Preharvest Spraying of CaCl₂ Alleviates the Scape Bending of Gerbera ‘Harmony’ Flowers by Strengthening the Pectin Crosslinks through Ca²⁺ Bonds

Abstract

Scape bending is the primary cause shortening the vase life of cut gerbera flowers (Gerbera jamesonii ‘Harmony’). A previous study showed that this bending is closely related to the scape hardness, which is attributed to cell wall rigidity maintained by calcium ion bonds. In this experiment, the developing gerbera scape was sprayed with 0.5 and 1% calcium chloride to determine whether exogenous calcium directly influences scape hardness and whether it is related to pectin crosslinks. The calcium spray hardened the scape by 12% and efficiently reduced bending, thereby prolonging the vase life of the cut gerbera flowers. A 9% increase in calcium ions (Ca²⁺) was detected in the CaCl₂-treated flowers compared to the control flowers. Additionally, the pectin content increased by up to 14% in the CaCl₂-treated flowers as compared to that in the control. Pectins are the main polysaccharides of cell walls that impact plant tissue integrity and rigidity, with calcium ions acting as bonds for pectin crosslinking. Calcium treatment efficiently increased the content of total pectin in the cell walls and slowed the conversion of insoluble pectin (IP) to water-soluble pectin (WSP) during vase life. The results suggest that exogenously applied calcium increases the Ca²⁺ in cellular tissue and affects the pectin levels, which may aid in increasing the scape hardness by strengthening the calcium–pectin combination in cell walls.

1. Introduction

Cut gerberas are one of the most popular cut flowers in the global flower market because of their colorful shiny ray florets, variable shapes, and colors [1]. However, consumers often experience a short vase life under room conditions [2]. The vase life of cut gerbera flowers is often cut short because of scape bending, even if the ray petals are not wilting. A previous study demonstrated that scape bending primarily occurred in the neck region of the gerbera stem, 12 cm below the floral head [3]. Several studies have reported that scape bending may also be related to a lack of mechanical support [4,5], adverse water relations, or lack of scape collenchyma development [6], considering that plant cell walls determine the mechanical strength of the plant structure [7].

Calcium is a crucial regulator of plant growth and development and is taken up as a divalent cation [8]. Moreover, it has a structural role in the cell walls and membranes, forming calcium pectates in the middle lamella during cell division [9]. Pectin is a significant structural polysaccharide that maintains the hardness of the cell wall [10]. Calcium ions preserve cell wall hardness by cross-linking pectin polymers (pectin), forming cell wall networks. The accumulation of calcium facilitates an increase in the mechanical strength of the cell wall [11,12].

CaCl₂ spray treatments have been effective for the physicochemical aspects of cell wall components in peaches [13]. They have also been suitable for increasing the calcium content in apples [14]. Pulse treatments with CaCl₂ have been reported to be effective against scape bending in gerbera ‘Tamara’ [15]. A preservative solution with CaCl₂ also delayed scape bending, increased the vase life, and positively affected the color of gerbera ‘Tamara’ [16]. Preharvest and postharvest treatment of gerbera with CaCl₂ increased its vase life and decreased scape bending, with the degree of response differing by the cultivar [17,18].

However, previous studies have not described the direct relationship between exogenously applied calcium and pectin in cut gerbera flowers, and the reasons for these positive effects on flower quality remain unclear; we hypothesize that these effects may be attributed to the binding of calcium with pectin and the consequent increase in cell wall hardness. This study, therefore, assessed the effects of exogenous calcium on scape quality, including bending symptoms, hardness, and vase life of cut gerbera flowers, and discussed the influence of internal calcium accumulation on the mechanical strength of the cell wall via pectin complex durability.

2. Materials and Methods

2.1. Experiment Design and Treatments

An experiment was designed that comprised preharvest CaCl₂ treatments and their postharvest results on the flower quality of cut gerbera. Three-year-old standard gerbera cultivar ‘Harmony’ plants with pink and semi-double inflorescences were bred by the National Institute of Horticultural and Herbal Science, Rural Development Administration (RDA) in 2010 [19]. Tissue-cultured plantlets were planted in the experimental greenhouse of the University of Seoul in October 2015 and then cultivated in coir media by drip irrigation with a Netherlands nutrient solution for gerbera (EC of 1.1 ± 0.1 dS∙m⁻² and pH of 5.9 ± 0.1). A 250 mL amount of nutrient solution was applied daily per plant with Ca of 6.0 me∙L⁻¹, at five to seven separate discharges per day. The calcium spray treatment was performed in June 2018 during the summer season when bending is known to often occur. The greenhouse environment (day/night) was 33.2 °C/20.7 °C AT and 44.7%/75.2% RH. Calcium chloride solutions of 0, 0.5, and 1% (w/v) were prepared with 95% anhydrous CaCl₂ (Yakuri Pure Chemicals Co., LTD., Kyoto, Japan). Each scape was sprayed once at each stage of scape development (three times in total) with 10 mL of CaCl₂ solution [20]. A developing scape was separated into three stages: ray flower size of less than 0.5 cm (S1), 2.5 cm long ray flower with starting to turn pink (S2), and fully opened flower with unmatured stamens (S3) (Figure 1). A calcium solution was sprayed on all sides in turns around the neck region of the scape, 12 cm below the floral head, where the scape usually bends [3].

The flowers were harvested at the optimum commercial stage when the outer 2 to 3 rows of disc florets were perpendicular to the stalk [21]. Harvested flowers were then dipped in distilled water and promptly transported to the laboratory in 10 min without environmental treatment. The postharvest test used uniformly cut flowers with an inflorescence diameter of 11 ± 1 cm and a scape length of 60 ± 5 cm. The harvested flowers were placed separately in glass bottles (1 L) filled with 250 mL of distilled water. The water covered 5 cm of the sides of the stems in the glass bottles. The flowers were monitored in an environmentally controlled growth chamber with the following room conditions: 25.1 ± 0.4 ℃ AT, 37.7 ± 5.2% RH, 20 µmol·m⁻²·s⁻¹ PPFD under fluorescent light, and a 12 h photoperiod.

2.2. Scape Bending and Vase Life

The degree of scape bending and hardness during the vase life test was measured. The vase life of cut flowers is determined when they lose their ornamental value, showing signs of ray flower drop, wilting, curling, de-coloration, and scape bending [22]. Scape bending, defined as a scape angle of over 90°, was measured daily until the end of vase life [3]. The scape hardness was measured with a penetrometer (FHM-1, Takemura, Tokyo, Japan). The penetrometer was perpendicular to the gerbera scape 12 cm below the floral head and measured by applying force until the surface of the scape was broken.

2.3. Calcium Content

The scape was sampled 12 cm from the floral head of harvested cut gerbera flowers. The samples were wet digested with 50% HclO₄-H₂SO₄. The calcium content was then measured using an atomic absorption spectrophotometer (Analyst 400, Perkin-Elmer Co., Waltham, MA, USA) adhering to the procedure described by RDA [23].

2.4. Pectin Extraction and Fraction

The cell wall material (CWM) of the sampled scape was extracted adhering to the procedure described by Campbell et al. [24], after which 4 mL of 95% ethanol per 1 g sample was boiled in water bath at 80 ℃ for 20 min to inactivate the cell wall-modifying enzymes. The sample was then homogenized and centrifuged at 10,500 RCF for 10 min, after which the supernatant was decanted. The pellet was resuspended by homogenization in 80% ethanol and re-centrifuged (10,500 RCF for 10 min), after which the supernatant was decanted again. This step was repeated until the supernatant became colorless. The pellet obtained from the last centrifuge was dried at room temperature and washed with acetone (10 mL). The redried pellet was referred to as CWM. The CWM was then fractionated as per the procedure described by Manganaris et al. [13], which is a modified method [25]. The CWM sample (5 mg) was suspended in distilled water for 1 h and centrifuged (10,500 RCF for 25 min), after which the supernatant was collected. The pellet was resuspended in distilled water for 1 h and centrifuged (10,500 RCF for 10 min). The two supernatants were combined and considered as the water-soluble pectin fraction (WSP). The pellet from the second centrifugation was dissolved in 2 mL concentrated H₂SO₄ and was considered as the insoluble pectin fraction (IP).

2.5. WSP and IP Analysis

The pectin content was analyzed from the WSP and IP fractions using Bitter and Muir’s modified carbazole colorimetric method [26]. Each sample (0.5 mL) of the WSP and IP fractions was mixed with 3 mL H₂SO₄, cooled in a refrigerator, and then heated in a water bath at 30 ℃. After 10 min, 0.1 mL of carbazole reagent was added, and the sample was then left for 2 h for maximal color development. The absorbance of the sample was measured using a UV-VIS spectrophotometer (UV-2450, Shimadzu, Kyoto, Japan) at 530 nm. D-(+)-Galacturonic acid monohydrate (0.04 g) was dissolved in 1 L distilled water to prepare a galacturonic acid standard (40 µg/mL). The standard was then divided into 0, 20, and 40 µg/mL and stabilized at 4 ℃ for one day. A calibration curve was prepared using the UV-VIS spectrophotometer at wavelength of 530 nm.

2.6. Statistical Analysis

Analysis of variance (ANOVA) was performed using the SAS statistical software package (ver. 9.4, SAS Institute Inc., Cary, NC, USA). The difference between the means was evaluated using the least significant difference (LSD, p = 0.05). A Pearson’s correlation analysis was additionally performed.

3. Results

3.1. Effect of Exogenous CaCl₂ on Flower Quality

The preharvest spraying of 0.5% and 1.0% CaCl₂ solutions increased the vase life of cut gerbera flowers by 49% and reduced the scape bending by 50% (

Table 1. Effects of preharvest spraying of CaCl₂ on the vase life and scape bending symptoms of gerbera ‘Harmony’ cut flowers.

Treatment (CaCl2)	Vase Life (Days)	Scape Bending (%)	Scape Angle (°)
Control	3.9 b z	50.0	75.1 b
0.5%	5.8 a	23.3	49.5 a
1%	5.7 a	26.7	50.6 a
Significance	**	-	*

^z Mean separation within columns by LSD at p = 0.05 (n = 30). *, ** mean significant at p < 0.05 or 0.01, respectively, ANOVA.

). The exogenous Ca²⁺ spray significantly strengthened scape hardness by 12%, increasing endogenous calcium content in the scape tissue before harvest (

Table 2. Effects of preharvest spraying of CaCl₂ on the scape hardness and Ca²⁺ content of gerbera ‘Harmony’ cut flowers at the start and end of vase life.

Treatment (CaCl2)	Scape Hardness (kg)		Calcium Content (Ca2+mg·L−1)
	Start	End	Start	End
Control	0.34 b z	0.29 b	17.99 b	16.93 b
0.5%	0.38 a	0.36 a	19.67 a	18.38 a
1%	0.38 a	0.35 a	19.86 a	18.48 a
Significance	**	**	**	*

^z Mean separation within columns by LSD at p = 0.05 (n = 30). *, ** mean significant at p < 0.05 or 0.01, respectively, ANOVA.

). The significant increase in the calcium content in the scape tissue indicates that the calcium ions were moved into the inner tissue of the scape due to the exogenous CaCl₂ spray. The scape hardness and calcium content were proportionally reduced during vase life and demonstrated almost the same patterns at the end of vase life. However, there was no statistically significant difference between the 0.5% and 1% CaCl₂ concentrations.

3.2. Effect of Exogenous CaCl₂ on Pectin Content

The exogenous calcium sprayed during the preharvest period effectively accumulated pectin in the tissue of the flowering scape (

Table 3. Effects of preharvest spraying of CaCl₂ on pectin content in the cell walls of gerbera ‘Harmony’ cut flowers at the start and end of vase life.

Treatment (CaCl2)	Pectin (μg·mg−1 CWM)
	WSP		IP		Total Pectin		IP/Total Pectin
	Start	End	Start	End	Start	End	Start	End
Control	0.91 c z	1.63	2.74 b	2.15 c	3.65 c	3.78 c	0.75 a	0.57 b
0.5%	1.09 b	1.53	2.77 a	2.41 b	3.86 b	3.94 b	0.72 a	0.61 a
1%	1.36 a	1.66	2.83 a	2.58 a	4.19 a	4.23 a	0.68 b	0.61 a
Significance	***	ns	*	***	***	***	*	**

CWM: cell wall material; WSP: water-soluble pectin; IP: insoluble pectin. ^z Mean separation within columns by LSD at p = 0.05 (n = 30). ns, non-significant; *, **, *** mean significant at p < 0.05, 0.01 or 0.001, respectively, ANOVA.

). Water-soluble and water-insoluble pectin contents (WSP or IP) and IP ratios were significantly higher in the scapes sprayed with CaCl₂. The effect of exogenous calcium was significantly concentration-dependent and more direct on WSP than IP. The WSP increased within the scape during vase life, while the IP decreased (Table 3). During the postharvest period, the WSP, IP, and IP ratios changed significantly. However, there was no significant change in the total pectin (WSP + IP). Considering that more calcium ions (Ca²⁺) were detected in the CaCl₂-treated flowers than in control flowers, the pectin content increased by up to 14% in the CaCl₂-treated flowers compared with that in the control.

The total pectin content increased by 14.8% with the application of 1% CaCl₂ and by 5.8% with 0.5% CaCl₂. Calcium ions (Ca²⁺) increased in the cell walls by exogenous calcium spray demonstrated a positive effect on vase life by enhancing the strength of the flowering scape through pectin increase, resulting in a significant decrease in scape bending (Figure 2). The exogenous calcium directly increased the cellular Ca²⁺ levels (r = 0.047 **), WSP (r = 0.96 ***), and total pectin (r = 0.86 ***). This consequently improved the flower quality in gerbera ‘Harmony’ by extending the vase life (r = 0.36 *) and decreasing the scape bending (r = −0.40 **) (

Table 4. Correlation between variables of flower quality trait of cut gerbera ‘Harmony’ treated with preharvest CaCl₂ spray.

Variable	VL	SB	SA	SH	Ca2+	WSP	IP	Total Pectin
CaCl2	0.36 *	−0.40	−0.30 *	0.50 **	0.47 **	0.96 ***	0.24 *	0.86 ***
VL		−0.38	0.52	0.35 *	0.33 *	0.31 *	0.52 *	0.48 *
SB			0.87 **	−0.31 **	−0.42 **	−0.16	−0.06	−0.15
SA				0.38 **	−0.31	−0.27 *	−0.09	−0.26 *
SH					0.55 *	0.45 **	0.11	0.39 **
Ca2+ WSP						0.42 **	0.10	0.38 **
							0.10	0.80 **
IP								0.68 **

VL: vase life; SB: scape bending; SA: scape angle; SH: scape hardness; Ca²⁺: calcium ion level; WSP: water-soluble pectin; IP: insoluble pectin. The data for WSP, IP, and total pectin are the values at the start of vase life. *, **, *** mean significant at p < 0.05, 0.01 or 0.001, respectively.

).

4. Discussion

Authors Park and Kim [3] reported shortening of the vase life of Gerbera jamesonii ‘Harmony’ with scape bending. In this experiment, preharvest spraying of exogenous CaCl₂ significantly improved the scape strength by 12%. It efficiently reduced the symptoms of scape bending by 50% and prolonged the vase life of cut gerbera flowers by 48.7% (Table 1). These results coincide with previous research showing that preharvest and postharvest treatment of gerbera with CaCl₂ extended the vase life and reduced the occurrence of scape bending, depending on the cultivar [16,17,18]. The flower longevity of cut roses was extended by applying CaCl₂ to the vase solution [27]. The scape angle was also improved by spraying with CaCl₂ during vase life. However, this was not significantly different among treatments before the vase life test. A previous study showed that scape bending is closely related to the scape hardness, which depends on the cell wall rigidity and its calcium ion bonds [12]. This result is similar to that of previous research showing that pulse treatment with CaCl₂ delayed the scape bending of cut gerbera flowers [15].

The effect of preharvest spraying of calcium chloride relied on the increased endogenous calcium content in the scape tissue before harvest. The Ca²⁺ content within the flowering scape increased by 10.4% as compared to that of the control. This effect lasted throughout the vase life (Table 2). A similar report showed that preharvest spraying with 4% CaCl₂ after bud emergence significantly strengthened the stem hardness in Paeonia lactiflora ‘Da Fugui’ [28]. At the end of their vase life, gerbera cultivars ‘Campitano’, ‘Dino’, and ‘Testarossa’, injected with 1.0% CaCl₂, followed by dipping in 1.0% CaCl₂ postharvest compared to control, showed a significant extension of vase life [17]. ‘Sangria’ sprayed with 1.0–1.5% CaCl₂ only demonstrated relatively high scape calcium concentrations compared to those in the control [17]. Treatments with 1.5% CaCl₂ considerably increased scape calcium content, but did not contribute any additional increase in the vase life of the flowers [17]. The leaf calcium content in the leaves of gerbera ‘G4′ and ‘OR’ increased after spraying with CaCl₂ until the first harvest [18].

As presented in Table 3, the calcium treatment efficiently increased the total pectin amount in the cell walls, especially the WSP. It was confirmed that IP was converted to WSP during the postharvest period in gerbera flowers. Preharvest calcium spraying delayed this conversion in this experiment. Manganaris et al. [13] reported that insoluble pectin decreased within the cell walls as the cell aged, and markedly decreased at the end of vase life. Calcium ions preserve cell wall hardness by crosslinking pectic polymers to form a cell wall network. Calcium accumulation thereby increases the mechanical strength of cell walls [11,12].

The results presented in Table 3 coincide with a previous study that demonstrated that preharvest spraying with CaCl₂ reduced the WSP content and significantly increased the IP content in peony stems [28]. Pectins have been shown to directly extend the vase life of carnation flowers because high levels of insoluble pectins in petal cell walls correspond with a long vase life [29]. Persimmon fruits with higher calcium concentrations demonstrate higher levels of calcium pectate, maintaining tissue resistance to degradation through polygalacturonase [30]. The results of this study demonstrated that greater reductions in calcium content, IP, and IP ratio and increases in WSP in control cut gerbera stems during vase life, representing the cell wall of control, resulted in somewhat faster aging (Table 3). In peach, the increase in cell wall calcium corresponded to an increase in IP calcium but not in WSP calcium [13]. It may, therefore, be confirmed that the positive effect of exogenous calcium spray in this experiment was due to calcium fortification in the IP. In this experiment, the Ca²⁺ that accumulated in the cell walls due to exogenous calcium spraying effectively extended the vase life by enhancing the flowering scape strength and increasing the amount of pectin; this resulted in a significant decrease in the scape bending phenomenon (Figure 2). Pectins are the primary polysaccharides of the cell walls impacting plant tissue integrity and rigidity, and calcium ions act as bonds for pectin crosslinking [10]. As presented in Table 4, calcium treatment efficiently increased the total pectin amount in cell walls and slowed the IP conversion of WSP during vase life.

5. Conclusions

In conclusion, exogenously applied calcium increases the Ca²⁺ in cellular tissue and affects pectin levels; this may aid in increasing the scape hardness by strengthening the calcium–pectin combination in the cell walls. However, the results were obtained from only one variety of cut gerbera, ‘Harmony’. Therefore, it is necessary to test more varieties of cut gerbera to confirm the effect of exogenous calcium in the future. Further studies are required to understand the mechanism of increasing WSP at vase-life start by preharvest calcium spray.