The Use of Callus Cultures for Searching Proteins Associated with Increased Freezing Tolerance During Cold Acclimation in Lonicera caerulea

Abstract

To elucidate the molecular events underlying cold acclimation in woody plants, we established an in vitro system that responds to cold treatment, using calli of blue-berried honeysuckle (Lonicera caerulea L. var. emphyllocaryx Nakai). The calli that had been exposed to low temperature at 5°C increased their cold tolerance after 8 days of the treatment, reaching a plateau on day 12. The cold-treated calli had an approximately 60% survival rate at -7.5°C, while the survival rate of non-acclimated calli at the same temperature was below 10%. The cold-treated calli lost their freezing tolerance within 2 days of incubation at 25°C. The proteins extracted from these calli were analyzed by 2-dimensional polyacrylamide gel electrophoresis. As a result, a 42kDa (pI5.4) protein was shown to accumulate in abundance during cold acclimation. The protein decreased by culture at 25°C. These results suggest that the callus of L. caerulea did cold-acclimate in response to low temperature conditions, and that it could be used for the investigation of proteins involved in the incremental development of freezing tolerance.