Somatic Embryogenesis, Variation And Their Proteomic Analysis In Cyclamen Persicum Mill.

Cyclamen persicum Mill. is economically significant and well-known pot species in the floriculture market. In this study, biology and proteomics about somatic embryos were studied to research variation and mechanism of somatic embryogenesis.The seedings were used as study materials and inoculated on 1/2MS medium supplemented with different hormones. Embryogenic callus was induced from the tubers of seedlings after culturing and subcultures. The regeneration of somatic embryos in high frequencies was observed after plating the embryogenic callus on hormone-free medium. The results showed that the tubers were better explants and 1/2MS+6-BA 0.2mg/L +2,4-D 2mg/L+sucrose 30g/L+agar 6.5 g/L+ inositolum 100mg/l+ casein 100mg/l was the best culture medium for induction of embryogenic callus.To produce highly uniform plantlets in a short period, suspension cultures in C. persicum Mill. were established. Growth curves for embryogenic and nonembryogenic suspensions were presented. The results showed that the starting density for embryogenic suspension is 8%(PCV), growth cycle was two weeks; growth cycle for nonembryogenic suspensions was related with the starting density.The embryogenic callus and somatic embryos in different stages were used as study materials. It was showed by histological sections that somatic embryos were initiated from a single cell or cell clusters of embryogenic callus. The embryogenic cell produced an embryonic cell and a suspensor cell by an asymmetrical division. Proembryos initiated from single cells had suspensors. Proembryos initiated from cell clusters had no suspensors. Embryos were sequentially differentiated through globular and torpedo-shaped embryo stages.Variation is the common phenomenon of somatic embryos. To observe variation of somatic embryos embryogenic callus was induced from the tubers of seedlings after cultures and subcultures. And the embryogenic callus was used as inocula for establishment of suspension culture system. The regeneration of somatic embryos in high frequencies was observed after plating the cultures from solid and liquid medium on hormone-free medium. According to characters of somatic embryos germinated, 10.9% were normal somatic embryos from solid medium and 6.25% from liquid medium, and the ratio of variation was 89.1% from solid and 93.7% from liquid medium. In addition, histological sections of abnormal somtic embryos wers done. The results showed that the variation should attribute to the abnormal polarity.Somatic embryogenesis is a process by which somatic cells undergo a developmental sequence similar to that seen in zygotic embryos. A comparative histological study of ontogenetic stages was carried out on zygotic and somatic embryos of C. persicum to further understand the development of somatic embryos. The results showed that the early segmentation of the embryo, the organization of the embryonic apex, formation epicotyl, the morphology and shape of the zygotic and somatic embryos of C. persicum at successive stages showed remarkable similarities in spite of the different environments in which they have developed and differentiated. The most striking difference between the zygotic and somatic embryogensis was that the former showed high synchronization, and much endosperm was developed. Moreover, variation of somatic embryos may be related with abnormal polarity from the somatic embryogenic single cell.In this study we studied cryopreservation of C. persicum Mill. callus to avoid variations produced by sub-culture. The callus in the logarithmic phase after sub-culture were used for experiments. Firstly, the callus were pre-cultured in culture-medium containing 4%, 6% or 8% sucrose for different time periods, transferred to different cryoprotectants to directly cryopreserve or incubated for 2 hours at -20°C, then submersed in liquid nitrogen, lastly thawed rapidly in a waterbath at 37°C, and washed with liquid culture-medium containing the corresponding concentration of sucrose. Cell survival rate was computed after stained by Neutral Red, and SPSS 13.0 software was used for statistical analysis. The results showed that sucrose concentration, pre-culture time, cryoprotectants had various impacts on cell survival rate. We have developed a simple but effective protocol for the cryopreservation of callus of C. persicum. Of the different protocols tested, 4% sucrose, pre-culturing for 3 days, NO. 9 cryoprotectant and freezing directly after 30 minutes at 0°C results in the highest cell survival rate.Plantlets regeneration derived from somtic embryos was studied using normal and mature torpedo-shaped somatic embryos as study materials. The results showed that generation ratio was 90%.Though mass production through somatic embryogenesis for cyclamens have been done, there are numerous biological unknowns regarding this complex developmental pathway. To understand the embryo developmental process, we applied a proteomic approach to analyze somatic embryogenesis of cyclamens. Proteins were separated by 2-DE, visualized by CBB staining, analyzed by Imagemaster software and digested with the site-specific protease trypsin. 35 somatic embryogenesis-related proteins could be displayed reproducibly across an isoelectric focussing range of 5–8, 5 proteins of those changed little, 13 proteins were up-regulated, 9 proteins were down- regulated, while 8 proteins were found to increase in torpedo-shaped somatic embryos. 12 protein spots were analyzed by matrixassisted laser desorption/ionization–time of flight mass spectrometry (MALDI-TOF-MS), and 4 of them was identified. Then the others spots were analyzed by MALDI-TOF-TOF-MS and 6 were identified. The results showed that somatic embryogenesis and maturation of somatic embryos were regulated by specific proteins.Proteomic approach was applied for primarily analysis of variation of somatic embryos in C. persicum. The results showed that 6 proteins may be related with differentiation of hypocotyl, 7 proteins may be related with differentiation of radicle and 3 proteins may be related with differentiation of both hypocotyl and radicle. However, it was difficult to ensure veracity of materials, so further study was needed.