Micropropagation of Brassavola nodosa (L.) Lindl. using SETIS™ bioreactor

Brassavola nodosa (L.) Lindl. is a tropical epiphytic orchid showing characteristics of interest for the ornamental nursery industry, as well as considered as an endangered species. Problems with traditional propagation methods limit the development of a large-scale commercial production system. The use of micropropagation has been investigated for this species, and the use of liquid in vitro systems showed potential for use of temporary immersion systems, also called temporary immersion bioreactors (TIB) for micropropagation of B. nodosa. This study evaluated the efficiency of the SETIS™ TIBs for the micropropagation of B. nodosa. Explants consisting of young shoots tips measuring between 0.3 and 0.5 cm were collected from 1-yr-old in vitro seedlings of B. nodosa and used for shoot multiplication. Four culture systems were evaluated: a semi-solid in vitro system consisting of 160 mm × 100 mm TP1600 Microbox containing 500 ml semi-solid medium as a control, and a liquid in vitro system consisting of 4 L temporary immersion bioreactor (SETIS™, VERVIT, Belgium) containing 500 ml liquid medium with immersion and aeration frequencies of 2 h, 4 h, and 8 h. For all bioreactor treatments, duration of immersion and aeration was 2 min. Results showed that temporary immersion of B. nodosa explants with a frequency of 2 h and duration of 2 min returned the highest multiplication rates, with 4.6 shoots produced per explant compared with 2.8 shoots per explant in semi-solid agar-based systems. The use of bioreactors also promoted increased growth and development and in vitro rooting, therefore improving survival, and facilitating acclimatization of in vitro-derived plantlets. This is the first study demonstrating a successful protocol for micropropagation of B. nodosa using SETIS™ bioreactors, which could have significant value and impact for the commercial production of this species as well as for conservation purposes. Micropropagation of Brassavola nodosa was achieved using SETIS temporary immersion bioreactors, showing good multiplication rates, increased growth and development and in vitro rooting, thus improving survival and acclimatization ex vitro.


Introduction
The expansion of the ornamental market and the increased demand for flowering plants associated with lower production costs, advanced production techniques, and an increased interest by growers and customers in new and improved orchid hybrids have placed orchids among the top flowering plants in the USA (Vendrame and Khoddamzadeh 2017). Brassavola nodosa (L.) Lindl. is a tropical epiphytic orchid distributed from Mexico through Central and South America (Mata-Rosas and Lastre-Puertos 2015) and produces very fragrant inflorescences rendering its common name as the "Lady of the Night". This species flowers throughout the year, has a unique flower shape, and requires low maintenance, making it desirable for the potted flower ornamental industry. However, the propagation of orchids in the genus Brassavola is difficult and seed germination in nature is very low while propagation by branching or pseudobulbs is a lengthy process (Mengarda et al. 2017). These factors limit commercial large-scale production. Furthermore, habitat destruction, the impact of climate change, and over-collection from native habitats has caused a reduction in population and distribution of these orchids (Swarts and Dixon 2009;Reed et al. 2011). Propagation and conservation strategies are required to guarantee population survival in nature and to provide plant material for commercial production. Micropropagation provides a feasible alternative for rapid mass clonal propagation of true-to-type plants with commercial potential, and it has been responsible for the success of the orchid industry (Chugh et al. 2009;Kumar and Reddy 2011;Bhoite and Palshikar 2014;Phillips 2019). Micropropagation of several orchid species and hybrids has been reported for various genera, including Anoectochillus, Arundina, Cymbidium, Dendrobium, Doritaenopsis, Phalaenopsis, Paphiopedilum, Vanda, and Vanilla, among many others (Arditti and Ernst 1993;Tokuhara and Mii 1993;Devi et al. 1997;Tisserat and Jones 1999;Kalimuthu et al. 2006;Wu et al. 2007;Neumann et al. 2009;Paek et al. 2011;Panwar et al. 2012;Divakaran et al. 2015;Teixeira da Silva et al. 2015;Rodrigues et al. 2015;Zeng et al. 2014;Kumar and Asthana 2020;Nowakowska et al. 2022).
However, micropropagation of Brassavola orchids remains a challenge (Xu et al. 2022) due to the limited number of studies in the genus Brassavola, including in vitro germination of a Brassavola perrinii hybrid (Chiapim et al. 2012) and B. tuberculata (Soares et al. 2020), micropropagation of a Brassavola hybrid (Villa et al. 2014), and acclimatization of in vitro derived plantlets (Sousa et al. 2015) and in vitro shoot production (Mengarda et al. 2017) The use of liquid cultures could be a feasible approach to improve orchid micropropagation (Young et al. 2000;Wu et al. 2007;Murthy et al. 2018;Shekhawat et al. 2022;Xu et al. 2022). Liquid culture systems offer advantages over traditional semi-solid agar-based systems in micropropagation including improved nutrient uptake and therefore enhanced plant growth and development, higher rates of multiplication, the potential of reduction of contamination, and reduced costs by automation of liquid systems (Ziv 2000;Xu et al. 2022). In addition, liquid cultures improve uniformity of culture conditions, such as easier renewal of media, the opportunity to use of larger containers, and the need for fewer frequent subcultures (Etienne and Berthouly 2002). However, the use of liquid culture systems is recent and not yet popular for the propagation of orchids. More recently, micropropagation of B. nodosa using liquid culture with partial immersion has been investigated and showed positive results creating an opportunity for the use of temporary immersion bioreactors (Xu et al. 2022).
Temporary immersion systems (TIS) also called temporary immersion bioreactors (TIBs) are advanced techniques for micropropagation using liquid media in bioreactors and are based on periodic immersion of plant tissues into the liquid medium for a pre-determined period of time, with alternating periods of aeration. This allows increased nutrient uptake, thus making TIB an efficient system for largescale micropropagation of plants (Georgiev et al. 2014;Ramírez-Mosqueda and Bello-Bello 2021). Various TIB systems have been designed, including ebb-and-flow bioreactor (Tisserat and Vandercook 1985), RITA® (Teisson and Alvard 1995), and Twin Flask System (Escalona et al. 1999). More recently, a new bioreactor system has been developed (SETIS™, VERVIT, Belgium), which allows for increased light irradiation, automation of cultivation systems, easy handling, large culture medium capacity, and successful protocols have been reported for the micropropagation of banana and vanilla Ramírez-Mosqueda and Bello-Bello 2021).
The objective of this study was to evaluate the efficiency of the SETIS™ bioreactor for the micropropagation of B. nodosa as compared to conventional semi-solid culture systems. The development of a large-scale micropropagation system for B. nodosa may aid in its commercialization and contribute for the conservation of the species.

Plant material
Seeds originated from a Brassavola hybrid, Brassavola nodosa 'Remar' x 'Mas Mejor' were selected for this study. Seeds were surface sterilized with 70% ethanol (Decon Labs, Inc., King of Prussia, PA) for 1 min followed by immersion in 0.8% sodium hypochlorite (NaClO, Thermo Fisher Scientific, Hampton, NH) with 2 drops of Tween 20 (Fisher Scientific, Pittsburgh, PA) for 5 min. Subsequently, seeds were rinsed three times in sterile autoclaved water. Seeds germinated in vitro on ½ strength Murashige and Skoog (MS) medium (Murashige and Skoog 1962, Phytotechnology Laboratories, Lenexa, KS) containing 15 g L −1 sucrose (Thermo Fisher Scientific Hampton, NH) and 7 g L −1 agar (Thermo Fisher Scientific Hampton, NH). The medium pH was adjusted to 5.7 and the medium was autoclaved at 121 °C and 15 lbs pressure for 20 min. About 15 mL of medium was dispensed in 100 mm × 15 mm disposable Petri dishes (Thermo Fisher Scientific Hampton, NH). Sixty days after seed germination, when protocorms had developed 1 to 2 leaves and roots, seedlings were transferred to 120 mm × 75 mm RA40 Microboxes (Sac O2, Deinze, Belgium) containing about 100 mL MS medium supplemented with 30 g L −1 sucrose and 7 g L −1 agar.

Effect of culture system on shoot multiplication
Explants consisting of young shoots tips measuring between 0.3 and 0.5 cm were collected from 1-yr-old in vitro seedlings and used for shoot multiplication. Shoot tips were cultured on MS medium containing 2 mg L −1 glycine, 0.1 mg L −1 NAA (naphthalene acetic acid), 2.0 mg L −1 BA (benzyladenine), 30 mg L −1 adenine sulfate, 10% coconut water, and 30 g L −1 sucrose. The medium pH was adjusted to 5.2 and autoclaved at 121 °C and 15 lbs pressure for 20 min.
Four different culture systems were used for in vitro culture of shoot tips: 1) in 160 mm × 100 mm TP1600 Microbox (Sac O2, Deinze, Belgium) containing 500 ml semi-solid medium (7 g L −1 agar) as a control, 2) in 4 L temporary immersion bioreactor (SETIS™, VERVIT, Beervelde, Belgium) with an immersion and aeration frequency of 2 h, 3) in 4 L temporary immersion bioreactor with an immersion and aeration frequency of 4 h, and 4) in 4L temporary immersion bioreactors with an immersion and aeration frequency of 8 h ( Fig. 1), all containing 500 ml liquid medium. This provided about 25 ml media per explant for both systems. For all treatments duration of immersion and aeration was 2 min. The experiment was comprised of 3 replicates per treatment, each replicate containing 20 explants. The cultures were maintained under controlled environment conditions with a 12-h photoperiod at light intensity of 50 μmol m −2 s −1 and temperature of 26 °C. Shoot multiplication, shoot length, number of leaves per shoot, rooting percentage, root number, root length, fresh weight, dry weight, chlorophyll relative content, stomata number, and stomata size were evaluated 60 days after culture establishment. The entire experiment was repeated once.

Chlorophyll relative content analysis
Fully expanded leaves were selected from 5 plants per treatment for chlorophyll relative content analysis. Chlorophyll relative content was measured as SPAD value by placing the third leaf of each plantlet, counted from top downwards in a portable SPAD-502 chlorophyll meter (SPAD-502, Minolta Co., Ltd., Japan).

Stomatal analysis
Five plantlets were randomly selected from each treatment for stomata analyses. The middle of the third fully expanded leaf was sectioned. Leaf sections were prepared on glass slides on top of a thin layer of super glue previously applied. After the glue was completely dry, the leaves were covered by a piece of translucent tape and the cuticle bind to the tape carefully removed, leaving a leaf impression. The stomata on the leaf impressions were visualized under an optical Leica DMLB microscope (Leica microsystems, Buffalo, NY, USA), at ×100 magnification. The stomata number on the abaxial and adaxial epidermis were counted within a diameter of 5 mm under the microscope from three different fields on the leaf surface. Results were expressed as means of counts per mm 2 .
The length and width of stomata were measured by randomly choosing 3 stomata on the adaxial and abaxial surface of each leaf in three different locations. The slides were observed and photographed under a light Leica DMLB microscope (Leica microsystems, Buffalo, NY, USA), coupled to a SPOT 4.7 IDEA (SPOT Imaging, Sterling Heights, MI, USA) digital camera. The images and the size of stomata were analyzed using the microscope imaging software SPOT basic (SPOT Imaging, Sterling Heights, MI, USA).

Acclimatization
Eighteen shoots were selected from each culture system for acclimatization. Shoots were treated with rooting hormone powder (TakeRoot® Rooting Hormone, Bridgeton, MO) containing 1.0 mg L −1 IBA. and transferred to 40-cell plastic trays with soilless media containing orchid bark (Sequoia Bark Sales, Reedley, CA). Plants were maintained in a greenhouse with mist system running for 20 s every 30 min. Plants were fertilized weekly with 300 ppm of 11-35-15 (% N-P 2 O 5 -K 2 O) orchid fertilizer (Better-Gro® Orchid Better-Bloom®, Arcadia, FL). The ex-vitro plant survival, shoot length, number of leaves per shoot, rooting percentage, root number, root length, fresh weight, and dry weight were evaluated for all treatments after 60 days.

Statistical analysis
A completely randomized experimental design was used for all experiments. Data were collected and submitted to analysis of variance (ANOVA) using the OriginPro® 2021b software (OriginLab, Northampton, MA). Data for root percentage was normalized by the OriginPro® 2021b software prior to ANOVA using arcsine transformation. Tukey's post hoc multiple comparison adjustment (α = 0.05) was used for all pairwise comparisons of means.

Evaluation of different culture systems on shoot multiplication
Significant differences were observed among the different culture systems for in vitro shoot multiplication ( Fig. 2A). The highest number of shoots (4.6 shoots/explant) occurred in SETIS™ bioreactor under immersion and aeration frequency of 2 h, followed by immersion and aeration frequency of 4 h (3.5 shoots/explant) and 8 h (3.5 shoots/ explant). The control in semi-solid medium had a multiplication rate of 2.8 shoots/explant. For leaf number and shoot length, there were no significant differences among culture systems (Fig. 2B, C). Rooting percentage (73.3% to 80.0%) and root number (2.0 to 2.6 roots per explant) in bioreactors were significantly higher than the explants cultured in semi-solid, with 6.7% rooting percentage and 0.1 roots per explant (Fig. 2D, E). The longest root length was recorded in the SETIS™ bioreactor under 2 h immersion frequency (1.58 cm), followed by 4 h (1.01 cm) and 8 h (0.97 cm), but were not significantly different (Fig. 2F). However, the control in semi-solid medium showed a significantly reduced root length of 0.03 cm (Fig. 2F).
Chlorophyll relative content was higher in explants cultured in bioreactors with SPAD values of 39.4 under 2 h immersion frequency, 37.7 under 4 h immersion frequency, and 30.6 under 8 h immersion frequency, but they were not significantly different. However, the control in semi-solid medium showed a significantly lower SPAD value of 26.5 (Fig. 3A). The largest fresh weigh was observed in bioreactors under 2 h immersion frequency (0.121 g), followed by 4 h (0.083 g), 8 h (0.079 g), and the control in semisolid medium (0.060 g) (Fig. 3B). The largest dry weight was recorded in bioreactors under 2 h immersion frequency (0.012 g), followed by 4 h (0.009 g), 8 h (0.010 g), and the control in semi-solid medium (0.006 g) (Fig. 3C). There were no significant differences in fresh weight between immersion frequency of 8 h and the control. However, dry weight in bioreactors under 8 h immersion frequency was higher than the control (Fig. 3B, C).

Effects of culture systems on stomata formation
The assessment of the effect of different culture systems on stomata formation showed that explants cultured in bioreactors under 2 h immersion frequency presented the highest adaxial and abaxial (95.0 and 75.3, respectively) stomata number, followed by 58.7 and 84.3 under 4 h, 52.7 and 61.7 under 8 h, and 53.3 and 60.0 under control, respectively (Table 1). Significant longest adaxial stomata length, adaxial stomata width, and abaxial stomata length were observed for plants in bioreactors under 2 h immersion frequency compared to other treatments. Both 2 h and 4 h immersion frequency treatments resulted in largest size of abaxial stomata width (Table 1). Explants cultured under 2 h immersion frequency produced spherical, wideopen stomata distributed on both adaxial and abaxial leaf surfaces (Fig. 4). Under 4 h immersion frequency, stomata morphology was similar to those under 2 h immersion frequency.  Fig. 5). There were no significant differences in shoot length and leaf number among the different culture systems, except for the control (Table 2). New roots were developed in plantlets derived from bioreactors under 2 h and 4 h immersion frequency but were reduced under 8 h (Fig. 5). The control showed no root development and low survival, therefore preventing the collection of data for other parameters (Table 2, Fig. 5). No morphological variations were observed for plants grown from treatments under 2 h, 4 h and 8 h immersion and they showed normal phenotype (Fig. 5).

Discussion
Micropropagation of orchids using TIS bioreactors has been reported to result in higher multiplication rates, increased biomass production, and healthier plant growth development compared to conventional culture systems using semi-solid  also reported higher shoot multiplication (6 shoots per explant) for the orchid Guarianthe skinneri when using temporary immersion bioreactors although the system used was different from the SETIS™ system. Specifically for the genus Brassavola, Mengarda et al. (2017), reported a 90-day period for the induction of 4 shoots per explant for Brassavola tuberculate micropropagated under semi-solid, agar-based medium, contrasting with our study whereby shoots were produced in a shorter period of time (60 d). These results demonstrate the efficiency of micropropagation of orchid species in the genus Brassavola using TIS bioreactors. Shoot multiplication can be achieved in a shorter period, which can also result in higher multiplication rates. In addition, studies on the optimization of culture conditions, including medium composition and adjustment of immersion parameters (frequency and duration) in bioreactors may improve multiplication rates. One interesting observation was that the explants in bioreactors generated roots during the multiplication stage, without the need to transfer shoots to a rooting medium. This represents time and cost savings during in vitro multiplication and is a factor that facilitated acclimatization of micropropagated plantlets in the greenhouse. A similar result was reported by Leyva-Ovalle et al. (2020) showing the presence of roots during the micropropagation of the orchid Guarianthe skinneri in TIS bioreactors. In contrast, B. nodosa explants cultured in semi-solid medium produced a reduced number of roots, possibly because of cytokinins in the medium, resulting in lower survival during acclimatization. Higher cytokinin has been reported to reduce auxin biosynthesis and inhibit root growth and development (Su et al. 2011). In addition, explants in semi-solid medium may require frequent subculturing to improve multiplication and transfer to a different medium composition to promote rooting (Leyva-Ovalle et al. 2020). The need for increased subculturing to promote rooting in semi-solid systems increases both labor and cost in the micropropagation process, therefore making the TIS bioreactor system more efficient and cost-effective.
Immersion and aeration frequency and duration in TIS bioreactors are factors that require adjustments for each species, and they play an important role in plant in vitro multiplication and plant development (Georgiev et al. 2014). In this study, we evaluated different immersion/aeration frequencies to determine the best parameters for highest shoot in vitro multiplication of B. nodosa. Results reported in the literature for other orchid species showed The optimization of micropropagation protocols using bioteactors by adjusting immersion/aeration frequency and duration parameters has been evaluated for different orchid species, such as for 3 h/10 min for Phalaenopsis (Hempfling and Preil 2005), 4 h/2 min for Vanilla planifolia (Ramírez-Mosqueda and Bello-Bello 2021), 6 h/3 min for Bletilla striata (Zhang et al. 2018), and 4 h/2 min for Guarianthe skinneri (Leyva-Ovalle et al. 2020). Similarly, our study shows that responses in vitro of B. nodosa are improved under high immersion and aeration frequencies and duration, such as 2 h/2 min. This demonstrates the effect of genotype in responses in vitro, and the need to evaluate and adjust immersion and aeration parameters accordingly for each species.
In our study, chlorophyll relative content was significantly higher for plantlets produced in TIS bioreactors as compared to those from semi-solid medium. One important factor in physiological processes that affects the synthesis of chlorophyll is irradiance (Mancilla-Álvarez et al. 2021). The increase in chlorophyll relative content in our study could be related to increased irradiance provided to explants by the SETIS™ bioreactor vessels as compared to the limited irradiance available in semi-solid system under microboxes. The increased aeration and air-exchange within the culture vessels can also improve photosynthetic activity by reducing ethylene concentration (Roels et al. 2006), recirculating CO 2 needed for photosynthesis (Aragón et al. 2010(Aragón et al. , 2014 and increasing leaf stomatal functionality compared to semisolid culture systems (Afreen 2008). These factors could have contributed to an increase in chlorophyll relative content in our study, and have also been reported when using temporary immersion for the micropropagation of Anthurium andreanum (Martínez-Estrada et al. 2019).
Stomata number, size, and functionality are affected by transpiration and availability of water (Larraburu et al. 2016). In our study, the higher stomata number and size, and number of open stomata in TIS bioreactors could be due to the higher relative humidity (RH) provided resulting in higher transpiration rates. The oppositive effect was observed under semi-solid medium where the filters in the lids of microboxes promoted air exchange, but resulted in reduced relative humidity thus affecting stomata functionality. The improved survival of plantlets during acclimatization is related to closed stomata and lower stomata numbers, which helps prevent dehydration (Ramirez-Mosqueda et al. 2019). However, in our study, plantlets that showed open stomata achieved highest survival during acclimatization. According to Joshi et al. (2006), although closed stomata are preferred for acclimatization of micropropagated plantlets ex vitro, high survival could still be obtained by gradually decreasing air humidity. This could explain the results reported for this study, as gradual reduction in relative humidity was promoted during acclimatization.
The high survival (93%) observed for micropropagated plantlets derived from TIS bioreactors under 2 h immersion frequency as compared to other treatments indicates that these parameters could be successfully applied for micropropagation of B. nodosa orchids using the SETIS bioreactor system. In contrast, lower survival of plantlets derived from semi-solid medium indicated the need for additional subculturing to promote root formation and therefore improve survival. Root formation during multiplication stages in bioreactors also contributed to the successful acclimatization and survival of plantlets and demonstrates the efficiency of temporary immersion bioreactors for the micropropagation of B. nodosa.

Conclusions
This is the first study reporting the use of the SETIS™ temporary immersion bioreactor system for the micropropagation of B. nodosa orchids. We determined that B. nodosa plantlets micropropagated under TIS bioreactors had higher multiplication rates compared to conventional culture systems using semi-solid agar-based medium. Root formation during the multiplication phase improved the efficiency of micropropagation, thus reducing labor and cost. Our study demonstrated a successful protocol for large-scale micropropagation of B. nodosa using bioreactors, which could have significant value and impact for the commercial production of this species as well as for conservation purposes. The evaluation and adjustment of immersion parameters is warranted for future studies to improve shoot multiplication.