The nucellar cells of navel orange(Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved by vitrification. In this method, cells were sufficiently dehydrated with highly concentrated cryoprotective solution(PVS2) prior to direct plunge in liquid nitrogen. The PVS2 contains(w/v) 30% glycerol, 15% ethylene glycol and 15% DMSO in Murashige-Tucker medium(MT) containing 0.15 M sucrose. Cells were treated with 60% PVS2 at 25°C for 5 min and then chilled PVS2 at 0°C for 3 min. The cell suspension of about 0.1 ml was loaded in a 0.5 ml transparent plastic straw and directly plunged in liquid nitrogen for 30 min. After rapid warming, the cell suspension was expelled in 2 ml of MT medium containing 1.2 M sucrose. The average rate of survival was about 80%. The vitrified cells regenerated plantlets. This method is very simple and the time required for cryopreservation is only about 10 min.
Dormant one—year—old twigs of about 70 tree species were collected during mid—winter from five tree regions in North America (Rocky and Western Mountain, Northern, Pacific Coast, Southeastern Coast, and Central and Eastern Lowlands). The twigs were artificially hardened in a regime of sub—freezing temperatures between —3 degrees C and —10 degrees C for 24 days to induce maximum freezing resistance. Four Northern species, Populus tremuloides, Populus balsamifera, Betula papyrifera and Larix laricina had the greatest freezing resistance of the species tested. They resisted freezing to —80 degrees C and even immersion in liquid nitrogen (—196 degrees C) following prefreezing to —15 degrees C. Most of the Northern and Rocky and Western Mountain conifers survived freezing between —60 degrees and —80 degrees C while several species from the Pacific and SoutheasternCoast regions, which have relatively mild humid winter climates, were hardy to only among —15 degrees C. The ranges of some Pacific Coast species such as Pseudotsuga menziesii, Thuja plicata and Tsuga heterophylla extend into the mountainous inland. Samples collected from inland sites were found to be much hardier than those from the coast. A similar trend was observed in the various collections of Tsuga heterophylla from northern, central and southern areas along the Pacific Coast. Winter minimal temperatures are among the important factors setting the northern boundaries of the natural ranges of many forest tree species. However, Populus deltoides and Salix nigra from locations with temperate or moderate winter climates survived freezing to at least —50 degrees C irrespective to their native habitats. In these species, winter minimal temperatures do not appear to be the principal factor governing geographic distribution.
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