واكنش گياه زعفران (.Crocus sativus L) به تنش يخ‌زدگي در شرايط كنترل‌شده

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

رشد و نمو گياه زعفران عمدتاً منطبق بر فصل‌هاي پاييز و زمستان است و گياه در طي اين مدت در معرض تنش سرما و يخ‌زدگي قرار مي‌گيرد. اين مطالعه باهدف بررسي اثر تنش يخ‌زدگي بر سه اكوتيپ زعفران (قائن، كاشمر و تربت‌حيدريه) در شرايط كنترل‌شده در آزمايشگاه فيزيولوژي گياهان زراعي، دانشكده كشاورزي دانشگاه فردوسي مشهد، به‌صورت فاكتوريل در قالب طرح بلوك‌ كامل تصادفي با سه تكرار انجام شد. به‌منظور ايجاد خوسرمايي گياهان در محيط طبيعي رشد داده شده و سپس براي اعمال دماهاي يخ‌زدگي به فريزر ترموگراديان منتقل شدند و در معرض شش دما (صفر، 4، 8، 12، 16، 20 درجه سانتي‌گراد) قرار گرفتند. ميزان پايداري غشاء سيتوپلاسمي با استفاده از شاخص نشت الكتروليت‌ها مورد ارزيابي قرار گرفت و دماي كشنده براي 50 درصد نمونه‌ها بر اساس آن تعيين گرديد. درصد بقاء گياهان سه هفته پس از تيمارهاي يخ‌زدگي و رشد آن‌ها در شاسي سرد از طريق شمارش تعداد بوته‌ها و تعيين نسبت آن‌ها به تعداد بوته قبل از تيمار يخ‌زدگي تعيين گرديد. درصد نشت الكتروليت‌ها، LT50el و درصد بقاء به‌طور معني‌داري تحت تأثير تيمارهاي آزمايشي قرار گرفتند. بيشترين و كمترين درصد نشت الكتروليت‌ها در دماي 20 درجه سانتي‌گراد به ترتيب مربوط به اكوتيپ كاشمر و اكوتيپ تربت‌حيدريه بود. نتايج اثر متقابل دما و اندام نشان داد كه بيشترين و كمترين درصد نشت الكتروليت‌ها به ترتيب مربوط به اندام برگ در دماي 20 درجه سانتي‌گراد و بنه در دماي صفر درجه سانتي‌گراد بود. اكوتيپ‌هاي قائن و كاشمر بيشترين و اكوتيپ تربت‌حيدريه كمترين مقدار LT50el را داشتند. همبستگي معني‌داري (**0/82 =r) بين درصد نشت الكتروليت‌ها و LT50el بنه زعفران مشاهده شد، به‌طوري‌كه با كاهش درصد نشت الكتروليت‌ها، دماي 50 درصد كشندگي كاهش يافت. هر سه اكوتيپ زعفران تا دماي 12 درجه سانتي‌گراد 100 درصد بقاء داشتند و با كاهش بيشتر دما درصد بقاء كاهش يافت و كمترين درصد بقا در دماي 20 درجه سانتي‌گراد مشاهده شد. نتايج اين آزمايش نشان داد كه اكوتيپ تربت‌حيدريه از كمترين درصد نشت الكتروليت‌ها، كمترين LT50elو بيشترين تحمل نسبت به تنش يخ‌زدگيبرخوردار بود.

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 IranTuranian 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 sles 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 (LT50el) 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 LT50el 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 LT50el. The lowest and highest LT50el was observed in Torbat heydarieh, Ghaen and Kashmar, respectively. Shashikumar and Nus (1993) reported that Bermuda grass cultivars were different in LT50el. The more tolerance cultivars showed the lower LT50el. There were significant correlation (r=0.82**) between %EL and corm’s LT50el where by reducing %EL, ecotypes’ LT50el 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 LT50el 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.