Obtaining Marker Free Transgenic Chrysanthemum Through Double T-DNA System

Chrysanthemum is the traditional famous flowers in our country.In order to make it blossom at proper time and satisfy the year-round production,the research of flowering time regulator was becoming one important aspect of breeding.As a floral meristem identity gene,LFY not only controls the transition from vegetative growth to inflorescence growth,its overexpression can induce the plant to flower early,so LFY gene was transformed into chrysanthemum to get early flowers.With the commercialization of transgenic products,the potential hazard on environment and food safety becomes the main public concern.It was one of the objectives and developing trends to cultivate transgenic plant varieties without marker gene.The co-transformation method creates a simple and efficient way; transgenic plants can be got through selection in the self-crossed progeny.The technology of eliminating marker gene attracted more and more attention and became the issue in focus.Biosafety issue of transgenic ornamental plants was considered for the first time in this study,it was supposed to get marker free transgenic chrysanthemum and reduce the safety risk.Double T-DNA vector was constructed and transformed into chrysanthemum by agrobacterium,transgenic plants without marker gene were selected,and gene stacking system was also explored.The main results were as follows:1.Construction of super binary vector pCAMBIA 1300-LFY-hptLFY gene was cloned from wild Arabidopsis,which can regulate the start-up of flower meristem and promote the blossom.The 1.5kb LFY gene fragment was cut from clone vector by BamHâ… and EcoRâ… and then blunt by Klenow fragment of DNA polymerase I.The acquired fragment was ligated to plasmid pCAMBIA 1300 on which hygromycin gene was digested and removed by Xhoâ… .And therefore the intermediate vector of pCAMBIA 1300-LFY was constructed.The plasmid pCAMBIA 1300 was digested by Hindâ…¢/EcoRâ… and self-linked to delete the multiple cloning sites.It was then digested by Sacâ…¡/Sphâ… and linked to the vector pCAMBIA1300-LFY which had been digested by Sacâ…¢,so the plant expression vector pCAMBIA1300-LFY-hpt with double T-DNA was finally obtained.In this vector,the maker gene hpt was in one T-DNA region and LFY gene was in another T-DNA.The results of the enzymes digestion demonstrated the correctness of the vector;the plasmid was then transformed into agrobacterium EHA105 by free-thaw method.2.Transformation system of 'Qi Yue Tao Hua' and 'Liu Jin Sui Yue' were established. Using 12 different concentration proportion of 6-BA and NAA which were based on MS culture medium to induce shoot regeneration,'Qi Yue Tao Hua' and 'Liu Jin Sui Yue' were selected from the ten varieties,the results indicated that the optimal media were MS+6-BA 2mg/L +NAA 0.5 mg/L and MS+6-BA 2mg/L,+NAA 0.1mg/L,the regeneration efficiency were 91.4%and 89.5%respectively. The variance analysis showed that the effects of 6-BA,NAA and their interaction to regeneration efficiency were significant.The results of antibiotic sensitivity test suggested that the critical concentrations of hygromycin for shoot regeneration and root formation were 8 mg/L and 10 mg/L respectively,to inhibit the over growth of agrobacterium,450 mg/L cefotaxime was optimal for the selection media while it was 200 mg/L for rooting medium.Studying the effects of pre-culture time,bacteria concentration,infection time,co-culture time and temperature to the transformation efficiency.The optimal transformation system was as follows:the leaf explants were pre-cultured for 12 hours,the optimal concentration of agrobacterium was OD₆₀₀ =0.4 and infecting time was 15 minutes,co-cultured at 25℃for two days in dark and then rinsed by sterile water for three times.The leaf discs were then transferred to the media MS+6-BA 2mg/L+NAA0.5mg/L+bygromycin 8mg/L+cefotaxime 450 mg/L and subcultured every 20 days.When the resistant shoot was one centimeter,it was cut and transferred to the media 1/2MS+ hygromycin 10mg/L +cefotaxime 200mg/L for rooting;transgenic plants were obtained after about 30 days.3.Obtaining marker free transgenic chrysanthemum varieties with early flowersIn the five independent experiments,900 leaf discs were used and after transformation,79 hygromycin resistant plants were got and marked as T₀ generation,only 68 plants were hygromycin positive by PCR test.The maximum and average transformation efficiency was 10.6%and 7.6%.Based on the PCR result of LFY and hpt gene in T₀ generation,21 plants were designated as the co-transformants,the average co-transformation efficiency was 2.3%,the To plants were self-crossed to get T₁ progeny,17 T₀ plants were randomly chosen to analyze the segregation of LFY and hpt gene. PCR was performed on the T₁ plants.There were four genotypes in this generation:hpt-/LFY-, hpt+/LFY+,hpt-/LFF+,hpt+/LFY- and in which 51 were hpt-/LFY+,that means these were plants without marker gene,the average efficiency was 9.31%.8 T₁ progenies were randomly chosen and self-crossed to get T₂ lines.Different traits were observed and analyzed in T₂ pure lines;the results showed that,there were not significant differences on height,crown width,internodes and diameter of flower between transgenic plants and control plants,but flowering time was different,only one lines blossomed 15 days earlier than control.4.Construction of the target vector,integrase vector and recombinase vector for gene stackingGene stacking rests on a concept that the integrating DNA brings along an extra recombination site, such that after insertion of the new recombination site into the genome,the extra recombination site then becomes the new target for the next round integration.This technology can get minimal variability in transgene expression and use the same locus for the multiple gene insertion,so the new traits can be stacked in the same plant step by step which also shorten the research cycle and release time of the new cultivars.In this experiment,the recombinase vector pMM-Caggs,pMM-Kozak and integrase vector pARTOS2 were cloned.The target vector pARTOS1 was transformed into tobacco by agrobacterium mediated method.Single copy transgenic lines were selected for the next round transformation.In this study,double T-DNA vector was transformed into chrysanthemum,the co-transformants were harvested then through the selection in the progenies,early blossom transgenic chrysanthemum without marker gene was obtained,which shortened censoring period and release time of new varieties, also facilitated the commercialization of transgenic product and improved the biosafety issue.The target vector,integrase vector and recombinase vector were constructed to explore the gene stacking system, which established a foundation for the precise gene integration.