Introduction
Phalaenopsis is an important genus in the Orchid family. Species in this genus have been extensively studied over the last decade because of the high demand for cut flowers and potted plants, especially in Germany, Japan, the Netherlands, Taiwan, and the United States of America (De et al. 2014;Kwon et al. 2018). Ornamental orchids from tissue culture flasks in this genus usually take more than 12-18 months to mature vegetatively and approximately three months thereafter to produce flowers; the blooms exhibit a variety of colors and last for 2-3 months (De et al. 2013). Thus, it takes more than a year or two for Phalaenopsis orchids to develop from the plantlet stage to marketable plants.
Owing to this long growth period, Phalaenopsis species are considered to be costly and energy-consuming (in a commercial sense) with uncertain returns on investment (Dueck et al. 2016). As a result, research on optimum growth conditions during the vegetative stage is important to reduce the production cost and shorten the cultivation period of this ornamental plant (Guo et al. 2012;Lee et al. 2015;Lopez and Runkle 2005).
The roots of Phalaenopsis spp. are not only absorptive organs but also contain chlorophyll and perform photosynthetic functions (Dole and Wilkins 2005, Martins et al. 2010). Hence, poor growth of the roots often affects the plant’s total growth performance, resulting in a longer growing period and decreased ornamental quality (Chomicki et al. 2015;Dycus and Knudson 1957;Lopez and Runkle 2005). To produce high quality and shorten the cultivation period of Phalaenopsis, it is necessary to focus on the early stage of plantlet, which has been reported to have a great influence on the subsequent growth and development of the plant (Susilo et al. 2014). Likewise, it has been reported that maintaining optimal temperature and fertilizer levels is crucial at this stage (Runkle et al. 2005).
Previous research has shown that temperature affects vegetative growth, flowering, and chlorophyll fluorescence in Phalaenopsis (Dueck et al. 2016;Ouzounis et al. 2014). In addition, mineral fertilization has been reported to improve flowering quality (Ruamrungsri et al. 2007) and flower induction (Lin et al. 2019) and accelerates vegetative growth (Wang 2010). The temperature levels and fertilizer application rates vary depending on the variety or cultivar; however, these factors are not yet well understood (Baker 2008). Plant growth is highly affected by the supply of carbohydrates which are essential in synthesizing new tissues (Gent and Seginer 2012), hence carbohydrate contents of different organs are common parameters taken to investigate treatment effects. Carbohydrate accumulation in Phalaenopsis have been associated with healthy plantlets (Nurcahyani et al. 2020), highly correlated with inflorescence initiation and may vary depending on certain conditions such as temperature (Chen et al. 1994).
Phalaenopsis ‘KS Little Gem’ is a new variety that has been reported to have long-lasting flowers that are considered desirable with excellent traits (Kwon et al. 2017). There is, to date, only limited information on the environmental factors in cultivation and best management practices for optimal growth and development of this cultivar (Lee et al. 2017;South et al. 2017). Accordingly, we aimed to evaluate the effects of temperature and fertilizer concentration on root growth and carbohydrate content of young plantlets of P. ‘KS Little Gem’.
Materials and Methods
Plant materials
Three-month-old after deflasking Phalaenopsis ‘KS Little Gem’ plantlets from Kangsan Orchids, Gangseo-gu, Busan, Korea, were used as experimental materials. Initial growth parameters were collected from experimental units. Leaf parameters included the number of leaves (7.0 ± 0.4), leaf length (6.5 ± 0.9 cm), width (3.4 ± 0.3 cm), thickness (1.6 ± 0.2 mm), and fresh shoot weight (6.3 ± 1.6 g), and root parameters included the number of roots (7.3 ± 1.4), root length (9.9 ± 3.3 mm), diameter (3.1 ± 0.7 cm), and fresh roots weight (2.7 ± 1.1 g).
Experimental design and treatment conditions
The experiment was laid out in a 3 × 3 factorial design in a completely randomized design. The variables were temperature level (20, 25, and 30°C) and fertilizer rate (0.33, 0.50, and 1.00 g･L-1). The experiments were replicated three times. Each replicate consisted of 13 plantlets. The environmental conditions were controlled using a growth chamber. Relative humidity was set to 60±5% and photosynthetic photon flux density to 210 μmol･m-2･s-1 (day/night: 12/12 h) with the use of fluorescent and high-pressure sodium lamps. Water soluble fertilizer (N:P:K = 20:20:20, Peters Professional Fertilizer, Everris NA Inc., USA) was applied with 20mL/pot by foliar spray to each treatment every 10 days. The fertilizer concentration was calculated based on the recommended application rate (0.5 g･L-1) under greenhouse cultivation. Each pot (7 cm in diameter), containing sphagnum moss substrate, was irrigated with 40 mL of water every three days.
Growth parameters
Prior to the treatments, parameters of the leaves (length, width, thickness, and fresh weight) and roots (number of roots, fresh root weight, length, and diameter) were measured. The longest leaf was tagged for measurement for each plant per replicationlongest. For root parameters, the longest root was tagged and measured. Two2 months after applying the treatments, data were collected at each stage to confirm their physiological stages after cultivation in the growth chamber (DF-95G-specialDooree ScienceKorea) for two months. Chlorophyll content was measured using a SPAD 502 chlorophyll meter (Konica Minolta, Japan).
Sugar content analysis
Frozen leaves and roots were analyzed using 2 g of sample per replication. These materials were applied in liquid nitrogen and ground in a mortar. The ground materials were then mixed with 20 mL of distilled water and centrifuged at 8,000 rpm for 20 min to extract the supernatant. The supernatant was filtered through filter paper (Whatman No. 541, UK) and run through 20 mL of distilled water. These were purified using a Sep-Pak C18 Cartridge (Waters Inc., USA) and filtered with a membrane filter (Millipore 0.20 μm) to create the final sample. High-performance liquid chromatography (HPLC) (600E, Waters Co., USA) was performed to analyze sucrose, glucose, and fructose, as described by Hunter et al. (1991). The operating equipment and conditions for the HPLC instrument included a column (Sugar-Park I) at 90°C, detector (RI 410) with a flow rate of 0.5 mL/min, and, for the mobile phase, Ca-EDTA buffer (50 mg Ca-EDTA/1L dH2O) was used.
Starch analysis
All starch contents were analyzed following the phenolsulfuric acid method (Dubois et al. 1956) using leaves and roots remaining after extracting free sugar. The dried residue was placed in a beaker and 1 mL of H2SO4 was added. Distilled water (0.5 mL) was then gradually added while rotating the magnetic bar. H2SO4 (1 mL) was added when the solution turned black during heating. After heating, distilled water (0.5 mL) was added to adjust the total volume to 10 mL. This solution was filtered and quantified in a 50 mL volumetric flask. A 0.5 mL of these samples was added with 0.5 mL of 5% phenol, and the mixture was shaken. Afterward, 2.5 mL H2SO4 was added, and the mixture was allowed to react at room temperature. Glucose solutions at standard concentrations (25, 50, 75, and 100 mg･L-1) were prepared, and the same procedure was applied to the sample. The starch content was measured at 490 nm using a UV-VIS spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan) with the phenol-sulfuric acid method, and the results are expressed as mg･g-1 dry weight.
Statistical analysis
Data were statistically analyzed using SAS package version 9.4 (SAS Institute, Cary, NC, USA). Two-way analysis of variance was used to determine significant differences between treatment groups, and Duncan’s multiple range test (DMRT) (α = 0.05) was performed to compare treatment means.
Results
Growth responses
The effects of temperature and fertilizer rate on the leaves and roots growth of young P. ‘KS Little Gem’ plantlets are shown in Table 1. The results revealed that temperature conditions significantly affected the leaves and roots growth parameters of the plantlets. Despite mean differences between treatments, fertilizer rates and interaction effects failed to statistically affect the said data.
The highest number of leaves was observed in plants grown at 25 and 30°C. Leaf length was greatest for plants grown at 25 (7.5 cm) and 20°C (7.3 cm), with no significant difference between the treatments. The thickest (3.8 cm) and widest (1.9 mm) leaf was obtained from plantlets grown at 25°C, followed by 20 and 30°C. Chlorophyll content was also found to be highest at 25°C, and its value significantly decreased at 30°C. Root length and width were found to be highest in plantlets grown at 20°C and 25°C, respectively. Although treatments were found to be non-significant, increased leaf length, chlorophyll content and root width was observed with increased fertilizer rate.
The effects of temperature and fertilizer rate on fresh leaves and roots weight of young P. ‘KS Little Gem’ plantlet are shown in Table 2. The fresh weight was significantly affected by temperature. The results revealed that lower temperatures (20 and 25°C) resulted in plantlets with higher fresh weights in both leaves and roots, which translated to a higher total fresh weight. The fresh weight of leaves and roots did not significantly differ at 20 and 25°C, but significantly decreased at 30°C. Among interaction effects, total fresh weight was significantly affected by combination of treatments. It was observed that plantlets treated at 20℃ + 0.33 g･L-1, 25℃ + 0.50 g･L-1, and 25°C + 1.00 g･L-1 had the highest total fresh weigh with 20.0, 20.4, 21.9 mg.g-1, while those treated at 30°C regardless of their fertilizer rates produced the lowest total fresh weight which is 50% less of the previously mentioned treatments.
Effects on the carbohydrate content
Table 3 shows the effects of temperature and fertilizer rate on free sugar content in the leaves of young plantlets of P. ‘KS Little Gem’. Sucrose, glucose, fructose, and total sugar content were significantly affected by temperature conditions for both leaves and roots. However, fertilizer rates and interaction of treatments did not significantly affect leaf and root sugar content.
The highest leaf sugar content was found in plants grown at 25°C, followed by 20 and 30°C. However, varying results were observed for the root sugar content. The highest sucrose content, 0.33 mg･g-1, was obtained from roots under 30°C, and the highest glucose content, 0.50 mg･g-1, was found in roots treated under 20°C, which significantly differed from results at higher temperatures. However, fructose content was found to be significantly higher in roots under 25°C (1.50 mg･g-1), which was significantly reduced from the lowest and highest temperature treatments.
Despite non-significance of fertilizer rates, higher amounts of sucrose (0.6 mg.g-1), fructose (0.2 mg.g-1) and total sugar content (0.9 mg.g-1) in leaves of P. ‘KS Little Gem’ plantlets were found in those fertilized with 1.00 g.L-1. In the roots, highest sucrose and glucose contents were found from plantlets fertilized with 0.33 g.L-1, while those fertilized with 1.00 g.L-1 had the highest fructose and total sugar content. Among combined treatments, plantlets grown under 25°C in all fertilizer rates produced the highest leaf and root sugar content. However, interaction effects were not statistically significant.
The total starch content in the leaves and roots of young plantlets of P. ‘KS Little Gem’ is shown in Table 4. Similar results were found for total starch content, wherein temperature treatment alone significantly affected starch content. The starch content in the leaves was highest at 25°C (53.5 mg･g-1) and was significantly different from results at 20 (42.9 mg･g-1) and 30°C (8.1 mg･g-1).
Discussion
Phalaenopsis hybrids are valuable ornamental crops, and there has been considerable, ongoing research to increase their potential in orchid breeding programs. Optimal cultivation practices for new cultivars need to be established in order for genetic modification to be fully expressed and thus increase production (Semiarti et al. 2007). The goal of the present study was to investigate the effects of temperature and fertilizer application on the growth of Phalaenopsis hybrid plantlets.
The growth of the plantlets was significantly affected by temperature whereas neither fertilizer nor the interaction of temperature and fertilizer had a significant effect on the plantlets. Data on growth parameters suggest that growing P. ‘KS Little Gem’ plantlets at 25°C produced better leaves and roots. Temperature is one of the primary factors affecting the rate of plant development (Ali et al. 2005;Dole and Wilkins 2005) and has been found to be important in orchids, including Phalaenopsis (Paradiso et al. 2012). At the optimum temperature, plant growth is accelerated, whereas low or high temperatures may induce lag or cessation. Our results show that Phalaenopsis, which is a tropical plant, is able to grow between temperatures ranging from 20°C to 30°C; however, the optimum temperature is 25°C, at which growth, sugar, and starch content were found to be highest in both leaves and roots. On the other hand, subjecting plantlets to a higher temperature of 30°C significantly affected these parameters. Ota et al. (1991) reported that Phalaenopsis hybrid leaves had increased net CO2 uptake with day/night fluctuation temperature of 25/15°C compared to those grown under constant 20°C. In general, at high temperatures, the volume of respiration (Q10) increases, decreasing net photosynthesis (Hopkins 1999). Several species of the genus Phalaenopsis reported that growing plants between 20-25°C were found to have optimal growth, flowering, and carbohydrate content, such as that of P. amabilis, during their vegetative phase (Blanchard and Runkle 2006;Chen et al. 1994;Sakanishi et al. 1980).
No significant differences were found among fertilizer rated for all plant parameters. Similar results were found from the studies of Rodriguez et al. (2005) of orchids in which they suggested that these non-significant responses using the application of commercial water-soluble fertilizers, e.g., Peters®, may be due to the deficiency of calcium and sulfur which are important in stem and root growth. However, when combined with other organic fertilizers, plants were highly responsive to its application (Rodrigues 2005).Likewise, results of Wang (1996) had similar results wherein Phalaenopsis orchids responded similarly despite fertilizer concentration, except for those of fresh weight. Ha et al. (2018) explained that Phalaenopsis orchids are slow-growing and would not likely need higher fertilization rates at specific times, hence they recommended the use of slow-release fertilizers to be supplemented at certain growth periods.
The decrease in photosynthetic rate can reduce the carbohydrate content of many plants (Araya et al. 2006;Paul and Driscoll 1997). The results showed that sugar and starch contents were remarkably higher in the leaves than in the roots. Likewise, sugar and carbohydrate contents were found to be higher in plants subjected to temperatures at 20°C. Pollet et al. (2011) reported that starch, glucose, and fructose are degraded by respiration at temperatures above 24°C in their study of energy efficiency in Phalaenopsis which indicated an increased respiratory CO2 production. Additionally, sugar is an indicator of organ activity, and plants move and accumulate sugars in areas where growth is vigorous (Zahara et al. 2016). Invertase, a sucrose hydrolase, is involved in the accumulation of sugars, and its activity has been reported to be high at these growth sites (Lin et al. 2015;Yuan et al. 2016). Kim and Suzuki (1989) reported that stem inhibition by topical treatment resulted from reduced sugar loading in the stem because of the inhibition of invertase activity. Invertase activity was not measured in this study, whereas in the case of Phalaenopsis plantlets at 30°C, invertase activity was inhibited in the roots more than leaves, leading to decreased sugar movement and inhibition of root growth. In contrast, at 25°C, invertase activity in the roots increased, suggesting that the accumulation of sugars and root growth were promoted. The decrease in growth of roots at a higher temperature compared to that of its leaves may be a physiological response of the plant to compensate for the decrease in sugar content.
The effect of temperature on the growth of Phalaenopsis has been reported previously (Ali et al. 2005;Chen et al. 2000;Hatfield and Prueger 2015;Song et al. 2015;Susilo et al. 2013) but was found to vary depending on the cultivar. In this study, we found that a high temperature of 30°C significantly inhibited the growth of Phalaenopsis roots. Additional studies are recommended to determine the mechanisms underlying these effects.
