Genetic Transformation With DFL And Its Expression Analysis In Chrysanthemum Lavandulifolium

Chrysanthemum×morifolium Ramat is one of the ten traditional famous flowers in china and the four famous cutting flowers in the world.There are many varieties planted everyyear.However,shading light and supplementing light will cost a lot in production of Chrysanthemum.The research on genetic and molecular mechanisms of the reproductive process on Chrysanthemum and get some new early flowering varieties which have independent intellectual property are very important.FLO/LFY homologue genes were initially characterized as floral meristem identity genes and play a key role in flower development among diverse species.It's not only controlling the switch from vegetative to reproductive development in plants,overexpression can induce the plant flower early. Inaddition to that,LFY has a second role in the activation of the floral homeotic genes that specify the identity of organs in the flower and is a key intergrator of the outputs of floral inductive pathways.DFL, a FLORICAULA/LEAFY homologous gene,which cloned from C.lavandulifolium,was a key gene of florescence for C.lavandulifolium.The DFL protein is 63%identical to LFY and 70%identical to FLO.The research of expression patterns of DFL in the process of inflorescence development serves as an important basis for further LFY expression analysis.This research provides reference for further research on genetic transformation of ornamentals' florescence.The success of the transformation establishes technical foundation for the transformation of chrysanthemum.Chrysanthemum lavandulifolium(Fisch.ex Trautv.) Makino,as one of the original species of Chrysanthemum,a shot day plant,could be a excellent pattern in the process of inflorescence development research.The establishment of integrated culture Methods for C.lavandulifolium will supply a need for further investigation both in C.lavandulifolium and in Chrysanthemum.This study established a solid foundation for breeding new chrysanthemum cultivars which bloom earlier by combining conventional breeding with molecular breeding.The main results are summarized as follows:1.Seed vitality of C.lavandulifolium in different medium and in different time were studied,the Seed should not be Storage more than 17 months.Based on the research of morphological characteristics and blossom characteristics of C.lavandulifolium,cultural Methods of Pot culture and cultivation room were developed.2.Susceptibility to antibiotics of C.lavandulifolium had been studied.C.lavandulifolium was susceptible to kanamycin.The critical concentration for shoot regeneration of d.lavandulifolium was 5mg/L Kan.20mg/L Kan was the critical concentration for radication.3.300-500mg/L carbenicillin was used in transformation protocol,which mediated by Agrobacterium tumefaciens of C.lavandulifolium was as follows.Sence and anti-sense expression were fused to a CaMV35S promoter of pBI-DFL to transform into EHA105.The leaves of C. lavandulifolium were pre-cultured for 2d on media.The suitable concentration of Agrobacterium tumefaciens was OD₆₀₀0.4-0.6.The leaves were immersed in the Agrobacterium tumefaciens for 5min then were co-cultivated for 2d on media(MS) at 20-25℃in darkness and delayed cultivated(MS+ 6-BA 1.0 mg/L+NAA0.2mg/L+Carb500mg/L) for 2d.Then the leaves were transferred to selection media(MS+6-BA 1.0 mg/L+NAA0.2mg/L+Carb500 mg/L+5 mg/L Hyg or kan) at 25℃. Secondary culture every 20d.Put the transgenic regeneration shoot into rooting media with 3 mg/L Hyg or 20 mg/L Kan.4.The florescence and morphological characters of the transgenic plants were observed.There were great phenomtypic differences in transgenic C.lavandulifolium plants.Compared to wild types, most of transgenic C.lavandulifoliums showed a higher growth rate and early flowering.The integration of DFL gene into C.lavandulifolium was identified by technology of PCR and PCR-Southern.The seeds of transformed plants are selected under antibiotic.The seeds could grow well,and also identified by PCR,It indicates that the DFL has been transmitted into the T₁ generation.