Genetic Transformation Of OSDLike Gene In Carnation

Carnation(Dianthus caryophyllus L.),also known as musk carnation,lion carnation,and flower boulder,is a perennial herb of Family Caryophyllaceae.As one of the main cultivars of flower industry in Yunnan Province,the common breeding methods are:hybrid breeding,induced mutation breeding,and molecular breeding.Among them,molecular breeding techniques are featured by breaking interspecific barriers,accelerating breeding process,and obtaining new peculiar variations.Therefore,using molecular breeding techniques in ornamental plants to enhance the quality of these plants and their market competitiveness,is an important goal of breeding research in carnation.In the present study,the regeneration system of carnation and the selection pressure of regeneration were optimized,and the OSD-like gene was introduced into the callus of carnation by Agrobacterium tumefaciens-mediated method.After resistance screening and PCR detection,we obtained the gene-carrying transgenic positive plants.The main findings are as follows:1.Optimized stem shoot and leaf regeneration systemsThe regeneration system of three cultivars,Master,Promesa,and Nogalte,were optimized using the aseptic tissue culture.The results showed that the requirement of rooting of TDZ was different for different parts of the same cultivar.The concentration requirement of TDZ was also different in different parts of the same variety.The concentration of TDZ for stem shoot material was 100 mg/L,while that for leaf material was 50 mg/L in variety Master.The concentration of TDZ for stem shoot material was 80 mg/L,while that for leaf material were 40 mg/L in variety Promesa.And the concentration of TDZ for stem shoot material was 90 mg/L,while that for leaf material was 50mg/L in variety Nogalte.2.Kanamycin selection pressure in three varietiesThe Kanamycin(Kan)selection pressure in the three cultivars of carnation was tested using the aseptic tissue culture,which would reduce the workload of subsequent detection.The results showed that the concentration of Kan in all three varieties was 90 mg/L.3.Genetic transformation in three carnation varietiesThe stem shoots were inoculated on the pre-culture medium for 4 d,and treated with Agrobacterium tumefaciens carrying the target gene for 25 min,then transferred to culture medium containing MS + TDZ 0.8 mg/L + NAA 0.5 mg/L + AS 30 mg/L for 4 d,and finally transferred to culture medium containing MS + TDZ 0.8 mg/L + NAA 0.1 mg/L+ Cef 400 mg/L + Kan 80 mg/L for 2 months.The adventitious buds were transferred to culture medium containing MS + NAA 0.1 mg/L + BA 0.1 mg/L + Kan 80 mg/L medium for subculture.The results showed that the callus differentiation rates in different varieties were different,the differentiation rate of variety Promesa was the highest,(average value 45%),while the differentiation rate of variety Nogalte was the most unstable(shifted between 10-63%).The callus regeneration rates in different varieties were also different,with that in variety Nogalte being the greatest(87%),while that in variety Promesa was the lowest(12%).PCR detection for transgenic plants were performed,producing 41 positive strains,among which were 16 strains of variety Master infected with vector OSDLa-PBI,6 strains of variety Nogalte infected with vector OSDLb-PBI,and 18 strains of variety Master infected with vector OSDLc-PBI Strain,and 1 vector of variety Promesa infected with vector OSDLc-RNAi.