Freezing Tolerance Test
Protocol III: Monitoring plant survival by whole plant freezing tests
The protocol described here is based on the protocol of Xin and Browse (1998). Although
this protocol was developed for seedlings grown in petri dishes, we have also
successfully used it for assessing tolerance of soil-grown Arabidopsis plants in pots (Guo
et al., 2002, Gong et al., 2002).
1. Plate sterilized Arabidopsis seeds in petri dishes containing sterile media (MS or
Gamborg’s medium), solidified with 0.9 % agar. Sucrose is omitted from the
media to minimize contamination. All genotypes to be tested should be plated in
different sectors of a single plate.
2. After 10-12 days of growth, plates can be transferred directly to a freezing
chamber set at -1 ± 0.1 oC, (dark) to check the basal cold tolerance. Alternatively,
if seedlings are to be cold-acclimated, the plates are first transferred to a cold
room set at 4 ± 2 oC, under constant light for 2 to 7 days.
Note: Light plays an important role during the process of cold
acclimation (Kim et al., 2002) and therefore cold acclimation should be
done under normal light conditions.
3. Sprinkle the plates with finely crushed ice and incubate for at least 16 h at -1 °C.
Note: Finely crushed ice can be made by grinding ice chips with a liquid
nitrogen-cooled mortar and pestle. Finely crushed ice should be used to
avoid physical injury to the seedlings by large ice crystals.
4. Decrease the temperature of the freezing chamber by 1 oC per h and remove petri
dishes at desired temperatures (usually at 1 °C or 2 °C intervals). After removal
from the freezing chamber, incubate the plates at 4 oC for 12 h in darkness.
5. Return the petri dishes to the original growth conditions.
6. After 2 days score the survival of seedlings visually and calculate the LT50.
Note: The experiment should be replicated at least three times with a
minimum of 3 plates per temperature.
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Freezing Tolerance Test
References
Gong Z, Lee H, Xiong L, Jagendorf A, Stevenson B, Zhu J-K (2002) RNA helicaselike protein as an early regulator of transcription factors for plant chilling and
freezing tolerance. Proceedings of The National Academy of Sciences USA 99:
11507-11512
Guo Y, Xiong L, Ishitani M, Zhu J-K (2002) An Arabidopsis mutation in translation
elongation factor 2 causes superinduction of CBF/DREB1 transcription factor
genes but blocks the induction of their downstream targets under low temperature.
Proceedings of The National Academy of Sciences USA 99: 7786-7791
Kim HJ, Kim YK, Park JY, Kim J (2002) Light signalling mediated by phytochrome
plays an important role in cold-induced gene expression through the Crepeat/dehydration responsive element (C/DRE) in Arabidopsis thaliana. Plant
Journal 6: 693-704
Xin Z, Browse J (1998) eskimo1 mutants of Arabidopsis are constitutively freezingtolerant. Proceedings of the National Academy of Sciences USA 95: 7799-7804
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