RAPD Analysis Of Genetic Diversity And In-Vitro Conservation Of 42 Accessions Of Compositae Germplasm Resources

In this study,42 accessions of compositae germplasm resources were collected for in vitro conservation. Stem-segments with axillary buds of compositae plants were used as the explants for establishing the in vitro regeneration system. The plantlets from"Zhuiyu"chrysanthemum were applied as the conservation materials to investigate the restricting growth conservation method, and the RAPD technique was used to detect the genetic stability of the conserved plantlets., which provided more scientific basis for the micropropagation and in-vitro preservation of compositae plants. 42 accessions of compositae plants were conserved in vitro and analysed by RAPD technique for discussing genetic diversity. The main results were as follows:1. Establishment of the regeneration system of Compositae plantsThe stem segments from the"Zhuiyu"chrysanthemum and its plantlets were used as materials to study the best conditions of the surface sterilization, proliferation, rooting and transplanting. The results showed that: the best method of surface sterilization was treated with 75% alcohol (30s) +0.1% of HgCl2 (3 min) + sterile water (24 h) +0.1% of HgCl2 (3 min); the best proliferation medium was the MS medium supplemented with 0.3 mg ? L-1 6-BA and0.15 mg ? L-1 NAA, and the proliferation coefficient was 9.05; the best rooting medium was 1/2MS +0.2㎎ / L NAA, NAA effect on root induction was better than IBA; and the best transplanting medium was the mixture of vermiculite and peat (1:2).2. Study on in vitro conservation of Compositae plantsThe plantlets of"Zhuiyu"chrysanthemum which had grown for 3 months were used as materials. The effects of different concentrations of sucrose, agar, sorbitol, growth inhibitor and light intensity on restricting growth conservation of plantlets were evaluated. As the results showed, MS media containing 40 g/L sucrose and 15g/L sorbitol was the optimal condition for conservation. The survival rate was up to 77.8% after preservation for 10 months. The optimal subculture time could be 8 months. At the same time, 30mg/LABA promoted conservation of plantlets as well. However, light intensity did not affect the in vitro conservation of"Zhui yu".3. Analysis of genetic stability of the conserved Compositae plantsUsing plantlets before conservation and plantlets conserved for 10 months as materials, the genetic variation between these two treatments was detected by RAPD-PCR marker techniques. The resulted demonstrated that the patterns of ISSP were absolutely identical, which showed that there were no variations of DNA levels and the genetic stability of the conserved plantlets were very stable. This also confirmed the feasibility of the restricting conservation.4. The in vitro restricting conservation of 42 accessions of Compositae germplasm resourcesAll collected compositae plants were preserved in vitro by advanced conservation method. All of them were sunbculured by every 7 months. The plantlets of Chrysanthemum and Wedelia were well preserved and growth of these plantlets was good after transplantation . Nevertheless, vitrification was easy to occur in the plantlets of Tagetes.5. RAPD analysis of genetic diversity of 42 accessions of Compositae germplasm resourcesIn this experiment, the genetic diversity of 42 accessions of compositae germplasm resources was assessed by using RAPD (Random amplified microsatellite polymorphism). As results showed, 127 amplified bands were produced by 11 random primers. The ratio of polymorphic bands reached to 99.2% with 126 polymorphic bands appeared, which illustrated that 42 accessions of compositae germplasm resources had abundant polymorphism and rich genetic resources. According to the diversity of the amplified bands, a dendrogram was constructed to indicate their genetic relationships. When similarity coefficient was 0.362, 42 accessions of compositae germplasm resource could be separated into 4 groups, Zinnia group, Indian blankets group, Coreopsis basalis group and another group consisting of Chrysanthemum, marigold, Wedelia chinensis. When similarity coefficient was 0.628, all Chrysanthemum species were put in the same group, the rest species were divided into other 9 groups. While, when similarity coefficient was 0.794, 22 groups were set up. 33 chrysanthemum species were divided into 13 groups which presented that 13 different chrysanthemum germplams were preserved.