Introduction

Orchid production has been increasing worldwide in China, Germany, Japan, the Netherlands, Taiwan, Thailand, and the United States (Griesbach 2000;Lopez and Runkle 2005). Despite this large-scale production, few genera, including Cymbidium, Phalaenopsis, Dendrobium, and Oncidium, are popular.

Most orchids grow slowly and take several years to flower. In particular, Cymbidium has 3 years of juvenility, and flowering cannot be induced by any treatment (Hew and Yong 2004). Thus, a strategy for shortening the Cymbidium cultivation period is needed. Previous studies showed that growth of Cymbidium was promoted by night interruption (NI), and flowering was hastened to within 2 years compared with 3 - 4 years of general cultivation (Kim et al. 2011). NI with 120 μmol･m^-2 ･s^-1 directly affected vegetative growth for ensuring flower initiation by increasing net photosynthesis (Kim et al. 2013). Oh et al. (2009) also reported that supplemental lighting (SL) at 200 μmol･m^-2 ･s^-1 during NI increased the number of leaves and dry weight in Cyclamen persicum. Not only NI, but also extended photoperiod by SL, such as day extension (DE), increased growth in lettuce (Martineau et al. 2012), sweet pepper (Dorais 2003), Campanula, and Lupinus (Cavins and Dole 2001).

Providing SL by NI or DE affects the mean photosynthetic daily light integral (DLI). Increasing DLI increases biomass accumulation, hastens development, and improves final plant quality in many floricultural plants (Oh et al. 2008). Additionally, high DLI can increase the growth rate by promoting photosynthesis (Nemali and Van Iersel 2004).

Markvart et al. (2009) reported that, under the same DLI, different SL timing resulted in no differences in vegetative growth of Chrysanthemum. Park et al. (2013) reported that NI from 02:00 to 06:00 was the most effective for development with no significant effect in net photosynthesis or subsequent growth compared to different timing in herbaceous plants. However, Dodd et al. (2005) suggested that the correct matching of the plant circadian clock with the environmental period positively influences net photosynthesis. SL by NI, which is an abnormal artificial light at midnight, might have an adverse effect on photosynthesis and growth even though it provides the same DLI compared to DE.

To our knowledge, a comparison of the effects of SL during night or DE on growth and photosynthesis has not been examined in Cymbidium. Moreover, it is unknown to what degree timing nighttime SL affects photosynthesis and vegetative growth. Thus, the objective of our study was to determine the best SL timing for enhancing photosynthesis and vegetative growth in Cymbidium.

Materials and Methods

Plant and growth conditions

28 plants of Nine-month-old Cymbidium hybrids ‘Yang Guifei’ and ‘Wine Shower’ (Mukoyama Orchids Co., Ltd., Yamanashiken, Japan) were transplanted into 12 cm pots filled with 100% pine bark. The plants were previously purchased from Haepyeung Orchid Farm (Gongju, Republic of Korea) at 2 months old and grown for 7 months (from Dec. to Jul.) at the Seoul National University Farm (Suwon, Republic of Korea). The temperature inside the greenhouse was maintained between min. 15 and max. 32°C. Photoperiod was determined based on natural daylength; when light intensity was low, additional SL was provided with high-pressure sodium (HPS, 312 μmol･m^-2 ･s^-1) lamps (SKL-01; GEO, Hwasung, Republic of Korea) from 9:00 to 10:30 and from 15:30 to 17:00 (from Dec. to Apr.). Plants were irrigated daily with tap water using a sprinkler. Additionally, water-soluble fertilizer (EC 1.0 mS･cm^-1; Technigro 20N–9P–20K, Sun-Gro Horticulture, Bellevue, WA, USA) was applied once a week. Four grams of slow-release fertilizer (11N–4.4P–15.7K+1.2Mg+TE, Everris Co., Geldermalsen, the Netherlands) was placed on the top of the substrate. Pesticides were applied at their recommended rates as needed throughout the growing period.

Light treatments

Uniform Cymbidium hybrids were then moved to a controlled-environment plant production system maintained at 20°C to identify growth by SL timing. Plants were treated with four different SL timings: from 22:00 to 02:00 (middle of the night, MN), from 17:00 to 21:00 (end of day extension, DE), from 07:00 to 09:00 plus 17:00 to 19:00 (both beginning and end of night as split day extension, SDE), and non-SL (8/16 h photoperiod; short day, SD). Among the SL treatments, DLI was set at the same value to determine growth under MN and DEs. All SL was provided by two types of 100% red LEDs (Stec LED C., Paju, Republic of Korea). The energy of red LEDs was peaked at 640 and 660 nm as measured with a spectroradiometer (Stellar Net, Tampa, FL, USA) because chlorophyll a and b efficiently absorb the energy around 640 and 675 nm, respectively (French et al. 1972). Daytime lighting averaged 350 μmol･m^-2 ･s^-1 for 8 h, and 150 μmol･m^-2 ･s^-1 was provided during SL application. Atmospheric CO₂ in the controlled-environment plant production system was supplied 800 μmol･mol^-1 during the night. The length of the SL treatments was 4 months (from Jul. to Nov.).

Data collection and analysis

Pseudobulb diameter, the number of leaves, leaf length, and leaf width were measured monthly during the experimental period. Pseudobulb diameter was measured at the widest point of the pseudobulb using a digital vernier caliper (ABS Digimatic Caliper; Mitutoyo Co., Ltd., Tsukuba, Japan). The longest leaf was measured from the base of the pseudobulb and was used to represent leaf length. Dry weights of leaves, pseudobulbs, and roots were measured after 7 days in a dry oven at 80°C at the end of treatment. Relative chlorophyll content of the third mature leaf from the top was measured monthly using a SPAD meter (SPAD 502; Konica Minolta Sensing Inc., Sakai, Osaka, Japan).

Net photosynthetic assimilation rate (A_n) of the plant was measured in both Cymbidium ‘Yang Guifei’ and ‘Wine Shower’ plants after 14 weeks of treatment using a portable photosynthesis measuring system (Li 6400; Li-Cor Co., Inc., Lincoln, NE, USA) equipped with an infrared gas analyzer. Three plants per treatment were randomly chosen and used for measurement. The third mature leaf from the top was clamped onto a 6 cm², clear-top head chamber. The stomatal ratio was input to be 1 because Cymbidium is a monocotyledon with equal stomata density on top and bottom. Relative humidity in the leaf chamber was 60%. The temperature was kept at 20°C during both day and night. The CO₂ concentration in the leaf chamber was maintained at approximately 400 and 800 μmol･mol^-1 during day and night, respectively, to match the growth conditions. A_n was measured every hour for 5 minutes over a 24 hour period. Daily A_n was calculated from the A_n data.

Completely randomized design was used in this study with 7 plants for each treatment. Data were analyzed using the SAS system for Windows version 9.3 (SAS Inst. Inc., Cary, NC, USA). Differences among treatment means were assessed by Duncan’s multiple range test at p < 0.05. Regression and graph module analyses were performed using Sigma Plot software version 8.0 (Systat Software, Inc., Chicago, IL, USA).

Results

Photosynthetic assimilation rate

A_n in response to SL timings was measured for 24 h after 14 weeks of SL treatment in both cultivars (Fig. 1). The SL for 4 hours prolonged the photosynthetic period, irrespective of application timing. The mean A_n increased during the nighttime, with a rate of approximately 1.55 and 1.24 μmol CO₂･m^-2 ･s^-1 compared to 0.54 and 0.44 μmol CO₂･m^-2 ･s^-1 under the SD condition, in Cymbidium ‘Yang Guifei’ and ‘Wine Shower’, respectively. Daily A_n was obtained after calculating A_n, which significantly (p < 0.001) increased in SL t reatments compared with SD (Fig. 2 ). Daily A_n had no statistical differences among SL timing in either cultivar. Daily A_n of SL treatments doubled, up to 96.86, 109.80, and 97.61 mol CO₂･m^-2·d^-1 in MN, DE, and SDE, respectively, compared with 47.83 under SD condition in ‘Yang Guifei’. Daily A_n of ‘Wine Shower’ was 103.32, 98.66, 98.37, and 59.15 mol CO₂·m^-2·d^-1 in MN, DE, SDE, and SD, respectively.

Vegetative growth

SL accelerated growth in both cultivars of Cymbidium regardless of SL timing (Table 1 and Fig. 3). Pseudobulb diameter was significantly (p < 0.05 and 0.01) greater under all SL treatments (MN, DE, and SDE) than SD in both cultivars. Among SL treatments, the pseudobulb diameter of Cymbidium ‘Yang Guifei’ was 26.68, 25.32, 25.42, and 23.11 in MN, DE, SDE, and SD respectively, and a similar trend was observed in ‘Wine Shower’. No differences among SL timings were found (Table 1 and Fig. 3). Other growth parameters, such as the number of new bulbs, the number of leaves, leaf length, leaf width, and chlorophyll content, also had no statistical differences among the treatments in either cultivar (Table 1). The growth of pseudobulb diameter corresponded to daily A_n (Fig. 2) regardless of SL timing. Dry weight was not significantly different among the treatments in ‘Yang Guifei’, though pseudobulb weight was higher in SL treatments compared with SD (Table 2). In ‘Wine Shower’, dry weight of pseudobulbs and roots increased significantly (p < 0.05) in the SL treatments.

Discussion

Prolonging the photoperiod with SL (increasing DLI) improves growth and yield for many horticultural crops. Although Cymbidium is a non-photoperiodic plant, it requires long days for rapid growth and pseudobulb maturity (Lopez and Runkle 2005). Dorais et al. (1996) reported that extending photoperiod by SL increased photosynthetic efficiency and carbon partitioning. In this study, A_n of both Cymbidium hybrids was increased under a 4 hours of extension in photoperiod regardless of timing. These results were similar to the previous study that NI with high light intensity increased plant growth in Cymbidium ‘Red fire’ and ‘Yokihi’ because it increased A_n compared with those of short-day conditions without NI (Kim et al. 2015).

Plant growth results when carbohydrates synthesized in photosynthesis exceed those lost in respiration (Nemali and Van Iersel 2004). Kim et al. (2013) also reported that a short-day with a long dark period is disadvantageous in the growth of long-day plants such as Cymbidium ‘Red Fire’ because of a high level of carbon loss with a short period of photosynthesis and a long period of dark respiration. These results are consistent with a study showing that prolonging the light duration by SL or shortening the dark period favored carbohydrate accumulation (Xu et al. 2004).

Correct matching of plant circadian clock with environmental period positively influences the net photosynthetic assimilation rate (Dodd et al. 2005; Hotta et al. 2007). Thus, we applied three different SL timings to evaluate the effect of the circadian clock during nighttime. Especially, MN, which interrupts nighttime, might reset circadian clock and influence in photosynthesis. However, there was no significant difference among SL timings in this study. MN as abnormal artificial light did not affect A_n and daily A_n in both Cymbidium hybrids. This result corresponds to the result of Markvart et al. (2009), where Chrysanthemum plants did not change their photosynthetic behavior under different SL timings. These results indicate that, if the net photosynthetic assimilation rate is not affected by SL timing, the photosynthetic assimilation rate is based only on the amount of light received (DLI). Since the DLI was identical in SL timings, there was no significant effect in net photosynthesis or growth in Cymbidium.

Several studies have suggested that SL accelerates growth when the light environment is poor (Shin et al. 2010;Treder 2003). In this study, only the pseudobulb diameter was increased by SL. Since the pseudobulb is the primary organ for storing photosynthates in Cymbidium during vegetative growth, a massive amount of carbohydrates made in leaves accumulates in this organ (Ng and Hew 2000). Thus, pseudobulb could be a major sink organ in the sink-source relationship, and its size is a significant parameter in determining the growth of Cymbidium. These results could be explained partially by the growth characteristics of Cymbidium, which grows rather slowly, with 3 years of vegetative growth (Hew and Yong 2004). Kim et al. (2011) reported that the value ranges of growth, such as the number of leaves, leaf length, and pseudobulb diameter, were much larger among treatments during the 2^nd year of NI treatment. Thus, if SL was provided for more years, the effect on vegetative growth would be more pronounced.

In conclusion, Increased DLI by SL promotes vegetative growth via enhanced photosynthetic assimilation rate. SL timing was not an important factor for growth, when the DLI is identical, in both Cymbidium hybrids. However, even though SL timing does not directly affect the photosynthesis and growth of Cymbidium hybrids, it can be considered in developing the SL strategy in Cymbidium production. SL has been widely used in northern latitudes, because of poor light conditions in winter. In terms of production costs, in Japan, electricity costs can be reduced by over 20% when using SL during the less expensive time (Kjaer et al. 2012). Also, if there was no additional CO₂, supplying SL late at night will be a more effective strategy because the amount of CO₂ is naturally increased through respiration at night in a greenhouse. These results could be useful in selecting artificial lighting in commercial greenhouses to effectively promote the vegetative growth of Cymbidium.