103 1. Introduction Cut foliage represents an important part of the flori- cultural industry. They are used as fillers in floral bou- quets especially for cut flowers without leaves such as gerberas. The vase life of cut foliage is usually longer than cut flowers, but it may become shorter when they are stored for long periods. Storage methods, wet or dry, affect the vase life of cut foliage (Ferrante et al., 2002 a) and play a crucial role for preserving quality: it is impor- tant, in particular, to delay the symptoms of leaf senes- cence considering that the intensity of leaf color is the most important quality parameter (Pacifici et al., 2007) and closely associated with the marketability of orna- mental cut foliage. During postharvest, quality losses of cut foliage are essentially represented by leaf senes- cence. The most common symptoms are leaf yellowing, leaf desiccation and/or leaf abscission, and weight losses (Pacifici et al., 2007). Leaf yellowing is common in many cut flowers such as Alstroemeria, Chrysantemum, lilies, tulips, but also in some cut foliage such as cut Danae racemosa foliage (van Doorn et al., 1992; Ferrante et al., 2002 b, 2003, 2005). Postharvest treatments with cytoki- nins are able to delay leaf yellowing in many cut flowers, but a substituted phenylurea, the Thidiazuron (N-phenyl- N’-1,2,3-thiadiazol-5-yl urea, TDZ), with cytokinin-like activity, has been found to be very effective in many cut flowers sensitive to leaf yellowing (Ferrante et al., 2002 b); application of low concentrations of TDZ delayed leaf yellowing (Ferrante et al., 2002 b, 2003; Mutui et al., 2005; Jiang et al., 2008). During the postharvest chain, another important prob- lem for cut foliage is weight loss. Since these ornamental items are sold by weight, any weight loss is directly trans- lated into economic loss. Cut foliage is essentially com- posed of branches with leaves, therefore, weight losses are due to water losses by transpiration. During postharvest, the hydraulic conductance of the branches or cut stems progressively declines due to vessel blockages from bacte- ria growth, embolism, etc. (van Doorn, 1997). Cut flowers and branches treated with glycerol are able to enhance osmotic potential and absorb great amounts of water, avoiding reduction of the water potential. Glycerol has been used to preserve ornamental plant materials (Du- bois and Joyce, 1992) as it causes a greater water removal due to an increment of osmotic potential. In this way, glyc- erol treatment reduces leaf weight loss, as well as water loss, which reflects on extending vase life (Shanan and Shalaby, 2011). In this study, the possibility to extend as much as pos- sible the storage of cut foliage in a cold room at 4°C for 60 days was evaluated with the aim of maximizing cut Danae availability on the market. This outlook has practical value for some cooperatives and commercialization companies as extention of the storage period could cover periods when production is lacking (Pacifici et al., 2013). Evaluation of postharvest storage and treatments in cut ruscus foliage R. Bulgari(*), M. Negri, A. Ferrante Dipartimento di Scienze Agrarie e Ambientali, Produzione, Territorio, Agroenergia, Università degli Studi di Milano, Via Celoria 2, 20133 Milano, Italy. Key words: Danae, glycerol, leaf senescence, Thidiazuron. Abstract: Danae racemosa (L.) Moench is an important cut foliage in the ornamental market. During postharvest, leaf senescence symptoms are leaf yellowing, weight loss and/or abscission of leaves. The aim of this work was to evaluate the effect of pre-treatments for 24 h with glycerol and Thidiazuron (TDZ) on Danae vase life before and after storage. Treatments were applied in vase water containing glycerol 0.1, 1 or 10 mM, 10 µM TDZ and a combined treatment of 10 µM TDZ plus 10 mM glycerol. The effect of treatments was evaluated through the determination of vase life, chlorophyll content, chlorophyll a fluorescence parameters, sucrose and total sugars content. The cut foliage were stored in sealed plastic bags and placed in a dark room at 4°C for two months. The vase life, before and after storage, was determined in a controlled chamber set to 20°C and RH 50-60% with a light intensity of 20 µmol m-2 s-1. Results demonstrated that 10 µM TDZ plus 10 mM glycerol was the most effective treatment in maintaining quality during vase life of stored and not stored cut foliage. Adv. Hort. Sci., 2015 29(2-3): 103-108 (*) Corresponding author: roberta.bulgari@unimi.it Received for publication 26 September 2014 Accepted for publication 26 March 2015 104 Adv. Hort. Sci., 2015 29(2-3): 103-108 2. Materials and Methods Plant materials Cut branches of Danae racemosa (L.) Moench. [=Rus- cus racemosus L.] were provided by a commercial compa- ny (Floratoscana, Pescia, PT, Italy) and transported to the postharvest laboratory of the Department of Agricultural and Environmental Sciences of the University of Milan. Cut branches were trimmed to a length of 70 cm to provide homogenous samples for the experiments. Chemical treatments and storage Cut foliage was pre-treated for 24 h with a solution con- taining 0.1, 1 or 10 mM glycerol, 10 µM TDZ and a com- bined pre-treatment of 10 µM TDZ plus 10 mM glycerol, while control samples were held in distilled water. After pre- treatments, the plant material was stored in vases containing distilled water at 20°C and 50-60% relative humidity. Each treatment was composed of eight replicates. A part of the cut branches treated with 10 mM glycerol, 10 µM TDZ and combined treatment 10 µM TDZ plus 10 mM glycerol were stored for 60 days in polyethylene bags at 4°C. The gas composition in terms of oxygen and carbon dioxide percentages was monitored by means of a “Binder Combigas GA-m3” (from Binder, D) portable gas analyzer equipped with an electrochemical cell for oxygen and CO 2 measurement and an infrared dispersion cell for NH 3 de- termination. Vase life evaluation After storage, cut branches were transferred to a growth chamber with controlled temperature (20°C), relative humid- ity (60-70%), and light intensity (10-15 μmol m-2 s-1 PPFD for 12 h per day). Non-stored cut branches (control) were used for vase life assessment immediately after harvest. Ten branches for each storage period were placed in individual bottles for vase life analysis. Vase life was determined as the number of days from when branches were placed in the wa- ter to the onset of leaf senescence, yellowing or abscission. Chlorophyll determination and chlorophyll a fluorescence Chlorophyll content was measured using a chlorophyll meter (CL-01, Hansatech, UK) that provides an indicator of green color of leaves. This device determines relative chlorophyll content in vivo using dual wavelength optical absorbance (620 and 940 nm wavelength). Chlorophyll a fluorescence was measured with a portable Handy Plant Efficiency Analyzer (PEA, Hansatech, UK). Leaves were dark-adapted for 30 min. Using a leaf clip (4 mm diameter), a rapid pulse of high intensity light of 3000 μmol m−2 s−1 (600 W m−2) was absorbed by the leaf inducing fluo- rescence, which was measured by the sensor. The fluores- cence parameters were calculated automatically. JIP analysis was performed to determine the Performance Index (PI). Sugars determination In order to quantify sucrose and total sugars content, about 0.5 g of leaves were ground in 10 ml of distilled water. The homogenate was centrifuged at 10000 rpm for 5 min. For sucrose determination, 0.2 ml of extract were added to 0.2 ml NaOH 2N and incubated at 100°C for 10 min; then 1.5 ml of resorcinol were added and incubated at 80°C for 10 min. A resorcinol solution was prepared by adding 35 mg of resorcinol and 90 mg of thiourea in 250 ml HCl 30%, mixed with 25 ml of acetic acid and 10 ml of distilled water. Samples were cooled at room temperature and spectropho- tometer readings were performed at 500 nm. A calibration curve was built with sucrose standards at 0, 0.5, 1, 1.5, 2 mM. Total sugars were calculated by anthrone method: 0.2 g of anthrone were melted in 100 ml of H 2 SO 4 and shacked for 30-40 min; 0.2 ml of diluted extract was added to 1 ml of anthrone solution, cooled in ice for 5 min and mixed thor- oughly. Samples were incubated at 95°C for 5 min and then cooled on ice. Absorbance readings were measured at 620 nm and a calibration curve was built with glucose standards at 0, 1, 2, 3 and 4 mM. Leaf extracts were diluted 1:10 for total sugars assay and used pure for sucrose assay. Statistical analysis Experiments were performed in a completely random- ized experimental design with eight replicates for each treatment. The data are reported as means with standard errors. Data presented in Table 2 were subjected to one- way ANOVA. 3. Results Gas compositions inside plastic bags The gas composition was determined after 30 and 60 days of storage. Results showed that oxygen was lower in the plastic bags containing cut branches treated with TDZ plus glycerol. At the same time, also higher values of NH 3 were found in the TDZ+Gly treatment (Table 1). Vase life and chlorophyll content The vase life was significantly improved by the com- bined treatment TDZ plus glycerol (33.6 days in average) in comparison with the control (average 21.2 days) and the other treatments (21.8 to 23.6 days) (Table 2). Vase life was significantly reduced in cut foliage stored for 60 days, but treatment with TDZ plus glycerol gave the longest vase life, 22.6 days on average. Vase life of the control was 13.8 days, while in the treatment with glycerol and TDZ alone it was 10.2 and 15.2 days, respectively (Table 2). The chlorophyll content was higher in all treatments compared to control during the first 15 days of vase life, then declined in 10 mM glycerol (this treatment had the best efficiency among the glycerol treatments) while cut foliage treated with TDZ showed chlorophyll reduction after 20 days. The combined TDZ plus glycerol treat- ment did not show any chlorophyll reduction until 22 days (Fig. 1). After storage the best results were obtained with TDZ and TDZ plus glycerol treatment. TDZ retained the 105 Bulgari et al., Evaluation of postharvest storage and treatments in cut ruscus foliage chlorophyll content in the treated cut foliage until 10 days, then chlorophyll started to decline. In the combined treat- ment TDZ plus glycerol, the chlorophyll values remained unchanged and similar to the fresh cut foliage (80 a.u.) until 20 days of vase life (Fig. 2). Chlorophyll a fluorescence measurements Chlorophyll a fluorescence was measured during stor- age by way of a non-destructive method to evaluate the leaf health status of Danae leaves during vase life, before and after storage. Among the different parameters and indexes calculated, the performance index (PI) is reported in figure Table 1 - Gas compositions inside the plastic bags during storage Days Gases Blank Treatments Control 10 mM Gly TDZ TDZ+Gly 30 CO 2 (%) 0 0.30 0.60 0.10 0.3 O 2 (%) 20 12.70 19.90 20.90 13 NH 3 (µL L-1) 2 15.00 0.00 12.00 17 60 CO 2 (%) 0.00 0.50 0.40 0.40 0.3 O 2 (%) 20.90 20.30 19.50 20.00 13 NH 3 (µL L-1) 0.00 8.00 8.00 8.00 17 Table 2 - Vase life before and after storage of cut foliage treated with glycerol (0.1, 1 or 10 mM), 10 µM TDZ or a combination 10 µM TDZ plus 10 mM glycerol Vase life of fresh harvest cut foliage Control Glycerol TDZ TDZ + glycerol 0.1 mM 1 mM 10 mM 10 µM 10 µM + 10 mM Vase life (d) 21.2±1.09 b 21.8±0.45 b 22.2±0.45 b 22±0 b 23.6±1.34 b 33.6±1.14 a Storage and vase life post-storage Control Glycerol TDZ TDZ + glycerol 10 mM 10 µM 10 µM + 10 mM Storage (d) 60 60 60 60 Post-storage vase life (d) 13.8±1.09 b 10.2±1.12 b 15.2±0.45 b 22.6±2.61 a Storage + vase life (d) 73.8±1.09 b 70.2±1.12 b 75.2±0.45 b 82.6±2.61 b Values are means with standard errors (n=5). Data were subjected to one-way ANOVA analysis and different letters indicate significant differences for P<0.05. Fig. 1 - Chlorophyll content measured from cut Danae branches dur- ing vase life with a chlorophyll meter and expressed as relative units. Values are means with standard errors (n=8). Fig. 2 - Chlorophyll content during vase life of cut Danae branches stored for 60 days. Chlorophyll was measured using a chloro- phyll meter and expressed as relative units. Values are means with standard errors (n=8). 106 Adv. Hort. Sci., 2015 29(2-3): 103-108 3 and 4. In particular, the PI measured during the vase life of non-stored cut foliage did not change in the combined treatment, while it declined in the control and glycerol treatments. Instead, in the TDZ treated cut branches, the PI declined after eight days of vase life (Fig. 3). After two months of storage, the PI was lower in all treatments and dropped faster in the control and TDZ, while in the TDZ plus glycerol it slightly declined after eight days and re- mained unchanged until 20 days of vase life (Fig. 4). Sugars content The sucrose content of cut foliage at harvest was 15 mg/g FW on average and declined during vase life. In glycerol treatments, and in particular in the 10 mM treatment, the reduction was faster and showed lower values. After 14 days of vase life, higher values were found in TDZ and TDZ plus glycerol. After 19 days only the cut branches treated with TDZ and glycerol were alive and the sucrose content was 4.5 mg/g FW on average (Fig. 5). In stored cut branches the sucrose content was five fold lower compared to the fresh harvested branches. The sucrose content declined in TDZ after eight days of storage and ranged from 1.5 to 2.2 mg/g FW until 15 days of vase life. In TDZ plus glycerol treat- ment the cut branches did not show reduction of sucrose content until 15 days of vase life and a significant decline was observed after 20 days of vase life (Fig. 6). Total sugars showed a similar trend of sucrose content during vase life before and after storage. The higher values were found in TDZ plus glycerol treatment, especially at the end of vase life (data not shown). 4. Discussion and Conclusions The visual appearance of cut foliage is the most im- portant quality parameter, and preservative treatments for Fig. 3 - Performance index (PI) measured on cut foliage during vase life determination. Values are means with standard errors (n=5). Fig. 4 - Performance index (PI) measured on cut foliage stored for 60 days and then transferred to 20 °C for vase life determination. Values are means with standard errors (n=5). Fig. 5 - Sucrose content measured in leaves of cut Danae branches treated with distilled water (control), 10 mM Glycerol, 10 µM TDZ or 10 µM TDZ plus 10 mM Glycerol during vase life. Values are means with standard errors (n=3). Fig. 6 - Sucrose content measured in leaves of stored cut Danae branch- es treated with distilled water (control), 10 µM TDZ or 10 µM TDZ plus 10 mM Glycerol during vase life. Values are means with standard errors (n=3). 107 Bulgari et al., Evaluation of postharvest storage and treatments in cut ruscus foliage these ornamental items are aimed at delaying leaf senes- cence or yellowing, which can occur during long-distance transportation or long storage periods. In addition to ex- ternal quality, another important parameter to control in cut foliage postharvest is weight loss. Since cut foliage is sold on the basis of weight, any weight losses translate into economic losses. Danae plants are in active vegeta- tive growth from September to May, thus there are three months without harvest and a lack of cut branches on the market. Flower markets and cooperatives are interested in storing as much cut foliage as possible. Cut Danae branches can be stored under mild vacuum packages or in water for two months while maintaining satisfactory vase life (Pacifici et al., 2014). However, preservative treat- ments are needed to delay leaf yellowing during post-stor- age vase life. The positive effect of cytokinin treatments on chlorophyll retention was observed in cut eucalyptus branches (Ferrante et al., 2002 b) and TDZ has been used in cut flowers to inhibit leaf yellowing in sensitive species such as Alstroemeria, chrysanthemum and tulips (Ferrante et al., 2002 a, 2003, 2005). Our results indicate that cut branches treated with TDZ plus glycerol provide the best post-storage performance. TDZ alone reduced chlorophyll decline but TDZ with glycerol showed a synergistic effect; the positive effect of the combination was observed in all parameters measured. Chlorophyll a fluorescence measurements can be used during postharvest to evaluate the health status of leaves. In the ornamental field, chlorophyll a fluorescence and rela- tive derived parameters have been used to evaluate quality losses in cut Eucalyptus and Danae branches (Pacifici et al., 2008, 2013). In potted Bougainvillea plants, the use of chlorophyll a fluorescence parameters were used to evalu- ate the efficiency of ethylene inhibitors during the post- production stage (Ferrante et al., 2012). In our experi- ments, chlorophyll a fluorescence showed that TDZ plus glycerol maintained leaf functionality as demonstrated by the Fv/Fm ratio (data not shown) and PI. Storage of cut Danae branches in polyethylene bags at 4°C for 60 days gave good results since the cut foliage was available on the market for more than 80 days from har- vest. The reduced gas exchanges avoided excessive water loss by transpiration and probably also conditioned respi- ration, as demonstrated by the gas compositions at the end of storage, especially in the bags containing cut branches treated with TDZ plus glycerol. Cut E. parvifolia branches treated with glycerol showed longer life compared with control (Ferrante et al., 2001) and analogous results were found in E. cinerea (Campbell et al., 2000). Glycerol up- loaded in the cell increases osmotic capacity and induces an initial stress which can be observed by measuring eth- ylene production (Ferrante et al., 2001), but it also helps maintain water balance. Sugars represent the energy source for stored products and are usually associated with vase life. Among the sug- ars, sucrose represents one of the most important energy reserves and its degradation provides a direct substrate for respiration (Reid, 1991; Ferrante and Reid, 2006). In the present study, the cut foliage treated with TDZ showed higher sugars content. Further studies are needed to un- derstand sugar metabolism in TDZ treated cut foliage after storage. These results can be further evaluated in combination with postharvest treatments and the use of passive refrig- eration systems, which have been applied to different per- ishable foods (Costa et al., 2013). In conclusion, the results obtained from this investiga- tion suggest that pulse treatment for 24 h with TDZ and glycerol can be used to extend the vase life of stored and non-stored cut Danae branches. However, further studies are required at physiological and biochemical level to eluci- date the biological pathways affected by TDZ and glycerol. 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