Innovations in micropropagation of the orchid Cattleya nobilior: the effect of bioreactor and enriched culture media

Inovações na micropropagação da orquídea Cattleya nobilior: efeito de biorreator e meios de cultura enriquecidos

ABSTRACT:

Cattleya nobilior Rchb.f. is an epiphytic species of ornamental value, facing risks due to environmental threats, indiscriminate collection, and habitat loss. In this context, in vitro cultivation presents a viable alternative for its commercial production and conservation efforts. This pioneering study evaluated the initial in vitro growth and ex vitro establishment of C. nobilior, using supplemented culture media and comparing conventional micropropagation with temporary immersion bioreactors. The experimental design was completely randomized, with treatments in a 4x2 factorial scheme, involving four culture media and two micropropagation systems. After 180 days of in vitro cultivation, the plants were assessed for the number of leaves, pseudobulbs, and roots, length of the largest leaf and root, plant height, pseudobulb diameter, and total fresh mass. Subsequently, they were transplanted into containers with mixed substrate and transferred to an ex vitro environment. After another 180 days, survival was recorded, and the same characteristics initially evaluated were measured again to calculate their increments. The results showed that, in in vitro cultivation, plants grown by the conventional method exhibited tillering, resulting in an increase in the number of leaves, shoots, and roots. However, those cultivated in a bioreactor showed an increase in leaf and root length, pseudobulb diameter, and plant height, especially when grown in medium enriched with banana pulp. During the ex vitro phase, plants originating from bioreactors continued to show the highest results, especially those cultivated in medium with the addition of banana. This study is the first to investigate the use of temporary immersion bioreactors in the micropropagation of C. nobilior, demonstrating that the combination of bioreactor and culture media supplemented with banana pulp provides optimal conditions for the robust growth of plants, both in vitro and ex vitro, offering new perspectives for the commercial production and conservation of the species.

Key words:
Orchidaceae; culture medium supplementation; ex vitro establishment; conservation; native species

RESUMO:

Cattleya nobilior Rchb.f. é uma espécie epífita de valor ornamental, enfrenta riscos devido a ameaças ambientais, coleta indiscriminada e perda de habitat. Nesse contexto, o cultivoin vitroé uma alternativa viável para sua produção comercial e esforços de conservação. Este estudo pioneiro avaliou o crescimento inicial in vitro e o estabelecimento ex vitro de C. nobilior, utilizando meios de cultura suplementados e comparando a micropropagação convencional com biorreatores de imersão temporária. O delineamento experimental adotado foi inteiramente casualizado, com tratamentos em esquema fatorial 4x2, envolvendo quatro meios de cultivo e dois sistemas de micropropagação. Após 180 dias de cultivo in vitro, as plantas foram avaliadas quanto ao número de folhas, pseudobulbos e raízes, comprimento da maior folha e raiz, altura de planta, diâmetro de pseudobulbo e massa fresca total. Em seguida, foram transplantadas para recipientes contendo substrato misto e transferidas para ambiente ex vitro. Após mais 180 dias, a sobrevivência foi registrada, e as mesmas características avaliadas inicialmente foram novamente mensuradas para calcular seus incrementos. Os resultados mostraram que, no cultivo in vitro, as plantas cultivadas pelo método convencional, apresentaram perfilhamento, resultando em um aumento no número de folhas, brotos e raízes. Contudo, aquelas cultivadas em biorreator apresentaram um incremento em comprimento de folha e raiz, diâmetro de pseudobulbo e altura de planta, principalmente quando cultivadas em meio enriquecido com polpa de banana. Durante a fase ex vitro, as plantas originárias de biorreatores continuaram apresentando os maiores resultados, principalmente aquelas cultivadas em meio com adição de banana. Este estudo é o primeiro a investigar o uso de biorreatores de imersão temporária na micropropagação C. nobilior, demonstrando que a combinação de biorreator e meio de cultura suplementado com polpa de banana proporciona condições ótimas para o maior crescimento robusto das plantas, tanto in vitro quanto ex vitro, oferecendo novas perspectivas para a produção comercial e conservação da espécie.

Palavras-chave:
Orchidaceae; suplementação de meio de cultura; estabelecimento ex vitro; conservação; espécie nativa

INTRODUCTION

The Orchidaceae family is recognized as the largest and most diverse among the Angiosperms. In Brazil, approximately 2,679 orchid species are recorded, of which 1,487 are endemic. The genus Cattleya, one of the most commercialized in the country, has about 48 species distributed throughout tropical America. This region is characterized by habitats that provide abundant light and high relative humidity, essential conditions for the development of these plants (FLORA & FUNGA OF BRAZIL, 2024; JUNQUEIRA & PEETZ, 2017).

Among the species of the genus, the native orchid Cattleya nobilior Rchb.f. stands out for its economic importance due to its ornamental characteristics, such as large, exuberant flowers with pink-purple coloration (ROMAHN, 2020; FLORA & FUNGA OF BRAZIL, 2024). These qualities, combined with the constant threats to its natural habitat and indiscriminate collection, place this species in a vulnerable position, making in vitro cultivation a promising strategy for both commercial seedling production and conservation efforts (STEFANO et al., 2022; VENDRAME et al., 2023).

In this scenario, the exploration of innovative and efficient cultivation systems becomes fundamental to overcome the limitations of conventional cultivation, especially regarding commercial viability. Cultivation techniques that use temporary immersion bioreactors are indicated as a viable alternative, standing out for their ability to optimize the control of essential physical parameters for plant development, in addition to allowing temporary and efficient contact of the plants with the cultivation medium, thus facilitating the uniform absorption of essential nutrients. Temporary immersion bioreactors have been used to propagate some orchid genera, such as Cattleya, Brassavola, Vanilla, Bletilla, Dendrobium and Phalaenopsis, using different media, growth regulators, and explants (VENDRAME et al., 2023; NONGDAM et al., 2023).

The composition of the cultivation medium, enriched with organic supplements such as coconut water and banana pulp, plays a fundamental role, acting as a rich source of vitamins, amino acids, fatty acids, carbohydrates, peptides, and growth factors. The addition of these compounds, whether in isolation or in combination, can enhance the germination of seeds and the development of seedlings, varying according to the species and genotype in question. (UTAMI & HARIYANTO, 2020; STEFANO et al., 2022).

Additionally, the acclimatization phase represents a challenge for the success of this type of propagation, requiring careful adaptation of the plants to a new environment (ex vitro). This process is fundamental to ensure the survival of micropropagated plants when they are transferred to heterotrophic conditions, highlighting the importance of well-developed multiplication protocols (STEFANO et al., 2022; SOARES et al., 2023).

Considering that there are no previous studies using temporary immersion bioreactors in the in vitro cultivation of the C. nobilior species (NONGDAM et al., 2023), this pioneering research investigated the use of this technology in the propagation of this species. Thus, with the objective of investigating the hypothesis that the use of bioreactors and the supplementation of the medium with banana pulp and coconut water have a positive influence both in the in vitro cultivation and in the acclimatization of the studied species, the present study evaluated the in vitro growth and ex vitro establishment of C. nobilior using different supplemented culture media and comparing them with conventional micropropagation systems and the use of temporary immersion bioreactor. This research contributed to the advancement of scientific knowledge but also provides valuable information for the sustainable production and conservation of threatened orchid species.

MATERIALS AND METHODS

The study material consisted of ripe fruits of Cattleya nobilior Rchb.f., obtained from manual pollination of matrices over fifteen years old, grown in a greenhouse screen under two overlapping layers of 50% shade cloth, providing an irradiance of 235 µmol m^-2 s^-1. The average conditions of temperature and relative humidity were maintained at 22.6 ± 5 ºC and 73.9 ± 10%, respectively, with irrigation performed by ballerina-type micro sprinklers located one meter above the plants, resulting in a water depth of 1 mm day^-1.

A sample of 0.005 g of seeds was weighed and subjected to the tetrazolium test, following the methodology of RIBEIRO et al. (2021), to confirm their viability. After confirmation, another sample of 0.005 g of seeds was taken to an aseptic environment and disinfected according to the methodology of SOARES et al. (2020), aiming to obtain the seed solution. Each culture flask was inoculated with 1 mL of this disinfected seed suspension for in vitro sowing. A total of 60 mL of Murashige & Skoog medium with half salt concentration (MS ½) was used per flask with a capacity of 600 mL. Subsequently, the cultures were placed in a growth room with controlled temperature and photoperiod (25 ± 2 ºC; 16 h) and irradiance of 22 µmol m^-2 s^-1 provided by two white fluorescent lamps (18.9 µmol m^-2 s^-1). The cultures remained under these conditions for 180 days, with one subculture performed during this period.

At the beginning of the experimental period, the plants, 180 days after in vitro sowing, were standardized in size (1.5 ± 0.3 cm) and transferred, in an aseptic environment, to the following culture media: 1- MS; 2- MS + apple banana pulp (BP) (100 g L^-1 of apple banana pulp); 3- MS + coconut water (CW) (100 mL L^-1); 4- MS + BP (100 g L^-1) + CW (100 mL L^-1). Subsequently, they were allocated to two micropropagation systems: 1) conventional system (CS); 2) temporary immersion system (TIS).

In the conventional cultivation, 40 mL of the medium solidified with 6.0 g L^-1 of bacteriological agar (Himedia^®, India) and 30 g L^-1 of sucrose were added to each flask. For the bioreactor, 200 mL of the same medium was used, but without a gelling agent, and the bioreactor was programmed with aeration cycles of 3 minutes every 5 hours and 57 minutes. The pH of all media was adjusted to 5.8 before autoclaving, carried out at 121 ºC and a pressure of 1.1 atm for 20 minutes. The inoculated material (25 plants per 200 mL of medium in each micropropagation system) was maintained under controlled conditions of photoperiod (16 hours), temperature (25±2 ºC), and irradiance (18.9 μmol m^-2 s^-1), under LED lighting composed of 50% white (6500K), 25% blue, and 25% red, for 180 days.

After this period, the plants were removed from the flasks, washed with water until the complete removal of the culture medium, and evaluated for the number of leaves (NL), number of roots (NR), number of shoots (NS), greatest leaf length (LL) (mm), greatest root length (RL) (mm), plant height (PHE) (mm), pseudobulb diameter (PD) (mm), and fresh mass (FM) (g). After the evaluations, the treatments were photographed using a camera attached to a mini photo studio.

Subsequently, the plants were transferred to transparent polypropylene containers (20 x 10 x 5 cm) with a capacity of 1000 mL with holes in the base and the lid, for substrate drainage and gas exchanges, respectively. The containers were filled with a substrate composed of ⅓ of its volume of pink sphagnum (Agrolink, Holambra - SP) and coconut fiber (Golden-Mix Chips, Amafibra) in a 1:2 (v:v) ratio. After planting, the containers were placed in a shaded greenhouse, under the same conditions as the parent plants. These containers remained covered during the first 15 days of acclimatization (pre-acclimatization) to minimize the initial effects of the transfer from the in vitro to the ex vitro environment, and then they were opened.

After 180 days of acclimatization, the plants were evaluated for survival and the same initial characteristics (NL, NR, NS, LL, RL, PHE, PD, and FM). To investigate the hypothesis of increased plant growth during the ex vitro phase, according to the treatments to which they were exposed in the in vitro phase, their increments (I) in relation to the initial values were calculated, using the expression proposed by RIBEIRO et al. (2019), $I=\left(VF-VI\right)$, where VI is the value of the variable before acclimatization and VF is the value after the ex vitro period. The values were expressed in percentage and submitted to analysis of variance.

The experimental design was completely randomized, and the treatments were arranged in a 4 x 2 factorial scheme (four culture media and two micropropagation systems), with five replicates of five plants each. The results were transformed to √(x+1) and subsequently subjected to analysis of variance. Differences between the micropropagation systems were verified by the Bonferroni t-test, while the Tukey test was used at a 5% probability level to check differences between the culture media, using the SISVAR software (Statistical Analysis Program v.5.3, Universidade Federal de Lavras (UFLA), MG).

RESULTS

In vitro growth

The analysis of variance showed a significant effect (P < 0.05) of the interaction between the culture media and the micropropagation systems on all evaluated variables in Cattleya nobilior. For the variables leaf number (NL), root number (NR), and shoot number (NS), the system x medium (S x M) interaction indicated that the highest values were observed in the conventional cultivation system (CS) combined with MS medium (52.40; 26.40; 11.80, respectively) (Table 1).

Table 1
Number of leaves (NL), number of roots (NR), and number of shoots (NS), longest leaf length (LL), longest root length (RL), plant height (PHE), pseudobulb diameter (PD), and fresh mass (FM) of Cattleya nobilior Rchb.f. according to the culture media [MS; MS + apple banana pulp (BP); MS + coconut water (CW); MS + BP + CW] and micropropagation system (CS = conventional system; TIS = temporary immersion system) 180 days after in vitro cultivation.

Culture Media	-------------NL-------------		-------------NR-------------		-------------NS-------------		---------LL (mm)----------
	CS	TIS	CS	TIS	CS	TIS	CS	TIS
MS	52.40 Aa	11.40 Ba	26.40 Aa	6.40 Ba	11.80 Aa	2.40 Ba	13.04 Ba	20.62 Ab
MS + BP	7.60 Ab	7.60 Aa	7.00 Ab	9.40 Aa	2.00 Ab	2.00 Aa	15.65 Ba	36.22 Aa
MS + CW	18.20 Ab	9.00 Aa	8.80 Ab	11.80 Aa	3.40 Ab	2.40 Aa	11.56 Ba	21.12 Ab
MS + BP + CW	8.50 Ab	5.16 Aa	7.50 Ab	6.33 Aa	2.00 Ab	1.33 Aa	18.84 Aa	13.34 Ab
Average	21.67	8.29	12.42	8.48	4.80	2.03	14.77	22.82
CV (%)	------------22.47-----------		-----------19.74------------		------------19.79-----------		------------18.48-----------
Culture Media	----------RL (mm)---------		--------PHE (mm)---------		----------PD (mm)---------		-----------FM (g)----------
	CS	TIS	CS	TIS	CS	TIS	CS	TIS
MS	30.95 Bb	65.30 Aa	52.60 Ba	94.27 Aab	1.97 Aa	1.39 Ab	1.78 Aa	0.73 Bb
MS + BP	58.84 Aa	57.55 Aa	79.64 Ba	105.67 Aa	1.72 Ba	2.44 Aa	1.06 Bb	1.63 Aa
MS + CW	39.95 Ab	39.49 Ab	61.30 Aa	67.82 Abc	2.11 Aa	1.46 Bb	0.73 Ab	0.80 Ab
MS + BP + CW	35.50 Ab	25.88 Ab	65.57 Aa	44.36 Ac	2.01 Aa	1.34 Bb	0.77 Ab	0.49 Ab
Average	41.31	47.05	64.77	78.03	1.95	1.65	1.08	0.91
CV (%)	------------18.23-----------		-----------19.02------------		-------------9.92------------		------------6.40-------------

• Means followed by the same uppercase letter in the row do not statistically differ from each other by the Bonferroni t-test (P ≥ 0.05), and means followed by the same lowercase letter in the column do not statistically differ from each other by the Tukey test (P ≥ 0.05) within the variables.

For the length of the longest leaf (LL), the highest averages were observed when the plants were grown in the temporary immersion system (TIS) with the MS + BP medium, reaching 36.22 mm. Similarly, the highest values for the length of the longest root (RL) were obtained in plants grown in a bioreactor with MS medium, with no significant statistical differences compared to the MS + BP medium (65.30; 57.55 mm, respectively) (Table 1).

C. nobilior showed the highest plant height values (PHE) when grown in MS + BP medium, although it was not statistically different from those grown in the original MS medium formulation (105.67; 94.27 mm, respectively) and the largest pseudobulb diameter (PD) was observed in MS + BP medium (2.44 mm), both using TIS. Conversely, the highest fresh mass values (1.78 g) were achieved when the CS combined with MS medium was used (Table 1).

Figure 1 illustrates the morphological variation of the plants as a function of the culture media and micropropagation systems. Notably, C. nobilior plants grown in CS showed tillering, resulting in an increase in the number of leaves, shoots, and roots, to the detriment of growth. However, it was visually observed that, in the TIS system, the plant organs increased in size, diameter, and length, especially when grown in MS+BP medium.

Figure 1
Cattleya nobilior Rchb.f. plants at 180 days after in vitro cultivation, as a function of the culture medium and micropropagation systems. MS = Murashige & Skoog; BP = apple banana pulp; CW = coconut water; CS = conventional system; TIS = temporary immersion system.

Ex vitro growth

After 180 days of acclimatization, the analysis of variance showed a significant effect (P < 0.05) of the interaction between the culture media and the micropropagation systems on all evaluated characteristics in the ex vitro cultivation of C. nobilior. At the end of the experimental period, the highest survival rate (%SURV) was observed in plants originating from in vitro cultivation in TIS with the MS + BP and MS + CW media, both achieving an average of 100% live plants (Figure 2).

Figure 2
Survival percentage of Cattleya nobilior Rchb.f. after 180 days of acclimatization as a function of the culture medium and micropropagation system. MS = Murashige & Skoog; BP = apple banana pulp; CW = coconut water; CS = conventional system; TIS = temporary immersion system. Uppercase letters compare different micropropagation systems in the same culture medium. Lowercase letters compare the same micropropagation system in different culture media.

Regarding the increases in NL and NR, the S x M interaction showed that the greatest increases were observed in plants from in vitro cultivation in MS + CW (27.40%) and MS (26.60%), both using TIS. For the increase in the number of shoots (NB), the greatest increase was observed in plants cultivated in the conventional system combined with MS + BP medium (19.80%) (Table 2).

Table 2
Number of leaves (NL), number of roots (NR), and number of shoots (NS), longest leaf length (LL), longest root length (RL), plant height (PHE) (mm), pseudobulb diameter (PD) (mm), and fresh mass (FM) (g) of Cattleya nobilior Rchb.f. according to the culture media [MS; MS + apple banana pulp (BP); MS + coconut water (CW); MS + BP + CW] and micropropagation system (CS = conventional system; TIS = temporary immersion system) 180 days after of acclimatization.

Culture media	-------------NL-------------		-------------NR-------------		-------------NS-------------		-------------LL-------------
	CS	TIS	CS	TIS	CS	TIS	CS	TIS
MS	0.00 Aa	0.00 Ab	12.40 Ba	26.60 Aa	0.00 Ab	0.00 Ab	33.96 Aa	15.78 Bb
MS + BP	0.00 Aa	0.00 Ab	0.00 Bb	4.40 Ab	19.80 Aa	0.00 Bb	22.49 Ab	7.90 Bc
MS + CW	0.00 Ba	27.40 Aa	0.00 Ab	0.00 Ab	0.00 Bb	19.60 Aa	14.31 Ac	11.10 Abc
MS + BP + CW	0.00 Aa	0.00 Ab	8.80 Aa	0.00 Bb	0.00 Ab	0.00 Ab	14.21 Bc	28.52 Aa
Average	0.00	6.85	5.30	7.75	4.95	4.90	21.24	15.82
CV (%)	------------12.58-----------		------------14.00-----------		-----------13.38-----------		-----------7.41-----------
Culture media	------------LR------------		---------PHE (mm)--------		----------PD (mm)---------		-----------FM (g)----------
	CS	TIS	CS	TIS	CS	TIS	CS	TIS
MS	0.32 Aa	0.00 Ab	4.00 Aa	0.00 Bb	0.00 Bc	18.72 Aab	3.53 Ac	1.61 Ac
MS + BP	0.00 Ba	9.75 Aa	3.70 Bab	8.92 Aa	55.18 Aa	13.37 Bc	12.41 Ab	14.30 Aa
MS + CW	0.00 Aa	0.00 Ab	0.00 Ac	0.00 Ab	0.00 Bc	20.19 Aa	0.00 Bc	12.51 Aab
MS + BP + CW	0.00 Aa	0.00 Ab	1.96 Ab	0.00 Bb	6.71 Bb	15.26 Abc	26.57 Aa	8.60 Bb
Average	0.08	2.43	2.41	2.23	15.47	16.89	10.62	9.26
CV (%)	------------7.42------------		------------13.49-----------		------------6.99------------		------------11.42-----------

• Means followed by the same uppercase letter in the row do not statistically differ from each other by the Bonferroni t-test (P ≥ 0.05), and means followed by the same lowercase letter in the column do not statistically differ from each other by the Tukey test (P ≥ 0.05) within the variables.

In terms of the increase in LL, the greatest increase was observed in plants from in vitro cultivation in CS using MS medium in its original formulation (33.96%), and for the increase in RL (Length of the Longest Root), in plants obtained through cultivation in TIS combined with MS + BP medium (9.75%) (Table 2).

Additionally, in the S x M interaction, the greatest increase in PHE (8.92%) was observed in plants cultivated in TIS using MS + BP medium. For the other variables, the greatest increase occurred in plants from in vitro cultivation in CS combined with MS + BP medium for the PD variable and MS + BP + CW for FM (Fresh Mass) (55.18; 26.57%, respectively) (Table 2).

Figure 3 shows that the in vitro cultivation conditions had a significant influence on plant growth during the ex vitro period. In general, as visually observed, plants cultivated in TIS presented the best results, especially in MS + BP medium. This cultivation system, associated with the culture medium supplemented with banana pulp, provided conditions for robust growth of C. nobilior both in vitro (Figure 1) and ex vitro (Figure 3).

Figure 3
Cattleya nobilior Rchb.f. plants at 180 days after of acclimatization, as a function of the culture medium and micropropagation systems. MS = Murashige & Skoog; BP = apple banana pulp; CW = coconut water; CS = conventional system; TIS = temporary immersion system.

DISCUSSION

In vitro growth

Plants grown in a conventional cultivation system, using hermetically sealed flasks and MS medium, showed higher values of NL, NR, and NS. This fact can be attributed to physiological disturbances and changes in tissue development, as the conventional cultivation system prevents gas exchanges between the internal microenvironment of the flask and the external environment. Such restriction results in reduced CO₂ concentrations and accumulation of ethylene (MIRANDA et al., 2020; FRITSCHE et al., 2022), conditions that can induce tillering in plants grown under these circumstances.

Conversely, C. nobilior plants grown in TIS showed increases in leaf length and root system, as well as greater height and pseudobulb diameter. Temporary immersion bioreactors offer a promising alternative, combining the benefits of liquid media with low operational costs, enhancing plant growth (EKMEKÇIGIL et al., 2019; NONGDAM et al., 2023). Temporary immersion provides greater contact of the plants with the culture medium, favoring nutrient absorption and; consequently, optimized plant development (NONGDAM et al., 2023). This could be one of the explanations for the observed results in the increase in leaf and root length. Previous studies support these findings, reporting success in the cultivation of various orchid species using TIS, including Cattleya walkeriana Gardner (MOREIRA et al., 2013), Brassavola nodosa Lindl.(VENDRAME et al., 2023), Vanilla planifolia Jacks. Ex Andrews (SPINOSO-CASTILLO et al., 2017; RAMÍREZ-MOSQUEDA et al., 2019; RAMÍREZ-MOSQUEDA & BELLO-BELLO, 2021), Cattleya tigrina (FRITSCHE et al., 2022), Cymbidium sinense (GAO et al., 2014), Dendrobium candidum Lindl. (CUI et al., 2014), Dendrobium nobile Lindl. (ZHANG et al., 2019), Epidendrum fungens Brongn. (FRITSCHE et al., 2022), Mokara leuen Berger (MINH, 2022), Paphiopedilum rothschildianum Rchb.f. (MASNODDIN et al., 2018), Paphiopedilum rothschildianum Rchb.f. (MASNODDIN et al., 2016), Phalaenopsis (SHEN & SHU, 2018), and Vanda tricolor Lindl. (ESYANTI et al., 2016), as cited by NONGDAM et al. (2023) in their review.

TIS is considered one of the most effective methods for in vitro micropropagation of plants on a commercial scale, offering advantages such as the reduction of subculture time and operational costs, which can be significantly lower, surpassing in some cases 40% of traditional costs. Moreover, it is crucial that the microenvironment provided by TIS is specifically idealized for the growth and development of plant tissues and organs, as these are specific requirements for large-scale plant micropropagation (NONGDAM et al., 2023).

The addition of organic supplements, such as banana pulp, to the culture media has proven beneficial for the in vitro growth of various orchid species, like Dendrobium nobile Lindl. (SU et al., 2012), Laeliocattleya (GONÇALVES et al., 2016; NONGDAM et al., 2023) and C. nobilior Rchb. f. (FREITAS et al., 2021). Compounds from the banana enrich the medium, providing essential vitamins, amino acids, and growth regulators, which are readily absorbed by the in vitro plants (DOLCE et al., 2020; STEFANO et al., 2022). This nutritional enrichment is reflected in accelerated growth and increased plant size, highly desirable characteristics for ex vitro cultivation, especially when the medium is supplemented with banana pulp, as evidenced in figure 3 and corroborated by FREITAS et al. (2021).

Ex vitro growth

Overall, the ex vitro establishment of C. nobilior revealed that the prior use of TIS with MS media enriched with banana pulp or coconut water provided a higher percentage of plant survival. Notably, the addition of BP not only favored survival but also promoted significant increases in plant size, both in height and root length, highly desirable characteristics during the acclimatization phase. The use of MS + BP medium combined with TIS resulted in good plant development, leading to better performance in ex vitro cultivation.

This fact can be attributed to the accumulation of carbohydrates in the culture medium, resulting from the supplementation with banana pulp. These carbohydrates serve as an energy source for the micropropagated seedlings, playing a fundamental role both in in vitro growth and development and in the acclimatization process (BADR et al., 2015). Banana pulp, a widely used organic additive in the in vitro propagation of orchids, acts as a rich source of potassium and a rooting stimulator. This is due to the presence of mineral salts and natural growth regulators, such as auxins, cytokinins, and gibberellins, which are essential for plant growth and development (DOLCE et al., 2020; MACHADO & ZAMARIAN, 2020; STEFANO et al., 2022).

Furthermore, the increase in plant growth rate during the acclimatization phase can be associated with the in vitro cultivation system employed. TIS facilitates and allows gas exchanges, leading to a decrease in relative humidity and an increase in transpiration and absorption of water and nutrients by the seedlings. This system also promoted the functionality of the stomata and reduces the accumulation of ethylene and CO₂, which favors plant growth in in vitro cultivation and provides subsequent hardening when transferred to the ex vitro phase (VENDRAME et al., 2023; NONGDAM et al., 2023).

Thus, during the multiplication phase using bioreactors, as stated by VENDRAME et al. (2023), root development is fundamental for the success of the establishment and survival of the seedlings. These authors reported a 93% survival rate when using TIS in the propagation of the species B. nodosa. Moreover, it is crucial to adapt the immersion and aeration parameters in TIS, as the frequency and duration of these processes directly influence in vitro multiplication and plant development according to the species (VENDRAME et al., 2023; NONGDAM et al., 2023). Thus, this fact highlighted the influence of genotype on in vitro responses, indicating the need to evaluate and adjust such parameters according to the specific characteristics of each species.

Therefore, the results of this study confirmed that the combination of TIS with the supplementation of banana pulp in the culture medium provided favorable conditions for the development of C. nobilior plants, both in vitro and ex vitro cultivation, as illustrated in figures 1 and 3.

CONCLUSION

This study is pioneering in the use of the temporary immersion bioreactor system for the micropropagation of the native orchid Cattleya nobilior Rchb.f. To promote in vitro growth and ex vitro establishment of this species, we recommend in vitro cultivation using the temporary immersion system with MS medium supplemented with 100 g L^-1 of apple banana pulp. This approach is recommended for both commercial and conservationist applications.

ACKNOWLEDGMENTS

The authors would like to thank the Universidade Federal Grande Dourados (UFGD) for the research incentive received, the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and the Fundação de Apoio ao Desenvolvimento da Educação, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) for the scholarship granted and financial support.

REFERENCES

• BADR, A. et al. Comprehensive analysis of in vitro to ex vitro transition of tissue cultured potato plantlets grown with or without sucrose using metabolic profiling technique. Plant cell, tissue and organ culture, v.122, p.491-508, 2015. Available from: <Available from: https://link.springer.com/article/10.1007/s11240-015-0786-3 > Accessed: Jan. 12, 2021. doi: 10.1007/s11240-015-0786-3.
  » https://doi.org/10.1007/s11240-015-0786-3.» https://link.springer.com/article/10.1007/s11240-015-0786-3
• CUI, H. Y. et al. Production of biomass and bioactive compounds in protocorm cultures of Dendrobium candidum Wall ex Lindl. using balloon type bubble bioreactors. Industrial Crops and Products (PCTOC), v.53, p.28-33, 2014. Available from: <Available from: https://www.sciencedirect.com/science/article/pii/S0926669013006754?via%3Dihub >. Accessed: Jan. 31, 2024. doi: 10.1016/j.indcrop.2013.11.049.
  » https://doi.org/10.1016/j.indcrop.2013.11.049.» https://www.sciencedirect.com/science/article/pii/S0926669013006754?via%3Dihub
• DOLCE, N. R. et al. In vitro propagation and germplasm conservation of wild orchids from South America. Orchid Biology: Recent Trends & Challenges, p.37-94, 2020. Available from: <Available from: https://link.springer.com/chapter/10.1007/978-981-32-9456-1_4 >. Accessed: Feb. 20, 2021. doi: 10.1007/978-981-32-9456-14.
  » https://doi.org/10.1007/978-981-32-9456-14.» https://link.springer.com/chapter/10.1007/978-981-32-9456-1_4
• ESYANTI, R. R. et al. Efficiency evaluation of Vanda tricolor growth in temporary immerse system bioreactor and thin layer culture system. Journal Advanced Agricultural Technologies, v.3, p.63-66, 2016. Available from: <Available from: https://www.joaat.com/uploadfile/2016/0224/20160224113358719.pdf >. Accessed: Jan. 31, 2024. doi: 10.18178/joaat.3.1.63-66.
  » https://doi.org/10.18178/joaat.3.1.63-66.» https://www.joaat.com/uploadfile/2016/0224/20160224113358719.pdf
• EKMEKÇIGIL, M. et al. High-frequency protocorm-like bodies and shoot regeneration through a combination of thin cell layer and RITA^® temporary immersion bioreactor in Cattleya forbesii Lindl. Plant Cell, Tissue and Organ Culture (PCTOC), v.136, p.451-464, 2019. Available from: <Available from: https://link.springer.com/article/10.1007/s11240-018-1526-2 >. Accessed: Jan. 31, 2024. doi: 10.1007/s11240-018-1526-2.
  » https://doi.org/10.1007/s11240-018-1526-2.» https://link.springer.com/article/10.1007/s11240-018-1526-2
• FLORA E FUNGA DO BRASIL. Jardim Botânico do Rio de Janeiro. Available from: <Available from: http://floradobrasil.jbrj.gov.br/ >. Accessed: Jan. 31, 2024.
  » http://floradobrasil.jbrj.gov.br/
• FRITSCHE, Y. et al. Cattleya tigrina (Orchidaceae) in vitro regeneration: Main factors for optimal protocorm-like body induction and multiplication, plantlet regeneration, and cytogenetic stability. South African Journal of Botany, v.149, p.96-108, 2022. Available from: <Available from: https://www.sciencedirect.com/science/article/abs/pii/S0254629922000667 >. Accessed: Feb. 02, 2024. doi: 10.1016/j.sajb.2022.02.019.
  » https://doi.org/10.1016/j.sajb.2022.02.019.» https://www.sciencedirect.com/science/article/abs/pii/S0254629922000667
• GAO, R. et al. Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems. Acta Physiologica Plantarum, v.36, p.117-124, 2014. Available from: <Available from: https://link.springer.com/article/10.1007/s11738-013-1392-9 >. Accessed: Feb. 02, 2024. doi: 10.1007/s11738-013-1392-9.
  » https://doi.org/10.1007/s11738-013-1392-9.» https://link.springer.com/article/10.1007/s11738-013-1392-9
• JUNQUEIRA, A. H.; PEETZ, M. S. Brazilian consumption of flowers and ornamental plants: habits, practices and trends. Ornamental Horticulture, v.23, p.178-184, 2017. Available from: <Available from: https://ornamentalhorticulture.emnuvens.com.br/rbho/article/view/1070/719 >. Accessed: Dec. 19, 2020. doi: 10.14295/oh.v23i2.1070.
  » https://doi.org/10.14295/oh.v23i2.1070.» https://ornamentalhorticulture.emnuvens.com.br/rbho/article/view/1070/719
• MACHADO, W.; ZAMARIAN, A. S. Polpa de banana, água de coco e carvão ativado no desenvolvimento in vitro de Lycaste sp. Ensaios e Ciência C Biológicas Agrárias e da Saúde, v.24, p.159-163, 2020. Available from: <Available from: https://ensaioseciencia.pgsskroton.com.br/article/view/8674 >. Accessed: Jan. 05, 2020. doi: 10.17921/1415-6938.2020v24n2p159-163.
  » https://doi.org/10.17921/1415-6938.2020v24n2p159-163.» https://ensaioseciencia.pgsskroton.com.br/article/view/8674
• MASNODDIN, M. et al. PLB regeneration of Paphiopedilum rothschildianum using callus and liquid culture system. Journal of Tropical Biology and Conservation, v.15, p.1-14, 2018. Available from: <Available from: https://jurcon.ums.edu.my/ojums/index.php/jtbc/article/view/1469 >. Accessed: Jan. 31, 2024. doi: 10.51200/jtbc.v15i.1469.
  » https://doi.org/10.51200/jtbc.v15i.1469.» https://jurcon.ums.edu.my/ojums/index.php/jtbc/article/view/1469
• MASNODDIN, M. et al. Micropropagation of an endangered borneo orchid, Paphiopedilum rothschildianum callus using temporary immersion bioreactor system. Thai Journal Agricultural Science, v.34, p.161-171, 2016. Available from: <Available from: https://li01.tci-thaijo.org/index.php/thaiagriculturalresearch/article/view/78448 >. Accessed: Dec. 10, 2023. doi: 10.14456/thaidoa-agres.2016.12.
  » https://doi.org/10.14456/thaidoa-agres.2016.12.» https://li01.tci-thaijo.org/index.php/thaiagriculturalresearch/article/view/78448
• MINH, T. V. Micropropagation of Mokara orchid by temporary immersion system technique. International Journal of Research and Innovation in Applied Science, v.7, p.54-58, 2022. Available from: <Available from: https://rsisinternational.org/journals/ijrias/DigitalLibrary/volume-7-issue-5/54-58.pdf >. Accessed: Jan. 31, 2024.
  » https://rsisinternational.org/journals/ijrias/DigitalLibrary/volume-7-issue-5/54-58.pdf
• MIRANDA, N. A. et al. Qualidade e intensidade de luz no desenvolvimento in vitro de microcepas de Eucalyptus urophylla em um sistema fotoautotrofico. Ciência Florestal, v.66, p.754-760. 2020. Available from: <Available from: https://academic.oup.com/forestscience/article/66/6/754/5892960 >. Accessed: Dec. 10, 2023. doi: 10.1093/forsci/fxaa027.
  » https://doi.org/10.1093/forsci/fxaa027.» https://academic.oup.com/forestscience/article/66/6/754/5892960
• MOREIRA, A. L. et al. Cattleya walkeriana growth in different micropropagation systems. Ciência Rural, v.43, p.1804-1810.2013. Available from: <Available from: https://www.scielo.br/j/cr/a/N6WZmcZqhH44rDtjKxHDF5D/?lang=en >. Accessed: Jan. 31, 2024. doi: 10.1590/S0103-84782013001000012.
  » https://doi.org/10.1590/S0103-84782013001000012.» https://www.scielo.br/j/cr/a/N6WZmcZqhH44rDtjKxHDF5D/?lang=en
• NONGDAM, P. et al. Orchid micropropagation using conventional semi-solid and temporary immersion systems: a review. Plants, v.12, p.1136.2023. Available from: <Available from: https://www.mdpi.com/2223-7747/12/5/1136 >. Accessed: Dec. 13, 2023. doi: 10.3390/plants12051136. PMid:36904000.
  » https://doi.org/10.3390/plants12051136. PMid:36904000.» https://www.mdpi.com/2223-7747/12/5/1136
• RAMÍREZ-MOSQUEDA, M. A. et al. Morphogenetic stability of variegated Vanilla planifolia Jacks. plants micropropagated in a temporary immersion system (TIB^®). Rendiconti Lincei Scienze Fisiche e Naturali, v.30, p.603-609, 2019. Available from: <Available from: https://link.springer.com/article/10.1007/s12210-019-00813-9 >. Accessed: Jan. 16, 2024. doi: 10.1007/s12210-019-00813-9.
  » https://doi.org/10.1007/s12210-019-00813-9.» https://link.springer.com/article/10.1007/s12210-019-00813-9
• RAMÍREZ-MOSQUEDA, M. A; BELLO-BELLO, J. J. SETIS^™ bioreactor increases in vitro multiplication and shoot length in Vanilla (Vanilla planifolia Jacks. Ex Andrews). Acta Physiologiae Plantarum, v.43, p.52, 2021. Available from: <Available from: https://link.springer.com/article/10.1007/s11738-021-03227-z >. Accessed: Jan. 16, 2024. doi: 10.1007/s11738-021-03227-z.
  » https://doi.org/10.1007/s11738-021-03227-z.» https://link.springer.com/article/10.1007/s11738-021-03227-z
• RIBEIRO, L. M. et al. Methodology of the tetrazolium test for identifying viable seeds of orchids of the genus Cattleya native to the Brazilian Cerrado. Semina: Ciências Agrárias, v.42, p.1351-1360, 2021. Available from: <Available from: https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/40227/29177 >. Accessed: Jul. 14, 2022. doi: 10.5433/1679-0359.2021v42n3p1351.
  » https://doi.org/10.5433/1679-0359.2021v42n3p1351.» https://ojs.uel.br/revistas/uel/index.php/semagrarias/article/view/40227/29177
• RIBEIRO, L. M. et al. Influência da luz, ventilação natural e tamanho do frasco no crescimento e desenvolvimento de Denphal (Orchidaceae). Revista Brasileira deCiências Agrárias, v.14, 2019. Availablefrom: <Availablefrom: http://www.agraria.pro.br/ojs32/index.php/RBCA/article/view/v14i3a5957/185 >. Accessed: Jan. 11, 2021. doi: 10.5039/agraria.v14i3a5957.
  » https://doi.org/10.5039/agraria.v14i3a5957.» http://www.agraria.pro.br/ojs32/index.php/RBCA/article/view/v14i3a5957/185
• ROMAHN, V. Enciclopédia das Orquídeas. São Paulo: Europa, 2020. 46p.
• SHEN, R S.; HSU, S. T. Virus elimination through meristem culture and rapid clonal propagation using a temporary immersion system. In Orchid Propagation: From Laboratories to Greenhouses-Methods and Protocols; Springer Protocols, 2018.
• SOARES, J. S. et al. Brassavola tuberculata Hook.: in vitro growth and ex vitro establishment as a function of the micropropagation system and sucrose. Brazilian Journal of Biology, v.83, 2023. Available from: <Available from: https://www.scielo.br/j/bjb/a/mPBHJ5PFVWyGx8NWFymfW7R/?lang=en >. Accessed: Jun. 11, 2023. doi: 10.1590/1519-6984.270892.
  » https://doi.org/10.1590/1519-6984.270892.» https://www.scielo.br/j/bjb/a/mPBHJ5PFVWyGx8NWFymfW7R/?lang=en
• SOARES, J. S. et al. Protocolo para germinação assimbiótica e desenvolvimento inicial de protocormos de orquídeas nativas do Cerrado brasileiro. Rodriguésia, v.71, 2020. Available from: <Available from: https://www.scielo.br/j/rod/a/fg6LFdpqGYSGTwSkxbmXyBB/?format=pdf⟨=pt >. Accessed: Feb. 28, 2021. doi: 10.1590/2175-7860202071095.
  » https://doi.org/10.1590/2175-7860202071095.» https://www.scielo.br/j/rod/a/fg6LFdpqGYSGTwSkxbmXyBB/?format=pdf⟨=pt
• SPINOSO-CASTILLO, J. L. et al. Antimicrobial and hormetic effects of silver nanoparticles on in vitro regeneration of Vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system. Plant Cell Tissue Organ Culture, v.129, p.195-207, 2017. Available from: <Available from: https://link.springer.com/article/10.1007/s11240-017-1169-8 >. Accessed: Apr. 18, 2021. doi: 10.1007/s11240-017-1169-8.
  » https://doi.org/10.1007/s11240-017-1169-8.» https://link.springer.com/article/10.1007/s11240-017-1169-8
• STEFANO, D. et al. In vitro micropropagation and acclimatization of an endangered native orchid using organic supplements. American Journal of Plant Sciences, v.13, p.380-393, 2022. Available from: <Available from: https://www.scirp.org/journal/ajps >. Accessed: Aug. 25. 2023. doi: 10.4236/ajps.2022.133023.
  » https://doi.org/10.4236/ajps.2022.133023.» https://www.scirp.org/journal/ajps
• SU, M. J. et al. Polpa de banana e fertilizantes comerciais no cultivo in vitro de orquídea. Científica, v.40, p.28-34. 2012. Available from: <Available from: http://cientifica.org.br/index.php/cientifica/article/view/345/pdf >. Accessed: Nov. 28, 2021.
  » http://cientifica.org.br/index.php/cientifica/article/view/345/pdf
• UTAMI, E. S.; HARIYANTO, S. Organic compounds: Contents and their role in improving seed germination and protocorm development in orchids. International Journal of Agronomy, v.2020, p.12, 2020. Available from: <Available from: https://www.hindawi.com/journals/ija/2020/2795108/ >. Accessed: Jun. 10, 2021. doi: 10.1155/2020/2795108.
  » https://doi.org/10.1155/2020/2795108.» https://www.hindawi.com/journals/ija/2020/2795108/
• VENDRAME, W. A. et al. Micropropagação de Brassavola nodosa (L.) Lindl. usando biorreator SETIS^™ Plant Cell, Tissue and Organ Culture (PCTOC), v.153, p.67-76, 2023. Available from: <Available from: https://link.springer.com/article/10.1007/s11240-022-02441-y >. Accessed: Dec. 14, 2023. doi: 10.1007/s11240-022-02441-y.
  » https://doi.org/10.1007/s11240-022-02441-y.» https://link.springer.com/article/10.1007/s11240-022-02441-y
• ZHANG, B. et al. Micropropagation of Dendrobium nobile Lindl. plantlets by temporary immersion bioreactor. Journal of Biobased Materials and Bioenergy, v.13, p.395-400, 2019. Available from: <Available from: https://www.ingentaconnect.com/content/asp/jbmb/2019/00000013/00000003/art00013 >. Accessed: Oct. 22, 2021. doi: 10.1166/jbmb.2019.1852.
  » https://doi.org/10.1166/jbmb.2019.1852.» https://www.ingentaconnect.com/content/asp/jbmb/2019/00000013/00000003/art00013

• CR-2024-0271.R1

Edited by

• Editors: Leandro Souza da Silva (0000-0002-1636-6643) Ana da Silva Lédo (0000-0002-4353-4788)

Publication Dates

• Publication in this collection
  24 Mar 2025
• Date of issue
  2025

History

• Received
  15 May 2024
• Accepted
  25 Sept 2024
• Reviewed
  16 Dec 2024