sac development at different culture times.The results were as follows:1.Study on in vitro culture system of unpollinated ovary of cucumberUsing different genotypes of F1 generation cucumber unpollinated ovary as donor material,the effects of genotype,culture medium,ovary development stage,sectioning method,sampling location and pre-cooling time on ovary culture were investigated.The effect of different genotype materials on ovary culture is significant.The effects of ovary culture under different media are quite different.The initiation rate of female nucleus in ovary slices 1～2 d before flowering was lower than that 3～4 d before flowering.Longitudinal and transverse cuts have no significant effect on the initiation rate of female nucleus.There is no significant difference in the initiation rate of female nucleus in the upper,middle,and lower parts of the same ovary.4℃ pre-cooling time has a significant effect on ovary culture,but the optimal treatment time varies for different genotype materials.In MS+0.04 mg·L-1 TDZ+1.0 mg·L-1 6-BA+0.1 mg·L-1 NAA+12 mg·L-1 AgNO3 medium(T medium),a large number of embryos are produced in the ovary structure,but no regenerated plants were obtained after differentiation culture.In MS+0.06 mg·L-1 TDZ medium(Z medium),three kinds of donor cucumbers were regenerated from unpollinated ovary,and the processes of plant regeneration by embryoid pathway and callus pathway were observed.2.Identification and character observation of regenerated plants in ovary cultureThe identification of regenerated plants includes ploidy identification and homozygosity testing.In this paper,ploidy of regenerated plants was identified by flow cytometry and root tip chromosome counting.Chromosomes can be accurately counted by DAPI staining and FISH probe labeling.In the SSR test,the homogeneity test of regenerated plants was performed based on screening polymorphic markers,which increased the reliability of the results.Regenerated plants were obtained from unpollinated ovary culture of three kinds of donor cucumber.The regenerated plants were verified to be haploid,double haploid,and autotetraploid,respectively.The seedling formation rate of embryo sac regeneration plants was calculated,which ranged from 0.1%to 1%.The leaves,flowers,and fruits of the haploid,double haploid,and autotetraploid transplanted field were compared and observed.The phenotypes of different ploidy plants were different,and it was difficult to obtain self-cross seed of regeneration plant.Some plants should be preserved in tissue culture before domestication and transplanting.3.Observation on the origin of regenerated plants and determination of hormone contentIn this experiment,the formation process and related physiological changes of embryo sac regeneration plants were investigated by paraffin section observation and hormone content determination.Paraffin sections showed that the embryo sac matured differently at different developmental stages of the ovary.On the day of flowering and 2 days before flowering,mature embryo sacs could be observed,embryo sac immature 4 d before flowering and the ovules at 6 d before flowering have not yet formed the integuments and the mature embryo sac.There was no obvious difference in the mature state of the embryo sac in the upper,middle and lower parts of the same ovary.The embryo sac was developed at 5 and 17 d in MS and Z medium.The hormone content was measured for 2,5,8,11,14,and 17 days.It was found that the contents of GA and IAA decreased first and then increased with the number of culture days.It reached the lowest at 8 d,and the GA content at 17 d was higher than that at 2 d,but there was no significant change in IAA contents.