Studies On Cryopreservation Of Chrysanthemum Shoot Tips And Genetic Stability Of Cryopreserved Plants

Chrysanthemum(Dethendrama moriforium) is one of the big four cut flowers in the world,China has abundant chrysanthemum resources.The germplasm resources of chrysanthemum are normally preserved by growing them in plant nursery year after year, but this is not a suitable way for long term preservation because it occupies lots of farmland and labor,and costs too much,and what is worse,it risks of being extinct from the earth as the result of drought,flood,cold,hot damage,plant diseases and insect pests. Cryopreservation is considered to be the safest and the lowest cost approach for conservation of plant germplasm.Under condition of ultra low temperature,the physiological and biochemistrial processes of plants are almost stopped,and the change of these processes could be limited to the minimum extend,it's the most promising way for long-term preservation.However,technical workflows of cryopreservation vary in different species of plant germplasm,and literatures on cryopreservation for a specific species like chrysanthemum is very limited.The study aimed at investigating the detailed techniques for cryopreservation of chrysanthemum(D.morifolium) based on the experiments with a Chinese chrysanthemum genotype(D.morifolium).The main results were as follows:Establishment of efficient in vitro propagation system suitable for cryopreservation of chrysanthemum(Dethendrama moriforium)Different explants were applied to induce chrysanthemum in-vitro plants.Results indicated that receptacle had the highest induction frequency,suggested that receptacle is the ideal explants for micro propagation system of chrysanthemum.Nodal segments cut from flowering stage were not suitable for propagation because of severe contamination. Experiments on hormones combination on MS medium showed that the cultures on MS+BA3mg/L+NAA0.01 mg/l obtained the highest in vitro plant induction rate of 300%.In order to get stronger in vitro plants,different illumination intensity and sucrose concentration in MS medium have been explored.The results reflected the in-vitro plants showed no significant difference under illumination intensity from 1800 to 3500 Lx; under the same illumination intensity,sub-culture onto MS medium supplemented with 0.2mol/L sucrose obtained stronger in vitro plants with better leaf color and plant type which were more suitable for cryopreservation.Cryopreservation technology of in vitro shoot-tips of chrysanthemum by virtrificationThe study of pre-culture revealed that it was essential for shoot-tips to be pre-cultured before cryopreservation.Pre-cultured shoot-tips on MS enriched with 0.4mol/L sucrose for 3d could induced the highest survival rate,while shoot tips without pre-culture had lower survival rate or none survival at all.Treat the shoot tips with LS before vitrification for varies time,the result showed 30min's treatment could improve the survival rate significantly.Experiments on vitrification solutions indicated that the apices treated with PVS2 for 15 min obtained a survival rate of 85.7%,while the longer that apices were immersed in PVS2,the lower survival rate it turned out.Compared with PVS3 and mPVS2,PVS2 could dehydrate the shoot tips more efficiently.Among different technical procedures of cryopreservation,such as vitrification, encapsulation-vitrification and controlled-rate-freezing,vitrification yielded the highest shoot regeneration rates of 85.7%.The encapsulation-vitrification was also successful in this aspect with the survival rate of 50%.Controlled-rate-freezing had no shoot tips survived after cryopreservation,maybe chrysanthemum shoot tips do not tolerate long-time treatment with ultra low temperature.For regeneration culture after thawing, the surviving meristems could grow better in solid medium than semi-solid medium. Successfully vitrified meristems developed shoots directly within 3-14d of culture.The regenerating rate could reach to 100%.In summary,the procedure of cryopreservation for chrysanthemum by vitrification can be described as followings:a) the 1-2 mm length in vitro-grown shoot tips were pre-cultured on MS medium enriched with 0.4 mol/L sucrose in darkness at 4℃for 2-3 d. b) pre-cultured shoot tips were immersed for 20 min in loading solution(LS) containing 2 mol/L glycerol and 0.4 mol/L sucrose at 25℃.c) treat the tips with ice-cooled PVS2 solution for 15 min,and then tips were put into cryo-tubes with fresh PVS2 and plunged into liquid nitrogen,d) rapid thawing the tips in 40℃water bath for 2 min and unloading the PVS2 wkh liquid MS medium contained 1.2 M sucrose for 20 min.Further recovery and growth took place on regeneration medium in the darkness for less than 3 days.The assessment of genetic stabilitySRAP molecular marker was used for detecting the genetic stability of plants recovered from cropreservd shoot-tips.Among 24 pairs of SRAP primers used in this study,3 pairs of them detected 9 different bands.Cluster analysis showed their minimum coefficient is 0.96 which suggested that the genetic stability was maintained after cryopresearvation.