Plant material and treatments

Forty flower stems from each of the Blush, Freedom and Topaz rose cultivars grown in a commercial greenhouse were harvested at 7:00. The average stem length was 70 cm. Immediately (phase I) the stems of each cultivar were separated into two lots of 20 stems each. The first batch was wrapped in kraft paper, placed in black polyethylene bags and kept in a greenhouse for 4 h (S_FI), the second batch was placed in containers with tap water (pH 7.5, EC 563 µS cm^-1); the water covered 10 cm of the base of the stem, and remained 4 h at the greenhouse (H_FI). The average environmental conditions in the greenhouse were: 22±3 °C and 77 % RH.

Afterwards, the two batches from the three cultivars were transported to a laboratory, each stem was weighed and had 5 cm from its base removed. Each batch was subdivided into two and stored at 1±1 °C and 85 % RH for 7 d (FII), one in containers with tap water (H_FII) and other, wrapped in kraft paper and stored in black polyethylene bags (S_FII). As a result, there were four management conditions or treatments (S_FI+S_FII, S_FI+H_FII, H_FI+S_FII, H_FI+H_FII) per cultivar.

At the end of the storage, 5 cm of the base of the stems were removed to help re-establish the water flow in the stems. The foliage was partially eliminated, leaving in each stem two trifoliate leaves and three pentafoliate. Subsequently, flower stems were individually placed in a 200 mL container with tap water, and randomly distributed in a room with a 12:12 h (illumination of 10 µmol m^-2 s^-1) photoperiod, at 20±3 °C and 56 % RH to be evaluated.

Life in vase (VF) and floral opening

The flower stems VF was evaluated, which corresponded to the number of days it took for any of the following symptoms of senescence to appear: necrotic spots on the periphery of the petals, turgor loss, stem bending, falling petals and yellowing or abscission of the leaves. Also, from the third day on, the apical diameter of the flower buds, the relation between the floral opening and the maximum opening were evaluated, which, according to ^{De La Cruz et al. (2015)} are 67.2 mm for Freedom, 88.7 mm for Topaz and 90.3 mm for Blush cultivars.

Fresh biomass (BF), water absorption and evapotranspiration rate

Each floral stem was daily weighed, and their percentage biomass gain or loss of was calculated. The water in each container was weighed and the water absorbed (mL g^-1) was assessed (^{Rezvanypour and Osfoori, 2011}). The total weight of the stem and the water absorption rate were used to calculate the evapotranspiration rate (TE) daily with the following Equation:

where TE: evapotranspiration rate (g g^-1); BFn: biomass of the floral stem on day 1, 2, 3, n; BFo: stem biomass on the previous day; PSn-1: biomass of the solution on the previous day; PSn: weight of the solution on day 1, 2, 3, n; Bit: initial biomass of the floral stem.

Colony forming units (CFU)

On the first and fourth day of VF, the CFUs were quantified at the vase water of each treatment; the control was the tap water without the floral stem. This evaluation was carried out by duplicate and by treatment on each evaluation day. For this, 1 mL vase solution was placed in the center of a plate for total aerobic counts (Petrifilm 3M™), kept at room temperature for 72 h and then the CFU was assessed.

Stomatal pore opening and number of stomata

The number of stomata and the opening of the stomatal pores was assessed on the first and fourth VF day on epidermal impressions of the second pentafoliate leaf. For this, a layer of transparent cosmetic varnish was applied between the ribs, allowed to dry for 30 min, the layer was detached and mounted on a slide, with the print side facing the microscope. Photographs were taken with a 6.3X objective in a photomicroscope (III, Carl Zeiss) with integrated digital camera for microscopy (PAXcam 3). The stomas number was quantified per square millimeter and the stomatal pore area was measured in the photographs taken with the 40X objective in the same microscope. Pores segmentation was done with the GIMP, 2.8.4 software, and the area was obtained with the Image tool (3.40) (^{Willcox et al., 2002}).

Botrytis sp. incidence

The incidence of Botrytis sp. by the seventh evaluation day in vase was determined via a visual scale that considered four damage levels: 0) absence of visible symptoms, 1) necrotic points on the petals (maximum three on one petal or five on several petals), 2) necrotic spots on maximum three petals, 3) brown spots at the apex of the petals and loss of turgor, 4) brown spots spread over most of the surface, including the center, wilting and falling petals (Figure 1). Floral stems turning level 3 were considered at the end of their VF, even if their leaves were turgid. The identification of Botrytis sp. was done through the isolation, purification and identification of the genus using the ^{Barnett and Hunter (1998)} keys.

Figure 1 Visual scale to qualify Botrytis sp. incidence in roses (Rosa hybrida). 

Experimental design and statistical analysis

The experimental design was completely randomized with a 3×2×2 factorial arrangement (three varieties × two treatments in phase I × two treatments in phase II). The experimental unit was a floral stem, and ten of them were included as repetitions. The evaluated variables were: life in vase, fresh biomass of the floral stem, floral opening, evapotranspiration rate and Botrytis sp. incidence. In addition, the number of stomata was counted and stomatal opening measured at the central area from five leaves, 15 fields were analyzed (three per sheet). The CFU were analyzed from a solution of two vases, by treatment, randomly selected. The data were analyzed with ANOVA and the significant differences were determined with the Tukey test (p≤0.05); besides, the interactions between the factors were obtained.

Life in vase and floral opening

On average, the VF of Topaz cultivar was the highest (14 %, p≤0.05) amongst the three evaluated cultivars (Table 1). In both postharvest phases of the stems in dry handling, out of the three cultivars had VF at least 20 % higher than with their similar in wet management. The cultivar-phase relation was highly significant and showed especially that in Blush and Topaz cultivars the dry condition during phase II was determinant for longer VF. Wet handling in both phases statistically reduced the VF of the Freedom cultivar (Table 1). Storage in humid condition (H_FII) kept the stems hydrated and maintained the metabolic activity of the tissues, the floral opening continued and reduced the VF after storage. This was the case of the flower buds of lisianthus (E. russellianum) stored in tap water, at 2±1 °C, from 1 to 3 weeks, which continued growing; however, when placed in a vase exhibit a premature 25 % buds drop (2.8 buds per floral stem) compared to dry handling, which resulted in the opening of 3.7 buds per floral stem (^{Ahmad et al., 2012}).

The floral opening index from the Topaz cultivar was 10 and 20 % higher than that of the Freedom and Blush. The cultivar × phase I relation had no effect on the floral opening; in contrast, the cultivar × phase II relation was highly significant, showing that floral opening in the Freedom stems was favored by the wet condition, unlike the Topaz cultivar (Table 2).

The opening index is related to the maturity of the flower bud, the sugars reserves and water absorption necessary for the expansion of the petals (^{Ichimura and Shimizuko-Yumoto, 2007}). The stems from the Freedom cultivar had lower fresh biomass (38.2±0.94 g) and VF than those from the Blush and Topaz cultivars; but its water absorption was intermediate (0.38 mL g^-1) between those two. This indirectly confirmed that the carbohydrates reserves in the stem and petals are important for flower opening (^{Evans and Reid, 1988}).

Total fresh biomass, absorption rate and evapotranspiration

Flower stems from the three cultivars increased their total biomass (102 %) the first day in the vase, the highest increase occurred in the treatments from the Freedom cultivar (108 %) which included the S_FI-S_FII conditions, and Topaz (109 %) at H_FI-S_FII and S_FI-S_FII conditions. From the second day on, the total biomass continuously decreased in all treatments. The stems in the two phases were maintained in dry condition, S_FI-S_FII significantly decreased less their wet biomass relative to those handled in wet conditions, H_FI-H_FII. These results are explainable since during the water storage stems were kept hydrated, but the stomas had 20 % greater opening compared with the stems stored in dry conditions. The open stomata maintain the transpiration gradient that favors the absorption of water at the base of the stems. On day 7, the total biomass of the Blush and Freedom cultivars was between 85 and 90 %, and in the Topaz cultivar remained between 92 and 95 %; so the latter showed greater floral openings and VF. In the stems of the three cultivars, weight losses greater than 15 % was related to evident wilting.

The absorption rate after storage was statistically higher (0.37, 0.48 and 0.65 mL g^-1) in the stems of the treatments included the S_FI-S_FII condition compared to H_FI-H_FII (0.26, 0.29 and 0.48 mL g^-1) in the Blush, Freedom and Topaz cultivars. The higher absorption in S_FI-S_FII was probably because the stress generated a lower water potential gradient compared to the one generated with the previous hydration in one of the two phases. Seven days after starting the vase period floral stems from the Topaz cultivar had 30 % more water absorption compared with Blush and Freedom (0.21 mL g^-1), which also had lower VF. When the water absorption rate is greater than that of transpiration, there is a positive water balance; the total biomass is maintained with minor changes for longer, and the VF increases. In contrast, if transpiration exceeds absorption, due to metabolic activity, vascular occlusion or both, there is a water deficit that, in turn, causes premature dulling or bending of the flower peduncle (neck bend) (^{Reddy and Singh, 1996}; ^{Macnish et al., 2009}; ^{Hussen and Hassen, 2013}).

Colony forming units (CFU)

The floral stems of the three cultivars in wet management in both phases on the first day of the VF had more CFU (136 to 189 CFU mL^-1), than those that had dry handling (63 to 134 CFU mL^-1). The same behavior was present in the evaluation on the fourth day, since it showed a significant increase in CFUs in the stems that remained wet in all the phases (Figure 2).

Figure 2 Colony forming units (CFU mL^-1 ± standard error; n=2) in three roses (Rosa hybrida) cultivars handled dry and/or wet before (phase I) and during storage (phase II). Different letters on the columns indicate statistical differences in each cultivar (Tukey, p≤0.05). 

The number of CFU in the vase solution is a limiting factor in the water absorption at the base of the stems. In this regard, the rose has been identified as one of the most susceptible to microorganism species presence that obstructs water entry and reduces VF (^{Bleeksma and van Doorn, 2003}, ^{Arévalo-Galarza et al., 2012}). Concentrations of 10⁷ UFC mL^-1 decreased the water conductivity (Kh) and lifespan in ‘Sonia’ roses in vase, while the occlusion of water flow occurred when at the basal section of the flowers stem the number of bacteria was equal to or greater than 10⁸ UFC mL^-1 (^{de Witte and van Doorn, 1988}). In the present study the vase solutions presented average values lower than 10⁷ UFC mL^-1; and confirmed the disadvantage of wet management, since, if the initial amount of bacteria at the base of the stems is significant and have poor hygiene during handling, bacteria occlusion risks is increased.

Number of stomata and opening of the stomatal pore

Topaz cultivar leaves showed 73.8 stomata mm^-2, this value was significantly higher than those of the Blush and Freedom cultivars (54 mm^-2 stomata). These values are close to those documented in the leaves of the Vega and Grand Gala of rose cultivars (57.6 and 60.4 stomata mm^-2) (^{Hernández-Hernández et al., 2009}). Stomata density depends on genetic characteristics of species, their cultivar and the growth conditions; however, the opening area of the stomata pore can rapidly vary due to treatment effect. The first day in vase, cultivars with dry handling at the first phase (S_FI-S_FII and S_FI-H_FII) had smaller stomatal pore aperture compared with those in wet management (H_FI-S_FII and H_FI-H_FII). This indicates that dry handling in the first phase determined stomatal opening during the post-harvest period and resulted in a larger vase lifespan. Thus, stomatal opening of the Freedom cultivar was the largest (89 to 102 µm²), with which it’s life in vase was the shortest; in contrast, the Topaz cultivar, which showed the smallest stomatal opening (22.7 to 32 µm²), had the longest VF (Figure 3). These results indicate that the effect of the management type modified stomatal opening during the postharvest stage.

Figure 3 Stomatal opening (± standard error; n=15) of three roses (Rosa hybrida) cultivars handled dry and/or wet before (phase I) and during storage (phase II). Different letters on the columns indicate statistical differences in each cultivar (Tukey, p≤0.05). 

Incidence of Botrytis sp.

The incidence of Botrytis sp. in petals of the three cultivars was lower when the flower stems were handled in dry conditions in both phases (S_FI-S_FII). Wet handling during phase II (S_FI-H_FII or H_FI-H_FII) increased the pathogen incidence in the Freedom cultivar by 50 % and 75 % in the Blush and Topaz ones. In contrast, when the flower stems were handled in wet conditions during the first phase and dry during the second (H_FI-S_FII) Botrytis sp. incidence was moderate (Figure 4).

Figure 4 Botrytis sp. incidence (± standard error; n=10) in three rose (Rosa hybrida) cultivars handled dry and/ or wet before (phase I) and during storage (phase II). The values presented as the mean ± standard error (n=10). Different letters on the columns indicate statistical differences in each cultivar (Tukey, p≤0.05). 

In this regard, B. cinerea conidia germination depends on the handling and temperature changes caused by condensed water on the petals surface and the cultivar (^{Harkema et al., 2013}). So, excess humidity and the refrigeration temperature in stage II in our study led to an increase in the condensed water on the petals and symptoms development thereon.