Impaginato 185 Adv. Hort. Sci., 2018 32(2): 185-191 DOI: 10.13128/ahs-21864 Effects of nano-silver pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut gerbera flowers S.-S. Shafiee-Masouleh Department of Genetics and Breeding, Ornamental Plants Research C e n t e r ( O P R C ) , H o r t i c u l t u r a l S c i e n c e s R e s e a r c h I n s t i t u t e ( H S R I ) , Agricultural Research Education and Extension (AREEO), Mahalatt, Iran. Key words: antioxidant, flavonoid, GA4+7, Gerbera jamesonii. Abbreviations: nano-silver= NS; deionized water= DI; calcium sulfate= CS; gib- berellin4+7 = GA; anthocyanin leakage= AL; total soluble solids= TSS. Abstract: The aim of this study was to evaluate interactions between NS cou- pled with CS and GA on flavonoid, cell membrane behavior and extending the vase life of cut gerbera. Pulse treatments of flowers were conducted in NS at concentrations of 0 (DI), 3 or 9 mg/l for 24 h. Then, flowers were treated with preservative solutions containing calcium sulfate (0, 10 or 20 mM) and GA4+7 (0 or 20 mg/l), plus 1.5% sucrose in all preservative solutions. Pulse treatments with 3 or 9 mg NS/l and holding in solution containing 20 mM CS compared to the control treatment (holding in the solution of sucrose following pulse treat- ment in DI) significantly extended vase life by 8 days. According to the antioxi- dant role of flavonoids, and lower amounts of flavonoid in the flowers that pre- treated with NS, therefore, it may be said that NS prevented from microbial attack. 1. Introduction Gerberas (Gerbera jamesonii) are well-known flowers for the variety of their colors, and are popular in the world flower trade (Liu et al., 2009 a; Solgi et al., 2009). However, often the growers and florists suffer further loss from short vase life of gerberas. For example, the mean of vase life in some cultivars of Gerbera (‘Bayadere’ and ‘Sunway’) are reported only between 6 to 10 days when water tap is used (Shabanian et al., 2018). Gerberas are ethylene insensitive, but bacterial plugging of the xylem is a main cause of early and rapid senescence in their cut flowers (Liu et al., 2009 a). The decrease of water uptake and consequently the increase of the ratio between transpiration and water uptake (i.e., high value of (*) Corresponding author: shafiee.masouleh@areeo.ac.ir Citation: SHAFIEE-MASOULEH S.-S., 2018 - Effects of nano- silver pulsing, calcium sulfate and gibberellin on an antioxidant molecule and vase life of cut ger- bera flowers. - Adv. Hort. Sci., 32(2): 185-191 Copyright: © 2018 Shafiee-Masouleh S.-S. 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 10 October 2017 Accepted for publication 12 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(2): 185-191 186 water balance) will be caused as a result of xylem obstruction, and will end the cut flower vase life. Nano-silver (NS) pulse and continuous treatments for cut flowers are newly used and are known as novel agents of anti-microbial (Liu et al., 2009 a; Solgi et al., 2009; Lü et al., 2010). As a novel antiseptic, NS is used in the medical industry, silver embedded fab- rics, water purification and vegetable disinfection. Due to their high surface area to volume ratio, among other unique chemical and physical proper- ties, NS formulations provide full contact with microorganisms and are highly effective as germi- cides. NS particles can connect the cell membranes and penetrate into bacteria. Then, NS can disrupt the respiration and cell division and cause the cell death. NS releases silver ions (Ag+) within bacterial cells, sil- ver ions have bactericidal activity (Liu et al., 2009 a; Solgi et al., 2009; Nair et al., 2010; Sharon et al., 2010; Lü et al., 2010; Liu et al., 2012). Naghsh (2010) described the inhibited meiosis in Aspergillus niger due to NS activity. Alavi and Dehpour (2010) report- ed that the nano-silver solution is effective on green- house cucumber downy mildew disease. Liu et al. (2009 a) and Lü et al. (2010) observed that NS pulse treatments extended vase life of the cut gerbera and the rose flowers. Also, Solgi et al. (2009) reported that NS continuous treatments inhibited the growth of bacteria in the solution and xylem vessels and increased vase life of cut gerbera flowers. Calcium increase postharvest longevity of fresh cut flowers (Gerasopoulos and Chebli, 1999; De C a p d e v i l l e e t a l . , 2 0 0 5 ; S o s a N a n , 2 0 0 7 ) . T h i s increased postharvest longevity may be due to a delay of physiological events related to senescence, such as a decrease in water uptake, increased water transpiration loss, decreased fresh weight, stem bending (Sosa Nan, 2007). Since the level of soluble carbohydrates will be maintained by the treatment of gibberellin (GA) (Ranwala and Miller, 2000; Whitman et al., 2001; Hatamzadeh et al., 2010), therefore, GA can have a positive effect on the water balance. Furthermore, sucrose in the preservative solutions maintains water balance, in addition to act as a food source (Solgi et al., 2009). The objective of this research was to evaluate the interactions of calcium sulfate and gibberellin contin- uous treatments by NS pulse treatments on flavonoid as an antioxidant component, and vase life of cut gerberas. Flavonoids have antioxidant effects and can be effective on the vase life. Antioxidant molecules can be efficient systems to protect cells against pathogen and water deficit-induced oxidative stress, and this prevents the senescence and cell death (Shabanian et al., 2018). 2. Materials and Methods Plant material Cut gerbera (Gerbera jamesonii cv. Pink Elegance) flowers that were grown in standard hydroponic greenhouse conditions were purchased from a flower and plant growing company (Pakdasht, Tehran, Iran). Flowers were harvested by pulling the stems off in the plants when 2-3 rows of stamens of the bisexual disc florets were mature (Gerasopoulos and Chebli, 1999; Solgi et al., 2009) in the morning. Stem bottom of harvested flowers was put in the flower capsule containing deionized water (DI). Flowers were packed and transported within 8 h to the laboratory. In the laboratory, stems were re-cut to a length of 45 cm into the DI to remove air emboli (Liu et al., 2009 a; Solgi et al., 2009). Flowers were re-cut 2-3 times, when it was necessary. The flowers were placed in a controlled environ- ment room at 20±2°C with 60±10% R.H. and 12 µmol/m.s. light intensity (cool white fluorescent lamps; 12 h/day). Experimental design and treatments Solutions of pulse treatment were prepared in two concentrations of NS (3 or 9 mg/ l), and DI was used as a control treatment. Flowers were treated f o r 2 4 h w i t h t h e t w o c o n c e n t r a t i o n s o f N S (Nanonasb-Pars Company, Iran) or DI. Each pulse treatment contained 18 flowers. Following pulse treatment, the flowers were individually kept into 1000 ml glass vases containing 500 ml of fresh solu- tions (as continuous treatment) that were prepared at second day of the experiment and were not renewed. In the continuous treatments, three con- centrations (0, 10 or 20 mM) of calcium sulfate, CS, (CaSO42H2O, Merck Company), and two concentra- tions (0 or 20 mg/ l) of GA4+7 (Serva Company, USA) were used. In the all continuous treatments, sucrose 1.5% was used. There were three replications and three samples per treatment in a completely randomized design as factorial experiment. Each sample was one flower per bottle. Data were analyzed using three-way analysis of variance (PROC GLM), and the means Shafiee-Masuleh - Antioxidant molecule and vase life of cut gerbera flowers 187 were compared by Tukey’s Test (HSD) at p≤0.05 using SAS (9.1) statistical software. Correlation coef- ficients between vase life and cell conditions and flavonoid were conducted by SPSS (version 11.5). Regression analysis (path analysis) was taken to determine the major factors that affect vase life (dependent variable). The independent explanatory variables were anthocyanin leakage, tissue pH, TSS and flavonoid. The software used for path analysis was SPSS/PC+ “Stepwise” (version 11.5). Vase life Vase life was recorded from harvest time by the time the flowers showed symptoms of petal wilting or curling, stem bending (≥90°) or breaking, there- fore, the flowers were visited daily. Total flavonoid assay In termination of the vase life, to extract total flavonoid, 20 ml of acidic methanol (1% HCl) was added to 0.2 g fresh weight of petals, and the mix- ture was stirred for 48 h in the dark. The extract was used to measure total flavonoid content immediately (Chang et al., 2002). Total flavonoid content was measured by aluminum chloride colorimetric assay (Chang et al., 2002; Kumar et al., 2008). An amount of 200 µl of plant extract was added to 600 µl of methanol, 40 µl AlCl3 (10%), 40 µl of potassium acetate (1 M) and was made to 2000 µl by distilled water. The solution was vigorously mixed and after keeping at room temperature in the dark for 30 min, the absorbance was measured against reagent blank at 510 nm with a spectrophotometer (T80+ UV/VIS Spectrometer, PG Instruments Ltd). The calibration curve of standard solutions of catechin (5-40 µg/ ml of 1% HCl in methanol) was drew (y= 0.0003x + 0.1654, R2= 0.9989). Total flavonoid content of flower was expressed as mg catechin equivalent per 100 g of fresh weight. Anthocyanin leakage To evaluate the effects of treatments on the cell membrane structure, anthocyanin leakage was mea- sured to observe the stability of plasma membrane. At tenth day of vase life, 0.5 g of petals was sliced to pieces of 1×1 cm, these pieces were washed in DI water two times and within a period of 2 h. Then 10 ml of DI water was added to samples. After 12 h in 25°C, the absorbance was recorded with a spec- trophotometer (T80+ UV/VIS Spectrometer, PG Instruments Ltd) at 525 nm (Poovaiah, 1979). Tissue pH measurement Tissue pH was measured according to the method of Hill (1999) to consider the treatment influences on the conditions of cell reactions. In termination of vase life, 2 g of petals was crushed in liquid N2, and then was placed at -80°C for 48 h. The frozen tissues were removed from -80°C, thawed at 20°C, then were frozen in liquid N2 again and placed at -80°C for a further 36 h. After thawing at 20°C again, 25 ml dis- tilled water was added to tissues in the test tube, then were frozen at -20°C for 24 h. The pH of filtered fluid was recorded after thawing with a pH meter. Total soluble solid Total soluble solid (TSS) of petal juice was mea- sured with a refractometer (CETI, Belgium) as °Brix (Roein et al., 2009). 3. Results and Discussion Interactions between NS and CS significantly extended the vase life (Fig. 1). NS pulse treatments and then holding in preservative solutions (10 or 20 mM CS) extended the vase life compared to DI puls- ing and preservative solution containing sucrose (control treatment) (Fig. 1). However, no significant (p<0.05) difference was found among various con- centrations of NS and CS to extend the vase life. Also, interactions between CS and GA4+7 had significant effect on the extension of the vase life (p<0.05) (Fig. 2). The longest vase life was found in the pulse treat- ment with 3 mg NS/l and preservative solution con- taining 20 mM CS. It was 7.5 days (i.e., 62.5%) added to vase life compared to the control (Fig. 1). Gerbera is insensitive to ethylene (Liu et al., 2009 a); there- Fig. 1 - Interactions between nano-silver (0, 3 or 9 mg/l) and cal- cium sulfate concentrations (= 0 mM; = 10 mM; ▲= 20 mM) on the vase life (days) in cut gerbera flowers. The means with same letters have no significant differen- ces (MSANOVA = 6.27; Type 1 Error, HSD0.05), N=3. Adv. Hort. Sci., 2018 32(2): 185-191 188 fore, NS effects for extending the vase life of gerbera are not related to anti-ethylene effects of NS. Therefore, it must be explained that the basic role of NS is to prevent from bacterial plugging of the xylem (Liu et al., 2009 a, b; Solgi et al., 2009; Chaloupka et al., 2010; Lü et al., 2010); then increasing water uptake and calcium. According to Gerasopoulos and Chebli (1999), post-calcium uptake prevents appear- ing the symptoms of the end of vase life in gerbera [wilting, petal curling, stem bending (in this cultivar was not observed) and breaking (in this cultivar was observed partially)]. The longest vase life was obtained in preservative solutions containing 10 mM CS without GA4+7 [approximately 19 days, i.e., 32.3% more than treatment with 20 mg/l GA, without CS, which had significant (p<0.05) differences with solu- tions containing 20 mg L-1 GA4+7 without CS] (Fig. 2). Gibberellin can enhance hydrolization of starch to glucose, and during enzymatic process sucrose will be produced. More production of sucrose causes strength of cell walls. Having more sugar in tissues preserves them of early disruption and increases their longevity (Halevy and Mayak, 1981). Also, Whitman et al. (2001) determined that GA4+7 sprayed t o L i l i u m l o n g i f l o r u m h a d a p o s i t i v e e f f e c t t o decrease foliar chlorosis and increased vase life con- trary to our results; moreover, it decreased the effect of CS. There were significant interactions (p<0.05) between GA, CS and NS on total flavonoid at the end of vase life (Table 1). The highest flavonoid content Table 1 - The effect of nano-silver pulsing and continuous treatments by calcium sulfate and gibberellin on the biochemistry factors of cut gerbera flowers Fig. 2 - Interactions between calcium sulfate (0, 10 or 20 mM) and GA4+7 concentrations (= 0 mg GA4+7/l; = 20 mg GA4+7/l) on the vase life (days) in cut gerbera flowers. The means with same letters have no significant differen- ces (MSANOVA = 1.41; Type 1 Error, HSD0.05), N=3. Treatments Flavonoid (mg equivalent catechin per 100 g F.W.) Anthocyanin leakage (Absorbance in 525 nm) Tissue pH TSS (° Brix)Pulse treatment Continuous treatment NS (mg/ l) CSz (mM) GA (mg/ l) 0 0 0 3933.3 bcd 0.165 a 4.98 abcd 11.2 a 20 2350.0 d 0.160 abc 5.15 a 10.8 ab 10 0 4516.7 abcd 0.161 abc 4.94 bcd 11.1 a 20 3877.8 bcd 0.161 abc 4.98 abcd 11.3 a 20 0 8183.3 ab 0.160 abc 4.91 bcd 11.2 a 20 8933.3 a 0.157 bc 4.94 bcd 9.9 ab 3 0 0 5711.1 abcd 0.159 abc 4.98 abcd 9.4 ab 20 2988.9 cd 0.163 ab 5.01 abc 10.0 ab 10 0 5655.6 abcd 0.155 c 4.88 bcd 9.0 ab 20 3433.3 bcd 0.157 bc 4.83 cd 8.9 ab 20 0 7544.4 abc 0.156 bc 4.86 cd 8.8 ab 20 5377.8 abcd 0.155 c 4.83 cd 7.9 b 9 0 0 2933.3 cd 0.156 bc 4.96 abcd 9.8 ab 20 3100.0 cd 0.157 bc 5.05 ab 10.4 ab 10 0 2766.7 cd 0.155 c 4.85 cd 8.3 ab 20 5322.2 abcd 0.156 bc 4.89 bcd 8.5 ab 20 0 6488.9 abcd 0.156 bc 4.82 d 9.5 ab 20 3711.1 bcd 0.157 bc 4.86 cd 8.9 ab ANOVA (Mean of Square) NS 815234.8 NS 0.0001 ** 0.04 ** 18.97 ** CS 50446502.1 ** 0.0001 ** 0.12 ** 4.47 * GA 12438400.2 * 0.00000002 NS 0.02 * 0.74 NS NS x CS 6853137.9 y 0.00002 * 0.0008 y 1.56 y NS x GA 696546.46 y 0.00002 * 0.01 y 0.47 y CS x GA 2485082.3 y 0.000003 y 0.01 y 1.52 y NS x CS x GA 5213477.41 y 0.00001 y 0.0009 y 0.1 y The means with different letters are significant (HSD0.05). N= 3.** = significant at p≤0.01; * = significant at p≤0.05; NS = not significant. y= Type I Error. Shafiee-Masuleh - Antioxidant molecule and vase life of cut gerbera flowers 189 was measured in the petals of flowers that were treat- ed with DI and kept in the solution containing 20 mM CS and 20 mg/l GA. Whereas treated flowers with DI and kept in the solution containing 20 mg/l GA showed the lowest total flavonoid. The antioxidant role of flavonoids was revealed for the flowers that were not pulsed by NS, because, the microbial attack might be a signal to synthesize the flavonoids (Khatiwora et al., 2010). When calcium (Meyer et al., 1973) and gib- berellin (Ranwala and Miller, 2000; Hatamzadeh et al., 2010) were made available, they activated the reduc- ing of nitrate to produce phenylalanin, and to form simple carbohydrates, respectively. Therefore, the pathway of flavonoid synthesis was completed and total flavonoid was increased. Phenylalanie transforms into 4-coumaroyl-CoA in the phenylpropanoid path- way, and finally enters the flavonoid synthesis path- way (Falcone Ferreyra et al., 2012). The most significant interactions (p<0.05) were observed between GA, CS and NS on the anthocyanin leakage. In the flowers which were not treated with NS and/or CS, anthocyanin leakage was highest (Table 1). The stability of cell membrane will have been decreased by factors as senescence, microbial (attacking by micro-organism) or no microbial (deficit of calcium) diseases. The measurement of antho- cyanin leakage at half of vase life could be a gauge to evaluate the stability of cell membrane. The accumu- lation of calcium in middle lamella of cell wall increases the stability of cell membrane and decreas- es anthocyanin leakage (Nikbakht et al., 2008). NS prevents microbial attack and decreases senescence and keeps stability (Liu et al., 2009 a; Solgi et al., 2009; Lü et al., 2010). The most tissue pH was recorded at pre-treat- ment with DI and keeping in solution containing 20 mg GA/l, and the least tissue pH was at pulsing with 9 mg NS/l and keeping in calcium sulfate solution (20 M m ) . G e n e r a l l y , s i g n i f i c a n t d i f f e r e n c e s w e r e observed between treatments (p<0.05) (Table 1). Schmitzer et al. (2010) explained that increasing the cell sap pH causes the developing flowers from the bud to senescence stage. The most TSS was measured in the flowers of con- trol treatment, and the least TSS was recorded in the flowers which were pulsed with 3 mg NS/l and kept in solution containing 20 mM CS and 20 mg GA/l. Significant differences (p<0.05) were observed between two mentioned treatments. However, no one has the significant differences with other treat- ments. Gebremedhin et al. (2013) interpreted that TSS will be increased by more water uptaking to pro- vide the required substrate for respiration. The analysis of correlation coefficients (Table 2) shows the negative significant correlation (p≤0.01) between vase life and anthocyanin leakage and TSS. Furthermore, there is positive significant correlation between anthocyanin leakage and TSS (Table 2). For the last parameter, the coefficient of multiple deter- minations (R2) was 0.479 in linear model for the vase life (Table 3). This coefficient gives the proportion of the total variation in the dependent variable (vase Parameters Vase life AL Tissue pH TSS Flavonoid Vase life 1 AL -0.656 ** 1 Tissue pH -0.353 0.412 1 TSS -0.692 ** 0.757 ** 0.375 1 Flavonoid -0.338 0.253 -0.373 0.206 1 Vase life Linear model Variable B SE B Standard β t Significance Constant 32.265 4.04 7.987 0 TSS -1.586 0.414 -0.692 -3.832 0.001 Multiple R 0.692 R2 0.479 Adjusted R2 0.446 Standard error 1.82 ANOVA Sum of squares df Mean squares F Significance Regression 48.834 1 48.834 14.688 0.001 Residual 53.197 16 3.325 Total 102.031 17 ** Significant in p<0.01 (two-tailed correlations), N=18. Table 3 - Vase life of gerbera flowers regressed (stepwise regression) against anthocyanin leakage, tissue pH, TSS and flavonoid Table 2 - Correlation coefficients between vase life (days), anthocyanin leakage (AL), tissue pH, total soluble solids (TSS) and flavonoid (mg equivalent catechin per 100 g F.W.), N=3 Adv. Hort. Sci., 2018 32(2): 185-191 190 life) explained by the predictors included in the model. Thus, from among four independent vari- ables, total soluble solids explained 47.9% of the observed total variation in the vase life, and other independent variable (anthocyanin leakage, tissue pH and flavonoid) had a lesser role in the vase life. Furthermore, the test statistic in linear model s h o w e d t h a t c o e f f i c i e n t o f T S S n e g a t i v e l y a n d significantly (p≤0.01) influenced vase life (Table 3, Fig 3). Therefore, the factors that cause the increased TSS and anthocyanin leakage lead to decrease vase life. This is also confirmed by other researchers (Gebremedhin et al., 2013). According to the results, we recommend pulse treatment with 3 mg NS/l and then, continuous treat- ment with 20 mM CS for increasing the vase life of cut gerbera by 8 days. 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