Studies On Establishment Of High Frequence Regeneration System And Agrobacterium-mediated Transformation Of Chrysanthemum With FT

Chrysanthemum is one of the ten famous flower which originated in our country. but most varieties of them bloom concentrated in November or so,at the same time only a few a little kinds of colors and smaller type of flowers can bloom at summer or early autumn. Therefore, the development of chrysanthemum production is severely constrained because of its flowering time. FT gene is a kind of protein kinase inhibitors. The flowering time can be promoted by the regulation of CO to the FT genes. The objective of this study is to transfer the FT gene to'Jinba'through the research of the efficient regeneration system and transformation system mediated by Agrobacterium tumefaciens of chrysanthemum to obtain new varieties of flowering time earlier chrysanthemums and established a foundation of wide application of improving chrysanthemum cultivars applying transgenic method.Chrysanthemum species 'Jinba' is used in this experiment as test material. The result showed that plant growth regulators can affect shoot regeneration and root inducement of chrysanthemum. A efficient and rapid regeneration system of chrysanthemum was established. In the study we found that shoots of cultivar'Jinba'can regenerated directly from the leaf disc on the media MS+NAA(1mg/L)+6-BA(1mg/L), after 20 days of tissue culture.At the same time the shoot regeneration rate can reach up to 95.5%.Transferred shoots onto the medium 1/2MS+NAA(0.7mg/L),we can foud that roots were obtained 10 days later, and the rooting percentage was 100%.Based on the present optimal efficient regeneration system which has been obtained of'Jinba', genetic transformation was studied by using leaf discs by co-cultivation with agrobacterium. The results showed that the efficiency of genetic transformation can be greatly affected by several factors, such as the time of preculturation,infecting,co-cultivation and delay-cultivation. We found that when infected leaf disc which has been preculturaion for 12 hours in bacterium liquid for 5min, then co-cultured for 3 days in dark and delay-cultivated for 3 days, we would obtain the highest genetic transformation rate. Transferred the leaf discs which has been co-cultured for 3 days in dark onto the media MS+NAA(1mg/L)+6-BA(1mg/L)+Km(20mg/L)for selection of the regeneration resistant buds. Shoots were tured on the selection medium 25 days later, and then transferred the shoots which were at around 1-2cm to 1/2MS+NAA(0.7mg/L) +Km 20mg/L for rooting selectin.After continuous selected on the medium with Km for more than 8 weeks, cut the resistant buds down and transferred them into the appropriate rooting medium with 10mg / L Km for a second screening. Extract DNA of the resistant buds which can generate roots normally, and monitored them by PCR analysis. The PCR results suggested that transgenie has 7 positive plantlets and the transformation rate is about 0.9%.It showed that the target gene of FT has been translated into genome of chrysanthemum.