Introduction
Rose (Rosa hybrida L.) is one of the most economically important cut flowers in the world. However, the short vase life of cut roses (Ichimura and Suto, 1999) often limits their commercial potential (G. van Doorn, 1995;Doi et al., 2000). Previous study has shown that water stress and/or microbial growth in the vase solution negatively affected on the postharvest longevity of cut roses (van Doorn et al., 1995). The accumulation of bacteria in the basal of the stem ends may be a major cause of early vase life termination (Woltering, 1987;van Doorn, 1989), as a result of water stress due to a low rate of water uptake (van Doorn et al., 1995). Water stress often causes premature wilting of leaves and petals of cut flowers. Previous studies have revealed that the high number of endogenous and exogenous bacteria in the basal stem and in vase solution decreased hydraulic conductance and water uptake of cut roses (van Doorn, 1989;van Doorn et al., 1995).
In order to alleviate stem blockage, improve water uptake, and extend cut flower longevity, the antibacterial compounds were applied in the vase solution (van Doorn et a l., 19 9;M0 acnish e t al., 2008;Lü et al., 2010;Mortazavi et al., 2011;Jędrzejuk et al., 2016). Numerous antibacterial agents have been recommended in the florist industry, and silver nitrate, silver thiosulfate, and nano-silver have received attention as an ethylene inhibitor and a promising antibacterial agent with silver ion activity (Lukaszewska et al., 1990;Lü et al., 2010;Li et al., 2012;Li et al., 2017).
Despite the successful use of various preservatives to improve postharvest quality of cut flowers, developing an easy-to-use, innocuous, and inexpensive new preservative solution is still important for the cut flower industry. The effect of natural plant extracts such as powder of green tea on postharvest longevity has been applied somewhat in cut roses or fresh-cut lettuce (Ichimura et al., 2005;Martín-Diana et al., 2008;Wu et al 2016). Scutellaria baicalensis Georgi (SC) is a species of flowering plants belonging the Lamiaceae family (Zhao et al., 2016). SC extract form roots contains many bioactive compounds such as baicalin, wogonoside, aglycones baicalein wogonin; these flavones have been reported to have various pharmacological functions, including anticancer, antibacterial, antiviral, antioxidant, and hepatoprotection (Zhao et al., 2016). Previous studies showed that SC has been applied as a natural antimicrobial against Escherichia coli, Staphylococcus aureus, Aspergillus fumigatus in medical industry (Yang et al., 2005;Bruzewicz et al., 2006;Shan et al., 2007). However, the application of SC as a preservative on the postharvest qualities of cut flowers has not been studied.
In the present study, a solution form of SC was applied to cut spray roses ‘Hessa’ to investigate its potential to improve postharvest quality and extend the vase life of cut flowers. In order to develop environmentally friendly preservatives that improve the postharvest quality of cut roses, the effectiveness of various preservative solutions was also determined by measuring bacterial contamination, physiological characteristics, and chlorophyll fluorescence parameters in leaves of cut rose flowers after pretreatments.
Materials and Methods
Plant materials
The spray roses ‘Hessa’ (Rosa hybrida L.) at the commercial maturity stage (loose pointed bud cylindrical (VBN, 2014)) were purchased from a commercial rose grower in Jangsu, Korea. After harvest, the cut flowers were immediately placed into buckets containing 20 L tap water and transported to the laboratory within 4 h. The flowers with uniform size, development stage, and color, and free of Botrytis cineria were used in the experiments. Three centimeters of the rose stem ends were trimmed under clean water before pretreatments.
Pretreatment of cut flowers
The Scutellaria baicalensis Georgi extract (SC) powder was provided by BiBiFlora Company (Gyeonggi-do, Korea). The concentration of SC used for the treatments was diluted from a 5000 μL·L-1 stock SC solution. Hydrosol is a natural antimicrobial solution, was extracted from Chrysanthemum by stream distillation method in BiBiFlora Company (Gyeonggi-do, Korea). Based on previous studies, we selected the optimal concentration of SC and preservatives for cut roses: Scutellaria baicalensis Georgi extract (SC) at 300 μL·L-1, hydrosol (H), Scutellaria baicalensis Georgi extract 300 μL·L-1 + sucrose 1% (SC + Suc), and hydrosol + sucrose 1% (H + Suc). Distilled water (DW) was used as a control in this study. The cut roses ‘Hessa’ were harvested on April 4, April 25, and May 16 in 2018. Twenty cut flowers per pretreatment of each cultivar were placed in the glass vase containing 1000 mL preservative solution and held for 10 h at 23 ± 1 °C and RH 40 - 60% in the dark. After pretreatments, the cut flowers were trimmed and held in the growth chambers at 23 ± 1 °C, RH 40 - 60%, and 12 h of light with a photon flux density of 20 μmol·m-2·s-1 for vase life assessment. Among the twenty cut flowers, nine flowers were used for vase life assessment and the remained 11 flowers were used for a soluble sucrose content and chlorophyll fluorescence measurements.
Measurements of morphological and physiological characteristics, and water relations
Changes in flower diameter, water uptake, and the fresh weight of flowers were measured daily at 9:30 am to determine the effects of preservative solutions on cut flowers. The water balance of cut flowers was determined by the difference between daily transpiration and daily water uptake values. The flower diameter was determined by measuring the maximum diameter and the diameter perpendicular to it (Fanourakis et al., 2012) using digital calipers.
The effects of pretreatments on the water stress status of cut roses was evaluated by measuring the chlorophyll fluorescence parameters in the leaves after 4 days of pretreatments using an Imaging Fluorometer (FluorCam 700MF, Photon Systems Instruments, Czech). Minimal fluorescence (F₀) was detected in 20 min dark-adapted leaves and maximal fluorescence (Fm) was also detected in the same leaves in full light adapted conditions. Maximal variable fluorescence in dark-adapted states (Fᵥ = Fm − F₀), and the maximal PSII quantum yield (Fv/Fm) were automatically calculated from the measured parameters. The Fv/Fm ratios in leaves represent the water stress status of cut roses.
The soluble sucrose content (brix) was determined in the uppermost leaves after 3 days of pretreatment. Leaf tissues (100 mg) were placed in an Eppendof containing 500 μL distilled water, then ground using a TissueLyser (TissueLyser II; Quiagen, Hilden, Germany). The brix (%) was measured by a portable refractometer (PR-104, Atago, Tokyo, Japan). The measurement of the chlorophyll content of the leaves was using a chlorophyll meter (SPAD-502Plus; Konica Minolta Sensinf, Inc., Osaka, Japan). The SPAD measurements were performed daily on the terminal leaflets of the uppermost leaves two times, throughout the vase life of the cut roses.
Measurements of antibacterial activity
To determine the antibacterial activity of preservative solutions, the total bacterial population was assessed at the base of the cut stem on day 1 and day 5 after pretreatments. The bacterial counts were performed using swab samples taken from the basal 3-cm of the cut stem ends. Each sample was diluted in 10 mL NaCl buffer (3M Pipette Swab; 3M Health Care, MN, USA), and 1 mL of each diluted sample was poured into an aerobic count plate (Petrifilm 6400; 3M Health Care, MN, USA). After two days incubation at 37 ± 1 °C, the bacterial count was assessed by counting the number of colonies formed on the plates.
Assessment of vase life
The vase life of cut flowers was determined as the number of days from the placement of the cut rose flower in the environmental controlled room after pretreatments to the end of vase life. Vase life evaluation was performed daily in accordance with the evaluation card for Rosa (VBN, 2014). Cut roses were considered to have reached a senescent stage when the flower showed at least one of the following senescence symptoms: petal or leaf abscission (≥ 50% petal or leaf drop), wilting of flower (the flower is visibly limp), or discoloration (the rose petals showed advanced fading at senescent stage) (Macnish et al., 2000).
Experiment design and data analysis
The vase life experiment was designed by a completely randomized block with 3 replicates for each pretreatment and nine flowers per replicate. Leaf chlorophyll fluorescence ratios, brix of leaf, and anti-microbial activity measurements were performed with six biological replicates for each treatment. Data were presented as mean ± standard error (SE). One-way analysis of variance (ANOVA) was performed using SPSS 16.0 (IBM, Somers, NY, USA). When significant effects were detected, post-hoc pairwise comparisons of group means were executed with Duncanʼs multiple range tests, with a significance level of p ˂ 0.05.
Results
Effect of preservative solutions on vase life and senescence of cut flowers
The postharvest longevity of cut flowers ‘Hessa’ was significantly prolonged by treatment with SC, H, and SC + Suc, compared with control flowers (Fig. 1A). The cut roses were pretreated with SC showed the longest vase life (14.1 d) and flowed by cut flowers pretreated with H (12.3 d), and SC + Suc (11.7 d) (Fig. 1A). Whereas, pretreatment with H + Suc extended vase life of cut flowers only slightly compared with control flowers (Fig. 1A).
In this experiment, petal abscission (66.7%) and wilting (33%) were the primary cause of vase life termination in cut roses ‘Hessa’ (Fig. 1B and C). However, pretreatment with preservative solutions reduced petal abscission in cut rose flowers (Fig. 1B and C). Notably, SC pretreatment completely reduced petal wilting in cut flowers (Fig. 1B and C).
Effect of preservative solutions on flower diameter and leaf brix
Pretreatments with SC increased the maximum flower diameter of cut roses (p < 0.05), compared with control and other preservative solutions. The maximum flower diameter was lowest during vase period in control flowers (Fig. 2A). Other pretreatments slightly increased flower opening as compared with control flowers (Fig. 2A).
The soluble sugar content in leaves was significantly highest in cut roses were pretreated with SC solution (0.3%). Pretreatments with H and SC + Suc also increased the soluble sugar content in leaves of cut flowers on day 5, compared with control flowers (Fig. 2B).
Effect of preservative solutions on bacterial number and water relations
The total bacterial population at the base of the cut rose stems on day 1 was reduced by SC and H pretreatments. SC significantly inhibited the bacterial proliferation after 5 days of pretreatments (p < 0.05) (Fig. 3).
Fresh weight of control and H + Suc flowers reached a maximum at day 4 and subsequently decreased thereafter (Fig. 4A). The cut flowers were pretreated with SC showed the highest fresh weight, compared with other treatments (p < 0.05). The reduction of fresh weight during the vase period was also effectively prevented by pretreatment with H and SC + Suc (Fig. 4A).
Pretreatment with preservative solutions also improved water uptake during vase life of cut flowers ‘Hessa’ (Fig. 4B). Among the pretreatments, SC effectively improved solution uptake (p < 0.05), followed by pretreatment with H, whereas SC + Suc, H + Suc and control flowers showed lower water uptake (Fig. 4B).
Water balance of cut roses was determined by the difference between water loss from leaves and water uptake by stems. SC significantly increased the number of days that flowers retained a positive water balance by 2.3, compared with control flowers (Fig. 4C). H pretreatment also increased the time that flowers retained a positive WB than control flowers (Fig. 4C). In this experiment, water balance of cut roses was not improved by pretreatments with SC + Suc and H + Suc (Fig. 4C).
Effect of preservative solutions on leaf chlorophyll fluorescence and chlorophyll content
Water stress of cut flowers generally leads wilting of petals and leaves, and plant emit decreased chlorophyll fluorescence (CF) ratio in response to the stress conditions. To evaluate the effects of preservatives on water stress status of cut roses, the CF ratio (Fv/Fm) were measured in leaves of cut flowers. Fv/Fm ratios in the leaves were most reduced in control and H + Suc pretreated flowers on day 4, whereas the reduction in Fv/Fm was significantly prevented by the treatment with SC, H, and SC + Suc (Fig. 5A). The results also showed that the Fv/Fm was highest in the flowers treated with SC. Interestingly, in this study, control and H + Suc pretreated flowers showed the leaf chlorophyll content values in accordance with the reduction in Fv/Fm at day 4 (Fig. 5A and B), indicating that the CF ratio may be closely related with leaf chlorophyll content. Pretreatments with SC, H, and SC + Suc retarded the degradation in leaf chlorophyll of cut rose flowers (Fig. 5B).
Discussion
It has been known that the long vase life and good postharvest quality are essential for commercialization of cut flowers (Mohd. Rafdi et al., 2014). The postharvest quality and longevity of cut flowers are determined by various factors such as harvest time, genetic factors, environmental factors, and management practices throughout the marketing chain (Pompodakis et al., 2005;Fanourakis et al., 2013; Mohd. Rafdi et al., 2014). One of the significant factor determining the postharvest longevity and quality of cut roses is water relations (In et al., 2017). The postharvest physiological of cut flowers is rapidly disordered by the microbial proliferation in vase solution (van Doorn, 1989;Macnish et al., 2008). The bacterial growth in vase solution leads to blockage of the xylem vessels and a reduction in water uptake of cut flowers. Therefore, evaluation of bacterial proliferation in the vase solutions provided important information on the antimicrobial activity of the preservative solutions in this study.
In the present study, 300 μL·L-1 SC solution was the most effectively inhibited microbial growth at the cut rose stem ends after 1 and 5 days of pretreatments. This result suggests that SC solution may be a promising germicide for the preservative solution of cut flowers. The antimicrobial effects of SC solution in this experiment agreed with the findings from previous studies which indicated that SC extracts are an antimicrobial agent against bacteria and fungi (Yang et al., 2005;Zhao et al., 2016). It has been shown that flavones such as baicalin and wogonoside are the major bioactivity compounds extracted from Scutellaria baicalensis Georgi. These compounds have been reported to have various functions, including antiviral, antibacterial, anticancer, and antioxidant (Gao et al., 1999;Li et al., 2000). The blockage of xylem vessels resulted in significant reduction in water uptake, leading to a decreased in flower opening, as well as in the postharvest longevity of cut roses (van Doorn, 1989). In this work, changes in daily fresh weight, water uptake, and the time that cut flowers maintained their positive water balance revealed that the antimicrobial activity of SC solution had a markedly positive effect, improving fresh weight as well as water uptake rate of cut flowers. These findings suggest that SC solution effectively inhibited microbial proliferation in the vase solution, thereby alleviation of xylem vessel blockage in cut rose stems so that the early wilting of cut roses due to decreased water absorption was completely reduced.
It is interesting to note that cut flowers with higher soluble sugar content in leaves had relatively longer vase life and larger flower diameter. Notably, in this study, the SC pretreated flowers had a higher soluble sugar content in leaves and larger flower diameter than other pretreated flowers. These results support the idea that the soluble sugar content may be important for providing the energy sources to facilitated flower opening and for the sustenance of vase life (Paulin, 1986;Kazuo et al., 2005;In et al., 2016). Although supply of exogenous sucrose retards flower senescence, sucrose promotes microbial growth in vase solution, which causes a shortening of vase life (Ichimura et al., 2002). The results from our study indicated that pretreatment with H + Suc did not increase vase life of cut roses, whereas SC + Suc pretreatment significantly prolonged vase life of cut flowers by 2.6 d as compared with control flowers. This results strongly supports to the antimicrobial activity of SC solution.
Chlorophyll fluorescence ratios (Fv/Fm) reflect the photosynthetic efficiency and stress conditions of plant tissues. An optimal chlorophyll fluorescence ratio in many plant species ranges from 0.79 to 0.84, the decrease in chlorophyll fluorescence ratio in leaves indicates plant stress such as water stress (Maxwell and Johnson, 2000). Previous studies indicated that chlorophyll content in plants affects positively on chlorophyll fluorescence (Mauromicale et al., 2006). The results from our study revealed that SC pretreatments retarded the degradation of chlorophyll and consequently maintained leaf chlorophyll fluorescence during vase periods and reduced water stress in cut roses.
In conclusion, the present study demonstrated that the 300 μL·L-1 of SC solution significantly increase the vase life and improve the postharvest quality of cut rose flowers. SC pretreatment extended the vase life of cut roses in association with an inhibition in the accumulation of bacteria at the basal of the flower stem ends, improved fresh weight and water uptake, and maintained chlorophyll fluorescence ratios in leaves. SC solution may therefore act as a natural, effective, and inexpensive preservative for the cut rose flower industry.
