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Plants 2020, 9, 1344 10 of 15 expressional level in both chi and fls gene (respectively, 1.18 and 1.21 times compared with FL). This might be one of the main reasons for the increasing of flavonoid contents in A. roxburghii cultured under BR LED light in comparison with those under FL or other LED lights (Figure 5c). Biological rhythms work based on the participation of many environmental induction factors, including light-sensing receptors. In particular, these receptors are affected by the lighting cycle as well as the characteristics of the lighting spectrum [58–60]. In this experiment, the different lighting conditions could influence the activity of those receptors and indirectly impact the activity of some genes that are controlled by circadian rhythms. However, further studies are required to reveal the correlation between light spectrum, light receptors, biological rhyme, and the flavonoid synthesis process in A. roxburghii. As the world attempts to exploit artificial lights to promote the secondary metabolites of vegetables and herbs, the big challenges remain regarding what the optimal time of the growth period of specific each plant under the optimal light intensity is and what wavelength light and how long irradiation of light should be completed [61]. Further research should be focused on the multiple adjustable combinations of LEDs in the different growth stages of the plant with varying light intensity, quality, and duration. 4. Materials and Methods 4.1. Plants Materials and Growth Conditions Two-month-old A. roxburghii rooted in vitro plants (with 2 roots, 3 leaves, and about 3 cm in height) were used as the initial materials. Plants were cultivated in hydroponic pots (17 cm long, 12 cm wide, and 7 cm deep) supplemented with 1/10 strength macro and minor salt of Murashige and Skoog (1962) medium. The LED light source was placed at the top of the steel frame structure of the cultivation rack. The distance between the plant canopy and LED lights fluctuated from 40 to 60 cm. The silver shading materials were used to insulate in the different treatments. The relative humidity in the growth room (2m in length × 0.6 m in width) was 80%, and the temperature was in the range of 22–24 ◦C with 12 h of lighting. The plants were subjected to different LED light for 2 months under following conditions: mono-wavelength (1) red (630 nm), (2) blue (465 nm), and mixed-wavelength, (3) BR (20% B light and 80% R light), (4) BRW 151 (13.8% B light, 72.4% R light, and 13.8% W light), and (5) BRW142 (13.8% B light, 58.6% R light, and 27.6% W light). Fluorescent lamp was a multiple wavelength and was used as the control (Supplementary Figure S1). All the LED lights, mixed LED lights were ordered as accurate above conditions and designed by Dien Quang Lamp Join Stock Company, Ho Chi Minh city, Vietnam. The average of light intensity of the distance from the LEDs to the plant canopy was about 30 ± 2.13 μmol.m−2 s−1 and measured by the LI-COR LI-250A Light Meter (Li-Cor Biosciences, Lincoln, NE, USA). 4.2. Morphological and Biomass Parameter Analyses For measurements of the morphological characteristics, 30 random plants were selected from each treatment. Plant height, stem diameter (3rd node), leaf numbers, leaf area, root length, root numbers, fresh weight, and dry weight were measured. Briefly, the fresh weight was determined by using the electronic balance (Vibra, Japan) and dried to a constant weight under 70 ◦C before dry weight measurement. The stem diameter was recorded using the digital vernier caliper. The root length was measured from the top of the root tip to the main stem. Leaf area was measured by using image analysis method with ImageJ software [62]. The values shown in Table 1 are the mean of 3 replicates, with the 30 random samples for each treatment. 4.3. Stem Anatomical Characteristics The second node of each plant was used to determine stem cell width and length. The 2nd node was excised from the stem and sectioned by using CS-05W Crimping cross-section monitorPDF Image | LED Lights Promote Growth and Flavonoid Accumulation of Anoectochilus roxburghii
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