Introduction

Cobalt (Co) is an essential element for humans, animals and prokaryotes. In higher plants, a physiological function of this element has not been identified so far; except for legumes that required for nitrogen-fixing performed by symbiotic bacteria (^{Witte et al., 2002}). Despite its non-essentiality, we have identified that Co levels in plant tissue, under 10 mg g^-1 dry weight, may be beneficial (^{Palit et al., 1994}).

In tomato (Solanum lycopersicum L.) grown in hydroponics, it was shown that the addition of Co at a concentration of 2.5 mg L^-1 of nutrient solution, favoured the absorption of nitrogen (N), phosphorus (P) and potassium (K) (^{Boureto et al., 2001}). ^{Gad (2005)} also in tomato, indicating that the supply of Co at a dose of 7.5 mg L^-1 of nutrient solution significantly increased growth parameters, yield and nutrient concentration; whereas fruits increased total soluble solids, total soluble sugar and ascorbic acid concentration and reduced acidity. In maize (Zea mays L.) established in soil, Co concentrations of 50 mg kg^-1 increased seedling vigor, number of pods per plant and seeds per pod was found; and also increase the concentration of photosynthetic pigments such as chlorophyll a, b and total, content of amino acids, proteins and starches (^{Jaleel et al., 2009}).

Under conditions of osmotic stress, it has also been reported positive effects of the addition of Co; e.g. in potato seedlings subjected to osmotic stress, the addition of it in concentrations of 25 mmol L^-1 inhibited ethylene production rate significantly decreased concentrations of oxygen free radicals and increased the activity of antioxidant enzymes (^{Li et al., 2005}). Also, the effect of Co on parameters of different post-harvest ornamental species has been studied. In rose (Rosa hybrida) cv. Samantha, inhibited the vascular blockade of Co stems, allowing a high flow of water through them, leading to a significant increase in water uptake of cut f lowers (Reddy, 1998). In carnation (Dianthus caryiohyllus L.), treatment with Co in concentrations of 50 to 100 mg L^-1, significantly increased vase life compared with the control; also observed a reduction in the rate of production of ethylene (^{Jamali and Rahemi, 2011}). In Lilium oriental hybrid 'Star Gazer "and" Star Fighter "Co concentrations of 0.1 and 0.2 mM showed delayed senescence symptoms of flower stalks (^{Mandujano-Piña et al., 2012}).

Gladiola is a species of high demand in our country, but the longevity of the flowers is quite short. The vase life of individual rods is between 4 and 6 days (^{Hatamzadeh et al., 2012}). Although there are studies that have shown that senescence of petals is not affected by exogenous ethylene, as well as inhibitors of ethylene (^{Ezhilmathi et al., 2007}), the latter response attributed to Co, this research aims to evaluate effects of mercury in other post-harvest parameters.

Materials and methods

Experimental conditions. This research was conducted under laboratory conditions, with average daytime temperatures of 25.8 ^°C and 25.3 ^°C atnight; relative humidity of 33.4 and 39.8% day and night, respectively. The average light intensity was 19 and two lumens, day and night, respectively

Plant material. Floral Rods of gladiola (Gladiolus grandiflorus Hort.) Cv. Borrega Roja were purchased in the local market, the flowers that make up the inf lorescence were closed entirely. The inflorescences were constituted by 12 to 16 flowers and its length was 92 cm.

Designing treatments and experimental design. From CoCl₂ 6H₂O (Sigma Aldrich, ACS Reagent 98%) were prepared with deionized water solutions with concentrations of 0.3 and 0.6 Co mM, using deionized water as a control. 250 ml of each of the solutions were placed in glass vases, with a flower stem, same as the experimental unit. Each treatment had 10 replications evaluated and distributed on tables completely random.

Results and discussion

Water absorption by floral rods. Absorption of floral stem water after 48, 96 and 144 h of vase set, shown in Table 1.

The term of the vase life for cut flowers is characterized by wilting associated with an imbalance between the development of water uptake through the xylem conduits in stems and water loss through the stomates and other structures in leaves and other organs (^{Lü et al., 2011}). Co is an element that has shown positive effects on water consumption of floral stems. In tuberose (Polianthes tuberosa L.), treatment in vase with CoCl₂ at a concentration of 300 mg L^-1 (2.3 mM Co), 9.3% increased the volume of water absorbed relative to stems that were not treated with this item (^{Mohammad et al., 2012}). So, in Canada goldenrod (Solidago canadensis L.) was reported in the Co concentration of 0.5 mM to 4% sucrose significantly increased water absorption (Reddy and Patil, 1997).

In Rosa hybrida cv. Samantha, ^{Mandujano-Piña et al. (2012)} reported 39% increase in water consumption of flowering stems of Lilium oriental hybrid 'Star Gazer'y' Star Fighter', when they were treated in vase for three days with 0.1 mM Co. This research observed positive effect of Co on the water uptake, the sampling performed only after 144 h of treatment (6 days). The rods treated with 0.3 and 0.6 mM Co absorbed a higher volume 21.11 and 31.48%, respectively, compared with the control. While in the assessments made after 48 and 96 h, there were no statistical differences in absorbed volumes between treatments, however these were always higher in Co-treated rods, regardless of the concentration (Table 1). The positive effect of Co in water absorption due to this element decreases the vascular blockade by suppressing microbial growth (Patil and Reddy, 1997).

Percentage weight loss of f lower rods. After 48 h of treatment, weight loss was 2.56, 1.86 and 1.64% in the control treatments, 0.3 and 0.6 mM of Co, respectively; however, there was no statistical difference. In the second evaluation (96 h) dtatistical differences between treatments were presented; and conclude that the higher weight loss (8.2%) occurred in rods treated with the highest concentration of Co; however, this treatment was not statistically different from the control. however, the lowest percentage of weight loss (5.28%) was observed in treatment with rods the lower dose of Co; but equally, this was not statistically different to the control (Figure 1).

Figure 1 Percentage weight loss of gladiola floral stem treated with different concentrations of cobalt. Mean ± SD with different letters at each measurement indicate significant differences (Tukey, p≤ 0.05) between treatments. 

In rose (Rosa hybrida) cv. Samantha, it was found that Co besides inhibiting the vascular blockage stems causes a partial closure of stomata, thus reducing the ratio of water loss/water absorption and maintains a high water potential on cut rose. This results in maintaining high fresh weights leading to an increased vase life (^{Reddy, 1988}).

In this research, it is clear that the low concentration of Co added were taken in the three samplings, the lowest percentages of weight loss in floral stem. In the control, after 144 h the higher loss weight recorded was by 10.5% of initial weight. ^{Moraes et al. (1999)} reported that losses in fresh weight of flowering stems between 10 and 15% can cause tissue death.

Kinetics anthesis. Two days after the establishment of the rods in vase, the flowers were observed in three states of openness, recording no statistical differences between treatments (Figure 2A). The opening phase A4 (flower opening to 100%) is after three days of treatment observed in 1.25% of the flowers for the control rods, without this significant percentage; the area A1, A2 and A3 phases were not statistically different between treatments (Figure 2B). In sampling conducted five days after the establishment of the research, statistical differences between treatments flowers were recorded in open state A1 (start of pigmentation in tight buttons), with a higher percentage of the control inflorescences; consequently, the three chronologically later stages (A2, A3 and A4), the control had a lower percentage of inflorescences but were not statistically different from other treatments. In this sampling took place initiated floral senescence (S), not being statistically different between treatments, with rates of 5.95, 7.09 and 7.43% in the control treatments, 0.3 and 0.6 mM of Co, respectively (Figure 2C). After 7 days in vase, no statistical differences between treatments in the stages of flower opening and senescence, noting that the treatment with Co, the percentage of flowers per inflorescence showed senescence was higher than 10%, while in the control 8.21% (Figure 2D).

Figure 2 Percentage of flower opening at 2, 3, 5 and 7 days (A, B, C and D respectively), gladiolus flowers treated rods with different concentrations of cobalt. A1= closed button in which we see starting pigmentation; A2= button with initial opening; A3= button with opening of 50%; A4= 100% button opening; and S= incipient onset senescence indicated by wilting petals margins. Mean ± SD with different letters at each measurement indicate significant differences (Tukey, p≤ 0.05) between treatments. 

In previous studies in gladiolus cv. Friendship, it was reported that treatment with CoCl₂ had a positive effect on longevity and bud opening (^{Murali and Reddy, 1993}); however, the authors do not consider the effect that could be the transport of sucrose added, the Co addition, since it has been shown that this element increases the absorption of water, in this case, consequently the absorption of sucrose.

Partition of dry in floral rods. After 7 days in vase, the weight of total dry matter (Figure 3A) was statistically higher in rods treated with 0.3 mM of Co; rods between the control treatment and those treated with 0.6 mM of Co had no statistical difference. With respect to weight of dry matter of leaves per rod, the highest average was recorded in the 0.3 mM treatment, exceeding 3.15% weight of leaves recorded in rods of the control, and this increase statistically not different to the control (Figure 3B). The dry matter weight of inflorescences was not statistically different between the treatments (Figure 3C). Treatments with Co, particularly at a concentration of 0.3 mM resulted in a significant increase in dry matter of the stem (Figure 3D).

Figure 3 Total dry biomass by flower stem and organ (leaf, stem and inflorescence) of gladiola treated for ten days in vase with different concentrations of cobalt. Mean ± SD with different letters in each sub-figure indicate significant differences (Tukey, p≤ 0.05) between treatments. 

The positive effect of Co on the weight of the dry biomass of cut flowers has been reported in other species as well. In carnation (Dianthus caryophyllus L.), Co concentrations of 75 and 100 mg L^-1 in post-harvest (1.27 and 1.7 mM of Co, respectively) did not affect the dry biomass of the stems; while 50 mg L^-1 of this (0.85 mM) element significantly reduced the weight of the f lower stems, compared with the absence of treatment with Co (^{Jamali and Rahemi, 2011}). Also ^{Noghani et al. (2012)} reported that, the treatment with 6.13 mM of Co (800 mg L^-1 CoCl₂) and 0.2% (NH₄)₂ SO⁴, significantly increased the dry biomass of floral stem of tuberose (Polianthes tuberose L.).

The evaluated treatments did not affect the dry weight of inflorescences; however, a negative relationship between it and the concentration of Co is seen. This trend has been reported in cut flowers treated with other metals are seen; such is the case of rose stems cv. Black Magic, which when treated with Al, Cu and Ag had lower petal dry weight than the control; however, these reductions were not significant at all (^{Hajizadeh et al., 2012}).

It is also noteworthy the inverse relationship between petals dry biomass (Figure 3) and the percentage of senescent flowers after 7 days in vase (Figure 2D); that is, the lower petals dry biomass is associated with a higher percentage of senescing flowers, because the petals are visibly wilted when epidermal cells lose turgor (^{Van Doorn, 2001}).

Chlorophyll leaf concentration. Figure 4 shows the results of concentration of chlorophyll in leaves of gladiolus cut flower treated with different levels of Co. Chlorophyll b was statistically higher in leaves treated rods Co, regardless of the concentration thereof. The concentrations of chlorophyll a and all are superior to treatment with 0.3 mM without presenting statistical difference with this treatment consisting of 0.6 mM Co; while, statistically superior to the control (Figure 4).

Figure 4 Foliar concentration of chlorophyll a, b and total in gladiola rods treated for 10 days in a vase with different concentrations of cobalt. Mean ± SD with different letters in each variable indicate significant differences (Tukey, p≤ 0.05) between treatments. 

The visible signs of leaf senescence is the loss of green color, caused by chlorophyll degradation; in particular, cut flowers senescence is closely related to the significant reduction of the energy necessary for synthesis reactions (^{Faraji et al., 2011}) process. In this research, the results confirm that, the Co is an element that delayed this process, especially in lower doses (Figure 4). In f lowering stems of Lilium "Star Gazer" and "Star Fighter", have also been reported positive effects on chlorophyll degradation during vase life after 13 days, where the addition of Co at concentrations of 0.2 and 0.8 mM submitted SPAD readings values ​significantly higher than those recorded without providing Co (^{Mandujano-Piña et al., 2012}).

Other metals such as Al, added from Al₂ (SO₄)₃ in rose cv. "Cherry Brandy" at a concentration of 100 mg L^-1, resulting in a higher value of SPAD readings compared with the control; concluded that this positive effect of Al in chlorophyll is due to improved water relations in the flower stems (^{Jowkar et al., 2012}.) matching results with those presented in the consumption of water and dry matter in Figures 1 and 3, respectively.

Concentration and N content. The concentration of N in leaves, flowers and inflorescences constituting floral stem of gladiola was not statistically different between treatments observed the following order of concentration for organ: inflorescence> leaves> stems (Table 2).

In Gladiolus caryophyllaceus during maturing (210 days after sprouting) N concentrations in stems and flowers were 11.19 and 4.12 mg kg^-1 of dry matter, respectively (^{Hocking, 1993}). Both concentrations were lower than those found in this study; particularly in the inflorescence. However, it is important to mention that the remobilization of nutrients in senescence is variable among those cut flower stems and stalks that remain on the plant (^{Jones, 2013}).

Coinciding with this investigation, Co had no significant effect on the concentration of N in flower stems of two cultivars of Lilium hybrid post-harvest; on the contrary, the addition of this metal in doses of 0.1, 0.2, 0.4 and 0.8 mM concentrations significantly increased N in leaves (Mandujano-Pineapple et al., 2012).

The results show that there is no relationship between leaf N concentration (Table 2) and leaf chlorophyll concentrations (Figure 3). In gladiola cultivated generally found a positive relationship between nitrogen and chlorophyll, since the former is a structural component of the latter; e.g. Gladiolus hybrida cv. Sancerre, a positive and highly significant relationship between the dose of nitrogen fertilization and foliar chlorophyll concentration was observed (^{Sewedan et al., 2012}).

The total N content in floral stem showed a significant reduction with treatment consisting of 0.6 Mm (Figure 5A). Also, leaf N content was significantly reduced when the rods were treated with Co; observing a negative relationship between it and the Co concentration supplied (Figure 5B). The N content in inflorescences was not statistically different between treatments (Figure 5C). The amount of N in stems, was statistically superior when floral stem were treated with 0.3 mM of Co (Figure 5D).

Figure 5 Content of total nitrogen and by organ of rods of gladiola treated for ten days in vase with different concentrations of cobalt. Mean ± SD with different letters in each sub-figure indicate significant differences (Tukey, p≤ 0.05) between treatments. 

It has been reported in Gladiolus grandiflorus that water deficit causes delay in the translocation of photo-assimilates from source leaves to sink organs (^{Robinson, 1983}). Considering this, the results confirm that while Co had no influence on the total N content of floral stem; this element does affect the partition of N because its positive effect on water status, promoting their translocation from leaves to stems (Figure 5); this statement supported by the significant increase in dry biomass (Figure 3).

Conclusions

The results indicate that, the Co has positive effects on gladiola post-harvest parameters; particularly those related to the water status of cut flowers: minor loss of fresh weight, dry matter weight and, partitioning of N. In addition, the Co retards the degradation of photosynthetic pigments.