Impaginato 63 Adv. Hort. Sci., 2024 38(1): 63­74 DOI: 10.36253/ahsc­14148 In vitro salt stress tolerance of ‘Sahand’ cultivar grafted on two wild almond rootstocks: An evaluation of physiologi­ cal and biochemical traits between rootstocks M. Asadi Zargh Abad, A. Shekafandeh (*) Department of Horticultural Science, College of Agriculture, Shiraz University, P.O. Box 65186, 71441 Shiraz, Iran. Keywords: Antioxidant enzymes, glycine betaine, grafting combination, proline. Abstract: The low salinity tolerance of almond cultivars can cause a significant setback in almond production. Therefore, selecting suitable cultivars and root­ stocks in salinity­affected areas can facilitate sustainable crop production. In this research, the effects of two wild almond species, Badamkohiand Arjan as rootstocks on the salinity tolerance of ‘Sahand’ as a scion were investigated through in vitro culture. A factorial experiment of 2 (species) × 4 (levels of salin­ ity) was conducted in a completely randomized design (CRD) with 4 replica­ tions. The results showed that ‘Sahand’ grafted on Badamkohi had the higher fresh and dry weight than grafted on Arjan in all level of salinity. The Na+ and Cl­ ions contents inthe shoots and root of both micrografting combinations increased with increasing salinity. However, their amount in the shoot and the root of ‘Sahand’/Arjan plants were significantly higher than those ions in ‘Sahand’/Badamkohi plants at 80 and 120 mM NaCl. The amount of total chlorophyll in ‘Sahand’ grafted on Badamkohi was 0.68 mg g­1 FW which was significantly higher than the total chlorophyll of the same scion grafted on Arjan rootstock (0.51 mg g­1 FW) at 120 mM NaCl. The highest leaf cell electrical leakage occurred in ‘Sahand’ grafted on Arjan which was significantly higher than leaf electrical leakage of the same scion grafted on Badamkohi at 120 mM NaCl. The grafting combination of ‘Sahand’/Badamkohi showed a higher pro­ line and glycine betaine content, compared to the grafting combination of ‘Sahand’/Arjan. The shoot and root antioxidant enzyme activities (SOD, POX and CAT) in micrografting combination of ‘Sahand’/ Badamkohi were also sig­ nificantly higher than those in ‘Sahand’/Arjan. It can be concluded that ‘Sahand/Badamkohi combination is a suitable choice for the regions with late spring frost and saline conditions. 1. Introduction Almond (Prunus dulcis Mill.) is one of the most important nut crops (Ansari and Gharaghan, 2019), with particular importance in the world. It (*) Corresponding author: shekafan@shirazu.ac.ir Citation: ASADI ZARGH ABAD M., SHEKAFANDEH A., 2024 ­ In vitro salt stress tolerance of ‘Sahand’ cultivar grafted on two wild almond rootstocks: an eva‐ luation of physiological and biochemical traits between rootstocks. ­ Adv. Hort. Sci., 38(1): 63­ 74. Copyright: © 2024 Asadi Zargh Abad M., Shekafandeh A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 3 January 2023 Accepted for publication 17 January 2024 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-14148 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2024 38(1): 63­74 64 is characterized by a high storage capacity, low degree of waste, ease of processing and transporta­ tion. The feasibility of its economic production has led to an increase in the total area of almond orchards (Bybordi, 2013). Salinity still remains the major abiotic stress that limits agricultural produc­ tion (Seleiman et al., 2020). Almonds can be affected by salinity stress through osmotic mechanisms and by enhanced levels of osmotic potential in the soil solution (Shrivastava and Kumar, 2015). High salinity in root zone not only reduces water uptake and tree growth, but also can cause nutritional imbalances and toxicity effects of the major saline ions (Na+ and Cl­). Meanwhile, the low salinity tolerance of almond trees can cause a significant setback in almond pro­ duction (Kaundal et al., 2019). Therefore, selecting suitable cultivars and rootstocks in salinity­affected areas can facilitate sustainable crop production. The dynamic nature of salinity with respect to time and space, as well as limited experimental designs restrict the complete study of genotype­envi­ ronment interactions (Sauvage et al. , 2014). Therefore, the crop breeding program can be com­ plemented with a suitable management option, such as grafting on appropriate salt tolerant rootstocks (Cuartero et al., 2006). Grafting has been reported as a rapid method for enhancing salt tolerance (Singh et al., 2020) that counteracts the salinity effects by maintaining low Na+/K+ ratios in the shoot and improves leaf stomatal conductance (Wei et al., 2017). The behavior of the rootstock in different plant species influences the metabolic processes of the scion leading to tolerance. Rootstocks are an essential component in modern fruit production (Shahkoomahally et al., 2020) and can provide sever­ al traits that may be absent in the scion, such as resistance to soil pest and disease, better root sys­ tems, enhanced nutritional uptake, better tolerance to soil salinity and water scarcity (Kumar et al., 2017). The cultivation of grafted plants has gradually increased in recent years since grafting enables the plant system to control important agronomic traits and offers a flexible pattern to the growth of a partic­ ular scion (Kumar et al., 2017). Grafting a scion on a suitable rootstock generally allows extensive use of the rootstocks (Gainza et al., 2015). While consider­ ing a wide range of salt­tolerant genotypes in the genus Prunus, the selection of more tolerant species as rootstock can lead to sustainable solutions in han­ dling commercial cultivars of almond and peach (Najafian et al., 2008). The use of wild almond species as rootstocks, has been considered feasible especially in arid and semi­arid regions (Karimi et al., 2015). Using these rootstocks can highlight the strat­ egy of allowing plants to overcome environmental stress, because of their adaptability and stimulated growth. Moreover, rootstocks affect the nutritional status of the scion and plant height (Aras and Eşitken, 2019). Trees have lengthy biological cycles, which implies prolonged time lapses until plants are produced for study, as well as evaluations that can last for the entire growing season or even more than one season (Bado et al., 2015). This is of particular importance in plant breeding programs where thousands of plants are handled yearly and the staff and land are restrict­ ed. However, the long juvenile periods and cost of maintaining all the seedlings until they are grown trees have encouraged researchers to develop early studies in young plants to discard those genotypes which do not fit in the breeding goals and minimize the cost and time of field evaluations (Vives­Peris et al., 2017). In recent years, tissue culture and in vitro selec­ tion have emerged as an effective tool in the further­ ance of efforts to develop stress­tolerant plants. In vitro cultivation techniques can largely assist with the study and selection of plant species, because more control is exerted on plant growth compared to the outside environment, and evaluations are usually conducive to good results in a confined space (Ghaleb et al., 2010; Rai et al., 2011). When the sta­ bility of medium culture enables a controlled condi­ tion, a uniform application of stress to all explants can create reliable results, because other intervene factors are eliminated (Seth and Kendurkar, 2015). The in vitro system can characterize the degree of salt tolerance of different genotypes at their primary growth phase within a short time, limited space and low cost (Ghaleb et al., 2010). This method has been applied for screening salt tolerant genotypes of some fruit species including cherry rootstocks (Erturk et al., 2007), citrus rootstocks (Ghaleb et al., 2010), grape rootstocks (Alizadeh et al., 2010), fig cultivars (Abdoli Nejad and Shekafandeh, 2014), apple varieties (Shibli et al., 2000), kiwifruit (Sotiropoulos and Dimassi, 2004) and pear (Sotiropoulos et al., 2006). The aim of this research was to study the influence of Badamkohi and Arjan as rootstocks on the salinity tolerance of ‘Sahand’ (a late­bloom almond cultivar) as a scion through certain morphological and bio­ http://https://www.frontiersin.org/articles/10.3389/fpls.2020.595055/full#ref43 Asadi Zargh Abad and Shekafandeh ‐ In vitro salt tolerance of different almond graft combinations 65 chemical responses in vitro condition. 2. Materials and Methods Establishment of in vitro micrografting According to Asadi and Shekafandeh (2021) pro­ cedure, the mature seeds of naturally grown wild almond trees, Prunus scoparia (C. Schneider) and Prunus elaengnifolia (E. Murrary) named Badamkohi and Arjun respectively, grow in arid and semi­arid regions were grown in vitro to produce seedlings. After removing the endocarps, they were surface­ sterilized by immersion in 70% alcohol for 1 min and then in 20% Whitex solution (sodium hypochlorite 5%) for 10 min. Subsequently, they were rinsed three times with sterile distilled water. The sterilized seeds were cultured on MS medium (Murashige and Skoog, 1962) supplemented with 30 g l­1 sucrose and 8 g l­1 agar as gelling agent (Fig. 1 a, b). For preparation of the micro­scion, young shoots of the late­blooming ‘Sahand’ were disinfected and cultured on MS medi­ um supplemented with 2.2 Μm benzyladenine (BA) and 0.54 μM naphthaleneacetic acid (NAA). After two weeks, the young offshoots have reached a suitable size to be used as scions (Fig. 1 c). Then, the two weeks old in vitro produced seedlings were decapi­ tated and by the help of a sharp scalpel a vertical slit (0.5 cm) was created on top of the stump (Fig. 1 d). The scion was cut into a “V” shaped wedge (Fig. 1 e) and inserted into the rootstock to form a micrograft (Fig. 1 f). The jars containing explants were maintained at 25±1°C under a 16 h photoperiod (light intensity of 4000 lux). Salinity treatments The graft combinations were allowed to grow for 4 weeks, and then they were cultured on agar free MS medium with a bridge paper and different con­ centrations of NaCl (0, 40, 80 and 120 mM). After 4 weeks of culture, certain morpho­physiological and biochemical characteristics of graft combinations were measured as follow. Fresh and dry weight of scion and rootstock After measuring the fresh weight of the shoot (scion) and the root (rootstock), they were dried in an oven for 24 h at 60°C and then re­measured for their dry weight (in mg). Sodium (Na+) and Chlorine (Cl‐) The samples (1 g) were dried in an oven at 500 to 550°C for 5 h, then reduced to ash. To each sample in the crucible was added 5 ml of 2 normal HCl. After passing the solution through filter paper, the filtered solution was transferred to a jug balloon. The volume of each sample was made up to 50 ml with hot dis­ tilled water and then sodium was measured using a flame photometer (Model Jenway PFP7 Bibby Scientific Ltd, Staffordshire, UK) and calculated in mg g­1 dry weight. To determine the chlorine of each sample, accord­ ing to the method of Chapman and Pratt (1961), one gram of sample was poured into a Chinese mortar and 250 mg of calcium oxide was added to each and was kneaded with distilled water. They were then placed in a kiln at 250°C for one h to remove all the soot from the initial burning. After, the kiln tempera­ ture was slowly raised to 550°C to reduce the sam­ ples to ashes. Then 15 mL of hot distilled water was added to the samples. After cooling, 5 drops of 5% potassium chromate were added to the solution and titrated with 0.05 N silver nitrate (2.12 g of silver nitrate in 250 mL of distilled water) to observe a red brick­colored precipitate. Finally, chlorine was calcu­ lated as mg g­1 dry weight. Total chlorophyll (Chl) Fresh leaf samples (0.1 g) were placed in test tubes and added 7 mL of dimethyl sulfoxide, then they were placed in an incubator for 30 minutes at 65°C. After extraction, the volume of extracts was made up to 10 mL by adding dimethyl sulfoxide. Fig. 1 ­ Performing in vitro micrograft. Germinated seeds of Arjan (a) and Badamkohi (b) using as rootstock. Proliferated shoots of ‘Sahand’ cv. on MS medium sup­ plemented with 2.2 μM BA and 0.54 μM NAA using as scion (c). An Arjan rootstock ready to be grafted (d). A scion ready for grafting (e). A micrograft combination of ‘Sahand’/Arjan (f). Adv. Hort. Sci., 2024 38(1): 63­74 66 Finally, the absorbance of the extracts at wave­ lengths of 645 and 663 nm was read using a spec­ trophotometer (USA Epoch Microplate, BioTek instruments, Inc) (Gross, 1991). Chlorophyll content was determined as follows: Chl (mg g­1) FW = [20.2 (OD645nm) + 8.02 (OD663nm)] × V/FW × 1000 Where V is final volume of solution (mL), FW the leaf fresh weight (mg), and OD the optical density. Electrolyte leakage(EL) EL was determined according to the method of Gulen and Eris (2004). Ten discs were cut from the fully developed leaves of the plants in each replica­ tion. Then, they were transferred to vials containing 5 mL deionized water and kept at 10°C for 24 h. After measuring their electrical conductivity (EC1) using a conductometer (Metrohm 644, Awess), the samples were then placed in a water bath at 95°C for 20 min, and after cooling at 25°C the electrical conductivity (EC2) was re­measured. The EL was calculated with the following formula: EL (%) = (EC1/EC2) × 100 Proline contents According to the modified method of Bates (1973), the proline content was determined. The leaf sample (0.5 g) was grinded in 10 mL sulfosalicylic acid (3%). The mixture was then centrifuged at 10,000×g for 10 min. Two ml of the supernatant was added to each test tube which contained freshly prepared acid­ninhydrinas a diluted solution (2 ml). The tubes were incubated in a water bath at 90°C for 30 min. Ultimately, the reaction ended in an ice bath. The reaction mixture was extracted using toluene (5 mL) and was vortexed for 15 s. The tubes were stored in darkness at room temperature for 20 min, thereby allowing the separation of toluene from the aqueous phase. The toluene phase was then carefully collect­ ed and the absorbance was measured at 520 nm by a spectrophotometer (model T60 USA). Glycine betaine Glycine betaine was measured by the method of Grattan and Griere (1985). The powdered sample of leaf (0.5 g) was mixed in a mortar with 20 mL of ion­ ized water. The specimens were placed on a shaker for 48 h at 25°C. They were passed through a Whatman filter paper ‘G42’ and diluted in a 1:1 ratio with two­molar sulfuric acid. Then, 0.5 mL of this solution was removed and poured into the Eppendorf tube. After cooling the samples for 2 h, 0.2 ml of potassium tri­iodide solution was added to each. Then, they were centrifuged for 20 min at 15,000 rpm at 0°C. The top phase discarded and the periodontal crystals were dissolved in the bottom of the container in 9 mL of 1­2 dichloroethane. Then, the absorption of the samples was measured at 365 nm with a spectrophotometer. Enzyme activity In order to estimate the enzymes activities, the leaf samples (0.5 g) were first homogenized in 50 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA, 3 mM 2­mercaptoethanol, and 2% (w/v) polyvinyl polypyrrolidone (PVPP) in a chilled mortar. The homogenate was then centrifuged at 16000 g for 30 min at 4°C and the supernatant was used for enzyme assays. Superoxide dismutase (SOD) The SOD (EC 1.15.1.1) can be measured by deter­ mining its ability to halt the photochemical reduction of nitro blue tetrazolium chloride (NBT) in the pres­ ence of light. In this method, the reaction mixture (3 mL) contained 50 μL enzyme extract, 50 mM potassi­ um phosphate buffer, 13 mM l­methionine, 75 μM NBT, 0.1 mM EDTA and 4 μM riboflavin. The reaction mixture was shaken and placed in a light chamber for 15 min to allow the reaction to take place. Eventually, the absorption rate of each specimen was recorded at 560 nm using a spectrophotometer (Biochrom WPA Biowave II UV/Visible Spectrophoto­ meter, England) against the non­irradiated blank (Dhindsa and Motowe, 1981). Catalase (CAT) The determination of the activities of CAT (EC 1.11.1.6) was based on the rate of H2O2 decomposi­ tion as measured by decreasing the absorbance at 240 nm (Dhindsa and Motowe, 1981). While the reaction mixture contained 50 mL potassium phos­ phate buffer (pH 7) and 15 mM hydrogen peroxide (H2O2), the reaction started by adding 1000 μL of the enzyme extract. One unit of activity is the amount of enzyme that could decompose 1 mM of H2O2 in 1 min. Peroxidase (POX) Peroxidase enzyme activity was read at 470 nm, based on an enhanced degree of light absorption as a result ofguaiacol oxidation in the presence of peroxi­ dase hydrogen. This was carried out by a spectropho­ Asadi Zargh Abad and Shekafandeh ‐ In vitro salt tolerance of different almond graft combinations 67 tometer (JENWAY model 7315 UK) in 1 min with a time interval of 10 s (Ozden et al., 2009). The activity of the enzyme was calculated based on the oxidized μmol of guaiacol per min and per g of fresh leaf weight. Statistical analyses The experiment was carried out as a factorial 2 (species) × 4 (levels of salinity) in a completely ran­ domized design (CRD) with 4 replications and 4 micrografted plants per replicate. A total of 122 micro­grafting combination of ‘Sahand’/Argan and ‘Sahand’/Badamkohi were used in this experiment. Data were analyzed using SAS 9.4 software and mean values were compared using LSD test (P≤0.05). 3. Results The results of analysis of variance showed that the interaction between species and salinity was sig­ nificant at 5% or 1% level in all measured traits. So, all the results were presented by the interactions. Effects of salinity on scion‐rootstock combination growth The results showed that, both rootstocks (Badamkohi and Argan) had significant difference in the root length of control (free salt medium). Badamkohi showed the highest root length of 263 mm that was significantly higher than Arjan (122 mm) (Table 1). This showed that they have different growth habit. However, in both rootstocks with increasing salinity to 120 mM in culture medium the root length decreased significantly. This reduction was 50% and 18% for Badamkohi and Arjan respectively compared to their controls. The highest length of scion (64 mm) was related to the salt­free treatment on Badamkohi rootstock which showed a significant difference with the same scion on Arjan rootstock (41 mm) in the same treatment. With increasing the concentration of sodium chlo­ ride from 0 (control) to 120 mM, the scion (‘Sahand’) length on both rootstocks was significantly reduced (Fig. 2 b, c, e, f), this reduction was 50.6% and 49% on Badamkohi and Arjan respectively. In both rootstocks, root fresh and dry weight decreased with increasing NaCl concentrations, how­ ever fresh weight loss in Badmkohi and Arjan was 62% and 76%, respectively, and also dry weight loss was 55% in Badamkohi and 51% in Arjan (Fig. 3 a and c). Fresh and dry weight of ‘Sahand’ grafted on Badamkohi at all salinity levels (except scion dry weight in 120 mM salt) was significantly higher than fresh and dry weight of ‘Sahand’ grafted on Arjan (Fig. 3 b and d). In both rootstocks, increasing the salt from 0 to 120 mM reduced the fresh weight of the scion by about 60% while this reduction in the dry weight of scion was 62% on Badamkohi rootstock and 55% on Arjan rootstock (Fig. 3 b and d). Effect of salinity on Na+ and Cl‐ contents in micro‐ grafting combinations The results showed that the Na+ and Cl­ contents in the roots and the shoots of both micrografting combinations increased with increasing salinity (Fig. 4 a, b, c, d,). However, the amount of Na+ and Cl­ in the shoot and the root of ‘Sahand’/Arjan plants were sig­ Fig. 2 ­ Growth of a micrografted combination of ‘Sahand’/ Badamkohi in control (a) and in 120 mM salinity (b and c); a micrografted combination of ‘Sahand’/Arjan in con­ trol, (d) and 120 mM salinity (e and f). Table 1 ­ Effect of sodium chloride on scion and root length in micrografting combinations of ‘Sahand’/Badamkohi and ‘Sahand’ /Arjan Micrografting combination NaCl mM Root length (mm) Shoot length (mm) ‘Sahand’/Badam kohi 0 263 a 64 a 40 202 b 44 b 80 148 c 38 bc 120 130 d 30 d ‘Sahand’/Arjan 0 122 ed 41 b 40 114 edf 33 cd 80 106 ef 29 d 120 100 f 21 e In each column, means with the same letters are not significantly different at 5% probability level using LSD test. 68 Adv. Hort. Sci., 2024 38(1): 63­74 nificantly higher than these ions in the shoot and the root of ‘Sahand’/Badamkohi plants at 80 and 120 mM NaCl. Total chlorophyll (Chl) The Chl of ‘Sahand’ grafted on both rootstocks decreased with increasing salt concentration. The highest amount of Chl was obtained in ‘Sahand’ (as scion) leaf grafted on Badamkohi rootstock in unsalt­ ed medium (control) which showed a significant dif­ ference compared to the same scion grafted on Arjan rootstock. The amount of Chlin ‘Sahand’ grafted on Badamkohi was 0.68 mg g­1 FW which was significant­ ly higher than the Chl of the same scion grafted on Arjan rootstock (0.51 mg g­1 FW) at 120 mM NaCl (Table 2). At the highest salinity level, the leaf chloro­ phyll content of ‘Sahand’ either grafted on Badamkohior Arjan was decreased by 51% and 63% respectively compared to their controls. Electrolyte leakage (EL) In all levels of salinity, the EL of ‘Sahand’ grafted on Arjan rootstock was significantly greater than the same cultivar grafted on Badamkohi. The highest leaf cell EL (72.29%) occurred in ‘Sahand’ grafted on Arjan rootstock which was significantly higher than leaf EL of the same scion grafted on Badamkohi rootstock at 120 mM NaCl (Table 2). Proline and glycine betaine (GB) in micrografting combination Increasing the level of salinity caused a significant rise in the proline content of both grafting combina­ tions (‘Sahand’/Badamkohi and ‘Sahand’/Arjan) (Table 3). The grafting combination of ‘Sahand’/ Badamkohishowed a higher proline content (in both parts, scion and rootstock) compared to the grafting combination of ‘Sahand’/Arjan in all salinity levels. The results also indicated that by increasing the salt concentration from 0 to 120 mM, the amount of GB increased in both micrografting combinations. The shoot and the root in micrografting combination of ‘Sahand’/Badamkohi showed significantly greater GB than those in micrografting combination of ‘Sahand’/Arjan in all level of salinity (except for root GB in salt free medium of both rootstocks). Fig. 3 ­ The effect of different concentrations of NaCl on fresh and dry weight of micrografting combinations of ‘Sahand’/ Badamkohi and ‘Sahand’/Arjan. Different let­ ters indicate significant difference at P ≤ 0.05 level of probability using LSD test. Table 2 ­ Effects of sodium chloride on leaf total chlorophyll and electrolyte leakage in micrografting combinations Fig. 4 ­ The Na+ and Cl­ contents of both rootstocks (a, b) and scion (c, d) in grafting combinations of ‘Sahand’ /Badamkohi and ‘Sahand’/Arjan in different concentra­ tion of NaCl. Different letters indicate significant differ­ ence at P ≤ 0.05 level of probability using LSD test. Micrografting combination NaCl mM Total chlorophyll (Mg g­1 FW) Electrolyte leakage (%) ‘Sahand’/Badam kohi 0 1.45 a 23.28 e 40 1.42 ab 31.32 de 80 0.95 bc 38.05 cd 120 0.68 d 43.86 c ‘Sahand’/Arjan 0 1.33 b 38.15 cd 40 1.25 bc 43.77 c 80 0.77 d 55.15 b 120 0.51 e 72.29 a In each column, means with the same letters are not sig­ nificantly different at 5% probability level using LSD test. Asadi Zargh Abad and Shekafandeh ‐ In vitro salt tolerance of different almond graft combinations 69 Superoxide dismutase (SOD) SOD activity in both‘Sahand’/Badamkohi and ‘Sahand/Arjan plants increased with increasing the levels of salinity (Table 4). The highest increase in the activity of SOD was obtained in the ‘Sahand’ /Badamkohi, combination with 101.27 and 103.30 (U g­1 FW min­1) in shoot and root respectively at 120 mM salt, which was significantly higher than those in ‘Sahand/Arjan plants. Peroxidase (POX) In the shoots and in the roots of both micrograft combinations, POX activity increased with increasing salt concentrations in the medium. Although, at all salinity levels, POX activity was higher in the shoot and the root of micrografting combination of ‘Sahand’/Bada Kohi than ‘Sahand’/Ajan, however the difference was only significant at 120 mM NaCl. Catalase (CAT) Both micrografting combinations showed the enhancement CAT activity in response to increasesalt concentrationfrom 0 to 120 mM NaCl. The highest CAT activity occurred in the shoot (63.70 U g­1 FW min­1) androots (59.70 U g­1 FW min­1) of ‘Sahand’/ Badamkohi plants at 120 mM NaCl which was signifi­ cantly higher than the activity of this enzyme in the ‘Sahand’/Arjan plants (Table 4). Table 4 ­ Effects of sodium chloride on the shoot and root enzymes activities (SOD, POX and CAT) in micrografting combinations of ‘Sahand’/ Badamkohi and ‘Sahand’/Arjan Table 3 ­ Effects of sodium chloride on the amounts of proline and glycine betaine (GB) in the shoots and roots of micrografting combi­ nations Micrografting combination NaCl levels (mM) Root GB µmol g­1 DW Shoot GB µmol g­1 DW Root proline µmol g­1 FW Shoot proline µmol g­1 FW ‘Sahand’/Bdam kohi 0 10.55 ef 6.88 cd 24.93 e 20.64 d 40 14.60 d 7.48 c 38.03 c 27.73 c 80 19.00 b 8.90 b 46.70 b 40.88 b 120 22.60 a 11.00 a 62.40 a 55.38 a ‘Sahand’/Arjan 0 9.30 f 5.70 e 14.80 f 13.22 e 40 11.47 e 6.30 de 21.70 ef 18.97 de 80 14.02 d 6.80 cd 28.9 de 24.06 cd 120 16.50 c 7.60 c 34.50 cd 30.40 c In each column, means with the same letters are not significantly different at 5% probability level using LSD test. Micrografting combination NaCl (mM) SOD shoot U g­1 FW min­1 SOD root U g­1 FW min­1 POX U g­1 FW min­1shoot POX root U g­1 FW min­1 CAT shoot U g­1 FW min­1 CAT root U g­1 FW min­1 ‘Sahand’/Badam kohi 0 90.10 e 76.60 c 31.62 ed 51.60 fe 40.60 cd 32.50 ef 40 92.90 d 77.30 bc 48.81 bc 60.30 cde 41.80 cd 44.90 bc 80 96.10 c 80.00 bc 50.70 b 69.80 bc 46.90 bc 48.70 b 120 101.27 a 103.30 a 76.99 a 95.00 a 63.70 a 59.70 a ‘Sahand’/Arjan 0 87.60 f 72.10 c 23.40 e 48.10 f 39.60 d 31.30 f 40 92.20 d 75.20 c 36.60 cd 58.80 de 41.10 cd 38.60 de 80 95.20 c 78.00 c 45.60 bc 67.20 bcd 43.60 bcd 42.40 cd 120 97.60 b 85.60 b 47.80 bc 73.50 b 50.50 b 46.20 bc In each column, means with the same letters are not significantly different at 5% probability level using LSD test. Adv. Hort. Sci., 2024 38(1): 63­74 70 4. Discussion and Conclusions Performance of grafted plants compared to non­ grafted or self­grafted plants under a stressful condi­ tion is often dependent on the rootstock’s root sys­ tem characteristics. A vigorous root system could be the most important criterion for increasing salt toler­ ance (Balliu et al., 2007). In this research, Badamkohi showed the highest root length of 263 mm that was significantly higher than Arjan (122 mm). The root­ stock’s root systems architecture specified by root length and density, root hairs and root surface area play a critical role in ion and water uptake, thus determining salt tolerance of grafted plants (Colla et al., 2010). A vigorous root system, for instance, pro­ duced more cytokinins and transported water to the shoot system by xylem sap, which positively affected plant growth and crop yield (Oztekin and Tuzel, 2011). Furthermore, hydraulic conductivity of the roots may control plant growth by manipulation of the water supply to epigeous plant parts (Gregory et al., 2013). The most immediate effect of salinity on plants is the inhibition of root and shoot development. Due to the imbalance of water potential between the apoplast and simplast, the osmotic potential decreas­ es and the absorption of water is hampered. Ultimately, this reduces plant growth by closing the stomata and weakening photosynthesis (Dustgeer et al., 2021). Fresh and dry weight of plants decrease with increasing salinity, which is usually due to ion toxicity and water stress (Arif et al., 2020; Corell et al., 2020). Salinity has the effects on metabolic activi­ ty and reduces the division of new cells and disrupt cellular processes, thus reduces plant growth com­ pared to normal conditions (Carillo et al., 2019). In this study, as mentioned before, the root system of Badamkohi was stronger than Arjan. However, in high level of salinity, the shoot length of the same scion (‘Sahand’) on Badamkohi was greater than on Arjan. It seems that the Arjan rootstock has a dwarf­ ing effect on the scion. On the other hand, at all salin­ ity levels, the fresh and dry weight of the scion on Badamkohi was more pronounced than on Arjan. This means that Badamkohi supports scion growth better than Arjan. It has been also reported some rootstocks are more capable of inducing tolerance to the scion against salt stress (Zrig et al., 2016; Aras and Eşitken, 2018). In this regard, adding sodium chloride to the growth medium of two graft combinations (‘Sahand’/Badamkohi and ‘Sahand’/Arjan) led to a decrease in scion growth, however this growth reduction of ‘Sahand’ subjected to increasing concen­ trations of NaCl was more acute when the rootstock was Arjan. This result is in consistence with the results of sweet almond grafted on different root­ stocks (Zrig et al., 2016). Sodium ion (Na+) is toxic to cellular metabolism and affects the activity of some enzymes, and high concentrations of Na+ causes ion imbalance (Roy et al., 2014). The ability of almond rootstocks varies in absorb­ ing or transferring sodium to the scion and there is a very close relationship between tolerance to salinity and the amount of sodium transferred to the leaves (Mickelbart and Arpaia, 2002). In the present research, Na+ concentration in the aerial parts was lower in ‘Sahand’/ Badamkohi compared to ‘Sahand’/Arjan. Such a prohibiting mechanism may explain, the higher shoot length and biomass of ‘Sahand’/Badamkohithat observed in our study. A higher Na+ concentration in the environment of root can depress K+/Na+ ratios in the plant, thereby, the plant becomes susceptible to specific ion injury as well as to nutritional disorders which may affect growth and yield. The exclusion of Cl­ from shoots is related to the ability of cell membranes to restrict the movement of Cl­ through the root to vascular tis­ sue and the degree of Cl­ accumulation in the roots (Walker and Douglas, 1983). In this experiment, Badamkohi rootstock was able to slow the accumula­ tion of Cl­ in the leaves. Similar results have been reported by García­Sánchez et al. (2002) in which the Cleopatra rootstock reduced the accumulation of Cl­ in the scion compared to the Carrizo rootstock. The finding of this experiment showed that Badamkohi rootstock hold up more the chloroplast integrity of ‘Sahand’ than Arjan rootstock. Chlorophyll depletion in salt stress can be linked to factors such as structural damage of chloroplasts due to the formation of reactive oxygen species and photo oxidation of chlorophyll (Taïbi et al., 2016), the destruction of chlorophyll synthesis precursors, the inhibition of biosynthesis of new chlorophylls, and hormone disorders (Sabzmeydani et al., 2020). This reduction could also be due to the increase in the activity of the enzyme chlorophyllase or to the insta­ bility of the protein pigment complexes by the ions (Saha et al., 2010). Surendar et al. (2013) reported a decrease in chlorophyll content under stress was caused to the destruction of the chloroplast mem­ Asadi Zargh Abad and Shekafandeh ‐ In vitro salt tolerance of different almond graft combinations 71 brane with increasing phosphatase activity, which is located on the membrane. Plasma membranes are the primary site of ion­ specific salt injury. Undesirable performance of the cell’s metabolism during periods of abiotic stress leads to the stimulation of reactive oxygen species which would damage the cell membrane and increase electrolyte leakage. Therefore, electrolyte leakage from plasma membranes is reported as one of the most important selection criteria for identifica­ tion of salt­tolerant plants (Besma and Denden, 2012). In this study, especially at higher levels of salinity, the ‘Sahand’ grafted on Arjan experienced a greater damage to the cell membrane of its leaves, than grafted on Badamkohi. This indicates Badamkohi rootstock’s ability to maintain the integri­ ty of scion cell membrane in salt stress conditions. In accordance with our finding, Colla et al. (2012) reported that in cucumber grafted plants, the amount of ion leakage in salinity stress is reduced compared to non­grafted plants and rootstock helps the maintenance of membrane function. In this study, the grafting combination of ‘Sahand’/Badamkohi accumulated more proline and glycine betaine in the root and shoot, compared to the same scion grafted on Arjan. Proline accumulation in salinity condition can play a role in stress tolerance mechanismsby stabilizing proteins at high ionic con­ centrations (Krasensky and Jonak, 2012). It also reduces damages caused by salinity via the preserva­ tion of water in cells and by diluting salts in the plant (Gulen et al., 2018). Proline has essential functions by osmoregulation, reducing the undesirable effects of ROSs under salinity stress.Therefore, higher proline accumulation can induce higher tolerance against salinity by the plant (Akbari et al., 2018). The proline content of micrografting combination significantly increased in response to an increase in the salinity level, in this regards ‘Sahand’/Badamkohi micrograft combination was more prominent than ‘Sahand’/Arjan. Our finding is in agreement with other reports on in vitro salt tolerance of pistachio (Raoufi et al., 2020) and fig (Abdoli Nejad and Shekafandeh, 2014). Glycine betaine was also found to play signifi­ cant roles in enhancing salt tolerance (Wei et al., 2017). It can maintain the osmotic regulation, improve the production, nutrients and water absorption, there­ by photosynthetic proteins are produced and mem­ brane peroxidation is reduced (Dustgeer et al., 2021). In this research, the grafting combination of ‘Sahand’/Badamkohi accumulated more glycine betaine in the root and shoot, compared to the graft­ ing combination of ‘Sahand’/Arjan, that means Badamkohi protects cell osmotic pressure, enhancing cell membrane integrity as well as maintaining photo­ synthetic apparatus (Niazian et al., 2021). As mentioned above, with increasing salinity lev­ els, Badamkohi rootstock enhanced SOD, POX and CAT activity in ‘Sahand’ Scion more than Arjan root­ stock. SOD plays a major role in ROS scavenging in plants and is considered as the first line of defense against the toxic effects of elevated ROS (Hou et al., 2019). SOD catalyzes the dismutation of superoxide radicals to H2O2 and O2 (Feng et al., 2015). Increasing the level of superoxide dismutase activity is impor­ tant for protecting chloroplasts and mitochondria from the stress of reactive oxygen species. In fact, under stress conditions, the chloroplast is where the majority of active oxygen species are produced and where is caused the highest degree of damage (Sofo et al., 2005; Kuşvuran et al., 2016). According to the results of the present study, salinity stress caused the SOD activity to increase sig­ nificantly in both micrograft combinations (‘Sahand’/Badamkohi and ‘Sahand’/Arjan). Nonetheless, Badamkohi rootstock enhanced the SOD activity in ‘Sahand’ more than Arjan rootstock. Enhanced activities of SOD enzyme usually reflect defensive responses to cellular damage induced by higher NaCl concentrations in the culture medium (Akbari et al., 2018; Kuşvuran et al., 2021). The enzyme hydrogen peroxidase (POX) also reduces oxidative stressby protecting the metabolic enhancers that sustain cell and plant survival (Aliakbarkhani et al, 2017). The results of this study showed that by increasing salinity levels in the growth medium of the two grafted combinations, there was an increase in the activity of peroxidase enzyme and this increment was more obvious in ‘Sahand’ grafted on Badamkohi rootstock. According to Fayek et al. (2018), the activity of POX enzyme changes with different scions graftedon different rootstocks. An increase in POX activity in grafted plants could be an indicator that the grafting process can rapidly induce a higher capacity to breakdown H2O2 in plant cells (Elsheery et al., 2020). Catalase is one of the most important enzymes that can inhibit ROS activity. It converts hydrogen peroxide to water and oxygen in the mitochondria, peroxisomes and cytosol (Acosta­Motos et al., 2017). Based on the results of this study, with increasing salinity levels, the activity of catalase increased in Adv. Hort. Sci., 2024 38(1): 63­74 72 both grafted combinations showing that tolerance to salinity corresponds with an increase in catalase enzyme of the plants, although Badamkohi rootstock was more tolerant than Arjan. The greater availability of CAT can enable the plant defense mechanism to increase the capability of eliminating reactive oxygen species (Madadkhah et al., 2018). Our finding corre­ sponds with those reported previously in pistachio (Akbari et al., 2018) and cherry (Chatzissavvidis et al., 2008). In all levels of salinity ‘Sahand’as scion grafted on Badamkohi had more shoot length, fresh and dry weight than grafted on Arjan. Badamkohi restricted the absorption of Na+ and Cl­ ions from the root medi­ um and reduced their transportation to aerial parts. The ‘Sahand’ leaf chlorophyll depletion and EL were higher on Arjan rootstock than on Badamkohi in all level of salinity condition. Badamkohi protected cell osmotic pressure, enhancing cell membrane integrity than Arjan by inducing more proline and GB as osmo­ protectants in the shoots. Badamkohi rootstock also enhanced the activities of antioxidant enzymes in ‘Sahand’ more than Arjan rootstock which reflect defensive responses to cellular damage induced by reactive oxygene species in higher NaCl concentra­ tions in the culture medium. In conclusion, Badamkohi could be a more suitable rootstock than Arjan for ‘Sahand’ scion under salinity conditions. Reference ABDOLINEJAD R., SHEKAFANDEH A., 2014 ­ Responses of two figs (Ficus carica L.) cultivars under salt stress via in vitro condition. ­ A. Sci. Dev., 3: 194­199. ACOSTA­MOTOS J.R., ORTUÑO M.R., BERNAL­VICENTE A., DIAZ­VIVANCOS P., SANCHEZ­BLANCO M.J., HERNAN­ DEZ J.A., 2017 ­ Plant responses to salt stress: adaptive mechanisms. ­ Agronomy, 7(1): 1­18. AKBARI M., MAHNA N., RAMESH K., BANDEHAGH A., MAZ­ ZUCA S., 2018 ­ Ion homeostasis, osmo regulation, and physiological changes in the roots and leaves of pista‐ chio rootstocks in response to salinity. ­ Protoplast, 255(5): 1349­1362. ALIAKBARKHANI S.T., FARAJPOUR M., ASADIAN A.H., AALI­ FAR M., AHMADI S., AKBARI M., 2017 ­ Variation 479 of nutrients and antioxidant activity in seed and exocarp layer of some Persian pistachio genotypes. ­ Ann. Agri. Sci., 62(1): 39­44. ALIZADEH M., SINGHS.K., PATELV.B., BHATTACHARYA R.C., YADAV B.P., 2010 ­ In vitro responses of grape root‐ stocks to NaCl. ­ Biol. Plant., 54(2): 381­385. ANSARI A., GHARAGHANI A., 2019 ­ A comparative study of genetic diversity, heritability and inter‐relationships of tree and nut attributes between Prunus scoparia and P. elaeagnifolia using multivariate statistical analysis. ­ Inter. J. Hort. Sci. Techn., 6(1): 137­150. ARAS S., EŞITKEN A., 2018 ­ Physiological responses of cherry rootstocks to short term salinity. ­ Erwerbs­ Obstbau, 60: 161­164. ARIF Y., SINGH P., SIDDIQUI H., BAJGUZ A., HAYAT S., 2020 ­ Salinity induced physiological and biochemical changes in plants: An omic approach towards salt stress tolerance. ­ Plant Physiol. Biochem., 156: 64­77. ASADI M., SHEKAFANDEH A., 2021 ­ In vitro grafting of ‘Sahand’ cultivar on two wild almond rootstocks and evaluation of its some physiological and biochemical traits vis‐a‐vis different rootstocks. ­ Plant Cell Tissue Org. Cult., (PCTOC) 145: 507­516. BADO S., FORSTER B.P., NIELEN S., ALI A.M., LAGODA P.J.G., TILL J., LAIMER M., 2015 ­ Plant mutation breed‐ ing: Current progress and future assessment, pp. 23­87. ­ In: JANICK J. (ed.) Plant Breeding Reviews 39. John Wiley & Sons, Hoboken, NJ, USA, pp. 462. BALLIU A., VUKSANI G., NASTO T., HAXHINASTO L., KAÇIU S., 2007 ­Grafting effects on tomato growth rate, yield and fruit quality under saline irrigation water. ­ Acta Horticulturae, 801: 1161­1166. BATES L., 1973 ­ Rapid determination of free proline for water stress studies. ­ Plant Soil, 39: 205­207. BESMA B.D., DENDEN M., 2012 ­ Effect of salt stress on growth, anthocyanins, membrane permeability and chlorophyll fluorescence of okra (Abelmoschus esculen­ tus L.) seedlings. ­ Am. J. Plant Physiol., 7: 174­183. BYBORDI B., 2013 ­ Evaluation tolerance of almond late flowering cultivar to salinity. ­ Crop Prod. Process, 3(3): 217­225. [In Arabic language]. CARILLO P., CIRILLO C., DE MICCO V., ARENA C., DE PAS­ CALE S., ROUPHAEL Y., 2019 ­ Morpho‐anatomical, physiological and biochemical adaptive responses to saline water of Bougainvillea spectabilis Wild. trained to different canopy shapes. ­ Agric. Water Manag., 212: 12­22. CHAPMAN H.D., PRATT P.F., 1961 ­ Methods of analysis for soils, plants, and waters. ­ Univ. of California, Div. Agr. Sci. Berkeley, CA, USA, pp. 309. CHATZISSAVVIDIS C., THERIOS I., ANTONOPOULOU C., DIMASSI K., 2008 ‐ Effect of high boron concentration and scion‐rootstock combination on growth and nutri‐ tional status of olive plant. ­ J. Plant Nut., 31: 638­658. COLLA G., ROUPHAEL Y., LEONARDI C., BIE Z., 2010 ­ Role of grafting in vegetable crops grown under saline con‐ ditions. ­ Sci. Hortic., 127: 147­155. COLLA G., ROUPHAEL Y., REA E., CARDARELLI M., 2012 ­ Grafting cucumber plants enhance tolerance to sodium chloride and sulfate salinization. ­ Sci. Hortic., 135: 177­ 185. CORELL M., MARTÍN­PALOMO M., GIRÓN I., ANDREU L., Asadi Zargh Abad and Shekafandeh ‐ In vitro salt tolerance of different almond graft combinations 73 GALINDO A., CENTENO A., PÉREZ­LÓPEZ D., MORIANA A., 2020 ‐ Stem water potential‐based regulated deficit irrigation scheduling for olive table trees. ­ Agr. Water Manage., 242: 106­418. CUARTERO J., CUARTERO J.,BOLARIN M.C., ASINS M.J., MORENO V., 2006 ­ Increasing salt tolerance in the tomato. ­ J. Exp. Bot., 57: 1045­1058. DHINDSA R.S., MOTOWE W., 1981 ­ Drought tolerance in two mosses: correlation with enzymatic defense against lipid peroxidation. ­ J. Exp. Bot., 32: 79­91. DUSTGEER A., SELEIMAN M., KHAN I., CHATTHA M., ALI E., ALHAMMAD A., JALAL R., REFAY Y., HASSAN M., 2021 ­ Glycine‐betaine induced salinity tolerance in maize by regulating the physiological attributes, antioxidant defense system and ionic homeostasis. ­ Not., Bot., Horti. Agrobo., 49(1): 12248. ELSHEERY N.I., HELALY M.N., OMAR S.A., JOHN S.V.S., ZABOCHNICKA­SWIĄTEK M., KALAJI H.M., RASTOGI A., 2020 ­ Physiological and molecular mechanisms of salinity tolerance in grafted cucumber. ­ South Afr. J. Bot., 130: 90­102. ERTURK U.N., SIVRITEPE C., YERLIKAYA M., BOR F., OZDEMIR I.T., 2007 ­ Responses of the cherry rootstock to salinity in vitro. ­ Biol. Plant., 51(3): 597­600. FAYEK M., ABD E., EBEED S., WALLA S., 2018 ­ Salt toler‐ ance of mango grafted on rootstocks and antioxidant enzyme responses. ­ Bioscience Res., 15(4): 3123­3130. FENG X., LAI Z., LIN Y., LAI G., LIAN C., 2015 ­ Genome‐wide identification and characterization of the superoxide dismutase gene family in Musa acuminata cv. Tianbaojiao (AAA group). ­ BMC Genomics, 16(1): 823. GAINZA F., OPAZO I., CARLOS M., 2015 ­ Graft incompati‐ bility in plants: metabolic changes during formation and establishment of the rootstock/scion union with emphasis on Prunus species. ­ Chilean J. Agri. Res., 75: 28­35. GARCÍA­SÁNCHEZ F., JIFON J.L., CARVAJAL M., SYVERTSEN J.P., 2002 ­ Gas exchange, chlorophyll and nutrient con‐ tents in relation to Na+ and Cl– accumulation in ‘Sunburst’ mandarin grafted on different rootstocks. ­ Plant Sci., 162: 705­712. GHALEB W.S.H., SAWWAN J.S., MUHANAD W.A., AKASH A., AL­ABDALLAT A.M., 2010 ­ In Vitro response of two citrus rootstocks to salt stress. ­ Inter. J. Fruit Sci., 10: 40­53. GRATTAN S.R., GRIEVE C.M., 1985 ­ Betaine status in rela‐ tion to nitrogen stress and transient stress. ­ Plant Soil, 85: 3­9. GREGORY P.J., ATKINSON C.J., BENGOUGH A.G., ELSE M.A., FERNÁNDEZ­FERNÁNDEZ F., HARRISON R.J., SCHMIDT S., 2013 ­ Contributions of roots and rootstocks to sus‐ tainable, intensified crop production. ­ J. Exp. Bot., 64: 1209­1222. GROSS J., 1991 ­ Pigments in Vegetables. ­ Van Nostrand Reinhold, New York, USA, pp. 351. GULEN H., ERIS A., 2004 ­ Effect of heat stress on peroxi‐ dase activity and total protein content in strawberry plants. ­ Plant Sci., 166: 739­744. GULEN H., KESICI H., CENTINKAYA C., ERGIN S., 2018 ­ Proline and antioxidant enzyme activities in some strawberry cultivars under drought and recovery. ­ Not. Bot. Horti.Agrobo., 46(2): 570­578. HOU X.L., HAN H., MENG F.R., CAI L.P., LIU A.Q., 2019 ­ Intermittent lead‐induced stress on antioxidant enzyme activity and subcellular distribution of Pb in Pogonatherum crinitum seedlings. ­ Plant Biol.,21(4): 634–642. KARIMI H.R., NASROLAHPOUR­MOGHADAM S., MOHAM­ MADIMIRIK A.A., 2015 ­ Variation and correlation between seeds and their seedlings traits in pistachio (Pistacia vera L.) in separation sex. ­ Plant Cell Biotech. Molecul. Biol., 19: 392­402. KAUNDAL A., SANDHU D., DUENAS M., FERREIRA J.F.S., 2019 ­ Expression of the high‐affinity K+ transporter 1 (PpHKT1) gene from almond rootstock “Nemaguard” improved salt tolerance of transgenic Arabidopsis. ­ PLoS ONE, 14(3): e0214473. KRASENSKY J., JONAK C., 2012 ­ Drought, salt, and temper‐ ature stress‐induced metabolic rearrangements and regulatory networks. ­ J. Exp. Bot., 63: 1593­1608. KUMAR P., ROUPHAEL Y., CARDARELLI M., COLLA G., 2017 ­ Vegetable grafting as a tool to improve drought resis‐ tance and water use efficiency. ­ Front. Plant Sci., 8: 1130. KUŞVURAN Ş., KAYA E., ELLIALTIOĞLU Ş.Ş., 2021 ­ Role of grafting in tolerance to salt stress in melon (Cucumis melo L.) Plants: Ion regulation and antioxidant defense systems. ­ Biotech. Studies, 30(1): 22­32. KUŞVURAN S., KIRAN S., ELLIALTIOGLU S.S., 2016 ­ Antioxidant enzyme activities and abiotic stress toler‐ ance relationship in vegetable crops, pp. 481­503. ­ In: SHANKER A.K., and C. SHANKER (eds.) Abiotic and Biotic stress in plants‐recent advances and future per‐ spectives. IntechOpen, London, UK. MADADKHAH E., BOLANDNAZAR S., OUSTAN S., 2018 ­ Effect of salt stress on growth, antioxidant enzymes activity, lipid peroxidation and photosystem II efficiency in cucumber grafted on cucurbit rootstock. ­ J. Hort. Sci., 49(2): 465­475. MICKELBART M.V., ARPAIA M.L., 2002 ­ Rootstock influ‐ ences changes in ion concentrations, growth, and pho‐ tosynthesis of ‘Hass’ avocado trees in response to salin‐ ity. ­ J. Am. Soc. Hort. Sci., 127: 649­655. MURASHIGET., SKOOG F., 1962 ­ A revised medium for rapid growth and bio assays with tobacco tissue cul‐ tures. ­ Plant Physiol., 15: 473­497. NAJAFIAN S., RAHEMI M., TAVALLALI V., 2008 ­ Effect of salinity on tolerance of two bitter almond rootstocks. ­ American­Eurasian. J. Agr. Environ. Sci., 3(2): 264­268. NIAZIAN M., SADAT­NOORI S.A., TOHIDFAR M., MORTAZA­ VIAN S.M.M., PAOLO SABBATINI P., 2021 ­ Betaine aldehyde dehydrogenase (BADH) vs. Flavodoxin (Fld): Two important genes for enhancing plants stress toler‐ ance and productivity. ­ Front. Plant Sci., 12: 650215. http://https://www.sid.ir/EN/JOURNAL/SearchPaper.aspx?writer=858646 http://https://www.sid.ir/EN/JOURNAL/SearchPaper.aspx?writer=696107 http://https://www.sid.ir/EN/JOURNAL/JournalList.aspx?ID=13078 http://https://www.sid.ir/EN/JOURNAL/JournalList.aspx?ID=13078 http://https://www.sid.ir/EN/JOURNAL/JournalList.aspx?ID=13078 http://https://www.sid.ir/EN/JOURNAL/JournalListPaper.aspx?ID=274534 Adv. Hort. Sci., 2024 38(1): 63­74 74 OZDEN M., DEMIREL U., KAHRAMAN A., 2009 ­ Effects of proline on antioxidant system in leaves of grapevine (Vitis vinifera L.) exposed to oxidative stress by H2O2. ­ Sci. Horti., 119: 163­168. OZTEKIN G.B., TUZEL Y., 2011 ­ Salinity response of some tomato rootstocks at seedling stage. ­ Afr. J. Agric. Res., 6: 4726­4735. RAI M.K., KALIA R K., SINGH R., GANGOLA M.P., DHAEAN A.K., 2011 ­ Developing stress tolerant plants through in vitro selection ‐ An overview of the recent progress. ­ Environ. Exp. Bot., 71(1): 89­98. RAOUFI A., SALEHI H., RAHEMI M., SHEKAFANDEH A., KHALILI S., 2020 ­ In vitro screening: The best method for salt tolerance selection among pistachio rootstocks. ­ J. Saudi Soc. Agr. Sci., 20(3): 146­154. ROY S.J., NIGRÃO S., TESTER M., 2014 ­ Salt resistant crop plants. ­ Curr. Opin. Biotechnol., 26: 115­124. SABZMEYDANI E., SEDAGHATHOOR S., HASHEMABADI D., 2020 ­ Salinity response of Kentucky bluegrass (Poa pratensis L.) as influenced by salicylic acid and proges‐ terone. ­ Rev. Chapingo Ser. Hortic., 26(1): 49­63. SAHA P., CHATTERJEE P., BISWAS A.K., 2010 ­ NaCl pre‐ treatment alleviates salt stress by enhancement of antioxidant defense system and osmolyte accumulation in mung bean (Vigna radiate L. Wilczek). ­ Indian J. Exp. Biol., 48: 593­600. SAUVAGE C., SEGURA V., BAUCHET G., STEVENS R., DO P.T., NIKOLOSKIZ., FERNIEA.R., CAUSSE M., 2014 ­ Genome‐wide association in tomato reveals candidate loci for fruit metabolic traits. ­ Plant Physiol., 165: 1120­1132. SELEIMAN M.F., SEMIDA W.M., RADY M.M., MOHAMMAD G.F., HEMIDA K.A., ALHAMMAD B.A., HASSAN M.M., SHAMI A., 2020 ­ Sequential application of antioxidants rectifies ion imbalance and strengthens antioxidant sys‐ tems in salt‐stressed cucumber. ­ Plants, 9: 1783. SETH R., KENDURKAR S.V., 2015 ­ In vitro screening: An effective method for evaluation of commercial cultivars of tomato towards salinity stress. ­ Int. J. Curr. Microbiol. Appl. Sci., 4(1): 725­730. SHAHKOOMAHALLY S., CHANGY., BRECHT J.K., CHAPARRO J.X., SARKHOSH A., 2020 ‐ Influence of rootstocks on fruit physical and chemical properties of peach cv. UFSun. ­ J. Food Sci. Nutri., 9(1): 401­413. SHIBLI R., MOHAMMAD M., ABU­EIN A., SHATNAWI M., 2000 ­ Growth and micronutrient acquisition of some apple varieties in response to gradual in vitro induced salinity. ­ J. Plant Nutr., 23(9): 1209­1215. SHRIVASTAVA P., KUMAR R., 2015 ­ Soil salinity: a serious environmental issue and plant growth promoting bac‐ teria as one of the tools for its alleviation. ­ Saudi J. Biol. Sci., 22: 123­131. SINGH H., KUMAR P., KUMAR A., KYRIACOU M.C., COLLA G., ROUPHAEL Y., 2020 ‐ Grafting tomato as a tool to improve salt tolerance. ­ Agronomy, 10: 263. SOFO A., TUZIO A.C., DICHIO B., XILOYANNIS C., 2005 ­ Influence of water deficit and rewatering on the com‐ ponents of the ascorbate‐glutathione cycle in four interspecific Prunus hybrids. ­ Plant Sci., 169: 403­412. SOTIROPOULOS T.E., DIMASSI K.N., 2004 ­ Response to increasing rates of boron and NaCl on shoot prolifera‐ tion and chemical composition of in vitro kiwifruit shoot cultures. ­ Plant Cell Tissue Organ Cult., 79(3): 285­289. SOTIROPOULOS T.E., FOTOPOULOS S., DIMASSI K.N., TSIR­ AKOGLOU V., THERIOS I.N., 2006 ­ Response of the pear rootstock to boron and salinity in vitro. ­ Biol. Plant., 50(4): 779­781. SURENDAR K.K., DEVID D.D., RAVI I., JEYAKUMER P., VELAYUDHAMK K., 2013 ­ Effect of water deficit on relationship between yield and physiological attributes of banana cultivars and hybrids. ­ Afr. J. Plant Sci., 7: 374­383. TAÏBI K., TAÏBI F., ABDERRAHIM L.A., ENNAJAH A., BELKHODJA M., MULET J.M., 2016 ­ Effect of salt stress on growth, chlorophyll content, lipid peroxidation and antioxidant defense systems in Phaseolus vulgaris L. ­ South Afr. J. Bot., 105: 306­312. VIVES­PERISV., GÓMEZ­CADENAS A., PÉREZ­CLEMENTE R.M., 2017 ­ Citrus plants exude proline and phytohor‐ mones under abiotic stress conditions. ­ Plant Cell Rep., 36: 1971­1984. WALKER R.R., DOUGLAS T.J., 1983 ­ Effects of salinity level on uptake and distribution of chloride, sodium and potassium ions in citrus plants. ­ Aust. J. Plant Physiol., 34: 145­153. WEI D.D.W., ZHANG C.C., WANG Q.W., LI M.G., CHEN T.H.H., YANG X.H., 2017 ­ Genetic engineering of the biosynthesis of glycine betaine leads to alleviate salt‐ induced potassium efflux and enhances salt tolerance in tomato plants. ­ Plant Sci., 257: 74­83. ZRIG A., BEN MOHAMED H., TOUNEKTIA T., KHEMIRA H., SERRANO M., VALERO D., VADEL A.M., 2016 ­ Effect of rootstock on salinity tolerance of sweet almond (cv. Mazzetto). ­ South Afr. J. Bot., 102: 50­59. http://https://www.sciencedirect.com/science/journal/1658077X http://https://www.sciencedirect.com/science/journal/1658077X/20/3 http://https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Chang%2C+Yuru http://https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Brecht%2C+Jeffrey+K http://https://onlinelibrary.wiley.com/action/doSearch?ContribAuthorStored=Sarkhosh%2C+Ali http://https://onlinelibrary.wiley.com/journal/20487177