131 1. Introduction Despite considerable advances in technology, agri- culture is exposed to climate changes in all parts of the world. Among climatic factors, rainfall is the most critical because 70% of the main areas under cultivation are still without irrigation (Wilhite, 2001). Water shortage is the main characteristic of agricul- ture in Mediterranean regions, inducing water stress dur- ing spring and summer. Fruit trees survive in this situation because they are prone to physiological or morphological changes which enable them to avoid drying damage, cast it back, or tolerate it (Torrecillas et al., 1999). Drought tol- erance is observed with different rates in almost all plant species. Understanding plant responses to the external en- vironment is an essential component for selection stress tolerance (Reddy et al., 2004). In recent years, in southern Iran, low and poor distribution of rainfall has caused great damage to plants. Water stress, particularly in rain-fed fig production areas (e.g. Estahban) has become a big issue. In the Estahban region not only the annual production has decreased, but also the highly productive trees are in dan- ger of destruction. One strategy to confront this problem is the identifica- tion and selection of more tolerant genotypes. Traditional breeding approaches for selection of tolerant plants are time consuming and complex processes. Imposing wa- ter stress in field-grown crops is difficult because of the unpredictability of rainfall and the possibility of seepage from adjoining plots and drought escape of deep roots from osmotic stress. Since field evaluation of drought ef- fects is highly correlated with environmental conditions, in vitro screening techniques allow for a better control of cul- ture conditions. Tissue culture also offers the possibility of screening many plants in limited space and time, assuming that there is a correlation between cellular, tissue, organ and in vivo plant responses (Mohammad et al., 2000). Polyethylene glycol (PEG) has been used to simulate water stress in plants. PEG of high molecular weight is a non-ionic osmoticum lowering the water potential of nu- trient solution without being taken up or being phytotoxic (Hassan et al., 2004). It has been shown that the shoot length decreases with increasing water stress via in vitro culture in cherry (Sivritepe et al., 2008) and mulberry (Tewary et al., 2000). In a study on mulberry, leaf rela- tive water content (RWC) decreased with increasing water stress (Ramanjulu et al., 1998). Accumulation of proline is the most common plant response to decreasing water potential (Helal Ragab and Samir Moustafa, 2008). It has been reported that with in- creasing PEG in date palm seedling culture, the rate of pro- line was increased in tolerant cultivar (Djibril et al., 2005). Carbohydrates have an important role in osmotic regu- lation in different plant parts (Masoudi-Sadaghiani et al., 2011). Simple sugars such as glucose and fructose were increased and the rate of sucrose and starch decreased un- der water stress (Sharp and Davies, 1979; Munns and Weir, 1981; Wang and Stutte, 1992; Nawar and Ezz, 1993; Clif- In vitro drought effects on morphological and physiological indices of two fig (Ficus carica L.) cultivars A. Shekafandeh(*), S. Hojati Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran. Key words: drought, growth parameters, polyethylene glycol, proline. Abstract: In vitro responses of two fig cultivars, ‘Sabz’ and ‘Siah’, were evaluated in MS media containing four levels of polyethylene glycol (PEG) (0, 2, 4, 6%) as a simulation of water stress. The results showed that in Sabz cultivar, shoot length, shoot fresh and dry weights were 43, 36 and 25%, respectively, lower than control in drought treatments caused by 4% PEG, while the leaf area and specific leaf area were not significantly (P>0.05) affected. In Siah cultivar, shoot length, fresh and dry weights were 57, 58 and 40%, respectively, lower in stressed media in comparison to control. In contrast to Sabz cultivar, leaf area and specific leaf area of Siah cultivar were significantly reduced by addition of 6% PEG (56 and 21.5%, respectively). Naturally, the amount of proline in ‘Sabz’ was higher than in ‘Siah’ (81.8 μmole g-1 versus 16.7 μmole g-1). However, in both cultivars, with addition of PEG in culture media, leaf proline content was increased, in com- parison to control. With increasing PEG% in culture media, the amount of leaf soluble sugars content increased and the amount of starch decreased. The result show that ‘Siah’ is more sensitive to drought than ‘Sabz’ and that in vitro culture can be used to evaluate drought tolerance of cultivars. Adv. Hort. Sci., 2012 26(3-4): 131-137 (*) Corresponding author: shekafan@shirazu.ac.ir. Received for publication 26 April 2012 Accepted for publication 12 September 2012 132 ford et al., 1998; Perez-Perez et al., 2007; Mafakheri et al., 2010). In Iran, Fars province with about 36,000 hectares under fig cultivation is the primary fig production area. Es- tahban district with 20,000 hectares under fig cultivation is the center of fig cultivation in Fars province, as well as in Iran. As mentioned above, due to drought in recent years, highly productive trees are in danger of destruction. The cultivars Sabz and Siah are the most desirable cultivars in Iran; there has not been report on water stress tolerance of these cultivars. Selection of more drought tolerant culti- vars can be used to establish of new orchards and a prom- ising rootstock for other interested cultivars. The aim of this investigation was to evaluate the ef- fects of drought created by polyethylene glycol (PEG) on morpho-physiological changes of two important fig culti- vars (‘Sabz’ and ‘Siah’) and leading to selection of more drought tolerant varieties using in vitro culture. 2. Materials and Methods Plant material and explant decontamination In this research, the in vitro-derived micro-shoots of two cultivars of Ficus carica L. ‘Sabz’ and ‘Siah’ were used. The branches (20~30 cm long) were cut from rain-fed, ma- ture mother trees established in Estanban Fig Research Sta- tion located at 29° 07’ N latitude and 54° 02’ E , 197 km southeast of Shiraz. Freshly grown stems with 2~6 nodes were cut from the above branches and washed in a solution of water-detergent (three drops of detergent in 100 ml of wa- ter) for 30 min, then placed in a solution of benomyl (3%) for 1 h (Torres, 1998). To prevent the secretion of phenolic compounds into the culture media, explants were treated in a solution of 2% ascorbic and citric acid for 45 min. At this point, the stems were transferred to a laminar air flow cabi- net. They were first dipped in 70% (V/V) ethanol for 45 s, then treated with 15% (V/V) Chlorox solution (a household bleach containing 5.25% sodium hypochlorite) for 15 min and rinsed three times with sterilized distilled water. Nodal segments of 1~1.5 cm long were cut and used as explants. The basal medium was MS (Murashige and Skoog, 1962), containing 3% sucrose (MERCK, LGaA 64271Darmstadt, Germany) and 1mg l-1 (4.4 µM) benzyl adenine (BA) and solidified by 0.8% agar-agar (MERCK, LGaA 64271Darm- stadt, Germany). The pH was adjusted to 5.7±0.05 prior to autoclaving at 1.2 atm pressure and 121˚C for 20 min. Cultures were maintained in a growth chamber at 25±2˚C, 58% relative humidity and a photon flux density of 40 μmol m-2 s-1 was provided by white fluorescent tubes, with a 16 h photoperiod. PEG treatments After four weeks, the micro-shoots (2 cm length) from the above cultures were transferred into 500 ml culture vessels containing 100 ml MS basal media supplemented with different levels of PEG (0, 2, 4 and 6%) as selective agent and maintained under the environmental conditions described above. Growth measurements Seven weeks after the beginning of experiments, shoot length and shoot fresh and dry weight were evaluated. Leaf area was measured by leaf area meter (Delta- Tdevices England) and specific leaf area was calculated as follows: specific leaf area = leaf area (cm2)/leaf dry weight (g) Relative water content Leaf relative water content (RWC) was estimated ac- cording to the method described by Whetherley (1950). Twenty healthy leaf discs of 0.7 cm diameter were cut from plants, using a leaf punch and washed three times with double distilled water. Leaf discs were weighed (FW), then placed into a 10 ml conical flask, immersed in 10 ml distilled water for 4 h in dark. Turgid weight (TW) of leaf discs were then measured and samples were dried in an oven (80ºC) until constant weight (DW) was achieved. RWC was calculated from the following equation: Relative water content % = FW - DW/TW - DW x 100 Chlorophyll content For chlorophyll determination, prior to extraction fresh leaf samples were washed with deionized water to re- move any surface contamination. One g leaf samples were ground in 80% acetone using a pestle and mortar. The mix- ture was centrifuged at 4800 rpm for 20 min. The optical density of the supernant was measured at 663 and 645 nm wavelengths and chlorophyll content was calculated using the following equation: Mg Chl g FW = [20.2 (OD645 nm) + 8.02 (od663 nm)] x V/FW x 1000 where V is final volume of solution (ml) and fw, leaf fresh weight (mg). Proline contents To determine proline content of the leaves, 0.5 g of plant material was homogenized in 10 ml of 3% aqueous of sulfo- salcylic acid and the homogenate filtered through Whatman # 2 filter paper. Two ml of filterate was reacted with 2 ml acid ninhydrin and 2 ml of glacial acetic acid in a test tube for 1 h at 100°C and the reaction terminated in an ice bath. The reaction mixture was extracted with 4 ml toluene, mixed for 15~20 seconds. Chromophore containing toluene was as- pirated from the aqueous phase and the absorbance was read at 520 nm. The proline concentration was determined based on standard curve and calculated as follows: μ moles proline/g FW = (μg proline/ml x ml toluene) / g sample/5 115.5 μg / μmole Electrolyte leakage Electrolyte leakage was measured as an assessment of cell wall permeability. Electrolyte leakage was measured using an Electrical Conductivity Meter. Two mature leaves per plant were taken and cut into 1 cm segments. After three washes to remove surface contamination, leaf sam- ples were placed in individual vials containing 10 ml of 133 distilled water. The samples were incubated at room tem- perature on a shaker (100 rpm) for 24 h. Electrical Con- ductivity (EC) of bathing solution (EC1) was read after incubation. Samples were then placed in an autoclave at 120°C for 20 min and the second reading (EC2) was de- termined after cooling the solution to room temperature. The electrolyte leakage was calculated as EC1/EC2 and expressed as percent. Soluble sugars content Soluble sugars were extracted from 0.1 g fresh leaves by heating with 5 ml of 80% ethanol in a water bath at 70°C for 30 min. The insoluble residue was removed by centrifuging at 5000 g for 10 min. One ml of the resulting extract was mixed with 1 ml of 5% phenol solution and 5 ml of sulfuric acid. The mixed solution was permitted to cool to room temperature, then vortexed. The absorbance was read at 490 nm using a spectrophotometer. The sol- uble sugar content of each sample was determined using standard curve for glucose and expressed as mg glucose g-1 FW (McCready et al., 1950; DuBois et al., 1956). Starch content The solid residue remaining in the centrifuge tube af- ter removal of all soluble sugars in the previous section was washed, re-extracted and re-centrifuged four times using 80% (V/V) ethanol. Starch content in the samples was determined colorimetrically using anthrone method (McCready et al., 1950). The absorbance was determined at 630 nm in a digital spectrophotometer (Spectronic 20 D+; Spectronic Instruments Inc., New York, USA.) as de- scribed by López et al. (2002). Statistical analysis The experiment was arranged as a 2×4 factorial ex- periment in completely randomized design (CRD) with 10 replicates, each consisting of three plants. Thus, there were 30 plants in each treatment and a total of 240 plants in the experiment. Data were subjected to analysis of vari- ance using the SPSS software (ver. 13.0) SPSS Inc. Mean differences were determined by Duncan’s multiple range tests at p≤0.05. 3. Results and Discussion Water stress caused a reduction in micro-shoot growth of the two fig cultivars (Table 1). In ‘Sabz’, shoot fresh and dry weights and shoot length were 36, 25, and 43% lower, respectively, in drought treatments caused by 4% PEG com- pared with the control, while in ‘Siah’, these traits were 58, 40 and 57% lower respectively, in the same stressed me- dia (4% PEG), than the control. In this latter cultivar, in contrast to ‘Sabz’, leaf area and specific leaf area were sig- nificantly affected by water deficit (56 and 21.5%, respec- tively) (Table 2). The findings of this study showed that the growth rate of 'Siah' in optimum conditions (control) was the same as Sabz cultivar, but with increasing intensity of water stress, the growth reduction rate in ‘Siah’ was more than in ‘Sabz’. The main effect of cultivars showed that without respect to different levels of PEG, ‘Sabz’ had a significantly higher leaf area (8.2 cm) than ‘Siah’ (5.51) (Table 2). Water stress significantly reduced vegetative growth indices of fig micro-shoots. This phenomenon has been previously report- ed (Oukabli et al., 2008). The long-term use of PEG in vitro on growth reduction and shoot regeneration in other plants has been well docu- mented (Bressan et al., 1982; Handa et al., 1982; Handa et al., 1983; Dami and Hughes, 1995; Al-Khayri and Al-Bah- rany, 2004). In most cases, PEG has been used to stimulate Table 1 - Interaction of water stress and cultivar on micro-shoot length, fresh and dry weight Drought stress (PEG%)  0 2 4 6 Cultivar Average fresh weight (g) Mean Siah 3.80 a (z) 2.1 bc 1.6 c (58) 2.10 bc 2.4 A Sabz 3.00 ab 2.3 bc 1.9 bc (36) 2.10 bc 2.3 A Mean 3.41 A 2.2 B 1.8 B 2.08 B  Average dry weight (g) Siah 0.50 a 0.30 bc 0.3 bc (40) 0.30 bc 0.34 A Sabz 0.40 ab 0.40 ab 0.3 bc (25) 0.30 bc 0.36 A Mean 0.46 A 0.34 B 0.3 B 0.31 B Shoot length (cm) Siah 2.60 a (z) 1.4 b 1.1 b (57) 1.20 b 1.57 A Sabz 2.33 a 1.4 b 1.3 b (43) 1.30 b 1.59 A Mean 2.47 A 1.4 B 1.2 B 1.25 B (z) In each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly dif- ferent using Duncan’s multiple rang test P≥0.05. The percentage of reduction with respect to control is reported in parentheses. Table 2 - Interaction of water stress and cultivar on leaf area (cm2) and specific leaf area (g cm-2) of micro shoots Drought stress (PEG%) 0 2 4 6 Cultivar Leaf area (cm2) Mean Siah 8.2 a (z) 6.9 ab 3.4 b 3.6 b (56) 5.51 B Sabz 12.4 a 8.2 a 6.9 ab 6.6 ab 8.52 A Mean 10.0 A 7.6 AB 5.4 B 5.4 B Specific leaf area (g cm-2) Siah 14.4 a 14.6 a 13.4 a 11.3 b (21.5) 13.4 A Sabz 16.8 a 15.3 a 15.1 a 13.1 a 15.1 A Mean 15.6 A 14.9 A 14.4 A 12.2 A (z) In each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly dif- ferent using Duncan’s multiple rang test P≥0.05. The percentage of reduction with respect to control is reported in parentheses. 134 water stress in plants. PEG with high molecular weight is a non-penetrating inert osmoticum which lowers the water potential of nutrient solutions without being taken up or being phytotoxic (Hassan et al., 2004). It has been shown that in vitro growth reduction of apple (Molassiotis et al., 2006) and cherry (Sivritepe et al., 2008) was due to the decrease in water potential created by PEG. Under water stress conditions, the reduction of mineral absorption has resulted in limited leaf growth and develop- ment and decreased plant water transpiration. Therefore, producing smaller leaves can be the first plant defense mechanism against water deficit. Hsiao (1973) reported that a decrease in leaf area results in lower light absorption and photosynthetic capacity; thereby reducing photosyn- thetate and plant growth. This can be true with plants in vivo, but probably this is less important in vitro, because in vitro plants are more heterotrophic, so decreases in their growth cannot be due to a deficiency of carbohydrates. In the present work it was clearly shown that the decrease in water availability is the main cause of decreasing growth. Specific leaf area (SLA) is a function of leaf area. The reduction of SLA in water deficit conditions is due to the fact that leaf area development is more affected than depo- sition of dry matter (Blum and Pneul, 1990). In both cultivars with increasing PEG% in culture me- dia, leaf relative water content (RWC) decreased and an in- crease in ion leakage was recorded (Table 3, Fig. 1). These findings are in agreement with the results obtained by other researchers in vitro (Sawwan et al., 2000; Al-Khayri and Al-Bahrany, 2004; Chai et al., 2005). Leaf yellowing and chlorosis were the consequence of structural damage to cell membranes in explants. Interestingly, in 6% PEG, although the rate of relative water content reduction in leaves of Sabz cultivar (50%) was more than ‘Siah’ (20%), the difference in their ion leakage was not significant. Leaves accumulated significant quantities of proline and, in contrast, the amount of chlorophyll was decreased with increasing PEG percentage in culture media (Fig. 2). Table 3 - Interaction of water stress and cultivar on leaf relative water content (%) and leaf ion leakage (%) Drought stress (PEG%) Leaf relative water content (%) Cultivar 0 2 4 6 Mean Siah 69.2 a (z) 68.6 a 59.3 ab 54.9 ab (20) 63.0 A Sabz 73.6 a 51.4 ab 61.7 a 36.7 b (50) 55.8 A Mean  71.4 A 60.6 AB 58.8 AB 45.7 B Leaf ion leakage (%) Siah 50.6 d 57.1 bcd 55.5 bcd 64.2 abc 56.9 B Sabz 54.1 cd 57.0 bcd 68.7 a 66.2 a 61.5 A Mean 52.3 C 57.0 BC 62.1 AB 65.2 A (z) In each row and column, means with similar letters are not signifi- cantly different using Duncan’s multiple rang test P≥0.05. The percentage of reduction with respect to control is reported in parentheses. Fig. 1 - Changes in leaf water content (RWC) and ion leakage in two fig cultivars: ‘Sabz’ (a) and ‘Siah’ (b). Fig. 2 - Changes in proline and total chlorophyll in two fig cultivars: Sabz (a) and Siah (b). 135 Naturally, the amount of proline in ‘Sabz’ was higher than in ‘Siah’ (81.8 µmole g-1 versus 16.7 µmole g-1) (Table 4). In both cultivars, with increasing PEG % in media, proline content increased. However, the results indicated that, in all levels of PEG treatments, the amount of proline in ‘Sabz’ was higher than in ‘Siah’. Plants produce and accumulate such compounds compatible to their metabolism to over- come adverse effects of drought stress (Zhu, 2001). Ac- cumulation of such substances would cause more negative water potential in plants, a necessary condition to absorb and keep the water in plant tissues (Sivritepe et al., 2008). Table 4 - Interaction of water stress and cultivars on leaf proline content (µm g-1 fresh weight) Drought stress (PEG%) Cultivar 0 2 4 6 Mean Siah 16.8 c (z) 50.5 b 73.7 b 121.0 b 65.5 B Sabz 81.8 b 320.7 a 293.6 a 289.5 a 246.4 A Mean 42.8 B 170.6 A 199.4 A 205.2 A (z) In each row and column, means with similar letters are not signifi- cantly different using Duncan’s multiple rang test P≥0.05 With increasing the severity of water stress, proline and soluble sugars (as compatible substances) accumulated sig- nificantly in explant leaves of both cultivars. The accumula- tion of these substances has been well documented in field (Clifford et al., 1998; Zamani et al., 2002; Mafakheri et al., 2010) and in vitro conditions (Handa et al., 1982; Brito et al., 2002; Al-Khayri and Al-Bahrany, 2004; Molassiotis et al., 2006; Sivritepe et al., 2008). A notable point in our results was the significant increase in proline accumulation in leaves of both cultivars. It has been reported that there is a direct positive relationship between drought tolerance and proline concentration in plant tissues (Al-Khayri and Al- Bahrany, 2004; Sivritepe et al., 2008). The role of proline in destroying reactive oxygen species (ROS) in water stress conditions has been reported in different plant species (Tur- kan et al., 2005; Verslues et al., 2006). Therefore, it can be expected that plants which accumulate more proline under water stress are more tolerant. Thus, it may be concluded that ‘Sabz’ is more tolerant than ‘Siah’, although in some plants a relationship was not found between proline accu- mulation and drought tolerance. The changes in leaf sugar and starch contents under dif- ferent levels of PEG are shown in Table 5 and figure 3. In both cultivars, with increasing PEG%, the amount of soluble sugar content increased and the amount of starch deceased. In drought conditions, soluble sugar can act as osmoticum and also osmoprotectant. In addition, accu- mulation of sugar may partly protect protein against the oxidative damage created by free radicals (Bohnert and Shen, 1999). In the present work, soluble sugars content in ‘Sabz’ was greater than in ‘Siah’, but it did not have sig- nificant effect on water absorption. Accumulating soluble sugars can be due to starch hydrolysis, which is in agree- ment with the results obtained by Shawky et al. (1997). Taylor et al. (1982) also reported that carbohydrates (re- ducing and non-reducing sugars) are the most abundant component in osmotic adjustment in tomato seedlings. Table 5 - Interaction of water stress and cultivar on leaf TSS and starch (mg g-1 dry weight) Drought stress (PEG%) 0 2 4 6 Cultivar TSS (mg g-1 dry weight) Mean Siah 170.3 c 168.9 c 256.6 bc 287.0 b 220.69 B Sabz 276.4 bc 344.5 b 357.5 b 522.2 a 375.2 A Mean  215.8 C 256.7 BC 311.6 B 404.6 A Starch (mg g-1 dry weight) Siah 133.5 ab 103.8 bc 73.7 bc 65.3c 94.1 A Sabz 167.0 a 124.2 abc 76.3 bc 80.3 bc 111.9 A Mean  152.56 A 112.83 B 75.4 B 73.9 B (z) In each row and column, means with similar letters (small letters for interaction and big letters for main effects) are not significantly dif- ferent using Duncan’s multiple rang test P≥0.05 In this study, with increasing PEG in culture media, the amount of total chlorophyll declined in leaves of the two fig cultivars. Effect of drought stress on reduction of chlorophyll content of other plants in vitro has previously Fig. 3 - Changes in leaf sugar and starch content in two fig cultivars: ‘Sabz’ (a) and ‘Siah’ (b). 136 been reported (Hernández-Sebastià et al., 2000; Brito et al., 2002; Molassiotis et al., 2006). Under field conditions, also chlorophyll content was reduced with increasing drought (Munne-Bosch and Penuelas, 2004). In the present experiment, with increasing water deficit (with addition of PEG), chlorophyll content was decreased along with ac- cumulation of leaf proline (Fig. 3). This may be due to the fact that chlorophyll and proline are synthesized through glutamate pathway, causing an increase in synthesis of proline under drought conditions, which resulted in reduc- tion of chlorophyll synthesis (Aspinall and Paleg, 1981). 4. 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