Increasing the Vase Life of Chrysanthemum Cut Flowers by Using Silver and Zinc Nanoparticles

Cut flowers are horticultural products that have great potential to be developed. Efforts to maintain quality and extend the shelf life of cut flowers are very important to obtain a product that is accepted in the market. The main problems of chrysanthemum cut flowers are the leaves easily turning yellow, wilting, and failure to fully open flowers. This study aimed to obtain the best pulsing solution formulation that increases vase life and maintains the freshness of chrysanthemum cut flowers. Pulsing solution treatment was carried out on chrysanthemum cut flowers during the evaluation period. Pulsing solution treatment consisted of control, AgNO3, nano-Ag (NAg), ZnO, and nano-Zn (NZn). The results showed that NAg20 treatment increased the vase life of chrysanthemum cut flowers up to 23 days, which was 19 days longer than the control. Nano-Ag inhibits bacterial growth, flower wilting, color degradation, and carotenoids. In addition, nano-Ag increased the size of the bloom-flower diameter. Considering the results of all postharvest quality parameters mentioned above, NAg20 extends the vase life of chrysanthemum cut flowers.


Introduction
Cut fowers are horticultural products that have great potential to be developed.Chrysanthemum (Chrysanthemum morifolium) is one of the leading cut fowers in the international market which is widely used as decoration which belongs to the Asteraceae family [1,2].In addition, many consumers like chrysanthemums because of their varied shapes, colors, and sizes [1].Eforts to maintain quality and extend the shelf life of cut fowers are very important to obtain a product that is accepted in the market [3,4].
Te vase life is an important parameter for evaluating the quality of cut fowers [5,6] for both domestic and export markets.Tis technique of extending the vase life will be a great asset to both the farmer and the consumer [7].Te diference in vase life among various chrysanthemum species and cultivars is one of the most valuable characteristics determining its quality, consumer preferences, and commercial value [8].Te results showed that good postharvest application resulted in the freshness of chrysanthemum cut fowers being maintained for up to 21 days [9,10], whereas poor postharvest handling resulted in the freshness of cut chrysanthemum fowers, only lasts up to 4-7 days [11,12].
Te main problems of chrysanthemum cut fowers are the leaves easily turning yellow, wilting, and failure of the fowers to fully open [13][14][15].One of the postharvest technologies to overcome this problem is applying a pulsing solution.Pulsing solutions enriched with AgNO 3 or ZnO have been shown to increase vase life and maintain cut fower quality [16][17][18].Te formulation of Ag particles as a pulsing solution can extend vase life, expand fower openings, and restore stem and fower size or petal color by balancing osmotic regulation [19].Zinc (Zn) is an activator of certain enzymes that can regulate the antioxidant activity and increase the life of cut fower vases [20].However, using these materials in a pulsing solution is more optimal for increasing vase life and maintaining the quality of potted fowers if the particle size of the material is nanosized [18].
Nanoparticles are expected to reduce the concentration of AgNO 3 or ZnO in the pulsing solution.Te application of nano AgNO 3 can improve relative water absorption, reduce microbial density at the tip of the stem and delay stem clogging, reduce electrolyte leakage, and suppress malondialdehyde (MDA), superoxide dismutase (SOD), and peroxide activity (POD) [21].Te accumulation of Zn nanoparticles on the cell surface can cause bacterial cell death, where the interaction between bacterial cells and Zn nanoparticles causes electrostatic pressure on the surface of the cell membrane and breaks the fow/transport of electrons across the membrane [22].However, research on applying nanoparticles in pulsing solutions to increase vase life and maintain the freshness of chrysanthemum cut fowers cultivated in the tropics still needs to be completed.Terefore, this study aims to obtain the best pulsing solution formulation that increases vase life and maintains the freshness of chrysanthemum cut fowers.

Plant Material.
In this study, the yellow spray chrysanthemum of the Zembla Sunny variety was harvested in the morning at 07.00 AM in the farmer's garden in Kawungluwuk village, Sukaresmi, Cianjur district, West Java province.Te criteria for harvesting fowers are 60% blooms, then sorting, and selecting those that are not defective, and the length of the stalks is uniform.Te fower stalks were cut obliquely.

Coating Material
Preparation.An aqueous solution of 0.04 M AgNO 3 (Sigma-Aldrich) and an aqueous solution of 0.02 M ZnO (Sigma-Aldrich) were prepared.A pulsing solution was made from the starter solution following the procedure of Rahayu et al. [23].Te pulsing solution was made at 5, 10, and 20 ppm for NZn and NAg following Carrillo-López et al.'s [9] formula with modifcations, and the pulsing solution was also added with commercial concentrations of sucrose 50 grams/liter and citric acid 200 ppm (Table 1).Sucrose as a source of carbohydrates and energy and organic acids were used to decrease the pH of the pulsing solution.Pulsing solutions with a low pH inhibited the growth of microbes so that they accelerated water and nutrients absorbed and maintained the freshness of cut fowers [6].Te pulsing solution was prepared according to the treatment (200 ml/glass).
Te fowers were then pulsed by immersing the part of the fower stalk that had been cut into the solution, with the condition that 4 cm of the stalk was submerged in the pulsing solution.Pulsing was carried out for 16 hours in a room with a temperature of 20-22 °C.When pulsing was complete, the cut fowers were transferred to another container flled with distilled water for display and placed in a room with an average temperature of 20-22 °C.Te scheme of study stages is presented in Figure 1.Each treatment was replicated three times in a randomized complete design.Observations were made during the display for each treatment on the absorbed pulsating solution (APS), water uptake, total bacteria, petal color, fower diameters, total carotenoid, and vase life.

Measurements of Absorbed Pulsating Solution (APS) and
Vase Life.Te APS was measured using the method proposed by Veluru et al. [24].Te fower stems were placed in a 250 ml conical fask containing a 200 ml pulsing solution.Te APS of the fower pieces was calculated by fnding the diference in the amount of the pulsing solution before and after 16 hours.Te APS was measured using a graduated glass cylinder.Finally, the APS was expressed in ml: Vf � the fnal volume of the solution after absorption and Vi � the initial volume of solution added.
Wilting of fower petals was used as a measure for determining the vase life of chrysanthemum cut fowers.Te average vase life was assessed to be terminated when 80% of fowers had senesced, which was characterized by the loss of turgor followed by petal wilting [25].Te vase life was calculated by counting the days from applying the treatment (frst day) until fower wilting [10].Ten, the vase life was expressed as the day.

Measurements of Water Uptake and Total Bacterial Count.
Te volume of the vases containing water without cut fowers was recorded every day during the vase life evaluation period [10].Total days of the water uptake rate were calculated at the end of the vase evaluation period of each treatment.Finally, the water uptakes were expressed in ml.
Te total bacteria were measured according to the method described by Carrillo-López et al. [9] with slight modifcations.At the end of vase life, an aliquot of 1 ml of the vase solution was taken and placed in a Petriflm Aerobic Count plate.Te results are expressed in CFU/ml.Te wilted leaves were calculated at the end of the vase life and expressed as percentages.Flower diameter was defned as the maximum width of each fower before and after blooming [25] and was measured with a ruler every day.

Measurements of Flower Color and Total Carotenoid.
Te color changes were measured according to the method described by Arif et al. [26,27] with slight modifcations.Te color measurement was carried out using CR-400 Chroma Meter (Minolta Japan) and expressed lightness (L) and hue values on fowers.Ten, the average was determined.Total carotenoid was determined by the method of Braniša et al. [28].Te extract preparation procedure is identical to the method described by Braniša et al. [28].A mixture of acetone water (4 : 1) was used as a solvent.Te maximum absorbance was read at 470 nm.

Statistical Analysis.
Te data were analyzed using the analysis of variance.Te efect of treatment on parameters was evaluated with the Duncan multiple range test (DMRT) to identify signifcant diferences at a signifcance level of 5%.All statistical analyses were carried out using SAS Portable 9.13 software.

Result and Discussion
3.1.Te APS and Vase Life.Te APS was not signifcantly diferent between treatments, ranging from 32.67 ml to 36.67 ml (Table 2).Tis shows that the fower conditions used for the study were relatively uniform and the APS for 16 hours went well.Kader [29] reported that the absorbed pulsating solution for 24 hours in cut roses did not difer between the AgNO 3 and NAg treatments.Te APS will afect the vase life of the cut fowers.
Te vase life is an important parameter for evaluating the quality of cut fowers.Te NAg20 treatment showed the highest vase life of 23 days, 19 days longer than that of the control (Table 2).Silver nanoparticles have been shown to inhibit ethylene production and increase postharvest longevity of the "First Red" rose cultivar [30,31].Other studies have also shown that silver nanoparticles signifcantly inhibit xylem blockage and reduce ethylene production by suppressing the level of transcripts of the ACS1 and ACO1 ethylene biosynthetic genes, thereby increasing the vase life of Dianthus caryophyllus [32][33][34].However, selecting of the optimum nanosilver concentrations for a particular genotype is essential to avoid phytotoxicity, which negatively impacts the vase life of cut fowers [35].In addition, in this study, the vase life of chrysanthemum cut fowers in the NAg20 treatment was four days longer than that in the Ag25 treatment.Te nanosilver treatment was more efective than Ag + in the vase life of cut carnation fower, where the accumulation of silver content was higher at the tips of the stems in the nanosilver treatment, which might be due to diferences in physical and chemical characteristics and/or concentration [36].Moreover, silver and nanosilver suppressed the expression of ACS and ACO, interacted with ethylene receptors, and modulated the ethylene response, so it increased the vase life of carnations [33].Tus, the NAg20 treatment can extend vase life up to 23 days in chrysanthemum cut fowers.
Te vase life of chrysanthemum cut fowers in the nanozinc (NZn) treatment was not diferent from that in the control, which was 4 days (Table 1).Tis is inconsistent with research reports showing that zinc treatment can increase vase life in Gerbera jamesonii fowers [16] and lisianthus [37].In this study, the nanozinc synthesis process did not use capping/doping materials, so resulting nanozinc became inefective and unstable.Nanozinc synthesized using a capping/doping agent (starch) increased the shelf life of cut fowers up to three times compared to controls whose charge was unstable and experienced agglomeration to a certain extent due to lack of surface passivation [16].

Te Water Uptake and Total
Bacteria.In this study, water absorption by chrysanthemum cut stems ranged from 5 to 7 ml per day, except for the control (Table 3).Te NAg20 treatment showed a stable water absorption of 147.89 ml for up to 23 days of the display period (Table 3).In this study, nanosilver particles kill bacteria that clog xylem vessels and maintain water balance.Nanosilver particles inhibit bacterial growth, enhance antioxidant activity, and promote the expression of the intrinsic plasma membrane protein genes  Te Scientifc World Journal PlPIP1: 2 and PlPIP2: 1, which help maintain water balance in Paeonia lactifora cut fowers [38].Tus, the NAg20 treatment indicated that it could maintain water balance in chrysanthemum cut fowers.
Water uptake is critical in improving cut fowers' vase life and quality [33].Water uptake is crucial to increase vase life and cut fower quality because it is needed to maintain water balance in the stem and for fower bud opening in cut fowers [36].Xylem is mainly responsible for transporting water to fower buds and fowers so that water absorption by cut fowers is inhibited by the proliferation and growth of microorganisms and the deposition of microbial residues in the lumen of xylem vessels [33,35].In addition to water uptake, total bacteria afect the vase life of cut fowers.In this study, total bacteria increased at the end of the display period (Table 4).Generally, the AgNO 3 and NAg treatments showed that the average of total bacteria per day was lower than that of the control and Zn treatments (Table 4).Te total bacteria in the NAg20 treatment on day 23 of the evaluation period were 6050 CFU/ml, which means 263 CFU/ml per day, where the total bacteria were the lowest compared to those in the other treatments (Table 4).Nanosilver particles have been shown to have stronger bacterial growth inhibition properties than the micrometric structure or oxidation state components of silver [39] due to their high surface area to volume ratio and large external area efect [40,41].Silver ions released by nanosilver particles interact with cytoplasmic components and nucleic acids to inhibit respiratory chain enzymes and disrupt membrane permeability, which can cause death in microorganisms [35].In addition, the nanosilver particles kill bacteria growing in the vase solution and enter the cut fower stems through the vascular tissue and inhibit bacterial colonization at the cut ends.Te proliferation of bacterial growth in xylem vessels is the leading cause of water transportation to cut fowers, so bacterial growth in xylem vessels can inhibit water uptake in vases [18,35,42].Microbial growth at the tips of the stems is the main cause of clogging of xylem vessels, which prevents water absorption and consequently reduces the vase life of cut fowers [43].Terefore, the NAg20 treatment was efective in inhibiting bacterial growth in chrysanthemum cut fowers.

Number of Wilted Leaves and Flower
Diameter.Te freshness of cut fower leaves is an important parameter that can afect the level of cut fower consumer preference.On the other hand, leaves on fower stalks treated with NAg or AgNO 3 began to wilt on the 5th day of the display period, and all wilted on day 7 to day 9 of the display period (Figures 2 and 3).Treatment with nanosilver particles reduced chlorophyll pigment degradation and lipid peroxidation (malondialdehyde) and increased SOD (superoxide dismutase) activity to prevent oxidative stress in Alstroemeria cut fower leaves [44].In addition, the treatment of nanosilver particles also improved the postharvest quality of cut fowers by reducing weight loss and increasing the greenness index of cut leaves [45].Tis shows that the nanosilver treatment inhibits leaf wilting in cut fowers.
In one sprig of spray-type chrysanthemum, there are several fower buds, namely, full bloom, half bloom, and buds.At the start of the display period, the average fower diameter at full bloom was 52-67 mm.At 4 days of the display period, the fowers in the control, ZnO, and NZn treatments showed wilted fowers, drooping petals, and decreased fower diameter (Figure 4).Tis is presumably due to the occurrence of barriers to water absorption and translocation of food reserves, especially carbohydrates, to the foral organs.Furthermore, pulsing treatment of AgNO 3 and NAg at various concentrations showed fower diameters of 63-67 mm up to 18-23 days.In the quarter-to-half blooms, the fower diameter was 23-30 mm and the AgNO 3 and NAg treatments showed an increase in a fower diameter of 34-44 mm (Figure 5).Flower diameter in the AgNO 3 and NAg treatments continued to increase until full bloom, where the fower diameter reached 51-56 mm.However, the diameter of the fower was smaller than the fower that was in full bloom from the start.Nanosilver particles kill bacteria that clog xylem vessels and support greater water uptake so that they can translocate water and carbohydrates to fowers [9].Leaves that quickly turn yellow and wither can cause smaller fower diameters, which eventually cause the fowers to wilt.Te promotion of fower opening, as evidenced by the yellowing and wilting of leaves, indicates increased transport of dissolved carbohydrates and sugars from leaves to fowers [9].Terefore, the NAg treatment increased fower size and delayed fower wilting.Te Scientifc World Journal

Flower Color and Total Carotenoid.
Color is an important visual parameter infuencing consumer acceptance of horticultural commodities [26,27].Te color of the petals on the Zembla Sunny spray chrysanthemum is yellow.Changes in the color of fower petals in the NAg20 treatment tended to show no signifcant changes during vase life (Figures 6-8).It is suspected that the NAg treatment afects the synthesis and production of ethylene.Naing et al. [33] reported that silver and nanosilver increased the vase life of carnations because they suppressed the expression of ACS and ACO, interacted with ethylene receptors, and modulated the ethylene response.Ethylene is a hormone that directly afects pigmentation and color degradation [26,27].Terefore, in this study, the lightness of the color of the fower petals in the NAg20 treatment was paler than that in the other treatments.

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Te Scientifc World Journal In addition, this study also observed the carotenoid content in chrysanthemum cut fowers during vase life.Carotenoids are pigments that are yellow, orange, or red.Te carotenoid content increased in control, ZnO, AgNO 3 , and NZn (Table 5).In contrast, the content of carotenoids in the NAg20 treatment tended to decrease (Table 5).Tis indicates that the process of carotenoid degradation in the NAg treatment is inhibited.Te inhibition of the color degradation process is caused by the ethylene synthesis process inhibited but increases the shelf life of most horticultural commodities [26,27].Te Scientifc World Journal

Conclusion
Te pulsing solution signifcantly afected postharvest physiological changes during vase life in chrysanthemum cut fowers.Te NAg20 treatment increased the vase life of the chrysanthemum cut fowers up to 23 days, which was 19 days longer than that in the control.NAg inhibits bacterial growth, fower wilting, color degradation, and carotenoids.In addition, nano-Ag increased the size of the bloom-fower diameter.Tese data are very important because they show the complexity in determining the appropriate pulsing solution treatment to extend the vase life of the chrysanthemum cut fower.Tis research can be used as a material for consideration for cut chrysanthemum industry players to determine the ideal pulsing solution treatment for the chrysanthemum cut fowers, which can meet consumer preferences and balance the interests of farmers.

Figure 2 :
Figure 2: Wilted leaves in chrysanthemum during vase life.

Figure 3 :
Figure 3: Te appearance of the fower buds in Ag-and Zn-pulsing treatments and the front view of the entire fower stalk on day 4 during the evaluation period.

Figure 4 :Figure 5 :
Figure 4: Changes in the diameter of blooming fowers during vase life.

Figure 6 :Figure 7 :
Figure 6: Changes in lightness L color of fowers during vase life.

Figure 8 :
Figure 8: Changes in b-value color of fowers during vase life.

Table 1 :
Te formula composition of the pulsing solution.
2.5.Measurements of the Wilted Leaves and Flower Diameter.

Table 2 :
Te APS and vase life of chrysanthemum cut fowers.Notes.Te numbers followed by the same letter in the same column indicate no signifcant diference by the Duncan extension multiple range test (P < 0.05).

Table 3 :
Water uptake in chrysanthemum cut fowers.Notes.Te numbers followed by the same letter in the same column indicate no signifcant diference by the Duncan extension multiple range test (P < 0.05).

Table 4 :
Total bacteria in chrysanthemum cut fowers.Te numbers followed by the same letter in the same column indicate no signifcant diference by the Duncan extension multiple range test (P < 0.05).

Table 5 :
Total carotenoids in chrysanthemum cut fowers.