Four systems of cyclic somatic embryogenesis of Siberian ginseng (Maxim) were
Four systems of cyclic somatic embryogenesis of Siberian ginseng (Maxim) were used to study the mechanism of embryonic cell cluster generation. cDNA library AS 602801 of DEC was constructed, and 1,948 gene clusters were obtained AS 602801 and used Rabbit Polyclonal to RFWD2 as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. and was more highly expressed in DEC than in other lines throughout the culture cycle, and expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that and may be the essential genes that play important roles during the induction of embryogenic cell clusters. Introduction Somatic embryos are embryos derived from somatic cells that are capable of developing into young plantlets via a series of morphological changes, a process that closely resembles that of zygotic embryos . Direct somatic embryogenesis (DSE) is usually a way of developing young plantlets by direct differentiation from explants without an intervening callus phase. During DSE, somatic cells turn into embryogenic cells, and then enter into a differentiation phase. Sometimes, the embryogenic cells do not differentiate directly, but proliferate for a period of time before the proliferated cells start to differentiate. These cells are called the embryogenic cell clusters, and the process is usually considered a type of DSE because of the direct generation of embryogenic cells. Studies of DSE and embryogenic cell cluster formation have been prevalent , , and cell proliferation is usually more conductive to increasing the heritability coefficient. AS 602801 One culture cycle can yield a large number of somatic embryos through embryogenic cell cluster process. Studies identified several genes that play regulatory roles either in specific phases of embryogenesis or during the entire process , , such as ((((Maxim) plantlets have been obtained through somatic embryogenesis using semisolid and suspension cultures both using DSE and ISE , . Siberian ginseng SEs has been successfully produced in bioreactors , . In this study, we compared the cellular structures and molecular mechanisms between four lines of repetitive somatic embryos of Siberian ginseng that were obtained by different inductive conditions. One condition was direct induction of secondary embryos from primary somatic embryos, and the other was DEC, where embryogenic cell clusters were induced from primary somatic embryos in agar medium. Our previous study found that somatic embryos, but not embryogenic cell clusters, were induced directly when DEC-derived somatic embryos were transferred into shaken flasks or a bioreactor to grow . Ultrastructural observation showed significant differences of epidermal cells among four lines of Siberian ginseng embryos developed in this study. We report here the screening, isolation, and functional prediction of candidate genes using EST, microarray, and differential expression analysis in controlling embryogenic cell cluster induction in Siberian ginseng. Materials and Methods Plant materials and growth conditions Seeds of Siberian ginseng were stratified in moist sand at 15C for 6 months; dehiscent seeds were chosen as the culture material. After removing the coat, seeds AS 602801 were sterilized in 70% ethyl alcohol for 30 sec followed by 1% NaClO for 10 min, then rinsed 5 times with sterile water. Sterilized seeds were transferred onto 1/3 MS (Murashige and Skoog) medium with 1% sucrose. Cultures were maintained under white fluorescent light [photosynthetic photon flux density (PPFD): 40 molm?2 s?1] and long day conditions (16 h light/8 h dark). For direct somatic embryo induction (DSEI), seeds whose cotyledon had been exposed for 1C3 days were incubated at 40C for 5 days, then transferred to plant growth regulator (PGR)-free 1/3 MS medium and cultured for 3 months without medium exchange. For direct embryogenic cell cluster induction (DEC), somatic embryos were transferred to 1/3 MS medium with 1 mgL?1 2,4-D (2,4-Dichlorophenoxyacetic acid); this medium possesses the ability to continuously generate embryogenic cell clusters. Embryogenic cell clusters were transferred onto 1/3 MS medium without PGR for somatic embryo development, and mature embryos eventually formed . Induction of secondary somatic embryos Somatic embryos from DEC and DSEI were used for generating secondary somatic embryos. The primary somatic embryos were cultured on 1/3 MS medium with 1% sucrose without PGR for secondary embryo induction. Secondary somatic embryos arising from the somatic embryos were used for further proliferation by repeated subculture onto fresh medium of the same composition. Secondary somatic embryos from DEC were transferred into 1/3.