Establishment of a Highly Efficient In Vitro Regeneration System for Rhododendron aureum

Abstract

Rhododendron aureum is identified as a vulnerable species in China. The establishment of an in vitro regeneration system assists significantly in species protection. Here, an in vitro regeneration system was developed using both direct and indirect organogenesis pathways. The role of thidiazuron (TDZ) in different developmental stages was also investigated. The leaves of wild-harvested R. aureum plants were used for callus induction after hydroponic cultivation. The optimal formula was found to be woody plant basal medium (WPM) supplemented with 0.5 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ 3-indolebutyric acid (IBA), while the optimal formula for the subculture and induction of adventitious buds was WPM containing 0.1 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA. The leaves from tissue-cultured seedlings were then used for the induction of bud clusters. An association was observed between the differentiation of bud clusters and the ratio of auxin and cytokinin. The optimal formula for the induction of bud clusters was WPM containing 0.5 mg·L⁻¹ TDZ and 0.1 mg·L⁻¹ IBA, yielding a 50% induction rate and the maximum number of buds. Higher concentrations of TDZ were found to be beneficial for bud proliferation, while a lower concentration was conducive to stem elongation. The optimal formula for subculture was WPM containing 0.1 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA, while that for stem elongation was WPM supplemented with 0.002 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA. Only IBA was found to be necessary for rooting, with increased IBA concentrations leading to an increased number of roots and earlier root formation, with larger callus areas; thus, an IBA concentration of 1.0 mg·L⁻¹ was found to be optimal for the rooting of R. aureum. After hardening the seedlings for two days, a substrate composed of vermiculite and peat soil in a 10:1 ratio was identified as a transplantation formula. This system provides directions both for the protection of endangered species and the promotion of industrial development.

1. Introduction

Azalea (Rhododendron L.), known as the king of woody flowers, is a generic term describing plants of the Rhododendron genus, and represents one of the ten traditional flowers of China. There are approximately 1000 recorded Rhododendron species distributed throughout Asia, Europe, and North America, with China being home to 580 species, 420 of which are unique to the country [1]. One of these species is R. aureum Georgi, an evergreen dwarf shrub that ranges from 10 cm to 50 cm in height, with well-branched trailing stems. It is typically found in alpine grassland or mossy layers at altitudes between 1000 and 2506 m in the Jilin, Liaoning, and Heilongjiang provinces of China. It is highly valued as an ideal bonsai plant and rare ornamental plant due to its light-yellow flowers and also has significant potential for development as a medicinal plant [2]. Despite its value, the distribution range of R. aureum is narrow, and its presence in the wild is limited due to its specific environmental requirements. Additionally, climate change and human interference have further restricted its distribution, leading to its identification as an endangered and vulnerable species [3].

Scientists in the 21st century are seeking effective methods to prevent the extinction of endangered species, and in vitro regeneration may be a useful tool for protection, preservation, sustainable management, restoration, and rewilding [4]. Two methods are available for the propagation of R. aureum, namely, sexual propagation (seeding) and cloning (layering, cutting, grafting). However, both methods have low propagation coefficients and require long growth cycles, with vulnerability to environmental factors. The use of in vitro regeneration technology, on the other hand, has numerous advantages, including high reproductive efficiency, consistent growth of seedlings, and unrestricted repropagation, making it especially well-suited for the breeding of alpine plants. In vitro regeneration (plant tissue culture) can also satisfy large-scale plant propagation needs and is a vital tool for the study of stress tolerance in plants [5]. The use of in vitro regeneration allows for both the rapid propagation of R. aureum and the ability to preserve germplasm in vitro to avoid plant extinction. To date, a variety of Rhododendron species have been propagated using in vitro systems, including R. micranthum [6], R. hainanense [7], R. moulmainense [8], and R. mucronulatum [9], amongst others. The micro-propagation of R. aureum was first investigated in 2008 using dormant buds as explants to determine the most suitable medium for germination, redifferentiation, rooting, and germplasm preservation in vitro [10]. That same year, Ding established an in vitro regeneration system using the stems and leaves of aseptic seedlings as explants, and explored the effects of the basal medium, phytohormone (zeatin [ZT]), and active carbon on plant regeneration [11]. Subsequently, a series of studies were conducted to optimize the tissue-culture system, resulting in the identification of the Murashige and Skoog (MS) basal medium and woody plant medium (WPM) as suitable media for the culture of R. aureum, with thidiazuron (TDZ), ZT, and isopentenyl adenine (2-iP) typically used as cytokinins, and 3-indolebutyric acid (IBA) as the auxin playing different roles at various stages of the tissue-culture process [12,13]. In R. simsii ‘Hellmut Vogel’, the shoots were found to be small after culture with TDZ, and a subsequent elongation step was required before rooting, while an elongation step was not required when ZT was used [14]. It is known that plant growth regulators play important roles in in vitro regeneration. In addition, Rhododendron species with different genotypes respond differently to cytokinins [15].

Considering the importance of biodiversity and the significant value of R. aureum as both an ornamental and medicinal plant, we developed a stable and efficient method for its culture using plant material collected from the Fenghuang Mountain Nature Reserve in Heilongjiang Province, China. The development of a comprehensive in vitro regeneration system for R. aureum is of vital significance for its germplasm preservation, protection, biodiversity, and commercial breeding.

2. Materials and Methods

2.1. Plant Materials

Semi-lignified stems with fresh leaves and buds were collected from Fenghuang Mountain Nature Reserve in May 2021. R. aureum grows in alpine meadow areas at altitudes of 1696.2 m in the Fenghuang Mountain Nature Reserve. These original materials were cultured hydroponically in the laboratory, changing the water daily (Figure 1a). Fresh tender leaves were sterilized for callus induction.

2.2. Components of the Medium

The components of the medium used in the study included: Lloyd & McCown Woody Plant Basal Medium with Vitamins (WPM) (L449); AGAR, PLANT TC (A111); Thidiazuron Solution (TDZ) (1 mg·mL⁻¹) (T818); Indole-3-Butric Acid Solution (IBA) (1 mg/mL) (I460): PhytoTechnology Laboratories (Lenexa, KS, USA). Sucrose (A502792) from Sangon Biotech (Shanghai, China).

2.3. Culture Condition

The WPM medium contained 25 g·L⁻¹ sucrose and 5.2 g·L⁻¹ plant agar, with a pH of 5.6 ± 0.02. The media used for different growth stages had the same composition except for the type and concentration of the plant growth regulator. The media were sterilized in an autoclave at 121 °C for 20 min. The culture temperature was 25 ± 1 °C, the light intensity was 40–50 μmol·m⁻²·s⁻¹, and a light/dark cycle of 16/8 h was used.

2.4. Sterilization of Explants

Fresh leaves were removed from the stems and washed under running water for 30 min before transfer to a tissue-culture bottle. The ensuing steps were performed on a vertical flow clean bench. All explants were disinfected with 75% ethanol for 20 s, followed by three to five thorough washes with sterile water to remove any residual ethanol. The disinfection process was then continued with 3% sodium hypochlorite for 10 min, followed by three to five washes with sterile water. Finally, the leaves were dried with sterile filter paper in preparation for callus induction.

2.5. Primary Cultures of R. aureum

After sterilization, the leaves were cut into blocks of 1 cm² to expose the leaf veins and placed in WPM medium containing varying concentrations of TDZ (0.2, 0.3, 0.5 mg·L⁻¹) and IBA (0.2, 0.5, 0.8 mg·L⁻¹). Five blocks were placed in each Petri dish (with three biological replicates), resulting in a total of 15 blocks per group. Callus induction was monitored for one month. The callus was then separated from the leaves before transfer to a WPM medium containing TDZ (0.01, 0.1, 0.3, 0.5 mg·L⁻¹) and 0.5 mg·L⁻¹ IBA for subculture. The status of the callus was monitored and recorded for 20 days. To obtain adventitious buds, the callus was subsequently transferred to the WPM medium containing varying concentrations of TDZ (0.1, 0.01, 0.05 mg·L⁻¹) and IBA (0.3, 0.5, 0.8 mg·L⁻¹), and the growth of buds was monitored and recorded for 60 days. Full details of these combinations are shown in

Table 1. Protocols used for the induction of adventitious buds.

Group	Purpose	TDZ Concentration (mg·L−1)	IBA Concentration (mg·L−1)
A	Induction of callus	0.2	0.2
B		0.5	0.5
C		0.3	0.5
D		0.5	0.2
E		0.3	0.8
B	Subculture of callus	0.5	0.5
B-1		0.3	0.5
B-2		0.1	0.5
B-3		0.01	0.5
B-2	Induction of adventitious buds	0.1	0.5
B-2-1		0.1	0.3
B-2-2		0.1	0.8
B-2-3		0.01	0.8
B-2-4		0.05	0.5

.

2.6. Induction of Bud Clusters from Tender Leaves

The tender leaves were carefully removed from the adventitious buds and transferred to the WPM medium with TDZ (0.5, 1.0, 1.5 mg·L⁻¹) and IBA (0.1, 0.5, 1.0 mg·L⁻¹) for the induction of bud clusters. Each Petri dish contained six tender leaves, with five replicates per group. The rate of bud cluster induction was calculated as the number of leaves that formed bud clusters divided by the number of leaves inoculated. The number of bud clusters was recorded after 40 days of cultivation.

2.7. Subculture of Bud Clusters

The bud clusters were transferred to the WPM medium containing TDZ (0.01, 0.1, 0.5 mg·L⁻¹) and 0.5 mg·L⁻¹ IBA for subculture. The subculture of bud clusters and subsequent procedures were implemented in tissue-culture bottles. Each bottle contained three blocks of bud clusters (about 20 buds), with three replicates per group. The proliferation and status of the buds were closely monitored and recorded after 30 days of cultivation. The multiplication coefficient was calculated as the increase in the number of buds divided by the number of buds inoculated.

2.8. Stem Elongation of Bud Clusters

As some of the bud clusters were found to be dwarfed, stem elongation was required before rooting. To achieve this, the bud clusters were carefully divided into blocks of 0.3 cm² each (about 15 buds) and were placed in the WPM medium containing TDZ (0.04, 0.02, 0.01, 0.005, 0.002 mg·L⁻¹) and 0.5 mg·L⁻¹ IBA to promote elongation of the stems. Each bottle included three blocks of bud clusters with four replicates per group. The height and status of the buds were monitored carefully and recorded for 30 days of cultivation. The stem elongation coefficient was calculated as the number of buds showing stem elongation divided by the number of buds inoculated.

2.9. Rooting Culture

Seedlings with heights of 1.5–2.5 cm were carefully selected and isolated from the bud clusters and were cultured in the WPM medium supplemented with varying concentrations of IBA (0.5, 0.75, 1.0, 1.25, 1.5 mg·L⁻¹). Each bottle contained two shoots, and each group consisted of eight seedlings with four biological replicates. The number and length of roots were recorded after 30 days of cultivation. The rooting rate was calculated as the number of rooting adventitious shoots divided by the number of adventitious shoots inoculated into the culture medium.

2.10. Seedling Training and Transplantation

Seedlings with well-developed roots were selected from the rooting culture for training. Appropriate amounts of distilled water were added to the bottles and left for 1 to 2 days, with gradual opening of the bottle cap during the training progress. The trained seedlings were carefully removed from the bottle, and their roots were gently washed to remove any remaining culture medium on the surface. The seedlings were then planted separately in substrates containing different proportions of vermiculite and peat soil. The growth status was observed and recorded for 30 days.

2.11. Statistical Analysis

The experimental data are presented as mean ± standard error (SE) and were analyzed in SPSS 25.0 using ANOVA followed by Duncan’s multiple range test. The significance level was set at p ≤ 0.05. Tables were constructed in Microsoft Excel 2010 and Microsoft Word 2010, and the figures were generated in PS 2019.

3. Results

3.1. Primary Culture of R. aureum

Callus induction was initially observed in group B with culture for 16 days. Successful callus induction was observed in groups A, B and E after one month of culture (information on the groups is provided in Table 1). In comparison, the callus in group A grew more slowly than those in group B (Figure 1b), and group E, and showed partial browning over time. Calluses in group E grew more slowly than those in group B, but faster than those in group A, and also showed browning to some degree. It is worth noting that the calluses showed slight vitrification and a loose texture. Calluses from group B showed close textures and were induced earlier than those in groups A and E. Based on these observations, the group B conditions were selected as optimal for the callus induction protocol. Next, the effects of TDZ concentrations on callus subculture and proliferation were explored, and the results showed that group B-2 (Figure 1c) and group B-1 were superior, where the calluses were green, with a close texture, rarely browned, and grew more rapidly than those in groups B and B-3. The most severe browning was visible in group B-3, followed by group B. However, browning did not differ significantly between groups B-1 and B-2 (Figure 1c). Thus, group B-2 was considered optimal for callus subculture and proliferation. On this basis, different combinations of TDZ and IBA were then tested for the induction of adventitious buds. It was found that only the group B-2 conditions resulted in successful induction (Figure 1d). It is worth noting that optimal conditions for the induction of adventitious buds were identical to those for the callus subculture.

3.2. Induction of Bud Clusters

The sterile tender leaves from adventitious buds were then used for the induction of bud clusters. It was found that the base of the leaves, along with a portion of the petiole, began to expand after two weeks. Bud clusters were observed in all five groups after approximately 20 days, with the first bud appearing at the base of the leaves, together with the subsequent appearance of adventitious buds on the leaf veins (Figure 2). As shown in

Table 2. Effects of different TDZ and IBA concentrations on the induction of bud clusters.

Group	TDZ Concentration (mg·L−1)	IBA Concentration (mg·L−1)	Bud Clusters Induction Rate (%)	Growth Condition
1	0.5	0.5	50.0 ± 0.05 ab	The number of adventitious buds was lower.
2	1.0	0.5	60.0 ± 0.04 a	Callus and adventitious buds were induced, but the number of adventitious buds was lower.
3	1.5	0.5	36.7 ± 0.03 b	Callus was more obvious than adventitious buds, adventitious buds were then formed from the callus.
4	0.5	0.1	50.0 ± 0.05 ab	Adventitious buds appeared early, and had the highest number of adventitious shoots.
5	0.5	1.0	50.0 ± 0.05 ab	Callus appeared early, and adventitious buds were formed from the callus.

Means followed by the same letters in rows are not significantly different at p ≤ 0.05.

, group 2, which contained 1.0 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA, showed the highest rate of bud cluster induction (60%) (Figure 2b). However, the number of adventitious buds in group 2 was lower than that in group 4 (Figure 2d). The lowest rate of bud cluster induction was observed in group 3. Increasing the IBA concentration while maintaining the TDZ concentration at 0.5 mg·L⁻¹ did not alter the rate of adventitious bud induction. It would thus appear that the ratio of auxin and cytokinin plays a crucial role in the process of adventitious bud formation, with the two substances showing synergistic effects in comparison to the use of individual plant growth regulators. Considering that there was no significant difference in the induction rates between groups 2 and 4, the conditions of group 4 were selected as the most suitable formula for the induction of bud clusters.

3.3. Subculture and Proliferation of Bud Clusters

After 30 days of differentiation culture, numerous buds were visible, and these were divided for subculture to obtain more buds according to demand. Choosing a suitable culture formula can improve the growth of buds and increase the efficiency of bud proliferation. As shown in

Table 3. The effects of TDZ concentration on proliferation of bud clusters.

Group	TDZ Concentration (mg·L−1)	IBA Concentration (mg·L−1)	Multiplication Coefficient	Growth Condition
3-1	0.5	0.5	1.02 ± 0.05 b	The buds were inconsistent in size, and some of leaves began to turn yellow.
3-2	0.1	0.5	1.78 ± 0.09 a	The buds were robust, and the leaves were large.
3-3	0.01	0.5	0.60 ± 0.09 c	The leaves were large, but several buds were observed to have elongated stems.

Means followed by the same letters in rows are not significantly different at p ≤ 0.05.

, the multiplication coefficient of the adventitious buds was highest in group 3-2, with large and robust leaves, as shown in Figure 3b. However, the conditions in group 3-3 resulted in the lowest multiplication coefficient (0.60), although a proportion of the buds increased in height by 1–1.5 cm, as seen in Figure 3c. Furthermore, Figure 3a shows that buds cultured in the group 3-1 conditions were prone to dormancy and showed inconsistent growth. Thus, after a comprehensive comparison, the conditions used in group 3-2 (WPM containing 0.1 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA) were considered optimal for the subculture and propagation of bud clusters.

3.4. Stem Elongation of Bud Clustes

The bud clusters were divided into several blocks (about 15 buds) for stem elongation. Seedling heights were recorded after 40 days. It can be seen in

Table 4. The effect of TDZ concentrations on the stem elongation of bud clusters.

Group	TDZ Concentration (mg·L−1)	IBA Concentration (mg·L−1)	Stem Elongation Coefficient	Growth Condition
3-2-1	0.002	0.5	0.17 ± 0.03 a	Stem lengths were up to 2.5–3 cm, the seedlings were healthy.
3-2-2	0.005	0.5	0.16 ± 0.03 a	The leaves showed a light redness, the stem length was inconsistent.
3-2-3	0.01	0.5	0.15 ± 0.02 a	The stem length was inconsistent, and the highest was approximately 1.5 cm, the leaves showed a light redness. The bud clusters had proliferation.
3-2-4	0.02	0.5	0	No stem elongation.
3-2-5	0.04	0.5	0	No stem elongation.

Means followed by the same letters in rows are not significantly different at p ≤ 0.05.

that, as the concentration of TDZ gradually increased, the stem elongation coefficient gradually decreased. No stem elongation was observed in groups 3-2-4 and 3-2-5 (Figure 4d,e). Additionally, there was no significant difference in the stem elongation coefficients between groups 3-2-1, 3-2-2, and 3-2-3. However, the stem lengths in group 3-2-1 reached 2.5–3 cm (Figure 4a), and the shoots were robust compared with those in groups 3-2-2 (Figure 4b) and 3-2-3 (Figure 4c). Based on these results, the conditions of group 3-2-1 (WPM + 0.002 mg·L⁻¹ TDZ + 0.5 mg·L⁻¹ IBA) were considered optimal for the stem elongation of bud clusters.

3.5. Induction of Adventitious Roots

The effects of IBA concentrations on adventitious root growth were analyzed using the indices shown in

Table 5. The effects of IBA concentration on the induction of adventitious roots.

Group	IBA Concentration (mg·L−1)	Days to Root Emergence (d)	Rooting Rate (%)	Adventitious Roots Number	Growth
S1	0.5	10	100	8.25 ± 0.41 a	Leaves turned red and stem did not elongate.
S2	0.75	10	100	9.88 ± 0.93 ab	New buds sprouted, with almost no callus, the average root length was about 0.5 cm.
S3	1.0	7	100	10.13 ± 1.01 ab	New buds sprouted, with almost no callus, the average root length was about 1 cm.
S4	1.25	6	87.5	10.13 ± 10.44 ab	The average root length was 0.5–2 cm, but the callus area was large.
S5	1.5	6	87.5	12.00 ± 0.60 a	The average root length was 0.5–1 cm but the callus area was large.

Means followed by the same letters in rows are not significantly different at p ≤ 0.05.

and the data shown in Figure 5. The adventitious roots of tissue-cultured seedlings were observed to be fibrous and mostly located on the surface of the culture medium. Secondary roots were visible after 30 days, and tertiary roots could be observed after 60 days (Figure 5f). Increased IBA concentrations led to the earlier appearance of adventitious roots, with the first roots seen after six days. However, at IBA concentrations greater than 1 mg·L⁻¹ (Figure 5c), the rooting rate gradually decreased although the length of the roots increased, with the longest roots seen in group S4 (1.25 mg·L⁻¹ IBA) (Figure 5d). Nevertheless, greater callus induction was seen in groups S4 (Figure 5d) and S5 (Figure 5e), with the roots in both groups usually growing from the callus, which was not suitable for seedling transplantation. The rooting rate in group S3 was higher than those in groups S4 and S5. Meanwhile, group S3 showed almost no callus formation around the bud base, and the seedlings grew vigorously and were suitable for later transplantation. Therefore, group S3 conditions (1.0 mg·L⁻¹ IBA) were used for the induction of adventitious roots.

3.6. Transplantation

This section explored the effect of the substrate on the transplantation of R. aureum tissue-cultured seedlings. After two days of hardening-seedling, the seedlings were transplanted into substrates containing different proportions of vermiculite and peat. It was found that seedlings grown in vermiculite showed better initial growth after transplantation; however, as the cultivation time increased, the leaf margins tended to become dry (Figure 6a). The seedlings grown in peat (Figure 6b) showed weaker growth during the early stages after transplantation, similar to those grown in a substrate of 10:1 vermiculite and peat (Figure 6c). However, the leaves became glossy, and the growth became robust after 30 days. It was thus preliminarily concluded that a 10:1 ratio of vermiculite and peat was the optimal substrate.

4. Discussion

Deterioration of the natural habitat of R. aureum has led to reduced numbers of mature individuals in the population. This situation is exacerbated by human excavation and logging, resulting in a continuous decline in the distribution of the plant and, ultimately, its declaration as an endangered species in China. Although the plant is normally propagated sexually, its large-scale propagation is limited by its low rate of germination and slow growth. However, in vitro regeneration of plant species can produce many robust seedlings with consistent growth in a relatively short time. In the present study, we established a complete in vitro regeneration system that included the stages of primary culture, adventitious bud induction, bud cluster induction, stem elongation, rooting, and transplantation. For primary culture, the formula used for group B (0.5 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA) was found to be optimal and was selected for callus induction. A low ratio of auxin and cytokinin was found to induce shoot regeneration [16]. Reduced TDZ concentrations were subsequently used for callus subculture and the induction of adventitious buds. However, during the callus subculture, specifically during the periodic transfer of the callus to the fresh culture medium, there is a possibility that genetic changes may accumulate in the cells due to various factors, including DNA methylation, chromosomal rearrangements, mutation, and epigenetic modifications, which may lead to the formation of somaclonal variants [16,17]. Because of this, sterile seedlings of R. aureum were used as explants for the induction of bud clusters, without the stage of callus induction.

We then explored the direct regeneration of buds to both reduce the working procedure and the likelihood of somaclonal variations. The tender leaves of adventitious buds from calli were used as explants, and we explored the effects of TDZ and IBA concentrations on the induction of bud clusters. TDZ is a diphenylurea synthetic herbicide that was originally developed as a cotton defoliant and has been widely used as a plant growth regulator for the induction of de novo regeneration, shoot organogenesis, somatic embryogenesis and callus formation in hundreds of species [18]. We evaluated the effects of TDZ concentrations on the in vitro regeneration of R. aureum. It was found that neither excessively high nor low concentrations of TDZ were suitable for bud cluster induction, and when the TDZ concentration remained constant, the bud induction rate did not change. There was a specific relationship between the differentiation of bud clusters and the auxin:cytokinin ratio. The optimal formulation for the induction of bud clusters was that used in group 4 (0.5 mg·L⁻¹ TDZ + 0.1 mg·L⁻¹ IBA). However, Ding et al. considered that 10 mg·L⁻¹ ZT + 3 mg·L⁻¹ IBA was optimal for the primary culture of leaves from aseptic seedlings [11]. An examination of the literature showed that the effects of TDZ on callus induction and the differentiation of R. ‘dongfanghong’, R. ‘ruyi’, and R. aureum were much greater than those of ZT [12,19]. In addition, the selection of the type of plant growth regulator and its concentrations are closely related to the original materials. The use of leaves as explants for primary culture was found to require higher IBA concentrations than when stem segments were used as explants [11]. It is interesting that, in the Rhododendron genus, floral reversion was obtained from anthers in a medium supplemented with 2-isopentenyladenine (2-ip) and TDZ, while TDZ–induced shoot regeneration was usually observed from the pedicel and in the area adjacent to the bud scales [20]. Subculture (propagation) is a very important stage in micropropagation. The metabolism of TDZ in plants is extremely slow, which can lead to an accumulation of cytokinin, and low ratios of auxin and cytokinin were found to be beneficial for bud induction [21]. Thus, decreasing the TDZ concentration from 0.5 mg·L⁻¹ to 0.1 mg·L⁻¹ (group 3-2) resulted in the highest multiplication coefficient and the development of robust buds. Nonetheless, an evaluation of the effects of various cytokinins (meta-Topolin, ZT, 6-BA, TDZ, 2-iP, and 2-iP + ZT) on the proliferation of buds in R. ‘Kazimierz Odnowiciel’ showed that both ZT and 2-iP increased the number of buds, as well as the chlorophyll, carotenoid, and total soluble sugar contents in micropropagated shoots, and strengthened plant defenses against oxidative stress in tissue culture [22]. Although the classical cytokinin used for the micropropagation of evergreen Rhododendron species is 2-iP [23], the use of ZT is preferable in some species [24]. 6-BA was found to be toxic to some Rhododendron species [25]. However, the substituted urea compound in TDZ is known not only to trigger higher regeneration rates in comparison to purine-based cytokinins but is also able to fulfill both the cytokinin and auxin requirements of regeneration responses in a number of woody plants [26]. We then investigated the effect of TDZ concentrations on stem elongation. It was found that the extent of stem elongation was inversely proportional to the TDZ concentration, and the stems became thinner as the TDZ concentration decreased. This result is similar to the findings on R. sichotense Pojark. and R. catawbiense cv. Grandiflorum, in which elongation shoots of R. sichotense were obtained only after cultivation with low concentrations of TDZ (0.1–1.0 μM) [27]. It should be noted that the results of the stem-elongation experiments in the present study were unexpected. The shoots in our experiment showed inconsistent growth with limited numbers of elongated stems. This may be a consequence of the original materials and the dwarf form of R. aureum used in the present study. Studies on the in vitro regeneration of R. aureum have usually used NAA, IBA, and IAA for rooting [2,11,28]. We conducted preliminary experiments to roughly evaluate the effects of these three plant growth regulators on rooting in R. aureum, and found that neither NAA nor NAA + IBA treatments induced root growth after one month. In our study, rooting required a medium supplemented with high IBA concentrations (group S3: 1.0 mg·L⁻¹). This is similar to the results found in R. ‘Ken Janeck’, where it was observed that IBA concentrations of 2 mg·L⁻¹ improved rooting and increased root length, with the greatest number of roots seen in the presence of 4 mg·L⁻¹ IBA [29]. Due to the slow growth of R. aureum, the present study only conducted a preliminary exploration of suitable transplantation substrates. In future studies, we intend to optimize this issue to provide a more comprehensive plan for the conservation of R. aureum, while promoting its industrial development and driving economic progress.

5. Conclusions

In this study, we established an in vitro regeneration system for R. aureum by both direct and indirect organogenesis. The leaves of plants collected in the wild and grown hydroponically were sterilized for primary culture. The suitable formula for callus induction was WPM medium supplemented with 0.5 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA, while for the subculture and induction of adventitious buds, the optimal formulation contained 0.1 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA. Tender leaves from tissue-cultured seedlings were used for the induction of clustered buds under the optimal conditions of 0.5 mg·L⁻¹ TDZ and 0.1 mg·L⁻¹ IBA, while for subculture and proliferation, the optimal concentrations were 0.1 mg·L⁻¹ TDZ and 0.5 mg·L⁻¹ IBA. For stem elongation, 0.002 mg·L⁻¹ TDZ + 0.5 mg·L⁻¹ IBA were found to be optimal, while a concentration of 1.0 mg·L⁻¹ IBA was best for rooting. Finally, the optimal substrate was found to be vermiculite and peat soil in a 10:1 ratio.