tion process in a number of plant species [17,18] and, when exogenously applied, it has been shown
to induce cold tolerance of plant cell cultures [19 – 21] as well as whole plants [17,18,22]. In fact,
exogenously applied ABA is known to induce greater cold tolerance more readily than the accli-
mating low temperatures do [18,19,23].
In this study we attempt to characterize the role of glycine betaine in cold acclimation and in in-
duction of cold tolerance in strawberry plants.
2. Materials and methods
2
.
1
. Plant materials Strawberry Fragaria X ananassa Duch. cv. Ear-
liglow plants were grown in the greenhouse at 2318°C daynight temperatures under natural day
light in 15 or 22.5 cm pots containing a sterile growing medium of peat, perlite, and soil 2:2:1
vv. Plants were irrigated once every two days to field capacity and fertilized weekly with Peat-Lite
Special Scotts-Sierra Horticultural Products Co., Marysville, OH with N-P-K of 20-10-20 ammo-
nium nitrate, potassium phosphate and potassium nitrate at 250 ppm of nitrogen in irrigation water.
2
.
2
. Cold acclimation Four to five-week old plants grown in the green-
house were transferred to walk-in cold chambers set at 42°C daynight with a 10 h photoperiod
200 mE m
− 2
s
− 1
and were held for up to 4 weeks for cold acclimation. Soil mix was always kept
moist during the cold acclimation treatment to avoid any water stress to the plants.
2
.
3
. E6aluation of cold tolerance Cold tolerance in leaves was determined follow-
ing the procedure described previously [24]. Whole leaves were first wrapped in a moist paper towel
and subsequently, partially wrapped with alu- minum foil and placed in 2.5 × 20 cm test tubes.
The samples were cooled at 2°Ch in a pro- grammable freezer Tenney Engineering, NJ with
ice nucleation of samples between − 1 and − 2°C. Samples were removed from the freezer after
reaching various test temperatures and allowed to thaw at 4°C for 12 h. The thawed samples were
evaluated for injury using electrolyte leakage. Conductivity was measured in the leachate using a
YSI conductance meter Model 32, YSI Co., OH after incubating samples in a 10 – 15 ml aliquot of
distilled water for 10 h. The total electrolytes were released from the leaves by autoclaving at 121°C
for 15 min. The final conductivity measurement was made after incubating the samples for 10 h.
The midpoint in the transition of electrolyte leak- age was considered the killing temperature as out-
lined
elsewhere [24].
The experiment
was conducted with four replications in a completely
randomized design. Survival and regrowth experiments were con-
ducted using unhardened, whole plants. Plants were subjected to cooling at 2°Ch in a pro-
grammable freezer. Plants were seeded with ice between − 1 and − 2°C. After reaching the test
temperatures, plants were removed from the freezer and allowed to thaw at 4°C for 12 h. Plants
were subsequently transferred to a growth cham- ber at 20 – 18°C with a 12 h photoperiod for
recovery and regrowth. Freezing injury was evalu- ated on the shoots two days after the freezing
tests. Evaluation of plant survival was based on lethal injury to the shoots which typically did not
reflect the survival of regenerative crowns. Evalua- tion of regrowth was conducted periodically over a
4 week period.
2
.
4
. Exogenous ABA and glycine betaine treatments
Plants were sprayed with 100 mM 9 ABA mixed isomers Sigma Chemical Co., MO in 0.1
mM CaCl
2
with 0.02 Tween-20 until the shoots and foliage were covered completely with the solu-
tion. Control plants were treated with a solution containing 0.1 mM CaCl
2
and 0.02 Tween-20 foliar spray under similar conditions. Glycine
betaine solution 2 mM was applied to the leaves at 4°C since uptake has been reported to be more
efficient in bacteria at low temperatures 11. Un- hardened plants were transferred to 4°C for 30
min for glycine betaine treatment and preliminary experiments showed that this exposure to low
temperature did not affect their cold tolerance. Leaf samples were collected periodically over a 72
h period and thoroughly washed in distilled water before estimating the time-course accumulation of
glycine betaine. Excised leaves were washed with 2
mM glycine betaine solution and the leaf washing was incubated for up to 72 h at 23°C. Incubated
leaf washings were used to determine the glycine betaine content to ascertain whether glycine be-
taine applied to the foliage was broken down by the microflora.
2
.
5
. Determination of glycine betaine content Samples consisting of leaves or aliquots of solu-
tion were frozen in liquid nitrogen immediately after collection. Glycine betaine was quantified
using NMR methods as outlined by Jones et al. [25] with some modifications. Thawed leaf samples
were used to express sap using a Carver Labora- tory Press Fred S. Carver, Inc., IN. Ten ml of the
extract was dried under a stream of nitrogen in a desiccator, and the final volume was made up to 1
ml with D
2
O. Glycine betaine was quantified using a Varian UNITY Plus 500 NMR spectrometer
operating at 11.75 T 499.869 MHz for
1
H NMR and spectra were measured at 30°C using a 5 mm
triple-resonance inverse detection probe. One di- mensional proton spectra were acquired with 16
transients, using 19.2 K data points, a pulse repeti- tion rate of 2.0 s, a flip angle of 60°, and a spectral
width of 8,000 Hz centered on the water peak. The free induction decays were zero filled to 64 K prior
to Fourier transformation using Varian NMR software VNMR 4.3b. The peak intensities were
measured digitally using the spectrometer’s inte- gration software.
3. Results
