Character Research Of Low-Temperature Tolerance And Transformation In Chrysanthemums For Outdoor Planting (Dendranthema × Grandiflora)

Chrysanthemum(Dendranthema×gandiflorum) used for outdoor planting is one of the most important flowers in the landscape greening.It has specific characteristics such as short height with a heavy bloom,planting need not careful management and possessing social and economic values.The blooming stage of chrysanthemum grown outdoors is usually at late autumn and its ornamental values deteriorate when sudden declining of temperature.In these cases,tolerance to low temperature of chrysanthemum for outdoor planting would be improved through transfering related genes to cold tolerance by genetic transformation.In this study, materials used were 11 major cultivars in northern parts of China(Jinbuhuan","Aihong", 'Fengwofen','Xuetao','Qiuyan','Fenmeigui','Ziqiushang','Fuli','Jinfenghuang', 'Donglinruixue',and 'Xiuzhenhong') and 16 lines selected in comprehensive evaluation from filial generations of maternd parents('Donglin 1','Donglin 2','Donglin 3','Donglin 4', 'Donglin 5','Donglin 6','Donglin 7','Donglin 8','Donglin 9','Donglin 10','Donglin 11', 'Donglin 12','Donglin 13','Donglin 14','Donglin 15','Donglin 16').After screening 27 lines of chrysanthemums under freezing treatments,four lines were selected to establish regeneration system by inducing direct production of adventitious buds from leaf explants.The plant expression vectors of AtproDH and P5CS genes,which were cloned from seedling of Arabidopsis thaliana,were constructed by Gateway clone technique.P5CS gene had been transformed to chrysanthemums.The main results are shown as follows:1.Screening freezing tolerant cultivars and lines of chrysanthemumTwenty seven lines of chrysanthemum grown outdoors were screened using 'injury rate' and 'injury index'.There were 7 cultivars and lines,which were relatively high tolerant to freezing treatment,i.e.,'Jinbuhuan','Xuetao','Fuli','Donglin 4','Donglin 6','Donglin 9', and 'Donglin 13'.There were 3 cultivars and lines,which were less tolerant,i.e.,'Fenmeigui', 'Donglin 1',and 'Donglin 2';The determination of electrolyte leakage,and contents of free proline,MDA,CAT,fructose,glucose and soluble sugars on 7 lines of chrysanthemums at different stages freezing showed that 'Xuetao' and 'Donglin 4' were more tolerant than others, and that especially 'Donglin 4' was able to regulate its osmotic pathway to alleviate the 'injury degree' caused by freezing conditions.2.Protein expression profile of chrysanthemum under low temperature stressTwo-dimensional liquid chromatography was applied to fractionate and analyze total protein taken from 'Donglin 4' leaves grown at low temperature and at normal condition. Chromatography peaks of fraction were transformed to gel graph by ProteoVue software.The 36 different protein points were found,and expression in 26 of them were enhanced.Three proteins correlation with low-temperature tolerance were identification by MS:Putative calcium-transporting ATPase 10,Ferredoxin-thioredoxin reductase and Putative cytochrome c oxidase subunitⅡPS17.3.Cloning freezing tolerant genes and constructing plant expression vectorsP5CS and AtproDH were cloned from cDNA of Arabidopsis thaliana by RT-PCR.The expression vectors of AtproDH and P5CS were constructed by Gateway clone technique.4.Constructing transformation system of chrysanthemumThe obvious difference in differentiation rate of leaf explants was revealed among gene types of 'Donglin 4','Donglin6' and 'Donglin9'.The highest differentiation rate was obtained in leaf explants of 'Donglin 9'.No adventitious bud produced from leaf explants of 'Donglin 13'.For 'Donglin 4',the most suitable culture medium(MSCM) was MS+3 mg/L BA+0.1 mg/L 2,4-D but the bud differentiation rate was only 10%.For 'Donglin 6',the bud differentiation rate was 82%,and MSCM was MS+2 mg/L BA+1 mg/L NAA.For 'Donglin 9', MSCM was MS+0.5 mg/L BA+1.5 mg/L NAA.The bud differentiation rate in upper young leaves of'Donglin 9' was as high as 95%,but the differentiation rate being lower than 50%for leaves at middle and lower parts of stems.Hygromycin reduced bud differentiation of leaf explants.'Donglin 9' lost ability to differentiation in the culture medium containing 5 mg/L hygromycin and differentiated buds died when soaked in solution at 30 mg/L hygromycin.Transformation system of P5CS in chrysanthemums for outdoor planting was established by A.tumefaciens LBA4404.Positive transgenic plants proved by PCR identification was obtained.