Introduction

Olive (Olea europaea L.) is one of the most important evergreen trees in the Mediterranean area. The olive tree grows mostly between latitudes of 30 and 45° in both hemispheres (Candev et al. 2009). However, in recent years increasing demand for better quantity and quality of olive oil has resulted in the cultivation of olive trees. This resulted in expanding olive cultivation areas into higher latitudes rather than its origin cultivation area in the Mediterranean basin (Mancuso, 2000)

A major limiting factor for growing olives at higher latitudes is exposure to minimum temperature especially in winter and early spring. When winter temperature drops below -7 °C, the aerial parts of olive tree can be damaged. This leads to yield reduction and in extreme situation threatens the life of the plant (Palliotti and Bongi, 1996). At temperatures below -12 °C, olive trees would face to severe damages which decrease its survival (Gomez del Campo and Barranco, 2005). Selection and use of tolerant cultivars are the most effective way for avoiding frost damage in olive trees. Plant physiological features such as stomatal density (Roselli et al., 1989), phenolic compound concentration (Roselli et al., 1992), ionic leakage (Barranco et al., 2005), and carbohydrate and starch concentrations (Lavee, 1986) have been used for selection of frost tolerant genotypes in olive.

In this study, changes in ionic leakage, reducing sugars, starch, proline and phenolic concentrations as biochemical features together with stomatal density as a morphological feature, were assessed in eight olive cultivars with contrasting levels of cold hardiness. Changes associated with freezing stress were analyzed to identify differences among cultivars in order to find suitable criteria for selection of tolerant cultivars in cold-winter regions.

Materials and Methods

Plant material and application of freezing stress

Eight olive cultivars (‘Amygdalolelia’, ‘Conservallia’, ‘Dakal’, ‘Shiraz’, ‘Dehghan’, ‘Zard’,‘Dezful’ and ‘Tokhme-Kabki’) were obtained from the Experimental Agricultural Station and Natural Resources of Kazeroon, Iran, in 2012. Two-year-old plants (three plants for each cultivar) were propagated by semi-hardwood cuttings and were grown in 2 l plastic bags under greenhouse condition before exposure to freezing temperature. The plants were exposed to low temperatures (4 °C, 0°C, -6 °C, -12 °C, and -18 °C) for one hour. The temperature was gradually decreased to -6 °C by approximately 1.5 °C h^-1 and by 5 °C h^-1 thereafter. To thaw slowly, plants were removed from each low temperature treatment and were exposed to 4 °C overnight. To estimate the extent of freezing injury to the leaves, electrolyte leakage and biochemical changes were determined.

Electrolye leakage

Ionic leakage was measured as described by Bartolozzi and Fontanszza (1999). Five discs with 10 mm diameter were collected from leaves of three plants that had been subjected to the different freezing treatments and placed in a test tube containing 25 ml manitol (0.2 M) and incubated in a shaker for 4 h. Electrolytic conductivity (EC1) was measured using digital conductivity meter (Model HI8633, USA). Solutions and samples were then autoclaved to kill the cells. Once the solution was cooled, conductance was again measured (EC2). Ionic leakage (EC%) was calculated based on the Equation (1).

EC% = EC1 / EC2 × 100          (1)

Proline concentration

Twenty four hour after removing from freezer, young fully-expanded leaves (0.2 g) were used for determination of proline. Samples were homogenized in 3% sulfosalicylic acid. After addition of acid-ninhydrin, proline content was determined according to the method described by Bates et al. (1973). The absorbance of the fraction with toluene was determined at 520 nm, using a spectrophotometer (Model UV-120-20, Japan).

Total Phenolic Concentration

Total phenolic concentration (TPC) was measured using Folin-Ciaceleture reagent. Five mg of dry leaf powder was mixed with distilled water: methanol (50:50) and extracted for 15 min. The extract was then centrifuged at 13400 rpm for 15 min and the remaining filtrate was re-extracted twice using 5% aqueous methanol (Gutfinger, 1981). One ml of extract was mixed with one ml of 2 % sodium carbonate (w/v) and was kept at room temperature for 30 min under dark conditions. The absorbance of the mixture was measured by spectrophometer at 750 nm. TPC was calculated as mass of gallic acid equivalents (GAE) per fresh weight (FW) mass of sample (g kg^-1).

Soluble carbohydrate concentration

To determine soluble carbohydrate concentration, 0.1 g of leaf powder was twice extracted with ethanol (80%). The samples were then centrifuged at 5000 rpm for 10 min and supernatant was adjusted to 25 ml. Soluble carbohydrate concentration was measured according to Dubois et al. (1956). One ml of 18% phenol and five ml of sulfuric acid were added to one ml supernatant in a test-tube. The mixture was immediately shacked and its absorption was recorded at 490 nm using a spectrophotometer (Model UV-120-20, Japan).

Starch concentration

Starch concentration in the leaf samples was measured using anthrone reagent (Mc Cready et al., 1950). To do this, five ml cold water and 6.5 ml perchloric acid (52%) were added to the pallet material collected from the sample which was used for sugar analysis and mixed for 15 min. Approximately, 20 ml water was added to the mixture and the samples was centrifuged at 5000 rpm for 10 min. The supernatant was separated and the same procedure was repeated. The supernatants were combined and left for 30 min at 0 °C. After filtration, the volumes of supernatants were adjusted to 100 ml. About 2.5 ml of cold anthrone solution (2%) was added and the sample was heated at 100 °C for 7.5 min, then transferred immediately to an ice bath and cooled down to the room temperature. Absorption at 630 nm was recorded using a spectrophotometer (Model UV-120-20, Japan).

Stomatal density

Stomatal density was measured according to Roselli et al. (1989). Twenty leaves from midpoint of one-year-old wood were taken from each of the 10 cultivars (previous cultivars + Derak and Roughani cultivars). The stellate hairs were removed from the lower surface of each leaf using adhesive tape. A thin film of Actrifix was then painted on the clean surface, allowed to dry at room temperature and then peeled from the leaves. The films from each leaf were mounted on a glass microscope slide. The stomatal density was recorded with a light microscope using a 10X acular, 40X objective in a field area of 0.49 mm². Three stomatal counts were made from each leaf position (apex, center and base) for nine observations per leaf, and in total 180 observations were recorded per each cultivar.

Determination of wood injury

To evaluate cold hardiness of olive cultivars, plants were held at 22±1 °C for 24 h after removing them from freezer (Whirl pool, IRAN). For this evaluation 10 percent of stem per each cultivar was randomly harvested at each temperature. Individual pieces of stem were sectioned with rozar blade with 10 mm diameter and examined under a binocular microscope. The samples were checked for necrosis of the wood and samples with dull, straw or black/brown appearances were considered as dead samples. This experiment was repeated three times for each cultivar (Rekika et al., 2004).

Statistical analysis of freezing treatments was conducted as a factorial experiment using a completely randomized design with three replications (plants). Measurements of stomatal density were conducted in a completely randomized design. Twenty leaves per cultivar and nine observations per leaf were analyzed using SAS software and means with significant differences ware compared using DMRT (P= 0.05)

Results

Electrolyte leakage

At -18 °C, the highest leakage rates were observed in ‘Dakal’ and ‘Shiraz’ while the lowest electrolyte leakage was observed in ‘Dehghan’ cultivar. At -12 °C, ‘Dehghan’ and ‘Zard’ showed the lowest electrolytic leakage (Table 1). Based on the obtained results for electrolytic leakage, olive cultivars can be ranked as the following:

 ‘Shiraz’ ≥ ‘Dakal’ ≥ ‘Dezful’ ≥ ‘Amygdalolelia’≥ ‘Tokhme-Kabki’≥ ‘Conservallia’ ≥ ‘Zard’ ≥ ‘Dehghan’.

Starch concentration

The analysis of results indicated that starch concentration in olive leaf tissue was 74.7 mg g^-1 DW following freezing stress at 0 °C, while it was 44.6 mg g^-1 DW following stress at -18 °C. Leaf tissue of ‘Shiraz’ cultivar had the highest starch concentration, whereas, ‘Dehghan’, ‘Tokhme-Kabki’, and ‘Zard’ had the lowest starch concentrations following freezing stress (Table 2). Based on the obtained results for starch concentration, olive cultivars can be ranked as the following:

‘Shiraz’> ‘Dakal’>‘Amygdalolelia’≥‘Dezful’> ‘Consercvallia’> ‘Zard’ ≥‘Tokhme-Kabki’ > ‘Dehghan’.

Reducing sugar concentration

Following freezing stress conditions, ‘Dehghan’ had the highest and ‘Shiraz’ had the lowest reducing sugar concentrations (Table 3). Reducing sugar concentrations were significantly higher (60.9 mg g^-1 DW) following stress at 0 °C, while the amount of reducing sugar was 90.2 mg g^-1 DW following freezing stress at -18 °C. Among the studied cultivars, ‘Shiraz’ had the lowest and ‘Dehghan’ had the highest reducing sugar concentrations (Table 3).

Based on the obtained results for concentration of reducing sugar, olive cultivars can be ranked as the following:

 ‘Dehghan’ > ‘Zard’ ≥ ‘Tokhme-Kabki’ ≥ ‘Conservallia’ >‘Amygdalolelia’ > ‘Dezful’ ≥ ‘Dakal’> ‘Shiraz’.

Proline concentration

Leaf proline concentrations following freezing stress were significantly increased as freezing temperature increased (Table 4). Proline concentration was 23.6 (µmol g^-1 FW) following stress at 0 °C and 79.9 µg mg^-1 FW at -18 °C. The highest proline concentration was recorded in ‘Dehghan’ (80.3 µg mg^-1 FW) while the lowest concentrations were observed in ‘Shiraz’ and ‘Dakal’ (27.7 and 34.5 µg mg^-1 FW, respectively) (Table 4).

Based on the obtained results for concentration of reducing sugar, olive cultivars can be ranked as the following:

‘Dehghan’ ≥ ‘Zard’ ≥ ‘Tokhme-Kabki’ ≥ ‘Conservallia’ ≥ ‘Dezful’ ≥ ‘Amigdalolelia’ ≥ ‘Dakal’ ≥ ‘Shiraz’.

Phenolic concentration

Following application of freezing stress, concentrations of total phenolic compounds in ‘Dehghan’ and ‘Zard’ cultivars were higher than their concentrations in other cultivars (Table 5). When freezing temperature increased from 0°C to -18°C, the subsequent phenolic concentrations in the leaves were increased from 12.1 to 18.6 mg g^-1 DW. Highest and lowest total phenolic concentrations were measured in ‘Dehghan’ and ‘Shiraz’, respectively (Table 5).

Based on the obtained results for concentration of phenolic compounds, olive cultivars can be ranked as the following:

‘Dehghan’ > ‘Zard’ > ‘Tokhme-Kabki’ > ‘Conservallia’> ‘Amygdalolelia’ > ‘Dezful’ > ‘Dakal’> ‘Shiraz’.

Stomatal density

Stomatal density was significantly different among the ten cultivars. The highest stomatal densities were found on the leaves of ‘Derak’ and ‘Amygdalolelia’ and the lowest Stomatal density was observed in ‘Zard’ cultivar (Table 6).

Based on the obtained results for stomatal density, olive cultivars can be ranked as the following:

‘Derak’ ≥ ‘Amygdalolelia’≥ ‘Shiraz’ > ‘Roughani’> ‘Dezful’> ‘Dehghan’ ≥ ‘Dakal’ > ‘Conservallia’ > ‘Tokhme-Kabki’ > ‘Zard’.