STUDY OF THE EFFECT OF LOW TEMPERATURES AND CALCIUM CHLORIDE TREATMENT ON THE GERMINATION OF IRANIAN AND EUROPEAN BARLEY CULTIVARS

Low temperature stress is one of the limiting factors of seed germination. In order to investigate the effect of low temperatures on germination of barley cultivars, identification of traits related to low temperature stress at germination stage and the effect of calcium chloride on these traits, 44 Iranian and European barley cultivars were evaluated in a factorial experiment within completely randomized design with 3 replications in the Laboratory of Plant Physiology, Agronomy and Plant Breeding department, Razi University. The first factor was 44 Iranian and European barley cultivars, the second factor included four temperature (0, 5, 10 and 20°C), and the third factor was the use of calcium chloride (10 mM) and its non-use (distilled water). Analysis of variance showed that there was a significant difference between cultivars for all traits except root length and seed vigor. Applying calcium chloride treatment at a concentration of 10 mM did not significantly affect the traits under the studied temperatures. Reducing temperature from 20°C to 10°C and 5°C reduced root length, shoot length, coleoptile length, root number, coefficient of velocity of germination, seed vigor and promptness index. The results of correlation analysis showed that there was a significant positive correlation between promptness index with average velocity of germination, coefficient of velocity of germination and seed vigor, germination percentage and root number in all studied temperatures. There was little differentiation between Iranian and European cultivars by both cluster and discriminant analysis.


INTRODUCTION
Barley as an agronomic plant compatible with drought stress and tolerant to adverse environmental conditions and possessing characteristics such as green grazing in the tillering, grain extraction and its use in food industry, has a special place in the agricultural systems of the arid regions of the world, including Iran (Rezaikalu et al., 2012). Barley is cultivated in many parts of the world due to its high resistance to environ-DOI: 10.1515/plass-2018-0012 mental stresses and less need for moisture and adaptation to the environment (Behnia, 1996). Barley is planted in an area of 1.8 million hectares in Iran, of which 60% is devoted to rainfed areas. Most of the rainfed lands are located in cold and humid areas. In cold regions, in addition to cold and drought stresses, most of the years, due to delays in precipitation in early autumn, seedling emergence due to cold occurrence is difficult causes decreasing the growth period and ultimately decreasing yield (Abdolrahmani et al., 2011). Low temperature stress is one of the limiting factors for plants germination. Germination plays an important role in grain quality and malt quality (Chloupek et al., 2003). Germination is a trait that varies greatly among populations (Baskin and Baskin, 1998).Cultivars with fast germination properties are more likely able to absorb more water and are more suitable for rainfed conditions due to resistance to winter cold (Rastegar, 1992). Calcium is an essential nutrient, and it plays an important role in the activation of metabolic activities, such as membrane stabilization, signal transduction through the second transducer, membrane preservation, and control of the transfer of ion particles and of the activity of enzymes in counteracting the conditions of environmental stresses (Arshi et al., 2006). When the plant is exposed to environmental stresses, including low temperature stress, calcium can withstand stress by regulate many physiological and cellular reactions (Hirschi, 2004). In a study by Perine et al. (2008), in order to increase the rate of germination and increase hormonal activity, instead of using hormones, NaOH, Mg(OH) 2 , Ca(OH) 2 and NaHCO 3 was used. The results showed that calcium hydroxide was effective in increasing germination by 60-66%. Sedaghathoor et al., 2015, to study the germination rate of seeds of three species of grass (Lolium perenne, Poa pratensis, Cynodon dactylon) used calcium chloride (2%). Calcium chloride treatment alone has had covert not a significant effect on the traits, but the effects of the type of grass and calcium chloride on the average daily germination had the most effect.
The aims of this study were to investigate the effect of low temperatures stress on germination of Iranian and European barley cultivars, to identify the traits related to tolerance to low temperatures in germination stage and to investigate the possible effect of calcium chloride on germination acceleration and other growth parameters of seedlings under low temperatures stress.

MATERIALS AND METHODS
In order to investigate the effect of low temperatures on germination of barley cultivars, identification of traits related to low temperature stress at germination stage and the effect of calcium chloride on these traits, 44 Iranian and European barley cultivars were evaluated in a factorial experiment within completely randomized design with 3 replications in the Laboratory of Plant Physiology, Agronomy and Plant Breeding department, Razi University. The first factor was 44 Iranian and European barley cultivars, the second factor included four temperature (0, 5, 10 and 20°C), and the third factor was the use of calcium chloride (10 mM) and its non-use (distilled water). Iranian cultivars recieved from Kermanshah Agricultural and Natural Resources Research Center and European cultivars seeds recieved from the Genomics and Post Genomics Institute (CRA-GPG) in Fiorenzola, Italy. Table 1 shows the name, source and some of the characteristics of the studied cultivars. Cultivars are named from 1 to 44. From each barley, for each experimental unit, 20 healthy seeds were selected and disinfected according to the following steps: First, the seeds were washed with distilled water and then disinfected with 70% alcohol for 1 minute and 3% hypochlorite for 3 minutes. Then, three times washed with distilled water for 1 minute, 3 minutes and 5 minutes. Seeds were then dressed with mancozeb fungicide (at a rate of 2 g a.i/kg) and cultured in Petri dishes under sterile conditions. The germinated seeds were counted daily for 10 days. The traits were measured based on the average of 10 seedlings including root length (cm), number of root, shoot length (cm), coleoptile length (cm) and the following traits: where GP -Germination percentage Ni -Number of germinated seeds S -Total number of seeds where AVG -Average Velocity of Germination in day / number: ΣNt -Total number of germinated seeds at time Σt -Total time (day), (Salehzade et al., 2009) where CVG -Coefficient of Velocity of Germination: N 1 to N X -the number of seeds germinated from the first day to the end of the test.
T 1 to T x are the time of counting This index is a characteristic of the seed germination rate (in day)., calculated from the following equation; (Scotte et al., 1984) where PI -Promptness Index: nd2, nd4, nd6, nd8 and nd10 -the number of germinated seeds on the second, fourth, sixth, eighth and tenth day (Bouslama and Schapaugh, 1984).
where SV -Seed Vigor: RL: Root length, SL: Shoot length, GP -Germination percentage; (Hamidi et al., 2009) where PCT-percentage change of traits Xn -the mean of trait in control conditions Xs -the mean of traits in the stress conditions Statistical analysis Data were analyzed based on based on a 44 × 2 × 4 factorial experiment within completely randomized design. Mean comparisons were determined with Least Significant Difference (LSD) test by the SAS software ver.9.2. Pearson's correlation coefficients between measured traits evaluated in all tempratures level and cluster analysis based on the Euclidean distance square using Ward's method were done by SPSS software (Ver. 16.0.1, SPSS Inc).

Analysis of variance
None of the studied cultivars germinated at 0°C temperature in all three replications, so the temperature level of 0°C was eliminated from the statistical analysis. Analysis of variance of germination traits in 44 barley cultivars showed that there was a significant difference between cultivars for all traits except root length and seed vigor index ( Table 2). The mean comparisons of 44 barley cultivars for the studied traits were done by using the least significant difference test (LSD). Considering the significance of the two and three way interactions for the studied traits, except for root length and seed vigor index, LSD test was performed only on these interactions, some of which are mentioned. Comparison of the significant interaction effect of calcium chloride and temperature for measured traits (Table 3) showed that at 5° C, except for the coleoptile length, other traits in the calcium chloride treatment decreased compared to distilled water. At 10°C, no significant difference was observed in the measured traits between calcium chloride and distilled water treatments. Only a significant decrease for coefficient of velocity of germination in distilled water treatment compared to calcium chloride was observed at 20°C (Table 3). The comparison of the mean of temperature effect (Table 4) for root length and seed vigor index indicated significant differences in these traits at 20°C compared to 10°C and 5°C.  Table 4 Results of mean comparison for main effect of temperature on root length and seed vigor Values followed by the same letter in the same column are not significantly different Percentage changes in traits at different temperatures compared to 20°C. Reducing the temperature from 20°C to 10°C and 5°C resulted in a significant decrease in coleoptile length ( Table 5). The roots number decreased by about 36.71 % compared to 20°C by reducing the temperature to 5°C. At the temperature of 10°C, the germination percentage and the average velocity of germination increased compared to the temperature of 20°C, but the reduction of temperature to 5°C reduced these traits. Coefficient of velocity of germination and promptness index decreased at 10°C and 5°C than 20°C. Pearson correlation analysis for all three temperatures are presented in Table 6. Correlation analysis of traits showed that there is a positive and significant correlation between root length and all traits measured at 5°C. Root number at 5°C had a positive and significant correlation with all traits except shoot and coleoptile length. There was a positive and significant correlation between shoot length and coefficient of velocity of germination at 5 and 10°C. Celoplite length showed a positive and significant correlation with root length, shoot length and seed vigor at 5°C. At 10°C, coleoptile length had significant positive correlation with coefficient of velocity of germination, shoot length and seed vigor and at 20°C with all traits except root length and seed vigor. Germination percentage showed positive and high correlation with seed vigor at all three temperatures. There was a positive and significant correlation between promptness index with coefficient of velocity of germination, average velocity of germination, seed vigor index, germination percentage and root number in all three temperatures. The cluster analysis for the data obtained from the germination test was performed using the Ward method based on the Euclidean distance square (Fig. 1). The results of the discriminant analysis divided the dendrogram into two groups and did not differentiate between Iranian and European cultivars ( Table 7). The mean of measured traits in each cluster is shown in Table 8. The first cluster consists of 8 Iranian cultivars and 15 European cultivars and the second cluster consists of 12 Iranian varieties and 9 European cultivars. The first cluster had the highest mean for all studied traits (

DISCUSSION
The effect of low temperature stress on reducing plant growth is one of the clearest response of plants. Analysis of variance showed that there was a significant difference between cultivars for all traits except root length and seed vigor. Applying calcium chloride treatment at a concentration of 10 mM did not significantly affect the traits under the studied temperatures. Askarian (2004) investigated the effect of CaCl2 on germination of two rangelands species namely Kochia prostrate and Elymus junceus, reported that with increasing CaCl2, germination decreases and even reaches zero. The results showed that the root and shoot length under low temperature stress are accompanied by a decrease, which is consistent with the results of Ghorbani et al., 2009. Abbasal-Ani andHay, 1983 reported that the growth rate of root and shoot in barley, oat, rye and wheat at low temperature (5 ° C) was low and at high temperature (15 and 25 ° C) is fast. It has been reported that at lower temperatures, the rooting of plants and roots grow decreases (Akbaraghdami et al., 2013). Macduff and Wild (1986) reported that the length and number of roots in germinated barley increased by 27 times, with increasing temperature from 3 to 25°C, after 20 days. The sensitivity of germination percentage and average velocity of germination were lower than other traits, so that at the temperature of 10°C, even the germination percentage and the average velocity of germination increased compared to the temperature of 20°C , but the reduction of temperature to 5°C reduced these traits. Mei and Song, 2010 reported the optimum temperature for the germination percentage in barley is 5-20°C. In the study conducted by Dinari and Meighani, 2014, the effect of cold stress on seed germination and growth of Hordeum spontaneum L (root and shoot length and weight) were studied. Reducing the temperature reduced seedling growth, but seed germination was more tolerant to cold stress than seedling growth. Klos and Brummer (2000) stated that the temperature of the environment determines the success of germination and seedling growth, and affects the capacity and velocity of germination. Particularly temperatures below the optimum can cause poor seed germination. Cultivars with fast velocity of germination are more likely to able to absorb water and adapt to snvironment and, due to their winter resistance, are more suitable for rainfed conditions (Rastegar, 1992). One of the most sensitive traits to low temperatures was seed vigor, which decreased by about 96.65% at 5°C. Due to changes induced by low temperatures, root capacity decreases for water absorption and ultimately plant growth reduces (Akbaraghdami et al., 2013). Root length showed significant positive correlation with all the traits measured at 5° C. Root length can be an important indicator for predicting the emergence of seedling in the field and it is also considered as the primary index of growth and development of seedlings and its changes as an indicator of seedling vigor are analyzed (Bagheri et al., 2012).
Root number at 5°C showed positive correlation with most of traits. Most cold-resistant plants, including barley, when exposed to low temperatures, show signs of water stress (low water potential and leaf inflammation), which is known as drought stress due to frostbite (Ghorbani et al., 2009. Creating a deep and widespread root system as a result of an increase in root number and length with fast growth rate resulted resistance to stress (Kafi, 1997). Tikonov (1973) studied the roots number in the germination stage in 40 wheat cultivars, and observed that the varieties with the highest roots number at germination time had the highest yield under rainfed conditions. Positive and significant correlation between shoot length and coefficient of velocity of germination at 5 and 10°C observed. Cultivars with high coefficient of velocity of germination and shoot length when exposed to cold stress, have better tolerance and better growth (Akbaraghdami et al., 2013).
Celoplite length showed a positive and significant correlation with most of traits at all tempratures. The importance of the coleoptile length in rapid emergence, early deployment and plant diameter, which protects the plant from environmental damage, such as cold and drought, has been reported (Shakeri et al., 2013). Positive and high correlation of germination percentage with seed vigor at all three temperatures indicated seeds that have better seed germination under stress conditions have stronger seedlings (Jajarmi, 2012). Promptness index showed correlation with coefficient of velocity of germination, average velocity of germination, seed vigor index, germination percentage and root number in all three temperatures. In plants such as barley, which are planted early in autumn, less germination time can result in faster seedling growth, and consequently rosseting and resistance to cold weather (Jajarmi, 2012).
Cluster analysis classified cultivars with more desirable germination characteristics under all studied temperatures into separate group. The cultivars in this cluster can be considered as resistant to low tempreratures during germination and seedling growth stages. The varieties grouped in the first cluster can be used in breeding programs for improvement of parameters related to germination in low temperatures. However, in order to be more reliable, the test should be repeated in a range of low temperatures and in field conditions. CONCLUSION  In general, the temperature decrease reduced the characteristics related to germination in the barley cultivars. The cultivars with desirable root number and length, shoot and coleoptiles,  Length showed better germination characteristics under low temperature conditions. Application of calcium chloride treatment with a concentration of 10 mM did not significantly influence the traits under the studied temperatures. It would seem that other concentrations should be considered. Cluster analysis classified cultivars with more desirable germination characteristics under all studied temperatures into separate group. Considering the importance of the ability of cultivars to face low temperature stress at the germination stage, it is recommended that the cultivars in this group be used for further studies and test confirmation.