Cryopreservation of Fraxinus mandshurica Rupr. by Using the Slow Cooling Method

Abstract

Cryopreservation is an important method for the excellent long-term preservation of plant germplasm. This study explores an optimal cryopreservation technology for the embryogenic callus of Fraxinus mandshurica to effectively maintain its genetic stability and morphogenesis potential. The optimal cryopreservation conditions were assessed using the embryogenic callus of F. mandshurica as the material, and the slow cooling method was optimized for its cryopreservation. The results indicated that the preculture of embryogenic callus in 0.4 mol·L−1 sorbitol solution for 20 h at room temperature, followed by its cryoprotection in 7.5% dimethyl sulfoxide solution at 0 °C for 90 min, constituted the optimal material treatment method. The freezing tube was placed in a −80 °C refrigerator for 2 h and then quickly put into liquid nitrogen for frozen storage. During thawing, the cryopreservation tube was taken out from liquid nitrogen, directly placed in a water bath at 40 °C for 2 min, and used for culturing on the woody plant media + 0.1 mg·L−1 6-benzyladenine + 0.15 mg·L−1 2, 4-dichlorophenoxyacetic acid. After cryopreservation using the slow cooling method, the highest survival rate of callus cells was 80.82%. The fresh weight reached 1.93 g after 60-day recovery culture. The regeneration rate and the proliferation coefficient of the callus were 100% and 2.79, respectively. The differentiation rate was 56.83%, and the emergence rate was 23.59%. The results provide a scientific basis for the long-term preservation of F. mandshurica germplasm resources.

Development of Cryopreservation Technique for Meristems of Syringa vulgaris L. Cultivars

“…Since the application of the droplet-vitrification method on lilac had some issues [28], it was decided to use a cryopreservation method focused more on the increase of plant cryoresistance. The technique developed in IPP RAS was tested in this study [35]. This method was already used in IPP RAS for the long-term storage of strawberries, blackberries, raspberries and rowan [36,41,42].…”

“…However, follow-up experiments revealed some issues: explants survived well after freezing, but they had problems with sprouting into shoots [29]. In addition, there are data on the application of the encapsulation/dehydration method for long-term lilac preservation [30] and published cryogenic protocols for other genera of the Oleaceae family [31][32][33][34][35]. This explains why the development of effective and comprehensive cryopreservation technique for Syringa remains a critical issue.…”

Current status of the cryopreservation of embryogenic material of woody species

“…Direct immersion in LN of embryogenic explants is frequently used, but positive results have also been reported after slow cooling (Jekkel et al, 1998;Vendrame et al, 2001;Scocchi et al, 2007;Ozudogru et al, 2010;Yu et al, 2022). Rapid thawing approach is employed in most protocols, whereby the cryopreserved tissues are plunged into water at 40-42°C for 2-3 min (Supplementary Material 3).…”

Improved Method for Cryopreservation of Embryogenic Callus of Fraxinus mandshurica Pupr. by Vitrification

“…The method of obtaining EC of F. mandshurica referred to the method of Yu et al [16]. Specific methods: Immature seeds were collected in early August from free-pollinated parent trees present on the campus of Northeast Forestry University, Harbin, Heilongjiang Province, China (126 • 37 55 E, 45 • 43 16 N).…”

“…This is why we are working on the development of cryoprotectants techniques for F. mandshurica. We have recently reported on a new system for the slowcooling of EC of F. mandshurica [16]. However, the lack of a programmable cooler to precisely control the temperature range and rate of cooling has led to some concerns about the stability of EC preservation results.…”