The limitations in the preservation of endemic plant species are variable because of their high diversity, geographic range, and/or accessibility. Research on this matter is urgent and the potential for future scientific studies on the cryopreservation of endemic plant species is enormous. Several factors and their interactions influence the success of any cryopreservation protocol. Understanding these factors, such as the length of dormant buds, preculture, plant physiology and dehydration period, will contribute to a faster and easier optimization of new cryopreservation procedures (Charoensub et al. 2003; Chen et al. 2011; Lambardi et al. 2000; Niino et al. 1992). In general, the small material can be seriously damaged in dehydration process which is difficult to regenerate; however, if the material is too large, the cryoprotectant solution is difficult to infiltrate into the interior cells, the material may not be protected well in the following freezing step, and the regeneration rate is also low. It is found that the optimal shoot-tip size of sweet potato [ Ipomoea batatas (L.) Lam. ] was 0.5-1.0 mm long (Pennycooke and Towill 2000). The survival rate of 1.5-3.0 mm long garlic shoot apices after cryopreservation was as high as 85%, and the regeneration rate was more than 73% (Baek et al. 2003). Wang et al. used in vitro-grown shoot tips of Vitis vinifera L. as experimental materials and found that the highest survival (about 65% ) of cryopreserved shoot tips was obtained in 1.0-1.5 mm length (Wang et al. 2003). In our study, high regeneration rate was obtained using 2–5 mm long dormant buds. Therefore, the optimal size of dormant buds need to be investigated depending on material and protocol for cryopreservation.
Materials pretreated can effectively reduce the water content of tissues, and enhance the resistance to low temperature, so that the materials can maintain high viability after cryopreservation (Salaj et al. 2010; Hong and Yin 2012; Sershen et al. 2012). Sucrose preculture and cold acclimation are the two most commonly used methods for improving plant recovery in cryopreservation. In many cryopreservation documents, such as Vitis (Volk et al. 2018), kiwifruit (Mathew et al. 2018), potato (Kaczmarczyk et al. 2008), Pyrus cordata (Chang and Reed 2001), Prunus cerasus (Barraco et al. 2012), etc all obtained good results using high concentration of sucrose preculture. In this study, both survival rate and regeneration rate of the dormant buds of F. przewalskii reached the highest (93%) when the buds were precultured on 0.5 M sucrose for 3 days. It indicated that high concentration of sucrose in the medium could induce cell dehydration by increasing osmotic pressure, thus reduced the content of free water in cells and improved its cold resistance (Bettoni et al. 2021; Chen et al. 2014; Kulus et al. 2018). Many studies have shown that cold acclimation can activate the cold resistance mechanism in plants and improve the survival of dormant buds in cryopreservation, especially for low-temperature sensitive plants (Channuntapipat et al. 2000; Engelmann 2011; Harding et al. 2009). For example, with cold hardening 6–14 weeks, the regrowth rate of the dormant buds of Malus domestica Borkh. had a maximum of 88% regrowth in the 6th and 8th week and were still capable of regenerating at 14 weeks (Bilavčík et al. 2012). The survival rate of blueberry was highest (91%) when the shoot tips were treated at low temperature for 2 to 4 weeks (Wang et al. 2017). In present study, both survival and regeneration rates of the dormant buds of F. przewalskii after cryopreservation were increased with the extension of time of cold acclimation.
Vitrification solution makes intracellular water too late to form ice crystals or ice crystals do not have enough time to grow, thus the cells turn a state of artificial complete vitrification (Sakai et al. 1990; Volk and Walters 2006; Rall 1987). At present, the most commonly used vitrification solution is PVS2, and the optimal dehydration time of PVS2 is varied among different plant species (Bilavčík 2012; Wu et al. 2006; Matsumoto et al. 2015; Sant et al. 2006). If the PVS2 treatment time is insufficient, the cell dehydration is not complete, and the cells are easy to form ice crystals when frozen in liquid nitrogen, and the cells will be seriously injured. On the other hand, if the treatment time is too long, the material can be poisoned by PVS2, the survival rate and regeneration rate will significantly reduce (Matsumoto et al. 2015; Wang et al. 2014). Therefore, the appropriate dehydration time in PVS2 can reduce cell damage and improve cell tolerance to freezing. The results of this experiment showed that both PVS2 and PVS3 were good vitrification solution.
Unloading is a normal step in cryopreservation process. But in our study unloading had little effect on the survival and regeneration of the dormant buds after cryopreservation, which is in accordance with cryopreservation for dormant buds of Codonopsis pilosula (Franch.) Nannf. (Zhang et al. 2020) and Astragalus membranaceus (Fisch) Bge. Var. Mongholicus (Bge) Hsiao (Dong et al. 2022). It indicated that dormant bud possessed strong resistance to cryopreservation.
The alternation and damage of dormant buds in process of cryopreservation can be observed by means of histological section. After dehydration of the dormant buds with the loading solution, the cells in each part lost water and showed slight plasmolysis. The cells were subjected to higher osmotic pressure and dehydrated severely by PVS2 treatment, which resulted in further plasmolysis (Popov et al. 2004). Cryopreserved cells were subjected to mechanical damage at the same time, presenting severe plasmolysis, cell membrane rupture, nuclear fragmentation and nucleolus disappearance. After direct cryopreservation of dormant buds, the cells in other parts were irreversibly damaged except for the apical meristem and leaf primordium, which still maintained integrity. The occurrence of cell damage is likely due to the extreme decline in water content, resulting in the inability of protoplasts to contract and concentrate cell fluid (Ganino et al. 2012). In addition, the freezing damage that causes cell death is caused by intracellular water crystallization during freezing or thawing. Therefore, the freezing and thawing process had severe damage to the buds, mainly manifested as the breakage of the cell wall of the bud axis, the blurring of the nuclear boundary, the ablation of the cytoplasm, and the disappearance of the nucleolus. Shoot apical meristem and leaf primordium are the least damaged parts, although there has slight plasmolysis in these cells, but the cell structure still maintains integrity. These two parts are the key parts of survival. Similar phenomena were also observed in the cryopreservation of apple (Feng et al. 2013), Rubus (Chang and Reed 1999), Dendrobium (Antony et al. 2011; Ching et al. 2012; Poobathy et al. 2013), Brassidium (Mubbarakh et al. 2014) and other plants.
The genetic stability of cryo-derived plants is the prerequisite that the technique applied to plant germplasm conservation. In our study, dormant buds directly produce bulblets without producing callus, so the risk of genetic variation is low. We did not found any phenotypic and DNA alteration in cryo-regenerated plantlets of F. przewalskii. Similarly, genetic stability has also confirmed in the shoot tips or dormant buds after cryopreservation in Dioscorea rotundata Poir (Mandal et al. 2008), apple (Liu et al. 2008), sugarcane (Kaya and Souza 2017), Dendranthema grandiflora Tzvelev (Martín and González-Benito 2005), grape and kiwi (Zhai et al. 2003). The genetic fidelity of regenerated plants ensures the established vitrification-cryopreservation protocol as a reliable technique for long term storage of F. przewalskii germplasm.