Brassavola tuberculata Hook.: in vitro growth and ex vitro establishment as a function of the micropropagation system and sucrose

Abstract This study examines the in vitro growth and ex vitro establishment of Brassavola tuberculata in relation to the micropropagation system and sucrose concentration employed in the in vitro culture. A completely randomized experimental design was utilized, employing a 2 x 5 factorial arrangement. The experimental period began with seedlings cultivated in vitro for 180 days, which were subsequently transferred to Murashige and Skoog culture media containing sucrose concentrations of 0, 15, 30, 45, or 60 g L-1. The cultures were subjected to two micropropagation systems: conventional and gas exchange. After 90 days of in vitro cultivation, the plants were evaluated, transplanted into a substrate, and placed in a screened nursery for ex vitro cultivation. After 300 days of ex vitro cultivation, the survival and initial characteristics of the plants were assessed. The micropropagation system allowing gas exchange and sucrose concentrations up to 30 g L-1 enhanced the shoot and root growth of in vitro propagated plants. No noticeable anatomical differences were observed after 90 days of in vitro culture among the different sucrose concentrations and micropropagation systems used. In the ex vitro establishment, irrespective of sucrose concentration, the micropropagation system facilitating gas exchange positively influenced all evaluated characteristics.


Introduction
The Orchidaceae family is recognized as one of the largest and most representative plant families among angiosperms, comprising species of high ornamental, nutritional, and pharmacological value.However, due to the extraction of native orchids driven by their captivating flowers, their populations have declined in various biomes.Consequently, many species have become vulnerable due to the systematic destruction of their habitats (Sasamori et al., 2020;Soares et al., 2020;Nongdam et al., 2023).

In vitro germination
The experiment was conducted at the In Vitro Culture Laboratory of Flowers and Ornamental Plants and the orchidarium of the School of Agricultural Sciences at the Universidade Federal da Grande Dourados.Ripe fruits of Brassavola tuberculata Hook.were used as the study material.The fruits were derived from manual pollination and obtained from matrices over ten years old grown in a nursery covered by two 50% shade screens.The irradiance level was maintained at 235 µmol m-2 s-1, with a mean temperature of 22.6 ± 5 ºC and relative humidity of 73.9 ± 10%.The plants were irrigated with rotary micro-sprinklers positioned one meter above the plants, providing a daily water depth of 1 mm.
A sample of 0.005 g of seeds was weighed, and a tetrazolium test was conducted following the methodology described by Soares et al. (2014) to confirm viability.After confirming viability, another sample of 0.005 g of seeds was taken to an aseptic environment and disinfected according to Soares et al. (2020) to obtain the seed solution.For in vitro sowing, 1.0 mL of the suspension of disinfected seeds was inoculated in a culture flask with 60 mL of Murashige and Skoog culture medium (MS) with half the salt concentration (MS ½).The flasks had a capacity of 600 mL.The cultures were placed in a growth room with controlled temperature and photoperiod (25 ± 2 °C; 16 h) and an irradiance of 22 µmol m-2 s-1 provided by two white fluorescent lamps (6,500K).

In vitro growth
After 180 days of cultivation, the plants were standardized to a size of 4.0 ± 0.5 cm and subcultured in an aseptic environment to initiate the experimental period.Flasks with a capacity of 600 mL containing 60 mL of Murashige and Skoog culture medium (MS) solidified with 7.0 g L -1 of bacteriological agar (Himedia®, India) were used.The culture medium was supplemented with different sucrose concentrations (0, 15, 30, 45, and 60 g L -1 ).The pH of the medium was adjusted to 5.8 using 0.1 M KOH before sterilization in an autoclave at 121 °C and 1 atm of pressure for 20 minutes.Each flask contained seven plants and was sealed with either a screw cap (conventional micropropagation system -CMS) or a screw cap with a hole and cotton filter (photoautotrophic or photomixotrophic micropropagation system with gas exchange -MSGE) to allow gas exchange.
After 90 days of subculture, the flasks were opened, and the plants were washed under running water to remove the culture medium.The plants were then evaluated using a digital caliper and precision scale to measure the number of leaves (NL), plant height (PLH) (mm), number of buds (NB), number of roots (NR), length of the largest root (LR) (mm), and fresh matter (FM) (g).The remaining culture medium in the flasks was heated until it returned to the liquid phase, and the pH was measured using a benchtop pH meter.
The genus Brassavola, widely distributed throughout Brazil, possesses remarkable ornamental potential and resilience.Consequently, it is among the genera most threatened by unregulated collection and holds commercial interest for hybrid production (Xu et al., 2022;Vendrame et al., 2023).Brassavola tuberculata Hook.stands out as a prominent species within this genus.
Therefore, strategies for propagation and conservation must ensure the survival of these plants in their natural habitats while providing plant material for commercial production.In this regard, in vitro cultivation presents a viable alternative as it enables the conservation of pathogen-free material, production of elite plants, and maintenance of genetic diversity in the short, medium, and long term (Teixeira da Silva et al., 2017;Soares et al., 2020;Nongdam et al., 2023;Vendrame et al., 2023).
In vitro propagation offers several advantages over conventional propagation methods.However, it still faces technical and economic limitations, including high production costs, shortage of skilled labor, and the inability of plants with morphophysiological changes to survive in ex vitro environments.Consequently, scaling up production becomes challenging (Miranda et al., 2020;Nongdam et al., 2023).
Despite the widespread use of in vitro cultivation techniques for orchids, the in vitro propagation of Brassavola species still lacks established protocols, primarily due to the limited number of studies focused on these species (Pereira et al., 2022;Xu et al., 2022;Vendrame et al., 2023).Regarding B. tuberculata, only a few scientific articles have been published on its in vitro germination (Herrmann et al., 2011;Soares et al., 2012Soares et al., , 2020;;Rosa et al., 2013), seed storage and viability (Macedo et al., 2014;Soares et al., 2014;Sousa et al., 2020), multiplication and in vitro rooting (Mengarda et al., 2017), and ex vitro acclimatization (Sousa et al., 2015).
Given the commercial, ornamental, and environmental demands associated with the Orchidaceae family, there is a current pursuit for sustainable production using advanced and cost-effective techniques (Teixeira da Silva et al., 2017;Nongdam et al., 2023).Consequently, there is a need for adaptations in in vitro cultivation protocols, particularly for native species.These adaptations should aim to enhance productivity and promote the development of plants with morphophysiological characteristics conducive to their ex vitro establishment.In this regard, modifications to micropropagation systems, commonly heterotrophic, and sucrose concentrations in culture media can lead to desirable traits in propagated plants (Ferreira et al., 2017;Ribeiro et al., 2019;Miranda et al., 2020;Santos et al., 2020;Nongdam et al., 2023).
Considering the above, numerous studies have explored the use of different micropropagation systems and sucrose concentrations in culture media for other cultivated species.However, this is the first study investigating the in vitro culture of B. tuberculata using photoheterotrophic, photoautotrophic, and photomixotrophic systems.Therefore, this study evaluates the in vitro growth and ex vitro establishment of B. tuberculata in relation to the micropropagation system and sucrose concentration employed in the culture.

Visual anatomical analysis
To analyze the effects of the treatments on leaf tissues, samples from plants subjected to different micropropagation systems and sucrose concentrations were collected.Leaf fragments measuring 5 mm were cut from the central portion of the leaves.These fragments were fixed in F.A.A. (formaldehyde 35%, glacial acetic acid, and 50% ethanol) and stored in a refrigerator.The fragments were then progressively dehydrated in an alcohol series with tertiary butyl alcohol (Dankin & Hussey, 1985).After the dehydration process, the leaf fragments were infiltrated with paraffin and paraplast.Cross sections of 10-µm thickness were made using a manual rotary microtome.The sections were stained with safranin-orange G-fast Green FCF (Hagquist, 1974).The images were analyzed using the AxioVision version 3.1 computational application coupled to the micrometer eyepiece microscope.

Ex vitro growth
For the evaluation of ex vitro growth, the plants were transplanted into disposable transparent polypropylene containers with a capacity of 1,000 mL (20 x 10 x 5 cm).The containers were filled with a substrate consisting of one-third pink sphagnum moss (Agrolink, Holambra, Brazil) and two-thirds coconut fiber (Golden-Mix Chips, Amafibra) (1:1, v:v).After transplantation, the containers were placed in a protected nursery and maintained under the same conditions as the parent plants for 300 days.
After 300 days of ex vitro cultivation, the plants were removed from the containers and washed under running water to remove the substrate.The plants were then evaluated for the same initial characteristics (NL, PLH, PD, NB, NR, LR, LF, and FM).In order to investigate the hypothesis of increased plant growth during the ex vitro stage, and according to the treatments to which plants were exposed in the in vitro stage, their increases (I) regarding the initial values were calculated using the expression ( ) Where VI is the value of the variable before the plant was acclimatized, and VF is the value of the same variable after the ex vitro period, with its values expressed as a percentage and subjected to analysis of variance (Ribeiro et al., 2019).

Experimental design
A completely randomized experimental design was used with a 2 x 5 factorial design (two micropropagation systems and five sucrose concentrations) and five replicates of a culture flask with five plants each.The results were transformed using √ (x + 1) and subjected to analysis of variance.The Tukey test (p < 0.05) was used to compare the means of the quantitative factors.Regression curves were fitted to the significant factors.The statistical analysis was performed using the SISVAR software (Statistical Analysis Program v.5.3., Universidade Federal de Lavras, MG).

In vitro growth
A significant effect was observed for the interaction between micropropagation systems and sucrose concentrations (p < 0.05) on the number of leaves (NL), buds (NB), length of the largest root (LR), fresh matter (FM), and pH of the culture media of B. tuberculata after 90 days of in vitro cultivation.Additionally, the micropropagation system had an isolated effect on plant height (PLH) and the number of roots (NR).The sucrose concentrations also had an isolated effect on NR.
Brassavola tuberculata plants exhibited higher NL, NB, FM, and pH when subjected to CMS.The calculated values included 26.95 leaves at a concentration of 17.12 g L -1 of sucrose, 9.28 shoots at a concentration of 17.59 g L -1 of sucrose, 0.47 g at a concentration of 17.16 g L -1 of sucrose, and a pH of 3.9 without the addition of sucrose.For LR, the highest calculated values were observed in MSGE when supplemented with 19.72 g L -1 of sucrose (32.21 mm) (Figure 1).Regarding the isolated effect of the micropropagation system, it was observed that flasks under MSGE had larger plants (53.75 ± 19.40 mm) and a greater number of roots (4.97 ± 1.58 roots) (Figure 2).
Concerning the isolated effect of sucrose concentrations, plants grown in a culture medium supplemented with an average value of 34.37 g L -1 of sucrose showed a higher number of roots, with 5.02 roots (Figure 2C).
Figure 3 shows the variation in the morphological aspects of plants as a function of micropropagation systems and sucrose concentrations.

Histological slides
According to Figure 4, the cross-section images of leaves indicated that the material was in good conservation condition.The different sucrose concentrations and micropropagation systems used did not visually show anatomical differences in plants after 90 days of in vitro cultivation.
The characteristics common to B. tuberculata were observed in all cross-sections, including a uni-stratified epidermis with irregularly shaped and sized cells, thick cell walls, and a smooth, continuous cuticle surface throughout the leaf.The secondary wall cells showed no thickening, and the fiber bundles were arranged from the leaf adaxial to the abaxial face, displaying good organization.The presence of chloroplasts was noted, with homogeneous mesophilic organization, equifacial and slightly elongated isodiametric epidermal cells in the paradermic view, and straight and thick anticlinal walls (Figure 4).
Although Brassavola leaves are terete, the adaxial face corresponds to the grooved surface area of the leaf, while the abaxial face corresponds to the convex and rounded region.The chlorenchyma is homogeneous, with polyhedral cells of irregular size and thin walls, without a clear distinction between lacunous and palisade cells.In the central rib region, the upper mesophile cells appear more isodiametric, while in the lower mesophile region throughout the leaf, the cells tend to lengthen their anticlinal walls.Moreover, regarding conduction, the layers of vascular bundles tend to follow the conduplicate shape of the leaf (Figure 4).

Ex vitro growth
After 300 days of ex vitro cultivation, the analysis of variance showed a significant effect of the interaction between micropropagation systems and sucrose concentrations (p < 0.05) on %SUR (survival), NL, NR, and LR.Furthermore, the micropropagation systems had an isolated effect on PLH and FM, while the effect of sucrose concentrations was observed only for PLH.
Regarding the interaction between micropropagation systems and sucrose concentrations, a higher %SUR was observed at the end of the experimental period when B. tuberculata plants were subjected to MSGE without sucrose in the culture medium, with %SUR = 82.32.MSGE without the addition of sucrose also provided the highest values for NL (46.73%).
For NR and LR, the highest calculated values were observed when using MSGE and a culture medium with 14.53 g L -1 of sucrose (169.85%) and 60.00 g L -1 of sucrose (234.40%),respectively (Figure 5).
Regarding the isolated effect of sucrose concentrations in the culture medium, the highest increases in PLH (89.06%) were observed using 44.90 g L -1 of this sugar (Figure 6C).
According to Figure 7, the conditions of in vitro cultivation did not limit plant growth during the ex vitro period.In general, MSGE showed the highest results for almost all parameters evaluated, suggesting that this micropropagation system may be suitable for cultivating this species.

In vitro growth
The observed results for NL in CMS suggest that its higher value may be related to the increased NB observed in the same treatment, leading to higher FM.This outcome could be attributed to the presence of ethylene gas and low CO 2 concentrations in the hermetic environment of CMS, which functions as a heterotrophic system.Consequently, it can reduce plant height and photosynthetic pigment content, thereby influencing the tillering of in vitro-grown plants (Teixeira da Silva et al., 2017;Miranda et al., 2020).
The results for LR, PLH, and NR in MSGE can be attributed to the utilization of this photomixotrophic propagation system, which enables gaseous exchange between the flask's interior and the atmospheric air, thus influencing the growth and development of in vitro plants (Silva et al., 2016;Fritsche et al., 2022) These findings align with those of Ribeiro et al. (2019), where Denphal plants cultivated in flasks with ventilation caps displayed superior growth characteristics compared to those grown in a hermetic environment.Employing micropropagation under natural ventilation conditions can be an alternative to improving morphophysiological issues arising from the conventional in vitro culture system.MSGE, by allowing gas exchange, enhances photochemical efficiency and photosynthetic carbon assimilation, resulting in plants with more efficient metabolism and robustness (Fuentes et al., 2007;Kozai, 2010;Xiao et al., 2011;Fritsche et al., 2022).
Regarding sucrose, the optimal results for in vitro cultivation of B. tuberculata were achieved with concentrations around 15 and 30 g L -1 .Studies on different sucrose concentrations in the in vitro culture of native orchids, such as Alatiglossum fuscopetalum (Ferreira et al., 2017), Oncidium flexuosum (Caovila et al., 2016), Cyrtopodium cachimboense (Paulino et al., 2021), Miltonia flavescens (Lemes et al., 2014), and Cattleya schilleriana (Galdiano-Júnior et al., 2018), support our results.However, in contrast to our findings, Silva et al. (2014) observed that Cattleya walkeriana Gardner plants exhibited increased root system growth when cultured with the highest tested sucrose concentration (45 g L -1 ) in flasks with ventilation caps.These observations suggest that the influence of sucrose may vary according to the orchid species studied, and the species examined here exhibits similar behavior to most reported in the scientific literature.
Regarding pH, the culture media were initially adjusted to 5.8 before the experimental period.However, a decrease in pH was observed for all treatments after 90 days of in vitro cultivation.The recommended pH range for adequate growth of most orchid species is between 5.0 and 6.5 (Faria et al., 2004).Additionally, plant tissue culture media often have low buffering capacity (Leifert et al., 1995).Therefore, some species can adjust the medium pH to values between 3.7 and 6.2 during the plant growth period, irrespective of the initial pH (Leifert et al., 1992).
In general, the pH decrease was more pronounced in MSGE.This decline may be attributed to water loss through gas exchange, occurring due to the different pressure and temperature gradients inside and outside the flask, concentration gradients of gases (CO 2 and H 2 O, among others), and the speed and pattern of air movement around the flask (Xiao et al., 2011).Additionally, gas exchange and increased CO 2 levels may promote H 2 CO 3 formation in the culture medium, leading to a decreased pH in this micropropagation system.However, Caovila et al. (2016) concluded in their research on sucrose concentrations and pH in the in vitro growth of Oncidium flexuosum that regardless of the pH value, the factor that most significantly influences the variables is the concentration of sucrose used.
This study demonstrates that photomixotrophic culture, with supplementation of up to 30 g L -1 of sucrose in the culture medium, provides favorable conditions for desirable characteristics in B. tuberculata plants during ex vitro cultivation, such as increased height and root system development.

Ex vitro growth
The results for %SUR and NL suggest that the use of photoautotrophic micropropagation systems, i.e., without adding carbohydrates to the culture medium and allowing gas exchange, promotes photosynthesis.However, most orchids are cultivated using a conventional micropropagation system (heterotrophic) characterized by the absence of gas exchange and high humidity, leading to physiological and morphological disorders in plants, particularly stomatal malfunction, increased water loss through leaf tissue, and potential decrease in survival of orchid seedlings during the ex vitro acclimatization process (Majada et al., 2002;Cha-Um et al., 2010;Silva et al., 2016;Teixeira da Silva et al., 2017).
In the photomixotrophic system (with carbohydrate addition and gas exchange), the characteristics of NR, RL, and FM can be attributed to the increased aeration facilitated by MSGE.Furthermore, this system promotes photosynthesis by allowing gas exchange within the flask, leading to normalized transpiration and regulation of stomatal function.Under these conditions, explants can utilize endogenous and exogenous carbohydrates as an energy source (Kozai, 2010;Santos et al., 2020).
These results are consistent with Silva et al. (2014), who observed that Cattleya walkeriana Gardner plants grown in flasks with ventilation caps exhibited superior growth characteristics compared to those grown in a hermetic environment.Regarding sucrose concentrations, Galdiano-Júnior et al. (2013), in their study on the effect of sucrose on the number of roots in Cattleya loddigesii Lindley, observed the highest values with a concentration of 20.7 g L -1 , similar to the findings in our study (14.53 g L -1 ).Additionally, the data obtained for LR align with those of Faria et al. (2004), who studied Dendrobium nobile Lindl.plants and observed the longest root length with a concentration of 60 g L -1 sucrose.
Regarding the isolated effect of sucrose on PLH, our results are similar to those reported by Besson et al. (2010), who observed a significant difference in shoot length of Miltonia flaviscens Lindl.with medium supplementation of 30 and 45 g L -1 of sucrose.However, Galdiano-Júnior et al. ( 2013) observed higher shoot length values for Cattleya loddigesii Lindl.with lower sucrose concentrations (21.5 g L -1 ).These observations indicate that the concentration of sucrose used in the culture media can influence different plant materials in distinct ways.
The higher PLH observed when using CMS alone may be attributed to the transfer to the ex vitro environment.These plants need to complete their autotrophism by increasing their metabolic rates (Teixeira da Silva et al., 2017), thus utilizing their photoassimilates for height growth.Therefore, plants grown in vitro using a conventional system exhibit an increased growth rate during the ex vitro phase.Meanwhile, plants from MSGE, which had already initiated rusting during in vitro cultivation, only maintained this growth rate (Ribeiro et al., 2019).
In orchid species propagation protocols, it is crucial to consider not only the number of micropropagated plants but also the morphophysiological quality of the resulting plants.Thus, the findings of this study, combined with those previously reported in the scientific literature for the Orchidaceae family, support the inference that photoautotrophic and photomixotrophic micropropagation systems promote the ex vitro establishment of plants when cultivated in vitro, making them recommended for the in vitro cultivation of B. tuberculata.

Conclusion
The use of a micropropagation system that allows gas exchange, along with sucrose concentrations of up to 30 g L -1 , enhances the shoot and root growth of B. tuberculata plants propagated in vitro.For ex vitro establishment, the micropropagation system that allows gas exchange is recommended regardless of the sucrose concentration used.

Figure 1 .
Figure 1.Number of leaves (NF), number of buds (NB), length of the largest root (LR), fresh matter (FM), and potential of hydrogen (pH) of Brassavola tuberculata Hook.. as a function of sucrose concentrations in the culture medium and micropropagation system.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE.

Figure 2 .
Figure 2. In vitro growth of Brassavola tuberculataHook.: A) plant height (PLH) (mm); B) number of roots (NR) after 90 days of in vitro cultivation as a function of the micropropagation system, and (C) NR after 90 days of in vitro cultivation as a function of sucrose concentrations in the culture medium.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE.

Figure 3 .
Figure 3. Brassavola tuberculata Hook.plants after 90 days of in vitro cultivation as a function of sucrose concentrations in the culture medium and micropropagation system.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE

Figure 5 .
Figure 5. Survival (%SUR) and increases (%) in the number of leaves (NL), number of roots (NR), and length of the largest root (LR) of Brassavola tuberculata Hook.. as a function of sucrose concentrations and micropropagation system.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE.

Figure 6 .
Figure 6.Increases in Brassavola tuberculata Hook..: A) plant height (PLH) (mm); B) fresh matter (FM) after 300 days of in vitro cultivation as a function of the micropropagation system, and (C) PLH after 300 days of in vitro cultivation as a function of sucrose concentrations in the culture medium.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE.

Figure 7 .
Figure 7. Brassavola tuberculata Hook.plants at 300 days of in vitro cultivation as a function of sucrose concentrations in the culture medium and micropropagation system.Conventional micropropagation system = CMS; micropropagation system with gas exchange = MSGE.