Light sources on the germination and initial in vitro establishment of Schomburgkia crispa Lindl., a species of the Brazilian Cerrado. Light sources on the germination and initial in vitro establishment

: Light is one of the factors that influence the germination and initial establishment of orchids under in vitro cultivation. This study evaluated the effect of different light sources on these stages in in vitro cultivation of Schomburgkia crispa Lindl. After sowing in an aseptic environment, we stored the cultures in a screened greenhouse (natural light) or in a growth room with the following light sources: 3,000 K yellow LED; 6,500 K white LED [1]; 6,500 K white LED [2]; or 6,500 K white fluorescent lamp (control). We assessed germination percentage and initial seedling establishment at 45 and 90 days after sowing. Light did not influence the germination of S. crispa. However, the use of 3,000 K LED provided a faster initial establishment of S. crispa when compared to the other light sources, also presenting lower seedling mortality. Thus, the light source 3,000 K LED is a potential substitute for the 6,500 K fluorescent lamps and LEDs used in growth rooms in in vitro culture laboratories.


INTRODUCTION
Schomburgkia crispa Lindl. is a species of the family Orchidaceae. It is an epiphyte that lives in gallery and dry forests of the Cerrado biome in Brazil (MENDONÇA et al., 2008;BARROS et al., 2018). In addition to their ornamental potential, Brazilian native orchids may also have some bioactive compounds with therapeutic applications. By means of phytochemical study in this species, the crispoic acid was isolated, which according to BELLOTO et al. (2017) has anticarcinogenic potential.
In vitro sowing of orchid seeds is a key tool in studies on biodiversity conservation because it maintains genetic variability, yielding a high germination percentage without symbiotic relationships (FAY, 2018). Orchid growth in natural environments is slow, which extends seedling production until the plants reach the reproductive stage. Using this type of cultivation is thus important to enable high-quality, large-scale production in a relatively short time, which contributes to reducing the risk of extinction (CARDOSO, 2014;HANUS-FAJERSKA & WOJCIECHOWSKA, 2017;FAY, 2018). Sorgato et al. Several abiotic factors influence the germination and initial establishment of in vitro plants. Of these, we highlighted spectral composition and irradiance since light is fundamental for photosynthesis and photomorphogenesis in plants (SORGATO et al., 2015;HANUS-FAJERSKA & WOJCIECHOWSKA, 2017).
An alternative to this type of cultivation is plant production in natural light environments, replacing artificial light. In this sense, DIGNART et al. (2009)

recommend cultivating Catteya walkeriana
Gardn under natural light conditions and reducing sucrose in the culture medium. SILVA JÚNIOR et al. (2012) concluded that Laelia purpurata Lindl. var. carnea plants grown in vitro in a greenhouse adapt better for subsequent ex vitro cultivation than those grown in a growth room. Plants grown in the first condition showed higher epidermal thickness, leaf mesophyll thickness, root velamen thickness, and stomatal density. Moreover, EBURNEO et al. (2017) reported that Catasetum x apolloi Benelli & Grade stomata responded better to water stress when the orchid grew in a natural light environment.
Light-emitting diode (LED) offers possibilities in horticultural lighting since it can separate and mix different light spectra, allowing irradiance adjustments for plant photoreceptors (SINGH et al., 2015). Thus, this light source can regulate characteristics of in vitro grown plants, including morphological, anatomical variations, and physiological attributes like elongation, axillary bud formation, somatic embryo induction, rhizogenesis, leaf anatomy, and photosynthetic abilities (GUPTA & JATOTHU, 2013).
Notwithstanding, there are still few scientific studies using natural light or LED lamps as light sources in in vitro orchid culture. Furthermore, the effects of spectral quality and irradiance levels on the germination, growth, and initial establishment of plants are still unclear, mainly for native species. Given the above, this study evaluates the effect of different light sources on the in vitro germination and initial establishment of Schomburgkia crispa Lindl.

MATERIALS AND METHODS
The plant material consisted of seeds of Schomburgkia crispa Lindl. fruits produced from manual self-pollination. After harvesting, we took the fruits to the in vitro culture laboratory. Subsequently, we disinfected the fruits with 70% ethyl alcohol solution and opened them with a scalpel. We homogenized seeds of different fruits, further assessing the viability of 9,756 seeds by the tetrazolium test (SOARES et al., 2014). A total of 6,956 (71%) seeds were viable. We then discarded the seeds used in the viability test and stored the rest in a desiccator with silica gel (25 ± 2 °C; 75% RH) for 14 days.
For seed germination, we used ½ MS medium (MURASHIGE & SKOOG, 1962) solidified with 7.0 g L -1 bacteriological agar (Himedia ® , India) and supplemented with 30 g L -1 sucrose. We adjusted the pH of the medium to 5.8 using 0.1M KOH. Then, we distributed the mixture in 50 mL screw-capped polypropylene vials (height and diameter = 5cm), considering a 20 mL culture medium volume per unit. Subsequently, we autoclaved the vials at 120 °C and 1 atm pressure for 20 minutes. After cooling (25 ± 2 ºC), we transferred the vials to a sterile environment.
We then weighed S. crispa seeds, further taking a 0.01 g portion to an aseptic environment and disinfecting it using 15 mL of 0.8% sodium hypochlorite solution for five minutes. After this period, we diluted the suspension to 100 mL using sterile distilled water. We washed the sample three times (80 mL per wash) and discarded the water after each wash. After this procedure, we completed the volume of the suspension to 100 mL with sterile distilled water for in vitro sowing, inoculating 1 mL of the seed suspension into each vial.
We assessed the germination percentage of the species at 45 days after sowing. For that, we washed the material contained in the vials with 3 mL of sterile distilled water; then, we placed it in 16cell (0.5 x 0.5 cm) acrylic plates (2 x 2 x 0.5 cm). We repeated this procedure until there was no seed or seedling in the growing units. Then, we used a binocular stereoscopic microscope to count non germinated seeds (NS) and chlorophyll propagules (CP). We calculated germination percentage (G%) according to modified ROSA et al. (2013), using the following expression: . We assessed the initial establishment at 45 and 90 days after sowing, following the methodology described by SUZUKI et al. (2009). We also counted the number of nonchlorophyll propagules (NCP). After the evaluations, we photographed all treatments with a camera coupled to the stereoscopic microscope with the aid of AxionVision software version 3.1 (Zeiss ® ).
The experimental design was completely randomized with five treatments (natural light; 3,000 K LED; 6,500 K LED [1]; 6,500 K LED [2]; and 6,500 K fluorescent lamp) and five replicates consisting of a culture vial. We transformed the results to and submitted them to analysis of variance and comparison by the Tukey test up to 5% probability with the aid of the SISVAR program version 5.3 (Statistical Analysis Program; Federal University of Lavras, MG).

RESULTS AND DISCUSSION
Light conditions affected all the characteristics (p<0.05) except germination percentage. All treatments provided high germination percentages, with an overall mean of 98% (Table 1).
The highest germination percentage occurred at 45 days with the use of 3,000 K LED, with no statistically significant difference between light sources, which is an important factor for in vitro cultured orchids (GUPTA & JATOTHU, 2013; HANUS-FAJERSKA & WOJCIECHOWSKA, 2017). However, these results suggested that light source is not a limiting factor for S. crispa seed germination, since all conditions led to low mortality, with an overall mean of chlorophyll propagules of 94%.
Regarding initial establishment, most of the propagules were in stage 1 (chlorophyll swollen protocorm) at 45 days. Natural light, 6,500 K LED [1], and 6,500 K LED [2] provided 100% of these protocorms, and seedlings developed up to stage 2 (first-leaf seedlings) only when subjecting the cultures to 3,000 K LED (20%) or 6,500 K fluorescent lamp (1%). Thus, the results allowed us to infer that the use of 3,000 K LED in growth rooms can more rapidly enhance the initial establishment of S. crispa seedlings (Table 1).
Similarly to the results at 45 days after sowing, at 90 days of in vitro culture there was a significant effect (p<0.05) of the different light conditions only for the initial establishment of S. crispa propagules, whose overall mean of germination was 100%.
In this evaluation, the light condition 6,500 K LED [2] provided the highest number of propagules in stage 1 (99%), which represented the smallest differentiation between stages (protocorms and seedlings) among all experimental conditions. This has to do with the hypothesis of light stress: when in understory conditions, natural environment orchids receive, on average, 10 µmol m -2 s -1 (SOARES et al., 2020), a lower irradiance when compared to growth room plants subjected to 6,500 K LED [2] ( Figure 1A). Prolonged exposure of plants or organelles to excess light can lead to photodestruction of photosynthetic pigments, as discoloration (photooxidation) depends on oxygen and light. This phenomenon can lead to cell death, or organism death, and may relate to the high mortality of propagules in this light condition at 90 days (ARAÚJO & DEMINICIS, 2009).
The conditions of 6,500 K fluorescent lamp and 3,000 K LED accelerated the initial establishment of S. crispa seedlings, with 23 and 43% stage 4 seedlings, respectively. Using 3,000 K LED provided a 46% increase in the number of seedlings in this stage of development compared to 6,500 K  However, the use of other spectral bands -for example, red, blue, green, yellow, or their combination -may improve or hinder the development of in vitro species (GUPTA & JATOTHU, 2013;HANUS-FAJERSKA & WOJCIECHOWSKA, 2017).
Although 3,000 K LED provides higher irradiance than 6,500 K LED [2], it has two winding peaks, at ≅ 460nm and ≅ 580nm, what probably contributes to the development of propagules (Figure 2). In addition, 3,000 K LED has more energy in the red region, which, from a physiological point of view, contributes more effectively to the excitation of photosynthetic pigments.
For plants cultured in screened greenhouses under natural light, 73% of seedlings were in stage 2 and 26% in stage 3. At 90 days of cultivation, only 0.2% of the seedlings reached stage 4. Thus, the initial in vitro establishment of the species takes longer under these conditions compared to growth room conditions. The greenhouse environment is not controlled like the growth room environment, which results in a temperature oscillation inside the culture vial, slowing down the initial establishment of protocorms and seedlings (DIGNART et al., 2009). Moreover, the light sources 6,500 K LED [1], 6,500 K LED [2], and 6,500 K fluorescent lamp led to a higher mortality of propagules, confirmed by the higher percentage of nonchlorophyll propagules: 87, 87, and 95%, respectively ( Figure 1B).
Growth rooms often use white fluorescent lamps (6,500 K), which are losing ground for white LED lamps (6,500 K). Notwithstanding, it is crucial to adjust the irradiance and spectral composition of the light sources of these rooms according to the cultivated species (HANUS-FAJERSKA & WOJCIECHOWSKA, 2017). The results of this study for species S. crispa showed that 6,500 K white fluorescent lamps and 6,500 K LEDs provided a higher percentage of nonchlorophyll propagules. This raises the hypothesis of replacing these lamps by 3,000 K LED lamps aiming for fast initial establishment, with stage 4 seedlings in shorter time and low percentage of nonchlorophyll propagules.

CONCLUSION
Light is not a limiting factor for germination of Schomburgkia crispa Lindl seeds. Using 3,000 K LED provides faster initial establishment of S. crispa, with lower seedling mortality. The light source 3,000 K LED is a potential substitute for 6,500 K fluorescent lamps and LEDs used in growth rooms in in vitro culture laboratories.