Cryo-scanning electron microscopic study on freezing behaviors of tissue cells in dormant buds of larch (Larix kaempferi)

Consideration of the reasons why dormant buds of trees have evolved extraorgan freezing as an adaptation for winter survival

“…2) but also the freezing response of primordial cells. Our previous cryo-SEM study indicated that primordial cells of whole dormant buds of larch slowly frozen (5 • C/day) to −30 • C exhibited only a slight shrinkage, although the shrunken cells included both cells exhibiting supercooling with incomplete dehydration and cells exhibiting extracellular freezing with a complete loss of freezable water (Endoh et al, 2009). The conditions in that study were similar to the freezing protocol used on isolated primordia in the current study.…”

“…Thus, no extracellular ice crystals were produced within primordial tissues (Endoh et al, 2009). A similar absence of extracellular ice in primordia has been reported in flower buds of blueberry exhibiting extraorgan freezing (Flinn and Ashworth, 1994).…”

“…All of these previous studies reported that isolated primordia exhibited a greater level of freezing injury than whole buds when they are subjected to freezing temperatures. Results of differential thermal analysis (DTA) indicated that the tissues in isolated primordia exhibited freezing at higher temperatures suggesting intracellular freezing (Sakai, 1979;Ishikawa and Sakai, 1981;Quamme et al, 1995;Kang et al, 1998), while cells in whole buds exhibited deep supercooling (George et al, 1974;Sakai, 1979;Kaku et al, 1980;Warmund et al, 1991;Sakai and Larcher, 1987;Quamme, 1995;Endoh et al, 2009). Direct evidence of the occurrence and possible causes of intracellular freezing in isolated primordia, however, have not been obtained.…”

“…For examining the presence or absence of freezable water in cells slowly frozen to given temperatures, a recrystallization experiment was performed (Kuroda et al, 2003;Endoh et al, 2009). Briefly, some of the samples that had been slowly frozen (5 • C/day) to a given temperature were put directly into liquid nitrogen (LN 2 ) for a few minutes and then rewarmed at −20 • C overnight and finally cryofixed at −20 • C. In the recrystallization experiment, when freezable water remained with slow freezing to a given temperature, freezing of such samples by LN 2 produced intracellular ice crystals, although such ice crystals were difficult to detect due to their very small size under the condition of freezing, and rewarming of such samples at −20 • C produced large intracellular ice crystals due to recrystallization of small ice crystals.…”

“…In such trees, bud development starts during the active growing period in late spring or early summer, continues during the summer, and the formation of a resulting in the development of large masses of extracellular ice crystals only in localized areas. No extracellular ice crystals are formed directly within vegetative or flower primordia of dormant buds, that exhibit extraorgan freezing (Flinn and Ashworth, 1994;Endoh et al, 2009).…”