Study of Cryopreservation and Freezing Behavior in Almond Winter Bud

Abstract

The aim of this study was to achieve the cryopreservation of almond winter dormant buds using a two-step freezing method and to investigate the freezing behaviors of almond cultivars. A two-step freezing cryo-method was used after desiccation to 18–28% moisture content, and recovery conditions such as dark incubation and rehydration in sterile moist moss grass for various durations were also used for enhancing survival. Cultivars such as ‘Almond-IXL,’ ‘Merced,’ ‘Pranyaj,’ ‘Primoriskij,’ and ‘Waris’ underwent initial recovery after cryopreservation. The highest recovery was observed in the ‘Merced’ cultivar, followed by ‘Pranyaj.’ The freezing impact in different cultivars of almond dormant buds was also investigated using scanning electron microscopy (SEM) and light microscopy. All living cells in bud tissues exhibited distinct shrinkage without intracellular ice formation as a result of slow cooling (5 °C/day) of dormant buds to −30 °C, detectable by SEM. However, the re-crystallization experiment of these slowly cooled tissue cells, which was carried out by further freezing of slowly cooled buds with liquid nitrogen (LN) and then rewarming to −10 °C, confirmed that some of the cells in the apical meristem lost freezable water with slow cooling to −30 °C, indicating adaptation of these cells by deep supercooling. Deep supercooling could not occur in dormant bud primordia if xylem vessels formed a continuous conduit connecting the dormant bud primordia with the remainder of the plant. If xylem continuity was established, ice could propagate via the vascular system and nucleate the water within the primordia. It is concluded that no extracellular ice crystals accumulated in such tissues containing deep supercooling cells with thin cell walls. Some cultivars in which ice crystallization was observed could not be successfully cryopreserved.