Cold-Hardiness Evaluation of Grapevine Buds and Cane Tissues

Cold-hardiness determination.

Dormant buds from nodes four through eight (to preserve the more basal nodes for spur-pruning) were cut from canes of field-grown grapevines (see below for cultivar and vineyard details). Approximately 2 mm of intact cane tissue surrounding and underlying the bud was left to allow the bud to supercool (Andrews et al. 1984, Wolf and Pool 1987). Four to five buds were placed directly on each TEM with the cut surface facing up; the attached cane tissue prevented the buds from flipping over. Approximately 35-mm long cylindrical sections of cane were cut from the internode area between nodes four and eight, and two to three such cane pieces were placed directly on each TEM. Cane pieces were left intact, since previous experience indicated that longitudinal sectioning did not improve the TEM output (unpublished data). One TEM in the top tray was left empty as a control for signal noise that was common to all TEMs. Preliminary tests showed that one empty TEM was sufficient for all four trays and that one empty TEM per tray did not improve accuracy. Foam insulation pads (4 cm x 4 cm x 9 mm thick) were placed on top of buds or cane pieces in each well to ensure adequate contact between the tissues and TEM. The chamber lid was then tightened (Figure 1B⇑) and the chambers placed in the freezer. Up to four chambers were stacked in the freezer (Figure 1D⇑) for a maximum of 35 TEMs loaded per run (175 buds or 105 cane pieces). The freezer was programmed to hold at 4°C for 1 hr, drop to −40°C in 11 hr (a cooling rate of 4°C/hr), hold at −40°C for 1 hr, then return to 4°C in 10 hr (a warming rate of 4.4°C/hr). The DAS recorded signals from each TEM at 15-sec intervals and downloaded voltage output directly to Excel. Exotherms were identified manually from a plot of thermistor output (x axis) versus loaded-TEM output minus empty-TEM output (y axis). Lethal temperatures for buds were reported as Bud LTE₁₀, Bud LTE₅₀, and Bud LTE₉₀, the temperatures at which 10%, 50%, and 90% of the buds were killed, respectively (Andrews et al. 1984). These values were determined from the bud exotherm range and distribution that were clearly visible as spikes on each TEM (Figure 2⇓). For canes, lethal temperatures were reported as phloem LTE₁₀ and xylem LTE₁₀. These values were determined by assuming the first range of small exotherms just below −10°C indicated death of phloem cells and the larger exotherms as the temperature continued to decline (Figure 2⇓) indicated death of xylem parenchyma and pith (Wample et al. 2001). Once these ranges were established, the temperatures at which 10% tissue damage occurred were then estimated for each TEM.

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Figure 2

Typical DTA profiles of low temperature exotherms (LTE), indicating lethal intracellular freezing, from two single TEM containing either five buds (lower plot) or two cane pieces (upper plot) of Malbec sampled on 23 Feb 2005, in Prosser, WA. High temperature exotherms (HTE), indicating nonlethal extracellular freezing, are shown to the right of the dashed −10°C line.

Comparison of DTA systems.

To test the updated system, bud and cane samples of the same grape cultivar, sampling date, and location were run simultaneously in both systems. Following dissection, 40 buds and 16 cane pieces were thoroughly mixed for each of eight cultivars (see below) and then randomly assigned to one of the two DTA systems with four replicate TEMs each (each TEM containing five buds or two canes). The two systems were run with identical temperature regimes (as described above) on each sampling date. This test was conducted from January through March 2005, as grapevines progressed from maximum cold hardiness to approaching budbreak, to compare as wide a range of LTEs as possible.

Relationship between LTE and cane damage.

Cane exotherm output of the updated DTA system was also compared with tissue browning (indicating tissue death). Because exotherms in cane tissues are more difficult to interpret than bud exotherms (Wample et al. 2001), it was important to confirm the accuracy of our TEM interpretation. Dormant canes from field-grown Chardonnay and Cabernet Sauvignon were collected on 9 March 2005, as described above. A portion of the canes were stored in a 2°C cooler and held for tissue damage verification following LTE analysis. Two internode pieces from the fourth to seventh nodes were placed on each of four TEMs (four replicates). The freezer was programmed for the standard 4°C/hr decline and LTE analysis was performed. Using the critical temperatures established from this LTE analysis, the remaining canes were exposed to a 4°C/hr cooling rate and then removed at the temperatures where LTEs indicated phloem and xylem damage (expected damage). Twelve two-bud cane pieces for each damage level (no damage, 20% phloem damage, 100% phloem damage, 20% xylem damage, and 50% xylem damage) were frozen to the predetermined temperatures. The temperature at which 20% damage of phloem and xylem occurred was used rather than the 10% damage (LTE₁₀) normally reported in order to ensure consistent damage. After freezing, the canes were allowed to set at room temperature for 24 hr, and then sectioned to confirm tissue browning visually (Figure 3⇓), as described by Andrews et al. (1984). One cross-section in the internode area of each cutting was made, and the fraction of brown tissue compared with the total surface area was estimated using a scale of zero (0% damage) to 10 (100% damage). The observed damage (determined from tissue browning) and expected damage (determined from exotherm output) were compared using χ² analysis.

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Figure 3

Cross sections of (A) uninjured grapevine composite bud, (B) bud killed by lethally low temperature, (C) cane with approximately 20% freezing-induced phloem injury, and (D) cane with 100% freezing-induced xylem injury.

Effect of surface moisture on freezing.

The influence of surface moisture on cold injury of bud and cane tissues was tested by performing LTE analysis on dormant canes collected on 15 March 2005 from field-grown Cabernet Sauvignon as described above. Canes were cut into three-bud sticks, and ends were sealed with household paraffin to prevent desiccation from or water infiltration through the cut ends. The canes were then assigned one of three treatments with 10 canes in each. The dry treatment was sealed in a plastic bag with drierite (W.A. Hammond Drierite Co., Xenia, OH), while the moist and untreated treatments were sealed in plastic bags. All three were stored in a 2°C cooler for 48 hr. One hour before samples were prepared for LTE analysis, the moist treatment was misted with water applied by a hand sprayer then held in the sealed plastic bag in the 2°C cooler. Low temperature exotherm analysis was performed as described above using four replicate TEMs per treatment.

Comparison of cultivars.

Once it was established that the two systems were detecting exotherms at essentially the same temperature (Figure 4⇓), data from both systems were combined to compare the seasonal bud and cane LTEs of selected winegrape cultivars. Samples were collected as described above from mature (>20 years old), own-rooted, field-grown, spur-pruned V. vinifera cvs. Riesling, Chardonnay, Cabernet Sauvignon, Merlot, and Malbec located at the Washington State University Irrigated Agriculture Research and Extension Center in Prosser (46°17′ N; 119°44′ W; elevation 270 m; for vineyard details see Keller et al. 2005) and from Viognier, Pinot gris, and Syrah located at adjoining vineyards with similar soil and management conditions. All eight cultivars had been run weekly in the standard system starting in Nov 2004, so data were available for the entire season. For each cultivar, four replicate TEMs with five buds each (20 buds total) and four TEMs with two canes each (eight cane pieces total) were run for each sample date as described above.

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Figure 4

Relationship between low temperature exotherm (LTE) values obtained using duplicate bud and cane samples collected from eight V. vinifera cultivars between January and March 2005 and run simultaneously on the standard and the updated DTA system for cold-hardiness evaluation. Each data point represents the mean output of four TEMs containing either five buds or two cane pieces each (r = 0.92, p < 0.001, n = 113).

Data analysis.

Statistical analyses were carried out using the Statistica software package (version 7.0; StatSoft, Tulsa, OK). Data for system comparison were tested using regression analysis. Data for cultivar comparison and the surface moisture experiment were tested using one-way analysis of variance. Duncan’s new multiple range test was performed for post-hoc comparison of means.

Interpretation of exotherm patterns.

A representative DTA profile (exotherm pattern) from the updated system for cold-hardiness evaluation, in this case using Malbec buds and canes, is shown in Figure 2⇑. The HTEs between −5°C and −10°C are associated with nonlethal freezing of extracellular water (Andrews et al. 1984). Bud LTEs between −19°C and −24°C correspond to intracellular ice formation, which results in lethal bud damage. The 5°C range over which these bud exotherms occurred clearly shows that not all buds freeze at the same temperature. This LTE range was generally wider (3°C to 7°C) during the acclimation and deacclimation periods in fall and spring, in contrast to a narrower range (2°C to 3°C) in winter when buds were fully acclimated. Phloem and xylem LTEs were much less pronounced than the bud LTEs, which is consistent with previous findings (Wample et al. 2000) and generally makes interpretation of cane damage difficult. Phloem damage was associated with the bumps between −14°C (injury beginning) and −18°C (injury complete; no live phloem tissue), whereas xylem damage began at ~ −22°C and was complete (no live xylem parenchyma) at ~ −32°C (Figure 4⇑). The “fuzzy” nature of these exotherms necessarily makes interpretation somewhat subjective and requires a skilled operator for consistent results (see the discussion below on the relationship between LTE and cane damage).

Comparison of DTA systems.

The two systems closely agreed (Figure 4⇑); the slope of the regression line was not significantly different from 1.0. This confirms that the updated system detected exotherms at the same temperatures as the standard system. Therefore, cold-hardiness values obtained with the updated system may be directly compared with those from the standard system. However, the updated system was much more efficient and user-friendly, since it was far easier to operate, permitted a more rapid sample throughput, was physically more compact, and allowed direct data transfer to Excel which streamlined the data analysis.

Relationship between LTE and cane damage.

Chardonnay and Cabernet Sauvignon had a small amount (5 to 8%) of phloem damage before being subjected to DTA analysis (Table 1⇓), which may have been caused by a slight freeze before sample collection or by mechanical injury. Not all canes showed phloem injury at exactly the same temperature. Similar to the buds, the range of phloem LTE₁₀ was broader in the fall and spring (≤7°C) than it was during midwinter (≤3°C). However, as expected, the severity of cold injury increased as temperature declined (Table 1⇓). There were only minor differences between expected damage and observed damage, indicating that actual extent of phloem and xylem injury coincided with damage predicted from the interpretation of LTEs. For instance, Chardonnay showed no xylem damage at −17°C but showed 20 to 30% damage at −18°C, regardless of measurement protocol. One exception occurred with Cabernet Sauvignon at −20°C, where only 80% phloem damage was observed even though LTE analysis predicted 100% damage. Nevertheless, considering the general difficulty of interpreting cane exotherms (see Figure 2⇑), the overall agreement between expected and actual injury levels (Table 1⇓) confirmed that reliable data can be consistently obtained by a trained operator.

Effect of surface moisture on freezing.

Bud LTEs occurred over a range of −14°C to −19°C in the moist treatment and a range of −15°C to −22°C in the untreated samples (Table 2⇓). The LTEs in the dry treatment ranged from −18°C to −23°C and were small because of the lower moisture content of the bud tissues. On average, surface moisture raised the temperature at which bud LTEs occurred by 1°C to 2°C when compared with the untreated buds and by 3°C to 4°C when compared with the dry buds (Table 2⇓). This is consistent with earlier results obtained under different conditions (Johnson and Howell 1981, Wolf and Pool 1987). The LTEs for phloem and xylem damage were similar for the untreated and moist samples but were about 2°C lower in the dry canes. Additionally, the greater amount of extracellular water in the moist samples led to high HTEs (~ −5°C) that were not detectable in the untreated and dry samples, which is in agreement with findings by Wolf and Pool (1987).

The present data confirmed that moist buds and canes may be damaged at a higher temperature than dry buds and canes. This may happen when the minimum temperature (at dawn) coincides with the presence of heavy dew. These results emphasize the importance of not allowing samples to dry out before and during LTE analysis. A temperature difference of 2°C in the vineyard could be the difference between live and dead buds. Thus a false LTE value could result in a grower waiting too long to start frost control, resulting in more bud and cane injury than expected.

Comparison of cultivars.

Bud cold hardiness of all cultivars tested increased with the overall trend of declining temperatures from November through January, after which the buds began to deacclimate and gradually lost cold hardiness (Figure 5⇓). The white winegrape cultivars were generally hardier in midwinter than their red counterparts. They also showed more pronounced cultivar differences in bud LTE₅₀ throughout the dormant season (Figure 5B⇓). Riesling was clearly the hardiest of all cultivars tested; while Chardonnay matched Riesling in midwinter, it was much less hardy in fall and late winter. Neither Riesling nor Chardonnay was able to regain hardiness levels in mid-March once deacclimation was underway. Bud hardiness of Pinot gris was similar to that of Viognier. Both of these cultivars were pruned in early March, so we were unable to follow their deacclimation patterns into late March. However, after midwinter their bud LTE₅₀ closely traced those of Chardonnay, which tends to deacclimate early and quickly. Viognier and Pinot gris may also be susceptible to late spring frosts, which should be confirmed with further investigation.

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Figure 5

Bud cold hardiness (based on LTE₅₀) of eight V. vinifera cultivars from November 2004 through March 2005 in Prosser, WA. Red winegrape cultivars (A), white winegrape cultivars (B), and daily maximum and minimum temperature (C). Asterisk (*) indicates dates at which differences in LTE₅₀ were significant at p ≤ 0.05 (n = 4).

Among the red winegrape cultivars, Syrah and Malbec buds were at least as hardy (based on bud LTE₅₀) if not hardier than Merlot buds through the entire season (Figure 5A⇑). Both Syrah and Malbec attained midwinter hardiness values as low as Cabernet Sauvignon but appeared to lose hardiness more rapidly in the spring, indicating they could be sensitive to spring frosts similar to Merlot. By mid-March all four cultivars were deacclimating as a result of the warming temperatures, but only Cabernet Sauvignon regained hardiness levels during a cooling period. Phloem and xylem LTEs (data not shown) showed similar trends and cultivar differences as those observed with buds. Again, among all V. vinifera cultivars, Riesling was very hardy in terms of cane phloem and xylem. Compared with the other cultivars, Riesling LTEs were ~ 3°C lower in fall, up to 6°C lower in midwinter (phloem LTE₁₀ = −22°C in early January), and 1°C to 2°C lower during the deacclimating phase beginning in late February. In contrast, Cabernet Sauvignon canes were relatively slow to harden off in the fall and were similar to the other cultivars through winter, but it was the only cultivar that matched Riesling during the deacclimation period.

These preliminary results suggest that buds and canes of Syrah, Malbec, and Pinot gris can withstand similar winter temperatures as the more established cultivars. In particular, fears that Malbec may not be able to survive typical winter temperatures in eastern Washington appear to be unfounded. However, the present results were obtained with >20-year-old vines. Although it seems unlikely, we cannot exclude the possibility that young vines might differ in their cold acclimation/deacclimation pattern and midwinter hardiness. There is a fair amount of published data on seasonal bud LTE₅₀ values of the commonly grown cultivars—Cabernet Sauvignon, Chardonnay, and Riesling (Hamman et al. 1996, Proebsting et al. 1980, Wample et al. 2001, Wolf and Cook 1994)—which compare closely with our results on these cultivars given climatic differences among regions and seasons. There has been far less research conducted on seasonal cane hardiness. However, the present phloem and xylem LTE₁₀ values on Cabernet Sauvignon and Chardonnay do compare closely with those reported by Wample et al. (2001). Cabernet Sauvignon, Chardonnay, and Riesling are excellent cultivars to be used as standards in future grape cold-hardiness research when testing potential new cultivars for areas prone to cold damage.