Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 AENSI Journals Advances in Environmental Biology ISSN-1995-0756 EISSN-1998-1066 Journal home page: http://www.aensiweb.com/AEB/ Heat shock effects on chlorophyll fluorescence, membrane stability, and metabolites accumulation in durum wheat (Triticum turgidum L. var. durum) seedlings and relationships with yield stress tolerance indices 1,2Sofia 3Mouna Hamli, 3Mostapha Labhilili, 2Kenza kadi, 2Abd El Hamid Khabthan, 3Meryem Alyadini, Tagouti, 3Rajae Manzeri, 3Fatima Gaboun and 1Hmenna Bouzerzour 1 Valorization of Biological Natural Resources Laboratory, Faculty of Life and Natural Sciences, Setif-1 University, 19000, Algeria Department of Agronomy, Faculty of Life and Natural Sciences, Khenchela University, 40000, Algeria 3 Center Regional of Agricultural Research, Biotechnology Unit, P.O. Box 415, Rabat, Morocco 2 ARTICLE INFO Article history: Received 28 January 2015 Accepted 25 February 2015 Available online 6 March 2015 Keywords: Triticum durum, RWC, Proline, Fluorescence, P index, heat shock, seedling ABSTRACT Physiological traits are receiving increasing attention as screening tools for stress resistance to enhance both yield potential and yield stability under stressful conditions. This study was undertaken to investigate the discriminating ability of bioassays applied to durum wheat lines subjected to heat shock at the seedling growth stage and to study their relationships with grain yield stress tolerance indices. Sufficient amount of variability was observed in the responses of the eight durum wheat genotypes evaluated. Heat shock increased proline, soluble sugars and damage to cell membrane and decreased Fv/Fm ratio, relative water content and chlorophyll content. Genotype responses varied according to stress intensity. Different genotypes were declared as tolerant to stress based on different parameters. Base on proline and soluble sugars results Korifla was identified as stress tolerant. Bousselam was declared stress tolerant based on membrane stability, relative water and chlorophyll contents results. Waha was tolerant based on Fv/Fm ratio. P index identified three varieties as stable and high yielding. Correlations study indicated that the relationships between bioassays parameters and with tolerance indices varied according to stress levels, suggesting that these physiological traits could not be recommended to replace grain yield field testing, under the Algerian conditions of durum production. © 2015AENSI Publisher All rights reserved. To Cite This Article: Sofia Hamli, Mostapha Labhilili, Kenza kadi, Abd El Hamid Khabthan, Meryem Alyadini, Mouna Tagouti, Rajae Manzeri, Fatima Gaboun and Hmenna Bouzerzour., Heat shock effects on chlorophyll fluorescence, membrane stability, and metabolites accumulation in durum wheat (Triticum turgidum L. var. durum) seedlings and relationships with yield stress tolerance indices. Adv. Environ. Biol., 9(8), 116-125, 2015 INTRODUCTION High temperature adversely affects wheat productivity in arid and semi-arid regions. This stress decreases photosynthesis, impairs respiration, inactivates enzymes and disrupts membranes, suppressing growth. This type of stress poses a challenge that can only be addressed through development of tolerant cultivars. The limited success in developing heat tolerant cultivars is imposed by the complex nature of heat stress effects on plant and the difficulty to separate the effects of this type of stress from the effects of other stresses . Selection of high yielding and stress tolerant genotypes is an important objective in these stressful environments, where breeding relies on direct selection for yield and few alternatives to direct selection have been adopted. To develop stress tolerant genotypes, reliable and efficient techniques must be made available to assess the plant response to the targeted stress. Lack of appropriate traits, to use in place of or along with grain yield, as selection criteria, prevents yield improvement in these environments. However examples of traits, correlated with stress tolerance, and which have been efficient in wheat breeding, are reported in the literature . Chlorophyll fluorescence, proline, water soluble sugars, relative water and chlorophyll contents and membrane stability, have been proposed as selection criteria to improve stress tolerance [3, 4, 5, 6, 7]. These traits, which had the capacity to discriminate between tolerant and sensitive genotypes, under stress conditions, would enable breeders to identify superior genotypes and devise breeding strategies for the improvement of heat tolerance. Heat stress causes chlorophyll breakdown Corresponding Author: Sofia Hamli, Deparment of Agronomy, Faculty of nature science and life, Khenchela University 40000, Algeria. E-mail: [email protected] 117 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 in sensitive genotypes and alters chlorophyll fluorescence pattern [8, 9]. Fv/Fm ratio is an indicator of photoinhibition under heat stress and as such it was found useful for screening wheat for chlorophyll stability [8, 10, 11]. Proline and water soluble sugars play an active role in the protection of plants by acting as cellular osmotic regulators between cytoplasm and vacuole . Heat stress leads to drastic change in the cell membrane stability whose structural properties are altered, allowing electrolyte leakage. The relative level of membrane injury can be estimated by the amount of leaked electrolyte following heat stress treatment. The membrane thermo-sensitivity assay has been used as a technique to screen for heat tolerance in various crops [13, 14]. Stress tolerance has been also approached, with some success, by developing tolerant varieties through traditional plant breeding techniques using field evaluation. Grain yield of tested cultivars is evaluated under both stress and stress free growth conditions, enabling a direct comparison of the performance and stability. Several indices were proposed to describe such yield variation for a given genotype [15, 16]. Fischer and Maurer  proposed a stress susceptibility index (S) based on the minimization of yield reduction from favorable to unfavorable environments. Lin and Binns  proposed an index (P) for comparing yield performances of genotypes across environments, where the highest-yielding genotype within each environment is taken as the reference. The objective of this study was to assess the discriminating ability of physiological screening tests applied to durum wheat lines subjected to heat shock at the seedling growth stage and to evaluate their relative utility as predictor of stress tolerance expressed under the form of grain yield index. MATERIALS AND METHODS Eight durum wheat (Triticum turgidum L. var. durum) genotypes namely Bousselam, Gaviota durum, Korifla, Mrb3, Ofanto, Tajdid, Vitron and Waha were used as plant material. The pedigrees of these varieties and cross origin were reported by Nouar et al., . Seeds were first cleaned and a uniform seed size lot of each genotype was obtained by sieving. Seeds were surface sterilized by immersion in a hypochlorite solution (1% available chlorine) for 2 minutes and then rinsed thoroughly with distilled water. Enough seeds per genotype were placed in Petri dishes on a germination paper moistened with distilled water, and germinated in an incubator for 6 days at 25°C. Germinated seeds were transferred to plastic pots filled with 1/3 soil and 2/3 sand (v/v) and irrigated daily. Sets of 3-leaf seedlings were up rooted, gently washed with distilled water and immersed in a water bath at 40°C for 0, 30 and 60 minutes. The following traits were measured. Chlorophyll content: Chlorophyll content measurements were made on the emerged blade of two seedlings per treatment. Total chlorophyll pigments were extracted from leaf samples of 0.1g fresh weight in 10 ml of 85% acetone. Absorbance of the extract was measured, at 645 and 663 nm, on a Sontays Techtron 635 spectrophotometer, and samples chlorophyll concentration deduced using the following equation due to Arnon : Chl (mg/g FW) = 20.2 A645 – 8.02 A663 Relative leaf water content: Harvested leaf-samples were weighted to obtain the fresh weight (FW) and immersed in glass test tubes containing 10 ml of distilled water and stored in a cold room, overnight, to allow for rehydration. Leaf-samples were re-weighted for turgid weight (TW) determination, then were dried at 65°C for 24 hours and weighted to get dry weight (DW). Relative water content was determined using the following formulae reported by Azimzadeh and Azimzadeh : RWC (%) = 100 x [(FW-DW)/(TW-DW)]. Membrane thermo stability: Blade leaf-samples were collected from each treatment, rinsed twice in deionized water and cut into segment, 1 cm long. Five segments per treatment were immersed in glass test tubes containing 10 ml of double deionized water. The test tubes were tightly capped and held overnight at room temperature, then an initial reading (T1) was done with a conductivity meter. The test tubes were immersed in water bath at 100°C temperature for 60 minutes, let to stand overnight, and a second reading was taken (T2). Membrane thermo stability was expressed in percentage units of the relative leakage, according to Ibrahim and Quick : % injury = 100 x (T1/T2). Proline accumulation: A second experiment, similar to the one described here above, was conducted to obtain enough plant material for the determination of the accumulated proline, according to the method of Bates et al., , and water soluble sugars, using the anthrone colorimetric assay . Proline was extracted from 0.5 g leaf samples tissue mixed with 1mL of 3% 5-sulfosalicylic acid (w/v) in test tubes and stirred for 20 minutes. One hundred μL aliquots were pipetted into test tubes and adjusted to 1mL with double-deionized water. One mL acid 118 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 ninhydrin (prepared by mixing 1.25 g ninhydrin + 30 mL glacial acetic acid+ 13.8 mL 85% phosphoric acid + 6.2 mL double deionized water) and 1.0mL glacial acetic acid were added to the test tubes. Test tubes were stirred for 1 minute and incubated in a water bath for 60 minutes at 100°C. The reaction was stopped after incubation by placing tubes in ice bath. Tubes were removed from the ice bath and 4 mL of toluene was added to each test tube. Tubes were again stirred for 1 minute and allowed to settle down for 5 minutes for phase separation. Three mL of the upper phase, containing toluene, were pipetted into cuvette and absorbance was read at 520 nm in a spectrophotometer (Sontays Techtron 635 spectrophotometer) using toluene as blank. A standard curve was prepared using known concentrations of proline. A calibration series, incremented by10 µg/mL from 10 to 100 µg/mL of proline, was run, and the standard curve plotted. The concentration of the unknown samples was estimated by referring to the standard curve and expressed on fresh weight basis. Sugars accumulation: Water soluble sugars were determined from 100 mg of plant material, extracted in 80% ethanol solution. The extract was incubated for 6 hours at 60°C. Anthrone reagent was prepared by dissolving 150 mg anthrone in 72% H2SO4 solution. Plant extract was pipetted in glass test tubes, and 6 ml of the anthrone reagent were added to each sample, heated in a water bath at 100°C for 10 minutes. The test tubes were ice cooled for 10 minutes and to settle at room temperature for another 30 minutes. Optical density was read at 625nm on a Sontays Techtron 635 spectrophotometer. Samples water soluble sugar concentrations were estimated by referring to the sugar standard curve and expressed on fresh weight basis. Leaf chlorophyll fluorescence: Fluorescence measurements were done on attached leaf blades using a portable fluorimeter (Opti-Science 30). Three measurements were done per treatment on the leaf adaxial side. Fluorescence signals were used to provide estimates of Fv/Fm ratio . The experiments were conducted at the plant physiology laboratory, Faculty of life and natural sciences, Khenchela University (Algeria). The chlorophyll fluorescence experiment was conducted at the Biotechnology Unit of the Regional Center of Agricultural Research (CRRA, INRA, Rabat, Morocco), during a stay of the first author in December 2012 and January 2013. Stress tolerance indices: Two stress tolerance indices, the stress susceptibility index (S)  and the genotypic superiority index (P) , were derived from grain yield data collected from field trials carried out during the 2009-10 cropping season at five locations . The S index was calculated for each genotype as follows: S = (1-ys/yns)/(1-Ȳs/Ȳns), where ys is the stressed and yns is the non-stressed genotype grain yield. Ȳs is the stressed and Ȳns is the nonstressed location mean yield. The P index was calculated for each genotype (i) as follows: Pi = [Σ (Xij-Mj)²]/2n, where n is the number of locations, Xij est the yield of the ith genotype in the jth location, and Mj is the yield of the best performing genotype in the j th location. Statistical analysis: A two-way analysis of variance with 3 replications was used to test the main effects and the interaction . When appropriate a heat shock susceptibility index (HSSI), similar to the salinity susceptibility index reported by Jiang et al.,  was calculated for each variety as: HSSI (%) = [(100Ys/Yc) -100], where Ys is the mean value of the parameter measured under heat shock conditions and Yc is the mean value of the same parameter measured in the absence of heat shock (control). Linear correlation analysis was used to determine the relationships between the measured traits. Data were analyzed using the free software Cropstat 7.2. 3 . RESULTS AND DISCUSSIONS Heat shock effect on the measured parameters: The analysis of variance indicated significant heat shock main effect and genotype x heat shock interaction of the measured traits (Table 1). Mean values of the measured traits, average over varieties, are indicated in table 2. Proline, sugars and ion-leakage increased; while relative water and chlorophyll contents and Fv/Fm ratio decreased under heat shock treatments (Table 2). The heat shock-induced increase, over control value, observed under 60 min heat-treatment, was 107.8%, 95.1% and 214.3%, for proline, water soluble sugars and ion-leakage, respectively. The control mean values of these traits were 26.03µg/ml proline, 50.79µg/ml water soluble sugars and 11.12% injury. The heat shock-induced decline, observed under 60 min treatment, was 15.40% for relative water content, 9.10% for chlorophyll content and 20.27% for the Fv/Fm ratio. The control mean values of these characters were 88.64% relative water content, 3.98 mg/g FW chlorophyll content and 0.74 Fv/Fm ratio (Table 2). 119 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 In the absence of heat stress (check treatment), the tested varieties showed inherent differences in the expression of the measured traits (Table 3). Waha accumulated significantly more proline (35.83µg/ml) and sugars (61.21µg/ml). Korifla accumulated significantly less proline (12.90 µg/ml) and sugars (30.47µg/ml). Ofanto had the highest mean values of electrolyte leakage (14.00%), relative water (91.40%) and chlorophyll (7.40 mg/g FW) contents. Gta durum had the lowest relative water content (82.07%) and Vitron exhibited the lowest chlorophyll content (2.30 mg/g FW). Values of the Fv/Fm ratio were not significantly different among the tested varieties (Table 3). The analysis of the relative changes in accumulated proline, under heat shock treatments, indicated that Korifla and Mrb3 were more sensitive to heat shock, and Tajdid and Waha were less responsive. The 60 min treatment was more discriminating among genotypes which clustered into three groups: high (Korifla and Mrb3), intermediate (Vitron and Ofanto) and low accumulating genotypes (Figure 1A). Korifla accumulated, relatively, more water soluble sugars compared to Ofanto which accumulated less. The 60 min treatment discriminated best among the tested genotypes which grouped into four clusters, with an averaged sugar HSSI of 293.2% (Korifla), 212.0% (Mrb3, Tajdid, Vitron, Bousselam), 177.6% (Waha, Gaviota durum), and 119.6% (Ofanto) (Figure 1B). Table 1: Analysis of variance mean squares of Fv/Fm ratio, proline, water soluble sugars, % injury to cell membrane, relative water and chlorophyll contents of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 minutes at the seedling stage. Sources of variation Heat shock (HS) Rep/HS Genotype (G) G x HS Error Ddl 2 6 7 14 42 Fv/Fm 0.14476* 0.00595 0.01972ns 0.01531** 0.00173 Pro (µg/ml) 3627.6** 17.5 696.8ns 486.4** 10.9 Suc (µg/ml) 14052.9** 89.9 1331.1* 380.9** 153.1 Chl (mg/g FW) 25.5** 0.1 12.1* 2.8** 0.1 Inj (%) 3415.5** 9.8 44.4ns 35.3** 6.0 RWC (%) 1198.4** 4.5 9.5ns 31.3** 6.1 Fv/Fm = quantum yield; Proc = proline, µg/ml; Suc = Soluble sugars, µg/ml; Inj= injury to cell membrane, %; RWC= relative water content, %; Chl= Chlorophyll content, mg/g FW. ns,* = non-significant and significant effect at 5% probability level, respectively. Table 2: Heat shock main effect of accumulated proline, soluble sugars, % injury to cell membrane, relative water and chlorophyll contents of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 minutes at the seedling stage. Heat shock duration at 40°C Parameters 0 30 60 Lsd5% Fv/Fm 0.74 0.68 0.59 0.05 Chl (mg/g FW) 3.98 2.81 1.92 0.18 RWC (%) 88.64 84.94 74.97 1.50 Pro (µg/ml) 26.03 39.25 54.09 2.95 Sug (µg/ml) 50.79 73.19 99.15 6.69 Inj (%) 11.12 23.82 34.96 2.21 Fv/Fm = quantum yield; Proc = proline, µg/ml; Sug = Soluble sugars, µg/ml; Inj= injury to cell membrane, %; RWC= relative water content, %; Chl= Chlorophyll content, mg/g FW. Relative water content was less affected by heat shock treatment in Bousselam (100.0 and 91.0%) and Gaviota durum (100.0 and 95.5%) than in Tajdid (90.8 and 78.8%) and Vitron (93.2 and 76.9µ), under both 30 and 60 min treatments, which ranked differently Ofanto and Korifla. No clear clustering of the studied varieties appeared for this trait which exhibited a large range under 60 min treatment (Figure 2A). Heat shock treatments classified differently the tested genotypes for ion-leakage. Vitron expressed high increase in ion-leakage (305.9%), under 30 min treatment, but under 60 min heat stress treatment, it is Korifla (385.0%) which exhibited the highest ion-leakage. Bousselam (140.0%), under 30 min treatment, and Ofanto (256.0%), under 60 min treatment, had the lowest % injury index (Figure 2B). 120 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 450 BOU A GTA Kori 400 Mrb OFA 350 Proline HSSI (%) TAJ Vit 300 WAH 250 200 150 100 0 30 Heat shock duration ( min) BOU 350 60 B GTA Sugars HSSI (%) Kori Mrb 300 OFA TAJ Vit 250 WAH 200 150 100 0 30 Heat shock duration (min) 60 Fig. 1: Proline (A) and water soluble sugars (B) HSSI (%) of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 min at the seedling stage. Table 3: Check mean value of proline (Proc, µg/ml); water soluble sugars (Sug, µg/ml); injury to cell membrane (Inj, %); relative water content (Rwc,%) ; Chlorophyll content (Chl, mg/g FW) and Fv/Fm ratio of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 minutes at the seedling stage. Genotype Pro Sug % Inj Rwc Chl Fv/Fm Bousselam 24.84 60.60 12.50 85.00 2.37 0.775 Gta durum 34.85 53.13 11.50 82.07 2.50 0.740 Korifla 12.90 30.47 10.00 88.87 6.22 0.730 Mrb3 14.87 37.20 11.00 88.33 4.72 0.747 Ofanto 35.29 58.76 14.00 91.40 7.40 0.732 Tajdid 28.60 57.28 11.50 92.00 2.67 0.759 Vitron 20.43 47.73 9.00 92.53 2.30 0.720 Waha 35.83 61.21 9.50 88.93 3.67 0.716 Lsd5% 5.5 20.4 4.0 4.1 0.5 0.07 Fv/Fm = quantum yield; Proc = proline, µg/ml; Sug = Soluble sugars, µg/ml; Inj= injury to cell membrane, %; RWC= relative water content, %; Chl= Chlorophyll content, mg/g FW. 121 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 Decline in chlorophyll content, at 30 min heat shock treatment, varied from 7.0% (Bousselam) to 54.1% (Ofanto), and from 22.8% (Mrb3) to 80.9% (Ofanto), at 60 min heat shock treatment. Bousselam, with a low initial chlorophyll content of 2.4 mg/g FW, was the least affected, expressing a relative decline of 7.0% and 26.8% under both stress treatments, while Ofanto, with a high inherent chlorophyll content of 7.4 mg/g FW, was the most affected by the stress. Chlorophyll content bioassay clustered the tested varieties into three groups: Bousselam and Mrb3, the least affected group, with an average chlorophyll decline of 24.5%, Gta durum, Korifla, Tajdid, Vitron and Waha, intermediate group, with an average chlorophyll reduction of 48.9%, and Ofanto, forms the sensitive group with a chlorophyll content reduction of 80.9%, under 60 min heat treatment (Figure 3). 105 A 100 RWC HSSI (%) 95 90 85 BOU GTA Kori Mrb OFA TAJ Vit WAH 80 75 70 0 20 40 60 Heat shock duration (min) 450 400 BOU GTA Kori Mrb OFA TAJ Vit WAH B 350 Inj HSSI (%) 300 250 200 150 100 50 0 0 10 20 30 40 50 60 Heat shock duration (min) Fig. 2: Relative water content (A) and ion-leakage (B) HSSI (%) of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 min at the seedling stage. 122 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 0 -10 -20 HSSI Chl (%) -30 BOU GTA Kori Mrb OFA TAJ Vit WAH -40 -50 -60 -70 -80 -90 0 30 60 heat shock duration min Fig. 3: Chlorophyll content HSSI (%) of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 min at the seedling stage. Concerning the decline in chlorophyll content under heat stress, the results of the present study corroborate those reported by Dash and Mohanty  who analyzed the responsiveness of bread wheat cultivars to growth at 40°C for 60 h and the potential of the heat stressed -seedlings to recover during 24 h post-stress growth at 25 °C. These authors mentioned that chlorophyll content in heat stressed-seedlings declined to 76% in HD-2643 cultivar and between 50 to 57% in HD-2327 and HD-2307 cultivars. The Fv/Fm genotype x heat shock interaction analysis suggested the high tolerance of Waha which showed no significant decline in its Fv/Fm ratio under both heat stress treatments. In comparison, Vitron and Korifla exhibited a significant reduction in the Fv/Fm ratio which declined by 7.0%, at 30 min, and by 45.0% at 60 min heat shock treatments. Bousselam and Gaviota durum maintained a constant Fv/Fm ratio at both treatments, showed an averaged decrease of 7.87 and 6.36%, at 30 and 60 minutes heat shock treatments, respectively. The 60 minutes heat shock treatment was the most discriminant between varieties, grouping them into three clusters: tolerant (Waha), moderately tolerant (Mrb3, Ofanto, Tajdid, Bousselam and Gaviota durum), and susceptible genotypes (Vitron and Korifla) (Figure 4). Concerning the variation in the decline of the Fv/Fm ratio, among genotypes, similar results to those of the present study were reported by Dash and Mohanty . These researchers analyzed the Fv/Fm ratio of control and heat stressed-seedlings, and found that heat-stress induced maximal decrease in the Fv/Fm ratio of PBN51 and HD-2643 cultivars and that among the studied cultivars, the Fv/Fm ratio was significantly reduced in C306, HD-2402, and HD-1553, but not in HD-2307cultivars. 110 Wah 100 Fv/Fm HSSI (%) Mrb 90 80 Bou Gta Of Tajd 70 Vit 60 Kor 50 0 10 20Heat shock 30 40 50 60 duration (min) Fig. 4: Fv/Fm HSSI (%) of eight durum wheat genotypes subjected to heat shock stress at 40°C during 0, 30 and 60 min at the seedling stage. 123 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 In general, the results of the present study corroborate those reported by Araus et al.,  who found that the Fv/Fm ratio, chlorophyll and relative water contents declined from the unstressed (irrigated) to the stressed (rainfed) environments. The reduction in the Fv/Fm ratio was interpreted as the occurrence of photo-damage rather than photoprotection. Similarly, Balochi  mentioned that stress decreased chlorophyll and relative water contents and Fv/Fm ratio, in all the tested varieties and that genetic variation was observed for the measured traits among varieties under stress conditions. Grain yield stress tolerance indices: Grain yield ranged from 1.05 t/ha, yield of Tajdid, expressed at HAR location to 5.59 t/ha, yield of the same genotype observed at KH location (Table 4). On average, Mrb 3, Bousselam and Waha were among the top grain yielding entries. P index values varied from 0.05 for Mrb 3 to 0.65 for Ofanto. Based on this index Mrb 3, Bousselam and to a lesser extent Waha are classified as stable and high yielding. While Ofanto, Vitron and Gaviota durum are classified as unstable and low yielding (Table 4). S index values varied from 0.92 for Ofanto to 1.11 for Tajdid. Since values of this index, less than unity, are suggestive, according to Fisher and Maurer , of tolerance to stress, then Ofanto, Gaviota durum, Vitron and Bousselam are tolerant to stress. These results suggested that Bousselam cumulates, both, above average grain yield and stress tolerance, while Ofanto, Gaviota durum and Vitron are stress tolerant and low yielding (Table 4). These results indicated that P index is best suited to select for higher and stable yield under both stressed and unstressed conditions. This index identified three genotypes as stable and high yielding among which two (Bousselam and Waha) have been released as cultivars . Table 4: Average grain yield per location and stress tolerance indices of the eight durum wheat genotypes Grain yield (t/ha) / location Génotype SET KH HAR SBA SAI Average Bousselam 4.37 5.01 1.47 2.71 3.68 3.45 Gaviota durum 3.55 4.26 1.33 2.29 4.10 3.10 Korifla 3.97 5.13 1.23 2.31 3.33 3.19 Mrb3 4.61 4.98 1.32 2.97 3.78 3.53 Ofanto 2.84 4.15 1.37 2.06 3.42 2.77 Tajdid 3.69 5.59 1.05 2.50 2.96 3.15 Vitron 3.22 4.53 1.37 1.95 4.16 3.05 Waha 3.82 5.04 1.25 2.78 3.76 3.33 Locations: SET= Setif, KH= Khroub, HAR= Harrouch, SBA= Sidi Bel Abbes, SAI= Saïda. Stress index P S 0.06 0.97 0.34 0.94 0.17 1.04 0.05 1.01 0.65 0.92 0.25 1.11 0.41 0.95 0.11 1.03 Relationships between tests parameters and grain yield stress tolerance indices: Using the relative values of the assays parameters, expressed as heat shock susceptibility index (HSSI), the analysis of the spearman's rank correlation indicated that under 30 minutes exposure at 40°C, soluble sugars were positively correlated with % damage to cell membrane (r= 0.881, P=0.007), and with Fv/Fm ratio (r= 0.762, P=0.037). Per cent damage to cell membrane was positively and significantly correlated with Fv/Fm ratio (r= 0.762, P=0.037) and negatively with relative water content, at 10% probability level (r= -0.643, P=0.083). Chlorophyll content showed a negative correlation with P index (r= -0.714, P=0.058). Under 60 minutes exposure to heat shock stress, soluble sugars were positively correlated with % damage to cell membrane (r= 0.850, P=0.011), but not with Fv/Fm ratio, as it was observed under 30 minutes exposure to heat shock stress. However soluble sugars showed significant correlation, negative with P index (r= -0.659, P=0.084), and positive with S index (r= 0.716, P=0.052). These relationships were not observed under 30 min exposure. These results indicated the inconsistency of the relationships between physiological tests. The relationships with stress indices appeared to be dependent on stress severity. These laboratory tests do not predict the genotypic response to stress experienced in the field. As such, they could not be recommended to replace field testing based on grain yield as screening tools for identification of tolerant genotypes to be used in crossing programs or applied directly to segregating populations. The results of the present study do not corroborate those of Talebi  who reported a significant correlation between chlorophyll content and S index, under moisture-stressed conditions, suggesting the potential of this index for selecting drought tolerant genotypes. Cseuz  reported that water retention capability, assimilates translocation and canopy temperature tests were closely correlated, in drier years, with multi-location yield tests, and suggested them as screening tools. Conclusion: The various laboratory tests assessed in this study indicated sufficient amount of variability in the responses of the seedling of the eight durum wheat genotypes evaluated. Heat shock affected significantly the measured parameters, increasing proline, soluble sugars and damage to cell membrane and decreasing Fv/Fm ratio, relative water content, and chlorophyll content. Genotype responses varied according to genetic background and stress intensity and could be grouped into tolerant and sensitive, based test results. P index appeared to be best suited to select for higher and stable yield under both stressed and unstressed field 124 Sofia Hamli et al, 2015 Advances in Environmental Biology, 9(8) May 2015, Pages: 116-125 conditions. This index suggested Mrb3, Bousselam and Waha as stable and high yielding. Spearman's rank correlations indicated that the relationships between bioassays results and with stress tolerance indices varied according to stress levels. As such, the laboratory tests assessed in the present study could not be recommended to replace grain yield field testing, under the conditions of the Algerian durum wheat production. 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