Studies On Cryopreservation Of Carnation (Dianthus Caryophyllus L.) Germplasm By Droplet-Virtrification

Carnation (Dianthus caryophyllus L.) is one of the world's four major cut flowers, and widely used in flower filed because of its beautiful flower-shaped and color. In this study, carnation variety 'Master' was used as the experimental materials, cryopreservation system was optimized and ultrastructure of meristem cells during cryopreservation were studied in our experiment. The main results are as follows:1. Shoot tips of 'Master' were successfully cryopreserved with the droplet virtrification technique. Procedure was established as follows:Shoot tips consisting 2-4 leaf primordia were excised from in vitro plants which had been subcultured in MS medium contained 2.5g/L activated caroon(AC) for about 1.5-2 month. Excised shoot tips were precultured in liquid MS medium supplemented with 0.3M sucrose for 2 days, soaked in liquid MS medium supplemented with 5% DMSO and 5% glycerol for 10min, osmoprotected in loading solution for 20min at 25℃, dehydrated with PVS2 solution for 60min at 0℃, frozen in the micro-droplets of vitrification solution placed on aluminium foils, which were immersed rapidly in liquid nitrogen. Rapid rewarming was conducted in the MS liquid medium contained 1.2M sucrose. Post-thawed shoot tips were transferred to regeneration medium and stored in the dark at 25℃for 1week, then cultured under white fluorescent light at an intensity of 2000 lux, with a 16h photoperiod at 25℃. Shoot tips were transferred to the fresh regeneration medium 15 days after post-thraw, then transferred to the medium which used to culture in vitro stock plants when developed normal shoots and leaves for about 1 month. The highest post-thaw surivival percentages and regeneration percentage of cultival'Master'was 91% and 73% respectively. The result showed a suitable method for carnation germplasm to cryopreserve.2. Applied this method to the other three carnation varieties.Applied the optimized programs to the other carnation varieties, the highest survival rates were 85%,50% and 47%; the highest regeneration rates were 62%,42%,42%. The results showed significant differences between varieties.3. Ultrastructure changes of apical meristem cells in the process of cryopreservation were observed using the transmission electron microscopy(TEM). The results showed that the biggest change occured in the stage of dehydration. The main changes were protoplast shrink, cytoplasm became denser, plasmolysis severely, mitochondrion were swelling and their matrix density were dropping, the cristae become well developed, the cisternae of the endoplasmic reticulum dilated. Membranous substance vesiculation, nucleus cisternal space expanded. After a recovery culture, some cells can repair the damage, owned an intact cell structure similar to the control. Others were lethally injured and can not repair.