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Article

Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’

by
Qin Zhou
1,†,
Yuexin Zhang
2,†,
Ruijie Li
2,
Miao Tian
2,
Changlong Zhong
1,
Xiongbo Jiang
1,* and
Wei Zhang
2,*
1
Landscape Construction College, Hubei Ecology Polytechnic College, Wuhan 430070, China
2
National Key Laboratory for Germplasm Innovation & Utilization of Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong Agricultural University, Wuhan 430070, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2025, 15(8), 1834; https://doi.org/10.3390/agronomy15081834
Submission received: 24 June 2025 / Revised: 24 July 2025 / Accepted: 25 July 2025 / Published: 29 July 2025
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

Roses are one of the most essential ornamental flowers in the world. At present, traditional techniques such as cross breeding are mainly used in rose breeding. The inefficiency of the in vitro regeneration system has become the limiting step for the innovation and genetic improvement of rose germplasm resources. A tissue culture rapid propagation system of Rosa ‘Pompon Veranda’ was established using the stem segments with shoots as the initial experimental material. The results showed that the best disinfection method was to soak the explants in 75% ethanol for 1 min, and then soak them in 15% sodium hypochlorite solution for 15 min. The contamination rate was only about 6%. The best rooting medium for tissue culture seedlings was 1/2MS with 0.1 mg∙L−1 NAA, and the rooting rate can reach around 95%. On this basis, calluses were induced by using leaflets of tissue-cultured seedlings as explants. The results showed that the optimal medium for inducing callus tissue was MS + 5.0 mg∙L−1 2,4-D, with an induction rate of 100%. The calluses were cultured in the medium of MS with 0.01 mg∙L−1 NAA, 1.5 mg∙L−1 TDZ and 0.1 mg∙L−1 GA3 for 12 days in the dark and then transferred to light conditions. The differentiation rate of callus was 10.87%. On the medium of MS with 0.5 mg∙L−1 6-BA, 0.004 mg∙L−1 NAA and 0.1 mg∙L−1 GA3, the shoots could regenerate into whole plants. This study has established an in vitro regeneration system of R. ‘Pompon Veranda’, which is a key perquisite for the subsequent establishment of its genetic transformation system. Moreover, this method will also be an important reference for studies on quality traits such as floral scent and prickles of Rosa plants.

1. Introduction

Rose (Rosa spp.) hailed as the ‘Queen of Flowers’ is an ornamental plant in the genus Rosa of the family Rosaceae. Due to the diverse categories, excellent flower forms, rich flower colors, and charming fragrance of roses, they have always held a prominent position in landscape horticulture. Meanwhile, its extended vase longevity and profound cultural significance have secured roses a substantial share in both the cut flower and potted plant markets. Additionally, roses possess high value in the food, pharmaceutical, spice, and cosmetics industries.
Modern roses (R. hybrida) are predominantly derived from hybridization or backcrossing of rose cultivars originating in China, Europe, and the Middle East [1]. Employing these conventional breeding approaches, breeders have created tens of thousands of new rose cultivars. However, repeated hybridization and backcrossing have resulted in offspring with a relatively narrow genetic background, leading to diminished fragrance, reduced disease resistance, and increasingly apparent limitations in this breeding approach. The advent of plant genetic engineering has transcended species boundaries, providing a novel pathway for targeted trait improvement in roses and enabling the creation of unique, high-quality varieties within shorter timeframes. Establishing efficient regeneration and genetic transformation systems for roses remains a fundamental prerequisite for advancing gene-editing research in this genus. To date, numerous attempts have been made in transgenic rose studies [2,3,4], yet regeneration and genetic transformation systems have been successfully established in only a limited number of Rosa hybrida cultivars.
Rose regeneration primarily relies on three pathways: organogenesis, somatic embryogenesis, and protocorm-like body (PLB) regeneration [5,6]. Hill [7] successfully induced regenerated plantlets via organogenesis using stem segments of Rosa odorata as explants. Dubois et al. [8] used leaves as explants to establish direct regeneration of shoots in cut-flower roses. Similarly, Meng et al. [9] and Ren et al. [10] also established a regeneration system through indirect organogenesis using the leaves of roses. Somatic embryogenesis also represents a vital regeneration route in roses. For example, Bao et al. [11] induced somatic embryos of R. ‘Samantha’ and established a relatively mature regeneration system. Other cultivars—such as R. chinensis ‘Old Blush’, R. hybrida ‘Eiffel Tower’, ‘Carefree BeautyTM’, ‘Italian Ice’ and ‘Ringo All-StarTM’—have also utilized somatic embryogenesis for regeneration system development [12,13,14]. Additionally, a limited number of rose varieties have achieved regeneration through the PLB pathway [15]. Although these pathways each have unique advantages, successful regeneration is markedly influenced by genotype, explant type, and culture conditions. Moreover, existing regeneration protocols generally exhibit low regeneration frequencies and recalcitrance to genetic transformation, underscoring the need to develop and refine more efficient regeneration systems.
R. ‘Pompon Veranda’ is a classic miniature rose cultivar distinguished by a pale-pink central eye, creamy outer petals, and a delicate tea fragrance. It is prized by consumers for its prolific flowering, extended bloom period, and strong disease resistance. However, the dense distribution of prickles on its stems poses significant challenges for field management and postharvest handling. In this study, nodal segments bearing shoots from seedling-derived R. ‘Pompon Veranda’ were used as initial explants to establish a rapid propagation system, and in vitro leaves were employed as explants to develop a complete organogenesis-based plant regeneration protocol (Figure 1). Therefore, the efficient regeneration system of R. ‘Pompon Veranda’ established in this study laid a solid foundation for the establishment of its genetic transformation system. The results can also be used for subsequent studies on quality traits such as floral scent and prickles of Rosa plants.

2. Materials and Methods

2.1. Experimental Materials

Branches with shoots were collected from the healthy, current-year apical shoots of R. ‘Pompon Veranda’ plants grown in a natural environment under field conditions for about 5 years in the Rosa germplasm repository of Huazhong Agriculture University, Wuhan, China. For in vitro leaf explants, the tender young leaves at the top that are not fully unfolded were harvested from tissue-cultured plantlets derived from the aforementioned nodal stem segments. These plantlets were initially cultured on shoot initiation medium for 30 days and subsequently subcultured three times prior to explant excision. The shoot initiation medium was slightly adjusted based on previous reports [16], with the specific formulation being Murashige and Skoog medium (MS, coolaber, Beijing, China) [17] + 1.0 mg∙L−1 N-(Phenylmethyl)-9H-purin-6-amine (6-BA, coolaber, Beijing, China) + 0.01 mg∙L−1 1-Naphthylacetic acid (NAA, coolaber, Beijing, China) + 0.1 mg∙L−1 Gibberellin A3 (GA3, coolaber, Beijing, China), pH = 5.8.

2.2. Experimental Methods

2.2.1. Explant Sterilization

The current-year apical branches of R. ‘Pompon Veranda’ were cut into stem segments containing one or two shoot points as the initial experimental explants. We first removed the leaves and soaked the stem segments in a 0.1% (w/v) carbendazim solution for 20 min and then rinsed them under running tap water for 2 h. Under aseptic conditions, explants were surface-sterilized by immersion in 75% ethanol for 1 min, followed by treatment with varying concentrations of sodium hypochlorite (NaClO) for different durations, according to nine sterilization combinations (Table 1). After each NaClO treatment, explants were rinsed 3 times with sterile distilled water (one minute per rinse). Throughout sterilization, wide-mouth flasks were gently agitated to ensure full contact between the explants and sterilant. Explants were then blotted dry on sterile filter paper and inoculated onto MS (Murashige and Skoog medium) with 1.0 mg∙L−1 6-BA, 0.01 mg∙L−1 NAA, 0.1 mg∙L−1 Gibberellin A3 (GA3, coolaber, Beijing, China). Each treatment comprised 15 explants and was replicated three times. Cultures were maintained at (22 ± 2 °C) under a 16 h light/8 h dark photoperiod with 40% relative humidity. After 30 days, contamination and mortality rates were recorded.

2.2.2. Proliferation Culture

Based on the shoot-multiplication protocol previously established for R. ‘Samantha’ [18], with minor modifications, the proliferation medium comprised MS with 0.5 mg∙L−1 6-BA, 0.01 mg∙L−1 NAA and 30 g∙L−1 Sucrose (coolaber, Beijing, China), pH = 5.8. Sterilized plantlets were inoculated onto this medium, with 15 explants per treatment and three biological replicates. Culture conditions were identical to those described in Section 2.2.1. After 30 days, the proliferation coefficient was calculated, and the growth status of the plantlets was recorded.

2.2.3. Rooting Culture

Uniform and vigorous plantlets were selected and transferred to rooting medium. The basal medium was MS with half-strength macronutrients (1/2MS, coolaber, Beijing, China), and the effects of different concentrations of NAA and 3-Indolebutyric acid (IBA, coolaber, Beijing, China) on root induction were evaluated. Four treatments were arranged, with the pH of each treatment medium at 5.8: 1/2MS with 0.1 mg∙L−1 NAA and 30 g∙L−1 Sucrose; 1/2MS with 0.5 mg∙L−1 NAA and 30 g∙L−1 Sucrose; 1/2MS with 0.1 mg∙L−1 IBA and 30 g∙L−1 Sucrose; 1/2MS with 0.5 mg∙L−1 IBA and 30 g∙L−1 Sucrose. Each treatment comprised 15 explants and was replicated three times. Culture conditions followed those in Section 2.2.1. After 30 days, rooting rate and mean number of roots per shoot were recorded, and root system morphology was documented.

2.2.4. Plantlet Acclimatization and Transplanting

We selected tissue-cultured seedlings of R. ‘Pompon Veranda’ with well-developed roots, opened the culture bottle cap, added a small amount of distilled water to the culture bottle, and acclimatized the seedlings under natural conditions for 3 days. We removed the tissue-cultured seedlings, washed the roots, and transplanted them into plastic pots containing a mixed substrate (peat:vermiculite:perlite = 1:1:1, v/v/v). We placed them in a greenhouse with an indoor temperature of (20 ± 5) °C and relative humidity of 50~60%. Water was added promptly to keep the soil moist, and we recorded the transplant survival rate of rooted seedlings after 4 weeks.

2.2.5. Effect of 2,4-D Concentration on Leaflets Callus Induction

Leaf explants (approximately 25-day-old subcultured plantlets) with intact petioles (1 mm in length) were excised from healthy in vitro shoots. The abaxial midveins were gently scored with a sterile scalpel to enhance wound response. Explants were placed on callus induction media consisting of MS basal medium supplemented with 30 g∙L−1 glucose, 3.0 g∙L−1 phytagel, and varying concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, coolaber, Beijing, China) (pH = 5.8): 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 mg∙L−1. Each treatment included 30 explants with three replicates. Cultures were incubated in darkness at 22 ± 2 °C and 40% relative humidity. Callus was induced by using leaflets of tissue-cultured seedlings as explants. The callus obtained in this way was called leaflets callus. After 30 days, leaflets callus induction rates were quantified, and callus morphology was documented.

2.2.6. Effect of TDZ Concentration on Callus Differentiation

Leaf explants of R. ‘Pompon Veranda’ were cultured on the optimal callus-induction medium (as determined in Section 2.2.4). After one month, calluses were gently peeled from the leaves, and healthy leaflets callus pieces were transferred to shoot-differentiation medium composed of MS basal salts supplemented with 0.01 mg∙L−1 NAA, 0.1 mg∙L−1 GA3, 30 g∙L−1 glucose, 3.0 g∙L−1 plant agar, and cytokinin-like compounds thidiazuron (TDZ, coolaber, Beijing, China) at 0.5, 1.0, or 1.5 mg∙L−1, at pH = 5.8. Each treatment included 30 explants and was replicated three times. Cultures were incubated in darkness at 22 ± 2 °C and 40% relative humidity for 12 days, and then transferred to 16 h light/8 h dark conditions. After four weeks, shoot-differentiation rates were recorded.

2.2.7. Effect of Different Shoot-Elongation Media on Shoot Growth

Calluses exhibiting abundant shoot-primordia development on induction medium were selected and transferred to one of five shoot-elongation media (Table 2). Each medium was tested with 30 explants, in triplicate. Culture conditions followed those described in Section 2.2.1. After four weeks, the impact of each medium on shoot elongation was assessed and recorded.

2.3. Statistical Analysis

The experimental data were analyzed using IBM SPSS Statistics 27 software for one-way analysis of variance (ANOVA) and significance testing (Duncan’s multiple comparison test, significance level α = 0.05). The results are presented as mean ± standard deviation (SD).

3. Results

3.1. Effect of Sterilization Treatments on Explant Contamination and Mortality

The efficacy of different sterilization protocols on explant disinfection is summarized in Table 3. The contamination is possibly fungal contamination. Treatment 1 exhibited the highest contamination rate (75.56%), indicating the poorest sterilization efficacy. In contrast, Treatments 7, 8 and 9 achieved markedly lower contamination rates of 6.67%, 8.89% and 8.89%, respectively, exhibiting superior sterilization performance. A general trend was observed where increasing NaClO concentrations correlated with reduced contamination rates. Furthermore, mortality rates across all treatments remained low, with no significant differences detected. Notably, Treatments 3, 8 and 9 achieved 0% mortality, confirming minimal phytotoxic effects on explants under these conditions. Based on the dual criteria of contamination and mortality rates, Treatment 7 (1 min immersion in 75% ethanol followed by 15 min exposure to 15% NaClO) was identified as the optimal sterilization protocol for R. ‘Pompon Veranda’, yielding a contamination rate of 6.67% and mortality rate of 2.22%.

3.2. Multiplication Coefficient of R. ‘Pompon Veranda’

R. ‘Pompon Veranda’ microshoots proliferated rapidly on the proliferation medium described in Section 2.2.2, producing numerous axillary shoots with vigorous growth and vibrant green coloration. Statistical results showed that the proliferation coefficient reached 4.04 after 30 days of culture.

3.3. Effect of Different Rooting Media on Rooting Performance of R. ‘Pompon Veranda’

Table 4 presents the effects of various auxin types and concentrations on rooting rate, mean number of roots per shoot, and root system morphology. The results indicate that both the type and concentration of auxin significantly influenced rooting percentage and root number. When supplemented with 0.1 mg∙L−1 NAA, the rooting rate and mean root number were significantly higher than with 0.5 mg∙L−1 NAA. Moreover, 0.1 mg∙L−1 NAA produced a well-developed root system characterized by long, robust roots, whereas 0.5 mg∙L−1 NAA resulted in shorter, weaker roots. For IBA treatments, the rooting rate at 0.1 mg∙L−1 IBA was significantly lower than at 0.5 mg∙L−1 IBA, although the average root number did not differ markedly between these two concentrations. The 0.5 mg∙L−1 IBA treatment yielded a denser and longer root network (Figure 2). Taken together, 1/2MS with 0.1 mg∙L−1 NAA was determined to be the optimal rooting medium for R. ‘Pompon Veranda’.

3.4. Plantlet Acclimatization and Transplanting

We selected tissue-cultured seedlings of ‘Pompon Veranda’ with well-developed roots, acclimatized them, and then transplanted them into the greenhouse. After 30 days, the survival rate of the tissue-cultured seedlings reached as high as 100%, with the transplanted seedlings showing vigorous growth, dark green leaves, and the emergence of multiple new leaves (Figure 3f).

3.5. Effect of 2,4-D Concentration on Callus Induction from Leaf Explants

Table 5 summarizes the impact of eight 2,4-D concentrations on callus-induction rate and callus morphology from R. “Pompon Veranda” leaf explants. All treatments successfully induced callus formation, with no significant differences in induction rates across concentrations, indicating that 2,4-D level had a minimal effect on induction percentage. However, callus appearance varied markedly: at 1.0 and 2.0 mg∙L−1 2,4-D, the calluses were small, yellowish-brown, and compact; at 3.0 and 4.0 mg∙L−1, the calluses were larger, yellow to yellowish-brown, and softer; at 5.0 mg∙L−1, the calluses were large, bright yellow, and friable; at 6.0 and 7.0 mg∙L−1, the calluses exhibited slight water-soaking, later turning from yellow to yellowish-brown; and at 8.0 mg∙L−1, callus characteristics resembled those at 5.0 mg∙L−1. Balancing callus quality and cost, 5.0 mg∙L−1 2,4-D was selected as the optimal concentration for callus induction (Figure 4).

3.6. Effect of TDZ Concentration on Callus Differentiation

Table 6 presents callus-differentiation rates of Pompon Veranda under three TDZ concentrations. After approximately 15 days in darkness, calluses in all TDZ treatments began to differentiate shoots. The differentiation rate increased progressively with TDZ concentration, i.e., 0.5, 1.0, to 1.5 mg∙L−1. The highest differentiation frequency of 10.87% was achieved at 1.5 mg∙L−1 TDZ, significantly exceeding lower concentrations. Accordingly, 1.5 mg∙L−1 TDZ was determined to be optimal for shoot differentiation from callus.

3.7. Effect of Shoot-Elongation Media on Shoot Growth

Table 7 shows the responses of shoots, differentiated from calluses, to five shoot-elongation media. After one month, shoots on Media 1, 2, and 3 remained at initial sizes, showing no progression toward complete plantlets. On Media 4, shoots reverted to hardened callus structures. Only on Media 5 did shoots continue to elongate vigorously, with leaves transitioning from light green to vibrant green and a distinct main stem formation. Subsequent transfer of these elongated shoots to rooting medium successfully induced roots (Figure 5l).

4. Discussion

4.1. Effects of Sterilization Duration and Agents on Explant Disinfection

The establishment of rapid plant propagation and regeneration systems can provide more efficient and convenient means for the innovation of planting resources and genetic improvement breeding [19]. In the establishment of rapid propagation systems for Rosa plants, stem segments with shoots points are usually selected as explants, and the rapid propagation system involves the following stages: (1) establishment of an aseptic system and initiation culture, (2) proliferation of tissue-cultured seedlings, and (3) rooting of tissue-cultured seedlings [20]. As the first step in establishing an aseptic system, sterilization treatment is crucial, as effective sterilization lays a solid foundation for subsequent experiments. Commonly used sterilizing agents include NaClO, HgCl2, and alcohol. Lu [21] used HgCl2 to sterilize Rosa wichuraiana ‘Basye’s thornless’ for 8 min, achieving the best sterilization effect. Although HgCl2 has a higher sterilization efficacy than NaClO, due to the significant harm HgCl2 poses to human health, NaClO is generally preferred for sterilization purposes. Sheikh-Assadi et al. [22], while exploring the rapid propagation of dog rose (Rosa canina L.), found that sterilization with 2.5% NaClO for 5 min or 5% NaClO for 2.5 min both achieved the best sterilization effect, with a 100% survival rate of explants.
The duration of disinfection and the concentration of the disinfectant both affect the sterilization efficacy and survival rate of explants. Short disinfection time and low disinfectant concentration may result in incomplete sterilization, while prolonged disinfection time and high disinfectant concentration can damage or even poison the explants. In this study, it was found that with protocols 1, 2 and 3 (the disinfectant concentration was 5% NaClO), the contamination rate and mortality rate gradually decreased as the disinfection time increased. With protocols 4, 5 and 6 (the disinfectant concentration was 10% NaClO), the contamination rate and mortality rate initially decreased but then increased with longer disinfection time. With protocols 7, 8 and 9 (the disinfectant concentration was 15% NaClO), both the contamination rate and mortality rate increased with longer disinfection time. However, when the disinfection time is the same, as the disinfection concentration increases, the contamination rate gradually decreases, while the mortality rate first rises and then declines. This indicates that the disinfection effect is not proportional to the disinfection time and concentration, similar to the findings of Sheikh-Assadi’s study [22].
Taking all factors into consideration, the optimal disinfection protocol for R. ‘Pompon Veranda’ was found to be treatment with 75% alcohol for 1 min followed by 15% NaClO for 15 min. This method effectively reduced fungal contamination and mortality. Although some fungal contamination still occurred with this treatment, the contamination rate remained within a controllable range compared to other protocols and did not affect subsequent experiments. In the future, we can further optimize the sterilization of explants by studying factors such as the sampling season (e.g., spring or autumn), sampling conditions (e.g., sunny weather, avoiding rainy days), and the state of the sampled branches (e.g., tender green shoots in spring).

4.2. Effects of Hormone Types and Concentrations on Shoot Proliferation

The number of shoots produced by tissue-cultured seedlings is an important indicator for measuring their proliferation. Although the proliferation coefficient is influenced by various factors, the most significant one is the ratio of auxin to cytokinin. A lower ratio of auxin to cytokinin can induce the formation of shoots in tissue-cultured seedlings. The most commonly used auxins for inducing shoot formation are NAA and IBA, while the cytokinin used is 6-BA. A concentration range of 6-BA between 0.5 and 2.0 mg∙L−1 promotes the elongation of shoots, whereas concentrations exceeding 3.0 mg∙L−1 can cause vitrification in tissue-cultured seedlings. NAA at concentrations of 0.05–0.2 mg∙L−1 promotes shoot production, while IBA at 0.1–0.5 mg∙L−1 enhances polar growth of shoots [23,24]. Cytokinins combined with low concentrations of auxins promote the formation and proliferation of shoots [25]. Noodezh et al. [26] found that the optimal proliferation medium for Rosa damascena Mill. tissue-cultured seedlings contained 4.0 mg∙L−1 of 6-BA and 0.25 mg∙L−1 of IAA, achieving a proliferation coefficient of 9.6. Zhang et al. [27] discovered that the ground cover rose ‘Royal Bassino’ exhibited the highest proliferation coefficient of 6.3 when the medium contained 0.5 mg∙L−1 of 6-BA and 0.05 mg∙L−1 of IBA. In the study of rapid propagation techniques for the Rosa hybrida ‘Carolla’, Yan et al. [28] observed that tissue-cultured seedlings grew vigorously and robustly with a proliferation coefficient of 4.48 in a medium containing 3.0 mg∙L−1 of 6-BA and 0.10 mg∙L−1 of NAA. This demonstrates that the optimal proliferation medium and coefficient vary among different varieties. This study found that the best proliferation medium for R. ‘Pompon Veranda’ contained 0.5 mg∙L−1 of 6-BA and 0.01 mg∙L−1 of NAA, achieving a proliferation coefficient of 4.04, with seedlings growing robustly and uniformly. These results are generally consistent with previous studies on rose tissue culture [29,30], though some differences exist.

4.3. Effects of Explant Types on Callus Induction

During the callus induction process, the type of explant significantly affects the induction efficiency. In previous studies on roses, researchers have attempted to induce callus using various explants such as leaves, stem segments, filaments, anthers, and petals. Most results indicate that leaf explants yield significantly better induction outcomes compared to other tissues. Kintzios et al. [31] conducted callus induction studies using mature leaves and stem segments from four rose varieties. Their results showed that only one variety produced embryogenic callus from mature leaves, while none of the stem segments from the four varieties yielded embryogenic callus. Rout et al. [32] used leaves of the R. ‘Landora’ variety as explants, culturing them on MS medium supplemented with 6-BA, NAA, and 2,4-D. After 7–12 days of inoculation, callus formation was observed at the petiole and midrib on the adaxial side, achieving a callus induction rate of 92%. Yi et al. [33] established a regeneration system using leaves from tissue-cultured seedlings of Rosa chinensis ‘Yueyuehong’ as explants. Meanwhile, Marchant et al. [34] studied callus induction using leaves, petioles, and roots of floribunda roses. Their findings revealed that only petioles and roots produced embryogenic calluses, while calluses derived from young leaves exhibited a dense state with poor growth. These studies demonstrate that the efficiency of callus induction varies depending on the explant type and rose variety.
It is worth mentioning that in this study, in addition to using leaflets of tissue-cultured seedlings as explants to induce callus, we also attempted to use anthers as explants for callus induction. The results showed that the highest callus induction rate using anthers as explants was only 45.45% (Appendix A Table A1 and Table A2). Moreover, during the cultivation process, the callus gradually became hard and turned brown (Appendix A Figure A1). Additionally, no differentiation of these callus tissues was observed in the later stages of cultivation. On the other hand, obtaining leaves from tissue-cultured seedlings is relatively simple and unaffected by seasons or environmental factors. Furthermore, the callus induction rate using leaves from tissue-cultured seedlings as explants can reach 100%, and the growth status of the callus is much better compared to that of callus induced from anthers. Therefore, leaflets from tissue-cultured seedlings were ultimately used as explants for callus induction of R. ‘Pompon Veranda’.

4.4. Effects of Plant Growth Regulators on Callus Induction and Differentiation

The type and concentration of plant growth regulators play a crucial role in the induction and differentiation of callus [35]. Auxin promotes significant changes in the transcriptome and chromatin, enabling plant cells to acquire totipotency [36]. Among these, 2,4-D is the most commonly used auxin for callus induction, as it facilitates the dedifferentiation of explants to form callus [37,38,39]. For example, Yan et al. [40] found that when inducing calluses in Rosa chinensis ‘Slater’s crimson China, the use of 5.0 mg∙L−1 2,4-D resulted in a higher induction rate and optimal callus condition. Zhu et al. [41] used 7.0 mg∙L−1 2,4-D during the induction of callus from the leaves of R. hybrida ‘Tineke’, achieving an induction rate of 83.34%. Li et al. [42] discovered that for R. chinensis minima cv. Red Sunblaze, the highest callus induction efficiency (70%) was achieved at 1.0 mg∙L−1 2,4-D, while for R. hybrida cv. Carefree Beauty, the optimal concentration was 0.25 mg∙L−1, yielding an efficiency of 66.7%. In contrast, for R. hybrida cv. Grand Gala, callus induction efficiency remained consistently high (93.3%) across a range of 2,4-D concentrations (0.25–4.0 mg∙L−1). A total of eight treatments were set up in this section. When the concentration of 2,4-D exceeded 5.0 mg∙L−1 (6.0 mg∙L−1 and 7.0 mg∙L−1), the callus exhibited slight waterlogging, which was unfavorable for its differentiation. Previous research suggested that high concentrations of 2,4-D disrupt Ga2+ signaling, hindering cell wall synthesis, while also promoting ethylene synthesis and increasing plasma membrane permeability. This leads to vitrification of the callus, making its surface transparent and waterlogged [24]. Interestingly, however, when the 2,4-D concentration was 8.0 mg∙L−1, its induction effect on calluses was nearly identical to that at 5.0 mg∙L−1. Taking into account both economic cost and induction efficiency, it is concluded that 2,4-D (5.0 mg∙L−1) is the optimal choice for inducing callus in R. ‘Pompon Veranda’, which aligns with the 2,4-D concentration previously determined for inducing callus in Rosa chinensis ‘Old Blush’ [12].
TDZ also plays a key role in callus induction and shoots formation. It possesses unique properties similar to both auxins and cytokinins, effectively inducing morphological changes in plants [43] and directly or indirectly altering endogenous hormones to generate responses necessary for regeneration in cells or tissues [44]. In experiments, TDZ can be used alone or in combination with other hormones (such as 2,4-D, 6-BA, and GA3) to induce embryogenic callus or somatic embryos. Yi et al. [33] successfully induced somatic embryos from the leaves of Rosa chinensis ‘Old Blush’ using a medium containing 3.0 mg∙L−1 2,4-D and 0.5 mg∙L−1 TDZ. Gao [18] cultured callus from R. ‘Samantha’ on a medium supplemented with 1.0 mg∙L-1 TDZ, 0.01 mg∙L−1 NAA, and 0.1 mg∙L−1 GA3, observing successful differentiation of shoots after 30 days. This study followed a similar approach to Gao, ultimately achieving shoot formation on a medium containing 1.5 mg∙L−1 TDZ, 0.01 mg∙L−1 NAA, and 0.1 mg∙L−1 GA3, with a differentiation efficiency of 10.87%.

4.5. Effects of Auxin on Rooting of Plant Tissue Culture Seedlings

During the process of inducing rooting in rose tissue culture seedlings, auxin is indispensable [45,46]. Effective rooting induction in roses depends on the type of auxin (commonly used auxins include NAA, IBA, and IAA) and its concentration. Previous studies have reported that using a single hormone alone can achieve good rooting results. Bhoomsiri et al. [25] found that rooting of Rosa damascena Mill. could only be achieved on a medium supplemented with 0.5 mg∙L−1 NAA. Al-Ali et al. [47] concluded that a medium supplemented with 0.2 mg∙L−1 NAA was the optimal condition for inducing rooting in Al-Taif Rose. Ambros et al. [48] observed the best rooting induction effect for Rosa canina L. on a medium containing 1.0 mg∙L−1 IAA. Some studies also reported that a combination treatment of 2,4-D and IAA yielded better rooting results in roses compared to treatment with a single hormone. However, this study found that using 0.1 mg∙L−1 NAA alone achieved a rooting rate of 95.55%, which contradicts the findings of Rezaneja et al. [49]. The reason for these discrepancies may be due to differences in genotypes.

5. Conclusions and Prospects

This study established an efficient rapid propagation system for R. ‘Pompon Veranda’ using nodal stem segments from seedlings (Figure 3). Furthermore, a regeneration system was developed via callus induction from in vitro leaf explants, followed by shoot differentiation, elongation, and root induction, ultimately generating intact plantlets (Figure 5a–l). This protocol provides a critical foundation for future genetic transformation studies to elucidate molecular mechanisms underlying floral architecture, fragrance biosynthesis, and prickle development in Rosa species.
The system is constrained by inefficient differentiation (<11%), long cycles (weeks), stringent technical regulation, and strong genotype dependence, resulting in low universality, high application costs, and difficulty in breaking through varietal limitations, especially in genetic improvement. Future efforts need to optimize callus quality by integrating molecular regulation (e.g., editing key regeneration genes).
At present, there are still many areas in which the system could be optimized. In the future, to further elevate the differentiation efficiency of callus, we can attempt to adjust the following factors: the basic medium types (WPM, SH, etc.), the ratio of different plant growth regulators, and culture methods (e.g., suspension culture). Furthermore, the regeneration system established in this study lays the foundation for the development of a genetic transformation system. Therefore, we need to conduct sensitivity tests of materials at different stages—callus proliferation, callus differentiation into shoots, and shoots elongation—to antibiotics (Kan, Hyg). We will then investigate the effects of the Agrobacterium strain, different OD values of Agrobacterium, infection time, and co-culture duration on the efficiency of genetic transformation. Furthermore, we can also try to employ plant developmental regulators (e.g., GROWTH-REGULATING FACTORs, BABYBOOM, etc.) to improve the efficiency of regeneration and genetic transformation or even overcome the barrier of genotype dependency.

Author Contributions

Conceptualization, X.J. and W.Z.; methodology, M.T. and C.Z.; formal analysis, Y.Z., Q.Z., R.L., M.T. and C.Z.; investigation, Y.Z., Q.Z. and R.L.; writing—original draft, Y.Z. and Q.Z.; writing—review and editing, X.J. and W.Z.; data curation, R.L., M.T. and C.Z.; project administration, X.J. and W.Z., Y.Z. and Q.Z. contributed equally to this work. All authors have read and agreed to the published version of the manuscript.

Funding

Scientific Research Project of Education Department of Hubei Province (No. B2023570).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

The authors wish to acknowledge Ying Bao at the Department of Life Science, Tangshan Normal University, for providing support and engaging in discussions.

Conflicts of Interest

The authors declare no conflicts of interest.

Correction Statement

This article has been republished with a minor correction to the existing affiliation information. This change does not affect the scientific content of the article.

Appendix A

Table A1. Medium used for induction of anthers.
Table A1. Medium used for induction of anthers.
Combination NumberComposition
1MS + 1.0 mg∙L−1 ZT + 1.0 mg∙L−1 2,4-D + 0.5 mg∙L−1 IAA
2MS + 0.6 mg∙L−1 TDZ + 0.4 mg∙L−1 2,4-D
3MS + 1.0 mg∙L−1 BA + 0.1 mg∙L−1 NAA
4MS + 1.0 mg∙L−1 BA + 0.1 mg∙L−1 NAA + 5.0 mg∙L−1 VC
5MS + 10.0 mg∙L−1 6-BA
6MS + 1.0 mg∙L−1 2,4-D + 0.1 mg∙L−1 6-BA
7MS + 0.2 mg∙L−1 2,4-D + 0.2 mg∙L−1 6-BA
8MS + 0.2 mg∙L−1 2,4-D + 5.0 mg∙L−1 6-BA
9MS + 1.0 mg∙L−1 6-BA + 0.2 mg∙L−1 IBA + 0.1 mg∙L−1 GA3
Table A2. Effect of different media components on anther induction of R. ‘Pompon Veranda’.
Table A2. Effect of different media components on anther induction of R. ‘Pompon Veranda’.
Process Combination NumberCallus Induction Rate (%)Callus Status
131.74 ± 3.64 bCompact callus, light yellow, large size
245.45 ± 6.18 aCompact callus, light yellow, large size
318.26 ± 2.75 cdLight yellow callus, the granularity is noticeably looser, large size
424.60 ± 4.95 cCallus yellow, the particle size is noticeably looser, smaller size
50.00 ± 0.00 eNo callus formation, browning and death of explants
634.12 ± 1.37 bCompact callus, yellow, white, or brown, larger size
742.07 ± 4.95 aCompact callus, light yellow, large in size, large size
815.87 ± 1.38 dCompact callus, light yellow, compact size
96.34 ± 3.64 eCompact callus, light yellow, compact size, some callus-forming anthers showed vitrified tissues around them, and gradually turn brown and die
Different lowercase letters in the same column indicate significant differences at 0.05 level.
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Figure A1. Different states of the anther callus in the R. ‘Pompon Veranda’. Note: (a) processing combination 1; (b) processing combination 2; (c) processing combination 3; (d) processing combination 4; (e) processing combination 5; (f) processing combination 6; (g) processing combination 7; (h) processing combination 8; (i) processing combination 9. Each treatment was inoculated with 40–50 explants, and the experiment was repeated three times. Bars = 1 cm.
Figure A1. Different states of the anther callus in the R. ‘Pompon Veranda’. Note: (a) processing combination 1; (b) processing combination 2; (c) processing combination 3; (d) processing combination 4; (e) processing combination 5; (f) processing combination 6; (g) processing combination 7; (h) processing combination 8; (i) processing combination 9. Each treatment was inoculated with 40–50 explants, and the experiment was repeated three times. Bars = 1 cm.
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Table A3. Some regeneration studies in Rosa spp.
Table A3. Some regeneration studies in Rosa spp.
GenotypesType of ExplantRegeneration PathwayReferences
96 different cultivars
(Rosa hybrida)		PetioleOrganogenesis [50]
‘Apollo’, ‘Black Baccara’, ‘Maroussia’ and ‘Amanda’ (Rosa hybrida)LeafOrganogenesis [51]
‘Samantha’ (Rosa hybrida)LeafOrganogenesis [18]
Rosa damascenaLeafOrganogenesis [30]
‘Charming’ (Rosa hybrida)Newly formed roots from the distal end of shootsCombination of somatic embryogenesis and organogenesis [38]
‘Carefree Beauty’, ‘Grand Gala’
(Rosa hybrida)
‘Red Sunblaze’
(Rosa chinensis) 		LeafCombination of somatic embryogenesis and organogenesis [42]
‘Carola’
(Rosa hybrida)		LeafSomatic embryogenesis [52]
‘John F. Kennedy’
(Rosa hybrida)		LeafSomatic embryogenesis[53]
‘Old Blush’
(Rosa chinensis)		LeafSomatic embryogenesis[12]
‘Yueyuehong’
(Rosa chinensis)		LeafSomatic embryogenesis[33]
‘Old Blush’
(Rosa chinensis)		LeafSomatic embryogenesis [54]
Rosa rugosaCotyledons without embryonic axes, and radicles excised from zygotic embryosSomatic embryogenesis[55]
‘Livin Easy’ (Rosa hybrida)LeafSomatic embryogenesis [56]

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Agronomy 15 01834 g001
Figure 1. Experimental flowchart.
Figure 1. Experimental flowchart.
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Figure 2. Root state of R. ‘Pompon Veranda’ on different rooting media. (a): 1/2MS + 0.1 mg∙L−1 NAA; (b): 1/2MS + 0.5 mg∙L−1 NAA; (c): 1/2MS + 0.1 mg∙L−1 IBA; (d): 1/2MS + 0.5 mg∙L−1 IBA. Bars = 1 cm. Each treatment is inoculated with 15 explants, and each treatment is repeated 3 times.
Figure 2. Root state of R. ‘Pompon Veranda’ on different rooting media. (a): 1/2MS + 0.1 mg∙L−1 NAA; (b): 1/2MS + 0.5 mg∙L−1 NAA; (c): 1/2MS + 0.1 mg∙L−1 IBA; (d): 1/2MS + 0.5 mg∙L−1 IBA. Bars = 1 cm. Each treatment is inoculated with 15 explants, and each treatment is repeated 3 times.
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Figure 3. Establishment process of rapid propagation system of R. ‘Pompon Veranda’. Note: (a,b) Morphological characteristics of R. ‘Pompon Veranda’. (a) Floral organ, (b) stem; (c) germination of axillary shoots on the stem segment; (d) proliferation of tissue culture seedlings; (e) rooting of tissue culture seedlings; (f) the regenerated plant after 20 days of transplanting. Bars = 1 cm.
Figure 3. Establishment process of rapid propagation system of R. ‘Pompon Veranda’. Note: (a,b) Morphological characteristics of R. ‘Pompon Veranda’. (a) Floral organ, (b) stem; (c) germination of axillary shoots on the stem segment; (d) proliferation of tissue culture seedlings; (e) rooting of tissue culture seedlings; (f) the regenerated plant after 20 days of transplanting. Bars = 1 cm.
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Figure 4. Callus status of R. ‘Pompon Veranda’ induced by different 2,4-D concentrations. Note: (1–8) represents the callus status at 2,4-D concentrations ranging from 1.0 mg∙L−1 to 8.0 mg∙L−1 (1 represents 1.0 mg∙L−1, and the same applies to other numbers). Bars = 1.0 cm.
Figure 4. Callus status of R. ‘Pompon Veranda’ induced by different 2,4-D concentrations. Note: (1–8) represents the callus status at 2,4-D concentrations ranging from 1.0 mg∙L−1 to 8.0 mg∙L−1 (1 represents 1.0 mg∙L−1, and the same applies to other numbers). Bars = 1.0 cm.
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Figure 5. Regeneration process of R. ‘Pompon Veranda’. Note: (a,b) Callus induction from leaves of tissue cultured seedling; (c,d) shoot differentiation of callus tissue (12-day cultivation in dark); (e–g) the state of shoots after being cultured under light (60 days cultivation); (h,i) shoot elongation; (j) the shoots regenerate into a whole plant; (k,l) root system status. Bars = 0.5 cm.
Figure 5. Regeneration process of R. ‘Pompon Veranda’. Note: (a,b) Callus induction from leaves of tissue cultured seedling; (c,d) shoot differentiation of callus tissue (12-day cultivation in dark); (e–g) the state of shoots after being cultured under light (60 days cultivation); (h,i) shoot elongation; (j) the shoots regenerate into a whole plant; (k,l) root system status. Bars = 0.5 cm.
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Table 1. Explant disinfection scheme.
Table 1. Explant disinfection scheme.
TreatmentsNaClO (%)Immersion Time (min)
1515
2520
3525
41015
51020
61025
71515
81520
91525
Table 2. Treatment schemes for the shoot-elongation culture of R. ‘Pompon Veranda’.
Table 2. Treatment schemes for the shoot-elongation culture of R. ‘Pompon Veranda’.
No.Medium Composition
1MS + 1.0 mg∙L−1 6-BA + 0.01 mg∙L−1 NAA
2MS + 0.5 mg∙L−1 6-BA + 0.01 mg∙L−1 NAA
3MS + 1.0 mg∙L−1 6-BA + 0.01 mg∙L−1 NAA + 0.1 mg∙L−1 GA3
4MS + 2.0 mg∙L−1 6-BA + 0.1 mg∙L−1 IBA + 0.1 mg∙L−1 GA3
5MS + 0.5 mg∙L−1 6-BA + 0.004 mg∙L−1 NAA + 0.1 mg∙L−1 GA3
Table 3. Effect of disinfection scheme on contamination rate and mortality rate of R. ‘Pompon Veranda’.
Table 3. Effect of disinfection scheme on contamination rate and mortality rate of R. ‘Pompon Veranda’.
No.Contamination Rate (%)Mortality Rate (%)
175.56 ± 7.70 a4.45 ± 3.85 a
260.00 ± 0.00 b2.22 ± 3.85 a
360.00 ± 6.67 b0.00 ± 0.00 a
437.78 ± 10.18 c8.89 ± 7.70 a
515.55 ± 3.85 de2.22 ± 3.85 a
624.45 ± 3.85 d4.45 ± 3.85 a
76.67 ± 0.00 e2.22 ± 3.85 a
88.89 ± 3.85 e0.00 ± 0.00 a
98.89 ± 3.85 e0.00 ± 0.00 a
Note: Different lowercase letters in the same column indicate significant differences at 0.05 level; the same as below.
Table 4. Effects of plant growth regulators on the rooting of R. ‘Pompon Veranda’.
Table 4. Effects of plant growth regulators on the rooting of R. ‘Pompon Veranda’.
No.Medium CompositionRooting Rate (%) Average Rooting NumberRoot Status
a1/2MS + 0.1 mg∙L−1 NAA95.55 ± 3.85 a24.09 ± 0.25 aDense, strong, well-developed and long
b1/2MS + 0.5 mg∙L−1 NAA60.00 ± 6.67 b15.13 ± 0.54 cSparse, weak and short
c1/2MS + 0.1 mg∙L−1 IBA48.88 ± 3.85 c16.35 ± 0.44 bSparse, weak and short
d1/2MS + 0.5 mg∙L−1 IBA62.22 ± 3.85 b16.22 ± 0.39 bDense, slender and long
Different lowercase letters in the same column indicate significant differences at 0.05 level.
Table 5. Effects of different concentrations of 2,4-D on callus induction of leaves.
Table 5. Effects of different concentrations of 2,4-D on callus induction of leaves.
2,4-D (mg∙L−1)Induction Rate (%)Callus Status
1.0100 aBrownish-yellow, small, hard texture
2.0100 a Brownish-yellow, small, hard texture
3.0100 aYellow or pale yellow, medium size, soft texture
4.0100 aBrownish-yellow, medium size, soft texture
5.0100 aYellow, loose, big, soft texture
6.0100 aYellow, loose, big, slight watery
7.0100 aYellow, loose, big, slight watery
8.0100 aYellow, loose, big, soft texture
Different lowercase letters in the same column indicate significant differences at 0.05 level.
Table 6. Effects of different concentrations of TDZ on callus differentiation.
Table 6. Effects of different concentrations of TDZ on callus differentiation.
TDZ (mg∙L−1)Shoots Differentiation Rate (%)Number of Shoots Generated Per Explant
0.56.50 ± 1.31 b1.00 ± 0.00 a
1.07.03 ± 1.19 b1.33 ± 0.58 a
1.510.87 ± 1.54 a1.67 ± 0.58 a
Different lowercase letters in the same column indicate significant differences at 0.05 level.
Table 7. Effects of different media on the elongation of shoots.
Table 7. Effects of different media on the elongation of shoots.
No.Shoot StatusAverage Shoot Length (cm)Number of Leaves Per Shoot
1Maintain the original state0.23 ± 0.06 a0.00 ± 0.00 b
2Maintain the original state0.20 ± 0.00 a0.00 ± 0.00 b
3Maintain the original state0.20 ± 0.01 a0.00 ± 0.00 b
4Change into hard callus tissue0.23 ± 0.06 a0.00 ± 0.00 b
5Develop into strong, bright-green plantlets with the main stem0.53 ± 0.06 a2.67 ± 0.58 a
Different lowercase letters in the same column indicate significant differences at 0.05 level.
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Zhou, Q.; Zhang, Y.; Li, R.; Tian, M.; Zhong, C.; Jiang, X.; Zhang, W. Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’. Agronomy 2025, 15, 1834. https://doi.org/10.3390/agronomy15081834

AMA Style

Zhou Q, Zhang Y, Li R, Tian M, Zhong C, Jiang X, Zhang W. Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’. Agronomy. 2025; 15(8):1834. https://doi.org/10.3390/agronomy15081834

Chicago/Turabian Style

Zhou, Qin, Yuexin Zhang, Ruijie Li, Miao Tian, Changlong Zhong, Xiongbo Jiang, and Wei Zhang. 2025. "Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’" Agronomy 15, no. 8: 1834. https://doi.org/10.3390/agronomy15081834

APA Style

Zhou, Q., Zhang, Y., Li, R., Tian, M., Zhong, C., Jiang, X., & Zhang, W. (2025). Establishment of an Efficient Regeneration System of Rosa ‘Pompon Veranda’. Agronomy, 15(8), 1834. https://doi.org/10.3390/agronomy15081834

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