| Potato is an important germplasm resource for food, vegetables and forages. The safe preservation of potato are benefit for the development of their utilization, new genes mining, new variety breeding. Field repository, in vitro genebank and cryopreservation were used in potato preservation. Cryopreservation has been considered as the critical method for long-term and safe preservation for clonal crops. Nowadays, vitrification-based techniques have been developed, improved and widely used for cryopreservation of potato; however, the mechanism of cryopreservation is only reported in several studies, In this study, in vitro plantlets of potato cultivars Huzi 81-213, Q504 and Kexin 4 were used. Survival percentages of potato shoot tips during droplet-vitrification procedure by in vitro recovery were investigated to study the effects of the critical steps during cryopreservation procedure on the survival of shoot tips; Secondly, the survival patterns of the potato meristems were investigated by TTC staining of the whole shoot tips at critical steps during droplet-vitrification; Finally, the water content, thermal analysis and ice formation of potato shoot tips at critical steps during droplet-vitrification were investigated by differential scanning calorimetry and low temperature scanning electron miscopy methods. Main results were listed as following:1. Survival percentages of three potato cultivars shoot tips at critical stesp during droplet-vitrification procedures by in vitro recovery were record. Results showed that the fresh and precultured (Murashige and Skoog liquid medium with 0.3 mol/L and 0.5 mol/L sucrose) shoot tips obtained survival percentages of 100%; After cryoprotection with Plant vitrification 2 (PVS2 solution) for 30 or 60 min, the survival percentages of potato shoot tips decreased; After cryo-storage in liquid nitrogen (LN), the cryopreserved shoot tips obtained lower survival percentages than before LN exposure and the cryopreserved shoot tips treated with PVS2 solution for 60 min achieve higher survival than PVS2 treatment of 30 min.2. TTC (2,3,5-Triphenyl tetrazolium chloride) vitality staining conditions for potato shoot tips of three potato cultivars were optimized in this study. The optimal staining temperature and duration were determined as 40℃ and 2h, respectively. The viability of potato shoot tips was investigated during droplet-vitrificaion cryopreservation by TTC staining under the optimal conditions. The fresh shoot tips and precultured shoot tips showed high vitality. However, after cryoprotection with PVS2 solution, the shoot tips had the temporal and spatial specificity of vitality loss and survival, and the inner meristem and the middle part of the leaf primordium kept high vitalities. Positive correlation were revealed between TTC staining vitality and survival percentages after recovery when the area ratio of the red-stained shoot tips was higher than 0.4. When the area ratio of the red-stained shoot tips was higher than 0.45, TTC staining results would predicte the survival percentages after recovery.3. Thermal analysis and histocytological investigations of potato shoot tips by DSC and Cryo-SEM methods were conducted at critical steps during droplet-vitrification procedure. Results showed that the moisture contents of the shoot tips decreased when the shoot tips were exposed to preculture solution and PVS2 solution. When cryopreserved shoot tips were recovered for 4 days, their moisture content increased as the level of fresh shoot tips. Furthermore, fresh shoot tips, the shoot tips precultured with sucrose solution, and the recovered shoot tips after cryopreserved in LN presented the water in the meristems which formed ice under Cryo-SEM investigation. Meanwhile, the glass state of shoot tips occurred when the shoot tips were cryoprotected by PVS2 solution.In conclusion, potato shoot tips had the temporal and spatial specificity of vitality loss and survival during droplet-vitrification, and PVS2 solution is considered as the critical steps to succeed the cryopreservation by decreasing the water content and forming the glass state of the meristem cells. |
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