The Effect of Freezing Stress on Percentage of Electrolytes Leakage and Survival of Flixweed (Descurainia sophia L.) Seedlings

Flixweed (Descurainia sophia L.) is a medicinal plant from Brassicaceae family which also known as a weed for winter cereals and oil seed rape. Low temperatures are one of the most important abiotic stresses that threat Flixweed growth and productivity. Therefore it is important to recognize the freeze tolerance of Flixweed for successful planting and utilization in cold regions such as Mashhad in Khorasan Razavi Province (Iran’s north. east). Among many laboratory methods which have been developed to estimate and to evaluate plants response or their tolerance to freez¬ing temperatures, electrolyte leakage (EL) test is widely used. This test is based on this principle that damage to the cell membranes results in enhanced leakage of solutes into the apoplastic water, hence recording the amount of leakage after stress treatments provides an estimation of tissue injury. Indeed continuing integration of plasma membrane is one important factor for survival of plants under freezing stress and any disturbance in membrane structure can lead to damage and death. So determination of LT50 point or critical temperature for electrolytes leakage and survival of plant is the most reliable, quantitative and simple methods for evaluating the cold tolerance of plants. The aim of this trial was to determine the LT50 according to the EL and SU% for Flixweed ecotypes.
In order to evaluate freeze tolerance in Flixweed, a factorial experiment based on completely randomized design with three replications was carried out in college of agriculture, Ferdowsi University of Mashhad. Experimental factors included five ecotypes of Flixweed (Eghlid, Sabzewar, Hamedan, Torbat-e-Jam and Neyshabour) and 10 freezing temperature levels (0, -2,-4, -6, -8,-10,-12,-14,-16 and -18°C). Flixweed seeds were cultivated in pots in autumn of 2008 and were grown until 5-7 leaf stage under natural weather conditions for acclimation. Then to apply freezing temperatures, they were transferred to a thermo gradient freezer. The initial temperature of programmable freezer was 5°C; but gradually decreased in a rate of 2°C.h-1 until reached to desired temperatures. When the temperature reached to -2°C, the plants were sprayed with the Ice Nucleation Active Bacteria (INAB) to help the formation of ice nucleus in them. As well this spraying was conducted to prevent from super-cooling of samples and to ensure that mechanism of freeze resistance is tolerance not avoidance as well. After reaching a desired freezing temperature happened, the samples were removed from the freezer and then were thawed slowly during 24 hours in a refrigerator at 5±1°C. Cytoplasmic membrane stability was evaluated by electrolyte leakage (EL) test, afterward lethal temperature was calculated for 50% of plants according to the electrolyte leakage % (LT50el). In order to evaluate correlation between EL% with survival percentage (SU%), remained plants were transferred to greenhouse and after 21 days recovery, SU% and then lethal temperature was determined for 50% of plants according to the SU% (LT50su). Analysis of variance performed by MSTAT-C software and correlation between data carried out by MINITAB 16 program. LT50el was determined by Slide write software. Mean separation was conducted by least significant difference (LSD) test at 1% probability level.
Results showed that the reduction of temperature to less than -8°C, led to increment of electrolyte leakage % and decline of survival %. Interaction effect of ecotype and temperature on electrolyte leakage and survival percentage was significant at 1% probability level. Enhancement of electrolyte leakage % for Neyshabour ecotype started from -8°C, while the others were affected from lower temperatures. In addition in Eghlid and Neyshabour ecotypes, decline of survival % begun from higher temperatures (-8 °C) compared with the other ecotypes (-10 °C). Cold hardiness is often reported as LT50el, means the temperature at which 50% of the electrolytes leaked from the cells. LT50el often is simply equaled to 50% sample mortality or LT50su. According to the LT50el and LT50su indices, Hamedan, Sabzewar and Torbat-e-Jam ecotypes had better freeze tolerance than Neyshabour and Eghlid ecotypes and according to the LT50su, Hamedan ecotype had potential to freezing tolerance up to -12°C.
In this survey, there was strong and negative correlation between EL% and SU% (r= -0.72***). Furthermore, there was high and positive correlation between LT50el and LT50su (r= 0.53*). These results indicate efficiency and alternative likelihood of each index in assessment of Flixweed freeze tolerance. Regarding to more rapid rate of EL method compared to SU evaluation, it seems better to utilize of this index.