Roses, belonging to the Rosaceae family, are widely cherished ornamental plants used for various purposes, including cut flowers, potted plants, and home gardens (Pati et al. 2006; Shabbir et al. 2009). Traditional rose propagation methods, such as grafting and budding, are complex and time-consuming, leading to low propagation efficiency (Jabbarzadeh and Morteza 2005). Consequently, the need for an efficient and rapid rose propagation method is evident.

In recent times, there has been a growing emphasis on the cultivation of native plants and Korean cultivars in gardens due to concerns about carbon footprints and the reduction of royalty payments for foreign cultivars (Oh et al. 2010). This trend has led to a steady increase in the localization rate of major horticultural crops in South Korea, resulting in reduced royalty expenditures. Notably, the localization rate for roses has surpassed 30% (MARFA 2022), and new cultivars are being selectively bred to meet consumer preferences for flower color, size, and thorniness. In essence, the development of propagation technologies is crucial to enhance the competitiveness of Korean-bred cultivars as garden materials (Kim et al. 2020).

Plant tissue culture technology plays an important role in the realm of plant research. It not only facilitates the study of various developmental physiologies of plants, but it also enables the production of superior seedlings and high value-added useful materials, as well as the development of new resistant and functional varieties through the introduction of beneficial genes and the ex-situ conservation of endangered plants (Pati et al. 2006). However, the efficiency of plant explant regeneration through tissue culture varies, contingent on factors such as the plant species and cultivar, explant positioning, and the type and concentration of plant growth regulators and inorganic salts in the medium (Farahani and Shaker 2012; Pati et al. 2010).

Rose micropropagation often results in the yellowing and shedding of leaves, and malformations due to excessive ethylene production by plant tissues (Park et al. 2016). Other factors that influence the growth of roses during micropropagation include light quality, which affects photosynthetic efficiency and pigment biosynthesis, CO₂ concentration, high humidity (≥ 95%) inside the Petri dish, air temperature, and other culture conditions (Naing et al 2021; Park et al. 2016).

A significant challenge in rose tissue culture is managing ethylene production, a naturally occurring plant hormone with pivotal roles in various physiological processes. However, its excessive production during tissue culture can result in adverse effects, including leaf yellowing and the accumulation of excessive phenolic compounds (Kumer et al. 2009), which can impact both the health and appearance of roses while complicating the cultivation process. Addressing this issue is crucial for achieving sustainable and economically viable rose production (Gaspar et al. 1989).

In this study, we aimed to inhibit ethylene production and enhance the growth of rose tissue cultures by treating them with silver nanoparticles (AgNPs) and antioxidants. AgNPs have been utilized to prolong post-harvest longevity and improve the quality of fresh flowers due to their ability to inhibit ethylene production and the growth and development of microorganisms (Naing and Kim 2020) AgNPs are increasingly used in plant biotechnology and as novel packaging materials for horticultural crops (Sarmast and Salehi 2016). The application of AgNPs enhanced the growth of Rosa hybrida and effectively inhibited ethylene production. The technology described here holds significant potential for revolutionizing the mass production of premium-quality roses.

Plant materials

In this study, 4-week-old in vitro shoots of Rosa hybrida ‘Sahara’ were obtained through axillary node culture of mother plants grown in a greenhouse at the National Institute of Horticultural & Herbal Science (Wanju, South Korea). These shoots were cultured on Murashige and Skoog (MS) medium supplemented with vitamins (Murashige and Skoog, 1962). The in vitro shoots were subcultured on MS medium containing 2.0 mg/L 6-benzylaminopurine (BAP) and 0.5 mg/L kinetin for 4 weeks to obtain young and uniform shoots for further experiments.

AgNPs, Silver Nitrate (AgNO₃), and Antioxidant Treatments

Four weeks after the node culture, uniformly grown plants were selected as the experimental material. The basal medium composition consisted of MS medium containing vitamins, 3% (w/v) sucrose, 0.75% plant agar, and 2.0 mg/L BAP. For the silver treatments, 1, 3, or 5 mg/L AgNPs or 5 mg/L AgNO₃ was added to the medium. The antioxidant treatments included single treatments of ascorbic acid (ASA) at 250 mg/L or citric acid (CIA) at 250 mg/L, as well as combined treatments consisting of 125 mg/L ASA and 125 mg/L CIA. Plants were immersed in a liquid medium containing the respective concentration of antioxidant/s for 1 h and then planted in the same medium composition. Following these treatments, the shoots were cultured in dark conditions for 2 days, followed by 16 h of light exposure (50 µmol/m²/s) for 4 weeks.

Measurement of Growth Parameters

Plant growth parameters, comprising plant height, shoot count, and fresh weight, were evaluated in both control and treated plants. Plant height was determined as the length from the crown to the tip of the plant shoot, while fresh weight was measured precisely to two decimal places using an electronic scale. Each growth parameter was assessed using four plants, and the analysis was replicated four times.

Measurement of SPAD Values

SPAD values were measured using a chlorophyll meter (SPAD-502, Minolta Co., Tokyo, Japan) in plants treated with AgNPs and antioxidants for 4 weeks. The values were taken directly from leaves attached to the plants, and five different plants were measured for each treatment.

Measurement of Superoxide Anion Radical (O₂^-)

A modified version of the procedure outlined by Doke et al. (1983) was employed to quantify the superoxide anion radical (O₂^-) content based on its ability to neutralize nitro blue tetrazolium (NBT). Specifically, 1.5 mL of a reaction mixture, comprising 10 mM potassium phosphate buffer (pH 7.8), 0.05% NBT, and 10 mM sodium azide (NaN₃), was combined with 0.1 g of liquid nitrogen-ground fresh leaf sample. After incubating the reaction for 20 min at room temperature, 0.5 mL of the solution was heated in a water bath at 85°C for 15 min and then rapidly cooled. The resultant absorbance at 580 nm was used to calculate the amount of O₂^- (A580 g-1 FW).

Determination of Ethylene Production

Plant ethylene production was assessed following the procedure outlined by Naing and Kim (2020). In summary, approximately 500 mg samples were obtained from plants subjected to treatments involving AgNPs, AgNO₃, antioxidants, and control conditions for a 16 h period (Naing et al. 2021). These samples were placed in 50 mL glass tubes, sealed with rubber stoppers, and kept in the dark at room temperature overnight. The ethylene produced in the glass tubes was extracted using a syringe, and its concentration was quantified using gas chromatography (GC-2010; Shimadzu, Tokyo, Japan). Ethylene production measurements were conducted on three plants from each treatment group, and the analysis was repeated three times.

RNA Extraction and Gene Expression Analysis

Total RNA was extracted from plants treated with AgNPs, AgNO₃, antioxidants, or control conditions after 4 weeks using a RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). cDNA synthesis was carried out using an oligo (dT) 20 primer and a reverse transcription kit (ReverTra Ace-á, Toyobo, Japan), following the method described by Naing and Kim (2020). Subsequently, the expression levels of ACS2, ACO1, ETR1, and CTR1 were determined using specific primers and quantitative real-time PCR (qRT-PCR). The primers and PCR conditions are listed in Supplementary Table 1. The Rh-tublin gene was used as a reference gene and analyses were performed using the Step One Plus Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Three independent biological samples per treatment were used for the gene expression analyses.

Statistical analysis

The results are presented as means (of three replicates) ± standard error (SE). Statistical analyses were performed using SPSS software version 11.09 (IBM corporation, Armonk, NY, USA). Tukey’s multiple range test and the least significant difference test were used to examine the significance of differences between means. Statistical significance was set at p < 0.05.

Plant Growth

After a 4-week culture period, the impact of AgNPs, AgNO₃, and antioxidants on plant growth was evaluated (Table 1, Fig. 1). Shoot height showed significant changes in response to treatments with 3 mg/L AgNPs (21.13 ± 0.78 mm) and 5 mg/L AgNO₃ (21.59 ± 0.81 mm) compared to other treatments. In both cases, the shoots displayed the greatest length, maintaining consistent size and robust health. Moreover, the leaves associated with these shoots were dark green and free from yellowing or defoliation. Conversely, the control group exhibited only a few regenerated shoots with signs of overhydration, including a glassy appearance, slender stems, and translucent leaves. Additionally, all antioxidant treatments yielded similar or lower plant growth values compared to the control.

Figure 1. Effect of AgNPs, AgNO₃, and antioxidants on Rosa hybrida ‘Sahara’ growth after 4 weeks of treatment. (A) Control, (B) 1 mg/L AgNPs, (C) 3 mg/L AgNPs, (D) 5 mg/L AgNPs, (E) 5 mg/L AgNO₃, (F) liquid medium pre-treatment with 125 mg/L ASA + 125 mg/L CIA for 1 h, (G) 125 mg/L ASA + 125 mg/L CIA, (H) 250 mg/L ASA, and (I) 250 mg/L CIA. AgNPs, silver nanoparticles; AgNO₃, silver nitrate; ASA, ascorbic acid; CIA, citric acid

Table 1 . Effect of AgNPs, AgNO₃, and antioxidant treatment on plant growth of Rosa hybrida ‘Sahara’.

Treatment				No. of shoots	Shoot height (mm)	Fresh weight (g)	SPAD (nmol/cm2)
AgNPs (mg/L)	AgNO3 (mg/L)	CIA (mg/L)	ASA (mg/L)
-	-	-	-	3.10 ± 0.15ns	16.75 ± 0.66bc	0.31 ± 0.08ab	17.48 ± 0.85bc
1	-	-	-	3.24 ± 0.18ns	19.22 ± 0.70ab	0.34 ± 0.08ab	12.5 ± 0.57c
3	-	-	-	3.13 ± 0.19ns	18.76 ± 1.14ab	0.35 ± 0.09ab	20.68 ± 2.32b
5	-	-	-	3.46 ± 0.22ns	21.13 ± 0.78a	0.41 ± 0.11a	27.72 ± 3.61a
-	5	-	-	2.76 ± 0.17ns	21.59 ± 0.81a	0.32 ± 0.09ab	32.44 ± 2.70a
-	-	250 (1 h)	250 (1 h)	3.14 ± 0.14ns	16.90 ± 0.60bc	0.34 ± 0.08ab	15.28 ± 2.46bc
-	-	250	250	2.89 ± 0.18ns	16.42 ± 0.55d	0.24 ± 0.06b	14.18 ± 0.60bc
-	-	-	500	2.94 ± 0.14ns	14.90 ± 0.69d	0.23 ± 0.07b	14.98 ± 1.22bc
-	-	500	-	3.00 ± 0.19ns	14.58 ± 0.74d	0.25 ± 0.09b	17.56 ± 3.53bc

Data are presented as the mean ± standard error of three replicates. Mean values with the same superscript letters are not significantly different. Statistical significance was set at p < 0.05. AgNPs, silver nanoparticles; AgNO₃, silver nitrate; CIA, citric acid; AIA, ascorbic acid; SPAD values, Soil Plant Analysis Development values displayed by a Konica Minolta chlorophyll meter and having a correlation with chlorophyll density..

The control group generated 3.10 ± 0.70 shoots, while the use of 1 mg/L and 3 mg/L AgNPs led to 3.24 ± 0.83 and 3.46 ± 0.78 shoots, respectively. These results indicate a tendency toward a higher number of shoots in the AgNPs treatment groups, but these differences did not reach statistical significance across all treatments. As a crucial metric for assessing overall plant growth and productivity, the fresh weight of the control group (0.31 ± 0.04 g) exhibited a statistically significant difference compared to that of the 3 mg/L AgNPs treatment group (0.41 ± 0.04 g). In contrast, the fresh weights of the other treatment groups did not exhibit statistically significant differences compared to those of the control. In the context of photosynthetic pigment synthesis, both AgNPs and AgNO₃ treatments led to an increase in chlorophyll content. However, the SPAD value was higher in the 5 mg/L AgNO₃ treatment (32.44 ± 2.70 nmol/cm²) group than in the AgNPs (27.72 ± 3.61 nmol/cm²) and control (17.48 ± 0.85 nmol/cm²) groups. These findings are consistent with those reported by Ha et al. (2020), who used 2 mg/L AgNPs for micropropagation.

ROS, such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), and hydroxyl radicals (OH^-), are generated in plants under stress conditions. As shown in Fig. 2, O₂^- levels were significantly lower in the AgNPs treatment groups than in all of the other treatment groups. AgNPs treatment had a minimal effect on O₂^- levels, especially when compared to AgNO₃. Notably, treatment with 3 mg/L AgNPs resulted in a significant reduction in O₂^- level (0.55 ± 0.01) compared to that of the control (0.83 ± 0.07). Furthermore, the effect of antioxidant treatment on the O₂^- level did not differ significantly from that of the control.

Figure 2. Effect of AgNPs, AgNO₃, and antioxidant treatments on (A) O₂^- level and (B) ethylene production in Rosa hybrida ‘Sahara.’ Data are presented as the means of three replicates ± standard error. Mean values with the same letters are not significantly different at p < 0.05. AgNPs, silver nanoparticles; AgNO₃, silver nitrate; ASA, ascorbic acid; CIA, citric acid; O₂^-, superoxide ion
Superoxide Anion Radical (O₂^-)

Ethylene Production

Ethylene production was observed in control plants, as well as in those treated with AgNPs or antioxidants. Although all treatments led to a decrease in ethylene production compared to the control plants, the reductions in ethylene production in the antioxidant-treated plants were not statistically significant (Fig. 2). Ethylene production significantly decreased compared with that of the control (0.21 µL/g/h) when higher concentrations of AgNPs were used (0.12, 0.10, and 0.09 µL/g/h ethylene when treated with 1, 2, and 3 mg/L AgNPs, respectively).

Expression of Ethylene Biosynthesis- and Signaling-Related Genes

The effects of AgNPs, AgNO₃, and antioxidants on the expression levels of genes related to ethylene biosynthesis and signal transduction were analyzed. Expression levels of the ethylene biosynthetic genes ACS2 and ACO1 were reduced by approximately half following treatment with AgNPs or AgNO₃ (Fig. 3). Moreover, there were no statistically significant differences between the two treatments. Similarly, the expression levels of the ethylene signaling gene ETR1 and the negative regulator of ethylene signaling gene CTR1 exhibited significant differences in all AgNPs and AgNO₃ treatment groups compared with that of the control plants (Fig. 3). For CTR1, treatments of AgNPs resulted in statistically significant differences. Furthermore, we found that the antioxidant treatment was not significantly different from the control in all genes.

Figure 3. Effect of AgNPs, AgNO₃, and antioxidant treatments on the expression levels of ethylene biosynthesis and signal transduction-related genes in Rosa hybrida ‘Sahara.’ Data are presented as the means of three replicates ± standard error. Mean values with the same letters are not significantly different at p < 0.05. AgNPs, silver nanoparticles; AgNO₃, silver nitrate; ASA, ascorbic acid; CIA, citric acid; ACS2, acetyl-CoA synthetase 2; ACO1, aconitase 1; ETR1, ethylene receptor 1; CTR1, serine/threonine-protein kinase CTR1

In Rosa hybrida tissue culture, overcoming challenges like basal browning caused by phenolic substances, leaf abscission, and chlorosis due to ethylene production is crucial for producing top-quality seedlings. Various in-medium treatments have been investigated to tackle these problems, often involving chemicals that inhibit ethylene synthesis in plants and microbial growth in the cuttings (basal) (LaRue and Gamborg 1971; Roh et al. 2013). However, in recent years, nanotechnology has emerged as a promising agricultural solution, showing the potential to improve productivity and reduce postharvest waste (Naing and Kim 2020).

In this study, we demonstrated the effectiveness of AgNPs in promoting growth and reducing ethylene production in Rosa hybrida ‘Sahara’ in vitro through nodal explant cultures. Additionally, recent studies have shown that AgNPs can mitigate issues such as yellowing and leaf shedding while improving the quality of roses in vitro (Ha et al. 2020).

Evaluations of shoot height, fresh weight, and SPAD values, as indicators of plant growth, demonstrated improvements in plants treated with AgNPs and AgNO₃ (Table 1). These enhancements imply that AgNPs, which penetrate the plant through plasmodesmata, facilitate nutrient absorption, increase trace element content, stimulate photosynthesis, and positively impact biomass (Yan and Chen 2019). Moreover, silver ions and NPs can induce alterations in the chemical composition of secondary compounds, indicating that AgNPs influence plant metabolism by releasing ions into the environment. When AgNPs enter the plant, they trigger a defense response that leads to transcriptional reprogramming and improved secondary metabolism (Rahmawati et al. 2022).

Despite being considered one of the less-reactive ROS, O₂^- can still be harmful because of its ability to diffuse into cellular compartments. Apoplastic ROS can alter enzyme activity, resulting in cell death by inducing oxidative damage to DNA, proteins, and cell membranes (Rahmawati et al. 2022; Sarmast and Salehi 2016). In this study, we observed that AgNPs treatment, regardless of the concentration, had an effect of reducing O₂^- levels. Compared with that of AgNPs treatment, AgNO₃ treatment had minimal effect on the production of O₂^- (Fig. 2). This observation aligns with a recent study in which AgNPs and AgNO₃ treatments in Brassica spp. showed that AgNO3 caused a more significant increase in ROS and H₂O₂ accumulation, possibly owing to the interaction of AgNO₃ with proteins in the cytoplasm and lipid bilayers, altering and impairing the composition of the antioxidant defense system (Vishwakarma et al. 2017).

Ethylene, one of the most important plant hormones, plays a critical role in several developmental processes, including seed germination, flower and leaf senescence, abscission, and fruit ripening (Abeles et al. 1992). Given that AgNPs reduced significant ethylene production in the shoots at 4 weeks after culture, we compared the expression of four genes involved in ethylene biosynthesis (ACS2 and ACO1) and signaling (ETR1 and CTR1) in these shoots (Fig. 3). The first step in ethylene production is the conversion of S-AdoMet to 1-Aminocyclopropane-1-carboxylate (ACC) by ACC synthase (ACS). Subsequently, ACC oxidase (ACO) converts ACC to ethylene (Kende 1993; Yang and Hoffman 1984). The expression of ACS genes controls the amount of ethylene produced (Vriezen et al. 1999). However, in certain circumstances, ACO gene expression can also control the amount of ethylene produced (Fernández-Otero et al. 2006; Vriezen et al. 1999; Wagstaff et al. 2005). The expression levels of ACS2 and ACO1 were reduced by both AgNPs and AgNO₃ treatments. Over the past few decades, significant research has been conducted to elucidate the ethylene signaling pathway in roses (Doke et al. 1983). This pathway involves five receptors (ETR1, ETR2, ERS1, ERS2, and EIN), which use copper cofactors for ethylene detection. Additionally, CTR1 proteins serve as inhibitors of ethylene response and interact with these receptors (Grefen et al. 2008; Tan et al. 2006). Several studies on cut roses have highlighted the importance of the expression of these genes related to ethylene signal transduction and their influence on ethylene sensitivity (Müller and Stummann 2003; Naing and Kim 2020; Naing et al. 2021; Tan et al. 2006). Notably, the expression of CTR1 exhibited a significant difference after AgNPs treatment, which could be attributed to its effect on the ability of copper ions (Cu₂⁺) to bind to the ETR1 receptor site (Oh et al. 2010; Pati et al. 2006; Roh et al. 2013). When Cu₂₊ cofactors are substituted with AgNPs, Ag⁺ binds to the receptor and prevents the signaling of inhibitory ethylene to the plant (Kumar et al. 2009; Naing and Kim 2020; Naing et al. 2021).

In summary, all AgNPs treatments effectively suppressed ethylene signal transduction in Rosa hybrida ‘Sahara.’ These treatments successfully alleviated leaf tip chlorosis during rose regeneration, leading to the inhibition of ethylene signaling and a decrease in associated ROS levels. Consequently, the overall ROS levels within the plant decreased, chlorophyll content increased, and plant growth was stimulated. However, the effects of antioxidant treatments were not significantly different from the control, warranting further investigation.

In this study, we developed an indirect organogenesis method for roses using nodal culture, considering various factors influencing healthy shoot regeneration. The optimized regeneration conditions included culturing on MS medium supplemented with 2.0 mg/L BAP and 3 mg/L AgNPs, which were employed to mitigate chlorosis caused by ethylene. We assessed multiple parameters, including the expression of ethylene-related genes, ROS levels, and chlorophyll content, to compare the control and treatment groups, revealing significant differences. These results provide valuable insights into the positive impact of AgNPs treatment on increasing chlorophyll levels in in-vitro plants, promoting their transformation into healthy green plants. Consequently, our study contributes to the advancement of biotechnological research for mass-manufacturing and/or modifying new rose varieties suitable for cultivation in Korea.

This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No RS-2022-RD010250)” Rural Development Administration, Republic of Korea.