Nuclear etic resoaace (NMR) relaxation times were studied in acclhnated and nonacclmated Kharkov It has been inferred by many investigators that membranes, primarily plasma membranes, are the site of freezing injury. This evidence is based upon loss of semipermeability properties of the membrane following freezing injury (7,19).Pulse NMR3 measurements of the longitudinal relaxation time (T1) and the transverse relaxation time (T2) have been used to study water and water interactions in biological systems (5,14,22 The objective of this study was to evaluate the effect of different freezing stresses (fast freezing versus slow freezing) on the plasma membrane of tender and hardy winter wheat (Triticum aestivum L.). Alterations to plasma water membrane permeability as a result of freezing stresses were studied by observing the effects of freezing on NMR relaxation times. Alterations to Mn2+ permeability were studied by observing the loss in the long T2 water signal as Mn2" diffused into the protoplast. MATERIALS AND METHODS PLANT MATERIALSCrown Tissue. Crowns of a hardy winter wheat cultivar, Kharkov MC22, were cold-acclimated under controlled environment conditions for 7 weeks (11). Fully acclimated plants were also collected from the field in late November and stored in a soilsand-peat mixture (1:1:1) at -3 C. The crowns were maintained under a photoperiod of 24 hr with a light intensity of 2,400 lux supplied from "Gw-Lux WS" fluorescent lamps. The cold hardiness level of these plants, as determined by artificial freeze tests, was -23 ± 1 C (12).Ced Tissue Cultures. Kharkov seeds were sterilized by shaking for 15 min in 10% NaOCl with 0.01% Tween -20. The seeds were washed five times with sterile distilled H20 and germinated in Petri dishes. Following 3 days germination, the mesocotyl of each seedling was dissected and transferred immediately to a flask containing 1-B5 agar medium (9). After 4 to 6 weeks the callus which formed was transferred to a 1-B5 liquid medium and shaken constantly. Cell suspension cultures were propagated by the subculturing method of Gamborg (9)
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