Studies On In Vitro Conservation Of Two Wild Chrysanthemum Species By Slow-Growth Method

Wild plant resources are material basis for human survival and development, it’s strategically important to keep their diversity for the Agriculture sustainable development. Growing in the situ place and Planting in the open field is the traditional method for the conservation of plant germplasm resources. However, because of diseases and insect pests, natural disasters and shortage of fund, large numbers of wild plant resources are endangered or extinction. Slow-growth conservation based on plant tissue culture is a widely used method, it restricts the growth of in vitro plantlets and reduces the frequency of subculture under the premise of the maintenance of genetic stability. In this research, we took wild Chrysanthemum species of Chrysanthemum shiwogiku var. kinokuniense and Chrysanthemum yoshinaganthum as study objects and studied the conservative effects of different culture temperatures and different supplements in the medium. Genetic stability of regenerated plantlets was examined by biological characteristics, isoenzyme and molecular markers analysis at the same time. The main results were as follows:1.Effects of low temperature on in vitro conservation of chrysanthemum indicum L. plantlets Stems of Chrysanthemum shiwogiku var. kinokuniense plantlets growed slowly in protration medium MS+0.5mg.L-1BA+0.1mg.L-1NAA+30g.L-1sucrose+6.5g.L-1agar, and plant height were significantly reduced, at4℃,no matter with or without10days preculture at normal culture cordition(25±2℃). The results showed that the survival rate was still high with85.17%and96.15%after360d. respectively,It showed that the condition of low temperature at4℃was suitable for the conservation of Chrysanthemum shiwogiku var. kinokuniense plantlets.2.Effects of different concentration of sucrose on in vitro conservation of chrysanthemum indicum L.plantletsAt normal culture condition (25±2℃),the growth rate of Chrysanthemum shiwogiku var. kinokuniense plantlets was not reduced obviously in protration medium (MS+0.5mg.L-1BA+0.1mg.L-1NAA+6.5g.L-1agar+15g.L-1sucrose)compared with CK protration medium with only30g.L-1sucrose. The conservation period of the plantlets could be prolonged for240d with28.57%plantlets survived in the medium with60g.L-1sucrose, therefore, it is suitable for the short-term in vitro conservation of Chrysanthemum shiwogiku var. kinokuniense plantlets. However, the conservation of Chrysanthemum shiwogiku var. kinokuniense plantlets in the condition of90g.L-1sucrose was difficult of because of the poisoning. effect of the high sucrose concentration.At normal culture conditions (25±2℃), Chrysanthemum yoshinaganthum plantlets growed slowly in protration medium MS+2.0mg.L-1KT+0.1mg.L-1NAA+6.5g.L-1agar+15g.L-1sucrose compared with CK protration medium with only30g.L-1sucrose. The conservation period of the plantlets could be prolonged for45days compared with CK. It was not suitable for the conservation of Chrysanthemum yoshinaganthum plantlets in the condition of high sucrose concentration(60-90g.L-1) because of its poisoning effect. The survival ratio were only29.58%-49.31%for the90days conservated plantlets, which growed really weak.3.Effects of different concentrations of sucrose and CCC on in vitro conservation of wild chrysanthemum plantletsAt normal culture conditions, the survival ratio of Chrysanthemum shiwogiku var. kinokuniense plantlets was improved in the protration medium with different concentrations of sucrose (15-90g.L-1) and CCC (1000-2000mg.L-1),which were added in protration medium MS+O.Smg.L-1BA+0.1mg.L0-NAA+6.5g.L-1agar. The conservation period of the plantlets could be prolonged for more than360d in the condition of low concentration of sucrose (30g.L-1) and high concentration of CCC (1500-2000mg.L-1),and the plant growth was robust, With suitable plant height,thickened blade and dark green leaf. Their survival ratio were92.86%-96.43%. Therefore; it was suitable for the medium-term in vitro conservation of Chrysanthemum shiwogiku var. kinokuniense plantlets. The basic medium supplemented with high concentrations of sucrose and CCC have different degrees of toxic effects on the plants.4. Effects of different concentrations of sucrose and PP333on in vitro conservation of wild chrysanthemum plantletsAt normal culture conditions, the growth of Chrysanthemum yoshinaganthum plantlets was inhibited in the protration medium with different concentrations of sucrose (15-90g.L-1) and PP333(3-9mg.L-1),which were added in protration medium MS+2.0mg.L-1KT+0.1mg.L-1NAA+6.5g.L-1agar. The conservation period of Chrysanthemum yoshinaganthum plantlets could be prolonged for360d in the condition of low concentration of sucrose (15g.L-1-30g.L-1) and high concentrations of PP333(6mg.L-1-9mg.L-1), and the survival ratio was93.53%-100%,The survival growth robust with dark green leaf, therefore, it was suitable for the medium-term in vitro conservation of Chrysanthemum yoshinaganthum plantlets.5.Histological observation of Chrysanthemum indicum L. Plantlets’in the process of slow-growingThe plantlets of Chrysanthemum shiwogiku var. kinokuniense and Chrysanthemum yoshinaganthum in the slow-growing process were observaed. The results showed that some changes have taken place for the plantlets with highest survival ratio compared with the control plantlets in cell organization structure in the presence of growth inhibiting substances or under low-temperature conditions. The mainly differences in mesophyll, included increased mesophyll cell layers, reduced intercellular space, increased cell density, more dense arrangement of leaf palisade tissue and thickened spongy tissue; Differences in seem cells included smaller stem cortical parenchyma cells, increased cell layers and reduced intercellular space. The changes above further proved the slow growth of plant cells, weakened metabolism and higher survival ratio was higher in condition of in vitro conservation, which could prolong the conservation period of the plantlets.6.Genetic stability of regenerated plantletsAfter cultured in recovering medium, all plantlets conserved with the above methods recovered normal growth ability. There is no obvious difference in morphological characteristics, isoenzyme zymogram of peroxidase and esterase, and amplified profiles of ISSR between the regenerated plantletsand the control plantlets, Suggesting that the regenerated plantlets maintained genetic stability, and the slow-growth method is a feasible way to conserve Chrysanthemum germplasm.