Study of saffron (Crosus sativus L.) reaction to freezing stress under controlled condition

Introduction
Saffron (Crosus sativus L.) is a perennial and herbs plant, belongs to the Iridaceae family and reproduces via corms (Behnia, 1991). Saffron is native to the mediterranean floristic region, extending eastward into the Iran-Turanian region and lie within the longitude 10°W to 80°E and latitude 30°N to 50°N. These areas are characterized by cool to cold winters and warm summers with very little rainfall. Iran is the most important saffron producers and exporters in the world market (Kafi et al, 2002). Saffron is cultivated in northeast of Iran which are characterized by cold winters and warm summers. The main part of the Saffron’s growing season is exposed to cold and winter frost, and low temperatures limits its production. Therefore, it is likely that a cold weather or frost during winter waste the saffron, so that it is necessary to identify cold tolerant ecotypes of saffron and determine the range of low temperatures that saffron may withst and without any considerable damage.
The objective of this experiment was to evaluate and compare the response of saffron ecotypes to freezing temperatures under controlled conditions.
 
Materials and methods
In order to evaluate the effect of freezing stress on three saffron ecotypes (Ghaen, Kashmar and Torbat heydarieh) a factorial experiment based on completely randomized block design with three replications was carried out under the controlled conditions in the Faculty of Agriculture, Ferdowsi University of Mashhad, Iran. Three native saffron ecotypes (Ghaen, Kashmar, and Torbate Heydarieh) were subjected to six different temperatures (0, -4, -8, -12, -16 and -20 ^oC). Two saffron’s corms were sown in plastic pots with 25 cm diameter and 23 cm hight to a depth of 8 cm. The pots were primarily filled with an equivalent ratio of sand, compost and soil. For cold acclimation, plants were grown in natural conditions and after that, transferred to the thermogradiant freezer with the six freezing temperatures. The seedlings were sprayed with the Ice Nucleation Active Bacteria (INAB) to help the formation of ice nuclei in the seedlings. Plants were kept at the nominated freezing temperature for 1 h. Following the freezing treatments, all samples were kept at 5±2°C for 24 h to decrease the rate of thawing. Leaf and corm cell membrane integrity were measured via electrolyte leakage percentage (%EL) index and lethal temperature 50 according to %EL (LT_50el) was determined. Three weeks after plants growth in cold frame, survival percentage of each saffron ecotype was measured by counting the plants and determining their proportional with the number of plants before freezing.
 
Results and discussion
Results showed that %EL was significantly affected by experimental treatments. The highest and the lowest electrolyte leakage values were observed in the Kashmar and Torbat heydarieh ecotypes at -20 ºC, respectively. Rezvan beydokhti et al (2011) showed that there were significant different amoung Persian shallot ecotypes for electrolyte leakage percentage. The EL increased markedly as temperatures decreased and the peak of EL% obtained at 20 ºC. The EL% of Organs (Leaf and corm) in response to the freezing temperature was different. It seems that the cell memberace of saffron’ leaf is more sensitive than corms to freezing stress. Interaction between temperature and organs showed that the highest and lowest %EL was observed in the leaf and corm at -20ºC and 0ºC, respectively. The temperature resulting in 50% leakage has been known as LT50_el i.e. freezing temperature that causes 50% mortality (Cardona et al., 1997). There was significant difference (P ≤0.01) among saffron ecotypes in terms of LT_50el. The lowest and highest LT_50el was observed in Torbat heydarieh, Ghaen and Kashmar, respectively. Shashikumar and Nus (1993) reported that Bermuda grass cultivars were different in LT_50el. The more tolerance cultivars showed the lower LT_50el. There were significant correlation (r=0.82^**) between %EL and corm’s LT_50el where by reducing %EL, ecotypes’ LT_50el decreased.
There were significant differences (P≤0.01) among freezing temperatures for the survival percentage. Survival percentage of all ecotypes did not affected up to -12°C, however, it is reduced by decreasing temperature, and the lowest survival percentage was obtained in -20°C.
Survival percentage varied among saffron ecotypes. Torbat heydarieh ecotype showed the highest (96.1%) and Kashmar ecotype had the lowest (94.4%) survived seedling.
The results of correlation analysis demonstrated a negative, significant correlation between leaf and crom’s EL% and survival percentage (r = -0.98**). Such results showed in other studies for different plants (Rife and Zeinali, 2003).
 
Conclusions
The results of this experiment showed significant effect of freezing temperatures on EL% where EL% increased as temperature decreased. Amoung ecotypes Torbat heydarieh ecotype showed the lowest %EL, lowest LT_50el and the most tolerant to the freezing stress. The saffron’s corm was more tolerant than leaf in freezing temperatures. Therefore, the EL method could be used as a fast and efficient method in evaluating the cold tolerance of saffron ecotypes.
 
 Acknowledgements
This study has been supported by the grant approval of the Ferdowsi University of Mashhad, Iran and the authors would like to appreciate it.