Utility of leaf disks cultured in vitro for studies on frost resistance

Abstract

Disks were isolated from young leaves of winter rape plants and grown in vitro at ambient (15°C) or low (2°C) temperatures for two weeks. In the control disks the growth cessation and beginning of chlorophyll degradation were observed after 1 week of culture. In the low-temperature treated disks the expansion of cells was slower than that in the control material but it continued for two weeks and was accompanied by a marked accumulation of dry matter. Practically, no chlorophyll degradation was observed. The low temperature treatment brought about the decrease in the frost killing temperature of the tissue which was associated with its increased capacity to subcool water. A short (18 h) exposure of the cold-grown leaf disks to slight frost (−5°C) increased further their resistance to freezing, despite the fact that the subcooling capacity of disks decreased in result of the treatment. Therefore, the two stages of hardening, observed previously for the whole plants, can also be detected in the isolated material. In the cold-grown disks, a transient accumulation of reducing sugars but a steady decrease in ATP and water-soluble protein contents were observed. These observations indicate that tissue isolation might affect processes involved in the functional adaptation of cells to cold.