Research On Cryopreservation Of Jerusalem Artichoke Shoot Tips

Jerusalem artichoke (Helianthus tuberous L.), belonging to Asteraceae family, is a perennial herb.It has a strong resistance, usually used to greening the highway and as a afforestation vegetation to improve desert and saline. Jerusalem artichoke is an important ornamental crop germplasm resource with high economic, ornamental, ecological and medicinal value. It’s an asexually propagated crops, so cryopreservation can conserve the Jerusalem artichoke germplasm for long term.In this study, Jerusalem artichoke variety’2009331028’was used as the experimental material. Cryopreservation system was optimized by one-factor experiments and then was used for the other3Jerusalem artichoke cultivars. The survival rate and regenerated rate were compered between vitrification and droplet vitrification. Differential Scanning Calorimetry and ultrastructure of shoot tips meristem cells during cryopreservation were studied in our experiment.Finally, the genetic stability was studied by microsatellites makers in our experiment. The main results were as follows:1. Shoot tips of’2009331028’were successfully cryopreserved with the droplet vitrification techniques. Procedure was established as follows:Shoot tips consisting2-3leaf primordia were excised from in vitro plants which had been subcultured in MS medium contained1.0mg/mL GA3and15g/L sucrose for about4-5weeks. Excised shoot tips were precultured in liquid MS medium supplement with0.4M sucrose for3days, osmoprotected in loading solution for30min at25℃, dehydrated with PVS2solution for15min at0℃, 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 contained1.2M sucrose for20min. Post-thawed shoot tips were transferred to regeneration medium and stored in the dark at25℃for3-5days, then cultured under white fluorescent light at an intensity1500lux, with a16h photoperiod at25℃, Shoot tips were transferred to the fresh regeneration medium1week after post-thraw, then transferred to the medium which used to culture in vitro stock plants when developed normal shoots and leaves for about2weeks. The highest post-thaw survival rate and regeneration rate of cultivar’2009330128’was93%and83%respectively.2. When shoot tips were cryopreserved by vitrification method and droplet vitrification method, the survival rate was34%and93%respectively, and the regeneration rate was26%and83%respectively. So, the best method was droplet vitrification.3. The other three Jerusalem artichoke cultivars’M6’,’Stampede’,’Relikt’were cryopreserved by the optimized procedure, the survival rate is44%,72%,50%respectively, The regeneration rate was37%,53%,37%respectively. The results showed significant differences among varieties.4. The results of DSC implied that part of the water contained in the system for specimens on the shoot tips of fresh, preculture and loading become frozen when cooled in LN. The frozen temperature, the volume of ice crystal and the frozen enthalpy were reduced with the treatment. Glass transition was detected when shoot tips were dehydrated with PVS2. The similarly results of four Jerusalem artichoke cultivars were detected by DSC.5. Ultrastructure changes of apical meristem cells in the process of cryopreservation were observed using the transmission electron microscopy(TEM). The results showed that the main dehydration were occured in loading and PVS2treatment before cryopreservation. The main changes were the plasmolysis, protoplast shrink, cytoplasm became denser, organelles such as mitochondrion, chloroplast were swelling, deformating even dissoluting. After a recovery culture, some cells could repair the damage, owned an intact cell structure similar to the control. Others were lethally injured and could not repaired.6. The genetic stability of the regenerated plantlets for Jerusalem artichoke after cryopreservation were detected by18pairs of microsatellite primers. PAGE gels showed that there was no obvious difference in the process of the tissue culture for once and no obvious differences between the regenerated samples and controls.