Studies On Regeneration System Of 4 Chrysanthemum Cultivars And Genetic Transformation With CDS Gene

Chrysanthemum was originated in China and was one of ten famous traditional flowers and one of the four most important cut flowers in the world. Chrysanthemum was susceptible to pests attack especially aphids. Pyrethrins was an ideal natural bio-pesticides extracted from Pyrethrum(Pyrethrum cinerariifolium). CDS (Chrysanthemyl Diphosphate Synthase) and CHS (Chrysanthemol Hydroxylate Synthase) were key genes which control metabolism of pyrethrins. Based on the research on aphids-resistance ability of Chrysanthemum, chose’Sanhuaxiandong’,’Ziyanxinzhuang’,’Huanglongzhao’and ’1581’to regeneration study; integrated CDS, CHS, GUS (CDS promoter), GFP (CDS promoter), CDS+CHS into chrysanthemum cultivar’1581’, CDS+CHS gene transgenetic shoots were analyed by GC-MS; test the indoor aphid-resistance ability of chrysanthemum cultivars:’Sanhuaxiandong’,’Ziyanxinzhuang’,’Ruizhugong’, ’Quanxiangmengzhiju’,’Jinsidie’,’Jinluanbaodian’and’1581’. The major results are as followings:1. Using’Sanhuaxiandong’,’Ziyanxinzhuang’,’Huanglongzhao’and’1581’as material, MS as medium, test the regeneration efficiency on different combinations of hormones. The results showed that,’1581’had high regeneration efficiency in all medium containing different hormone concentrations, the highest regeneration efficiency is 97.5%. The average regeneration efficiency of’Ziyanxinzhuang’reached 70% on MS+6-BA5.0 mg/L+NAA0.5 mg/L, the average propagation rate was 2.0; the average regeneration efficiency of’Sanhuaxiandong’was 47.50% on MS+6-BA0.1 mg/L+NAA1.0 mg/L, but ’Huanglongzhao’showed poor result.2. CDS gene, CHS gene, GUS (CDS promoter) gene, GFP (CDS promoter) gene, CDS+ CHS double gene were integrated into chrysanthemum cultivar’1581. The transformation conditions were:pre-cultured for 2 days in kanamycin concentration of 30mg/L, infected for 2 minutes and co-cultivated for 60 hours. Transgenetic plants were identified by PCR. The transformation efficiency of the each genes were noted, CDS, CHS, GUS (CDS promoter), GFP (CDS promoter) and CDS+CHS gene’s transformation efficiency were 1.40%; 0.35%; 1.70%; gene’s,5.00%; and 3.10% respectively.3. The content and volatile compounds of CDS+CHS transgenetic plants were analyzed by GC-MS, the results showed that:the n-hexane extract of leaves of transgenic plants content had no acid group metabolism of pyrethrins intermediate, while using solid-phase microextraction, chrysanthemol, which is pyrethrins metabolism intermediate product, was detected t in transgenic plants leaves.4. Stem segments with leaf from different varieties of chrysanthemum, newly born aphids were inoculated to the Chrysanthemum leaves and then placed in artificial climate chamber with an adjusted climate chamber temperature of 25℃, humidity 80%, illumination time was 14L/10D. Ten young aphids were put on chrysanthemum plant, repeated five times for each cultivar. Based on the aphid-resistance ability of chrysanthemum cultivars range from low to high, the grades for aphid-resistance ability of chrysanthemum cultivars was set as grade 1, grade 2, grade 3, grade 4 and grade 5 in order to compare the different cultivars of leaf aphid-resistance. The results showed that: ’1581’was grade 1;’Quanxiangchongtian’was grade 2;’Quanxiangmengzhiju’was grade 3;’Jinsidie’and’Sanhuaxiandong’aphid resistance ability were closer, was grade 4; ’Jinluanbaodian’and’Ziyanxinzhuang’was grade 5.5. In this experiment,5-8 ray florets from different Chrysanthemum cultivars were used as materials. They were placed on petri dish containing 7% agar culture medium,10 adult aphids on ray florets by brush, each cultivars was repeated 10 times.The cultivar was placed in the artificial incubator at temperature 25℃, humidity was 80%, illumination time was 14L/10D. Based on the aphid-resistance ability of chrysanthemum cultivars range from low to high, grades were set based on aphid-resistance ability of chrysanthemum cultivars as grade 1, grade 2, grade 3, grade 4 and grade 5. The results showed:’Quanxiangchongtian’was grade 1;’Jinluanbaodian’was grade 2;’Ruizhugong’ was grade 3;’1581’,’Sanhuaxiandong’and’Jinsidie’had no difference on aphid-resistance ability, were grade 4;’and Quanxiangmengzhiju’was grade 5. This study recommends further research on the function of CDS gene and CHS gene, evaluation of chrysanthemum aphids-resistance and breeding new transgenic aphids-resistant chrysanthemum cultivar.