Studies On Regulation Of Somatic Embryogenesis And Cloning And Expression Of SERK Gene In Camellia Nitidissima Chi.

In this experiment the in vitro regenerating system originated from the adult stem-tips was used as the material for synchronization of somatic embryos and optimization of MCFS (embryo with multiple cotyledons and flower-like shape) embryogenesis multiplication systemic in Camellia nitidissima Chi.; cloning of SERK gene from somatic embryos, expression analysis of SERK gene by real-time quantitative PCR technology during somatic embryogenesis in Camellia nitidissima Chi.. The main results were as follows:1. Synchronization of somatic embryos and induction of anther embryogenic callus in Camellia nitidissima Chi.In this experiment,somatic embryos originated from the adult stem-tips were used as the material for synchronization of somatic embryos. The results indicated that somatic embryos grew well on MS+4%sucrose +2% Sorbitol +6% agar(C1) and MS+4% sucrose +2% Mannitol +6%agar medium(C2), somatic embryos were highly synchronized in globular embryos and heart-shape embryos .The synchronization of somatic embryos was obtained by culturing and picking means in cotyledon embryos and mature embryos. Induction of anther embryogenic callus was conducted in this experiment. The results indicated that after 4 degrees Celsius temperature pretreatment for 24h, authers were cultured on the medium MS+2.0 mg·L-12, 4-D+0.5mg·L-1KT, callus could be induced. When the callus was cultured on the differential medium MS+1.5 mg·L-16-BA+1.0 mg·L-1IAA, white callus became green. After screening and morphological observation, it was preliminary considered as embryogenic callus.2. Optimization of MCFS embryogenesis multiplication systemic in Camellia nitidissima Chi.The experimental research that different concentrations PEG6000, betaine, hormone combination affected induction MCFS embryos. The results indicated that MCFS embryos could be obtained on medium MS+0.8 g·L-1betaine +20 g·L-1 mannitol, and inductivity reached 86.6%; and this condition also promoted secondary embryo proliferation. MCFS embryos could be obtained on medium MS+PEG60007.5%+20 g·L-1mannitol and the inductivity reached 76.9%. Relatively mature embryo selected from the above induction medium were transferred onto the Y0 medium 1/2MS+NAA 0.2mg/L+BA 2mg/L, then the somatic embryos turned magenta or even became brown; and finally, the browned body generated nearly synchronized and identical secondary MCFS embryos on the surface. Going back to the first step for more than 40 days, the secondary MCFS embryos could repeat the process. 3. Cloning of SERK gene in Camellia nitidissima Chi. somatic embryosThe globular embryos were used as the material for cloning SERK gene. The primers were designed based on conserved sequences from the given other plants'SERK gene sequences reported in GenBank. The partial conserved and 3'end sequences were obtained by homology cloning combined with RACE technology. Sequencing of the purpose fragment got 1219 bp. The results showed the SERK gene sequence from Camellia nitidissima Chi. somatic embryos was 85-73% homologous with other plants reported in GenBank; sequence analysis revealed that splicing of the cDNA encoded 277 amino acids. Amino acid alignment showed that the SERK align in Camellia nitidissima Chi with SERK genes from other Species reported in GenBank had high similarity. The result showed that SERK gene from Camellia nitidissima Chi EC existed conservatism.4. SERK gene expression analysis during somatic embryogenesis in Camellia nitidissima Chi. by real-time quantitative PCR18S rRNA gene was used as the reference gene for SERK gene expression analysis by real-time quantitative PCR technology during somatic embryogenesis in Camellia nitidissima Chi.. The results showed that transcriptional expression levels of SERK gene at different stages during somatic embryogenesis were expressed at different degree, the expression level was the highest at the stage of cotyledon embryos; the next was at the stage of mature embryos and MCFS embryo., and the lowest was at the stage of globular embryo and heart-shape embryos, which suggested that SERK gene expression played an important role during somatic embryogenesis in Camellia nitidissima Chi., specially in maintaining cotyledon embryos morphogenesis.