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Article

In Vitro Cultivation of the Orchid Hybrid Rhyncattleanthe Queen Bee JLA 1 and Its Propagation Under Different Systems

by
Luis Alberto Solano-Rodríguez
1,
María Elena Galindo-Tovar
1,
Odon Castañeda-Castro
2,
Juan Valente Hidalgo-Contreras
3,
Joaquín Murguía-González
1,
Gabriela Lucero Cuatra-Xicalhua
1,
José Guadalupe Vián-Pérez
2,
Pablo Antonio Mendoza del Ángel
1 and
Miriam Cristina Pastelín-Solano
2,*
1
Facultad de Ciencias Biológicas y Agropecuarias, Universidad Veracruzana, Carretera Peñuela-Amatlán Kilómetro 177, Córdoba 94500, Veracruz, Mexico
2
Facultad de Ciencias Químicas, Universidad Veracruzana, Ote. 6 1009, Rafael Alvarado, Orizaba 94340, Veracruz, Mexico
3
Colegio de Postgraduados Campus Córdoba, Carretera Córdoba-Veracruz km 348, Congregación Manuel León, Amatlán de los Reyes 94961, Veracruz, Mexico
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 722; https://doi.org/10.3390/horticulturae11070722 (registering DOI)
Submission received: 14 May 2025 / Revised: 13 June 2025 / Accepted: 17 June 2025 / Published: 21 June 2025
(This article belongs to the Special Issue The Role of Plant Growth Regulators in Horticulture)
Browse Figures
Versions Notes

Abstract

:
The Orchidaceae family is of significant decorative, pharmaceutical, alimentary, and cultural importance worldwide. This family is very vulnerable due to illegal looting, habitat destruction, and climate change. The development of new hybrids helps meet the demand for specimens that possess outstanding appearance, fragrance, and resistance characteristics and may reduce illegal looting. The objective of this research was to investigate the in vitro propagation of the hybrid Rhyncattleanthe Queen Bee JLA 1 (Rth. Queen Bee JLA 1). Shoot induction was performed with germinated seedlings that were 1 cm in length on semi-solid MS medium with different 6-Benzylaminopurine (BAP), 1-Naphthaleneacetic acid (NAA), 3-Indoleacetic acid (IAA), and 3-indolebutyric acid (IBA) concentrations. Micropropagation was conducted using a temporary immersion system (TIS), a liquid continuous immersion system (CIS), and a conventional semi-solid system (SSS). Afterwards, all regenerated seedlings underwent an acclimatization stage. The highest numbers of shoots (7.04) and leaves (14.28) were obtained with the combination of 1.5 mg L−1 BAP and 0.4 mg L−1 NAA, while the addition of 0.4 mg L−1 IBA in combination with 1.5 mg L−1 BAP enhanced the length of stems (2.12 cm) and leaves (1.88 cm). TIS produced the highest number of shoots (15.68), leaves (22.92), stem length (5.94 cm), and number of leaves (3.50) in seedlings analyzed. The combination of growth regulators BAP and NAA together with the temporary immersion system influenced both the development of the vitroplants and their vegetative development after acclimatization of the hybrid Rth. Queen Bee JLA1 orchid.

1. Introduction

Orchids are a unique group distinguished by their extensive diversity, particularly in phenotypic characteristics [1,2]. The latest update documents 736 genera, from which five sub-families and more than 28,000 species are derived [3,4]. Mainly due to their ornamental characteristics, orchids are marketed as cut flowers or potted plants [5]. Additionally, they are rich in polysaccharides, alkaloids, and various other components, making them a crop in high demand at the global level [6].
Even with a wide diversity, orchids are among the most vulnerable plants due to various factors such as loss of habitat, illegal trafficking, and climate change [7].
The growing demand for various specimens adds to the indiscriminate plundering of several endemic species [5]. The generation of hybrids with characteristics of commercial and ornamental interest represents an alternative to supply the current demand [4]; however, the generation of a new hybrid entails the development of an efficient technique for its propagation [8].
In economic terms, the Netherlands is the world’s leading exporter with 37%, and the USA is the leading importer with 6%, with Mexico listed as a new competitor [9]. However, despite having an ideal geographic location for vegetative and commercial development, it is necessary to develop efficient strategies to stand out as a global producer [7].
The use of efficient propagation techniques represents a sustainable tool for the production of orchid specimens [10].
The Cattleya genus is renowned for producing excellent commercial hybrids with outstanding ornamental characteristics [11], characterized by large, wide petals, as well as elongated sepals [12].
Despite the existence of novel techniques for genetic improvement, such as gene insertion using agrobacterium and particle bombardment [13], traditional methods remain the main approach for the generation of orchid hybrids [4].
The propagation of orchids by conventional methods is time-consuming [14]. In addition, during the embryonic period, they lack functional endosperm and cotyledons [15]. Some seedlings require symbiosis with a mycorrhizal fungus for germination [16]. As a result, germination rates in natural environments are below 25% [17].
In vitro cultivation has become a powerful tool for germination, propagation, and regeneration of various orchid genera [18]. There is an extensive database for in vitro propagation of different orchid genera, including Phalaenopsis, Cymbidium, Cattleyas, and Dendrobium. Most research has focused on types of explants to be used, composition of culture media, effect generated in the explant, etc.; however, no information is available for the intergeneric hybrid Ryncattleanthe [19,20,21].
After the generation of a hybrid for commercial purposes, an efficient propagation technique will determine the time to market [22]. In addition, each specimen needs efficient protocols for its propagation [8].
The development of efficient protocols is largely governed by the interaction of growth regulators [23], which influence various metabolic processes in order to maintain intracellular and extracellular balance [24]. Combinations of growth regulators at different concentrations can have stimulatory or inhibitory effects [25].
The optimization of an in vitro culture propagation technique is mainly moderated by understanding the hormonal interactions that may be expressed [26]. Auxin–cytokinin interaction has been shown to be an efficient regulator of the in vitro propagation of different species of orchids and a great diversity of plant tissues [27].
In vitro culture techniques in semi-solid media consist of various phases, which require extensive labor [28]. Various investigations using bioreactors as a temporary immersion system (TIS) have described it as an efficient and cost-effective method for in vitro propagation of many plant species [29].
Specifically, temporary immersion bioreactors (BIT®) are recommended as low-cost, high-capacity production tools for a variety of crops [29,30,31].
This study focused on the determination of efficient techniques for in vitro propagation of the orchid hybrid Rhyncattleanthe Queen Bee JLA 1 (Rth. Queen Bee JLA 1), recently registered with the Royal Horticultural Society [32].

2. Materials and Methods

2.1. Hybridization

Hybridization was carried out manually during the flowering season of the parent plants, in May and June. The ovule of {Rlc.} [{Blc.}] Hwa Yuan Grace was used as the recipient (Figure 1a), and the male gametophytes of {Ctt.} [{C.}] Chocolate Drop were the donors (Figure 1b). After pollination, the mother plant was maintained in greenhouse conditions with 30% shade, receiving irrigation of 500 cc every 3 days and fertilization based on Morard’s general formula with an electrical conductivity of 2.5 mS/cm. After 6 months, the mature capsule, identified by the appearance of striae, was collected and cut with sterile pruning tweezers.

2.2. In Vitro Establishment

The capsule was transferred to the laboratory, flushed with tap water for 10 min, and washed with commercial liquid soap. In a laminar flow cabinet, the capsule was immersed in a 70% alcohol solution for 10 min. Then, the excess solution was removed by rinsing three times with sterile distilled water. After that, they were immersed in a 30% NaClO solution for 20 min. The excess solution was removed by rinsing three times with sterile distilled water.
Subsequently, a transverse cut was made to the capsule, dividing it into 2 sections. The embryos inserted in the sides of the capsule were extracted using a sterile stainless-steel spatula and scalpel. They were placed in 81 mm diameter glass bottles of 250 mL with transparent silicone lids. Each flask contained 40 mL of 50% basal Murashige and Skoog (MS) culture medium supplemented with 30 g L−1 sucrose, 0.04 g L−1 thiamine, 0.1 g L−1 myo-inositol, 2 g L−1 activated carbon and 3 g L−1 Phytagel, with a pH of 5.7 Sterilization was performed in an autoclave at 1.5 Kg cm2 pressure and 121 °C for 20 min.
After 60 days, the germinated embryos were transferred to 250 mL culture flasks containing 40 mL of 100% MS culture medium and incubated under a 16 h light/8 h dark cycle at a temperature of 24 ± 1 °C.

2.2.1. Preliminary Evaluation of Combinations of Growth Regulators

To evaluate the effects of different growth regulator combinations on shoot elongation, shoot production, and leaf formation, 6-benzylaminopurine (BAP) was used as the cytokinin, combined with three types of auxins: 3-indoleacetic acid (IAA), indole-3-butyric acid (IBA), and 1-naphthaleneacetic acid (NAA). A total of 21 different combinations were tested, establishing 8 vitroplants for treatment (Table 1). The cultures were incubated incubated under a 16 h light/8 h dark cycle at a temperature of 24 ± 1 °C, under controlled conditions for 60 days, with 3 subcultures, in culture medium MS supplemented with 30 g L−1 sucrose, 0.04 g L−1 thiamine, 0.1 g L−1 myo-inositol, 2 g L−1 activated carbon and 3 g L−1 Phytagel, with a pH of 5.7. Sterilization was performed in an autoclave at 1.5 Kg cm2 pressure and 121 °C for 20 min.

2.2.2. Selection of Treatments for Elongation and Generation of Shoots and Leaves

After the analysis of the 21 combinations of growth regulators, the treatments—T3, T4, T7, T9, T10, T11—obtained superior averages; therefore, they were selected to evaluate in a new subculture the morphometric variables generation of new shoots and leaves in vitroplants of the hybrid.
Similarly, six outstanding treatments were selected to evaluate the morphometric variables stem and leaf elongation in vitro plants: T6, T13, T16, T18, T19, T20.
In each case, for the six treatments and the control treatment, 25 vitroplants that were 1 cm in length were placed in 25 × 150 mm PIREX® culture tubes. The tubes contained 10 mL of culture medium. The treatments consisted of the different selected combinations of BAP as cytokinin and IAA, IBA, NAA as auxin sources, and GR. The hybrid vitroplants were cultivated in MS culture medium supplemented with 30 g L−1 sucrose, 0.04 g L−1 thiamine, 0.1 g L−1 myo-inositol, 2 g L−1 activated carbon and 3 g L−1 Phytagel, with a pH of 5.7 Sterilization was performed in an autoclave at 1.5 Kg cm2 pressure and 121 °C for 20 min.
After 60 days of in vitro incubation for the different treatments, the number of new shoots, the number of new leaves, stem length (cm), and leaf length (cm) were evaluated for each experiment.

2.2.3. In Vitro Multiplication with Different Systems

Vitroplants of 1 cm in length were cultivated in a conventional semi-solid system (SSS), a temporary immersion system (TIS) using a 1.5 L Temporary Immersion Bioreactor (BIT®), and a continuous liquid immersion system (CIS) [28].
For the SSS and CIS, 25 × 150 mm culture tubes containing 25 mL of MS basal culture medium were used, and one vitroplant was placed per tube.
For TIS 1.5 L Temporary Immersion Bioreactors, with immersion frequencies of five minutes every eight hours, 25 mL of MS basal culture per vitroplant were used, and 25 vitroplants were placed per bioreactor.
For the CIS, the same characteristics were used as for SSS without the addition of the gelling agent Phytagel.
In each system, the medium was supplemented with 30 g L−1 sucrose, 0.04 g L−1 thiamine, 0.1 g L−1 myo-inositol, 2 g L−1 activated carbon, 1.5 mg L−1 BAP, and 0.4 mg L−1 NAA, with the pH adjusted to 5.7. Sterilization was performed in an autoclave at 1.5 kg cm2 pressure and 121 °C for 20 min. In addition, for SSS, 2.5 g L−1 phytagel as a gelling agent was added. After 60 days of in vitro incubation, the number of new shoots, new leaves, stem length (cm), and leaf length (cm) were evaluated.

2.3. Data Analysis

An exploratory analysis was carried out, and the data obtained were analyzed using descriptive statistics of eight units per treatment, including the arithmetic mean, and plotted using a radar graph. Subsequently, the highlighted results were analyzed separately.
To analyze the combinations of growth regulators with superior means and the comparative data of the in vitro propagation systems, 25 experimental units were set up, both cases following a completely randomized design. An analysis of variance (ANOVA) was performed, followed by a comparison of means using Tukey’s test, p ≤ 0.05. Data were processed with R statistical software, version 2024. The data were plotted with Origin Pro®, version 2024.

2.4. Acclimatization of Vitroplants of the Rth. Queen Bee JAL 1 Hybrid

The developed vitroplants were acclimatized in trays with 72 cavities, each with a 45 cc volume and a transparent polypropylene dome. They were placed in a substrate composed of 70% pine bark, 20% sphagnum moss, and 10% agrolite under greenhouse conditions, with 50% shade mesh. Relative humidity was maintained at 80%. They were preserved under these conditions for 60 days, during which the levels of relative humidity (Hr), light intensity (lx), and temperature (°C) were monitored. After 60 days, the domes were removed, and the survival percentage was evaluated. The seedlings were transplanted into 3” transparent pots, containing a substrate composed of 70% pine bark, 20% agrolite, and 10% coconut fiber.

3. Results

3.1. In Vitro Evaluation of Combinations of Growth Regulators on the Rth. Queen Bee JLA 1 Hybrid in Three Subcultures

The application of different growth regulators at different concentrations led to outstanding results. Treatments 3, 4, 7, 9, 10, and 11 generated a greater number of shoots and leaves (Figure 2a,b). The vitroplants from treatments 6, 13, 16, 18, 19, and 20 developed longer leaves and stems (Figure 2c,d). Additionally, it was observed that the vitroplants showed a tendency to increase in the values of the analyzed variables with the application of a new subculture, a common effect observed in several species of plants cultivated in vitro. The auxin/cytokinin interaction is reflected in T10, where a greater generation of shoots and foliar structures is clearly observed from the first subculture.

3.2. Effect of Combinations of Prominent Growth Regulators

The combination of BAP with NAA stimulated the development of new shoots in vitroplants of the hybrid, specifically the concentrations of 1.5 mg L−1 of BAP and 0.4 mg L−1 of NAA (Figure 3), generating an average of (7.04 ± 2.80) new shoots per vitroplant. The same concentration promoted the development of new leaves, generating an average of (14.28 ± 5.74) new leaves per vitroplant (Figure 4a). The mean comparison analysis showed statistical superiority (p ˂ 0.05) to the control treatment and the rest of the treatments evaluated (Figure 4b), these two excellent growth parameters being used to evaluate the behavior of the hybrid under different concentrations of the cytokinin/auxin interaction.
When substituting the auxin source with IBA 0.5 mg L−1, we observed a decrease in the generation of new structures and slightly elongated vitroplants in leaf and stem structures, but no significant differences were found between treatments; the development of vitroplants of adequate size is an important factor during the acclimatization process of in vitro cultures.

3.3. Effect of Semi-Solid System (SSS), Continuous Immersion System (ICS), and Temporary Immersion System (TIS) Type BIT on the In Vitro Propagation of the Orchid Hybrid

In vitro propagation of the hybrid by TIS (Figure 5b) stimulated the generation of new shoots (15.68 ± 3.95) and new leaves (22.92 ± 2.4). The characteristics of the vitroplants obtained by TIS were superior compared to those obtained by the SSS (Figure 5a) and CIS (Figure 5c), developing stems and leaf structures with greater length, undoubtedly important factors for the acclimatization process.
It should be noted that no tissues with hyperhydricity were observed in TIS. This effect could be due to the optimal immersion intervals or to the characteristics of the hybrid, since the tissues exposed to constant immersion suffered slight vitrification effects, contrasting with the expected effects.
In addition to superior results in the variables analyzed during in vitro development, the hybrid vitroplants under TIS propagation exhibited greater vigor and size (Figure 6b) compared to those under the SSS (Figure 6a) and CIS (Figure 6c).
Another characteristic of the vitroplants obtained by TIS was the development of the root system, which could be observed more prominently compared to the other two propagation methods, undoubtedly a factor of utmost importance for acclimatization and further development of this process.
Another observation from this part of the experiment was that the vitro plants established in the CIS showed a slight yellowing of the leaves, the growth was very slow; however, the loss during acclimatization was low.
During greenhouse evaluation, Rth. Queen Bee JLA 1 hybrid seedlings, from the three in vitro propagation systems, showed different growth patterns, with those from the TIS (b) presenting accelerated development compared to seedlings from the CIS (c) and SSS (a) (Figure 7).

4. Discussion

Analyzing subcultures and growth regulator combinations is crucial for determining efficient in vitro propagation models, especially for recently created specimens with no previous studies on their behavior under in vitro conditions [33]. An efficient propagation model will determine when a specimen can reach the acclimatization stage and begin development in the greenhouse [34]. However, it is not enough to produce a large number of plantlets in a relatively short time; the vitroplants must also have good vegetative development, which will contribute to physiological efficiency and seedling adaptation to ex vitro conditions [35].
Each subculture applied to the Rth. Queen Bee JLA1 hybrid showed different effects. In terms of shoot and leaf generation (Figure 1a,b), an increasing trend was observed between each subculture. The authors of [36] reported a similar trend of increasing values during subcultures one and two of the orchid Hadrolaelia grandis, obtaining vitroplants with a greater number of shoots and leaf structures during each subculture. In the current study, treatments T3, T4, T7, T9, T10 and T11 stood out. These results provided a basis for further analysis, focusing on the doses of key growth regulators with an incubation interval of 60 days.
Experimentation with the key treatments showed that the addition of 1.5 mg L−1 BAP + 0.4 mg L−1 NAA was statistically superior (p ˂ 0.05) to the other treatments, generating a greater number of shoots and leaves. The physiological effects could be due mainly to the interaction of the growth regulators and the concentrations at which they are added to the culture medium [37]. The present results align with research on the propagation of Brassolaeliocattleya by [38], who reported that the addition of 1.5 mg L−1 BAP resulted in vitroplants with a greater number of leaves. They also proposed further investigation of BAP with the addition of auxins. Shoot generation in the orchid hybrid Rth. Queen Bee JLA 1 responds to the typical effects in plant tissues of cytokinin interaction in the presence of lower concentrations of auxins, which promote cell division by expressing in the formation of new structures [23]. Similarly, during the propagation of the orchid species Aerides multiflora, the combination of BAP and NAA at similar concentrations generated a higher propagation rate [21].
The analysis of variance indicated that the number of outbreaks was significantly higher (p ˂ 0.05) under propagation in the TIS (15.68 ± 3.95 a) compared to the SSS (6.48 ± 0.87 b) and CIS (4.96 ± 2.93 b). These results agree with those of [28], who found that propagation of the orchid Epipactis flava, variety Seidenf, in BIT-type systems produced the highest number of buds and leaves. Another study reported similar findings, where vitroplants inoculated in a TIS developed a greater number of leaf structures, although the growth regulators used were promoters of cell division [39]. Additionally, the renewal of the incubation atmosphere, as well as constant aeration, acts as a catalyst, increasing the proliferation of shoots and leaves [40,41,42].
During the analysis of stem length and leaf length, a significant difference (p ˂ 0.05) was found in vitroplants developed in the TIS compared to those grown in the SSS and CIS (Table 2). Although different combinations and concentrations of growth regulators did not result in significantly greater lengths, the TIS generated vitroplants with superior development. This can be attributed to gas exchange, as various authors have reported that the dynamics of gas exchange allow the expulsion of certain gases [43], such as ethylene [44].
Some research has highlighted the importance of generating longer vitroplants, as this characteristic is associated with a higher survival rate during acclimatization [45,46,47,48]. Additionally, it has been shown that gas exchange improves the mixotrophic capacity of plants, which significantly helps the acclimatization and development phases of seedlings [8].

5. Conclusions

The results obtained demonstrate that the combination of 1.5 mg L−1 BAP with 0.4 mg L−1 NAA is efficient for the in vitro propagation of the Rth. Queen Bee JAL 1 orchid hybrid in MS semi-solid media.
The use of BIT-type temporary immersion systems increases the production of vitroplants with excellent quality, even increasing the length of stems and leaves.
The combination of plant growth regulators in combination with the use of BIT systems proved to be viable tools for the production and seedling development of the Ryncattleanthe Queen Bee JLA 1 hybrid.
With an efficient protocol for the propagation of the hybrid, it is recommended to analyze the following stages of development under greenhouse conditions.

Author Contributions

Conceptualization, L.A.S.-R., M.E.G.-T., O.C.-C., J.M.-G. and M.C.P.-S.; Methodology, M.E.G.-T., O.C.-C., J.V.H.-C., J.M.-G., G.L.C.-X., J.G.V.-P., P.A.M.d.Á. and M.C.P.-S.; Software, L.A.S.-R.; Validation, P.A.M.d.Á.; Investigation, L.A.S.-R., M.E.G.-T., O.C.-C., J.V.H.-C. and M.C.P.-S.; Resources, L.A.S.-R.; Data curation, J.G.V.-P.; Writing—original draft, L.A.S.-R.; Writing—review & editing, M.E.G.-T., O.C.-C., J.V.H.-C., J.M.-G., G.L.C.-X., P.A.M.d.Á. and M.C.P.-S.; Visualization, O.C.-C. and M.C.P.-S.; Supervision, M.E.G.-T., O.C.-C. and M.C.P.-S.; Funding acquisition, L.A.S.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by Queen Bee Flower Market S.A de C.V. Genetic improvement department, project; Rhyncattleanthe Queen Bee 1510-2021.

Data Availability Statement

Data is contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

The following abbreviations are used in this manuscript:
BAP6-Bencylaminopurine
CISContinuous immersion system
IAA3-indolacetic acid
IBAindole-3-butyric acid
NAA1-naphthalenacetic acid
Rth.Rhyncattleanthe
TISTemporary immersion system
RHSRoyal Horticultural Society
SSSSemi-solid System
BITTemporary immersion system bioreactors

References

  1. Manu, P.; Ayushi, N.; Arundhati, S.; Aarushi, G.; Megha, R. Cattelya Orchids: A Mini Review. J. Crit. Rev. 2020, 7, 4592–4599. [Google Scholar] [CrossRef]
  2. Khasim, S.M.; Hegde, S.N.; González-Arnao, M.T.; Thammasiri, K. Orchid Biology: Recent Trends & Challenges; Springer: Singapore, 2020. [Google Scholar] [CrossRef]
  3. Chase, M.W.; Cameron, K.M.; Freudenstein, J.V.; Pridgeon, A.M.; Salazar, G.; Van den Berg, C.; Schuiteman, A. An updated classification of Orchidaceae. Bot. J. Linn. Soc. 2015, 177, 151–174. [Google Scholar] [CrossRef]
  4. Li, C.; Dong, N.; Zhao, Y.; Wu, S.; Liu, Z.; Zhai, J. A review for the breeding of orchids: Current achievements and prospects. Hortic. Plant J. 2021, 7, 380–392. [Google Scholar] [CrossRef]
  5. Hinsley, A.; de Boer, H.J.; Fay, M.F.; Gale, S.W.; Gardiner, L.M.; Gunasekara, R.S.; Kumar, P.; Masters, S.; Metusala, D.; Roberts, D.L.; et al. A review of the trade in orchids and its implications for conservation. Bot. J. Linn. Soc. 2017, 186, 435–455. [Google Scholar] [CrossRef]
  6. Vilcherrez-Atoche, J.A.; Silva, J.C.; Clarindo, W.R.; Mondin, M.; Cardoso, J.C. In Vitro Polyploidization of Brassolaeliocattleya Hybrid Orchid. Plants 2023, 12, 281. [Google Scholar] [CrossRef]
  7. Murguía-González, J.; Leyva-Ovalle, O.R.; Lee-Espinosa, H.E.; Galindo-Tovar, M.E.; Pardío-Sedas, V.T.; Llarena-Hernández, R.C. Orchid production systems (Orquidaceae) from Veracruz, México. Agroproductividad 2016, 9, 62–66. [Google Scholar]
  8. Bello-Bello, J.J.; Cruz-Cruz, C.A.; Pérez-Guerra, J.C. A new temporary immersion system for commercial micropropagation of banana (Musa AAA cv. Grand Naine). Vitr. Cell. Dev. Biol.-Plant 2019, 55, 313–320. [Google Scholar] [CrossRef]
  9. De, L.C.; Pathak, P.; Rao, A.N.; Rajeevan, P.K. 2 Global Orchid Industry. Commer. Orchid. 2019, 13–19. [Google Scholar] [CrossRef]
  10. Téllez-Casas, J.M.; López-Peralta, M.C.G.; Hernández-Meneses, E.; Cruz-Huerta, N. Direct organogenesis of spouts from Rhyncholaelia glauca (LINDLEY) schlechter seedlings germinated in vitro. J. Fitotec. Mex. 2024, 47, 293. [Google Scholar] [CrossRef]
  11. Cardoso, J.C.; Martinelli, A.P.; da Silva, J.A.T. A novel approach for the selection of Cattleya hybrids for precocious and season-independent flowering. Euphytica 2016, 210, 143–150. [Google Scholar] [CrossRef]
  12. De, L.C.; Senjam, B. Flower Drying Techniques for Orchids. In Geography, Earth Science and Environment: Research Highlights; BP International: Hong Kong, China, 2025; Volume 6, pp. 177–199. [Google Scholar] [CrossRef]
  13. Azzam, C.R. Genetic Resources, Breeding, and Molecular Genetic Markers for Orchard Improvement and Management. In Handbook of Research on Principles and Practices for Orchards Management; IGI Global: Hershey, PA, USA, 2022; pp. 70–115. [Google Scholar] [CrossRef]
  14. Sabastian, K.S.; Hemanta, L.; Ariina, M.M.S.; Khamrang, M.; Devi, M.P. Micropropagation of orchids. In Recent Trends in Forest and Horticultural Crop Propagation; Taran publication: Haryana, India; Volume 68, pp. 75–76. Available online: https://www.researchgate.net/publication/355031740_Micropropagation_of_Orchids2021 (accessed on 19 April 2025).
  15. Castillo-Pérez, L.J.; Maldonado-Miranda, Á.J.; Alonso-Castro, J.J.; Candy, C.-Á. Effect of 6-benzylaminopurine and potassium nitrate on the in vitro micropropagation of Laelia anceps subsp. anceps (Orchidaceae). Biotecnia 2020, 22, 32–38. [Google Scholar] [CrossRef]
  16. Hanun, A.; Karend, A.; Setiari, N.; Nurchayati, Y.; Azzahra, F.; Rashid, A. Somatic Embryo Enhancing of Phalaenopsis amabilis (L.) Blume Orchid with 6-Benzyl Amino Purine (BAP). J. Ris. Biol. Dan Aplikasinya 2025, 7, 88–98. [Google Scholar] [CrossRef]
  17. Hira, A.; Bijaya, P. In vitro seed germination and seedling growth of the orchid Dendrobium primulinum Lindl. Afr. J. Plant Sci. 2019, 13, 324–331. [Google Scholar] [CrossRef]
  18. Jaime, K.A.F.; Zacarías, M.d.C.O.; Gómez, O.G.A.; Zambrano, E.A.G.; Fuentes, H.R.; Pérez, G.R. Inducción de brotes a partir de varas florales de la orquídea Phalaenopsis spp. (Blume) in vitro. Rev. Mex. Cienc. Agric. 2019, 10, 1207–1218. [Google Scholar] [CrossRef]
  19. Timilsina, S.; Poudel, A.; Ghimire, N.; Neupane, S.; Poudel, P.R. Micropropagation of ornamental plants: A review on Nepalese protocols. Cogent Food Agric. 2025, 11, 2512165. [Google Scholar] [CrossRef]
  20. Su’UDi, M.; Rosida, W.; Ravitamala, E.S.; Siddiq, A.M.; Setiawan, R.; Setyati, D.; Ningrum, A.Y.; Roziqoh, W. Plant Breeding Techniques in Tissue Culture to Improve the Quality of Orchids. J. Ilmu Pertan. Indones. 2024, 30, 116–122. [Google Scholar] [CrossRef]
  21. Chugh, S.; Guha, S.; Rao, I.U. Micropropagation of orchids: A review on the potential of different explants. Sci. Hortic. 2009, 122, 507–520. [Google Scholar] [CrossRef]
  22. JCardoso, J.C.; Zanello, C.A.; Chen, J.-T. An Overview of Orchid Protocorm-Like Bodies: Mass Propagation, Biotechnology, Molecular Aspects, and Breeding. Int. J. Mol. Sci. 2020, 21, 985. [Google Scholar] [CrossRef]
  23. Alcántara, J.; Godoy, A.; Alcantara, J.; Sánchez, R. Main hormone regulators and their interactions in plant growth. Nova 2019, 32, 109–129. [Google Scholar]
  24. Garay-Arroyo, A.; de la Paz Sánchez, M.; García-Ponce, B.; Álvarez-Buylla, E.; Gutiérrez, C. Auxin homeostasis and its importance in the development of Arabidopsis thaliana. Rev. Educ. Bioquímica 2014, 33, 13–22. [Google Scholar]
  25. Celedón, P.V.; Martinez, H.C.; González, M.; Seeger, M. Review Biosynthesis of indole-3-acetic acid and plant growth promoting by bacteria. Cultiv. Trop. 2016, 37, 31–37. [Google Scholar] [CrossRef]
  26. Ramírez-Mosqueda, M.; Cruz-Cruz, C.; Atlahua-Temoxtle, J.; Bello-Bello, J. In vitro conservation and regeneration of Laelia anceps Lindl. S. Afr. J. Bot. 2018, 121, 219–223. [Google Scholar] [CrossRef]
  27. Cruz, K.Z.C.M.; Alencar, A.A.d.S.; Santana, J.G.S.; Alves, L.E.O. The Auxin and Cytokinin promotes the regeneration of shoots in Phalaenopsis Golden Peoker ‘BL’ HCC/AOS (Orchidaceae). Braz. J. Dev. 2024, 10, e68953. [Google Scholar] [CrossRef]
  28. Kunakhonnuruk, B.; Inthima, P.; Kongbangkerd, A. In vitro propagation of rheophytic orchid, Epipactis flava seidenf.—A comparison of semi-solid, continuous immersion and temporary immersion systems. Biology 2019, 8, 72. [Google Scholar] [CrossRef]
  29. Hwang, H.-D.; Kwon, S.-H.; Murthy, H.N.; Yun, S.-W.; Pyo, S.-S.; Park, S.-Y. Temporary Immersion Bioreactor System as an Efficient Method for Mass Production of In Vitro Plants in Horticulture and Medicinal Plants. Agronomy 2022, 12, 346. [Google Scholar] [CrossRef]
  30. Rico, S.; Garrido, J.; Sánchez, C.; Ferreiro-Vera, C.; Codesido, V.; Vidal, N. A Temporary Immersion System to Improve Cannabis sativa Micropropagation. Front. Plant Sci. 2022, 13, 895971. [Google Scholar] [CrossRef] [PubMed]
  31. Thanonkeo, S.; Kitwetcharoen, H.; Thanonkeo, P.; Klanrit, P. Temporary Immersion Bioreactor (TIB) System for Large-Scale Micropropagation of Musa sp. cv Kluai Numwa Pakchong 50. Horticulturae 2024, 10, 1030. [Google Scholar] [CrossRef]
  32. Your Personal Gardening Coach–And More for RHS Members/RHS. Available online: https://apps.rhs.org.uk/horticulturaldatabase/orchidregister/orchiddetails.asp?ID=1041072 (accessed on 29 January 2025).
  33. Xu, J.; Beleski, D.G.; Vendrame, W.A. Effects of culture methods and plant growth regulators on in vitro propagation of Brassavola nodosa (L.) Lindl. hybrid. Vitr. Cell. Dev. Biol.-Plant 2022, 58, 931–941. [Google Scholar] [CrossRef]
  34. Hazarika, B.N.; da Silva, J.A.T.; Talukdar, A. Effective Acclimatization of in Vitro Cultured Plants: Methods, Physiology and Genetics. 2006. Available online: https://www.researchgate.net/publication/283300426 (accessed on 20 February 2025).
  35. Castañeda, N.B.; Barrera, C.A.C.; Perez, M.M. Revisión sistemática sobre tipos de sustratos utilizados en la propagación de orquídeas bajo invernadero. Rev. Mutis 2022, 13, 1–18. [Google Scholar] [CrossRef]
  36. Vudala, S.; Padial, A.; Ribas, L. Micropropagation of Hadrolaelia grandis through transverse and longitudinal thin cell layer culture. S. Afr. J. Bot. 2018, 121, 76–82. [Google Scholar] [CrossRef]
  37. Aremu, A.O.; Plačková, L.; Pěnčík, A.; Novák, O.; Doležal, K.; Van Staden, J. Auxin-cytokinin interaction and variations in their metabolic products in the regulation of organogenesis in two Eucomis species. New Biotechnol. 2016, 33, 883–890. [Google Scholar] [CrossRef]
  38. Cavallaro, V.; Pellegrino, A.; Muleo, R.; Forgione, I. Light and Plant Growth Regulators on In Vitro Proliferation. Plants 2022, 11, 844. [Google Scholar] [CrossRef] [PubMed]
  39. Saravia-Castillo, G.; Figueroa, L.T.Y.; Borjas-Ventura, R. Auxins and Cytokinins elicit a differentiated response in the formation of shoots and roots in Cattleya maxima Lindl and Phalaenopsis amabilis (L.) Blume. Sci. Agropecu. 2022, 13, 63–69. [Google Scholar] [CrossRef]
  40. Aragón, C.E.; Escalona, M.; Rodríguez, R.; Cañal, M.J.; Capote, I.; Pina, D.; González-Olmedo, J. Effect of sucrose, light, and carbon dioxide on plantain micropropagation in temporary immersion bioreactors. Vitr. Cell. Dev. Biol.-Plant 2010, 46, 89–94. [Google Scholar] [CrossRef]
  41. Hvoslef-Eide, A.K.; Preil, W. Liquid Culture Systems for In Vitro Plant Propagation; Springer: Dordrecht, The Netherlands, 2005. [Google Scholar] [CrossRef]
  42. Zhao, Y.; Sun, W.; Wang, Y.; Saxena, P.K.; Liu, C.-Z. Improved mass multiplication of Rhodiola crenulata shoots using temporary immersion bioreactor with forced ventilation. Appl. Biochem. Biotechnol. 2012, 166, 1480–1490. [Google Scholar] [CrossRef] [PubMed]
  43. Solís-Zanotelli, F.Y.; Baltazar-Bernal, O. In vitro germination and development of ‘Canelita’ (Lycaste aromatica (Graham) Lindl.) in gravity immersion bioreactors. Res. Sq. 2022, 1–11. [Google Scholar] [CrossRef]
  44. Cuba-Díaz, M.; Acuña, D.; Cordero, C.M.; Klagges, M. Optimización de parámetros para la propagación in vitro de Colobanthus quitensis (Kunth) Bartl. Gayana Bot. 2014, 71, 58–67. [Google Scholar] [CrossRef]
  45. Delgado-Aceves, L.; Corona, S.; Marin-Castro, U.R.; Rascón-Díaz, M.P.; Portillo, L.; Gutiérrez-Mora, A.; González-Arnao, M.T. Comparative Studies for Cryopreservation of Agave Shoot Tips by Droplet-Vitrification. Plants 2024, 13, 2609. [Google Scholar] [CrossRef]
  46. Sánchez-Vidaña, M.R.; Tejeda-Sartorius, O.; Hernández-Anguiano, A.M.; Trejo-Téllez, L.I.; Soto-Hernández, R.M.; Gaytán-Acuña, E.A. Ambiente y antecedentes de floración en el crecimiento, inducción y desarrollo floral de Laelia anceps subesp. anceps (Orchidaceae). Agrociencia 2018, 52, 35–54. [Google Scholar]
  47. Bazzicalupo, M.; Calevo, J.; Adamo, M.; Giovannini, A.; Copetta, A.; Cornara, L. Micromorfología de semillas, germinación in vitro y características morfológicas de plántulas en etapa temprana de Cattleya purpurata (Lindl. y Paxton) Van den Berg. Horticulturae 2021, 7, 480. [Google Scholar] [CrossRef]
  48. Hernández, A.M.; de la O, J.L.R.; Cruz-Castillo, J.G.; Mascorro-Gallardo, J.O.; Juárez-Hernández, M.d.J.; Barrera-Guzmán, L.Á.; Mójica-Zárate, H.T. Micropropagation and phytopathology of calla lily (Zantedeschia spp.). Rev. Colomb. Cienc. Hortic. 2024, 18, e17623. [Google Scholar] [CrossRef]
Horticulturae 11 00722 g001
Figure 1. Recipient parent Rhyncholaeliocattleya Hwa Yuan Grace (a). Progenitor donor of male gametophytes Cattlianthe Chocolate Drop (b).
Figure 1. Recipient parent Rhyncholaeliocattleya Hwa Yuan Grace (a). Progenitor donor of male gametophytes Cattlianthe Chocolate Drop (b).
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Figure 2. The graphs represent the values obtained by the analysis of the arithmetic mean of each 8 vitroplants in the variables: number of shoots (a); leaves (b); stem length in cm (c); and leaves in cm (d). The different colored lines represent the effect of the subcultures applied to the orchid hybrid: red line (subculture 1), blue line (subculture 2), and green line (subculture 3). The combinations of growth regulators used control treatment. The outstanding treatments are shown in red for each of the variables analyzed; T1 is the control treatment.
Figure 2. The graphs represent the values obtained by the analysis of the arithmetic mean of each 8 vitroplants in the variables: number of shoots (a); leaves (b); stem length in cm (c); and leaves in cm (d). The different colored lines represent the effect of the subcultures applied to the orchid hybrid: red line (subculture 1), blue line (subculture 2), and green line (subculture 3). The combinations of growth regulators used control treatment. The outstanding treatments are shown in red for each of the variables analyzed; T1 is the control treatment.
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Figure 3. Effect of the combination of BAP/NAA on hybrid Rth. Queen Bee JLA 1. Horizontal bars represent the mean number of new structures and elongation, each per 25 vitroplants with standard deviation, asterisks indicate significant differences (p < 0.05).
Figure 3. Effect of the combination of BAP/NAA on hybrid Rth. Queen Bee JLA 1. Horizontal bars represent the mean number of new structures and elongation, each per 25 vitroplants with standard deviation, asterisks indicate significant differences (p < 0.05).
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Figure 4. Vitroplants incubated for 60 days in culture MS media added with 1.5 mg L−1 BAP and 0.4 mg L−1 NAA (a); control treatment MS without PGRs (b).
Figure 4. Vitroplants incubated for 60 days in culture MS media added with 1.5 mg L−1 BAP and 0.4 mg L−1 NAA (a); control treatment MS without PGRs (b).
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Figure 5. Propagation of hybrid Rth. Queen Bee JLA 1, by different systems (a); semi-solid systems (b); temporary immersion system (c); and continuous immersion systems.
Figure 5. Propagation of hybrid Rth. Queen Bee JLA 1, by different systems (a); semi-solid systems (b); temporary immersion system (c); and continuous immersion systems.
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Figure 6. Development of vitroplants in 60 days, under three in vitro propagation systems: semi-solid system (a); temporary immersion system (b); continuous immersion system (c).
Figure 6. Development of vitroplants in 60 days, under three in vitro propagation systems: semi-solid system (a); temporary immersion system (b); continuous immersion system (c).
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Figure 7. Seedlings of the Rth. Queen Bee JLA1 hybrid, four months into development under greenhouse conditions.
Figure 7. Seedlings of the Rth. Queen Bee JLA1 hybrid, four months into development under greenhouse conditions.
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Table 1. Concentrations and combinations of the growth regulators BAP/NAA, IAA, and IBA.
Table 1. Concentrations and combinations of the growth regulators BAP/NAA, IAA, and IBA.
TreatmentsBAPNAAIAAIBA
mg L−1
T10000
T20.1
T30.5
T41.0
T51.5
T62.0
T70.10.1
T80.50.2
T91.00.3
T101.50.4
T112.00.5
T120.1 0.1
T130.5 0.2
T141.0 0.3
T151.5 0.4
T162.0 0.5
T170.1 0.1
T180.5 0.2
T191.0 0.3
T201.5 0.4
T212.0 0.5
Combination of cytokinin, 6-benzylaminopurine (BAP), and different concentrations of auxins; 1-naphthaleneacetic acid (NAA), 3-idoleacetic acid (IAA), indole-3-butyric acid (IBA), for preliminary analysis on the Rth. hybrid. Queen Bee JLA 1, 21 treatments.
Table 2. Propagation results of the Rth. Queen Bee JLA 1 hybrid, using three different systems: temporary immersion, semi-solid, and continuous immersion.
Table 2. Propagation results of the Rth. Queen Bee JLA 1 hybrid, using three different systems: temporary immersion, semi-solid, and continuous immersion.
Propagation SystemsNumber of ShootsNumber of LeavesLeaf Length (cm)Stem Length (cm)Survival in Acclimatization (%)
TIS15.68 ± 3.95 a22.92 ± 2.40 a5.94 ± 1.00 a3.50 ± 1.17 a100 ± 0.00 a
SSS6.48 ± 0.87 b5.40 ± 6.50 b2.14 ± 1.57 b1.93 ± 0.17 b96.66 ± 3.33 a
CIS4.96 ± 2.93 b4.12 ± 0.50 b1.60 ± 0.73 b1.64 ± 0.61 b92.66 ± 7.34 a
Values in the table are the mean of 25 repetitions, equal letters do not have a significant difference (p ≤ 0.05). Propagation types (treatments): TIS—Temporary immersion systems, SSS—Semi-solid systems, CIS—Continuous immersion systems. Means ± standard error with different letters in the columns indicate significant statistical difference (Tukey, p ≤ 0.05).
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MDPI and ACS Style

Solano-Rodríguez, L.A.; Galindo-Tovar, M.E.; Castañeda-Castro, O.; Hidalgo-Contreras, J.V.; Murguía-González, J.; Cuatra-Xicalhua, G.L.; Vián-Pérez, J.G.; Mendoza del Ángel, P.A.; Pastelín-Solano, M.C. In Vitro Cultivation of the Orchid Hybrid Rhyncattleanthe Queen Bee JLA 1 and Its Propagation Under Different Systems. Horticulturae 2025, 11, 722. https://doi.org/10.3390/horticulturae11070722

AMA Style

Solano-Rodríguez LA, Galindo-Tovar ME, Castañeda-Castro O, Hidalgo-Contreras JV, Murguía-González J, Cuatra-Xicalhua GL, Vián-Pérez JG, Mendoza del Ángel PA, Pastelín-Solano MC. In Vitro Cultivation of the Orchid Hybrid Rhyncattleanthe Queen Bee JLA 1 and Its Propagation Under Different Systems. Horticulturae. 2025; 11(7):722. https://doi.org/10.3390/horticulturae11070722

Chicago/Turabian Style

Solano-Rodríguez, Luis Alberto, María Elena Galindo-Tovar, Odon Castañeda-Castro, Juan Valente Hidalgo-Contreras, Joaquín Murguía-González, Gabriela Lucero Cuatra-Xicalhua, José Guadalupe Vián-Pérez, Pablo Antonio Mendoza del Ángel, and Miriam Cristina Pastelín-Solano. 2025. "In Vitro Cultivation of the Orchid Hybrid Rhyncattleanthe Queen Bee JLA 1 and Its Propagation Under Different Systems" Horticulturae 11, no. 7: 722. https://doi.org/10.3390/horticulturae11070722

APA Style

Solano-Rodríguez, L. A., Galindo-Tovar, M. E., Castañeda-Castro, O., Hidalgo-Contreras, J. V., Murguía-González, J., Cuatra-Xicalhua, G. L., Vián-Pérez, J. G., Mendoza del Ángel, P. A., & Pastelín-Solano, M. C. (2025). In Vitro Cultivation of the Orchid Hybrid Rhyncattleanthe Queen Bee JLA 1 and Its Propagation Under Different Systems. Horticulturae, 11(7), 722. https://doi.org/10.3390/horticulturae11070722

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