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Supplementary Light Source Affects Growth and Development of Carnation ‘Dreambyul’ Cuttings

Supplementary Light Source Affects Growth and Development of Carnation ‘Dreambyul’ Cuttings agronomy Article Supplementary Light Source A ects Growth and Development of Carnation ‘Dreambyul’ Cuttings 1 1 1 1 , 2 , 3 , Mengzhao Wang , Jie Xiao , Hao Wei and Byoung Ryong Jeong * Department of Horticulture, Division of Applied Life Science (BK21 Plus Program), Graduate School of Gyeongsang National University, Jinju 52828, Korea; meng.zhao@163.com (M.W.); xiaojsicau@163.com (J.X.); oahiew@gmail.com (H.W.) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea * Correspondence: brjeong@gnu.ac.kr; Tel.: +82-55-772-1913 Received: 22 July 2020; Accepted: 17 August 2020; Published: 18 August 2020 Abstract: Carnation (Dianthus caryophyllus L.) is a major floricultural crop, cultivated widely for cut flowers. This study was conducted to determine the optimal supplementary light source for the cutting propagation of carnation ‘Dreambyul’ cuttings. Terminal cuttings were propagated in a 2 1 glasshouse with an average of 260 molm s photosynthetic photon flux density (PPFD) coming from the sun (the control), supplemented with one of three artificial light sources: mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix), metal halide (MH) lamps, or high-pressure sodium 2 1 (HPS) lamps. The supplementary light was provided from 7:00 to 17:00 h at 100 molm s PPFD during propagation. The cuttings were kept on a fogged bench in a glasshouse for 25 days with 24/15 C day/night temperatures and 88% relative humidity. Compared with the control, better root formation was observed from cuttings grown under the supplementary lights after 10 days. After 25 days of propagation, MH significantly increased the root length, root number, root fresh and dry weights, and shoot biomass, shoot length, and shoot fresh and dry weights. The best root ball formation and the highest root activities were also found in cuttings propagated with supplementary MH light. Supplementary light increased the plant temperature, quantum yield, stomatal conductance, and the contents of chlorophyll, soluble proteins, and carbohydrates. Overall, the root formation and development of carnation ‘Dreambyul’ cuttings were significantly promoted by the three supplementary light sources. Of the three, MH was identified as the optimal supplementary light source. Keywords: adventitious root formation; artificial light; biomass; quantum yield; root activities 1. Introduction The economic value of ornamental plants has increased significantly worldwide, by 8–10% annually [1]. After rose, the most sought-after species in the global market is carnation (Dianthus caryophyllus L.), due to its excellent characteristics [2]. In Korea, carnations were cultivated over 42.9 hectares, with the total market value of 6.7 billion KRW in 2016 [3]. Carnations in the Korean market have a wide range of attractive shapes, colors, sizes, and delicate fragrances [4]. Vegetative propagation is common and rapid for several ornamental plant species. It retains the genetic traits of stock plants by creating clones with the same characteristics [5]. Ecient adventitious rooting is a key process in the vegetative propagation of horticultural species. Well-rooted cuttings are a requisite for the optimal growth of high-quality plants. Carnation is primarily propagated as cuttings in Korea [6], and profitable carnation cultivars are propagated from terminal stem cuttings collected from elite mother plants [7]. Several studies have investigated internal and external factors that a ect Agronomy 2020, 10, 1217; doi:10.3390/agronomy10081217 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1217 2 of 15 the rooting of carnation cuttings during propagation. Such factors include the rooting substrate [8], rooting hormones [9], propagation season [10], cutting types [11], and endogenous hormone levels in stem cuttings [12]. However, not many studies have been conducted to investigate the e ects of light on the rooting of carnation cuttings during the propagation process. Light is the foremost energy source for plant growth. Supplementary lights are broadly used in agricultural production for the advancement in crop growth, yield, and quality, which has been confirmed by many researchers [13–15]. In a glasshouse, the daily light integral (DLI) may be reduced by the glazing materials, interior structures, and hanging baskets suspended above benches. Supplementary light is the only way to substantially increase the DLI in such environments [16], and it has been used to enhance plant growth and development in horticulture [17]. Supplementary lighting can aid plant growth by providing the necessary light when insucient natural light is present in a greenhouse. Improving the light conditions in greenhouses and optimizing the phytochemical concentrations in vegetables produced in controlled environments enhance the growth and yield [18]. Therefore, supplementary lighting is an important horticultural strategy to improve crop growth, maintain high yields all year round, and produce superior-quality plants [19,20]. The morphology, physiology, biochemistry, and transcriptional expression vary in the light spectrum [21–23]. Changes in the light spectrum can a ect the leaf structure, palisade mesophyll cell thickness, and epidermis [24,25]. The supplementary light source is a key factor of the changes in the light spectrum. High-pressure sodium (HPS) and metal halide (MH) lamps are two conventional artificial light sources commonly applied in greenhouse horticulture due to their long service life, reasonable light spectrum, and low price [24,26–28]. Light-emitting diodes (LEDs) have gained widespread attention, since they provide a narrow wavelength band with a high eciency [29–31]. However, data are still scarce regarding the choice of the light source and how the supplementary light source influences the adventitious rooting of carnations during cutting propagation. This work aimed to study the e ects of supplementary light provided by LED-mix, MH, and HPS on carnation cuttings and identify the optimal supplementary light source for greenhouse cutting propagation of carnations. 2. Materials and Methods 2.1. Plant Materials and Culture Conditions The mother plants of carnation ‘Dreambyul’ used in the study were acclimated after 0  0 micropropagation in vitro and then grown in a Venlo-type glasshouse (35 09 N, 128 06 E, Jinju, Korea) for 3 months. Then terminal cuttings from these mother plants were harvested and stuck in 128 square-cell plug trays containing a commercial BVB medium (Bas Van Buuren Substrates, EN-12580, De Lier, The Netherlands). The cuttings were kept on a mist (fogged for 30 min every 30 min) propagation bench with a mean air temperature of 24/15 C day/night and relative humidity of 88% 2 1 in a Venlo-type glasshouse with an average daily light intensity of 260 molm s photosynthetic 2 1 photon flux density (PPFD) coming from the sun. The daily light integral (DLI) was 9.36 molm d . This experiment was carried out from 22 October to 16 November 2019. 2.2. Supplementary Light Treatments This study used mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, FL300, Senmatic A/S, Søndersø, Denmark), metal halide lamps (MH, SunLumen Lighting, Gyeongju, Korea), and high-pressure sodium lamps (HPS, BLV Licht- und Vakuumtechnik, Steinhöring, Germany) as supplementary light sources, and a group without any supplementary light was set as the control. The lamps were placed above the bench, and the supplementary light intensity was set at 2 1 100 molm s PPFD from 07:00 to 17:00 by adjusting the position of the lamps after measuring multipoint averages in each treatment area. The supplementary light intensity was measured with a Agronomy 2020, 10, 1217 3 of 15 quantum radiation probe (FLA 623 PS, ALMEMO, Holzkirchen, Germany) at the top leaf level of the cuttings at night to avoid the e ects of sunlight, and the spectrum of the light sources was measured with spectroradiometers (International Light Technologies, Peabody, MA, USA). The experiment was laid out in a completely randomized design with three replications per treatment, and the treatment locations in a controlled environment were randomly laid out in order to minimize location e ects. In addition, enough space was reserved, and two layers of 0.3-mm-thick black non-woven fabric curtain was used between lamps to prevent light from one treatment from interacting with another treatment. 2.3. Measurement of Plant Temperatures Before the end of the experiment, infrared measurements with a thermographic camera (testo 882, Testo SE & Co. KGaA, Titisee-Neustadt, Germany) were taken to estimate the plant temperature on a sunny day. The thermograms were taken 1 m above the cuttings at 13:00 when the supplementary light was on and were analyzed with IRSoft (version 4.6; available online: https://www.testo.com/en/ services/download). 2.4. Determine of Root Activities To determine the root activities, 0.5 g of fresh roots were completely immersed in a solution containing 5 mL of 0.4% triphenyl-tetrazolium chloride (TTC) and 5 mL of 66 mmolL phosphate bu er (pH 7.0). The fresh roots were then incubated at 37 C for 3 h, and 2 mL of 1 molL H SO was 2 4 subsequently added to end the reaction. The roots were then taken out and the moisture was carefully wiped o . The roots were then thoroughly ground with 3 mL of ethyl acetate and a small amount of quartz sand to extract triphenyl formazan. After filtering the red extract into a 10-mL volumetric flask, the residue was washed 3 times with 2 mL of ethyl acetate before being added to the volumetric flask, and then the volume of the solution was brought up to 10 mL with ethyl acetate. The absorbance at 485 nm was recorded with a UV spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The root activities were expressed as the amount of TTC reduction (g) per g of root fresh weight per hour. 2.5. Stomatal Conductance and Plant Chlorophyll Fluorescence The stomatal conductance and photosynthesis were sampled and averaged from three cuttings within each repetition. Measurements were taken on fully expanded leaves after 2 h of the supplementary light treatments. The stomatal conductance was assessed using a Decagon Leaf Porometer SC-1 (Decagon Device Inc., Pullman, WA, USA), and the photosynthesis was assessed with a FluorPen FP 100 (Photon Systems Instruments, PSI, Drásov, Czech Republic). 2.6. Chlorophyll A and B, Soluble Protein, and Carbohydrate Analyses The contents of chlorophyll a and b were estimated according to the method described by Arnon [32] with some modifications. In brief, 0.1 g of fresh leaves were cut into small pieces and kept in 2.0 mL tubes, where 2 mL of 80% acetone was added. The tubes were wrapped with tin foil and kept in a 4 C refrigerator for 12 h before extraction. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was transferred to a colorimeter tube, then the absorbance was measured at 645 and 663 nm, against a bu er blank in a UV spectrophotometer. The contents of chlorophyll a and b were determined using the following equations: ( ) 12.72 OD at 663 nm 2.59 OD at 645 nm  V Chlorophyll a = (1) Sample fresh weight ( ) 22.88 OD at 645 nm 4.67 OD at 663 nm  V Chlorophyll b = (2) Sample fresh weight Agronomy 2020, 10, 1217 4 of 15 The chlorophyll content was expressed as mg of chlorophyll per g of fresh leaf weight. The V is the volume of the extract solution. Soluble proteins were extracted with a sodium phosphate bu er and measured calorimetrically Agronomy 2020, 10, x FOR PEER REVIEW 4 of 16 using a Bradford Reagent (Sigma-Aldrich, St. Louis, MO, USA), according to Bradford’s method [33]. The soluble sugar and starch contents were determined by the Anthrone colorimetric method according The soluble sugar and starch contents were determined by the Anthrone colorimetric method to Wei et al. [15]. according to Wei et al. [15]. 2.7. Measurements of Growth Parameters 2.7. Measurements of Growth Parameters After 10 days of the supplementary light treatments, the mid-measurement was carried out to After 10 days of the supplementary light treatments, the mid-measurement was carried out to record the rooting ratio and compare the initial growth of roots and shoots of the cuttings. After 25 days record the rooting ratio and compare the initial growth of roots and shoots of the cuttings. After 25 of the supplementary light treatments, the cuttings treated with a supplementary light source had days of the supplementary light treatments, the cuttings treated with a supplementary light source formed a well-developed root ball that was easily pulled out of the plug tray. At this stage, the final had formed a well-developed root ball that was easily pulled out of the plug tray. At this stage, the measurement of the growth parameters was collected. For measuring the growth parameters, whole final measurement of the growth parameters was collected. For measuring the growth parameters, cuttings were harvested, and roots were washed carefully using tap water. Growth parameters, such whole cuttings were harvested, and roots were washed carefully using tap water. Growth as parameters, such as the fresh weight, the fresh weight, length, and the number length, an of roots, d th wer e number o e measurfed roots, wer directly, wher e measured d eas the dry irectly, weights of whereas the dry weights o shoots and roots were measur f shoots edand r after oots were me drying for 72 ash ured at 60 afte C r d in rying an oven. for 72 h The atchlor 60 °C ophyll in an oven. level was The chlorophyll level was measured using a Plus Chlorophyll Meter (SPAD 502, Spectrum measured using a Plus Chlorophyll Meter (SPAD 502, Spectrum technologies, Korea). The cuttings technologies, Korea). The cuttings were placed in liquid N2 in a −80 °C refrigerator for further were placed in liquid N in a80 C refrigerator for further analysis. analysis. 2.8. Data Collection and Analysis 2.8. Data Collection and Analysis The experimental assays used to obtain all results were repeated three times and are presented The experimental assays used to obtain all results were repeated three times and are presented as the mean standard error. The collected data were subjected to an analysis of variance (ANOVA) as the mean ± standard error. The collected data were subjected to an analysis of variance (ANOVA) followed by Duncan’s multiple range test at p < 0.05 to find the statistical significance among the followed by Duncan’s multiple range test at p < 0.05 to find the statistical significance among the treatments by the Statistical Package for the Social Sciences version 21 (SPSS Inc., Chicago, IL, USA). treatments by the Statistical Package for the Social Sciences version 21 (SPSS Inc., Chicago, IL, USA). All figures were made with the OriginPro software version 9.0 (OriginLab Corporation, Northampton, All figures were made with the OriginPro software version 9.0 (OriginLab Corporation, MA, USA). Northampton, MA, USA). 3. Results 3. Results 3.1. Morphology and Growth Parameters Analysis 3.1. Morphology and Growth Parameters Analysis Di erent supplementary light sources provide various spectral characteristics that may di er Different supplementary light sources provide various spectral characteristics that may differ from those of sunlight. LED-mix consisted of a mixture of red, blue, and white lights that occupy a from those of sunlight. LED-mix consisted of a mixture of red, blue, and white lights that occupy a narrow spectrum with two peaks at 434 and 662 nm (Figure 1A). The accumulated irradiance was narrow spectrum with two peaks at 434 and 662 nm (Figure 1A). The accumulated irradiance was mainly distributed in red (62.8%), blue (17.4%), and green (18.2%) lights (Table 1). The MH lamps were mainly distributed in red (62.8%), blue (17.4%), and green (18.2%) lights (Table 1). The MH lamps white in appearance and strong emission lines were largely dispersed in all ranges with about 32.7 and were white in appearance and strong emission lines were largely dispersed in all ranges with about 54.9% of the total irradiance focused on blue and green lights (Figure 1B, Table 1). The HPS lamps 32.7 and 54.9% of the total irradiance focused on blue and green lights (Figure 1B, Table 1). The HPS produced a characteristic golden-orange light, and the largest accumulated irradiance was mainly lamps produced a characteristic golden-orange light, and the largest accumulated irradiance was observed in green light (53.7%), followed by red (36.5%) and blue lights (6.2%) (Table 1). mainly observed in green light (53.7%), followed by red (36.5%) and blue lights (6.2%) (Table 1). Figure 1. The spectral distribution of mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, Figure 1. The spectral distribution of mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, (( A A )), )), meta metall halide lamps (MH halide lamps (MH, , (( B B )), )), and hig and high-pr h-pressu essur re sod e sodium ium lam lamps ps (HP (HPS, S, (C)). (C)). Agronomy 2020, 10, 1217 5 of 15 Table 1. The irradiance distributions of the supplementary light sources: LED-mix, mixed (red: blue: white = 6:1:1) light-emitting diodes; MH, metal halide lamps; and HPS, high-pressure sodium lamps. Irradiance (Wcm ) Supplementary UV Blue Green Red FR IR Light Source (250–400 nm) (401–500 nm) (501–600 nm) (601–700 nm) (701–800 nm) (801–1050 nm) LED-mix 5.1 267.5 279.4 966.8 16.6 2.5 MH 59.3 503.2 843.9 144.3 16.7 4.8 HPS 0.9 89.8 783.9 532.9 37.9 14.2 Observations revealed varied morphology di erences in the roots of the cuttings after 10 days of propagation (Figure 2A). The rooting ratio was greatly influenced by the supplementary light source (Table 2). Cuttings supplied with MH light exhibited the highest rooting ratio (100%), followed by those supplied with HPS light (91.7%) and LED-mix light (75%), while the control had lowest rooting ratio (50%). There were no significant di erences in the shoot length, shoot fresh and dry weights, chlorophyll level, and specific leaf weight among cuttings with or without supplementary light treatments. However, the roots of cuttings were vividly enhanced with supplementary light treatments, evidenced by the improvement in root parameters including the root length, number of roots, and root fresh and dry weights. Cuttings grown under MH light had the highest number of roots, followed by those grown under HPS light. The MH and HPS supplementary lights resulted in similar root fresh weights, but MH yielded the greatest root dry weight. Agronomy 2020, 10, x FOR PEER REVIEW 6 of 16 Figure 2. The morphology of carnation ‘Dreambyul’ cuttings affected by the supplementary light Figure 2. The morphology of carnation ‘Dreambyul’ cuttings a ected by the supplementary light source after 10 (A) and 25 days (B) of propagation. source after 10 (A) and 25 days (B) of propagation. Agronomy 2020, 10, 1217 6 of 15 Table 2. The growth parameters, measured at 10 and 25 days after treatment initiation, of carnation ‘Dreambyul’ a ected by the supplementary light source. Shoot Root Supplementary Cultivation Rooting Fresh Dry Specific Leaf Survival (%) Length Fresh Weight Dry Weight Chlorophyll Length Light Source Duration (%) Number Weight Weight Weight (cm) (mg) (mg) Level (SPAD) (cm) (mg) (mg) (mgcm ) Control 100.0 50.0 c 7.6 600.3 90.6 35.8 59.6 0.8 b 8 c 7.2 c 0.6 c LED 100.0 75.0 b 7.7 633.3 100.5 33.2 60.8 1.4 a 10 b 15.2 b 1.7 b 10 days MH 100.0 100.0 a 7.8 676.7 104.1 37.4 60.8 1.6 a 16 a 19.6 a 2.2 a HPS 100.0 91.7 ab 7.6 606.7 91.8 34.2 56.2 1.4 a 12 ab 17.7 a 1.8 b Control 100.0 100.0 7.8 734.3 104.0 b 38.1 b 59.6 7.3 b 20 b 142.3 c 13.1 c LED 100.0 100.0 8.1 750.6 117.6 ab 45.9 ab 57.0 7.9 ab 29 ab 173.7 b 18.7 b 25 days MH 100.0 100.0 8.1 781.3 126.8 a 50.8 a 60.2 9.1 a 36 a 267.8 a 28.7 a HPS 100.0 100.0 8.0 738.5 104.1 b 41.9 ab 57.9 8.0 ab 31 ab 199.4 ab 20.7 b Di erent letters indicate significant separation within columns by the Duncan’s multiple range test at a 0.05 level. Agronomy 2020, 10, 1217 7 of 15 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 16 Furthermore, all cuttings grown with di erent treatments survived and rooted after 25 days of Furthermore, all cuttings grown with different treatments survived and rooted after 25 days of propagation (Table 2). However, cuttings grown with supplementary lights had a higher number propagation (Table 2). However, cuttings grown with supplementary lights had a higher number and and longer roots, as well as a better root ball formation compared to those in the control (Figure 2B). longer roots, as well as a better root ball formation compared to those in the control (Figure 2B). There There were no di erences observed in the shoot length, shoot fresh weight, and specific leaf weight of were no differences observed in the shoot length, shoot fresh weight, and specific leaf weight of cuttings grown with the di erent supplementary light sources. However, the shoot dry weight was cuttings grown with the different supplementary light sources. However, the shoot dry weight was enhanced by MH light. Cuttings grown with supplementary light had higher chlorophyll contents enhanced by MH light. Cuttings grown with supplementary light had higher chlorophyll contents than the control, with MH yielding the highest value, followed by LED-mix and HPS. Moreover, the than the control, with MH yielding the highest value, followed by LED-mix and HPS. Moreover, the root length, number of roots, and root fresh and dry weights were significantly increased for cuttings root length, number of roots, and root fresh and dry weights were significantly increased for cuttings grown under the di erent supplementary lights. MH lamps resulted in the highest improvements of grown under the different supplementary lights. MH lamps resulted in the highest improvements of root parameters, followed by HPS lamps and LED-mix. root parameters, followed by HPS lamps and LED-mix. 3.2. Plant Temperature 3.2. Plant Temperature Thermal images of the cuttings under di erent treatments showed a huge di erence (Figure 3A). Thermal images of the cuttings under different treatments showed a huge difference (Figure The plant temperature was increased significantly by the supplementary light, and di erences were 3A). The plant temperature was increased significantly by the supplementary light, and differences observed among di erent supplementary light sources. The highest plant temperature was found in were observed among different supplementary light sources. The highest plant temperature was the cuttings under MH lamps, where the temperature ranged from 22.0 C to 31.8 C with an average found in the cuttings under MH lamps, where the temperature ranged from 22.0 °C to 31.8 °C with temperature of 25.0 C, which was 3.7 C higher than that of the control. The average plant temperature an average temperature of 25.0 °C, which was 3.7 °C higher than that of the control. The average plant of cuttings exposed to HPS lamps and LED-mix were, respectively, 24.0 C and 22.6 C, which were temperature of cuttings exposed to HPS lamps and LED-mix were, respectively, 24.0 °C and 22.6 °C, 2.7 C and 1.3 C higher than that of the control, respectively (Figure 3B). which were 2.7 °C and 1.3 °C higher than that of the control, respectively (Figure 3B). Figure 3. Thermal images of carnation ‘Dreambyul’ a ected by the supplementary light source (A) and Figure 3. Thermal images of carnation ‘Dreambyul’ affected by the supplementary light source (A) the temperature profiles (B). The images were taken 1 m above the cuttings at 13:00. and the temperature profiles (B). The images were taken 1 m above the cuttings at 13:00. 3.3. Root Activities 3.3. Root Activities As shown in Figure 4, the root activities of the cuttings significantly increased with the As shown in Figure 4, the root activities of the cuttings significantly increased with the supplementary light treatments. Cuttings grown with MH lamps had the highest root activities, while supplementary light treatments. Cuttings grown with MH lamps had the highest root activities, while cuttings grown with LED-mix and HPS lamps displayed no significant di erences and had a lower cuttings grown with LED-mix and HPS lamps displayed no significant differences and had a lower level of root activities. Cuttings in the control had the lowest level of root activities. level of root activities. Cuttings in the control had the lowest level of root activities. Agronomy 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/agronomy Agronomy 2020, 10, x FOR PEER REVIEW 8 of 16 Agronomy 2020, 10, x FOR PEER REVIEW 8 of 16 Agronomy 2020, 10, 1217 8 of 15 Figure 4. Effect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data are shown as the mean ± SEs, and different letters on the bars indicate significant differences according Figure 4. E ect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data Figure 4. Effect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data to the Duncan’s multiple range test at p ≤ 0.05. are shown as the mean SEs, and di erent letters on the bars indicate significant di erences according are shown as the mean ± SEs, and different letters on the bars indicate significant differences according to the Duncan’s multiple range test at p 0.05. to the Duncan’s multiple range test at p ≤ 0.05. 3.4. Quantum Yield and Stomatal Conductance 3.4. Quantum Yield and Stomatal Conductance 3.4. Quantum Yield and Stomatal Conductance The chlorophyll fluorescence ability was measured after the supplementary light treatments. The chlorophyll fluorescence ability was measured after the supplementary light treatments. The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly The chlorophyll fluorescence ability was measured after the supplementary light treatments. The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in the the stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the greatest in greatest in cuttings grown with supplementary MH light, which was almost twice that of the control. the stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the cuttings grown with supplementary MH light, which was almost twice that of the control. The stomatal The stomatal conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 greatest in cuttings grown with supplementary MH light, which was almost twice that of the control. 2 1 −2 −1 conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 mmolm s mmol·m ·s higher than that of the control, respectively (Figure 5). The stomatal conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 higher than that of the control, respectively (Figure 5). −2 −1 mmol·m ·s higher than that of the control, respectively (Figure 5). Figure 5. E ect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal Figure 5. Effect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal conductance of carnation ‘Dreambyul’. Data are shown as mean SEs, and di erent letters on the bars conductance of carnation ‘Dreambyul’. Data are shown as mean ± SEs, and different letters on the Figure 5. Effect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal indicate significant di erences according to the Duncan’s multiple range test at p 0.05. bars indicate significant differences according to the Duncan’s multiple range test at p ≤ 0.05. conductance of carnation ‘Dreambyul’. Data are shown as mean ± SEs, and different letters on the 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses bars indicate significant differences according to the Duncan’s multiple range test at p ≤ 0.05. 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly a ected by 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly the supplementary light source (Figure 6). The highest chlorophyll a and b contents were found in affected by the supplementary light source (Figure 6). The highest chlorophyll a and b cuttings grown under MH lamps. The cuttings treated with HPS lamps had the lowest contents of The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly contents were found in cuttings grown under MH lamps. The cuttings treated with HPS chlorophyll a and b among those treated with the three supplementary light sources. The chlorophyll affected by the supplementary light source (Figure 6). The highest chlorophyll a and b a/b ratio in the control group was 2.0, which was significantly lower than that of cuttings grown with lamps had the lowest contents of chlorophyll a and b among those treated with the three contents were found in cuttings grown under MH lamps. The cuttings treated with HPS the supplementary lights. However, the chlorophyll a/b ratio of cuttings grown with the di erent supplementary light sources. The chlorophyll a/b ratio in the control group was 2.0, which lamps had the lowest contents of chlorophyll a and b among those treated with the three supplementary light sources exhibited no significant di erences. Soluble protein contents in leaves, supplementary light sources. The chlorophyll a/b ratio in the control group was 2.0, which Agronomy 2020, 10, x FOR PEER REVIEW 9 of 16 was significantly lower than that of cuttings grown with the supplementary lights. However, the chlorophyll a/b ratio of cuttings grown with the different supplementary light sources exhibited no significant differences. Soluble protein contents in leaves, stems, and Agronomy 2020, 10, 1217 9 of 15 roots were significantly higher for cuttings grown under MH lamps than those of the control. Cuttings grown with LED-mix had higher protein contents in the leaves but lower protein contents in the stems and roots compared to those grown with HPS lamps. For the stems, and roots were significantly higher for cuttings grown under MH lamps than those of the contents of soluble sugars, no significant differences were observed in the stems among the control. Cuttings grown with LED-mix had higher protein contents in the leaves but lower protein contents in the stems and roots compared to those grown with HPS lamps. For the contents of soluble four groups. The soluble sugar contents in the leaves and roots of cuttings were promoted sugars, no significant di erences were observed in the stems among the four groups. The soluble by the supplementary lights, where MH yielded the highest level, followed by LED-mix and sugar contents in the leaves and roots of cuttings were promoted by the supplementary lights, where HPS. There were no significant differences in the starch content of stems among cuttings MH yielded the highest level, followed by LED-mix and HPS. There were no significant di erences in grown with the supplementary lights, which were higher compared to that of the control the starch content of stems among cuttings grown with the supplementary lights, which were higher group. MH resulted in the highest starch content in leaves and roots, followed by LED-mix compared to that of the control group. MH resulted in the highest starch content in leaves and roots, and HPS. followed by LED-mix and HPS. Figure 6. E ect of the supplementary light source on contents of chlorophyll a and b, soluble proteins, Figure 6. Effect of the supplementary light source on contents of chlorophyll a and b, soluble proteins, soluble sugars, and starch of carnation ‘Dreambyul’. Data are shown as the mean SEs, and di erent soluble sugars, and starch of carnation ‘Dreambyul’. Data are shown as the mean ± SEs, and different letters within columns indicate significant di erences according to the Duncan’s multiple range test at letters within columns indicate significant differences according to the Duncan’s multiple range test p 0.05. at p ≤ 0.05. 4. Discussion 4. Discussion Many studies have been conducted on in vitro or in vivo adventitious rooting utilizing diverse Many studies have been conducted on in vitro or in vivo adventitious rooting utilizing diverse light qualities, but few studies have been done on in vivo adventitious rooting in greenhouses with light qualities, but few studies have been done on in vivo adventitious rooting in greenhouses with the application of di erent supplementary light sources in daylight situations. Bielenin [34] found the application of different supplementary light sources in daylight situations. Bielenin [34] found no no significant di erences in rooting for Juniperus and Thuja cuttings when supplementary lighting significant differences in rooting for Juniperus and Thuja cuttings when supplementary lighting was 2 1 was provided at 20 molm s PPFD. However, 70% red + 30% blue LEDs resulted in a somewhat −2 −1 provided at 20 µmol·m ·s PPFD. However, 70% red + 30% blue LEDs resulted in a somewhat 2 1 enhanced root mass for Petunia when the supplementary light intensity was 70 molm s PPFD [16]. −2 −1 enhanced root mass for Petunia when the supplementary light intensity was 70 µmol·m ·s PPFD 2 1 In our study, 100 molm s PPFD supplementary lighting from LED-mix, MH, or HPS lamps −2 −1 [16]. In our study, 100 µmol·m ·s PPFD supplementary lighting from LED-mix, MH, or HPS lamps strongly enhanced the rooting of carnation ‘Dreambyul’. After 10 days of propagation, all of the strongly enhanced the rooting of carnation ‘Dreambyul’. After 10 days of propagation, all of the cuttings grown with supplementary MH lighting rooted and cuttings exposed to LED-mix and HPS cuttings grown with supplementary MH lighting rooted and cuttings exposed to LED-mix and HPS rooted at a lower level, while the control group showed the weakest root formation, where only 50% of rooted at a lower level, while the control group showed the weakest root formation, where only 50% the cuttings rooted. Additionally, the use of supplementary light enhanced the root biomass, including of the cuttings rooted. Additionally, the use of supplementary light enhanced the root biomass, root length, number, and fresh and dry weights, compared to control. Though all the cuttings survived and rooted after 25 days of cultivation, the cuttings propagated under the di erent supplementary lights had dramatically enhanced shoot and root biomasses. Moreover, they had developed a better root ball that was more suitable for transplanting. All the observations indicated that the utilization of Agronomy 2020, 10, 1217 10 of 15 supplementary lights could promote the growth and development of shoots and roots of carnation cuttings and shorten the cultivation duration. It is reported that only 30 and 24% of the consumed electricity is converted to light by HPS and MH lamps, respectively, while the rest of the used electricity vanishes as heat [26,35]. Our results showed that when supplied at the same intensity, MH and HPS lamps resulted in a vividly increased plant temperature, compared to the control and LED-mix (Figure 3). Images taken at 10:00 and 16:00 showed similar results (data not shown). Temperature can potentially stimulate adventitious rooting by promoting the nutrient/water uptake and influencing certain enzymatic actions [36]. With some exceptions, the optimal rooting temperature would be between 20 and 30 C, and the number of roots, rooting ratio, and the speed of root formation increased with increasing temperature [37–39]. In this study, the heat released by MH lamps resulted in the highest average plant temperature, 3.7 C higher than that of the control. Cuttings grown with supplementary MH lighting had the highest shoot biomass, number of roots, root fresh and dry weights, and the fastest root formation. However, the plant temperature amplified by HPS was higher than LED-mix, but the shoot dry weight of cuttings grown under LED-mix was slightly higher than that of cuttings grown under HPS. In addition, there were no significant di erences in the root number, length, and dry weight amongst the cuttings grown under LED-mix and HPS. This phenomenon may reflect an important role the light spectrum plays in addition to the temperature in the rooting and growth of cuttings. Rooting of Tsuru-rindo (Tripterospermum japonicum) cultured in vitro was promoted by red light but was inhibited by blue light [40]. On the other hand, Shin [41] observed that root growth was enhanced by blue light and hindered by red light for in-vitro-cultured Doritaenopsis plants. The contrary observations may indicate that the e ects of the light spectrum on the root growth is species-dependent. In terms of root physiology research, TTC reduction has been used extensively to di erentiate between living and lifeless roots and vitality in plants [42–45]. TTC is colorless until it is reduced to triphenyl formazan, a bright red pigment that is simply extracted and photometrically measured [46]. Our study exhibited increased TTC reduction in all cuttings grown with supplementary lighting compared to that of the control, reflecting that supplementary light enhances root activities. The MH lamps induced the highest level of root activities among the three supplementary light sources, while no significant di erences were observed in the root activities of cuttings grown with LED-mix and HPS. It was reported that the root activities are dependent on the soil temperature [47]. Our studies revealed MH lamps results in the highest ambient temperature around the cuttings, followed by HPS and LED-mix. However, the root activities of cuttings grown with supplementary HPS lighting was the same as those grown with LED-mix, which was consistent with how the two supplementary light sources a ected the shoot and root biomasses. The results of the study leads us to conclude that the root activities were influenced by the combined e ects of the temperature and light spectrum. The Fv/Fm ratio, the maximum quantum yield of PSII photochemistry, is frequently used as an indicator of the photoinhibition or other kinds of stresses caused by the PSII system [48–50]. The Fv/Fm value of the vast majority of C plants is from 0.8 to 0.84 under normal physiological conditions. When the Fv/Fm value of plants is below this range, the plant is exposed to environmental stresses [51]. In our research, the supplementary light treatments increased the Fv/Fm value compared to the control. These results implied that MH, as well as LED-mix and HPS, had a positive e ect on the photosynthetic eciency compared to the control. Additionally, the cuttings grown under supplementary lights had a higher light utilization eciency and a stronger ability to adapt to low-light conditions. However, the Fv/Fm values presented in this study were lower than a healthy range, which implied that the cuttings were stressed. This could be explained by the light intensity during the cultivation period. The average 2 1 daily light intensity of control (sunlight) and supplementary light, 260 and 360 molm s PPFD, respectively, were lower than that in the growing season. A similar trend was observed in the change of the stomatal conductance. Stomata are important in regulating the gas exchange with the atmosphere and influence two basic physiological processes: photosynthesis and transpiration [52,53]. Previous studies have reported that a supplementary light source influences the formation, development, and Agronomy 2020, 10, 1217 11 of 15 functioning of stomata [54–56]. Compared with red light, blue light increased the stomatal conductance in Cordyline australis, Ficus benjamina, and Sinningia speciosa [57]. An increasing share of blue light also amplified the stomatal conductance and the net photosynthetic rate in Alternanthera brasiliana [24]. Consistent with the results of previous studies, the highest upsurge of stomatal conductance was found with supplementary MH lighting, followed by LED-mix and HPS. MH lamps had the highest proportion of blue light, followed by LED-mix and HPS. Plant pigments have specific wavelength absorption patterns known as the absorption spectra, and biosynthetic wavelengths for the production of plant pigments are referred to as the action spectra [22]. Cuttings grown under all supplementary light treatments displayed a higher chlorophyll a content, as it has a wider spectrum compared to that of chlorophyll b. Chlorophyll a is what enables photosynthesis [58]. Compared with HPS, MH and LED-mix used in the current study had a higher proportion of blue light, which lead to an increase in the chlorophyll content, which agrees with the results of previous studies that blue light is favorable to chlorophyll formation [22,59]. Furthermore, we found that the chlorophyll a/b ratio was largely increased with the application of supplementary light. This was in agreement with the reports that plants grown under high-intensity lights and the sun exhibit larger ratios of chlorophyll a/b than plants grown under a low light intensity [60,61], and the ratio increased with higher light intensity during growth [62,63]. An increased rooting capacity is related to an upsurge in the synthesis of soluble proteins during the root regeneration process [64]. Adventitious rooting relies on an adequate supply of carbohydrates in the root regeneration zone, where they can encourage root instigation and expansion [65,66]. In this study, the contents of soluble proteins, soluble sugars, and starch in leaves, stems, and roots were measured. The trends in soluble proteins and carbohydrates in the cuttings were similar to those of the root biomass and root activities (Figure 6 and Table 2). Light—especially red and blue—can stimulate photosynthetic enzyme activities and improves stomatal opening, which is the foundation of photosynthesis [67]. Compared to HPS lamps, MH and LED-mix provide a higher percentage of red and blue lights, which resulted in a greater increase of soluble proteins and carbohydrates. Terfa et al. [68] showed higher levels of soluble carbohydrates in the plants grown under LEDs (red:blue = 8:2) than those grown under HPS in Rosa  hybrid. However, a logical, rigorous mechanism to discover the impact soluble proteins and carbohydrates have on adventitious rooting remains to be studied. 5. Conclusions This study demonstrated that the application of supplementary light was very e ective in improving the adventitious root formation during the propagation of carnation ‘Dreambyul’ cuttings. Application of supplementary light increased the plant temperature, quantum yield, stomatal conductance, chlorophyll a and b contents, root activities, as well as higher levels of soluble proteins and carbohydrates. The MH supplementary light was found to be the best to increase shoot and root biomasses, as well as accelerating root formation. Therefore, MH lamps can be strategically used to enhance the root formation during propagation of carnation cuttings. Author Contributions: Conceptualization, B.R.J. and H.W.; methodology, B.R.J. and M.W.; software, M.W.; validation, B.R.J.; formal analysis, B.R.J. and M.W.; investigation, M.W. and J.X.; resources, B.R.J.; data curation, M.W. and J.X.; writing—original draft preparation, M.W.; writing—review and editing, B.R.J., M.W., H.W., and J.X.; visualization, M.W. and J.X.; supervision, B.R.J.; project administration, B.R.J. and M.W.; funding acquisition, B.R.J. and M.W. All authors have read and agreed to the published version of the manuscript. 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Supplementary Light Source Affects Growth and Development of Carnation ‘Dreambyul’ Cuttings

Wang, Mengzhao; Xiao, Jie; Wei, Hao; Jeong, Byoung Ryong
Agronomy , Volume 10 (8) – Aug 18, 2020
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Abstract

agronomy Article Supplementary Light Source A ects Growth and Development of Carnation ‘Dreambyul’ Cuttings 1 1 1 1 , 2 , 3 , Mengzhao Wang , Jie Xiao , Hao Wei and Byoung Ryong Jeong * Department of Horticulture, Division of Applied Life Science (BK21 Plus Program), Graduate School of Gyeongsang National University, Jinju 52828, Korea; meng.zhao@163.com (M.W.); xiaojsicau@163.com (J.X.); oahiew@gmail.com (H.W.) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Korea Research Institute of Life Science, Gyeongsang National University, Jinju 52828, Korea * Correspondence: brjeong@gnu.ac.kr; Tel.: +82-55-772-1913 Received: 22 July 2020; Accepted: 17 August 2020; Published: 18 August 2020 Abstract: Carnation (Dianthus caryophyllus L.) is a major floricultural crop, cultivated widely for cut flowers. This study was conducted to determine the optimal supplementary light source for the cutting propagation of carnation ‘Dreambyul’ cuttings. Terminal cuttings were propagated in a 2 1 glasshouse with an average of 260 molm s photosynthetic photon flux density (PPFD) coming from the sun (the control), supplemented with one of three artificial light sources: mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix), metal halide (MH) lamps, or high-pressure sodium 2 1 (HPS) lamps. The supplementary light was provided from 7:00 to 17:00 h at 100 molm s PPFD during propagation. The cuttings were kept on a fogged bench in a glasshouse for 25 days with 24/15 C day/night temperatures and 88% relative humidity. Compared with the control, better root formation was observed from cuttings grown under the supplementary lights after 10 days. After 25 days of propagation, MH significantly increased the root length, root number, root fresh and dry weights, and shoot biomass, shoot length, and shoot fresh and dry weights. The best root ball formation and the highest root activities were also found in cuttings propagated with supplementary MH light. Supplementary light increased the plant temperature, quantum yield, stomatal conductance, and the contents of chlorophyll, soluble proteins, and carbohydrates. Overall, the root formation and development of carnation ‘Dreambyul’ cuttings were significantly promoted by the three supplementary light sources. Of the three, MH was identified as the optimal supplementary light source. Keywords: adventitious root formation; artificial light; biomass; quantum yield; root activities 1. Introduction The economic value of ornamental plants has increased significantly worldwide, by 8–10% annually [1]. After rose, the most sought-after species in the global market is carnation (Dianthus caryophyllus L.), due to its excellent characteristics [2]. In Korea, carnations were cultivated over 42.9 hectares, with the total market value of 6.7 billion KRW in 2016 [3]. Carnations in the Korean market have a wide range of attractive shapes, colors, sizes, and delicate fragrances [4]. Vegetative propagation is common and rapid for several ornamental plant species. It retains the genetic traits of stock plants by creating clones with the same characteristics [5]. Ecient adventitious rooting is a key process in the vegetative propagation of horticultural species. Well-rooted cuttings are a requisite for the optimal growth of high-quality plants. Carnation is primarily propagated as cuttings in Korea [6], and profitable carnation cultivars are propagated from terminal stem cuttings collected from elite mother plants [7]. Several studies have investigated internal and external factors that a ect Agronomy 2020, 10, 1217; doi:10.3390/agronomy10081217 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1217 2 of 15 the rooting of carnation cuttings during propagation. Such factors include the rooting substrate [8], rooting hormones [9], propagation season [10], cutting types [11], and endogenous hormone levels in stem cuttings [12]. However, not many studies have been conducted to investigate the e ects of light on the rooting of carnation cuttings during the propagation process. Light is the foremost energy source for plant growth. Supplementary lights are broadly used in agricultural production for the advancement in crop growth, yield, and quality, which has been confirmed by many researchers [13–15]. In a glasshouse, the daily light integral (DLI) may be reduced by the glazing materials, interior structures, and hanging baskets suspended above benches. Supplementary light is the only way to substantially increase the DLI in such environments [16], and it has been used to enhance plant growth and development in horticulture [17]. Supplementary lighting can aid plant growth by providing the necessary light when insucient natural light is present in a greenhouse. Improving the light conditions in greenhouses and optimizing the phytochemical concentrations in vegetables produced in controlled environments enhance the growth and yield [18]. Therefore, supplementary lighting is an important horticultural strategy to improve crop growth, maintain high yields all year round, and produce superior-quality plants [19,20]. The morphology, physiology, biochemistry, and transcriptional expression vary in the light spectrum [21–23]. Changes in the light spectrum can a ect the leaf structure, palisade mesophyll cell thickness, and epidermis [24,25]. The supplementary light source is a key factor of the changes in the light spectrum. High-pressure sodium (HPS) and metal halide (MH) lamps are two conventional artificial light sources commonly applied in greenhouse horticulture due to their long service life, reasonable light spectrum, and low price [24,26–28]. Light-emitting diodes (LEDs) have gained widespread attention, since they provide a narrow wavelength band with a high eciency [29–31]. However, data are still scarce regarding the choice of the light source and how the supplementary light source influences the adventitious rooting of carnations during cutting propagation. This work aimed to study the e ects of supplementary light provided by LED-mix, MH, and HPS on carnation cuttings and identify the optimal supplementary light source for greenhouse cutting propagation of carnations. 2. Materials and Methods 2.1. Plant Materials and Culture Conditions The mother plants of carnation ‘Dreambyul’ used in the study were acclimated after 0  0 micropropagation in vitro and then grown in a Venlo-type glasshouse (35 09 N, 128 06 E, Jinju, Korea) for 3 months. Then terminal cuttings from these mother plants were harvested and stuck in 128 square-cell plug trays containing a commercial BVB medium (Bas Van Buuren Substrates, EN-12580, De Lier, The Netherlands). The cuttings were kept on a mist (fogged for 30 min every 30 min) propagation bench with a mean air temperature of 24/15 C day/night and relative humidity of 88% 2 1 in a Venlo-type glasshouse with an average daily light intensity of 260 molm s photosynthetic 2 1 photon flux density (PPFD) coming from the sun. The daily light integral (DLI) was 9.36 molm d . This experiment was carried out from 22 October to 16 November 2019. 2.2. Supplementary Light Treatments This study used mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, FL300, Senmatic A/S, Søndersø, Denmark), metal halide lamps (MH, SunLumen Lighting, Gyeongju, Korea), and high-pressure sodium lamps (HPS, BLV Licht- und Vakuumtechnik, Steinhöring, Germany) as supplementary light sources, and a group without any supplementary light was set as the control. The lamps were placed above the bench, and the supplementary light intensity was set at 2 1 100 molm s PPFD from 07:00 to 17:00 by adjusting the position of the lamps after measuring multipoint averages in each treatment area. The supplementary light intensity was measured with a Agronomy 2020, 10, 1217 3 of 15 quantum radiation probe (FLA 623 PS, ALMEMO, Holzkirchen, Germany) at the top leaf level of the cuttings at night to avoid the e ects of sunlight, and the spectrum of the light sources was measured with spectroradiometers (International Light Technologies, Peabody, MA, USA). The experiment was laid out in a completely randomized design with three replications per treatment, and the treatment locations in a controlled environment were randomly laid out in order to minimize location e ects. In addition, enough space was reserved, and two layers of 0.3-mm-thick black non-woven fabric curtain was used between lamps to prevent light from one treatment from interacting with another treatment. 2.3. Measurement of Plant Temperatures Before the end of the experiment, infrared measurements with a thermographic camera (testo 882, Testo SE & Co. KGaA, Titisee-Neustadt, Germany) were taken to estimate the plant temperature on a sunny day. The thermograms were taken 1 m above the cuttings at 13:00 when the supplementary light was on and were analyzed with IRSoft (version 4.6; available online: https://www.testo.com/en/ services/download). 2.4. Determine of Root Activities To determine the root activities, 0.5 g of fresh roots were completely immersed in a solution containing 5 mL of 0.4% triphenyl-tetrazolium chloride (TTC) and 5 mL of 66 mmolL phosphate bu er (pH 7.0). The fresh roots were then incubated at 37 C for 3 h, and 2 mL of 1 molL H SO was 2 4 subsequently added to end the reaction. The roots were then taken out and the moisture was carefully wiped o . The roots were then thoroughly ground with 3 mL of ethyl acetate and a small amount of quartz sand to extract triphenyl formazan. After filtering the red extract into a 10-mL volumetric flask, the residue was washed 3 times with 2 mL of ethyl acetate before being added to the volumetric flask, and then the volume of the solution was brought up to 10 mL with ethyl acetate. The absorbance at 485 nm was recorded with a UV spectrophotometer (Libra S22, Biochrom Ltd., Cambridge, UK). The root activities were expressed as the amount of TTC reduction (g) per g of root fresh weight per hour. 2.5. Stomatal Conductance and Plant Chlorophyll Fluorescence The stomatal conductance and photosynthesis were sampled and averaged from three cuttings within each repetition. Measurements were taken on fully expanded leaves after 2 h of the supplementary light treatments. The stomatal conductance was assessed using a Decagon Leaf Porometer SC-1 (Decagon Device Inc., Pullman, WA, USA), and the photosynthesis was assessed with a FluorPen FP 100 (Photon Systems Instruments, PSI, Drásov, Czech Republic). 2.6. Chlorophyll A and B, Soluble Protein, and Carbohydrate Analyses The contents of chlorophyll a and b were estimated according to the method described by Arnon [32] with some modifications. In brief, 0.1 g of fresh leaves were cut into small pieces and kept in 2.0 mL tubes, where 2 mL of 80% acetone was added. The tubes were wrapped with tin foil and kept in a 4 C refrigerator for 12 h before extraction. The extract was centrifuged at 5000 rpm for 10 min, and the supernatant was transferred to a colorimeter tube, then the absorbance was measured at 645 and 663 nm, against a bu er blank in a UV spectrophotometer. The contents of chlorophyll a and b were determined using the following equations: ( ) 12.72 OD at 663 nm 2.59 OD at 645 nm  V Chlorophyll a = (1) Sample fresh weight ( ) 22.88 OD at 645 nm 4.67 OD at 663 nm  V Chlorophyll b = (2) Sample fresh weight Agronomy 2020, 10, 1217 4 of 15 The chlorophyll content was expressed as mg of chlorophyll per g of fresh leaf weight. The V is the volume of the extract solution. Soluble proteins were extracted with a sodium phosphate bu er and measured calorimetrically Agronomy 2020, 10, x FOR PEER REVIEW 4 of 16 using a Bradford Reagent (Sigma-Aldrich, St. Louis, MO, USA), according to Bradford’s method [33]. The soluble sugar and starch contents were determined by the Anthrone colorimetric method according The soluble sugar and starch contents were determined by the Anthrone colorimetric method to Wei et al. [15]. according to Wei et al. [15]. 2.7. Measurements of Growth Parameters 2.7. Measurements of Growth Parameters After 10 days of the supplementary light treatments, the mid-measurement was carried out to After 10 days of the supplementary light treatments, the mid-measurement was carried out to record the rooting ratio and compare the initial growth of roots and shoots of the cuttings. After 25 days record the rooting ratio and compare the initial growth of roots and shoots of the cuttings. After 25 of the supplementary light treatments, the cuttings treated with a supplementary light source had days of the supplementary light treatments, the cuttings treated with a supplementary light source formed a well-developed root ball that was easily pulled out of the plug tray. At this stage, the final had formed a well-developed root ball that was easily pulled out of the plug tray. At this stage, the measurement of the growth parameters was collected. For measuring the growth parameters, whole final measurement of the growth parameters was collected. For measuring the growth parameters, cuttings were harvested, and roots were washed carefully using tap water. Growth parameters, such whole cuttings were harvested, and roots were washed carefully using tap water. Growth as parameters, such as the fresh weight, the fresh weight, length, and the number length, an of roots, d th wer e number o e measurfed roots, wer directly, wher e measured d eas the dry irectly, weights of whereas the dry weights o shoots and roots were measur f shoots edand r after oots were me drying for 72 ash ured at 60 afte C r d in rying an oven. for 72 h The atchlor 60 °C ophyll in an oven. level was The chlorophyll level was measured using a Plus Chlorophyll Meter (SPAD 502, Spectrum measured using a Plus Chlorophyll Meter (SPAD 502, Spectrum technologies, Korea). The cuttings technologies, Korea). The cuttings were placed in liquid N2 in a −80 °C refrigerator for further were placed in liquid N in a80 C refrigerator for further analysis. analysis. 2.8. Data Collection and Analysis 2.8. Data Collection and Analysis The experimental assays used to obtain all results were repeated three times and are presented The experimental assays used to obtain all results were repeated three times and are presented as the mean standard error. The collected data were subjected to an analysis of variance (ANOVA) as the mean ± standard error. The collected data were subjected to an analysis of variance (ANOVA) followed by Duncan’s multiple range test at p < 0.05 to find the statistical significance among the followed by Duncan’s multiple range test at p < 0.05 to find the statistical significance among the treatments by the Statistical Package for the Social Sciences version 21 (SPSS Inc., Chicago, IL, USA). treatments by the Statistical Package for the Social Sciences version 21 (SPSS Inc., Chicago, IL, USA). All figures were made with the OriginPro software version 9.0 (OriginLab Corporation, Northampton, All figures were made with the OriginPro software version 9.0 (OriginLab Corporation, MA, USA). Northampton, MA, USA). 3. Results 3. Results 3.1. Morphology and Growth Parameters Analysis 3.1. Morphology and Growth Parameters Analysis Di erent supplementary light sources provide various spectral characteristics that may di er Different supplementary light sources provide various spectral characteristics that may differ from those of sunlight. LED-mix consisted of a mixture of red, blue, and white lights that occupy a from those of sunlight. LED-mix consisted of a mixture of red, blue, and white lights that occupy a narrow spectrum with two peaks at 434 and 662 nm (Figure 1A). The accumulated irradiance was narrow spectrum with two peaks at 434 and 662 nm (Figure 1A). The accumulated irradiance was mainly distributed in red (62.8%), blue (17.4%), and green (18.2%) lights (Table 1). The MH lamps were mainly distributed in red (62.8%), blue (17.4%), and green (18.2%) lights (Table 1). The MH lamps white in appearance and strong emission lines were largely dispersed in all ranges with about 32.7 and were white in appearance and strong emission lines were largely dispersed in all ranges with about 54.9% of the total irradiance focused on blue and green lights (Figure 1B, Table 1). The HPS lamps 32.7 and 54.9% of the total irradiance focused on blue and green lights (Figure 1B, Table 1). The HPS produced a characteristic golden-orange light, and the largest accumulated irradiance was mainly lamps produced a characteristic golden-orange light, and the largest accumulated irradiance was observed in green light (53.7%), followed by red (36.5%) and blue lights (6.2%) (Table 1). mainly observed in green light (53.7%), followed by red (36.5%) and blue lights (6.2%) (Table 1). Figure 1. The spectral distribution of mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, Figure 1. The spectral distribution of mixed (red: blue: white = 6:1:1) light-emitting diodes (LED-mix, (( A A )), )), meta metall halide lamps (MH halide lamps (MH, , (( B B )), )), and hig and high-pr h-pressu essur re sod e sodium ium lam lamps ps (HP (HPS, S, (C)). (C)). Agronomy 2020, 10, 1217 5 of 15 Table 1. The irradiance distributions of the supplementary light sources: LED-mix, mixed (red: blue: white = 6:1:1) light-emitting diodes; MH, metal halide lamps; and HPS, high-pressure sodium lamps. Irradiance (Wcm ) Supplementary UV Blue Green Red FR IR Light Source (250–400 nm) (401–500 nm) (501–600 nm) (601–700 nm) (701–800 nm) (801–1050 nm) LED-mix 5.1 267.5 279.4 966.8 16.6 2.5 MH 59.3 503.2 843.9 144.3 16.7 4.8 HPS 0.9 89.8 783.9 532.9 37.9 14.2 Observations revealed varied morphology di erences in the roots of the cuttings after 10 days of propagation (Figure 2A). The rooting ratio was greatly influenced by the supplementary light source (Table 2). Cuttings supplied with MH light exhibited the highest rooting ratio (100%), followed by those supplied with HPS light (91.7%) and LED-mix light (75%), while the control had lowest rooting ratio (50%). There were no significant di erences in the shoot length, shoot fresh and dry weights, chlorophyll level, and specific leaf weight among cuttings with or without supplementary light treatments. However, the roots of cuttings were vividly enhanced with supplementary light treatments, evidenced by the improvement in root parameters including the root length, number of roots, and root fresh and dry weights. Cuttings grown under MH light had the highest number of roots, followed by those grown under HPS light. The MH and HPS supplementary lights resulted in similar root fresh weights, but MH yielded the greatest root dry weight. Agronomy 2020, 10, x FOR PEER REVIEW 6 of 16 Figure 2. The morphology of carnation ‘Dreambyul’ cuttings affected by the supplementary light Figure 2. The morphology of carnation ‘Dreambyul’ cuttings a ected by the supplementary light source after 10 (A) and 25 days (B) of propagation. source after 10 (A) and 25 days (B) of propagation. Agronomy 2020, 10, 1217 6 of 15 Table 2. The growth parameters, measured at 10 and 25 days after treatment initiation, of carnation ‘Dreambyul’ a ected by the supplementary light source. Shoot Root Supplementary Cultivation Rooting Fresh Dry Specific Leaf Survival (%) Length Fresh Weight Dry Weight Chlorophyll Length Light Source Duration (%) Number Weight Weight Weight (cm) (mg) (mg) Level (SPAD) (cm) (mg) (mg) (mgcm ) Control 100.0 50.0 c 7.6 600.3 90.6 35.8 59.6 0.8 b 8 c 7.2 c 0.6 c LED 100.0 75.0 b 7.7 633.3 100.5 33.2 60.8 1.4 a 10 b 15.2 b 1.7 b 10 days MH 100.0 100.0 a 7.8 676.7 104.1 37.4 60.8 1.6 a 16 a 19.6 a 2.2 a HPS 100.0 91.7 ab 7.6 606.7 91.8 34.2 56.2 1.4 a 12 ab 17.7 a 1.8 b Control 100.0 100.0 7.8 734.3 104.0 b 38.1 b 59.6 7.3 b 20 b 142.3 c 13.1 c LED 100.0 100.0 8.1 750.6 117.6 ab 45.9 ab 57.0 7.9 ab 29 ab 173.7 b 18.7 b 25 days MH 100.0 100.0 8.1 781.3 126.8 a 50.8 a 60.2 9.1 a 36 a 267.8 a 28.7 a HPS 100.0 100.0 8.0 738.5 104.1 b 41.9 ab 57.9 8.0 ab 31 ab 199.4 ab 20.7 b Di erent letters indicate significant separation within columns by the Duncan’s multiple range test at a 0.05 level. Agronomy 2020, 10, 1217 7 of 15 Agronomy 2020, 10, x FOR PEER REVIEW 7 of 16 Furthermore, all cuttings grown with di erent treatments survived and rooted after 25 days of Furthermore, all cuttings grown with different treatments survived and rooted after 25 days of propagation (Table 2). However, cuttings grown with supplementary lights had a higher number propagation (Table 2). However, cuttings grown with supplementary lights had a higher number and and longer roots, as well as a better root ball formation compared to those in the control (Figure 2B). longer roots, as well as a better root ball formation compared to those in the control (Figure 2B). There There were no di erences observed in the shoot length, shoot fresh weight, and specific leaf weight of were no differences observed in the shoot length, shoot fresh weight, and specific leaf weight of cuttings grown with the di erent supplementary light sources. However, the shoot dry weight was cuttings grown with the different supplementary light sources. However, the shoot dry weight was enhanced by MH light. Cuttings grown with supplementary light had higher chlorophyll contents enhanced by MH light. Cuttings grown with supplementary light had higher chlorophyll contents than the control, with MH yielding the highest value, followed by LED-mix and HPS. Moreover, the than the control, with MH yielding the highest value, followed by LED-mix and HPS. Moreover, the root length, number of roots, and root fresh and dry weights were significantly increased for cuttings root length, number of roots, and root fresh and dry weights were significantly increased for cuttings grown under the di erent supplementary lights. MH lamps resulted in the highest improvements of grown under the different supplementary lights. MH lamps resulted in the highest improvements of root parameters, followed by HPS lamps and LED-mix. root parameters, followed by HPS lamps and LED-mix. 3.2. Plant Temperature 3.2. Plant Temperature Thermal images of the cuttings under di erent treatments showed a huge di erence (Figure 3A). Thermal images of the cuttings under different treatments showed a huge difference (Figure The plant temperature was increased significantly by the supplementary light, and di erences were 3A). The plant temperature was increased significantly by the supplementary light, and differences observed among di erent supplementary light sources. The highest plant temperature was found in were observed among different supplementary light sources. The highest plant temperature was the cuttings under MH lamps, where the temperature ranged from 22.0 C to 31.8 C with an average found in the cuttings under MH lamps, where the temperature ranged from 22.0 °C to 31.8 °C with temperature of 25.0 C, which was 3.7 C higher than that of the control. The average plant temperature an average temperature of 25.0 °C, which was 3.7 °C higher than that of the control. The average plant of cuttings exposed to HPS lamps and LED-mix were, respectively, 24.0 C and 22.6 C, which were temperature of cuttings exposed to HPS lamps and LED-mix were, respectively, 24.0 °C and 22.6 °C, 2.7 C and 1.3 C higher than that of the control, respectively (Figure 3B). which were 2.7 °C and 1.3 °C higher than that of the control, respectively (Figure 3B). Figure 3. Thermal images of carnation ‘Dreambyul’ a ected by the supplementary light source (A) and Figure 3. Thermal images of carnation ‘Dreambyul’ affected by the supplementary light source (A) the temperature profiles (B). The images were taken 1 m above the cuttings at 13:00. and the temperature profiles (B). The images were taken 1 m above the cuttings at 13:00. 3.3. Root Activities 3.3. Root Activities As shown in Figure 4, the root activities of the cuttings significantly increased with the As shown in Figure 4, the root activities of the cuttings significantly increased with the supplementary light treatments. Cuttings grown with MH lamps had the highest root activities, while supplementary light treatments. Cuttings grown with MH lamps had the highest root activities, while cuttings grown with LED-mix and HPS lamps displayed no significant di erences and had a lower cuttings grown with LED-mix and HPS lamps displayed no significant differences and had a lower level of root activities. Cuttings in the control had the lowest level of root activities. level of root activities. Cuttings in the control had the lowest level of root activities. Agronomy 2020, 10, x; doi: FOR PEER REVIEW www.mdpi.com/journal/agronomy Agronomy 2020, 10, x FOR PEER REVIEW 8 of 16 Agronomy 2020, 10, x FOR PEER REVIEW 8 of 16 Agronomy 2020, 10, 1217 8 of 15 Figure 4. Effect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data are shown as the mean ± SEs, and different letters on the bars indicate significant differences according Figure 4. E ect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data Figure 4. Effect of the supplementary light source on the root activities of carnation ‘Dreambyul’. Data to the Duncan’s multiple range test at p ≤ 0.05. are shown as the mean SEs, and di erent letters on the bars indicate significant di erences according are shown as the mean ± SEs, and different letters on the bars indicate significant differences according to the Duncan’s multiple range test at p 0.05. to the Duncan’s multiple range test at p ≤ 0.05. 3.4. Quantum Yield and Stomatal Conductance 3.4. Quantum Yield and Stomatal Conductance 3.4. Quantum Yield and Stomatal Conductance The chlorophyll fluorescence ability was measured after the supplementary light treatments. The chlorophyll fluorescence ability was measured after the supplementary light treatments. The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly The chlorophyll fluorescence ability was measured after the supplementary light treatments. The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS The quantum yield (Fv/Fm) of cuttings treated with the supplementary lights were significantly promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in promoted (Figure 5). The MH yielded the highest value (0.75), followed by LED-mix (0.73) and HPS (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in the the stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the (0.71). The control group exhibited the lowest quantum yield (0.67). Similar results were observed in stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the greatest in greatest in cuttings grown with supplementary MH light, which was almost twice that of the control. the stomatal conductance of carnation ‘Dreambyul’ cuttings. The stomatal conductance was the cuttings grown with supplementary MH light, which was almost twice that of the control. The stomatal The stomatal conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 greatest in cuttings grown with supplementary MH light, which was almost twice that of the control. 2 1 −2 −1 conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 mmolm s mmol·m ·s higher than that of the control, respectively (Figure 5). The stomatal conductance of the cuttings grown with LED-mix and HPS lamps were 28.7 and 20.2 higher than that of the control, respectively (Figure 5). −2 −1 mmol·m ·s higher than that of the control, respectively (Figure 5). Figure 5. E ect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal Figure 5. Effect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal conductance of carnation ‘Dreambyul’. Data are shown as mean SEs, and di erent letters on the bars conductance of carnation ‘Dreambyul’. Data are shown as mean ± SEs, and different letters on the Figure 5. Effect of the supplementary light source on the quantum yield (Fv/Fm) and the stomatal indicate significant di erences according to the Duncan’s multiple range test at p 0.05. bars indicate significant differences according to the Duncan’s multiple range test at p ≤ 0.05. conductance of carnation ‘Dreambyul’. Data are shown as mean ± SEs, and different letters on the 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses bars indicate significant differences according to the Duncan’s multiple range test at p ≤ 0.05. 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly a ected by 3.5. Chlorophyll a and b, Soluble Proteins, and Carbohydrates Analyses The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly the supplementary light source (Figure 6). The highest chlorophyll a and b contents were found in affected by the supplementary light source (Figure 6). The highest chlorophyll a and b cuttings grown under MH lamps. The cuttings treated with HPS lamps had the lowest contents of The contents of chlorophyll a and b, carbohydrates, and soluble proteins were highly contents were found in cuttings grown under MH lamps. The cuttings treated with HPS chlorophyll a and b among those treated with the three supplementary light sources. The chlorophyll affected by the supplementary light source (Figure 6). The highest chlorophyll a and b a/b ratio in the control group was 2.0, which was significantly lower than that of cuttings grown with lamps had the lowest contents of chlorophyll a and b among those treated with the three contents were found in cuttings grown under MH lamps. The cuttings treated with HPS the supplementary lights. However, the chlorophyll a/b ratio of cuttings grown with the di erent supplementary light sources. The chlorophyll a/b ratio in the control group was 2.0, which lamps had the lowest contents of chlorophyll a and b among those treated with the three supplementary light sources exhibited no significant di erences. Soluble protein contents in leaves, supplementary light sources. The chlorophyll a/b ratio in the control group was 2.0, which Agronomy 2020, 10, x FOR PEER REVIEW 9 of 16 was significantly lower than that of cuttings grown with the supplementary lights. However, the chlorophyll a/b ratio of cuttings grown with the different supplementary light sources exhibited no significant differences. Soluble protein contents in leaves, stems, and Agronomy 2020, 10, 1217 9 of 15 roots were significantly higher for cuttings grown under MH lamps than those of the control. Cuttings grown with LED-mix had higher protein contents in the leaves but lower protein contents in the stems and roots compared to those grown with HPS lamps. For the stems, and roots were significantly higher for cuttings grown under MH lamps than those of the contents of soluble sugars, no significant differences were observed in the stems among the control. Cuttings grown with LED-mix had higher protein contents in the leaves but lower protein contents in the stems and roots compared to those grown with HPS lamps. For the contents of soluble four groups. The soluble sugar contents in the leaves and roots of cuttings were promoted sugars, no significant di erences were observed in the stems among the four groups. The soluble by the supplementary lights, where MH yielded the highest level, followed by LED-mix and sugar contents in the leaves and roots of cuttings were promoted by the supplementary lights, where HPS. There were no significant differences in the starch content of stems among cuttings MH yielded the highest level, followed by LED-mix and HPS. There were no significant di erences in grown with the supplementary lights, which were higher compared to that of the control the starch content of stems among cuttings grown with the supplementary lights, which were higher group. MH resulted in the highest starch content in leaves and roots, followed by LED-mix compared to that of the control group. MH resulted in the highest starch content in leaves and roots, and HPS. followed by LED-mix and HPS. Figure 6. E ect of the supplementary light source on contents of chlorophyll a and b, soluble proteins, Figure 6. Effect of the supplementary light source on contents of chlorophyll a and b, soluble proteins, soluble sugars, and starch of carnation ‘Dreambyul’. Data are shown as the mean SEs, and di erent soluble sugars, and starch of carnation ‘Dreambyul’. Data are shown as the mean ± SEs, and different letters within columns indicate significant di erences according to the Duncan’s multiple range test at letters within columns indicate significant differences according to the Duncan’s multiple range test p 0.05. at p ≤ 0.05. 4. Discussion 4. Discussion Many studies have been conducted on in vitro or in vivo adventitious rooting utilizing diverse Many studies have been conducted on in vitro or in vivo adventitious rooting utilizing diverse light qualities, but few studies have been done on in vivo adventitious rooting in greenhouses with light qualities, but few studies have been done on in vivo adventitious rooting in greenhouses with the application of di erent supplementary light sources in daylight situations. Bielenin [34] found the application of different supplementary light sources in daylight situations. Bielenin [34] found no no significant di erences in rooting for Juniperus and Thuja cuttings when supplementary lighting significant differences in rooting for Juniperus and Thuja cuttings when supplementary lighting was 2 1 was provided at 20 molm s PPFD. However, 70% red + 30% blue LEDs resulted in a somewhat −2 −1 provided at 20 µmol·m ·s PPFD. However, 70% red + 30% blue LEDs resulted in a somewhat 2 1 enhanced root mass for Petunia when the supplementary light intensity was 70 molm s PPFD [16]. −2 −1 enhanced root mass for Petunia when the supplementary light intensity was 70 µmol·m ·s PPFD 2 1 In our study, 100 molm s PPFD supplementary lighting from LED-mix, MH, or HPS lamps −2 −1 [16]. In our study, 100 µmol·m ·s PPFD supplementary lighting from LED-mix, MH, or HPS lamps strongly enhanced the rooting of carnation ‘Dreambyul’. After 10 days of propagation, all of the strongly enhanced the rooting of carnation ‘Dreambyul’. After 10 days of propagation, all of the cuttings grown with supplementary MH lighting rooted and cuttings exposed to LED-mix and HPS cuttings grown with supplementary MH lighting rooted and cuttings exposed to LED-mix and HPS rooted at a lower level, while the control group showed the weakest root formation, where only 50% of rooted at a lower level, while the control group showed the weakest root formation, where only 50% the cuttings rooted. Additionally, the use of supplementary light enhanced the root biomass, including of the cuttings rooted. Additionally, the use of supplementary light enhanced the root biomass, root length, number, and fresh and dry weights, compared to control. Though all the cuttings survived and rooted after 25 days of cultivation, the cuttings propagated under the di erent supplementary lights had dramatically enhanced shoot and root biomasses. Moreover, they had developed a better root ball that was more suitable for transplanting. All the observations indicated that the utilization of Agronomy 2020, 10, 1217 10 of 15 supplementary lights could promote the growth and development of shoots and roots of carnation cuttings and shorten the cultivation duration. It is reported that only 30 and 24% of the consumed electricity is converted to light by HPS and MH lamps, respectively, while the rest of the used electricity vanishes as heat [26,35]. Our results showed that when supplied at the same intensity, MH and HPS lamps resulted in a vividly increased plant temperature, compared to the control and LED-mix (Figure 3). Images taken at 10:00 and 16:00 showed similar results (data not shown). Temperature can potentially stimulate adventitious rooting by promoting the nutrient/water uptake and influencing certain enzymatic actions [36]. With some exceptions, the optimal rooting temperature would be between 20 and 30 C, and the number of roots, rooting ratio, and the speed of root formation increased with increasing temperature [37–39]. In this study, the heat released by MH lamps resulted in the highest average plant temperature, 3.7 C higher than that of the control. Cuttings grown with supplementary MH lighting had the highest shoot biomass, number of roots, root fresh and dry weights, and the fastest root formation. However, the plant temperature amplified by HPS was higher than LED-mix, but the shoot dry weight of cuttings grown under LED-mix was slightly higher than that of cuttings grown under HPS. In addition, there were no significant di erences in the root number, length, and dry weight amongst the cuttings grown under LED-mix and HPS. This phenomenon may reflect an important role the light spectrum plays in addition to the temperature in the rooting and growth of cuttings. Rooting of Tsuru-rindo (Tripterospermum japonicum) cultured in vitro was promoted by red light but was inhibited by blue light [40]. On the other hand, Shin [41] observed that root growth was enhanced by blue light and hindered by red light for in-vitro-cultured Doritaenopsis plants. The contrary observations may indicate that the e ects of the light spectrum on the root growth is species-dependent. In terms of root physiology research, TTC reduction has been used extensively to di erentiate between living and lifeless roots and vitality in plants [42–45]. TTC is colorless until it is reduced to triphenyl formazan, a bright red pigment that is simply extracted and photometrically measured [46]. Our study exhibited increased TTC reduction in all cuttings grown with supplementary lighting compared to that of the control, reflecting that supplementary light enhances root activities. The MH lamps induced the highest level of root activities among the three supplementary light sources, while no significant di erences were observed in the root activities of cuttings grown with LED-mix and HPS. It was reported that the root activities are dependent on the soil temperature [47]. Our studies revealed MH lamps results in the highest ambient temperature around the cuttings, followed by HPS and LED-mix. However, the root activities of cuttings grown with supplementary HPS lighting was the same as those grown with LED-mix, which was consistent with how the two supplementary light sources a ected the shoot and root biomasses. The results of the study leads us to conclude that the root activities were influenced by the combined e ects of the temperature and light spectrum. The Fv/Fm ratio, the maximum quantum yield of PSII photochemistry, is frequently used as an indicator of the photoinhibition or other kinds of stresses caused by the PSII system [48–50]. The Fv/Fm value of the vast majority of C plants is from 0.8 to 0.84 under normal physiological conditions. When the Fv/Fm value of plants is below this range, the plant is exposed to environmental stresses [51]. In our research, the supplementary light treatments increased the Fv/Fm value compared to the control. These results implied that MH, as well as LED-mix and HPS, had a positive e ect on the photosynthetic eciency compared to the control. Additionally, the cuttings grown under supplementary lights had a higher light utilization eciency and a stronger ability to adapt to low-light conditions. However, the Fv/Fm values presented in this study were lower than a healthy range, which implied that the cuttings were stressed. This could be explained by the light intensity during the cultivation period. The average 2 1 daily light intensity of control (sunlight) and supplementary light, 260 and 360 molm s PPFD, respectively, were lower than that in the growing season. A similar trend was observed in the change of the stomatal conductance. Stomata are important in regulating the gas exchange with the atmosphere and influence two basic physiological processes: photosynthesis and transpiration [52,53]. Previous studies have reported that a supplementary light source influences the formation, development, and Agronomy 2020, 10, 1217 11 of 15 functioning of stomata [54–56]. Compared with red light, blue light increased the stomatal conductance in Cordyline australis, Ficus benjamina, and Sinningia speciosa [57]. An increasing share of blue light also amplified the stomatal conductance and the net photosynthetic rate in Alternanthera brasiliana [24]. Consistent with the results of previous studies, the highest upsurge of stomatal conductance was found with supplementary MH lighting, followed by LED-mix and HPS. MH lamps had the highest proportion of blue light, followed by LED-mix and HPS. Plant pigments have specific wavelength absorption patterns known as the absorption spectra, and biosynthetic wavelengths for the production of plant pigments are referred to as the action spectra [22]. Cuttings grown under all supplementary light treatments displayed a higher chlorophyll a content, as it has a wider spectrum compared to that of chlorophyll b. Chlorophyll a is what enables photosynthesis [58]. Compared with HPS, MH and LED-mix used in the current study had a higher proportion of blue light, which lead to an increase in the chlorophyll content, which agrees with the results of previous studies that blue light is favorable to chlorophyll formation [22,59]. Furthermore, we found that the chlorophyll a/b ratio was largely increased with the application of supplementary light. This was in agreement with the reports that plants grown under high-intensity lights and the sun exhibit larger ratios of chlorophyll a/b than plants grown under a low light intensity [60,61], and the ratio increased with higher light intensity during growth [62,63]. An increased rooting capacity is related to an upsurge in the synthesis of soluble proteins during the root regeneration process [64]. Adventitious rooting relies on an adequate supply of carbohydrates in the root regeneration zone, where they can encourage root instigation and expansion [65,66]. In this study, the contents of soluble proteins, soluble sugars, and starch in leaves, stems, and roots were measured. The trends in soluble proteins and carbohydrates in the cuttings were similar to those of the root biomass and root activities (Figure 6 and Table 2). Light—especially red and blue—can stimulate photosynthetic enzyme activities and improves stomatal opening, which is the foundation of photosynthesis [67]. Compared to HPS lamps, MH and LED-mix provide a higher percentage of red and blue lights, which resulted in a greater increase of soluble proteins and carbohydrates. Terfa et al. [68] showed higher levels of soluble carbohydrates in the plants grown under LEDs (red:blue = 8:2) than those grown under HPS in Rosa  hybrid. However, a logical, rigorous mechanism to discover the impact soluble proteins and carbohydrates have on adventitious rooting remains to be studied. 5. Conclusions This study demonstrated that the application of supplementary light was very e ective in improving the adventitious root formation during the propagation of carnation ‘Dreambyul’ cuttings. Application of supplementary light increased the plant temperature, quantum yield, stomatal conductance, chlorophyll a and b contents, root activities, as well as higher levels of soluble proteins and carbohydrates. The MH supplementary light was found to be the best to increase shoot and root biomasses, as well as accelerating root formation. Therefore, MH lamps can be strategically used to enhance the root formation during propagation of carnation cuttings. Author Contributions: Conceptualization, B.R.J. and H.W.; methodology, B.R.J. and M.W.; software, M.W.; validation, B.R.J.; formal analysis, B.R.J. and M.W.; investigation, M.W. and J.X.; resources, B.R.J.; data curation, M.W. and J.X.; writing—original draft preparation, M.W.; writing—review and editing, B.R.J., M.W., H.W., and J.X.; visualization, M.W. and J.X.; supervision, B.R.J.; project administration, B.R.J. and M.W.; funding acquisition, B.R.J. and M.W. All authors have read and agreed to the published version of the manuscript. 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