Pits of a softwood (Pinus wallichiana) and a hardwood (Mallotus japonicus) were studied by Cryo-scanning electron microscopy (Cryo-SEM). The samples were chemically untreated and fully hydrated during freezing. Tori of P. wallichiana were frequently aspirated. It is not clear whether torus aspiration was caused by the freezing process or other factors during preparation. Aspirated tori had a turgid appearance. Offcenter aspirated tori which did not completely cover the pit pore could be detected. The margo strands were usually quite large and fanned out into fibrillar structures at the pit border. Pit membranes of M. japonicus had a very granular appearance. No fibrillar structures, different layers or pores could be detected. The granular structures may have developed from a continuous layer covering the pit membrane during freezing. Cryo-SEM is discussed as a further suitable tool for obtaining novel information about the native state of pit membranes. Future studies are needed to validate whether all observed features represent characteristics of the native state.
In this study, the ice nucleation activity (INA) and ice nucleation temperature (INT) as well as extracellular ice formation within the bark were determined for three woody species with different degrees of frost resistance, Betula nana, Betula albosinensis and Castanea sativa. Current-year stems and at least 2-year old stems of B. nana and C. sativa as well as current-year stems of B. albosinensis were compared, during summer (non-acclimated state) and winter (acclimated state), to evaluate possible ontogenetic and seasonal differences. Acclimated plant parts of the selected species revealed nearly similar results, with an INT from -7.52 to -8.43°C. The current-year stems of B. nana had a somewhat higher INT than the older stems. Microscopic analysis showed that extra-cellular ice formation occurred in the intercellular spaces within the bark of stems of B. nana, B. albosinensis and C. sativa. Size of the intercellular spaces of the bark were species-specific, and B. nana showed the largest intercellular space volume. While freezing behavior and extracellular ice formation thus followed principally the same pattern in all considered species, B. nana is obviously capable of dealing with large masses of extracellular ice which accumulate over extended periods of frost, making B. nana capable of protecting living tissue in colder regions from freezing damage.