Effect of short-term versus prolonged freezing on freeze-thaw injury and post-thaw recovery in spinach: Importance in laboratory freeze-thaw protocols
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Spinach (Spinacia oleracea L.) is a cool-season vegetable crop which can be damaged by intermittent spring frost that is needed to better understand freezing tolerance of spinach. This thesis focused on the physiology of spinach's freezing tolerance through duration of freezing, including an ability of recovery, using freeze-thaw injured spinach tissues exposed to various durations of freezing at two sub-lethal temperatures. Various physiological parameters [electrolyte leakage, Fv/Fm ratio (efficiency of PSII), MDA test (an indicator of lipid peroxidation), and histochemical detection of ROS (i.e. superoxide (O2-) and hydrogen peroxide (H2O2)] were quantified. Based on the LT50 of spinach (approximately - 5.5 Â°C) , -4 and -4.5 Â°C were selected as two sub-lethal temperatures for prolonged freezing test, but only -4.5 Â°C for post-thaw recovery test. Enhanced ion-leakage, increased MDA content, accumulated ROS and decreased Fv/Fm ratio were observed in freeze-thaw injured spinach tissues subjected to four duration (0.5, 3.0, 5.5 and 10.5 h) of freezing at both -4 and -4.5 Â°C; however, all measurements were shown to be worse at -4.5 Â°C than -4.0 Â°C during four durations of freezing. For the post-thaw recovery test, spinach tissues frozen for relatively short-term duration of freezing at -4.5 Â°C was recoverable during post-thaw periods, but was irrecoverable when stressed by a longer duration of freezing at the same temperature. In summary, depending upon duration of freezing, temperatures interpreted as sub-lethal based on LT50 (short-term freezing assays) could be actual lethal. Therefore, "duration of freezing" should be considered as one of critical factors influencing on plant freeze-thaw tolerance when using artificial freeze-thaw protocols.