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Growth and Cell Division of Lettuce Plants under Various Ratios of Red to Far-red Light-emitting Diodes. Myung-Jin Lee1,2, So-Young Park1,2, and Myung-Min ...
Hort. Environ. Biotechnol. 56(2):186-194. 2015. DOI 10.1007/s13580-015-0130-1

ISSN (p rint) : 2211-3452 ISSN (online) : 2211-3460

Research Report

Growth and Cell Division of Lettuce Plants under Various Ratios of Red to Far-red Light-emitting Diodes 1,2

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Myung-Jin Lee , So-Young Park , and Myung-Min Oh 1

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Division of Animal, Horticultural and Food Sciences, Chungbuk National University, Cheongju 361-763, Korea Brain Korea 21 Center for Bio-Resource Development, Chungbuk National University, Cheongju 361-763, Korea *Corresponding author: [email protected]

Received October 28, 2014 / Revised February 2, 2015 / Accepted April 9, 2015 GKorean Society for Horticultural Science and Springer 2015

Abstract. We investigated the effects of various ratios of red to far-red light-emitting diodes (LEDs) on growth characteristics, physiological response, and cell division of red leaf lettuce. Sixteen-day-old lettuce seedlings were transferred into growth chambers and cultivated under various ratios of red (R) and far-red (FR) LEDs (R/FR = 0.7, 1.2, 4.1, and 8.6), only red LEDs (RED), or fluorescent lamps (control) for 22 days. Growth characteristics were measured at 11 and 22 days of treatment. In addition, cell division rate, epidermal cell density, chlorophyll fluorescence, and photosynthesis of leaves were analyzed. Fresh and dry weights and leaf area in all R/FR treatments were higher than those in the control at 22 days of treatment. The R/FR 1.2 had the highest values among R/FR treatments. The number of leaves appeared to increase as R/FR ratio increased. The specific leaf weights in the R/FR ratio of 0.7, 1.2, and 8.6 were similar to the control at 22 days of treatment. The SPAD values in all R/FR treatments were lower than that in the control. All R/FR treatments led to a longer leaf shape than the control. The percentage of cells in the G2M phase, indicating the cell division rate, increased in the R/FR treatments after 4 days of treatment, which supported the growth improvement in the R/FR treatments. The Fv/Fm and the photosynthetic rate in all treatments decreased due to the absence of blue light. The results of this study suggest that the supplementation with far-red LEDs should be considered when designing artificial lighting systems for closed-type plant factories since far-red light affects the vegetative growth of leafy vegetables such as lettuce. Additional key words: G2M phase, light quality, phytochromes, plant factory

Introduction Recently, the usage of light-emitting diodes (LEDs) has been increased as a potential lighting source in closed-type plant production systems (Zakir, 2007). The LEDs have various advantages over existing lighting sources such as fluorescent, metal halide, high-pressure sodium lamps, and incandescent bulbs. The LEDs have a small volume, semipermanent life span, and high emission efficiency and can conveniently control light quality due to their narrow band of the spectrum (Lin et al., 2013). The main trend in LED application to plant cultivation is that red and blue LEDs have been used as major wavelengths, and related research has frequently been reported (Li et al., 2013; Son et al., 2012). In addition, a positive effect of green LEDs on plant growth has been found; red and blue LEDs combined with green LEDs or white LEDs, including three wavelength ranges, are being applied to plant cultivation (Kim et al., 2004a; Lin

et al., 2013). Besides visible light, ultra-violet (UV) is a major research subject, but there had been few studies using UV LEDs. Many previous studies reported that irradiation with UV-A or B lamps stimulated the accumulation of phytochemicals in plants (Alothman et al., 2009; Jeong et al., 2009; Lee et al., 2013). Studies on far-red light have also largely been conducted using fluorescent lamps and incandescent bulbs similar to UV experiments (Shibuya et al., 2010, 2011). However, few studies using far-red LEDs have been conducted. Phytochromes, a type of photoreceptors, have two forms of proteins, Pr, which absorbs red light (660 nm), and Pfr, which absorbs far-red (730 nm). These two forms are converted into each other based on the amount of red and far-red light, which is called photoconversion (Rockwell et al., 2006). In other words, the portion of red and far-red absorption determines the status of phytochrome. Irradiation with more red light than far-red light results in the formation of Pfr, and the phytochrome affects various plant responses

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(Stutte, 2009). Increasing R/FR using photoselective film inhibited stem elongation in chrysanthemum and bell pepper seedlings (Li et al., 2000). In addition, short internode and delayed flowering resulted from a high R/FR by using several light sources in Eustoma grandiflorum (Yamada et al., 2009). Comparison of several different R/FR revealed that a relatively high R/FR delayed flowering and internode elongation, but a low R/FR by using incandescent lamps did not affect flowering and further inhibited internode length in pea plants (Cummings et al., 2007). To date, it has been difficult to control light wavelengths besides R/FR because most studies related to R/FR have used fluorescent lamps, incandescent bulbs, or filters under natural light. Thus, it has been difficult to examine the pure effect of R/FR on plant growth and development. Furthermore, due to the limited range of R/FR using conventional light sources such as fluorescent lamps and incandescent bulbs, most previous studies could not represent the availability of a broad range of R/FR ratios in plant cultivation. However, commercially available LEDs with a specific wavelength have become an alternative solving the two problems mentioned above. In other words, the usage of red LEDs and far-red LEDs makes it possible to represent a wide range of R/FR ratios (Craig and Runkle, 2013; Yang et al., 2013). Thus, the objective of this study was to explore the effect of various R/FR ratios on the basic physiological responses of a leafy vegetable, lettuce, under the same photosynthetic photon flux (PPF).

Materials and Methods 3ODQW 0DWHULDOV DQG *URZWK &RQGLWLRQV Two or three red lettuce seeds (Lactuca sativa ‘Sunmang’, Nongwoo Bio, Suwon, Korea) were sown in cells of a 105cell plug tray containing the growing medium (Myung-Moon, Dongbu Hannong, Seoul, Korea). The seedlings germinated were cultivated under a growth chamber (DS-12BPLH, Dasol Scientific, Hwaseong, Korea) with normal growth conditions (air temperature 20°C, relative humidity 60%, PPF 130 ± 5 ȝmol·m-2·s-1, photoperiod 12 hours) for 16 days. One lettuce seedling per cell was maintained and the remaining seedlings were thinned out 7 days after sowing. Each seedling was transplanted into an individual pot (10.6 cm× 10.6 cm × 11.5 cm, L × W × H) containing growing medium at 16 days after sowing. Sixteen pots were placed on a tray and six trays were used in this study. Transplanted lettuce plants were cultivated for 22 days in the growth chambers installed with red and far-red LEDs. The growth conditions were the same as for the seedling stage except for light conditions. The pots in a tray were systemically rotated every day to reduce unbalanced light distribution. Two liters of nutrient solution

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for lettuce (pH 5.5, EC 1.2 dS·m , N:P:K = 17.3:4.0:8.0) was subirrigated to pots once a week after the cotyledons fully developed. Distilled water (1 L) was also supplied every 2 or 3 days during the entire growing period. 5HG DQG )DUUHG /('V 7UHDWPHQWV Red (660 nm, HanaMicron, Asan, Korea) and far-red (732 nm, HanaMicron, Asan, Korea) LEDs were used to make various combination of R/FR ratios. Five ratios of R/FR (0.7, 1.2, 4.1 and 8.6 based on light intensity) and 100% red (RED) were made by adjusting the number of red and far-red LEDs in plate-type LED lighting systems, and fluorescent lamps were used as the control. The light spectrum of each lighting source was measured at an interval of 0.7 nm by a spectrophotometer (JAZ-EL 200, Ocean Optics, Dunedin, FL, USA) and was represented as a relative wavelength (Fig. 1). The PPF of the light treatments, including the control, was measured at the crossing points of 4 pots in a tray, out of the 9 points in each tray, using a quantum sensor (LI-190, Li-Cor, Lincoln, NE, USA). To exclude the effect of different PPFs of red LEDs on the photosynthetic rate, the PPF for all of the treatments was adjusted to 132 ± 7 μmol· -2 -1 m ·s . 3ODQW *URZWK Growth characteristics such as fresh and dry weights of shoots and roots, leaf length, leaf width, leaf area, and SPAD value were measured at 11 and 22 days of treatment. Fresh weights of shoots and roots of lettuce were measured using a digital scale (Si-234, Denver Instrument, Denver, CO, USA), and then the lettuce samples were dried in an oven (VS-1202D3, Vision Scientific, Daejeon, Korea) at 70°C for 72 hours for dry mass. The leaf shape index of the third leaf from the basal part was calculated by dividing leaf length by leaf width. The leaf area of intact lettuce plants was measured using a leaf area meter (LI-3100C, Li-Cor, Lincoln, NE, USA). Chlorophyll content was represented as the SPAD value, which was measured by a portable chlorophyll meter (SPAD-502, Konica Minolta, Tokyo, Japan). &HOO 'LYLVLRQ Completely unfolded young leaves were selected for cell division analysis and collected at 4, 8, 14, and 20 days of treatment. Collected leaf samples were examined after cutting sections of 5 mm × 5 mm. A 400 ȝL nuclear extraction buffer of a reagent kit (Partec Cystain UV Precise P, Partec, Münster, Germany) was added to a scale containing the leaf sample and subsequently chopped using a sharp razor blade, and then the solution was filtered through a 30-μm nylon sieve (Partec Cell Trics, Partec, Münster, Germany). Then, the filtered solution was mixed with staining buffer (1.6 mL) of the

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Myung-Jin Lee, So-Young Park, and Myung-Min Oh

Fig. 1. Spectrum of the control (fluorescent lamps) (A), various ratios of red to far-red LEDs (B-E), and 100% red LEDs (F). Photosynthetic photon flux of all the treatments was 132 ± 7 μmol฀m-2฀s-1. Spectral scans were recorded at the top of the pots and averaged at 9 points of each treatment.

reagent kit. The degree of cell division of the solution was determined by a PA flow cytometry (Ploidy Analyser, Partec, Münster, Germany). (SLGHUPDO &HOO 'HQVLW\ The epidermal cell density of lettuce leaves was measured at 22 days of treatment. The section to the right of the main vein of the fourth leaf from the bottom was used as a sample for microscope observation. Colorless nail polish was applied to the leaf sample, and after 1 minute, epidermal cells were peeled out using tape. Epidermal cells were observed by a fluorescence microscopy (JSB-F40, SamWon Scientific, Goyang, Korea) and epidermal cell density was calculated by the method described in Ceulemans et al. (1995) with minor modifications. Epidermal cell density was expressed as epidermal cell number per unit leaf area. &KORURSK\OO )OXRUHVFHQFH The maximum PS II quantum yield (Fv/Fm), which is an indicator of stress level, was measured in lettuce leaves subjected to various R/FR LEDs. The fifth leaf from the top was used and the leaf was stabilized using a clip to expose it to dark conditions for 30 minutes before the measurement. The Fv/Fm of the stabilized leaf was determined by a chlorophyll fluorescence meter (PAM 2000, Heinz Walz, Effeltrich, Germany). 3KRWRV\QWKHWLF 5DWH The photosynthetic rate of lettuce leaves was measured at

10, 14, and 19 days of R/FR treatments. The photosynthetic rate of the fifth leaf from the top, a completely unfolded leaf, was tested using a portable photosynthesis system (LI6400, Li-Cor, Lincoln, NE, USA). The measurement conditions for the fluorescence chamber attached to the infrared gas -1 analyzer of the system were: air flow rate, 400 μmol·s ; -1 CO2 concentration, 400 μmol·mol ; block temperature, 20°C; -2 -1 and PPF, 400 μmol·m ·s , the previously determined light saturation point. To reduce the variation according to the time of the day, the measurementswere taken from 10:00 a.m. within 2 hours. 'DWD &ROOHFWLRQ DQG 6WDWLVWLFDO $QDO\VLV All measurements were replicated four times, and the experiment was repeated twice to check the reproducibility of results. For statistical analysis, the SAS program (SAS 9.2, SAS Institute, Cary, NC, USA) was used. ANOVA and Duncan’s multiple range tests were used to analyze the differences in data and to compare means among the treatments, respectively.

Results and Discussion 3ODQW *URZWK Fresh and dry weights of shoots and roots and shoot/root (S/R) ratios of red leaf lettuce were measured at 11 and 22 days of treatment (Table 1). At 11 days, all the treatments except for R/FR 4.1 had higher fresh and dry weights of shoot than the control (fluorescent lamps). The R/FR 0.7 and

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Table 1. Growth characteristics of lettuce plants grown under various ratios of red to far-red LEDs at 11 and 22 days of treatment. 'D\

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R/FR 8.6 had maximums of 2.0 times and 1.6 times higher fresh and dry weights of shoots, respectively, than the control. The lowest fresh and dry weights of shoots were in R/FR 4.1, and the weights tended to increase from there with increasing or decreasing R/FR. As R/FR increased the root fresh weight also increased, which was a different trend from the shoot results. At 22 days of treatment, the fresh and dry weights of shoots in all the treatments were significantly higher than in the control. Especially, R/FR 1.2 induced about 2.8 and 2.7 times higher fresh and dry matter accumulation, respectively, than the control. There was no specific pattern in the fresh and dry weights of shoots according to R/FR change, and no significant differences were observed among treatments except for R/FR 1.2. There was a similar trend in the fresh and dry weights of roots. R/FR 1.2 lead to about 2.3 times higher values in both parameters than the control. R/FR 0.7 had significantly higher shoot/root ratio (S/R) than other treatments, which was similar to the results at 11 days, while the lowest value was observed in the control. R/FR 0.7 stimulated stem elongation in lettuce plants. The growth pattern changed from 11 days of treatment to 22 days of treatment. This implies that the effect of R/FR on lettuce growth varies depending on the growth stage. According to Yamada et al. (2009), the budding and flowering time of Eustoma grandiflorum was different in various R/FR irradiation treatments. Meanwhile, R/FR 1.2 yielded the highest shoot and root growth at 22 days of treatment. In general, the R/FR ratio of solar light is 1.1 without shading (Shibuya et

al., 2010), and the lettuce seeds used in this study was bred under field conditions. Thus, these facts support our results mentioned above. In a previous study using chrysanthemums and bell peppers, R/FR of 1.2 generated by photoselective plastic films led to the best shoot dry weight among several R/FR ratios (Li et al., 2000). The fact that R/FR 1.2 had the best growth among the treatments suggests another interesting point. Although our study excluded blue and green light and used only red light to determine the pure effect of R/FR, the growth promotion by supplementation with far-red light suggests that far-red LEDs should be used in closed-type plant production systems that previously had used only the visible light spectrum, such as red, blue, and/or green. Franklin and Whitelam (2005) reported that a low R/FR increased the length of the stem and petiole in Arabidopsis thaliana. Similarly, a low R/FR, such as 0.7, resulted in a longer stem length in lettuce in this study. On both measurement days, the leaf area and leaf number in most of R/FR treatments were significantly higher than those in the control (Figs. 2A, 2B). The R/FR 4.1 had the lowest leaf area and leaf number, and the leaf number was also low in R/FR 1.2 at 11 days of treatment. This trend was similar to the results for the fresh and dry weights of shoots. At 22 days of treatment, leaf number increased as R/FR increased. In the case of leaf area, although no significant difference was observed among treatments except for the control, the values of R/FR 1.2 and 8.6 were numerically higher than those of the other treatments, which was also

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Day of treatment Fig. 2. Leaf area (A), number of leaves (B), leaf area/number of leaves (C), and specific leaf weight (D) of lettuce plants grown under various ratios of red to far-red LEDs at 11 and 22 days of treatment. The control and RED represent fluorescent lamps and red LEDs 100%, respectively. The data indicate the means ± SE (n = 4). Significant at *p = 0.05 **p = 0.01 and ***p = 0.001.

similar to the results for the fresh and dry weights of shoots. After 11 days of treatments, the average leaf area per leaf was not significantly different between treatments, but R/FR 4.1 had the lowest value, similar to the results for leaf area and number of leaves (Fig. 2C). The R/FR 1.2 and 0.7 had relatively high values at 22 days of treatment, although no significant difference was observed at p = 0.001. This indicates that the low R/FR induced larger individual leaves. Specific leaf weight was significantly the highest in the control, followed by R/FR 8.6 and RED at 11 days of treatment (Fig. 2D). In other words, lettuce plants subjected to low R/FR had relatively thin leaves. At 22 days of treatment, specific leaf weight was the lowest in R/FR 4.1,which had poor shoot growth, and was the highest in R/FR 1.2, which showed outstanding shoot growth. The trend for the results of leaf area, number of leaves, and specific leaf weight changed in accordance with growth stage, but the changed pattern was similar to that of shoot growth, as suggested in Table 1. This suggests that the shoot growth of lettuce with dwarf stems was mainly determined by the growth and development of leaves. Thus, it can be concluded that vigorous shoot growth in R/FR 1.2 and 8.6 resulted from the large, thick leaves in both treatments

at 22 days of treatment. In contrast, lettuce subjected to R/FR 4.1 had small and thin leaves, which was directly connected to poor shoot growth. In a previous study on pepper plants irradiated at various R/FR, R/FR 1.1 stimulated the enlargement of leaves, which was consistent with our results (Li et al., 2000). Figure 3 shows SPAD values and leaf shape indices. All R/FR treatments had SPAD values that were significantly lower by about 1.9 times compared to the control, and no significant difference was observed between the treatments at 11 days of treatment. The change of SPAD value at 22 days had a similar trend to those at 11 days (Fig. 3A). Leaf shape indices in all treatments (except for R/FR 8.6 at 11 days) were significantly higher by at least 1.7-fold compared to the control at 11 and 22 days of treatment (Fig. 3B). Blue light is known to typically stimulate the biosynthesis of pigments, including chlorophyll (Carvalho et al., 2011; Li and Kubota, 2009). In an experiment growing two lettuce cultivars under monochromatic LEDs, green and red LEDs produced lower chlorophyll contents than blue LEDs (Son et al., 2012). In addition, the supplementation of red LEDs with blue LEDs yielded higher chlorophyll contents than red

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Fig. 3. SPAD value (A) and leaf shape index (B) of lettuce plants at 11 and 22 days of treatment and picture of lettuce (C) grown under various ratios of red to far-red LEDs at 22 days of treatment. The control and RED represent fluorescent lamps and red LEDs 100%, respectively. The data indicate the means ± SE (n = 4). Significant at *p = 0.05 and ***p = 0.001.

LEDs alone in red leaf lettuce (Son and Oh, 2013). In this study, we used only red LEDs as a photosynthetically usable light source to identify the effect of R/FR excluding other wavelengths. Thus, the low chlorophyll levels in all R/FR treatments were reasonable, because of the absence of blue light, compared to the normal chlorophyll level in the control (fluorescent lamps) containing both red and blue lights. At both growth stages, leaf shape indices of all the treatments were higher than the control, indicating longer leaves. In addition, leaf shape indices were lower at 22 days than at 11 days regardless of the treatment, which indicates that lettuce leaves grew wider as the plants grew. Leaf shape index results are shown in Fig. 3C. Furthermore, the large leaf size in R/FR 1.2 corresponded with the results for shoot growth and leaf area mentioned above. &HOO 'LYLVLRQ Several treatments had more cells in the mitotic phase, G2M phase, than the control at 4, 8, 14, and 20 days of treatment (Fig. 4). In particular, at 4 days of treatment, the percentage of G2M phase of young leaves around the growing point had high values in RED, followed by R/FR

4.1, 8.6, 0.7, and 1.2, which had a relatively low value. The control had the lowest value. The percentage of G2M phase gradually increased with increasing R/FR, and this trend became clearer as the growing period increased from 14 days to 20 days. The complete process of cell division consists of G1, S, G2, and M phases. The G1 phase involves the growth of cells, and DNA duplication occurs in the S phase. Cells that have completed DNA duplication are ready for cell division in the G2 phase. In the M phase, a cell is actually divided into two cells through mitotic cell division. One cycle of cell division takes place via these processes (Vázquez-Ramos and Sánchez, 2003). At 4 days of treatment, the percentage of G2M phase in the R/FR and RED treatments was higher than in the control, suggesting that all the treatments stimulated cell division. The results for cell division at 4 days of treatment may affect the early growth of lettuce plants. The fact that the number of leaves at 11 days of treatment had a similar trend supports this supposition. Overall, treatments except for R/FR 0.7 also had more cells in the G2M phase than the control, and the cell division rate increased with increasing R/FR at 14 days of treatment. This pattern was consistent with that of the number of leaves at 22 days of treatment, where

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increased R/FR led to more leaves (Fig. 2B). (SLGHUPDO &HOO 'HQVLW\ Although the number of epidermal cells at 22 days of treatment was not significantly different between the control and any treatments, RED and R/FR 4.1 had numerically more epidermal cells than R/FR 0.7, 1.2, or 8.6 (Fig. 5). In a study on stoloniferous herbs grown under high light or shade, an increased petiole length in herbs grown in shade was caused by increasing cell numbers (Weijschede et al., 2008). Similarly, in our study, RED, with the highest number of cells, had the highest number of leaves (Fig. 2B). The small number of epidermal cells per unit area indicates that the number of cells is small but the size of cells may be large. Therefore, expanded epidermal cell size was associated with the enlargement of leaf area; the results of the leaf area per leaf number were consistent with the results of epidermal cell number (Fig. 2C). &KORURSK\OO )OXRUHVFHQFH DQG 3KRWRV\QWKHVLV The changes in the chlorophyll fluorescence in the treatments

Fig. 5. Epidermal cell density of lettuce plants grown under various ratios of red to far-red LEDs at 22 days of treatments. The control and RED represent fluorescent lamps and red LEDs 100%, respectively. The data indicate the means ± SE (n = 4).

and the control are shown in Table 2. At 11 days of treatment, the Fv/Fm of lettuce in the control was 0.81, while all the treatments had Fv/Fm below 0.8. In particular, R/FR 1.2 and

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Table 2. Chlorophyll fluorescence (Fv/Fm) and photosynthetic rate of lettuce plants grown under various ratios of red to far-red LEDs at 11 and 22 days and 10, 14, and 19 days of treatment, respectively. &KORURSK\OO IOXRUHVFHQFH )Y)P

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8.6 and RED had significantly lower values. No significant difference was observed between the control and treatments at 22 days, but the pattern of change was similar to that at 11 days. The Fv/Fm was calculated by (Fm-Fo)/Fm, where Fm is the maximum fluorescence irradiated from light and Fo is the fluorescence value under non-photochemical quenching. Therefore, Fv/Fm represents the potential quantum efficiency of PSII. In general, Fv/Fm of normal plants is about 0.83, and lower values indicate photoinhibition and the degree of stress (Maxwell and Johnson, 2000). In this study, Fv/Fm of all treatments was below 0.8, which was lower than the control, suggesting that lettuce plants in the R/FR treatments were stressed. This may be explained by the absence of blue light. The control had a significant higher photosynthetic rate than any of the treatments at 10 and 19 days of treatment (Table 2). There was no significant difference in the photosynthetic rate between the control and treatments, but overall, treatments had lower values at 14 days of treatment. The decrease in the photosynthetic rate in R/FR treatments throughout the whole growth stage resulted from the absence of blue light, which was similar to the result for Fv/Fm, while fluorescent lamps containing a variety of light wavelengths had a higher photosynthetic rate. Kim et al. (2004b) reported that the best photosynthetic rate of chrysanthemum plantlets was under a combination of red and blue light, followed by fluorescent lamps and a combination of red and far-red light. In summary, the R/FR treatments and RED stimulated the accumulation of biomass in lettuce compared to the control. In particular, R/FR 1.2 had the highest fresh and dry weights of shoots and roots and leaf area. The growth promotion of the lettuce in the treatments was confirmed by the cell division results. Unlike the control, the R/FR treatments and RED excluding other light wavelengths had lower values for SPAD, chlorophyll fluorescence, and photosynthetic rate because of the absence of blue light. Our results can be used as basic

information for future studies using R and FR LEDs. Furthermore, it is expected that the supplementation with far-red LEDs will improve crop growth and yields in closed-type plant production systems. Acknowledgement: This work was supported by the research grant of Chungbuk National University in 2014.

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