Evaluation of Winter Hardiness in Peppermint (Mentha piperita L.) by Electrolyte Leakage Indicator

Peppermint or Mentha is an aromatic, medicinal and perennial herb from Lamiaceae family which has been used for healing a variety of diseases such as common cold, bronchitis, nausea, flatulence, diarrhea, vomiting, indigestion, stomach cramps, menstrual cramps and parasitoids. Peppermint is largely cultivated in Indiana, Mexican and California for the production of peppermint oil. Mentha reveals suitable winter hardiness in warm and temperate regions, But in cold areas, it confronts with winter stresses particularly freezing stress. So recognizing the freeze tolerance of peppermint for successful planting and using of this plant in cold regions such as Mashhad, Iran where peppermint is cultivated now is important. Among the many laboratory methods which have been developed to evaluate freez¬ing tolerance of plants, electrolyte leakage (EL) test is widely used. This test is based on this principle that any damage to the cell membranes results in enhanced leakage of solutes into the apoplastic water, hence measuring the amount of leakage after stress treatments provides an estimation of tissue injury. Often, the 50% level of relative EL, or index of injury, is simply equaled to 50% sample mortality. This study was done to evaluate the freeze tolerance of peppermint organs by electrolyte leakage test and also to determine the winter survival ability of this plant by lethal temperature at which 50% of electrolytes leaked from the cell (LT50el).
In order to evaluate the cold tolerance of peppermint, a factorial experiment based on completely randomized design with four replications was carried out under controlled conditions. For this aim samples from stolon and rhizome of peppermint were selected monthly (December 2010 to April 2011) from Research Field, College of Agriculture, Ferdowsi University of Mashhad and were exposed to low temperatures (from 0 to -20°C with 4°C intervals) in a thermo gradient freezer at laboratory. 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 nuclei in them. As well the spraying had been conducted to prevent from super-cooling of samples and to ensure that the mechanism of freeze resistance is tolerance not avoidance. After a desired freezing temperature was reached, the samples were removed from the freezer and then were thawed slowly during 24 h in a refrigerator at 5±1°C. In order to assess plasma membrane stability, four freeze stressed samples from stolon and rhizome were incubated in vials which containing 50 ml of double distilled water and the initial electrolyte leakage (E1) was measured by an electrical conductivity meter next day. Afterward for determining of final electrolyte leakage due to the death of whole sample, accessions were boiled in autoclave with pressure near to 1.2 bar and temperature around 110°C for 20 minutes. E2 was measured next day similar to E1. Electrolyte leakage percentage was expressed as E1 to E2 ratio. Afterward lethal temperature for 50% of samples according to the EL% (LT50el) was calculated to estimate the freeze tolerance of peppermint organs during different sampling times.
Results showed that by decreasing of temperatures, EL% increased in both organs and at -20°C, EL% was 50 percent more than control (0°C) treatment. Moreover at -12°C, EL% from stolons was eight % less than rhizomes. Studies showed that cold sensitive plants or organs showed further amount of ions leakage from their cells. So further leaked material from rhizomes should be interpreted as more sensitivity of this organ to freezing temperatures in comparison to stolon. The least and the most EL% was observed in January and April, respectively. And the least and the most value of LT50el was achieved in February and April, respectively. It seems that due to the occurrence of cold hardening in both organs during cold months of year, stability of membranes have been increased, so EL% has been decreased. Stabilization of membranes to cold stress damage is a key role of cold hardening. In addition it could be stated because of occurrence of de-hardening in samples during warm months of year, freeze tolerance level of organs have been declined based on LT50el. LT50el for stolons depend on sampling date varied between -8.4 to -14.5 °C and for rhizome LT50el ranged between -8.8 to -13.9 °C. Interaction effect of organs, temperature and sampling date on EL% was significant. The most EL% belonged to stolon in April at -20°C and the lowest EL% was seen in this organ in December at -4°C. Similarity in rhizome the highest EL% was recorded in April at -20 °C and the least EL% was observed at 0 °C in February.
According to the electrolyte leakage and LT50el indices, peppermint can tolerate freezing temperature up to -14°C during the cold months of year. Despite this for complete understanding of peppermint response to freezing stress, further studies and reaserches under controlled and field conditions are required.