| Cucumber(Cucumis sativus L.)is a kind of Cucurbitaceae which has important economic value.It has large planting area and wide distribution.Cucumber is a cross-pollinating plant,which has obvious heterosis.The varieties used in production are almost all hybrids.However,the process of hybrid breeding is relatively long,due to the extensive purification time of parents.It takes 5-8 generations to develop a stable inbred line by artificial self-crossing.In order to accelerate the purification of cucumber parents and improve the breeding efficiency,double haploid(DH)is effective way to obtain homozygous DH in 1-2 years,and haploid has significant value in genetic research.However,the lack of effective regeneration technology,the mechanism of embryogenesis is unclear and the regeneration rate is low,which disrupts the application of this technique in cucumber.In this paper,30 cucumber cultivars of North China type,South China type and Greenhouse type in Europe were used as materials for ovary culture,and genotypes that could regenerate plants were screened.Genotype effect,pretreatment time at 4℃ and TDZ concentration were studied.Combining with high temperature heat shock culture at 33℃ for 2 days,an effective method of ovary culture directly forming plants was discussed.The ploidy analysis was performed by counting the chloroplast number of guard cells and by flow cytometry.The haploid plantlets were doubled by 0.1%colchicine to study the optimal treatment time.SSR molecular markers and field character observation were used to determine the homozygosity of the next generation of diploid from ovary culture.An efficient ovary culture technology system was set up.At the same time,the process of embryogenesis induced by in vitro gynogenesis in cucumber was studied.On this basis,six time points of the samples were subjected to transcriptome sequencing at the early stage of embryo development,embryo maturation stage and shoot formation stage,which laid an important theoretical and technical foundation for the application of the haploid production technology of the cucumber in breeding practice.The results of the study are as follows:1.The DH induction system of cucumber ovary culture was establishedCucumber ovaries were pretreated at 4 C for 4 days,and a medium(MS+0.06 mg.L-1 TDZ+sucrose 3%+agar 7%)was carried out.The plant regeneration was induced by dark heat shock at 33℃ for 2 days and then directly induced by light at 25℃ for 16 h and dark for 8 h).The chromosome ploidy level was identified by flow cytometry and counting chloroplast number of guard cells,the diploid plants were identified as DH by SSR markers with a natural doubling rate of 44%.The haploid shoot tip was treated with 0.1%colchicine for 1 h,and the doubling rate was 72.5%.Ovaries were pretreated low temperature,a medium combined with high temperature heat shock culture were used to culture 30 cucumber varieties.60%of the genotypes were successful and the highest regeneration rate was 73.3%.Combining with effective ploidy identification,chromosome doubling and SSR molecular marker technology,a technical system of cucumber double haploid induction was constructed.2.The process of embryogenesis in vitro gynogenesis of cucumber ovary culture was preliminarily revealedMegasporogenesis of cucumber belongs to the monosporal pattern and the embryo sac belongs to the common polygonum-type embryo sac.There were two nuclei,four nuclei and eight nuclei at the same time.The ratio of mature embryo sac was 15%,45%,85%,respectively in the ovary at 2 d,1 d and 6 h before anthesis.The corresponding ovary petals were green,green yellow and yellow.The embryo rate of ovary culture reached 92%at 6 h before anthesis.The color of ovary petals was closely related to the development of the embryo sac,which could be used as a material selection index for ovary culture.The embryoid of cucumber ovary culture is mainly derived from the synergid and the central cell,which reveals the cytological mechanism of embryogenesis induced by ovary culture of cucumber.3.Several key genes of regulating gynogenesis were identified preliminaryIn the early stage of embryonic development,the enlargement of the embryo sac was a sign of obtaining embryonic potential and transforming gametophyte development pathway into sporophyte development pathway.3468 up-regulated genes were identified by RNA-seq,including hormone signal transduction,hormone response and stress-induced genes,and the reported embryogenesis-related genes BBM,HSP90 and AGL15 were also up expressed at this stage.At the stage of embryo maturation,480 differentially expressed genes were continuously expressed.These genes were mostly involved in microtubule movement,cell or subcellular biological processes.They could promote protein complex binding,microtubule binding,tetrapyrrole binding,microtubule binding and other microtubule active molecular functions,mainly distributed in microtubules,supramolecular fiber and polymerized cytoskeleton fiber cell components.These results suggest that microtubule genes continued to expression during embryo development,maintained the cytoskeleton structure and participated in embryo morphogenesis.In the shoot formation stage,1383 genes were up-regulated,mainly in phenylalanine biosynthesis,plant hormone signal transduction,phenylalanine metabolism,starch and sucrose metabolism,and so on.The shoot formation might be regulated by six transcription factors encoding B3 domain,nine AP2/ERF family genes and two WUS homologous domain protein genes. |
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