Preliminary Study On Tissue Culture And Flower Colour Genetic Transformation Of Jasminum Sambac (Linn.) Ait

Jasmine [Jasminum sambac (Linn.) Ait] is a subtropical plant belonging to oleaceae gelsemium (or jasmin). It is city flower of Fuzhou.The main cultivated varieties in Fuzhou generally divided into single and double jasmines.An aseptic system of Fuzhou jasmine was established by using its stems as explants in this experiment. The results showed that the best conditions for Jasmine explants inducing were 8 minutes of 0.1% HgCl2 surface disinfection,3000-4000 lx of light densities and the medium of ’MS+KT 4.0 mg/L+NAA 0.04 mg/L+sucrose 30 g/L+agar 6 g/L’. The initial culture medium with high concentration or low concentration phytohormone resulted in low browning ratio of the explants.The jasmine callus induction, subculture and long-term maintenance were vaccinated on media with different concentrations of phytohormone. The results indicated that the suitable light densities for callus growth were 1500-2000 lx, and different phytohormones could form six types of callus. The medium of ’MS+BA 2.0 mg/L+NAA 0.2 mg/L+sucrose 30 g/L’ was the best for callus inducing, with an average induction rate of 100%. The medium of’MS+BA 2.0 mg/L+NAA 0.2 mg/L+sucrose 30 g/L" was the best for the callus subculture multiplication, and the callus with good growth state could be obtained.The DFR cDNA sequence was cloned from the callus using RT-PCR techniques in Vitis davidii var. davidii, and the accession number was KF915803 in GenBank. The open reading frame was 1014 bp, encoding a polypeptide of 337 amino acids. Bioinformatics analysis showed that the protein was a Stable hydrophilic protein located in cytoplasm position. The secondary structure was made of the alpha helix, beta sheets and coils. Dendrogram and phylogenetic analysis indicated that the DFR of V. davidii var. davidii was close to Vitis amurensis in genetic relationship, with the reported higher homology dicotyledonous plants.The pCAMBIA1302-35S-DFR-NOS expression vector was constructed by double digestion technology, and transformed into Agrobacterium EHA105. The agrobacterium was injected in to tabacum plants, and the transient expression was conducted. Jasmine putative transformants via agrobacterium-mediated transformation of JC2-type callus were dectected, and then PCR, GFP and SDS-PAGE detection of resistant callus after hygromycin selection were performed. The results showed that the GFP protein was transiently expressed in tobacco leaves. Using GFP transient expression system, EHA105 strains with pCAMBIA1302-35S-DFR-NOS were transferred into the JC2-type callus, and the resistant callus were obtained. After screening, the detection test showed that the target gene was successfully transformed into callus and it was expressed in little increaseing quantities.