Primary Study On Direct Introduction Of Exogenous Genes To Viola Spp.

Viola spp.is regeneration recalcitrant,thus the efficient transformation based on regeneration system could not be used to conduct molecular analysis and genetic modification.AtWUS and CcBBM were regeneration-promoting genes found in Arabidopsis thaliana and Cinnamomum camphora,respectively.In this study,direct introduction of exogenous genes AtWUS and CcBBM was investigated,together with different transformation media,methods and species.Completely randomized design was employed.Results show that direct introduction is expected to be used in molecular analysis and genetic improvement of Viola spp.Main research results are as follows:1.Screening of Viola tricolor 'No.21' kanamycin resistance concentration.Sprayed 6 concentration gradients of kanamycin on Viola tricolor 'No.21' wild type,with the rising of concentration,etiolation percentage went up and survival percentage down.Survival percentage was 12.5%when concentration was 350mg/L.2.Data analysis of harvest seeds of pollen tube pathway transformation treatments.In direct introduction of exogenous genes plasmid through pollen tube pathway transformation in Vila tricolor 'No.21',for fructification number,only transformation methods had highly significant differences,for single fructification seeds number,genes and transformation methods had highly significant differences,interactions between factors were significant or higher,for hundred-grain weight,neither genes,transformation methods or plasmid concentrations had effects.In direct introduction of exogenous genes plasmid through.pollen tube pathway transformation in Viola xwittrockiana,for fructification number,inbred lines and transformation methods had significant differences respectively,inbred lines had significant interactions with transformation methods,the three had highly significant interactions,for single fructification seeds number,inbred lines had significant differences and it had significant interactions with genes.In direct introduction of exogenous genes Agrobacterium tumefaciens through pollen tube pathway transformation in Viola tricolor 'No.21',for fructification number,neither factors had significant differences,for single fructification seeds number,genes and transformation methods had highly significant differences and they had highly significant interactions,for hundred-grain weight,genes and transformation methods had significant differences.3.Indentification of positive plants and analysis of direct introduction factors.In direct introduction of exogenous genes Agrobacterium tumefaciens through soaking germinating seeds transformation in Viola tricolor 'No.21',6 treatments,positive percentage was 2.78%with AtWUS and 1.0 hours of soaking germinating seeds introduction with Agrobacterium tumefaciens,7.41%with AtWUS and 1.5 hours.In direct introduction of exogenous genes plasmid through pollen tube pathway transformation in Viola tricolor 'No.21',12 treatments,positive percentage was 4.63%with AtWUS,pollen tube pathway transformation without stigma or stylus and 200ng/?L,1.85%with AtWUS,pollen tube pathway transformation without stigma or stylus and 400ng/?L.In direct introduction of exogenous genes plasmid through pollen tube pathway transformation in Viola×wittrockiana,12 treatments,positive percentage was 2.78%with 'Bai 15',CcBBM and pollen tube pathway transformation without stigma or stylus,3.70%with 'Bai 15',AtWUS and 6 hours of pollen tube pathway transformation with plasmid,4.63%with 'Bai 15',AtWUS and pollen tube pathway transformation without stigma or stylus.4.Phenotype observation and callus induced from in vitro leaves of Viola tricolor positive plants.Leaves' length and width curving degree of Viola tricolor positive plants had highly significant differences.Leaves' length curving degree of HH-7 was 0.390,width curving degree of L-20 was 0.443.In the whole plant observations,HH-5 cut some apical buds itself,L-20 was small,but apical dominance was very obvious,only HH-11 and HH-18 were flowering,but stalkers were shorter than CK.Using leaves as explants of 8 positive plants,percentage of callus had highly significant differences between CK and positive plants,E-10 was 66.67%,the highest.