sodininkystė ir daržininkystė 25(4)

For the Z experiment, root pieces (2–4 mm diameter) were randomly selected
from apple rootstocks and cut into 3–4 cm long segments. The samples were prepared
and frozen as described by Privé and Zhang (1996). Cooling rates were programmed
at 4.8°C·hr -1 and held for one hour at the pre-selected test temperatures of
-3, -6, -9, and -12°C, then removed and allowed to thaw for 20 minutes at 20°C
before being analysed using Z (this is considered a 1X freeze-thaw treatment). One
advantage of measuring tissue impedance (Æ) as an indicator of physiological status
is that the tissue is not destroyed while useful data are being obtained. This permitted
the same root segments to be analyzed for multiple freeze-thaw events. Hence, our
samples were again placed into the test tubes, re-frozen at the pre-selected test
temperatures for a second time, re-thawed and re-analysed for a second time (treatment
2X). This procedure was repeated once again (treatment 3X).
For the rootstock seedling experiments, seeds were stratified in moist sand at 3
to 5°C for 9 weeks. At the first sign of radicle emergence, 60 seeds for each of the
three genotypes were planted in a soilless mixture in Styro seedling trays (240 cells/
tray, Beaver Plastics Ltd., Edmonton AB, Canada). All exterior cells of the tray were
filled with seedlings and acted as buffers for the treatment seedlings. Plants were
grown in a greenhouse for 6–8 weeks under good growing conditions (16 h, 20–
30°C, 900 μmol·m -2·s -1 , fertilised with 200 ppm of 10N-52P-10K once a week for 3
weeks followed by 200 ppm of 20N-20P-20K at 3x/week for another 3 weeks until
plants were 5–7 cm.). Seedlings were then acclimated in controlled environment
chambers (8 h of 600 μmol·m -2·s -1 , 12-15°C/3–4°C D/N, 100 ppm 20N-20P-20K
once a week for 6 weeks). Two frost events (-3°C for 16 h) were then given in the
next two weeks while growing at 10/3°C D/N and 8 h light periods. Following this,
the trays were placed in insulated boxes with vermiculite and heating cables to prevent
the roots from freezing and placed in programmable freezers. Cooling rates
were programmed at 2°C·hr -1 and held for one hour at the pre-selected test temperatures
of -10, -20, -25, -30, and -40°C, removed and held in cold storage (3–5°C)
overnight.
Cold hardiness evaluations. For the whole-plant controlled freezing experiment,
tree mortality was recorded, final root volume and trunk cross-sectional area (TCSA)
were measured to calculate the growth increment over the season, and new shoot
growth dry weight was determined after oven drying at 80°C for one week. Root
and shoot regrowth data were only taken from surviving trees at each of the selected
temperatures. Validation of mortality was done by comparing our laboratory results
with field results (percent survival) from the Champlain Valley in New York, USA in
2004.
For the electrical impedance experiment, the spectra between 100 Hz and 800
kHz were measured by a computer-controlled Hewlett Packard 4284A Precision
LCR Meter using needle electrodes (Zhang and Willison, 1992). The impedance
spectra were analyzed by a complex nonlinear least squares program called BIA,
which was written in Visual Basic (available upon request from Dr. Zhang). The
formula used in the program was: Z = R ∞
+ (R o
- R ∞
) / [1 + (jωτ) ψ ], where Z is
impedance; R o
corresponds to extracellular resistance or the resistance at extremely
low frequency; R ∞
corresponds to total tissue resistance or the resistance at
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