Roses, with their vibrant colors, delicate petals, and captivating fragrances, have remained a beloved choice for ornamental purposes across various cultures and regions worldwide for hundreds of years. Rose is a significant commercial ornamental crop of the Rosa genus, which belongs to the subfamily Rosoideae in the Rosaceae family (Leus et al. 2018). In vitro flowering refers to the process of inducing and studying the flowering of plants in a controlled laboratory environment, rather than in their natural habitat. This method provides researchers with a valuable tool for dissecting the intricate processes involved in plant flowering, such as the transition from vegetative growth to reproductive growth, the development of floral structures, and senescence (Nguyen and Van 2020). By examining these phenomena in a controlled setting, scientists can understand the genetic, hormonal, and environmental factors that influence plant flowering, ultimately contributing to advancements in agriculture, horticulture, and plant biology. Many previous studies have demonstrated that the addition of auxin and cytokinin to the culture medium increases the flowering rate (Vu et al. 2006; Wang et al. 2002, Zeng et al. 2013) using a sucrose concentration of 50 g/L, 3.0 mg/L BA, and 0.1 mg/L NAA, resulting in a 68.33% in vitro flowering shoot percentage (Zeng et al. 2013). Other studies focused on obtaining the highest percentage of flowering on MS medium supplemented with 3.0 mg/L BA, 0.1 mg/L NAA, and 30 g/L sucrose (Vu et al. 2006). At a concentration of 2.0 mg/L, AgNO₃ effectively induced 50% of rose shoots to undergo in vitro flowering. After this, higher concentrations also exhibited induction of flowering, albeit accompanied by imperfect flower development (Matos et al. 2021). The preceding in vitro flowering studies in ornamental plants, such as Dendrobium Sonia 17 (Tee et al. 2008), Gentiana species (Zhang and Leung 2000), and Rosa species (Zeng et al. 2013), indicate that the various cytokinins investigated, BA demonstrates significant potential as an inducer of in vitro floral buds (Sreelekshmi and Siril 2021).

In vitro flowering studies have many limitations such as ethylene biosynthesis, causing leaf drop and yellowing... Therefore,

chemical inhibitors of ethylene action can be used in the culture medium to reduce these symptoms. Silver (Ag) is added to culture media to prevent bacterial contamination and to address abnormalities during micropropagation. The Ag⁺ ions found in silver nitrate (AgNO₃) and silver thiosulfate (Ag₂SO₄) are typically incorporated into culture media. The ethylene receptor (ETR1) has a binding site for copper ions (Cu²⁺) that is crucial for ethylene signaling. When Cu²⁺ is replaced with Ag⁺, it blocks the receptor and inhibits the ethylene signals in plants (Kumar et al. 2009). Sugars produced through photosynthesis regulate essential biological processes during growth and development, such as flowering time, initiation of senescence, embryogenesis, seed germination, seedling growth, and tuber formation (Wingler et al. 2018; Yoon et al. 2020). Sucrose plays a crucial role in governing various developmental and metabolic processes in plants. In some other reports, the role of sucrose in the in vitro flowering process was also studied. For example, Vu et al. (2006) showed that a sucrose concentration of 45 g/L gave the highest flowering rate of 33.3% (Zeng et al. 2013), Murashige and Skoog (MS) medium (Murashige and Skoog 1962) containg 50 g/L sucrose resulted in a higher in vitro flowering rate than medium with other sucrose concentrations (Saritha and Naidu 2007).

Changes in medium composition, plant growth regulators (PGRs), or changes in culture medium can accelerate growth, shorten the vegetative period, and lead to early flowering. Additional research is required to understand better these phenomena related to the physiology of flowering. This study aimed to determine the role of some factors, such as sugar, PGRs, and AgNO₃, in the in vitro flowering process of Rosa hybrida L.

Selection of explant material

The nodal explants, approximately 1.5 cm long and containing one axillary bud, were selected from the mid-stem region of 3-month-old stems in Da Lat Hasfarm Company (VietNam). The explants were initially washed under running tap water, followed by a rinse with 70% ethanol for 30 seconds, then rinsed in sterile distilled water. Next, the explants were sterilized for 3 minutes with 0.2% mercuric chloride (HgCl₂). After each treatment, the explants were gently washed with sterile distilled water 3-4 times.

Experimental design

Establishment and shoot multiplication stages. Surface sterilized nodal explants (about 0.5 cm in length) were individually cultured on MS medium with BA (0.0, 0.5; 1.0, 1.5 mg/L) and 30 g/L sucrose.

Floral induction media

After 30 days in culture, the axillary buds were transferred to flower media. Individual shoots (2-3 cm in height) were collected from the shoot multiplication medium, which was found to be the most suitable for in vitro flowering based on preliminary experiments. Shoots were transferred to MS media that included individual plant growth regulators such as BA (0.5, 1.0, 1.5, 2.0 mg/L), sucrose (30, 40, 50, 60 g/L), AgNO₃ (0.5, 1.0, 1.5, 2.0 mg/L), or a combination of BA, sucrose, and AgNO₃. The MS medium was selected as the base (control) establishment medium from which various modifications were made. The culture medium was adjusted to a pH of 5.8 before autoclaving at 121°C and 1 atm for 20 minutes. Cultures containing explants per tube were incubated at 22 ± 1°C, the humidity 55-60%, in a controlled growth chamber under a 12-hour photoperiod with a light intensity of 3000 lux. Data were recorded on the number of shoots (shoots/explant), shoot height (cm), fresh weight (mg), dry weight (mg), and flowering percentage (%) after 60 days of culture.

Data analysis

All experiments were set up using a completely randomized design and were repeated three times. Each treatment consisted of three replicates, with 10 tubes per replicate. The data were analyzed using SPSS 20.0 for Windows, with a one-way ANOVA followed by Duncan’s multiple range test (DMRT) to determine significant differences between means at P ≤ 0.05.

Effects of BA in MS culture on in vitro multiplication of rose

When treated with different concentrations of BA, the number of shoots gradually increased with concentration, reaching the highest at BA concentrations of 1.0 and 1.5 mg/L. The tallest shoot height was at BA concentration of 0.5 (2.02 cm), then gradually decreased at BA concentrations of 1.0 and 1.5 mg/L (1.83 and 1.31cm, respectively). In contrast, the shoot multiplication rate at BA concentrations of 1.0 and 1.5 was highest after 30 days of culture. Table 1 shows that BA at a concentration of 0.5 mg/L resulted in the highest shoot height and a high shoot multiplication rate. Therefore, BA was used at a concentration of 0.5 for the flowering experiments.

Table 1 . Effect of BA in Murashige and Skoog basal medium containing 30 g/L sucrose on the in vitro multiplication of rose after 30 d of culture.

BA (mg/L)	Shoot height (cm)	Multiplication ratio
Control	1.50 ± 0.07c	1.78 ± 0.38c
0.5	2.02 ± 0.10a	2.44 ± 0.38b
1.0	1.83 ± 0.09b	3.44 ± 0.19a
1.5	1.31 ± 0.10d	3.67 ± 0.33a

Different letters indicate statistically significant differences (ANOVA, Duncan’s test)..

BA, 6-benzyladenine..

Effects of sucrose on in vitro flowering

The concentration of sucrose significantly affected in vitro flowering, as shown in Table 2. Medium MS supplemented with 40 and 50 g/L sucrose produced the tallest shoot height and the highest dry weight. The fresh weight also increased with sucrose treatment compared to the control group. The highest flowering rates were observed at sucrose concentrations of 40 and 50 g/L, which were 53.33% and 51.48%, respectively. However, at sucrose concentrations of 50 and 60 g/L, wilting of leaves and flowers at the shoot occurred (Fig. 1).

Figure 1. Effect of sucrose on in vitro flowering after 60 d of culture in MS medium. (A) MS medium with no flower buds. (B) In vitro flowering in MS medium containing 40 g/L sucrose. (C) Flower buds with wilted leaves in MS medium supplemented with 50 g/L sucrose. (D) Wilted flower buds and leaves in MS medium supplemented with 60 g/L sucrose. (Scale bar = 1 cm). MS, Murashige and Skoog

Table 2 . Effect of sucrose in Murashige and Skoog basal medium on the in vitro flowering of roses after 60 d of culture.

Sucrose (mg/L)	Shoot height (cm)	Fresh weight (mg)	Dry weight (mg)	Flowering (%)
Control	1.66 ± 0.11c	121.17 ± 0.85b	9.50 ± 0.22c	-
40	3.35 ± 0.22a	150.08 ± 1.06a	14.93 ± 0.32a	53.33 ± 8.01a
50	3.29 ± 0.16a	149.66 ± 4.10a	15.14 ± 0.34a	51.48 ± 7.88a
60	2.49 ± 0.08b	147.43 ± 2.06a	14.25 ± 0.29b	26.67 ±11.55b

Different letters indicate statistically significant differences (ANOVA, Duncan’s test)..

Effects of cytokinin on in vitro flowering

When treated with different concentrations of BA, the shoot height reached the highest value at a BA concentration of 1.0 mg/L (2.93 cm) (Fig. 2B), while the fresh weight and dry weight gradually increased with increasing BA concentration. The treatment with BA 1.0 and 1.5 mg/L resulted in the highest flowering rate (45.18 and 38.15%, respectively), whereas no flowers were formed in the control experiment (Table 3). Increasing BA concentration enhances flower size and improves color in the culture medium (Fig. 2).

Figure 2. Effects of cytokinin on in vitro flowering after 60 d of culture in Murashige and Skoog medium. Treatment with (A) 0.5 mg/L BA, (B) 1.0 mg/L BA, (C) 1.5 mg/L BA, and (D) 2.0 mg/L BA (scale bar = 1 cm). BA, 6-benzyladenine

Table 3 . Effect of BA in Murashige and Skoog basal medium containing 30 g/L sucrose on the in vitro flowering of rose after 60 d of culture.

BA (mg/L)	Shoot height (cm)	Fresh weight (mg)	Dry weight (mg)	Flowering (%)
Control	1.65 ± 0.10c	121.17 ± 0.85e	9.50 ± 0.22e	-
0.5	2.20 ± 0.14b	135.06 ± 2.50d	11.30 ± 0.21d	13.70 ± 5.48b
1.0	2.93 ± 0.15a	142.23 ± 1.41c	12.38 ± 0.34c	45.18 ± 8.98a
1.5	2.07 ± 0.12b	156.25 ± 3.79b	14.14 ± 0.51b	38.15 ± 7.40a
2.0	1.63 ± 0.19c	187.71 ± 1.97a	17.90 ± 0.76a	18.70 ± 7.04b

Different letters indicate statistically significant differences (ANOVA, Duncan’s test)..

BA, 6-benzyladenine..

Effects of AgNO₃ on in vitro flowering

When treated with AgNO₃ 1.0 mg/L, the shoot height (3.06 cm), fresh weight (147.25 mg), dry weight (13.66 mg), and flowering percentage reached their highest values (56.67%). Treatment with AgNO₃ at 1.5 mg/L resulted in a lower flowering rate of 18.52% compared to the 1.0 mg/L treatment (Table 4). In contrast, the culture medium containing AgNO₃ at a concentration of 0.5 mg/L yielded the lowest flowering rate at 17.04%. Higher concentrations of AgNO₃ are associated with the development of larger and more vibrant flowers in the culture medium (Fig. 3).

Figure 3. Effect of AgNO₃ on in vitro flowering after 60 d of culture in Murashige and Skoog medium. Treatment with (A) 0.5 mg/L AgNO₃, (B) 1.0 mg/L AgNO₃, (C) 1.5 mg/L AgNO₃, and (D) 2.0 mg/L AgNO₃ (scale bar = 1 cm). AgNO₃, silver nitrate

Table 4 . Effect of AgNO₃ in Murashige and Skoog basal medium containing 30 g/L sucrose on in vitro flowering of rose after 60 d of culture.

AgNO3 (mg/L)	Shoot height (cm)	Fresh weight (mg)	Dry weight (mg)	Flowering (%)
0	1.65 ± 0.10d	121.17 ± 0.85d	9.50 ± 0.22c	-
0.5	2.37 ± 0.10b	128.06 ± 2.50c	10.91 ± 0.18b	17.04 ± 5.13c
1	3.06 ± 0.11a	147.25 ± 3.58ab	13.66 ± 0.49a	56.67 ± 5.77a
1.5	2.49 ± 0.08b	143.23 ± 2.51b	13.15 ± 0.39a	38.15 ± 7.40b
2.0	2.10 ± 0.14c	149.44 ± 4.44a	13.27 ± 0.36a	18.33 ± 7.64c

Different letters indicate statistically significant differences (ANOVA, Duncan’s test)..

AgNO₃, silver nitrate..

Effects of sucrose, BA, and AgNO₃ on in vitro flowering

In all treatments containing sucrose, BA, and AgNO₃ in the culture medium, flowering occurred. When BA concentrations were varied, BA 1.0 mg/L gave the highest shoot height (3.49 cm) and flowering percentage (60.74%). The dry weight and fresh weight increased with higher BA concentrations, with BA 2.0 mg/L recorded at 192.44 mg/L for fresh weight and 18.90 mg/L for dry weight. The lowest flowering percentage was at BA 2.0 mg/L (26.67%) (Table 5). In addition to flower formation in the culture media, root formation was observed in all media when treated in combination, except for the BA concentration of 0.5 mg/L (Fig. 4)

Figure 4. In vitro flowering in MS medium containing 40 g/L sucrose, 1.0 mg/L silver nitrate, and cytokinin at different concentrations. (A) Small flower bud in MS medium without BA. (B) Small flower bud with roots in MS medium containing 0.5 mg/L BA. (C) Flower bud with roots in MS medium containing 1.0 mg/L BA. (D) Flower bud with roots in MS medium containing 1.5 mg/L BA. (E) Flower bud with roots in MS medium containing 2.0 mg/L BA (Scale bar = 1 cm). MS, Murashige and Skoog; BA, 6-benzyladenine

Table 5 . Effect of BA in Murashige and Skoog basal medium containing 1.0 mg/L AgNO₃ and 40 g/L sucrose on the in vitro flowering of rose after 60 d of culture.

Treatment			Shoot height (cm)	Fresh weight (mg)	Dry weight (mg)	Flowering (%)
Sucrose (g/L)	AgNO3 (mg/L)	BA (mg/L)
40	1.0	0	1.90 ± 0.10c	134.77 ± 1.90e	11.07 ± 0.22d	41.10 ± 8.40b
		0.5	2.58 ± 0.11b	141.06 ± 2.50d	12.21 ± 0.18c	44.81 ± 5.01b
		1.0	3.49 ± 0.16a	162.92 ± 2.97c	15.68 ± 0.63b	60.74 ± 5.60a
		1.5	2.69 ± 0.08b	156.23 ± 2.51b	15.92 ± 0.16b	44.81 ± 5.01b
		2.0	1.71 ± 0.08c	192.44 ± 4.44a	18.90 ± 0.49a	26.67 ± 2.89c

Different letters indicate statistically significant differences (ANOVA, Duncan’s test)..

BA, 6-benzyladenine; AgNO₃, silver nitrate..

In tissue culture systems, sugar plays two primary roles: as an energy source (Gago et al. 2014; Lavie et al. 2024) and as a signal for flower induction (Yoon et al. 2020). Carbohydrates are essential for floral meristem production. Raising the levels of endogenous sucrose also promotes flowering in Solanum lycopersicum (tomato) (Micallef et al. 1995). Applying 50 mM sucrose to the leaves of Floral Yuuka induces flowering when plants are grown under short-day conditions (Sun et al. 2017). In species like Sinapis alba, Rudbeckia bicolor, Perilla nankinensis, and Xanthium strumarium, sucrose levels in the phloem sap and shoot apical meristem rise before floral induction (Bodson and Outlaw 1985; Yoon et al. 2020). Sucrose addition induces flowering in Brassica campestris grown in vitro, especially under low light (Friend et al. 1984). In this study, treatments with 40 and 50 g/L sucrose concentrations resulted in high flowering rates and good flower quality. Our findings show that sucrose significantly impacted growth and in vitro flowering. This suggests that increased sugar flow to the shoot apical meristems before flowering may regulate floral induction. However, at a concentration of 50 g/L and 60 g/L, flower buds began to wilt due to the excessive sugar levels (Fig. 1).

Plant growth regulators have a significant impact on morphogenesis in vitro. Cytokinins are known to stimulate cell division and work alongside other hormones to regulate various biochemical, physiological, and morphological processes in plants (Hallmark and Rashotte 2019; Nowakowska et al. 2022). Cytokinin is an important component of the flowering induction medium, which can promote in vitro flowering in several rose cultivars (Saritha and Naidu 2007; Vu et al. 2006; Zeng et al. 2013; Zhang et al. 2007). According to Yuan et al. (2024), adding hormones, sugars, and nutrients to the culture medium improved the flowering of roses in vitro, with cytokinin having the strongest effect. At 1.5 mg/L, floral bud induction and flowering rates reached 91.11% and 79.73%, respectively (Yuan et al. 2024). In this study, MS medium supplemented with different concentrations of BA was shown to induce in vitro flowering compared to the control. When the concentration of BA increased to 2.0 mg/L, the plant's ability to stimulate flowering decreased, but the number of shoots on the medium increased, leading to an increase in plant weight (Fig. 2). Therefore, when BA concentration increases, it will stimulate more shoot formation instead of flower development on the main stem. According to the results from Table 3, 1.0 and 1.5 mg/L BA treatment gave the highest flowering rate. Additionally, flower formation in the culture encountered some unusual problems, including issues with shape, number of petals, and flower color (Fig. 5).

Figure 5. Main flower abnormalities observed during in vitro flower induction. (A) The flower bud is a small green-petal rose. (B) Leaves have developed into flowers. (C) Undeveloped petals (Scale bar = 1 cm)

Applying silver ions as AgNO₃ in plant tissue culture media significantly regulates ethylene activity in various plant systems. Silver nitrate has significant physiological effects in plants, including organogenesis, somatic embryogenesis, in vitro rooting of micro shoots, and control of flowering and leaf abscission. Another study also showed that the accumulation of ethylene gas was significantly reduced when AgNO₃ was used as an inhibitor in the micropropagation of cherry (Sarropoulou et al. 2016). Applying silver ions in the form of AgNO₃ to the plant tissue culture medium has been shown to reduce ethylene activity and induce flowering in rose buds. According to Matos et al. (2021), a concentration of 2.0 mg/L of AgNO₃ resulted in a flowering rate of 50%. In this paper, however, a concentration of 1.0 mg/L of AgNO₃ produced the highest flowering rate at 56.67%. Conversely, higher concentrations of AgNO₃ led to abnormal flowering in rose buds due to the accumulation of excessive silver ions in the plant tissue, similar to findings in a study on tobacco (Cardoso et al. 2019). When treated together, cytokinin, AgNO₃, and sucrose induced flowering in rose varieties. Cytokinins stimulated the flowering induction process, and Ag⁺ ion reduced the sensitivity of rose buds to ethylene under in vitro conditions, leading to the induction of flowering. In addition, sucrose is considered an essential carbon source in the culture medium for flower induction and development. Besides sucrose, hormones can also play a role in increasing the number of flowers. Cytokinins can impact meristem size, enhancing flower production (Han et al. 2014). Additionally, cytokinins are facilitative in tissue culture transitioning from vegetative to floral meristems (Taylor et al. 2005).

However, research results on PGRs and AgNO₃ are still quite limited, especially in overcoming the abnormal phenomenon of roses cultured in vitro. In this study, BA, sucrose, and AgNO₃ were added separately or in combination to the culture medium to increase growth and improve flowering ability in vitro, overcoming the abnormal phenomenon of increasing flower quality. This result can enhance the understanding of flowering physiology in rose species and help improve flowering induction.

This research is funded by University of Science, VNU-HCM under grant number T2024-41.