細胞外凍結するシラカンバ冬芽の超低温保存.

Japanese white birch (Betula platyphylla) is an economically important tree in boreal forests of Japan; therefore, it is desirable to clone this species for long-term ex situ conservation. We investigated the cellular tolerance of B. platyphylla winter buds to slow-freezing cryopreservation. Leaf buds were frozen from -3°C to -30°C at a cooling rate of 0.2°C/min. Cryo-scanning electron microscopy revealed the formation of extracellular ice crystals in intercellular spaces within the leaf primordia and bud scales, and freezing-induced dehydration was observed in all cells within frozen bud tissues. In a recrystallization experiment, no freezable water was observed in dehydrated cells within winter buds frozen below -30°C. After slow freezing to -40°C, leaf buds survived immersion into liquid nitrogen (-196°C), and shoots were successfully grown in vitro from the cryopreserved buds. We conclude that B. platyphylla buds have very high freezing tolerance and were protected from immersion in liquid nitrogen by extracellular freezing. Therefore, slow freezing of winter buds is a feasible method for preparing B. platyphylla buds for cryopreservation. [ABSTRACT FROM AUTHOR]