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Changes in the Histology of Cold-Hardened Oat Crowns during Recovery from Freezing

^a USDA and North Carolina State Univ., 840 Method Rd., Unit 3, Raleigh, NC 27695
^b North Carolina State Univ., 840 Method Rd., Unit 3, Raleigh, NC 27695
^c Center for Advanced Microscopy, B7 CIPS Bldg., Michigan State Univ., East Lansing, MI 48824
^d Dep. of Crop and Soil Sciences, Plant and Soil Sciences Bldg., Michigan State Univ., East Lansing, MI 48824

View larger version (103K): [in a new window]   Fig. 1. (A–F) Plants used in the subsequent five figures. (A, B) Nonhardened and cold-hardened plants, respectively, shown in Fig. 2. (C) Cold-hardened plant that was frozen at –11°C and allowed to recover for 3 d (Fig. 3). Note new growth at the top of the two tillers on the left. (D) Frozen plant after 10 d of recovery (Fig. 4). (E) Frozen plant at 21 d of recovery that was rated as a nonsurvivor (Fig. 5). (F) Frozen plant at 21 d of recovery that completely survived (Fig. 6). (G) Fractionated ‘Wintok’ oat crown. The whole crown is shown on the left, the crown meristem (CM) complex (I) in the center, and the lower portion of the CM complex consisting of mostly crown core tissue (J) on the right after removal of the apical region (H) from the CM complex by razor blade. Roots were trimmed as close to the base of the crown core as possible before calorimetric measurements.

View larger version (152K): [in a new window]   Fig. 2. (A) Longitudinal section stained with Safranin, Fast Green, and Orange G, of a 5-wk-old oat plant grown at 13°C under controlled conditions; this was a nonhardened plant corresponding to the plant "A" in Fig. 1. (B) Longitudinal section of an oat plant grown for 5 wk at 13°C and then 3 wk at 3°C to induce cold-hardening; this section corresponds to the plant "B" in Fig. 1 and is the control tissue to which all other sections were compared. (C) Part of the crown core showing parenchyma, tracheids, metaxylem, and fibers. (D) Part of the crown core showing a longitudinally sectioned vessel bundle. Note longitudinal metaxylem vessels adjacent to those in cross section. The unlabeled bar in each subpanel is 100 µm.

View larger version (173K): [in a new window]   Fig. 3. (A) Longitudinal section stained with Safranin, Fast Green, and Orange G, of a cold-hardened oat crown that had been frozen at –11°C and then allowed to recover for 3 d at 13°C in planting medium. This section is from the plant in Fig. 1C. (B) Normal nuclei showing the somewhat grainy purple-blue color of the chromatin, and adjacent cells that contain abnormally condensed chromatin that stained a dark red color and lacked any clear internal definition. With a few exceptions, abnormal nuclei were primarily found in cells on the outside of stem bases. Inset: a closer view of two adjacent cells, one with a normal nucleus and the other with an abnormal one. (C) Phloem with abnormal nuclei and metaxylem beginning to plug with dark red material. Note tearing in the wall of the metaxylem vessel. (D) Tearing of tissue within the crown core. The unlabeled bar in each subpanel is 100 µm.

View larger version (149K): [in a new window]   Fig. 4. (A) Longitudinal section stained with Safranin, Fast Green, and Orange G, of a cold-hardened oat crown frozen at –13°C and then allowed to recover for 10 d at 13°C in planting medium. This section is from the plant in Fig. 1D. (B) A large root with plugged phloem, but in this case with no plugging in the xylem. (C) Abnormal phloem in the central part of the crown core. Note the lack of nuclei and reddish color as compared with the control (Fig. 2D). (D) Plugged metaxylem and parenchyma with abnormal nuclei as compared with control (Fig. 2C). (E) Cross-section of a small root from a different section of the same plant with several xylem vessels plugged. Most of the phloem vessels in a ring outside the xylem also appear plugged. The unlabeled bar in each subpanel is 100 µm.

View larger version (172K): [in a new window]   Fig. 5. (A) Longitudinal section stained with Safranin, Fast Green, and Orange G, of a cold-hardened oat crown frozen at –13°C and then allowed to recover for 3 wk at 13°C in planting medium. This section is from the plant in Fig. 1E that was rated as a nonsurvivor. Note that the whole plant appeared dead (Fig. 1E) but at 3 wk a seemingly new shoot had emerged. Note the dark staining region that forms what looks like a barrier between disintegrated tissue and normal tissue. (B) Abnormal phloem from leaf-base tissue. (C) Abnormal phloem from the leaf base of a tiller that had not been damaged as badly as the leaf base from the opposing tiller. (D) A vascular bundle in cross-section showing what appear to be plugged vessels as compared with controls in Fig. 2C and 2D. (E) Interior portion of the crown core showing disintegrated cells with little evidence of structure. Inset: A closer view of the putative barrier from a different section of the same plant. Note the abnormal nuclei in the parenchyma cells outside the barrier. In this instance the barrier clearly did not prevent the occurrence of nuclear damage. The unlabeled bar in each subpanel is 100 µm.

View larger version (162K): [in a new window]   Fig. 6. (A) Longitudinal section stained with Safranin, Fast Green, and Orange G, of a cold-hardened oat crown frozen at –13°C and then allowed to recover for 3 wk at 13°C in planting medium. This section is from the plant in Fig. 1F. This plant survived the freeze test and would have produced functional reproductive tissue, despite extensive freeze-damage to the crown core (B). (C, D) Normal metaxylem, and procambium (immature xylem or phloem), from various parts of the crown core. (E) Normal phloem and parenchyma from a tiller that appeared to be elongating. The unlabeled bar in each subpanel is 100 µm.

View larger version (20K): [in a new window]   Fig. 7. A comparison between the apical region and crown core of the change in dry weight as a percentage of the fresh weight during cold-hardening. Each data point is the average of three experiments, each experiment consisting of tissue from 16 separate oat plants cold-hardened at 3°C. Error bars represent the LSD at p = 0.05.

View larger version (21K): [in a new window]   Fig. 8. Percentage of the total plant water that froze at –2°C in fractionated tissue from oat crowns. Bars represent the LSD at p = 0.05.

View larger version (21K): [in a new window]   Fig. 9. Freezing curves of fractionated tissue from oat crowns (see Fig. 1) showing the latent heat of fusion at –2°C. For comparison, a separate freezing curve for 0.59 g of water was included with the freezing curves of the apical region and crown core. (A) The first 3 h of the 6-h freezing event. (B) The first 20 min of the same freezing event in (A) to show differences in initial freezing rates.