Evaluation of Physiological and Biochemical Changes of Pistachio (Pistacia vera L. cv. Ahmad-Aghaii) on Cold Tolerant and Sensitive Rootstocks under Freezing Stress Conditions

Spring cold injury is one of the main limiting factors to production and distribution of pistachio. Pistachio is one of the most valuable and exported agricultural crops of Iran. Since, spring frosts results to considerabe damage to this plant, hence, it is important to investigate methods for reducing freezing damage. For this reason, selection of rootstocks and cultivars are an important objective in breeding programs. Freezing temperatures (below 0ºC) cause the movement of water from the protoplast to the extracellular space, resulting in the growth of extracellular ice crystals and ultimately, cell dehydration. Plants have developed complex processes to survive and recover from unfavorable conditions. To tolerate cold stresses, plants develop multiple mechanisms, including the accumulation of cryoprotective molecules and proteins, alterations in membrane lipid composition, and primary and secondary metabolite composition, as well as changes in global gene and protein expression Frost affects cell membranes, which become less permeable, and even break, giving rise to the leakage of solute from damaged cells. There is often a good correlation between ion leakage and freezing tolerance (22). Sugars may depress the freezing point of the tissue and act as a nutrient and energy reserve, alter phase properties of membranes in the dry state and act as cryoprotectants to preserve protein structure and function. Other compounds acting similarly are lipids, soluble proteins and free proline (44). Proline seems to have diverse roles under osmotic stress conditions, such as stabilization of proteins, membranes and subcellular structures and protecting cellular functions by scavenging reactive oxygen species (23). The aim of the present study was to evaluate different degrees of sensitivity to low temperatures in different genotypes and ‘Ahmad-Aghaii’ cultivar in relation to physiological and biochemical changes in field conditions. In order to determine the effects of rootstock on pistachio cultivar ‘Ahmad-Aghaii’ under freezing stress conditions, an experiment was carried out as factorial based on a randomized completely design (RCD) with four replications. Treatments consisted of two levels: 1- rootstock genotype (four cold sensitive and tolerant rootstocks) and 2- temperatures (-2 and -4 ºC). The sampling was performed in full bloom stage from apical branches of pistachio cultivar ‘Ahmad-Aghaii’ budded on these rootstock genotypes. The branches in pots contain distillted water treated under -2 and -4 °C for 2 h. After treatment, the chilling index was determined. Flower clusters were used for measuring physiological and biochemical parameters. All determinations were carried out in four triplicates and data were subjected to analysis of variance. Analysis of variance was performed using the ANOVA procedure. Statistical analyses were performed according to the SAS software. Significant differences between means were determined by Duncan’s multiple range tests. P values less than 0. 05 were considered statistically significant. The results showed that chilling index was significantly lower in the cold-tolerant rootstocks than cold-sensitive rootstocks at -2 and -4 ºC. Also, Results indicated that electrolyte leakage, hydrogen peroxide (H2O2) and malondialdehyde (MDA) were significantly lower in tolerant rootstocks than sensitive ones. The content of soluble carbohydrate, total protein and proline were significantly higher intolerant rootstocks than sensitive ones. The activity of anti-oxidant enzymes ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT) in tolerant rootstocks was greater than sensitive rootstocks. The reaction of temperature and rootstock indicated that electronic leakage, proline, H2O2, و and MDA significantly increased in -4 ºC. The activity of anti-oxidant enzymes APX, GPX and CAT decreased in -4 ºC as compared to -2 °C especially in cold-sensitive rootstocks. Some researchers believe that the accumulation of proline is as an index to select the drought-resistance varieties (26, 48). Stated that there was no comprehensive information about the relationship between the accumulation of proline and tension resistance. Research on apricot and peach confirmed the results of the present study because this pattern is also seen in their proline level (26 and 41). While the starch concentration decreases during the dormancy, the amount of proline increases which is in accordance with their results (36). In this study, the damage of the membrane increased with decreasing temperature. The results showed that the rootstocks could increase the resistance to cold by increasing the amount of soluble sugars, protein, proline and the activity of the antioxidant system in the shoots and leaves of the scion. Regarding physiological and biochemical studies, it was determined that ‘Ahmad-Aghaii’ cultivar budded on cold tolerant rootstocks had higher soluble sugars, total protein, proline and CAT, APX and GPX enzymes activity and had less chilling index, ion leakage, H2O2 and MDA, which indicates less damage to the membrane of the cell and its contents compared with the cultivar 'Ahmad-Aghaii' budding to sensitive rootstocks. Consequently, the findings of this study selected TR1 as the most tolerant rootstock compared to other ones.