2.2. Freeze test
The effects of growth temperature, photoperiod and cold acclimatisation
on frost tolerance were assessed by electrolyte leakage
from leaf discs of kale leaves. Discs (diameter 11 mm) were cut out
between main veins of the leaf number eight from the top leaf (the
second fully extended leaf). Sixteen leaf discs from each treatment
were placed between sheets of moistened paper towels and packed
in moist sand in 2 l plastic boxes. The boxes were placed at 0.5 C
overnight before being moved to a temperature-regulated freezer
at 3 C for 17 h. Temperature was lowered at a rate of 1 C h from
3 to 10 C and then 3 C h until 17 C, and the boxes were
removed each second degree. The temperature in the boxes was
recorded with a thermocouple probe. After freezing, plants were
thawed overnight at 4 C. Leaf discs were then removed from the
sand, and four leaf discs were placed in 5 ml of distilled water
for 1.5 h in tubes with a lid. Leaf discs were also placed at continuous
0.5 C for comparison. The electrical conductivity of the
extract was measured by a digital conductivity metre (712 Conductometer,
Metrohm, Switzerland) at room temperature. Each treatment
had four replicates at each freezing temperature.