Polyamine Titers Change And Biosynthetic Genes Expression During Citrus Somatic Embryogenesis

Somatic embryogenesis is the main morphological way in tissue culture, genetic transformation and so on. So creat a new high efficiency somatic embryogenesis system is the key factor for citrus callus somatic embryogenesis research that biotechnology applied to citrus genetic improvement. Polyamine is a kind of nitrogen containing organic compounds character with strongly basic and aliphatic polycation. It is important to plant morphology, growth and development, response to stress. There are no evidence in polyamine affects to citrus in vitro embryogenesis so far. In present research, we investigated polyamine biosynthesis during citrus (Valencia orange) somatic embryogenesis. The main results show as follows:1. somatic embryogenesis induction from calli of Valencia orange, there are amount of globular embryo at the 10th days induction, heart-shaped and torpedo embryo happended at 20th days and at 30th days cotyledon embryo can be found。Treatment with inhibitor can inhibit somatic embryogenesis, expecially inhibited cotyledon embryo development. the inhibition can be reversed by exogenous putrescine addition.2. embryoids of Valencia orange at different stages during somatic embryogenesis in paraffin section were observed under microscope, unorganized calli structure changed in to organized embryo structure when somatic embryo development. Protocambium can be observed in heart-shaped embryo.3. Three fragments of polyamines biosynthetic genes (ODC, SPDS, SPMS) were cloned according to the homologous genes, they are 522 bp, 595bp and 307bp respectively.4. Polyamine content was analyzed by HPLC, the result show that compared with basal media the level of putrescine is lower on induction media, sprmidine and spermine is higher during the fist 20 days, and there is no difference during the last 20 days. putrescine and spermindine discreased when treated with inhibitor, but spermine seems a little accumulation. the level of three polyamines increased with exogenous putrescine addition.5. Semi-quantitative RT-PCR was used to analysed gene expression involved in polyamine biosynthesis. The results show that the 5 genes expression increased at 10 days intervals along with somatic embryogenesis induction. ADC,SAMDC,SPMS were expressed in high level while SPDS in low level, and ODC didn't expressed during embryo development. ADC was fully inhibited at the hightest concentration of D-arginine treatment and DFMO shows no affect to ODC. SPDS increased with hightest concentration inhibitor treatment, the level of ODC expression increased during the treatment with exogenous putrescine addition, and other genes have different expression pattern.