In vitro and ex vitro production of Schomburgkia crispa: effect of flask sealing systems and different light sources

Abstract The extraction of native orchids from natural habitats is relevant for the reduction of populations in the Cerrado biome, making it necessary to establish practices aiming their production both for reintroduction and commercialization. The objective here is to evaluate light sources and sealing systems on the in vitro and ex vitro growth of Schomburgkia crispa. Two flask sealing systems were tested: conventional (CSS) and with gas exchange (SSGE), and eight light sources: FL1-100% white LED, FL2-100% blue LED, FL3-100% red LED, FL4-50% white + 25% red + 25% blue LED, FL5-50% red + 50% blue LED, FL6-25% red + 75% blue LED, FL7-75% red + 25% blue LED, and FL8- with fluorescent lamp, with five replications in each treatment. A completely randomized design was adopted with a 2x8 factorial scheme (vial sealing system x light sources). After 120 days of cultivation in vitro and 180 days ex vitro, the plants were evaluated as for number of leaves, roots and shoots, plant height, pseudobulb diameter, length of the largest root, largest leaf, and fresh mass. For the in vitro growth, the use of SSGE together with the light sources blue and red favors the cultivation of S. crispa. For the ex vitro growth, the cultivation in vitro in SSGE together with FL4 affects the acclimatization of plants.


Introduction
Orchidaceae contains a large number of genera and species.It is the second largest family among angiosperms.They are distributed in all continents, with a greater concentration and diversity in tropical and subtropical regions (Ferreira et al. 2022).In Brazil, there are about 2,677 species of orchids, of which 1,484 are endemic to it (Flora & Funga of Brazil 2022, continuously updated).These plants stand out in the flower and ornamental plant sector due to genetic combinations and exuberance of flowers, in addition to their appeal for food and pharmacological purposes (Teixeira da Silva et al. 2015;Belloto et al. 2017;De Stefano et al. 2022).
The Schomburgkia crispa Lindl. is a species of epiphytic habit found both in gallery forests and dry forests of the Cerrado, including the state of Mato Grosso do Sul (Ostetto 2015; Barros et al. 2018).Morphologically, it presents bifoliate pseudobulbs with a length of 8 to 10 cm, leaf length from 24 to 26 cm, and leaf width from 5 to 7 cm.The inflorescences are, on average, 95 to 110 cm long, with brown flowers, petals and sepals, yellow borders, and a lilac lip (Sorgato et al. 2021b).In January 2017, this species was included in the Appendix II of the CITES (Cites 2017), which includes species not necessarily threatened with extinction but whose trade must be controlled in order to avoid uses incompatible with its survival.
As an alternative to a rapid and efficient multiplication of orchids and aiming conservation and production on a commercial scale, in vitro cultivation techniques are used (Ribeiro et al. 2019;Sorgato et al. 2020;Soares et al. 2020;Castilho-Pérez et al. 2021;Kumar et al. 2022).Among the techniques, the in vitro germination of orchid seeds stands out since this type of sowing leads to high percentages of germination compared to germination under natural conditions, which occurs only in the presence of mycorrhizal fungi (Zhang et al. 2018;Soares et al. 2020;Kumar et al. 2022).In this sense, the production of orchids by that method is important, as it allows a large number of seedlings to grow in a small space, in a short time, and with a high sanitary quality (Teixeira da Silva et al. 2015Silva et al. , 2017a;;Ferreira et al. 2022;Pereira et al. 2022;Nowakowska et al. 2022).
Several factors affect the growth and development of the in vitro culture.Among them culture medium, flask sealing system, and light stand out.Culture media must meet the needs of plants in terms of mineral nutrition.Thus, there may be changes in formulations to meet the requirements of each species cultivated in vitro (Galdiano Júnior et al. 2013;Silva et al. 2015;Miler et al. 2019).
The conventional sealing system provides, inside in vitro cultivation flasks, a high relative humidity and low gas exchange.These factors may lead to anatomical and metabolic disorders in plants subjected to this system (Silva et al. 2016).Therefore, different strategies can be used for sealing, such as a natural ventilation of flasks (Silva et al. 2014(Silva et al. , 2016;;Ribeiro et al. 2019;Santos et al. 2020).
The light-emitting diode (LED) technology offers countless possibilities in horticultural lighting due to its ability to mix and separate different light spectra, allowing appropriate irradiance adjustments to the plant's photoreceptors, in addition to reducing energy consumption in growth rooms and causing less impacts on the environment (Singh et al. 2015;Loconsole et al. 2019).It is also possible to regulate parameters of plants cultivated in vitro: morphological and anatomical variations and physiological attributes, such as elongation, formation of axillary shoots, induction of somatic embryos, rhizogenesis, leaf anatomy, and photosynthetic abilities (Gupta & Jatothu 2013).
Cultivation protocols in vitro may also affect survival and establishment ex vitro.Thus, for the success of this cultivation, it is important to ensure the plant's adjustment to ex vitro conditions during acclimatization since these plants need to complete the autotrophism after transfer (Teixeira da Silva et al. 2017b;Santos et al. 2020).
However, there are still few scientific studies analyzing sealing systems and using LED lamps as a source of light energy in in vitro cultivation considering its influence on the acclimatization of native species, such as S. crispa.Thus, the objective here is to evaluate the effects of these two parameters in the in vitro and ex vitro growth of the orchid Schomburgkia crispa Lindl., native to the state of Mato Grosso do Sul, Brazil.

Experimental environment and biological material
The study material was ripe fruits of Schomburgkia crispa grown by hand pollination and matrices more than ten years old cultivated at the Orchid Garden of the Faculty of Agrarian Sciences (22°11'53.2"S;54°56'02.3"W).The nursery is covered by an overlap of two 50% shading screens, providing a shading of 10% and an irradiance of 235 µmol m -2 s -1 , under average temperature and relative humidity of 22.6 ± 5 ºC and 73.9 ± 10%, respectively.Irrigation was performed by ballerina-type microsprinklers positioned one meter above the plants, totaling a water depth of 1 mm day -1 .
To meet the objective proposed here, the plants were submitted to in vitro cultivation in the LabCFPO and ex vitro cultivations in a nursery.

Cultivation in vitro -120 days
A sample of 0.005 g of seeds was collected, and the tetrazolium test was performed according to the methodology of Soares et al. (2014).After confirming viability, another sample of 0.005 g of seeds was collected in an aseptic environment and disinfected according to the methodology of Soares et al. (2020) to obtain the seed solution.For in vitro sowing, 1.0 mL of the disinfested seed suspension was inoculated per culture flask.60 ml of Murashige & Skoog (1962) culture medium were used at half the salt concentration (MS 1/2) per flask, which has a capacity of 600 ml.Then, the cultures were placed in a growth room with controlled temperature and photoperiod (25±2 °C; 16 h) and irradiance of 22 µmol m -2 s -1 provided by two white fluorescent lamps (6500K), remaining under these conditions for up to 120 days.Three subcultures were conducted.
After this period, the seedlings were standardized in terms of size (1.5 cm) and subcultured for the beginning of the experimental period.The MS culture medium solidified with 7.0 g L -1 bacteriological agar (Himedia ® , India) and topped up with 30 g L -1 sucrose.The pH of the medium was measured and adjusted to 5.8 using KOH (0.1M) before sterilization in autoclave (121 °C and 1.1 atm pressure) for 20 minutes.60 mL of the medium were distributed into flasks with a capacity of 600 mL.There were four seedlings per culture flask inoculated in an aseptic environment.Subsequently, half the flasks was hermetically sealed with polyvinyl chloride (PVC) film (conventional sealing system -CSS) and the other half with PVC with cotton filter (sealing system allowing gas exchange -SSGE).
Spectral distribution measurements were taken with an Ocean Optics portable spectrometer (Model MMO with fiber optics) at room temperature with an integration time of 10 ms.
After 120 days of cultivation, the flasks were removed from the growth room and opened.The seedlings were removed and washed under running water until the culture medium was completely removed.The following factors were evaluated with a digital caliper and a precision scale: survival (%SUR), number of leaves (NL), number of roots (NR), plant height (PHe) (mm), length of the largest leaf (LL) (mm), length of the largest root (LR) (mm), and fresh mass (TFM) (g).After the evaluations, the seedlings were photographed with a camera attached to a mini photographic studio.

Cultivation ex vitro -180 days
For the evaluation of ex vitro growth, the plants were transferred to disposable transparent polypropylene containers with a capacity of 1,000 mL (20 × 10 × 5 cm).There were holes in the lid for gas exchange and holes in the base for substrate drainage, with 1/3 of its volume filled with pink sphagnum (Agrolink, Holambra, SP) plus coconut fiber (Golden-Mix Chips, Amafibra) (1:1, v:v -1 ).After transplantation, they were placed in a screened nursery and remained there for 180 days under the same conditions as those used for mother plants.In the first 15 days, the containers remained with the lids closed in order to minimize stress caused by the change in environment (in vitro to ex vitro).After this period, the lids were opened.Irrigation during the experimental period was performed by ballerina-type microsprinklers positioned one meter above the plants, totaling a water depth of 1 mm day -1 .
After this, the plants were removed from the containers and washed under running water until the substrate was completely removed.Then, plants were evaluated as for the same initial characteristics (NL, PHe, NR, LR, LL, TFM, and %SUR).
In order to investigate the hypothesis of increased plant growth during the ex vitro phase according to the treatment to which plants were initially exposed, the increases in values (I) were calculated in relation to the initial values using the expression I = (FV -IV), where IV is the value of the characteristic before the plant is acclimatized and FV is the value of the same variable after the ex vitro period.The values were expressed in percentage and submitted to analysis of variance.

Experimental design and statistical analysis
The experimental design was completely randomized with a 2x8 factorial scheme (two sealing systems and eight light conditions).There were five replications.Each experimental unit consisted of a flask containing four seedlings.The data of the sealing system were submitted to analysis of variance, and the means were compared by F test.Light condition data were analyzed by Scott-Knott test at 5% probability using the SISVAR statistical program (Program of Statistical Analysis, v. 5.3, Federal University of Lavras, MG) (Ferreira 2011).

In vitro growth -120 days
There was an isolated effect of light sources and sealing system on the number of leaves (NL) and number of roots (NR).There was also a significant effect of the interaction between light sources and sealing systems as for the characteristics plant height (PHe), length of the largest leaf (LL), length of the largest root (LR), total fresh mass (TFM), and percentage of survival (%SUR) after four months of in vitro cultivation (Fig. 2).
We noted that, for the isolated effect of light sources on the variable NL, plants of S. crispa, when cultivated in FL7, had a higher number of leaves (18.67) but no significant difference between FL8 and FL6, with means of 16.17 and 14.46 leaves, respectively.As for the sealing system, the plants, when cultivated under the CSS, had the highest NL: 14.16 leaves.The highest NR (8.71) occurred when plants were submitted to 100% white LED lamp, with a mean of 8.71  roots but no significant difference between FL8 and FL7: 7.88 and 8.33, respectively (Fig. 2a).The NR was higher in plants grown in a conventional sealing system, with a mean of 7.64 roots (Fig. 2b).
In this work, S. crispa showed an increase both in number of leaves and number of roots when grown under lamps that contain a red wavelength in a light composition of up to 75%.Hung et al. (2016) reported that the wavelength of the red light promotes leaf growth, anatomical changes, and carbohydrate accumulation in plant material.However, other authors pointed out that each cultivated species reacts differently to the light conditions provided and that this directly affects plant growth and development (Taiz et al. 2017).
Both the highest NR and NL occurred when CSS was used since the conditions established by this system are related to plant tillering, which is affected by the greater number of shoots (Ribeiro et al. 2019).Freitas et al. (2021) observed a similar result for Cattleya nobilior Rchb.f.cultivated in vitro.It presented a greater number of leaves and a greater fresh mass in plants cultivated under conventional system due to their tillering setting.This system interferes with gas exchange since the hermetic sealing of flasks results in an increase in CO 2 and ethylene gas.This may cause physiological changes in plants, tissue growth, and induction and differentiation of plant organs (Silva et al. 2014;Teixeira da Silva et al. 2017a).
For the interaction between light sources and sealing systems, plants grown in SSGE and submitted to FL6 showed a higher PHe (54.37 mm) and no significant difference from FL7 (47.01 mm).The plants of S. crispa showed a higher LL (40.51 mm) when cultivated in FL6 with SSGE but no significant difference to FL5 and FL7 (32.54 and 35.85,respectively).Regarding the LR (57.14 mm), the highest means occurred when plants were cultivated under FL4 using CSS (Tab.1).
The highest FM values (0.999 g) occurred when plants were grown under FL1 using SSGE, although there was no significant difference to the FL6 treatment (0.939 g).Regarding the percentage of survival, all treatments showed satisfactory results, with 100% survival.However, the FL5 under SSGE resulted in 91.67% of live plants (Tab.1).
When analyzing the results of this study, in general the SSGE and the FL6 light source are beneficial for the survival and in vitro growth of S. crispa.
This may be related to the use of SSGE because this system allows gas exchange between the internal and external environments to the flask, which results in a decrease in the accumulation of ethylene gas and CO 2 , thus contributing to plant growth during in vitro cultivation (Silva et al. 2016).
Furthermore, according to Silva et al. (2014), the conditions of gas exchange in the in vitro cultivation of plants allow an approximate cultivation of photoautotrophy, promoting changes in tissues and favoring the growth of plant organs.
As for light sources, the best results observed in the combinations of blue and red may be related to the ability of these wavelengths to better excite photoreceptors (phytochromes, phototropins, and cryptochromes), thus increasing photosynthetic activity (Dou et al. 2017).Furthermore, the greater proportion of blue to red wavelengths (3:1) absorbed by phototropin and cryptochromes may be associated with leaf expansion since phototropin is related to this function (Macedo et al. 2011;Cunha et al. 2019).Taiz et al. (2017) reported that the quality of light provided to plant material is considered important because it regulates plant morphogenesis and growth.However, white light is a mixture of low intensities of red and blue and other wavelengths of low-efficiency light (Cunha et al. 2019).This may be related to the higher values observed in white light treatments and in combinations of blue and red.
The results found in this study show that the light condition of the cultivation room for Orchidaceae species are, in addition to speciesspecific, also related to the stage of development of the plant.The results found in the literature differ according to the stage of seedlings used (Freitas et al. 2021;Sorgato et al. 2021a).
F i g u r e 3 s h o w s t h e v a r i a t i o n i n morphological aspects of plants as a function of sealing systems and irradiance.As can be visually assessed, plants grown in CSS appear to be smaller than those grown under SSGE.They showed a higher PHe, a higher LL, and a higher LR.Visually, it can be seen that plants grown in CSS show shoots with a higher number of leaves and roots compared to the growth of these vegetative structures under SSGE, which showed a greater growth, especially when plants were grown under the light sources FL2, FL4, FL6 and FL7.
Table 1 -Plant height (PHe mm), length of largest leaf (LL mm), length of largest root (LR mm), total fresh mass (TFM) (g), and percentage of survival (%SUR) of Schomburgkia crispa in function of sealing system (CSS = conventional sealing system; SSGE = sealing system with gas exchange) and different light sources after four months of in vitro cultivation.

Ex vitro growth -six months
For the 180 days of ex vitro cultivation of S. crispa, there was an interaction between light conditions and sealing systems previously used in in vitro cultivation (p < 0.05) for all characteristics evaluated.As for %SUR, the best results occurred when plants were previously cultivated under the SSGE system and the light sources FL1, FL3, FL4, FL5, FL6, and FL8, providing 100% of survival (Tab.2).
At the end of the experimental period ex vitro, the highest PHe values occurred when the SSGE was previously used in conjunction with the light source FL4, leading to a 62.48% increase.For NL, higher increases occurred in SSGE under white LED (FL1) (6.17%), differing only from FL2 (0.17%) and FL7 (1.75%).Regarding the NR, the highest percentage of increase (6.50%) occurred when plants were previously submitted to CSS + FL2 (Tab.2).
Table 2 -Increases (%) in plant height (PHe, mm), number of leaves (NL), number of roots (NR), length of largest leaf (LL mm), length of largest root (LR mm), total fresh mass (TFM), and percent survival (%SUR) of Schomburgkia crispa in function of sealing system (CSS = conventional sealing system; SSGE = sealing system with gas exchange) and different light sources after six months of ex vitro cultivation.
The results for the ex vitro growth and establishment of S. crispa, in general, showed that when the SSGE and the light source FL4 were previously used, all characteristics obtained the best performance.
Such growth characteristics may be related to a greater aeration that SSGE allows.These results corroborate those of Silva et al. (2014) and those of Ribeiro et al. (2019).Plants of Cattleya walkeriana Gardner and Denphal, respectively, when cultivated with lids that allowed gas exchange, showed, when transferred to an ex vitro environment, growth attributes superior to those of plants cultivated in a sealed environment.
Furthermore, the results of this work suggest that the use of SSGE together with a mixture of white + blue + red LED light may have provided conditions that favored the ex vitro growth of S. crispa.These treatments may have affected plant physiology, so that it started its hardening while still in an in vitro cultivation, resulting in a better performance in ex vitro cultivation.Silva et al. (2016) explained that the benefits of cultivation in SSGE result from the decreases in in vitro humidity and from increased aeration, providing a later hardening in plants when transferred to ex vitro conditions.Lazzarini et al. (2017) also report that, in plants, the absorption of blue and red lights emitted by LED lamps is around 90% of the light emitted.This indicates that the development of plants and plant physiology are strongly affected by these wavelengths.Thus, both red and blue lights may be effective in inducing photomorphogenic responses, as occurred with S. crispa plants.
In addition, plants need a broad spectrum of light to optimize photosynthetic processes.Such need varies according to plant species.For S. crispa, the highest results show that, in addition to the use of blue and red LEDs, the addition of a white LED contributed to the ex vitro growth of plants.The white LED (460-560 nm) has a higher proportion of blue and green in its spectrum and a lower ratio of red and extreme red compared to fluorescent lamps (Fraszczak et al. 2014).The use of this LED in crops may increase plant growth, as it allows light to penetrate the leaves better than monochromatic blue and red lights do, as observed for hydroponic Lactuca sativa (Lin et al. 2013).
Research with LED shows that plants need a broad spectrum of light to optimize photosynthetic processes.Such need may vary according to the species cultivated.Based on the results obtained for S. crispa, the combination of blue and red wavelengths is beneficial for an in vitro cultivation.However, for the ex vitro growth and establishment of these plants, it is necessary to cultivate them in white LED associated with red and blue LEDs.Therefore, further studies on ornamental species and the use of LEDs are needed since the use of this technology promotes benefits to several physiological aspects of cultivated plants.
This is even more relevant in studies on native species.The recent technologies used for the production of flowers and ornamental plants require the improvement of techniques and better cultivation conditions in vitro, as they are essential biotechnological tools in the production and propagation of plants.Ornamental horticulture seeks to obtain better quality seedlings and large-scale production in less time aiming both commercialization and species conservation.
Figure 4 shows that the in vitro cultivation conditions limited plant growth and survival in the ex vitro period.In general, the SSGE showed the highest results in all parameters visually observed, mainly under the light sources FL3, FL4, FL5 and FL6.This allows inferring that this sealing system, associated with light sources that use blue and red wavelength, may be appropriate for the in vitro cultivation of this species.
For in vitro growth, the use of a sealing system allowing gas exchange together with light sources combining blue and red wavelengths at different proportions favors the cultivation of Schomburgkia crispa.

Figure 1 -
Figure 1 -Spectral energy distribution of LEDs and of the fluorescent lamp.