Impaginato 99 Adv. Hort. Sci., 2021 35(2): 99­110 DOI: 10.36253/ahsc­10414 ‘Momordica charantia’ introducing a new rootstock for grafted cucumber under low­temperature stress S. Mohammadnia, M. Haghighi (*) Department of Horticulture, College of Agriculture, Isfahan University of Technology, Isfahan, Iran. Key words: Cucurbita maxima, female flower, Karela, photosynthesis, root­ stock, yield. Abbreviation: Rma= grafted onto Cucurbita maxima; Rmo= grafted onto Momordica charantia; Rn= non­grafted; Rs= self­grafted; Tc= con­ trol temperature; Ts= stress temperature. Abstract: Cucumber is a sensitive vegetable to low temperatures. Grafting veg­ etables on different rootstocks can decrease the harmful effects of environ­ mental stresses, including low­temperature stress. An experiment was per­ formed to evaluate grafting cucumbers on different rootstocks at low tempera­ tures. Cucumber growth and yield and photosynthesis traits were examined. Treatments were the optimum temperature (25±2°C), and cold temperature (15±3°C, Ts), and rootstocks, were Momordica charantia (Rmo), Cucurbita max‐ ima (Rma), non­grafted (Rn) and self­ grafted (Rs) with 4 replications. Shoot fresh and dry weight, chlorophyll, RWC, transpiration, decreased with tempera­ ture stress. The number of female flowers, electrolyte leakage, photosynthesis, stomatal conductance increased with Ts. First fruit emergence per plant, N, P, K, Mg concentration decreased with Ts stress. Transpiration, female flower, RWC, and stomata conductance, N, P, K, Ca, and phenol increased in Rma and Rmo. Mg was at the highest concentration in Rma and Na in Rn. All in all, using Rmo as well as Rma is recommended for rootstock as it causes more reproduc­ tive growth. 1. Introduction Recently, the greenhouse culture of plants has widely expanded (Yan et al., 2013). Cucumbers (Cucumis sativus) are commonly grown in the greenhouse. The optimal temperature for aerial growth and roots are about 28­23/18­15°C (day/night) and 20°C, respectively (Ikeda and Kawashiro, 2005). Thus, cucumbers are cold­sensitive vegetables. Low­ temperature stress can occur in plastic culture or non­equipped green­ houses, which are very common in Iran. It may also affect field culture cucumbers and spring culture cucumbers. Low temperatures have differ­ (*) Corresponding author: mhaghighi@iut.ac.ir Citation: MOHAMMADNIA S., HAGHIGHI M., 2021 ­ ‘Momordica charantia’ introducing a new root‐ stock for grafted cucumber under low‐ temperature stress. ­ Adv. Hort. Sci., 35(2): 99­ 110 Copyright: © 2021 Mohammadnia S., Haghighi M. 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 February 2021 Accepted for publication 12 February 2021 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-10414 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., 2021 35(2): 99­110 100 ent deleterious effects on cucumbers, including affecting the nutrient uptake of plants (Pregitzer and King, 2005), and inducing greater H2O2, MDA, and sol­ uble sugar content in cucumbers, causing damage to plants and inhibiting plant growth (Qiu­yan et al., 2013). There are several ways to control low­tempera­ ture damage to plants, including controlling root temperatures by warming nutrient solution or soil. Still, these ways require excess energy (Willits and Peet, 1998). Finally, excess chemical nutrient applica­ tions for promoting growth can prevent damage to plants, which unfortunately involves environmental risks. Therefore, vegetable grafting can be a proper way to increase plant resistance to environmental stresses in addition to expanding its yield, quality, and growth. Furthermore, if any energy­efficient way is introduced, which can raise the growth rate of plants in greenhouse conditions at lower tempera­ tures, vast amounts of energy and money will be saved. Vegetable grafting has been used to increase plant tolerance under environmental stresses, includ­ ing high temperatures (Rivero et al., 2003), low tem­ peratures (Rivero et al., 2003), salinity stresses (Estan et al., 2005), drought stresses (Bhatt et al., 2002) and heavy metal stresses (Edelstein et al., 2005). Fig leaf gourd and bur cucumbers (Sicyos angula‐ tus) are used for increasing resistance against low­ temperature stresses for cucumber rootstocks (Venema et al., 2008). These rootstocks increase plant resistance using different ways. First, low­tem­ perature stresses decrease Co2 assimilation in cucum­ bers and this reduction is improved by grafting (Zhou et al., 2009). Secondly, a previous study done by Zhou et al. (2007) showed that fig leaf gourd root­ stocks enhanced vegetative growth and yield of cucumbers under low­temperature stresses. There is a hypothesis stating that grafted plant responses to temperature stresses are related to scion species (Venema et al. , 2005). Many researchers have shown beneficial effects of veg­ etable grafting on the growth, fruit yield, and quality of plants. Some of the studies have used different rootstocks for cucumbers under different stresses showing better growth rate for grafted plants under stress conditions compared to non­grafted ones like: heavy metal absorption (Rouphael et al., 2008; Kumar et al. , 2015), low soil temperatures (Tachibana, 1982), salinity (Huang et al., 2010), improve NaCl and CaCl2 tolerance in Cucumber (Colla et al., 2013), Al toxicity in cucumber (Rouphael et al., 2016). Different rootstocks were used in normal and stress conditions to improve yield and growth traits like dif­ ferent kinds of squash (Yang et al., 2006), (Massai et al., 2004), (Rouphael et al., 2008), Fig leaf guard (Tachibana, 1982), pumpkins and bottle gourd and P360 (Cucurbita maxima Duch.×Cucurbita moschata Duch.) (Colla et al., 2010 and 2013). To the best of our knowledge, few studies have investigated the use of Momordica charantia as a rootstock for cucurbits, or more explicitly grafting, to examine the possibility of cucumber cultivation at low temperatures in the whole growth period. Hence, the goal of the present study was the use of Momordica charantia and Cucurbita maxima as rootstocks and Cucumis sativus var. DavosII, which is a common variety of cucumbers in Iran as a scion under low­temperature stress. 2. Materials and Methods Experimental design and plant preparation This experiment was conducted in a plastic green­ house in the Department of Horticulture Science at the Isfahan University of Technology, Isfahan, Iran. An investigation was arranged as a combined analysis involving data from two locations, simultaneously collected, based on CRD with 4 replications. Treatments were optimum (25±2°C, Tc) and low tem­ peratures (15±3°C, Ts). Cucumbers (Cucumis sativus var. DavosII) were grafted to Momordica charantia (Rmo), Cucurbita maxima (Rma). Non­grafted (Rn) and self­grafted (Rs) consider as a control. Scion seeds had been cultivated 10 days before rootstock seeds in cocopeat: perlite 1:1. Scion plants and root­ stocks were cut beneath and above the first true leaves, respectively. Hole­insertion grafting was used and grafted plants were transferred to a recovery greenhouse with high relative humidity. Plants were kept for 2 weeks in recovery conditions and gradually adapted to normal greenhouse conditions. Grafted plants were transferred to 5kg pots, including soil. Irrigation was used when the plant needed it. Chemical fertilizer NPK (20, 20, 20) 2 mg/pot was applied every 10 days. Plants were conducted to wire above the greenhouse, and there was no use of any pesticide. Plant growth and fruit properties The male and female flower, node numbers, and shoot numbers were counted during the experiment; Mohammadnia et al. ‐ ‘Momordica charantia’ a new rootstock for grafted cucumbers 101 root and internode length was determined with a ruler. The time and node of male and female flower emergence were recorded. Fruit diameter, fruit firm­ ness, and TSS were measured with a caliper (Mitutoyo Corp, Japan), Pentameter (model OSK­I­ 10576), and portable Refractometer (PAL­1 Brix, Japan), respectively (Raeisi et al., 2014). Shoots were excised from the roots using a steel blade and then fresh weights of roots and shoots were measured. All the samples were oven­dried at 70˚C for 48 hours and the dry weights were estimat­ ed. During the experiment and finally 124 days after transplanting, fruits were harvested and washed using tap water and were weighed by an analytical balance. SPAD value and photosynthesis trait assay Chlorophyll content was measured using a chloro­ phyll meter (SPAD­502 plus, Minolta, Japan). Fv/Fm was measured by chlorophyll fluorescence (OS­ 30, USA) after 3 weeks. Photosynthetic properties were determined from the youngest fully­expanded leaf for 3 replications per treatment by Portable photo­ synthesis systems for gas exchange and chlorophyll fluorescence measurements (LI­COR­6400, USA) from 10:00 to 11:00 AM on a clear day (without clouds). The measurements were conducted with photosyn­ thetically active radiation (PAR) intensity of 1000 μmol m­2 s­1 and CO2 concentration of 350 μmol·mol. Mesophyll conductance (μmol m­2s­1) was calculated by dividing the photosynthetic rate by the sub­stom­ atal CO2 level (Ahmadi and Siosemardeh, 2005). Antioxidant activity Antioxidant activity was measured and expressed as gallic acid (equivalents l of gallic acid/g) with UV­ VIS spectrophotometer (Shimadzu UV160A­Japan). Three mg of sample were dissolved in 5 mL methanol stock, and 1.4 ml of this solution was blended with 0.6 mL of DPPH solution. After 30 min, the absorbance of the solution was recorded at 515 nm by the spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan) against methanol as a blank. The 0.2 mM of DPPH solution in methanol was used as a stock of DPPH for the determination of the free radi­ cal scavenging activity of the samples (Koleva et al., 2002). Phenolic content Total phenolic content was determined using the Folin­Ciocalteu method. The absorbance was mea­ sured at 725 nm with a spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan). The results were expressed in gallic acid equivalents (mg/100 g fresh weight) using gallic acid (0­0.1 mg/mL) standard curve (Singleton et al., 1965). Proline Proline accumulation was determined using the method proposed by Bates et al. (1973). After the extraction of toluene, the clear phase was recovered and spectrophotometrically estimated at 520 nm using toluene as a blank. Purified proline was used for standardization (0­50 mg/mL) and was expressed as mol proline g­1 fresh weight. Electrolyte leakage Electrolyte leakage (EL) was measured using an electrical conductivity meter employing the method described by Lutts et al. (1995) Relative water content Relative water content (RWC%) was determined using ten 7 mm­diameter leaf discs. The leaf discs of each treatment were weighed (FW). They were then hydrated until saturation was reached (constant weight) for 48 h at 5°C in darkness (TW). The leaf discs were dried in an oven at 105°C for 24 h (DW). Relative water content was calculated according to the following equation (Filella et al., 1998): RWC% = (FW − DW)/(TW − DW)× 100 The determination of total nitrogen in the leaf samples was based on the Kjeldahl method (Estan et al., 2005). The concentrations of K, Ca, Mg, and P were measured (Shield Torch System, Agilent 7500a). Meanwhile, phosphorus was estimated by the vanadomolybdo phosphoric acid colorimetric method at 460 nm (Estan et al., 2005). P was colori­ metrically determined using a spectrophotometer (UV 160A­ Shimadzu Corp., Kyoto, Japan). Statistical analysis All data were analyzed using two­way ANOVA (Statistix 8 software) (Tallahassee FL, USA) and the means were compared for the significance by the least significant difference (LSD) test at P<0.05. 3. Results and Discussion Analysis of variance of temperature and rootstock on some characteristics of cucumber Temperature effect shoot fresh and dry weight, chlorophyll index, and the number of the female flower, photosynthetic rate, transpiration, stomatal Adv. Hort. Sci., 2021 35(2): 99­110 102 conductance, RWC, EL, phenol content, and all nutri­ ent concentration (Table 1 a, b). Rootstock changes all cucumber characteristics except for average fruit weight, fruit diameter, TSS, firmness, mesophyll con­ ductance, proline, and antioxidant (Table 1 a, b). The interactive effect of temperature and rootstock showed that all measured parameters change signifi­ cantly (Table 1 a, b). The main effect of temperature on cucumber Shoot fresh and dry weight, chlorophyll, RWC, transpiration, decreased with temperature stress. The number of the female flower, EL, photosynthesis, and stomatal conductance increased with Ts (Table 2 a). First fruit emergence per plant, N, P, K, Mg con­ centration decreased with Ts stress (Table 2 b). Shoot fresh and dry weight and fresh root weight was high­ Table 2 a ­The effect of temperature on some characteristics of cucumber Tc= optimum temperature, Ts= low temperature. Within a column means followed by the same letter are not significantly different at P<5% according to LSD test. Table 2 b ­ The effect of temperature on some characteristics of cucumber Tc= optimum temperature; Ts= low temperature. Within a column means followed by the same letter are not significantly different at P<5% according to LSD test. Table 1 a ­ Analysis of variance of the effect of temperature and rootstock on some characteristics of cucumber NS= no significant, * significant at 5% and ** significant at 1%. Table 1 b ­ Analysis of variance of the effect of temperature and rootstock on some characteristics of cucumber NS= no significant, * significant at 5% and ** significant at 1%. Source df Shoot fresh weight Shoot dry weight Root fresh weight ChI Number of female flowers Number of male flowers Fruit number Fruit yield First fruit per plant Average fruit weight Fruit diameter TSS Firmness Temperature 1 3203.42 * 230. 9 ** 244.56 NS 43.12 ** 78.12 * 6.12 ns 230. 9 NS 1441.5 NS 152.91 * 1971.09 NS 0.002 NS 0.30 NS 7.65 NS Rootstock 3 2467.99 * 23.45 * 150.01 * 35.93 ** 602.45 ** 13.08 ** 23.45 * 19335 * 92.57 * 2094.59 NS 0.10 NS 0.18 NS 2.07 NS T×R 3 1327.25 * 26.54 ** 111.77 ** 4.65 ** 81.12 ** 4.20 * 26.54 ** 11769.1 ** 4.81 ** 2133.88 * 0.04 ** 0.01 ** 1.23 ** Error 18 708.05 5.27 67.38 3.23 76.32 1.35 5.27 5110.2 17.23 1292.36 0.04 0.12 4.26 CV 10.30 25.62 13.04 15.50 13.97 14.38 25.62 18.03 26.05 18.65 6.77 13.40 25.25 Source df Photo­ synthetic rate Tran­ spiration Stomata conduc­ tance Mesophyll conduc­ tance RWC EL Proline Anti­ oxidant activity Phenol content N Conc. P Conc. K Conc. Ca Conc. Mg Conc. Na Conc. Temperature 1 47.52 * 10.52 ** 0.002 ** 240063 NS 0.006 ** 257.36 ** 3.27 NS 0.06 NS 55824.1 ** 0.72 ** 0.036 ** 0.06 ** 0.15 ** 0.16 ** 0.039 ** Rootstock 3 25.08 * 2 ** 0.001 * 26980 NS 0.001 ** 60.38 ** 1.31 NS 0.006 NS 5561.8 ** 1.39 ** 0.03 ** 2.06 ** 0.65 ** 0.07 ** 0.01 ** T×R 3 14.25 ** 1.12 ** 0.002 ** 4898 ** 0.0008 ** 72.71 ** 2.82 ** 0.02 ** 4553.6 ** 0.54 ** 0.01 ** 0.62 ** 0.54 ** 0.06 ** 0.008 ** Error 18 6.17 0.97 0.001 14354 0.00003 9.38 1.05 0.03 52.2 6.53 4.71 5.08 3.85 1.05 2.78 CV 14.41 14.82 17.80 38.05 0.74 7.56 11.03 5.81 5.01 12.1 26.7 21.5 7.87 6.23 16.9 Temperature (° C) Shoot fresh weight (g) Shoot dry weight (g) ChI (SPAD value) Number of the female flower El (%) RWC (%) Transpiration (mmol H2O m­2 s­1) Photosynthetic rate (mmol H2Om­2 s­1) Stomata conductance (mmol H2Om­2 s­1) Tc 76.52 a 11.71 a 12.7 a 2.18 b 37.1 b 0.79 a 2.83 a 4.16 b 61.31 b Ts 55.53 b 6.21 b 10.4 b 3.06 a 43.91 a 0.75 b 1.57 b 7.02 a 177.34 a Temperature (° C) First fruit emergence per plant (day) Phenol (mg g ̵¹ FW ) N (%) P (%) K (%) Ca (%) Mg (%) Na (%) Tc 19.57 a 17.69 b 3.51 a 0.43 a 3.11 a 1.54 b 0.74 a 0.14 b Ts 12.30 b 271.06 a 3.37 b 0.39 b 2.54 b 1.66 a 0.68 b 0.21 a Mohammadnia et al. ‐ ‘Momordica charantia’ a new rootstock for grafted cucumbers 103 est in Rma, the number of male flower increased in Rn and Rs and female flower increased in Rmo and Rma. The main effect of rootstock on cucumber The highest El and photosynthesis were observed in Rmo. The RWC and stomata conductance increased in Rma and Rmo, however, transpiration increased in Rs, Rmo, and Rma (Table 3 a). N, P, K, Ca, and phenol increased in Rma and Rmo. Mg was at the highest concentration in Rma and Na in Rn (Table 3 b). Rma has the highest fruit number, fruit yield, although, first fruit emerges in Rmo (Table 4). The interaction effect of temperature and cucumber grafting on some growth characteristics of cucumber K concentration was the highest in Rmo in both temperatures and P concentration increased in Rma×Ts. The highest N, K and Ca, concentrations were seen in Rma×Ts. P concentration increased in Rma and Rmo at Ts. Mg increased in Rs×Tc and Na in Rs×Ts (Table 5). Shoot fresh weights decreased in Rn and Rs at low temperatures (Ts), but shoot dry weights did not sig­ nificantly change in Rma and Rmo at both tempera­ tures (Fig. 1). Shoot dry weights followed the same trend as the fresh shoot weight was the highest in Rn, Rs, Rma at Tc (Fig. 1). Root fresh weights decreased in Rn, Rs, and Rma at Ts compared with Tc, but did not significantly change in Rmo at both temperatures, although the fresh root weight was the lowest in Rmo at Tc. The highest root fresh weight was seen in Rma×Tc (Fig. 2 a). SPAD value was the highest in Rs, Rma, and Rmo at Tc (Fig. 2 b). The interaction effect of the tempera­ Table 3 a ­The effect of rootstock on some characteristics of cucumber Rn= nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Within a column means followed by the same letter are not significantly different at P<5% according to LSD test. Rootstock Shoot fresh weight (g) Shoot dry weight (g) Root fresh weight (g) Number of male flowers Number of female flower El (%) RWC (%) Photosynthetic rate (µmol CO2 m ­2 s­1) Transpiration (mmol H2O m–2 s–1) Stomata conductance (mmol H2O m–2 s–1) Rn 61.28 b 8.23 b 16.78 b 23.50 a 2.37 b 38.98 bc 0.76 b 4.94 b 1.65 b 78.79 b Rs 55.48 b 9.29 ab 17.96 ab 23 a 1.75 b 41.67 ab 0.76 b 3.83 b 2.69 a 71.94 b Rma 92.34 a 11.17 a 18.21 a 5.5 b 4.5 a 37.10 c 0.78 a 4.84 b 1.86 ab 89.96 ab Rmo 55b 7.14 b 17.95 b 12.75 b 3.87 ab 44.38 a 0.78 a 8.75 a 2.61 ab 126.62 a Table 3 b ­ The effect of rootstock on some characteristics of cucumber Rn= nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Within a column means followed by the same letter are not significantly different at P<5% according to LSD test. Rootstock Phenol (mg g ̵¹ FW) N (%) P (%) K (%) Ca (%) Mg (%) Na (%) Rn 124.54 bc 3.19 c 0.45 b 2.52 d 1.05 d 0.70 b 0.19 a Rs 112.19 c 3.08 d 0.38 c 2.55 c 1.32 c 0.69 c 0.20 a Rma 133.90 b 3.85 a 0.47 a 3.65 a 2.11 a 0.51 d 0.14 c Rmo 206.87 a 3.64 b 0.33 d 2.57 b 1.89 b 0.93 a 0.18 b Table 4 ­ The effect of rootstock on some characteristics of cucumber Rootstock Fruit number Fruit yield (g) First fruit emergence per plant (day) Rn 0.8 b 63.88 b 21.51 a Rma 2.00 a 159.50 a 15.29 ab Rmo 1.37 ab 91.88 ab 11.00 b Rn= nongrafted (Cucumis sativus var. DavosII); Rs= self grafted did not have any fruit; Rma= Cucurbita maxima; Rmo= Momordica charantia. Within a column means followed by the same letter are not signi­ ficantly different at P<5% according to LSD test. 104 Adv. Hort. Sci., 2021 35(2): 99­110 ture on grafted cucumber did not significantly affect the time of the emergence of the first female flowers (data did not show). The highest and the lowest num­ ber of the male flower were seen in Rs×Tc and Rma, respectively. The female flower was affected by tem­ perature variations in different rootstocks. The female flower increased in the Rn and Rma at Ts and did not change in Rs and Rmo at Tc and Ts (Fig. 3). Fruit number was the highest in Rma×Tc, although the same results were seen in Rmo×Tc and Rmo and Rma at Ts (Fig. 4 a). Fruit yield was the highest in Rma×Tc; the same result was statically seen in Rma ×Ts (Fig. 4 b). First, fruit emergence time significantly decreased in grafted plants at this temperature (Fig. 5). The highest fruit weight was in Rma×Tc; fruit diameter did not significantly change between treatments (Fig. 6). TSS increased in Rn and Rmo at Ts (Fig. 7 a). Firmness decreased in Rmo×Ts (Fig. 7 b) results, Goreta et al. (2008) found that shoot weight reduc­ tion in watermelon grafted onto Cucurbita maxima Duch.× Cucurbita moschata Duch was less) than non­ grafted plants under salt stresses. The grafted cucum­ ber plants showed less change in shoot and root weights under Cu stress conditions and this might be due to the lower accumulation of Cu in leaves through their squash rootstocks (Rouphael et al., 2008). The lowest male flower was seen in Rma at both temperatures (Fig. 3). Conversely, the highest num­ ber of female flowers was observed in Rma at Ts and Tc compared with other rootstocks at each tempera­ ture. On the other hand, at the optimum tempera­ ture (Tc), each rootstock had a lower female flower Table 5 ­ The interaction effect of temperature and cucumber grafting on some elements concentration (%) of leaves Fig. 1 ­ The interaction effect of temperature and cucumber grafting on shoot fresh and dry weight. Tc= optimum temperature; Ts= low temperature; Rn = nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Fig. 2 ­ The interaction effect of temperature and cucumber grafting on root fresh weight (a) and SPAD value (b) Tc= optimum temperature; Ts= low temperature; Rn = non­ grafted (Cucumis sativus var. Davos II); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Rootstock N P K Ca Mg Na Tc Ts Tc Ts Tc Ts Tc Ts Tc Ts Tc Ts Rn 3.36 d 3.03 e 0.37 c 0.35 c 2.50 f 2.75 e 1.28 c 0.83 g 0.81 b 0.58 c 0.18 c 0.21 b Rs 3.08 e 3.08 e 0.33 c 0.34 c 3.00 d 2.15 g 1.65 b 1.00 e 0.88 a 0.51 e 0.12 f 0.28 a Rma 3.78 c 4.2 a 0.34 c 0.52 a 3.45 b 3.69 a 1.24 d 1.75 a 0.51 e 0.57 d 0.12 f 0.14 e Rmo 3.98 b 3.78 c 0.42 b 0.48 a 3.25 c 3.10 cd 0.83 g 0.87 f 0.56 d 0.50 e 0.09 g 0.16 d Rn= nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Within a column means followed by the same letter are not significantly different at P<5% according to LSD test. Mohammadnia et al. ‐ ‘Momordica charantia’ a new rootstock for grafted cucumbers 105 showed the highest male flower. Van der Ploeg and Heuvelink (2005) reported that low temperatures reduced the tomato fruit set through poor pollen quality and increased the period between anthesis and fruit maturity resulting in lower fruit yields. The result of Khah et al. (2006) showed that tomatoes cv. Big Red grafted onto cv. Heman and Primavera pro­ duced more fruit at low temperatures compared to non­grafted plants in greenhouse conditions. In our study, fruit number decreased at Ts and grafting had a beneficial effect on fruit number, but did not influ­ ence fruit yields. In agreement with our study, com­ mercial tomato grafting was not able to improve the reduction of tomato yields under low light stresses by shading (Krumbein and Schwarz, 2013). The economic increase of yield imparted by select vigorous commercial rootstocks (Kyriacou et al., 2017) like increasing tomato yields was observed by Fig. 3 ­ The interaction effect of temperature and cucumber grafting male and female flower number. Tc= optimum temperature; Ts= low temperature; Rn = nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Fig. 4 ­ The interaction effect of temperature and cucumber grafting on fruit number (a) and fruit yield (b) Momordica charantia Rs did not have fruit. Tc= optimum temperatu­ re; Ts= low temperature; Rn = nongrafted (Cucumis sati‐ vus var. DavosII); Rs= self grafted; Rma= Cucurbita maxi‐ ma; Rmo= Momordica charantia. Fig. 5 ­ The interaction effect of temperature and cucumber grafting on the first fruit emergence of a plant. Tc= opti­ mum temperature; Ts= low temperature; Rn = nongraf­ ted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Fig. 6 ­ The interaction effect of temperature and cucumber grafting on average fruit weight and fruit diameter. Tc= optimum temperature; Ts= low temperature; Rn = non­ grafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Adv. Hort. Sci., 2021 35(2): 99­110 106 grafting tomatoes onto Kagemusia and Helper’s root­ stocks by Lee et al. (2007). Santa­Cruz et al. (2001) grafted tomato cv. Moneymaker onto Pera and observed that growth and yield of tomato increased under salt stress conditions. Similar results were observed by Estan et al. (2005), who found that tomato yield increased through grafting under salt stresses and also reported that fruit yield in hetro­ grafted plants increased more compared with self­ grafted plants. Ruiz et al. (1997) believed that increase yield and growth of the grafted plants might be due to an increase in the nutrient and water uptake fed by vigorous rootstock. Huang et al. (2010) reported that cucumbers grafted had a higher fruit yield than non­grafted plants under salt stresses. Colla et al. (2010) found that increasing CO2 assimila­ tion by cucumber grafting increased fruit yields. The interaction effect of temperature and cucumber grafting on some physiological characteristics and nutrient concentration of cucumber The photosynthesis rate decreased in Rn and Rs at Ts compared with Tc. However, it did not significantly change in Rma and Rmo (Fig. 8 a). Transpiration decreased at Ts in all rootstocks compared with Tc (Fig. 8 b). Stomata conductance gradually increased in Rs, Rma, and Rmo at Ts compared with Tc and reached the highest in Rmo×Ts (Fig. 8 c). Mesophyll conductance did not significantly change in each rootstock at Tc and Ts, except for ungrafted cucum­ bers in which it increased at Ts compared with Tc (Fig. 8 d). El and RWC were generally the lowest and the highest at TS, respectively. the highest EL was seen in Rmo×Ts. The maximum RWC, at Ts, was observed in Rn, Rma, and Rmo (Fig. 9 a, b). Antioxidant increased in Rn and Rs at Ts and did not change in other treat­ Fig. 7 ­ The interaction effect of temperature and cucumber grafting on TSS (a) and firmness (b). Tc= optimum tempe­ rature, Ts= low temperature; Rn = nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Fig. 8 ­ The interaction effect of temperature and cucumber grafting on photosynthesis (a), transpiration (b), stomata conductance (c), mesophyll conductance (d) Tc= opti­ mum temperature; Ts= low temperature; Rn = nongraf­ ted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Mohammadnia et al. ‐ ‘Momordica charantia’ a new rootstock for grafted cucumbers 107 ments (Fig. 10 a). Phenol content increased in all plants at Ts and reached the highest point in Rmo×Ts (Fig. 10 b). Proline did not significantly change among treatments (Fig. 10 c). Increasing antioxidant content with using root­ stock was reported by Rouphael et al. (2018); this may help grafted plant to have a better function under different stress. The photosynthesis rate and SPAD value showed that the SPAD value did not change in Rmo. Thus, photosynthesis did not change. However, the same result was not seen in Rma. Therefore, it seems that another mechanism interfered with the stability of photosynthesis in Rma in this condition rather than changes of chlorophyll content (Fig. 6 a and 7a). Some researchers demonstrated that the photo­ synthesis rate was influenced by leaf area, stomatal conductance, and chlorophyll content. In our study, low­temperature stress caused a reduction in SPAD value and resulted in photosynthesis reduction. On the other hand, the photosynthesis in grafted cucum­ bers having high SPAD value and stomata conduc­ tance improved under low­temperature stresses. Davis et al. (2008) reported that grafting increased photosynthesis under salt stress conditions too. Grafted cucumbers showed higher photosynthesis rates, stomatal conductance, and intercellular CO2 under salt stresses compared with non­grafted plants (Yang et al., 2006). Massai et al. (2004) and Moya et al. (2002) reported that grafting improved photosyn­ thesis under salt stress conditions, too. Rootstock­ induced changes in stomatal development, as reduced transpiration relates to lower stomatal den­ sity in grafted plants (Kumar et al., 2017). Zhou et al. (2009) reported that increasing the photosynthesis rate by grafting plants onto a differ­ ent rootstock might be due to a decrease in the ROS concentration in the leaves. Beside, grafting increased water and nutrient uptake of the plant and could enhance its photosynthesis (Martinez­Ballesta Fig. 9 ­ The interaction effect of temperature and cucumber grafting on El (a) and RWC (b) Tc= optimum temperature, Ts= low temperature, Rn = nongrafted (Cucumis sativus var. DavosII); Rs= (self grafted); Rma= Cucurbita maxima, Rmo= Momordica charantia. Fig. 10 ­ The interaction effect of temperature and cucumber grafting on antioxidant (a) and phenol (b) and proline (c) Tc= optimum temperature; Ts= low temperature; Rn = nongrafted (Cucumis sativus var. DavosII); Rs= self grafted; Rma= Cucurbita maxima; Rmo= Momordica charantia. Adv. Hort. Sci., 2021 35(2): 99­110 108 et al., 2010). Salehi et al. (2010) reported that melon grafting had high CO2 assimilation due to increasing stomata conductance and Ci and resulted in an increase in photosynthesis. The photosynthesis rate and the fresh shoot weight of Rmo and Rma did not significantly change at both temperatures. Thus, low temperatures could not affect the photosynthesis rate in Rmo and Rma. On the other hand, decreasing the fresh weight was seen at Ts in Rn due to a decrease of photosynthesis rate in these rootstocks (Fig. 7). Overall, low stomata and mesophyll conductance of un­grafted cucumbers which have adverse effects on water relation, mineral nutrient uptake and trans­ port (Kumar et al., 2017), carbohydrate and hormone relationship, photosynthesis and respiration rates result in a decline in yields (Kozlowiski, 1984; Barrick and Noble, 1993; Bacanamwo and Purcell, 1999). These results were in line with our results at low­ temperature stresses. Still, when cucumbers were grafted onto Karella they were kept under proper conditions for more efficient photosynthesis, and consequently, a better yield was produced. The result of the study showed that salinity stress­ es reduced the K concentration in melons and cucumbers and grafted plants had higher nutrient concentrations than non­grafted plants. Many root­ stocks are capable of increasing the uptake and translocation of nutrients (Kumar et al., 2017). In agreement with our result, Colla et al. (2013) report­ ed that cucumber grafting had no influence on K and P concentrations in the leaves, root, and fruit under salt stresses. Our result showed that the highest K concentration was in Rmo at both temperatures and P concentration increased by grafting at Tc. 4. Conclusions It seems that the photosynthetic traits of grafted cucumbers were not affected mainly by Rmo and Rma at stress conditions. 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