Impaginato 61 Adv. Hort. Sci., 2018 32(1): 61-69 DOI: 10.13128/ahs-21860 Extending vase life of cut rose (Rosa hybrida L.) cv. Bacara by essential oils M.R. Salehi Salmi (*), M. Falehi Hoseini, M. Heidari, M.H. Daneshvar Department of Horticulture Science, Khuzestan Ramin Agriculture and Natural Resources University, Khuzestan, Iran. Key words: antioxidant, bacteria, bent-neck, hold solutions. Abstract: Recent studies showed that some essential oils functions as antibac- terial compounds. In this study results showed treatment with essential oils promoted vase life of cut roses via decreasing bacteria number inside the stem. We investigated components in the hydrodistilled essential oils of Bunium per- sicum Bioss, Mentha spicata L., Thymus vulgaris L. and Satureja hortensis L., as hold solutions, and their effects on relative fresh weight, water uptake, vase life, electrolyte leakage, anthocyanin content, soluble sugar content and num- ber of bacteria at stem end of cut flowers of rose. GC-MS analysis of the extracted essential oil of B. persicum, M. spicata, Th. vulgaris and S. hortensis L. led to the identification of 14, 20, 13 and 14 major compounds, respectively. In cut rose, the treatment containing the essential oils extended flower opening period longer than the control. The 200 µl l-1 essential oil of M. spicata treat- ment almost doubled the vase life of cut roses. Hence these essential oils might be powerful, environmentally friendly substitutes for the chemical compounds currently added to vase waters to control bacterial content. 1. Introduction Rose (Rosa hybrida L.) cut flowers play an important role in the florist trade (Cairns et al., 2000). The cut flowers can have limited marketable value because they dehydrate during vase life as a result of decreased water uptake and the vase life is often very short. The cut flowers wilt and the floral axis becomes bent just under the flower head (bent neck). The appearance of such symptoms is considered to be caused by various fac- tors such as bacteria, physiological responses of stems to cutting and air emboli. The development of this occlusion is correlated with the growth of bacteria at the cut surface and inside the stem (Van Doorn et al., 1989, 1990) and accumulation of bacteria in vase water shortens the vase life of cut rose flowers (De Witte and Van Doorn, 1988). The addition of chemi- cals, including some trace elements such as silver nitrate, aluminum sul- phate and 8-hydroxyquinoline sulphate into holding solutions has been tried with various accomplishments in efforts to prolong the vase life of cut roses (Van Doorn et al., 1990). The use of essential oils has recently become a common practice (*) Corresponding author: mrsalehisalmi@gmail.com Citation: SALEHI SALMI M.R., FALEHI HOSEINI M., HEIDARI M., DANESHVAR M.H., 2018 - Extending vase life of cut rose (Rosa hybrida L.) cv. Bacara by essen- tial oils. - Adv. Hort. Sci., 32(1): 61-69 Copyright: © 2018 Salehi Salmi M.R., Falehi Hoseini M., Heidari M., Daneshvar M.H. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distribuited 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 6 October 2017 Accepted for publication 11 January 2018 AHS Advances in Horticultural Science Adv. Hort. Sci., 2018 32(1): 61-69 62 because of strong antimicrobial activities against sev- eral pathogens. Some essential oils of plants have been reported to be effective in extending the vase life of gerbera cut flowers (Solgi et al., 2009). These natural organic substances have high levels of pheno- lic compounds such as carvacrol (Sharififar et al., 2007). Essential oils indicate a wide range of protec- tive properties against disease states, oxidative stress, and microbial infection (Botelho et al., 2007; Yahyazadeh et al., 2008). And have reportedly been used for controlling plant diseases, particularly on fruits (Yahyazadeh et al., 2008; Ramezanian et al., 2016). However, there is little available information (Solgi et al., 2009) on the use of essential oils for the control of microbial contaminations and extending the vase life of cut flowers. Appropriate use of chemi- cal fungicides during the postharvest could help mini- mize fungal infections; however, the use of chemical compounds is being discouraged for economic aims and because of growing concern about environment safety issues (Wagacha and Muthomi, 2008). In this study, an attempt has been made to inves- tigate the protective role of carvacrol during vase life in ‘Bacara’ cut-roses. This information will help to elucidate the best concentrations of these essential oils to be used in holding solutions to obtain remark- able beneficial results. 2. Materials and Methods Extraction, analysis and antioxidant activity of essen- tial oils The essential oils from fresh leaves and flowers of Bunium persicum Bioss, Mentha spicata L., Thymus vulgaris L. and Satureja hortensis L. were extracted following hydro-distillation method. The leaves and flowers (100 g) were hydrodistilled in a clevenger apparatus with 400 ml of water. The extraction was carried out for 3.5 h. The extracted oils were collect- ed from the graduated receiver. For the deletion of water traces from the oil, the extracts were dried over anhydrous Na2SO4 and were stored in sealed ampoule bottle in a refrigerator at 4°C until for analy- sis. Gas chromatographic analysis was performed with Agilent 6890A with helium as a carrier gas with a linear velocity of 30 cm/s on HP-5 column (30 m × 0.25 mm i.d, 0.25 µm film thickness). The oven was programmed to rise 50°C (3 min) isotherm, and then to 265°C at a rate of 5°C/min. Injector and detector temperatures were 300°C and 265°C, respectively. The GC mass analysis was carried out on Agilent 5975 equipped with a HP-5 column with the same charac- teristics as the one used in GC. Unknown essential oil was recognized by comparing its GC retention time to that of known compounds and by comparison of its mass spectra either with identified compounds or available spectra in the literature. The antioxidant activity of essential oils was determined based on the DPPH (1,1-Diphenyl-2- picrylhydrazyl) free radical scavenging capacity by the method described by Brand-Williams et al. (1995). The amount of 180 µl DPPH reagent and 20 µl sam- ples were mixed and shocked. Decline of absorbance of tested fusions was monitored for 30 min at 517 nm. For each essential oil, the tests were repeated three times. Plant material and general processing Rose flowers (Rosa hybrida L.) ‘Bacara’ grown under standard commercial conditions in a glass cov- ered greenhouse were used as plant material. Flowers were harvested at commercial stage to 0.45 m in length. Flower stems were placed in tap water after harvest and transported by non-refrigerated car to the laboratory within 2 h. The flowers were placed randomly in 3 L glass vases containing 1 L of essential oils solutions of 100, 200 and 400 µl.l-1 concentra- tions for 24 h. Distilled water was used as control. Sucrose (4%, w/v) was added to all solutions and the concentrations of essential oils were prepared in Tween 20 (0.1%). Then, flowers were individually placed in glass bottles of 25 cm height. Each bottle contained approximately 200 ml of distilled water. Bottles were standing in a controlled room under the following conditions: 12 h photoperiod (06.00-18.00) at photosynthetically activated radiation of 12 µmol m-2 s-1 provided by fluorescent lamps, 25±1°C and relative humidity of 60-70%. In total, nine floin three replications were placed in Bottles. Measurements Relative fresh weight was calculated by the for- mula: RFW (% initial fresh weight)= (FWt/FWt=0)×100 where RFW was Relative fresh weight, FWt was the fresh weight of flower (g) at t (in day) = 0, 1, 2, 3, etc., and FWt=0 was the fresh weight of the same flower (g) at t (in day) = 0 (He et al., 2006). Total vase solution uptake. Weight of vases con- taining vase solution without the cut stems were measured during the experiment period and flower opening was measured daily until the flowers Salehi Salmi et al. - Effect of essential oils on vase life of cut rose 63 attained their largest size in the vase and began loss their size. Cut flower longevity was documented as days vase life from the stage flowers were placed into glass bottles till the end of vase life defined as the time that flowers showed indications of petal wilting or curling, fall of one or more petals (Van der Sman et al., 1996). Electrolyte leakage was measured using an elec- trical conductivity meter. Leaves were excised and washed with deionized water. After drying with tis- sue paper, 1 g fresh weight of leaves were cut into small pieces (about 1 cm2) and then immersed in 20 ml deionized water and incubated at 25°C. After 24 h, electrical conductivity (EC1) and after 48 h (EC2) of the bathing solution were recorded for the samples (Lutts et al., 1996). Determination of anthocyanins. Samples of petal (100 mg) were powdered in a pre-chilled pestle and extracted into methanol: HCl (99:1). Samples were incubated overnight at 4°C in darkness. The content of anthocyanins determined spectrophotometrically at 535 nm using an extinction coefficient (ε) of 29,600. The final concentration of anthocyanin was calculated based on weight of sample used and total volume of the extract. For determination of soluble sugars, fresh petals (0.30 g) were put into test tubes with 10 mL distilled water and sealed. The tubes were incubated in a water bath at 90°C for 30 min, then the tubes were removed and the volume set at 25 ml. The amount of 0.5 ml of supernatant was mixed with 1.5 ml distilled water, 5 ml concentrated sulfuric acid and 0.5 ml anthrone. The mixed solution was read at 620 nm (Frohlich and Kutscherah, 1995). Ascorbate peroxidase activity. The fresh leaves (1 g) were mixed in 100 mM potassium phosphate buffer (pH 7.8) containing 1% (w:v) PVP (polyvinyl- pyrrolidone), 0.1 mM EDTA (ethylenediamine- tetraacetic acid), and 0.5% (v:v) Triton X-100 at 4°C, except that in the case of APX activity leaves were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 1 mM EDTA and 5 mM ascorbate. The homogenate was filtered through 4 layers of cheesecloth and centrifuged at 18000 g for 20 min at 4°C. Peroxidase activity was assayed as per the method of Kar and Mishra (1976). Determination of bacterial numbers. The lower- most 2 cm (about 0.5 g in weight) of the stems were cut. The samples were washed 3 times with sterile deionized water. They were ground and then dilution was made with a 0.9% normal salt solution. Liquid extract (0.1 ml) was spread on Plate Count Agar (PCA) plates. Before counting of bacteria, PCA plates were incubated at 38°C for 2 days (Balestra et al., 2005). Statistical analysis Completely randomized experiment designs were used. Statistical significance between mean values was assessed using analysis of variance (ANOVA) and a conventional Tukey’s test at p≤0.05 using SAS (9.1) statistical software. For data of maximum flower opening, standard deviation (SD) was calculated and data are expressed in mean±SD of three replicates. 3. Results and Discussion GC-MS analysis of the extracted essential oil of B. persicum, M. spicata, Th. vulgaris and S. hortensis L. led to the identification of 14, 20, 13 and 14 major compounds, respectively (Table 1). The results revealed that 5 major components of essential oil from B. persicum contained γ-Terpinene, β- Caryophyllene, Cuminyl acetate, p-Cymene and β- Pinene. Investigation is limited about the chemical composition of the B. persicum essential oil. It has been reported that γ-Terpinene and β-Caryophyllene are main components (Shahsavari et al., 2008). Five major components of Mentha spicata L. essential oil were Carvone, 1,8-Cineole, Borneol, Limonene and Pulegone. Mentha essential oil was characterized by the dominant presence of carvone (56.9%) in agree- ment with results from other authors, who reported the presence of 76.65% in samples collected from India (Chauhan et al., 2009) and 64.4% in samples collected in Montenegro (Scherer et al., 2013). Carvacrol (67.3%), Thymol (12.7%), α-Pinene (4.25%), γ-Terpinene (3.53%) and Eucalyptol (3.32%) were most abundant in essential oil of Th. vulgaris. Similar results have been obtained by Ben El Hadj Ali et al., (2014) with Th. numidicus. The principle compounds identified in essential oil of Satureja hortensis L. were Carvacrol (66.4%), p-Cymene (18.1%), Linalool (4.5%), γ-Terpinene (3.95%) and Borneol (1.8%). A compari- son of our study with a previous report (Ghasemi- Pirbalouti et al., 2014) suggests that the few differ- ences in the volatile composition of the plant materi- al could be attributed to a series of factors such as the genotype, plant developmental stage, environ- mental conditions and the methods of extraction. All assayed essential oils were able to reduce Adv. Hort. Sci., 2018 32(1): 61-69 64 DPPH, reaching 50% of reduction with IC50 values ranging 54.19±0.87 mg/mL for M. spicata, to 258.16 ± 1.53 mg/mL for S. hortensis (Table 2). The variance analysis performed on the DPPH scavenging activity of the essential oils showed significant differences among species (p≤0.05) and that the essential oil of M. spicata was the most potent of all the oils. Therefore, some compounds such as compound Carvone, 1,8-Cineole and Borneol were responsible for the DPPH scavenging effects of M. spicata (Mahdavikia and Saharkhiz, 2015). The essential oil of Th. vulgaris possessed slightly higher DPPH scaveng- ing effects than that of B. persicum and S. hortensis. This meant that the common compound Thymol and high value of Carvacrol played a leading role (Ghasemi-Pirbalouti and Dadfar, 2013). Bacterial numbers Essential oils treatment, particularly 200, 400 µl l-1 M. spicata and 200, 400 µl.l-1 Th. vulgaris, had negative effect on bacterial numbers. Preventing bacterial division in vase water can reduce the occur- rence of stem bending (Solgi et al., 2009), suggesting that bacteria are a main cause. A positive correlation between the number of bacteria and water uptake of the flower stem have been reported (Van Doorn et al., 1989). Vascular occlusions in cut rose flower usually develop when the number of bacteria in vase water reach 7-11 Log10 CFU/ml (Van Doorn et al., 1990). Some antimicrobial compounds have been used in tests with cut flowers. These compounds included silver nitrate (Nair et al., 2003), nano-Silver (Nazemi Rafi and Ramezanian, 2013), 8-hydrox- Table 1 - Major natural volatile components in the hydrodistilled essential oils of B. persicum, M. spicata, Th. vulgaris and S. hortensis No. Component Retention indices Percentage (%) B. persicum M. spicata Th. vulgaris S. hortensis 1 α-Thujene 924 0.05 0.03 0.18 0.3 2 α-Pinene 932 1.75 1.09 4.25 0.5 3 Camphene 946 - 0.56 - - 4 Sabinene 971 0.8 0.74 - - 5 β-Pinene 977 3.68 1.59 0.44 0.1 6 Myrcene 989 0.71 0.41 0.86 0.6 7 3-Octanol 922 - 0.21 - 0.1 8 Eucalyptol 1018 - - 3.32 - 9 p-Cymene 1021 6.91 0.83 - 18.1 10 Limonene 1036 3.28 5.69 0.44 - 11 1,8-Cineole 1039 0.1 13.53 0.2 - 12 γ-Terpinene 1059 27.61 0.58 3.53 3.95 13 Linalool 1086 1.1 - 0.71 4.5 14 Menthone 1154 - 0.26 - - 15 Borneol 1159 - 8.15 - 1.8 16 α-Terpineol 1169 0.98 3.25 0.54 1.02 17 Pulegone 1231 0.2 3.28 - - 18 Carvone 1237 - 56.94 - - 19 Thymol 1287 3.4 0.67 12.79 0.3 20 Carvacrol 1293 0.35 0.47 67.36 66.46 21 β-Caryophyllene 1415 25.1 0.93 - 0.85 22 Cuminyl acetate 1434 16.68 - - - 23 Caryophyllene oxide 1580 0.47 0.16 1.23 1.2 Mean separation by Tukey Test, P≤0.05. Table 2 - Antioxidant activity of essential oil extracts of B. persicum, M. spicata, Th. vulgaris and S. hortensis DPPH B. persicum M. spicata Th. vulgaris S. hortensis IC 50 (mg/mL) 183.5 ± 2.15 b 54.19 ± 0.87 d 153.52 ± 2.66 c 258.16 ± 1.53 a Salehi Salmi et al. - Effect of essential oils on vase life of cut rose 65 yquinoline citrate (Solgi et al., 2009), chlorine bleach, dichloroisocyanuric acid (Jones and Hill, 1993) and essential oils such as carvacrol (Nazemi Rafi and Ramezanian, 2013) and thymol (Solgi et al., 2009). The inhibitory effect of some essential oils on bacter- ial growth has been attributed to alcohols, esters, aldehydes, phenols, carvacrol, thymol, and eugenol (Bassole and Juliani, 2012). Burt (2004) reported that essential oil components including thymol and car- vacrol partition in the lipids of the cell membrane, rendering the membrane permeable and leading to leakage of cell contents, thus exerting their antibac- terial action. Relative fresh weight and total water uptake As expected, relative fresh weight continuously increased early days of experiment (Table 3). Nevertheless, such increases were significantly pro- longed by using essential oils in hold solution. The relative fresh weight in 200 µl.l-1 M. spicata essential oils treated cut flowers was initially about 105.2% (day 1), and increased to 130.1% over 6 days of hold- ing, but in untreated (control) cut flowers, relative fresh weight was initially about 111.3% (day 1), and decreased over 4 days of holding to 105%. Similarly, as with different concentrations of M. spicata essen- tial oil, a positive effect of other essential oils on rela- tive fresh weight was confirmed by their impact on duration of increase and percentage of fresh weight. Total water uptake of cut flowers under various treatments from day 1 to 8 in control as well as in all treated samples (Table 3). The maximum uptake of vase solution was found 1.14-fold increase in the treated flower with 200 µl.l-1 Th. vulgaris as com- pared to untreated (control) cut flowers. All treated flowers always took up water more than the control. Water status is a factor directly correlated with the vase life of rose cut flower. This factor is deter- mined by the balance between water uptake and loss due to transpiration (Fanourakis et al., 2016). Water uptake depends on variation of cultivars (Fanourakis et al., 2016), the viscosity of the vase solution, vascu- lar conductivity and the osmosis gradient between vase solution and stem solute (Alaey et al., 2011). The improved water uptake in this study may be due to possible antibacterial activity of essential oils by inhibiting vascular blockage and/or increasing osmo- sis gradient. The water lose was affected by treat- ments and essential oils increased water retaining capacity compared to control treatment (without essential oil). It may be due to positive regulatory role of essential oils on stomatal closure which regu- lates the rates of transpiration and decreases the water loss of leaves and petals (Solgi et al., 2009). Flower opening Generally, the opening of cut flowers kept in essential oils vase solutions was more than those flowers kept in distilled water (control). Also maxi- mum opening of control flowers occurred earlier. Time and diameters of maximum opening flower were different between treatments. However, maxi- Table 3 - Vase life, total solution uptake, electrolyte leakage, petal anthocyanin content, soluble sugars of petal and bacterial numbers of cut rose (Rosa hybrida L.) cv. Bacara treated with B. persicum, M. spicata, Th. vulgaris and S. hortensis Mean separation for each parameter within rows by Tukey Test, P≤0.05. Characteristic Vase life (days) Total solution uptake (g) Electrolyte leakage (%) Petal anthocyanins content (mg g-1 FW) Soluble sugars of petal (mg g-1 FW) Ascorbate peroxidase activity (Units min-1 mg-1 protein) Bacterial numbers (Log10 CFU/ml) Relative FW in 10th day (%) Control 6.70 e 98.350 f 60.6 a 5.24 d 1.41 f 22.6 h 8.2 a 79.41 g B. persicum 100 µl.l-1 8.70 bc 103.25 e 51.4 b 6.52 bc 1.70 f 25.1 g 8.1 a 94.72 cd B. persicum 200 µl.l-1 9.80 b 106.44 b 48.5 bc 7.37 ab 2.29 e 24.6 gh 7.8 ab 100.80 ab B. persicum 400 µl.l-1 7.90 cd 100.23 e 45.7 c 6.07 c 2.14 e 22.9 h 7.3 d 84.22 f M. spicata 100 µl.l-1 11.2 a 110.31 a 19.8 i 7.95 a 4.52 b 54.9 a 6.1 f 104.50 a M. spicata 200 µl.l-1 10.4 ab 108.65 ab 28.3 g 7.68 a 4.05 bc 46.7 b 5.7 g 95.00 c M. spicata 400 µl.l-1 8.70 bc 106.71 b 33.1 f 6.03 c 3.77 c 43.3 bc 5.5 gh 91.48 de Th. Vulgaris 100 µl.l-1 8.30 c 105.94 c 22.4 h 7.19 b 4.35 b 38.6 d 7.5 cd 91.71 de Th. Vulgaris 200 µl.l-1 10.6 ab 112.67 a 17.4 j 7.70 a 5.18 a 40.8 cd 5.5 gh 104.50 a Th. Vulgaris 400 µl.l-1 9.20 b 107.36 b 32.0 f 7.36 ab 3.95 c 41.0 c 5.3 h 98.54 bc S. hortensis 100 µl.l-1 7.80 d 98.670 f 41.5 d 5.83 c 2.35 e 33.7 ef 7.8 bc 83.54 fg S. hortensis 200 µl.l-1 8.60 bc 103.68 de 38.5 e 6.44 bc 3.28 d 32.1 f 7.2 d 87.83 ef S. hortensis 400 µl.l-1 9.10 bc 104.86 d 37.5 e 6.87 b 3.46 cd 35.9 e 6.8 e 97.87 bc Adv. Hort. Sci., 2018 32(1): 61-69 66 mum diameter was observed with flowers kept in 100 µl.l-1 M. spicata essential oil vase solution (Fig. 1). Mechanisms of flower opening vary for different flowers and are sensitive to various environmental conditions such as temperature, light, carbohydrate supply (Kumar et al., 2008) and water relations. During the opening, numerous proceedings take place in a well-defined sequence, representing all parts of plant development, such as division, differ- entiation and elongation of cell (Kumar et al., 2008) or gene expression (Hoeberichts et al., 2005). In an experiment, essential oils at some concentrations had a noticeable effect on promoting flower opening of cut rose flowers. However, the treatments with a relatively higher concentration of essential oils showed a less effect on flower opening. According to Mahdavikia and Saharkhiz (2015) the function of essential oils as an effective antioxidant mainly depends on its concentration. It has been demon- strated that a relatively high dose of essential oils may be associated with injury of membranes and nucleic acids and cell death (Ghasemi-Pirbalouti and Dadfar, 2013). The results suggest that essential oils at effective concentrations maintained water uptake and inhibited vascular occlusions development. Thus, essential oils may improve water relations and sup- plementation of carbon sources, leading to cell expansion and flower opening. Petal anthocyanin content Petal anthocyanin content in cut flowers was determined. Cut flowers treated with 100 µl.l-1 M. spicata essential oil in hold solution were observed to accumulate the highest rates of anthocyanin in the petals. Other treatments led to higher amounts of anthocyanins compared to untreated (control) cut flowers. Generally, all cut flowers treated with essen- tial oils showed a similar tendency to maintain antho- cyanin Petal, however, essential oils of Th. vulgaris and M. spicata were more efficient than B. persicum and S. hortensis. One of plant defense system is the presence of endogenous antioxidative compounds such as antho- cyanins (Apel and Hirt, 2004). Singlet oxygen pro- duced can be detoxified by anthocyanins and affect the vase life. Accumulation of these pigments in cell vacuoles, their hue and intensity depend on external conditions. Anthocyanin biosynthesis is an essential part of flower development (Dela et al., 2003). In the primary developmental phases of the flower, the anther produces a signal or GA, which promotes petal pigmentation (Kumar et al., 2008). So, in this study, flower opening and development could be effect on anthocyanins accumulation. Additionally, earlier studies have indicated that sugars such as sucrose are required as substrates for anthocyanin biosynthesis (Nagira and Ozeki, 2004). Here, we showed that essential oils increased petal soluble sugar content. It seems that there is an interaction between the soluble sugars concentration and antho- cyanins. Ascorbate peroxidase activity With cut roses, the highest ascorbate peroxidase activity in petals occurred in those treated with M. spicata essential oils at the concentration of 100 µl l-1. As shown in Table 3, the activity of ascorbate per- oxidase in control flowers was less than that of the flowers treated with essential oils, however, there were no significant difference between control flow- ers and flowers treated with different concentrations of B. persicum essential oils. Ascorbate peroxidase enzyme is considered to play significant roles in cellu- lar defense against stresses (Sevillano et al., 2009). During the later stages of vase life, petals contain higher levels of reactive oxygen species leading to oxidative damage (Hossain et al., 2006). Generally, treatment with essential oils increased ascorbate peroxidase activity compared the control. Higher lev- els of the enzymatic activity in treated flowers were likely to counteract the oxidative stress and to scav- enge the active oxygen species alleviate to the oxida- tive stress induced in cut roses and thus delayed flower senescence. These results are in agreement with the findings of Hasan et al., (2014) who men- tioned that cut roses were of an efficient antioxidant defense system. Electrolyte leakage The ability to increase membrane stability is a characteristic of drought tolerance strategy (Turner, 1986). With control flowers, a significant increase in Fig. 1 - Time of maximum flower opening. Values are given as mean±SD of three replicates. Salehi Salmi et al. - Effect of essential oils on vase life of cut rose 67 electrolyte leakage was detected; however, treat- ment with any essential oils level significantly inhibit- ed electrolyte leakage increase relative to the control (Table 3). The treatment with 200 µl l-1 Th. vulgaris essential oils resulted in the lowest electrolyte leak- age followed by 100 µl l-1 M. spicata essential oils. Wilting in cut flower is often accompanied by mem- brane damage resulting in the leakage of solutes (Ye et al., 2000). With studied cut flower, such as day lily (Panavas et al., 1998), the most changes in the rate of electrolyte leakage were observed during the late stage of vase life. Maintenance of membrane stability in response to essential oils application was most likely due to induced reduction of lipid peroxidation. This is supported by a lower level of electrolyte leak- age in essential oil treatments. Soluble sugars Another factor controlling vase life of cut rose flower is sugar content, as the carbon supply is cut (Halevy and Mayak, 1979). The essential oils had a strong influence on the soluble sugars concentration in the petal of cut flowers and all treatments led to significantly more soluble sugars. Soluble sugars increased approximately 3.6 fold from 1.41 mg g-1 FW for the control to 5.18 mg g-1 FW for pulsing flowers in solutions containing essential oil of Th. vul- garis (200 µl l-1) and to 4.52 mg g-1 FW using 100 µl l-1 M. spicata essential oil. With cut flowers treated with preservative solution, minimum soluble sugar contents were observed in flowers kept in solutions containing 100 µl l-1 B. persicum essential oils. It seems that essential oils can change the capacity for sugar uptake in petals and stimulate active sucrose uptake. Translocation of soluble sugars is considered as a key factor affecting the vase life (Khayat and Zieslin, 1989). The addition of chemicals including some componds such as silver nitrate, aluminum sul- phate and 8-hydroxyquinoline sulphate to holding solutions has been tried with varied success in efforts to control vascular blockage and prolong the vase life of cut roses (Van Doorn et al., 1990). However, there is no available evidence on the use of essential oils for control of microbial contaminations and holding soluble sugars petal of cut flowers such as rose. Vase life Cut flower wilting is a widely reported problem during vase life of rose, particularly when xylem ves- sels are blocked by microorganisms (Damunupola and Joyce, 2008). In this study, longevity of treated cut flowers was significantly improved compared with the untreated (control) cut flower. Also, signifi- cant differences were found in longevity conferred by the essential oils of species and concentrations. However, B. persicum, M. spicata and Th. vulgaris decreased vase life at concentrations more than 200 µl l-1, 100 µl l-1 and 200 µl l-1, respectively. The treat- ment with M. spicata 200 µl l-1 essential oil almost doubled the vase life of cut roses. Proper doses of some essential oils at proper dose could delay senes- cence in selected fruits (Ramezanian et al., 2016) and cut flowers (Solgi et al., 2009). For example, Solgi et al. (2009) concluded that the vase life of gerbera flowers was extended by the 50 or 100 mg l-1 car- vacrol from 8.3 to 16 days. In the present study, simi- lar results was obtained with the vase life of cut rose. 4. Conclusions The naturally occurring compounds in essential oils, such as thymol, carvone, carvacrol and menthol showed different levels of antibacterial activity. The major qualitative trait of cut roses is their vase life, which mainly depends on the water uptake of the stems after harvest and is hampered by the presence of bacteria. Also, at relatively high concentration of the essential oils tested, early flower opening and senes- cence were related to an effect other than bacteria. This effect was correlated with low relative fresh weight and water uptake. It cannot be excluded at pre- sent that this early senescence might be due to a toxic effect. More information regarding the action of essen- tial oils during vase life is required for better under- standing of the mechanism of petal senescence of rose. Acknowledgements This work was granted by Khuzestan Ramin University of Agriculture and Natural Resources. References ALAEY M., BABALAR M., NADERI R., KAFI M., 2011 - Effect of pre - and postharvest salicylic acid treatment on physio-chemical attributes in relation to vase-life of rose cut flowers. - Postharvest Biol. Technol., 61: 91-94. APEL K., HIRT H., 2004 - Reactive oxygen species: metabo- lism, oxidative stress and signal transduction. - Ann. Rev. Plant Biol., 55: 373-399. BALESTRA G.M., AGOSTINI R., BELLINCONTRO A., MEN- CARELLI F., VARVARO L., 2005 - Bacterial populations related to gerbera (Gerbera jamesonii L.) stem break. - Adv. Hort. Sci., 2018 32(1): 61-69 68 Phytopathol. Mediterr., 44: 291-299. BASSOLE I.H.N., JULIANI H., 2012 - Essential oils in combi- nation and their antimicrobial properties. - Molecules, 17: 3989-4006. BEN EL HADJ ALI I., BAHRI R., CHAOUACHI M., BOUSSAID M., HARZALLAH-SKHIRI F., 2014 - Phenolic content: antioxidant and allelopathic activities of various extracts of Thymus numidicus Poir.- organs. - Ind. Crops. Prod., 62: 188-195. BOTELHO M.A., NOGUEIRA N.A.P., BASTOS G.M., FONSECA S.G.C., LEMOS T.L.G., MATOS F.J.A., MONTENEGRO D., HEUKELBACH J., RAO V.S., BRITO G.A.C., 2007 - Antimicrobial activity of the essential oil from Lippia sidoides, carvacrol and thymol against oral pathogens. - Braz. J. Med. Biol. Res., 40: 349-356. BRAND-WILLIAMS W., CUVELIER M.E., BERSET C., 1995 - Use of a free radical method toevaluate antioxidant activity. - LWT-Food Sci. Technol., 28: 25-30. BURT S., 2004 - Essential oils: their antibacterial properties and potential applications in foods - A review. - Int. J. Food Microbiol., 94: 223-253. CAIRNS T., YOUNG M., ADAMS J., EDBERG B., 2000 - Modern roses XI: The world encyclopedia of roses. - Academic Press, Cambridge, MA, USA, pp. 11-12. CHAUHAN R.S., KAUL M.K., SHAHI A.K., KUMAR A., RAM G., TAWA A., 2009 - Chemical composition of essential oils in Mentha spicata L. accessions from Northwest Himalayan region, India. - Ind. Crops Prod., 29: 654- 656. DAMUNUPOLA J.W., JOYCE D.C., 2008 - When is a vase solution biocide not, or not only, antimicrobial? - Jpn. Soc. Hortic. Sci., 77: 211-228. DELA G., OR E., OVADIA R., NISSIM-LEVI A., WEISS D., OREN-SHAMIR M., 2003 - Changes in anthocyanin con- centration and composition in ‘Jaguar’ rose flowers due to transient high air temperature conditions. - Plant Sci., 164: 333-340. DE WITTE Y., VAN DOORN W.G., 1988 - Identification of bacteria in the vase water of roses, and the effect of the isolated strains on water uptake. - Sci. Hort., 35: 285-291. FANOURAKIS D., GIDAY H., LI T., KAMBOURAKIS E., LIGOXI- GAKIS E.K., PAPADIMITRIOU M., STRATARIDAKI A., BOURANIS D., HEUVELINK E., OTTOSEN C., 2016 - Antitranspirant compounds alleviate the mild-desicca- tion-induced reduction of vase life in cut roses. - Postharvest Biol. Technol., 117: 110-117. FROHLICH M., KUTSCHERAH U., 1995 - Changes in soluble sugars and proteins during. Development of rye coleop- tiles. - J. Plant Physiol., 146: 121-125. GHASEMI-PIRBALOUTI A., DADFAR S., 2013 - Chemical con- stituents and antibacterial activity of essential oil of Satureja bachtiarica (Lamiaceae). - Acta Pol. Pharm., 70: 933-938. GHASEMI-PIRBALOUTI A., VOSOGHI N., CRAKER L., SHIR- MARDI H.A., 2014 - Chemical composition of the essen- tial oil of Satureja kallarica Jamzad. - J. Essent. Oil Res., 26: 228-231. HALEVY A.H., MAYAK S., 1979 - Senescence and posthar- vest physiology of cut flowers, part 1. - Hortic. Rev., 1: 204-236. HASAN F.A.S., ALI E.F., EL-DEEB B., 2014 - Improvement of post harvest quality of cut rose cv. ‘First Red’ by biolog- ically synthesized silver nanoparticles. - Sci. Hortic., 179: 340-348. HE S.G., JOYCE D.C., IRVING D.E., FARAGHER J.D., 2006 - Stem-end blockage in cut Grevillea ‘Crimson Yul-lo’ inflorescences. - Postharvest Biol. Technol., 41: 78-84. HOEBERICHTS F.A., DE JONG A.J., WOLTERING E.J., 2005 - Apoptotic like cell death marks the early stages of gyp- sophilla (Gypsophila paniculata) petal senescence. - Postharvest Biol. Technol., 35: 229-236. HOSSAIN Z., MANDAL A.K.A., DATTA S.K., BISWAS A.K., 2006 - Decline in ascorbate peroxidase activity-a pre- requisite factor for tepal senescence in gladiolus. - J. Plant Physiol., 163: 186-194. JONES R.B., HILL M., 1993 - The effect of germicides on the longevity of cut flowers. - J. Amer. Soc. Hortic. Sci., 118: 350-354. KAR M., MISHRA D., 1976 - Catalase, peroxidase and polyphenoloxidase activities during rice leaf senes- cence. - Plant Physiol., 57: 315-319. KHAYAT E., ZIESLIN N., 1989 - Translocation of 14C carbohy- drate content and activity of the enzymes of sucrose metabolism in rose petals at different night tempera- tures.- Physiol. Plant., 76: 581-585. KUMAR N., SRIVASTAVA G.C., DIXIT K., 2008 - Flower bud opening and senescence in roses (Rosa hybrida L.). - Plant Growth Regul., 55: 81-99. LUTTS S., KINET J.M., BOUHARMONT J., 1996 - NaCl- induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. - Annals. Bot., 78: 389-398. MAHDAVIKIA F., SAHARKHIZ M.J., 2015 - Phytotoxic activi- ty of essential oil and water extract of peppermint (Mentha × piperita L. cv. Mitcham). - J. Appl. Res. Med. Aromatic Plants, 2: 146-153. NAGIRA Y., OZEKI Y., 2004 - A system in which anthocyanin synthesis is induced in regenerated torenia shoots. - J. Plant Res., 117: 377-383. NAIR S.A., SINGH V., SHARMA T.V.R.S., 2003 - Effect of chemical preservatives on enhancing vase-life of ger- bera flowers. - J. Trop. Agric., 41: 56-58. NAZEMI RAFI Z., RAMEZANIAN A., 2013 - Vase life of cut rose cultivars ‘Avalanche’ and ‘Fiesta’ as affected by nano-silver and S-carvone treatments. - S. African J. Bot., 86: 68-72. PANAVAS T., WALKER E.L., RUBINSTEIN B., 1998 - Possible involvement of abscisic acid in senescence of daylily petals. - J. Exp. Bot., 49: 1987-1997. RAMEZANIAN A., AZADI M., MOSTOWFIZADEH-GHALAM- FARSA R., SAHARKHIZ M.J., 2016 - Effect of Zataria mul- Salehi Salmi et al. - Effect of essential oils on vase life of cut rose 69 tiflora Boiss and Thymus vulgaris L. essential oils on black rot of ‘Washington Navel’ orange fruit. - Postharvest Biol. Technol., 112: 152-158. SCHERER R., LEMOS M.F., LEMOS M.F., MARTINELLI G.C., MARTINS J.D.L., DA SILVA A.J., 2013 - Antioxidant and antibacterial activities and composition of Brazilian spearmint (Mentha spicata L.). - Ind. Crops Prod., 50: 408-413. SEVILLANO L., SANCHEZ-BALLESTA M.T., ROMOJARO F., FLORES F.B., 2009 - Physiological, hormonal and molec- ular mechanisms regulating chilling injury in horticul- tural species. Postharvest technologies applied to reduce its impact. - J. Sci. Food Agric., 89: 555-573. SHAHSAVARI N., BARZEGAR M., SAHARI M.A., NAGHDIBA- DI H., 2008 - Antioxidant activity and chemical charac- terization of essential oil of Bunium persicum. - Plant Foods Hum. Nutr., 63: 183-188. SHARIFIFAR F., MOSHAFI M.H., MANSOURI S.H., KHO- DASHENAS M., KHOSHNOODI M., 2007 - In vitro evalu- ation of antibacterial and antioxidant activities of the essential oil and methanol extract of endemic Zataria multiflora Boiss. - Food Control., 18: 800-805. SOLGI M., KAFI M., TAGHAVI T.S., NADERI R., 2009 - Essential oils and silver nanoparticles (SNP) as novel agents to extend vase-life of gerbera (Gerbera jamesonii cv. ‘Dune’) flowers. - Postharvest Biol. Technol., 53: 155-158. TURNER N.C., 1986 - Crop water deficit: a decade of progress. - Adv. Agron., 39: 1-51. VAN DER SMAN R.G.M., EVELO R.G., WILKINSON E.C., VAN DOORN W.G., 1996 - Quality loss in packed rose flowers due to Botrytis cinerea infection as related to tempera- ture regimes and packaging design. - Postharvest Biol. Technol., 7: 341-350. VAN DOORN W.G., DE WITTE Y., PERIK R.J., 1990 - Effect of antimicrobial compounds on the number of bacteria in stems of cut rose flowers. - J. Appl. Bact., 68: 117-122. VAN DOORN W.G., SCHURER K., DE WITTE Y., 1989 - Role of endogenous bacteria in vascular blockage of cut rose flowers. - J. Plant Physiol., 134: 375-381. WAGACHA J.M., MUTHOMI J.W., 2008 - Mycotoxin prob- lem in Africa: current status, implications to food safety and health and possible management strategies. - Int. J. Food Microbiol., 124: 1-12. YAHYAZADEH M., OMID-BAIGI R., ZARE R., TAHERI H., 2008 - Effect of some essential oils onmycelial growth of Penicillium digitatum Sacc. - World J. Microbiol. Biotechnol., 24: 1445-2145. YE Z., RODRIGUEZ R., TRAN A., HOANG H., DE LOS SANTOS D., BROWN S., VELLANOWETH L., 2000 - The develop- mental transition to flowering repress ascorbate peroxi- dase activity and induces enzymatic lipid peroxidation in leaf tissue in Arabiodopsis thaliana. - Plant Sci., 58: 115-127.