ABSTRACT: Background. Ovary culture has been a useful way to generate double haploid (DH) plant in cucumber (Cucumis sativus L.). However, the rate of embryo induction and the ability for induced embryo to grow into normal embryo are quite low. Moreover, the s mechanism of cucumber embryogenesis remains ambiguous. In this study, the molecular basis for cucumber embryogenesis was explored to set up basis for a more efficient ovary culture method. Differentially expressed genes during embryogenesis process, including the early stages of embryo formation, embryo maturation and shoot formation, were investigated using transcriptomic sequencing. Methods. Based on the cytological observation of cucumber ovary culture, the ovary culture can be divided into three stages:early embryo development, embryo maturation (from pre-embryos to cotyledon embryos) and the shoot formation stage. six key time points were selected for transcriptome sequencing and analysis. Results. We firstly conducted cytological observations which suggest that cell enlargement is the symbol for gametophytes to switch to sporophyte development pathway during early embryogenesis stage. In this stage, RNA-seq revealed 3468 up-regulated genes, including hormone signal transduction genes, hormone response genes and stress-induced genes. The reported embryogenesis-related genes BBM, HSP90 and AGL were also actively expressed during this stage. The total 480 genes that function in protein complex binding, microtubule binding, tetrapyrrole binding, tubulin binding and other microtubule activities were continuously up-regulated during the embryo maturation stage, indicating that the cytoskeleton structure was continuously being built and maintained by the action of microtubule-binding proteins and enzyme modification during embryo development. In shoot formation stage, 1383 genes were up-regulated, which were mainly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, phenylalanine metabolism, and starch and sucrose metabolism. The shoot formation stage might be regulated by 6 transcription factors that contained a B3 domain, 9 genes in the AP2/ERF family and 2 genes encoded WUS homologous domain proteins. Conclusions. These findings offer a valuable framework for explaining the transcriptional regulatory mechanism underlying embryogenesis during cucumber ovary culture.
