| Clematis L.belonging to Ranunculaceae,is a kind of vine plants with high ornamental value and a variety of resistance.Based on collecting different clematis germplasm resources,using ISSR technology combined with field observation,the genetic diversity of clematis was studied in this experiment.Different parent combinations of hybrid were selected to study the hybrid technology and hybrid seed germination technology,and traits of hybrid progenies were observed.At the same time,rapid propagation in vitro technology of clematis wasinvestigated.The results in this experiment will provide scientific basis for the classification of clematis germplasm resources and the new variety breeding.1.Through optimizing the reaction parameters,the ISSR-PCR reaction system was established:the 25ul PCR system included 40ng template DNA,0.4uM forward primer,0.4uM reverse primer,0.20mM dNTPs,1U Taq polymerase and 2?L 10×Buffer.The optimum annealing temperature of different ISSR primers was studied,13 primers with high polymorphism were selected.PCR was carried out with an initial denaturation 94? for 5min,following amplication by 40 cycles of 94?for 50s,51.8-54? for 1 min,72? for 1 min and a final 10min extension at 72?.Then it was stored at 4?.ISSR analysis was conducted with 32 clematis materials and related 2 materials.172 polymorphism bands were obtained.On average,each primer amplified 13.23 bands.A total of 168 polymorphic loci was amplifiedby 13 primers,the average of each primer amplified 12.92 loci,percentage of polymorphic loci was 98.24%.ISSR analysis results showed that the genetic similarity distance of 34 accessions tested was between 0.22 and 1.21.32 clematis materials are divided into three classes in the genetic distance of 1.01 and divided into 13 groups at the genetic distance of 0.77.2.In this study 5 combinations of parents of clematis were selected to do hybrid.Among them,no seeds were obtained from the hybrid 'Utopia '(female)with ' Dan '(male)The other 4 combinations got 105 seeds totally,the average seed number of 5 combinations was 21,the average seed setting rate was 70.93%.There were 92 of clematis seed germination,the average germination percentage was 69.02%.The number of flowering of Fi generation seedlings were 33 trees,7 plants with high ornamental values were selected from the flowering plants.3.Rapid propagation of clematis 'King's dream' were explored.The explant disinfection method and the most suitable culture medium were studied..The bulb plates of 'King's dream' were sterilized best when it was soaked in 0.1%mercuric chloride solution for about 10 minutes to 12 minutes.The optimal stemperature of adventitious bud induction was 30?.MS basal medium with 3.0 mg/L 6-BA and 0.5 mg/LNAA is the best medium for the shoot subculture.The optimum rooting medium for is MS basal medium with 0.3 mg/L IBA and 0.1 mg/L NAA. |
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