Impaginato 271 Adv. Hort. Sci., 2019 33(2): 271-281 DOI: 10.13128/ahs-23815 Nanosilver, salicylic acid and essential oils effects on water relations of gerbera ‘Rosalin’ cut flowers M.S. Motaghayer (*), M. Azizi, A. Teheranifar Department of Horticultural Science and Landscape, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran. Key words: clove, hydraulic conductivity, peppermint, thyme, vase life. Abstract: The effects of pulse and permanent treatments were studied on vase life, water content and hydraulic conductivity of gerbera cut flower cv. Rosalin. This study was conducted as a factorial experiment based on completely ran- domized design with three replications. The first factor was pulse treatments, using nanosilver (NS) 5 and 10 mg/L, salicylic acid (SA) 50 and 100 mg/L and dis- tilled water as control, and the second factor was permanent treatments apply- ing distilled water, sucrose, peppermint, thyme and clove essential oils (EO). The results showed that NS 10 mg/L + peppermint EO 100 mg/L and NS 10 mg/L + thyme EO 100 mg/L treatments had the best effect on longevity and main- taining the water content and hydraulic conductance of Rosalin cut flower, compare to other treatments. These solutions enhanced life of gerbera cut flowers to about 14 days. Flower water content was high (about 90%) except in 4% sucrose permanent treatment flowers which decreased more rapidly during the vase life. The effective hydraulic conductivity was observed in NS 10 mg/L + peppermint EO 100 mg/L (0.16 cm/min) and NS 10 mg/L + thyme EO 100 mg/L (0.21 cm/min) solutions and had nearly stable trend even in day 8 after pulsing. 1. Introduction Gerbera (Gerbera jamesonii Bolus, Asteraceae) commonly known as Transvaal Daisy, Barberton Daisy or African Daisy, is one of the ten most popular and important commercial cut flowers grown in a wide range of climatic conditions. Gerbera is a perennial, tropical, herbaceous plant with colorful and attractive flowers that are widely used as a decorative garden plant or cut flowers. Cut gerbera flowers consist of a terminal composite floral head (inflorescence), called the capitulum, and a stem, which called scape and has no leaves (Dole and Wilkins, 2006). Gerbera has the fourth place in the international cut flower market. The flowers are hardy and resist against transport conditions. However, the most important problem of the gerbera cut flowers is short vase life. The end of vase life of cut gerbera flowers is often due to bending of the scape, which precedes wilting of the ray florets (Nair et al., 2003; Van Son, 2007; Ansari et al., 2011; Perik et al., 2012; Kilic and Cetin, 2014; Aghajani and Jafarpour, 2016). However, postharvest life of cut flowers could be affect- (*) Corresponding author: mahroo.motaghayer@gmail.com Citation: MOTAGHAYER M.S., AZIZI M., TEHERANIFAR A., 2019 - Nanosilver, salicylic acid and essential oils effects on water relations of gerbera ‘Rosalin’ cut flowers. - Adv. Hort. Sci., 33(2): 271-281 Copyright: © 2019 Motaghayer M.S., Azizi M., Teheranifar 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 19 August 2018 Accepted for publication 6 March 2019 AHS Advances in Horticultural Science http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(2): 271-281 272 ed by the application of various chemicals as preserv- atives (Nair et al., 2003; Prashanth et al., 2010). Insufficient water uptake is one of the main rea- sons for water deficit and wilting during the vase life (Knee, 2000; Van Ieperen et al., 2002). Stem end blockage is a main factor in the imbalance between water uptake and water loss from cut flowers (He et al . , 2009). Researches showed that bacteria (microbes) (Van Meeteren, 1978; He et al., 2006) or bacteria and decay products (Liu et al., 2009) cause the blockage of cut gerbera flower. Bacteria in vase water can block the vessels in the surface of cut stems (Ferrante et al., 2007). Bacterial inhibitors such as silver nanoparticles or salicylic acid could extend vase life of cut flowers (Loubaud and Van Doorn, 2004; Solgi et al., 2009; Vahdati Mashhadian et al., 2012). Physiological substances such as lignin, mucilage or gum (Van Doorn and Cruz, 2000; Loubaud and Van Doorn, 2004, Wang et al., 2014) and cavitation (Van Meeteren et al., 2006) decrease the vase life of some cut flowers. The air emboli or cavitation would be reduced if cut flowers were put into water after cutting immediately (Van Ieperen et al., 2002). Cut flowers are sensitive to microbial cont- amination at the stem end and this determines a reduction of their vase life (Van Meeteren, 1978, Van Doorn and De Witte 1994, Balestra et al., 2005). Nano technology is based on engineered particle of 1-100 nm (diameter). Nanosilver (NS) is included in this technology and can have more chemical and bio- logical activities in order to reduce size. In recent years, NS is being used as a new antiseptic for many industrial processes like medical industry, water purification and vegetable disinfection (Rai et al., 2009). In addition, NS treatment has been proposed for improving the postharvest life of cut flowers (Liu et al., 2009; Solgi et al., 2009; Ansari et al., 2011; Danaee et al., 2013). The role of salicylic acid (SA), as an internal growth regulator and a natural phenolic compound, has been completely proved in multiple physiological processes like ethylene biosynthesis, stomatal con- ductance, respiration, senescence and the activation of defense systems against different pathogens. By activating antioxidant enzymes, SA delays the process of senescence in flowers. In addition, SA inhibits ethylene synthesis and action (Raskin, 1992; Hayat et al., 2010; An and Mou, 2011; Jamshidi et al., 2012). Exogenous supply of carbohydrate can play an important role in lengthening the vase life and postharvest conditions of cut flowers. The gerbera cut flowers have short postharvest life. Sucrose effect on enhancing the vase life of cut flowers is associated with water balance. The application of sucrose treatment and sugars accumulated in the flowers increase the sugar and osmotic concentra- tion, improve water absorption and flower turgidity (Reddy and Singh, 1996; Prashant et al., 2010; Bhanusree et al., 2015). Researches also showed that the combined use of NS 5 mg/L with 4% sucrose and 2.5 mg/L gibberellic acid increase postharvest life of gerbera (Ansari et al., 2011). Many chemicals have been used in cut flowers vase solutions for inhibiting microorganisms’ growth and extending the vase life by improving water uptake. These chemicals include silver nitrate, 8- hydroxyquinoline sulfate and 8-hydroxyquinoline cit- rate, which are expensive and harmful for the envi- ronment and human health (Nowak et al., 1990; Ichimura et al., 1999; Nair et al., 2003; Motaghayer and Esna-Ashari, 2009; Solgi et al., 2009; Ansari et al., 2011). It is crucial to use natural, safe and inexpen- sive compounds for the large-scale application of preservatives improving cut flower vase life (Kilic and Cetin, 2014). Essential oils (EO) are organic, natural, safe and eco-friendly substances that have strong anti-inflammatory, antibacterial, antifungal, antioxi- dant and anticarcinogenic effects. These properties are attributed to the high levels of phenolic com- pounds (Solgi et al., 2009; Bayat et al., 2011; Raut and Karuppayil, 2014). The application of different medicinal plants EOs on increasing the vase life of cut flowers have been studied by many researches. The effect of pepper- mint (Mentha pipperita L.) EO has increased fresh- ness and quality of flower color and prevented the discoloration in alstroemeria (Babarabie et al., 2016), flower’s quality and delay of leaf and flower senes- cence of tuberose cv. Pearl (Hoseini and Korehpaz, 2015) and vase life of ‘Utopia’ rose cut flowers (Saghazadeh et al., 2014). Thyme (Thymus vulgaris L.) EO (Solgi et al., 2009) and water extract of thyme (Amini et al., 2014) was added to the preserving solu- tion for extending the vase life of gerbera ‘Dune’ cut flower and essence containing Thymus vulgaris and Cuminum cyminum increased solution uptake and quality of gerbera ‘Sorbet’ cut flowers (Dareini et al., 2014). Clove (Eugenia caryophyllata Thunb.) EO increased lisianthus cut flower vase life (Kazemi et al., 2014) and clove EO and water extract increased gerbera ‘Ecco’ vase life (Ziyaei Movahed et al., 2010). There are two different ways for treating cut flow- ers; pulse and permanent treatment. Pulsing is a Motaghayer et al. - Nanosilver and essential oils effects on gerbera ‘Rosalin’ cut flowers 273 short-term treatment that can be done by producers and it helps postharvest vase life and flowering after storage period. Permanent treatment mostly is a long-term treatment, which can be done by con- sumers for enhancing cut flower vase life (Abdel- Kader and Rogers 1986; Nowak et al., 1990; Arora and Singh, 2002). Sucrose can maintain the cell’s turgor pressure and provide energy for cellular respiration, also is an important nutrient for microorganisms. Therefore, it should not be used without anti-microbial agents in preservatives (Nowak et al., 1990). The effect of NS (Liu et al., 2009) and SA (Jamshidi et al., 2012) treat- ments alone on extending cut flowers vase life was assessed in different researches. Since the effects of different concentrations of various preservative solu- tions on the postharvest life of cut flowers are alter- ing depend on plant species, the applied chemicals and interaction of their compounds in vase solution and the method of treatment, the determination of the effective preservatives as well as the method of application is very important. Therefore the aim of this study was to screen the effects of NS and SA as pulse treatment and sucrose and thyme, clove and peppermint EOs as permanent treatment on vase life and hydraulic conductivity of gerbera ‘Rosalin’ cut flowers. 2. Materials and Methods Plant growth conditions and treatments Gerbera (G. jamesonii cv. ‘Rosalin’) flowers were grown in standard hydroponic greenhouse conditions in Ferdowsi University of Mashhad, Iran. The flowers were harvested during morning by pulling out the stems from the plants when 2-3 rows of stamens of the bisexual disc florets were mature. Stems were pulled, not cut and the base of stem was removed before hydration (Dole and Wilkins, 2006). The stems were taken immediately to the laboratory and recut under water to 35 cm length. The cut flowers were immediately immersed individually into 500 ml vase solutions. In order to simulate the domestic use, the vase solutions were not changed and the stems were not recut during the experiment. The end of gerbera cut flower vase life was considered as the time in which more than one third of the outer petals of inflorescence start to be brown or wilted or curled or stem bending (≥90°) or breaking was occurred (Dole and Wilkins, 2006). This study was conducted as a factorial experiment based on completely random- ized design with three replications and four stems in each replicate. The first factor was pulse treatments: distilled water (D), salicylic acid (SA) 50 and 100 mg/L (Merck Company), Nanosilver (NS) 5 and 10 mg/L (Nanocid Company, Iran). The second factor was per- manent treatments: distilled water, sucrose 4% (Merck Company), peppermint EO 100 mg/L, thyme EO 100 mg/L, clove EO 300 mg/L (Zardband Company, Iran) (Table 1). Pulse treatments were applied for 24 h. Treated stems were then stood into vases containing permanent treatments. Vase solu- tions were freshly prepared at the beginning of the experiment and not renewed during of the study. The EOs constituents were determined by Zardband Company (Iran) using GC-MS analysis Table 1 - Pulse and permanent treatments used in the experi- ment Pulse treatment/Permanent treatment Distilled water/Distilled water Distilled water/Sucrose Distilled water/Peppermint EO Distilled water/Thyme EO Distilled water/Clove EO SA 50 mg.L-1/Distilled water SA 50 mg.L-1/Sucrose SA 50 mg.L-1/Peppermint EO SA 50 mg.L-1/Thyme EO SA 50 mg.L-1/ Clove EO SA 100 mg.L-1/Distilled water SA 100 mg.L-1/Sucrose SA 100 mg.L-1/Peppermint EO SA 100 mg.L-1/Thyme EO SA 100 mg.L-1/ Clove EO NS 5 mg.L-1/Distilled water NS 5 mg.L-1/Sucrose NS 5 mg.L-1/Peppermint EO NS 5 mg.L-1/Thyme EO NS 5 mg.L-1/ Clove EO NS 10 mg.L-1/Distilled water NS 10 mg.L-1/Sucrose NS 10 mg.L-1/Peppermint EO NS 10 mg.L-1/Thyme EO NS 10 mg.L-1/ Clove EO Adv. Hort. Sci., 2019 33(2): 271-281 274 (Table 2). GC-MS analysis revealed that the major constituents of the EOs were: thymol (53.5%) in thyme EO; 1-menthol (41.22%) and menthone (24.01%) in peppermint EO and eugenol (62.4%) in clove EO. Measuring hydraulic conductivity Hydraulic conductivity was measured by a slight modification in the method of Melcher et al. (2012) (Fig. 1). A piece of 15 cm of flower stem end was cut with a sharp blade under distilled water. The upper part of the stem (part 1 in Fig. 1) was inserted into a silicon tube (part 2 in Fig. 1) (internal diameter 4 mm) filled with degas distilled water and the basal part of the stem (part 3 in Fig. 1) was kept in the degas dis- tilled water. Using a three-way glass valve (part 4 in Fig. 1), the silicon tube was connected from one side to the degassed distilled water tank (part 5 in Fig. 1) and from the other side was attached into a U- shaped pipe (part 6 in Fig. 1) below the stems end. The whole set (stem, three-way glass valve, degas distilled water tank and U-shaped pipe) was fixed (Van Ieperen et al., 2002). The stem vase was placed on a digital scale (part 7 in Fig. 1) connected to the computer and the stem and degas distilled water weight changes were recorded at time regular inter- vals (30 minutes). Fifty cm head pressure of water (h; which made 5 kPa pressure) was applied, so that water had passed through the segments. The flow rate was then determined by measuring the volume of the passed water. Three stem segments were used for each treatment (Ichimura et al., 2005). Measurements of stem hydraulic conductivity involves measuring the flux for a given driving force (Q⁄ΔP; where ΔP is the pressure drop across the seg- ment), normalized by the length of the stem segment and referenced either to the cross-sectional area of the stem (Melcher et al., 2012). In equation 1, K (cm/min) is hydraulic conductivity, Q (cm3/min) is the recorded flux (gravimetric or volumetric flow rate), L (cm) is the length of the measured segment, A (cm2) is the cross-sectional area of the stem segment and h (cm) is head pressure of water height. The data were collected at days 2, 4, 6 and 8 after pulse treatment. K = qL/Ah) (1) To observe the microscopic effects of chemicals and EOs on stem closure and hydraulic conductivity during the vase life, 2 cm of treated stem was used for histological study. The cut stem segments (3-5 cm in length) were stored in a solution of FAA (formalin (40%): glacial acetic acid (50%): ethyl alcohol (70%): Fig. 1 - Hydraulic conductivity system scheme; the upper part of the stem (1); silicon tube (2); the basal part of the stem (3); the three-way glass valve (4); the degas distilled water tank (5); the U-shaped pipe (6); the digital scale (7); h: water height. Table 2 - Major chemical constituents of the EOs Peppermint EO Thyme EO Clove EO GCMS Analysis (%) GCMS Analysis (%) GCMS Analysis (%) Limonene 2.25 Terpinene gamma 7.20 Alpha Copaene 0.04 Cineole 4.59 Para-Cymene 27.4 Beta Caryophyllene 3.79 Menthone 24.01 Thymol 53.50 Alpha Humulene 0.45 Isomenthone 3.83 Oxyde De Caryophyllene 0.29 1-Methyl acetate 4.38 Eugenol 81.83 Neomenthol 2.84 Isoeugenol 0.13 1-Menthol 41.22 Acetate De Eugenyle 12.50 Pulegone 1.56 Methyl Eugenol 0.01 Menthofuran 2.98 Density (20°C) 0.9036 0.923 10.636 Refractive Index(20°C) 14.605 1.502 15.335 Optical Rotation (°) -23.68 -1.0 -0.35 Batch 13/47/23 45109 68382 Motaghayer et al. - Nanosilver and essential oils effects on gerbera ‘Rosalin’ cut flowers 275 and permanent solutions had remarkable effect on flower longevity. The best treatments were NS 10 mg/L + thyme EO 100 mg/L (14.25 days) and NS 10 mg/L + peppermint EO 100 mg/L (14 days) which had significant difference with other treatments (Table 4). It has been observed that SA 50 mg/L + 4% sucrose was less effective than control on flower postharvest life and sucrose had negative effect on gerbera flow- ers life (Table 4). Flower water content Flower water contents for the pulse treatments with distilled water and 50 mg/L SA were a little high- er than other treatments (Table 5). In 4% sucrose per- [13:5:200]) to preserve the tissue before sectioning. Stem transverse sections, at 16-µm thickness, were made using a manual rotary microtome (Leitz 1512, Germany) after fixing in FAA and permanent mounts were prepared in paraffin wax. Cross sections were stained with Safranin O/Fast Green Stain method and embedded on microscope slides. Digital images were made at 10X magnification with a digital camera (Olympus DP71, Japan) attached to a light micro- scope (Olympus BH2, Japan) and computer. Flower water content Flower water content (WC) was measured as men- tioned in equation 2. Flower fresh weights (FW) were assessed at the beginning of the experiment and flower dry weights (DW) were recorded after drying to constant weight in an oven for at least 48 h at 85°C. Water content was calculated for three repli- cates (He et al., 2006; Lu et al., 2012). WC = (FW-DW)/DW *100 (2) The experiment was conducted in the laboratory at 20-22°C, 40-50% RH, and 15 µmol/m2s light inten- sity (cool white florescent tubes) under a daily light period of 12 hours. The obtained data were analyzed using MSTAT-C program and mean comparison was done using LSD range test. 3. Results Flower vase life Results of this study showed that in single effect of applied treatments, all NS pulse treatments markedly (P<0.01) extended vase life of gerbera ‘Rosalin’ cut flowers. The 10 mg/L NS pulse treatment gave the longest vase life (12.20 days) as compared to the other treatments (Table 3). SA 100 mg/L (9.08 days) pulse treatment significantly increased flower vase life compared to the control (8.13 days). However, there was no significant difference between NS 5 mg/L and SA 100 mg/L. The single effect of applied treatments indicated that peppermint and thyme EOs (100 mg/L) applica- tion in preservative solutions as permanent treat- ment (Fig. 2) could extend the vase life of gerbera cut flowers to 9.98 and 10.35 days respectively (Table 3). However, there was no significant difference among these two treatments and control in extending vase life. Flowers placed in sucrose 4% and clove EO 300 mg/L had the least (8.83 days) vase life (Table 3). The results showed that the interaction of pulse Table 3 - The simple effect of pulse and permanent treatments on ‘Rosalin’ gerbera cut flower vase life (day) The means showing similar letters in each column have no signi- ficant difference according to the LSD range test (P<0.01). Treatments Vase life (Day) Pulse treatment Distilled water 8.13 d SA 50 mg.L-1 8.30 cd SA 100 mg.L-1 9.08 bc NS 5 mg.L-1 10.02 b NS 10 mg.L-1 12.20 a Permanent treatment Distilled water 10.02 a Sucrose 8.55 b Peppermint EO 9.98 a Thyme EO 10.35 a Clove EO 8.83 b Fig. 2 - Gerbera ‘Rosalin’ cut flowers treatment by NS (10 mg/L) and peppermint EO (100 mg/L). During pulse treatment, vase solution was covered by dark plastic coverage to prevent undesirable light reaction in NS. 276 Adv. Hort. Sci., 2019 33(2): 271-281 manent treatment flowers, water content declined more rapidly during the vase life period and was sig- nificantly different from the others. Generally, the interaction of pulse and permanent treatment showed that 4% sucrose had negative effect especial- ly after 10 mg/L NS application as pulse treatment (Table 4). Hydraulic conductivity The hydraulic conductance of the stem end seg- ments did not change over the first 2 days after puls- ing and had very low rates. Thereafter it changed over time and increased slightly on day 4. The rate of stem flower hydraulic conductivity sharply increased at day 6 and 8 after pulse treatment application. Hydraulic conductivity of stems treated with 5 and 10 mg/L NS pulse markedly showed lower rate during the experiment compared to other treatments (Table 6). In addition, permanent treatments had significant effect on hydraulic conductance throughout assess- ment. Result showed that peppermint EO 100 mg/L had lower look rate in hydraulic conductance than other solutions (Table 7). However, hydraulic conductance of the stem seg- ment was nearly the same at the initial day of the experiment. The interaction of pulse and permanent treatment determined that NS 10 mg/L + peppermint EO 100 mg/L and NS 10 mg/L + thyme EO 100 mg/L had the lowest rate even in day 8 after pulsing (Table 8). In the NS 10 mg/L + peppermint EO 100 mg/L flowers, the hydraulic conductance of the stem seg- ments slightly increased thereafter. Hydraulic con- Table 6 - The effect of pulse treatments on trends of hydraulic conductivity of gerbera cut flower stem on day 2, 4, 6 and 8 of the expe- riment Table 4 - The effect of different treatments’ interactions on ‘Rosalin’ gerbera cut flower vase life (day) and water content (%) Table 5 - The effect of pulse and permanent treatments on ‘Rosalin’ gerbera cut flower water content (%) The means showing similar letters have no significant difference according to the LSD range test (P<0.01). Treatment Vase life (Day) Water content (%) Distilled water/Distilled water 9.58 efg 90.95 ab Distilled water/Sucrose 8.08 i 86.15 c Distilled water/Peppermint EO 7.83 ij 91.29 a Distilled water/Thyme EO 8 i 91.39 a Distilled water/Clove EO 7.17 jk 91.21 a SA 50 mg.L-1/Distilled water 9.08 fgh 91.31 a SA 50 mg.L-1/Sucrose 7 k 85.02 c SA 50 mg.L-1/Peppermint EO 8.08 i 91.09 a SA 50 mg.L-1/Thyme EO 8.92 gh 90.97 a SA 50 mg.L-1/ Clove EO 8.42 hi 91.15 a SA 100 mg.L-1/Distilled water 9.83 def 90.54 ab SA 100 mg.L-1/Sucrose 8.58 hi 83.44 d SA 100 mg.L-1/Peppermint EO 9.17 fgh 90.45 ab SA 100 mg.L-1/Thyme EO 9.83 def 90.73 ab SA 100 mg.L-1/ Clove EO 8 i 89.46 b NS 5 mg.L-1/Distilled water 9.67 efg 90.55 ab NS 5 mg.L-1/Sucrose 8.58 hi 82.68 d NS 5 mg.L-1/Peppermint EO 10.83 c 90.81 ab NS 5 mg.L-1/Thyme EO 10.75 c 90.56 ab NS 5 mg.L-1/ Clove EO 10.25 cde 90.42 ab NS 10 mg.L-1/Distilled water 11.92 b 90.52 ab NS 10 mg.L-1/Sucrose 10.50 cd 81.1 e NS 10 mg.L-1/Peppermint EO 14 a 91.36 a NS 10 mg.L-1/Thyme EO 14.25 a 91.22 a NS 10 mg.L-1/ Clove EO 10.33 cde 90.99 a The means showing similar letters in each column have no signi- ficant difference according to the LSD range test (P<0.01). Treatment Water content (%) Pulse Treatment Distilled water 90.20 a SA 50 mg.L-1 89.91 a SA 100 mg.L-1 88.93 b NS 5 mg.L-1 89.00 b NS 10 mg.L-1 89.04 b Permanent treatment Distilled water 90.77 a Sucrose 83.68 b Peppermint EO 91.00 a Thyme EO 90.97 a Clove EO 90.65 a The means showing similar letters in each column have no significant difference according to the LSD range test (P<0.01). Pulse treatment K (cm/min) Day 2 Day 4 Day 6 Day 8 Distilled water 0.57 d 5.42 a 7.58 b 8.95 c SA 50 mg.L-1 1.04 b 5.35 a 12.96 a 9.17 c SA 100 mg.L-1 1.72 a 6.44 a 10.42 a 15.47 a NS 5 mg.L-1 0.84 c 2.50 b 4.45 c 13.08 b NS 10 mg.L-1 0.57 d 0.91 c 2.05 d 4.97 d Motaghayer et al. - Nanosilver and essential oils effects on gerbera ‘Rosalin’ cut flowers 277 ductivity of other treated flowers increased sharply after day 6 during the rest of the vase life (Table 8). The survey of slope trend of each treatment dur- ing the experiment’s period, showed that the lowest slope was observed in NS 10 mg/L + peppermint EO 100 mg/L and NS 10 mg/L + thyme EO 100 mg/L respectively. While in other treatments, slope trend was enhanced so that the most slope trend was con- sidered in 50 mg/L SA + 4% sucrose. In addition, histological study showed that in the NS 10 mg/L + peppermint and thyme 100 mg/L EOs treated flowers stem remained healthy for longer period while the other stems became hollow after a few days (Fig. 3 and 4). Table 7 - The effect of permanent treatments on trends of hydraulic conductivity of gerbera cut flower stem on day 2, 4, 6 and 8 of the experiment The means showing similar letters in each column have no significant difference according to the LSD range test (P<0.01). Permanent treatment K (cm/min) Day 2 Day 4 Day 6 Day 8 Distilled water 0.81 c 5.14 a 8.66 b 14.62 a Sucrose 1.46 a 4.93 ab 13.50 a 12.76 a Peppermint EO 0.84 c 2.74 c 3.61 d 6.25 c Thyme EO 0.69 d 3.65 bc 6.00 c 8.77 b Clove EO 0.94 b 3.52 bc 5.07 cd 9.25 b Table 8 - The trends of hydraulic conductivity of gerbera cut flower stem in all treatment on day 2, 4, 6 and 8 of the experiment The means showing similar letters in each column (Day) have no significant difference according to the LSD range test (P < 0.01). Treatment K (cm/min) Slope trend Day 2 Day 4 Day 6 Day 8 Distilled water/Distilled water 0.42 ghi 5.60 bcd 9.41 cde 17.04 cd 53.664 Distilled water/Sucrose 0.64 fg 5.96 abcd 8.88 def 14.04 ef 43.117 Distilled water/Peppermint EO 0.21 hi 4.89 cde 4.62 ghi 0.00 j 5.16 Distilled water/Thyme EO 0.62 fg 4.52 de 6.41 fg 0.00 j 52.384 Distilled water/Clove EO 0.96 de 6.14 abcd 9.23 cde 13.69 f 41.263 SA 50 mg.L-1/Distilled water 0.76 ef 7.91 a 11.28 cd 14.95 def 45.931 SA 50 mg.L-1/Sucrose 1.14 d 7.58 ab 43.58 a 0.00 j 16.337 SA 50 mg.L-1/Peppermint EO 1.51 bc 1.94 fgh 2.74 ij 0.00 j 12.728 SA 50 mg.L-1/Thyme EO 0.57 fg 4.39 de 9.54 cd 14.21 ef 46.081 SA 50 mg.L-1/ Clove EO 1.19 cd 5.67 bcd 12.34 c 16.71 cde 53.215 SA 100 mg.L-1/Distilled water 1.53 b 7.30 ab 11.33 cd 14.69 def 43.499 SA 100 mg.L-1/Sucrose 1.67 ab 6.87 abc 21.41 b 21.51 a 74.065 SA 100 mg.L-1/Peppermint EO 1.83 ab 5.17 cd 6.60 efg 20.43 ab 57.224 SA 100 mg.L-1/Thyme EO 1.67 ab 7.95 a 10.99 cd 20.72 ab 60.198 SA 100 mg.L-1/ Clove EO 1.94 a 5.10 cd 5.31 gh 0.00 j 5.769 NS 5 mg.L-1/Distilled water 1.04 de 4.22 de 8.54 def 18.74 bc 5.741 NS 5 mg.L-1/Sucrose 1.91 a 3.18 ef 5.28 gh 22.59 a 64.126 NS 5 mg.L-1/Peppermint EO 0.52 fgh 2.27 fg 4.69 ghi 10.40 g 32.056 NS 5 mg.L-1/Thyme EO 0.45 ghi 1.97 fgh 4.26 ghi 7.48 hi 23.375 NS 5 mg.L-1/ Clove EO 0.40 ghi 1.07 ghi 0.67 k 6.19 i 16.963 NS 10 mg.L-1/Distilled water 0.40 ghi 1.51 ghi 3.93 hi 7.68 ghi 24.278 NS 10 mg.L-1/Sucrose 1.91 a 1.86 fgh 4.79 ghi 5.65 i 14.158 NS 10 mg.L-1/Peppermint EO 0.16 i 0.12 i 0.28 k 0.41 j 0.0904 NS 10 mg.L-1/Thyme EO 0.21 i 0.57 hi 0.94 jk 1.42 j 0.4012 NS 10 mg.L-1/ Clove EO 0.23 hi 0.67 hi 1.02 jk 9.65 gh 28.601 Fig. 3 - The gerbera cut flower healthy stem. Adv. Hort. Sci., 2019 33(2): 271-281 278 3. Discussion and Conclusions Flower vase life NS particles enter into cell, tissue and organs, so they can replace with silver salts (such as silver nitrate or silver thiosulfate) in preservative solutions. NS inhibits the respiration and electron transfer sys- tem and material transfer in microbial cell membrane (Paull and Lyons, 2008). Various researches indicated that flowers treated with NS solution, had more vase life. Silver ions, because of small size, have more con- tact with outer space and influence more on their environment. NS, in comparison with silver ions, showed antimicrobial property at inferior concentra- tion (Solgi et al., 2009; Ansari et al., 2011) Different studies determined antimicrobial effects of main components of thyme (Nikolić et al., 2014 a), peppermint (Kazem Alvandi et al., 2011; Nikolić et al., 2014 b) and clove EO (Boukaew et al., 2017). In addi- tion, Amini et al. (2014) reported that thyme EO in pulsing with distilled water treatment showed the best results for extending cut gerbera flower vase life and preventing more weight loss. Hydrophobicity is an important characteristic of thyme and peppermint EOs. This enables them to separate the lipid compo- nents of the bacterial cell membrane and mitochon- dria, binding to membrane proteins and releasing lipopolysaccharides, which results in disturbing cell wall structures (Solgi et al., 2009). Researches indicated also that SA pulse treat- ment, followed by NS as permanent solution (Danaee et al., 2013), SA utilization as permanent treatment (Jamshidi et al., 2012) significantly promoted the vase life of gerbera cut flowers. However, in this study, SA had no special effect on flower longevity. Although Ziyaei Movahed et al. (2010) reported that clove EO increased gerbera vase life. In this study, it had the least effect on postharvest life of gerbera cut flowers. Despite of sucrose important role in extend- ing the vase life of cut flowers, Ansari et al. (2011) reported a negative effect on ‘Rosalin’ gerbera cut flowers. The main reason could be severe bacterial growth in vase solution. Flower water content Cut flowers and foliage can have limited commer- cial value because they dehydrate during vase life because of water uptake decrease. Water deficit could develop even when cut flowers are placed in water (Nazari Deljou, et al., 2012). Gerbera cut flow- ers stem break of is mainly caused by water shortage in the flowers due to the increased difficulty of water flow from the water source to petals. It is also sup- posed to be a competition for available water between flower heads and stems. The increase in flow resistance leads to stem break as a result of microbial activity in the vase water (Balestra et al., 2005). It could be concluded from the results of this study that in all treatments with antimicrobial agents such as NS, SA and thyme, peppermint and clove EOs flower water contents were high and had no harmful decrease. Hydraulic conductivity Many experiments were performed to find out the cause of stem bend in gerbera cultivars. Research showed that removal of the floral head prevented stem bending, indicating that bending is physically due to the gravitational pull on the floral head. Stem bending in cut gerbera can be due to lack of mechan- ical support. Bending might relate to lack of wall thickening, particularly in the xylem. At least two other factors might contribute to mechanical stem strength. The first factor is gerbera stems elongation during vase life. Elongation zones usually have weak- ly developed xylem and sclerenchyma. Since the stems are usually placed in water under an angle, stem elongation will increase the gravitational pull of the floral head result in earlier stem bending. The second factor is the presence of a cavity in the center of the gerbera stem. Observations showed the cavity at the time of harvest, in several cultivars (Perik et al., 2012). Other factors affecting stem bending could be adverse water relations such as lack of turgor. After a few days of vase life, there are many bacteria in the vase solution. Stem bending can be due to xylem blockage by bacteria, which results in low water uptake. As transpiration is not inhibited as Fig. 4 - The gerbera cut flower hollow stem. Motaghayer et al. - Nanosilver and essential oils effects on gerbera ‘Rosalin’ cut flowers 279 much as water uptake, net water loss occurs fol- lowed by the loss of turgor and stem bending (Van Meeteren, 1978). Generally, in cut flowers fresh weight decreased before stem bent occurred, and this is accompanied by a decline in absorption of water by the flowers. Stem break could be prevented by pretreatment of the stems with NS by adding to the vase water. Van Meeteren (1978) suggested that there are two differ- ent pathways for water uptake: a direct one through the xylem vessels at the cut surface and an indirect one through the cavity in the stem. Only the direct water uptake is strongly inhibited by growth of bac- teria in the vase water. Stem bend occurs when the direct water uptake is inhibited by bacterial activity. Van Meeteren (1978) suggested that the minimum concentration of silver nitrate could avoid stem bending and inhibit bacterial growth in the water. There is an association between a high population density of bacteria in the water and scape bending (Van Doorn and De Witte, 1994). The results of this experiment also showed that bacteria would block the main water pathway (xylem vessels) over the time and the stem would become hollow (Fig. 3 and 4). So that active water uptake is effectively prevent- ed. Bacterial activity could be significantly inhibited by adding NS (as pulse treatment), peppermint (1- menthol and menthone) and thyme (thymol) EOs (as permanent treatment) to vase solution. Based on the results of this study, new antimicro- bial agents such as NS, thyme and peppermint EOs had a positive effect on flower vase life and water content. It might be due to this fact that these are very effective antimicrobial agents, which inhibited the microbial growth and prevented bacterial plug- ging in conducting tissues. 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