In vitro cold acclimation is required for successful cryopreservation of Juglans regia L. shoot tips.

Background: Persian walnut (Juglans regia L.) is one of the most economically important nut crops. In the Western Tien Shan in Kazakhstan, walnut forests are the northernmost in the natural range of this species. Shoot tip cryopreservation is an important strategy to ensure long-term clonal conservation of plant genetic resources.

Objective: To determine the effect of cold acclimation duration (0-6 weeks) with alternating temperatures (8 h at 22 degree C, light intensity 10 μmol m^-2 s^-1/16 h at -1 degree C, in the dark) and of plant vitrification solution 2 (PVS2) exposure duration (30, 50, 80 or 100 min) on shoot tip regrowth after cryopreservation by vitrification.

Materials and methods: In vitro-grown shoots of three wild Juglans regia accessions from a native population of Sairam-Ugam National Natural Park in the south of Kazakhstan and of cultivar Milotai 10 were used as sources of plant material. Shoot tips (1.8-2.0 mm with five to six leaf primordia) were excised from in vitro-grown shoots and cryopreserved using PVS2 vitrification technique.

Results: Regrowth of cryopreserved shoot tips increased and was significantly higher (P < 0.05) after 4-6 weeks cold acclimation with the highest regrowth between 59.9-67.8 degree after 5 weeks for the four genotypes tested. The highest regrowth level was obtained after 80 min of PVS2 exposure, which was significantly better (P < 0.05) compared to 30, 50 or 100 min exposure.

Conclusion: The PVS2 vitrification protocol established is very effective for cryopreservation of both unique wild J. regia germplasm and of walnut cultivars. Doi: 10.54680/fr23410110612.