Introduction

Carnation (Dianthus caryophyllus L.) is an essential floricultural crop with a high global market interest (Burchi et al. 1996), and are popular as cuttings and potted flowers for its ornamental value (Birlanga et al. 2015). In Korea, carnations are planted over 42.9 hectares, mostly in Gyeongnam and Busan regions, with the total market value at 6.7 billion KRW in 2016 (MAFRA 2017). The area and economic value of carnations continue to increase each year, projecting a continued high demand for carnation plantlets. Carnations have traditionally been propagated by cuttings, which occupied about 20% of those cultivated in Korea (Lee et al. 2006). In addition, many researchers have studied the application of tissue culture methods for the rapid micropropagation of carnations (Nakano et al. 1994;Kallak et al. 1997;Jain et al. 2001;Karami and Kordestani 2017).

Plants need light as the energy source for photosynthesis and morphogenesis. Light also stimulates the primary and secondary metabolism in plants (Carvalho et al. 2011). The application of different light qualities have been studied for plants in greenhouses, but not much for plants in vitro (Gupta and Sahoo 2015). Light emitting diodes (LEDs) have recently become popular, due to their low heat radiation, compact size, long lifespan, low environmental impact, low energy/electricity consumption, high efficiency, and varied spectral qualities (Samuolienė et al. 2012). The availability of LEDs with the ability to specify spectral outputs to match the action spectra of photosynthesis and photomorphogenesis created the platform for LED-based plant illumination systems (Tamulaitis et al. 2005). Blue and red lights absorbed by the chlorophyll are responsible for photosynthesis and the metabolism of primary metabolites, and improve plant biomass (Kim et al. 2004). Muneer et al. (2017) showed that r ed a nd blue lights from L EDs affected the g rowth of carnations in vitro, reduced hyperhydricity and enabled normal photosynthetic metabolism. Incorporating blue and red LEDs similarly enhanced the quality of carnations ‘Green Beauty’ and ‘Purple Beauty’ propagated in vitro (Manivannan et al. 2017). Besides, Kozai (1991) explained that the carbon dioxide concentration is very important for the photosynthetic activity and growth of plants in vitro. Explants (leafy, single node cuttings) of carnation were cultured for 30 days in test tubes with soft plastic caps in a growth chamber at an enriched CO₂ concentration, which resulted in better growth of the in vitro plantlets (Kozai and Iwanami 1988). In this study, we investigated the effects of the light quality and container ventilation on the growth, physiology, antioxidant metabolism, and mineral contents of carnations in vitro.

Materials and Methods

Plant materials and culture conditions

The single node cuttings with a pair of leaves of carnation ‘Marble Beauty’ were obtained from plantlets grown in vitro. Explants were cultured in a MS medium (Murashige and Skoog 1962) supplemented with 3% (w/v) sucrose and 0.8% (w/v) agar. The pH of the medium was adjusted to 5.8 and autoclaved at 1 atm and 121°C for 15 minutes. A ll plants were p laced in the growth c hamber a t 25/18°C (day/night) under a 16-hour photoperiod, 33 μmol·m^-2·s^-1 photosynthetic photon flux density (PPFD), 80% relative humidity (RH), and 350 μmol·mol-1 CO₂.

Experimental design

Carnation explants were cultured in two container types: (1) containers covered without regular caps, and (2) containers covered with caps having one ventilation filter (Nhon Millipore Ltd., Yonezawa, Japan) each against a hole of 1 cm diameter. After planted, all explants were cultured under white LEDs for 28 days before being placed under different light quality treatments, including white (control), red, blue, or blue + red (1:2 ratio) LEDs for additional 28 days. This experiment utilized a completely randomized design for two factors with three replications per treatment, where each replication had two containers with seven explants inside.

Measurements of physiological, biochemical, and elemental properties

On day 56 after being planted, the carnations were harvested for measurements of the growth parameters, which include the hyperhydricity percentage, number of shoots and roots, shoot length, total number of nodes per plant, root length, and fresh and dry weights of shoots and roots. Leaves were analyzed using a scanning electron microscope ( SEM) a ccord ing to the method of Mujib et a l. (2014) with some modifications. Initially, the third or fourth leaf from the shoot tip was cut and fixed in 2.5% glutaraldehyde overnight at 4°C and washed with a 0.1 M phosphate buffered saline (PBS, pH 7.0). Then, the samples were dipped in a buffered 1% osmium tetroxide solution (pH 7.0) f or 2 h ours a t 4°C, f ollowed by washing three times with the PBS for 10 minutes to remove osmium tetroxide solution. Samples were then rinsed with a graded ethanol series (30, 50, 70, and 90 sec) for 10 minutes each. After being dipped in acetone for 20 minutes, samples were taken out and put in a drying oven for 10 minutes at 60℃ for dehydration. All samples were finally gold-coated and observed with a scanning electron microscope (JSM-6380, JEOL, Tokyo, Japan).

The chlorophyll contents of leaves in vitro were measured according to the method of Arnon (1949). Fresh carnation leaves, at the third position from the shoot tip, were cut into p ieces and d ipped i n 80% ( v/v) a cetone f or 7 2 hours for pigment extraction. The total chlorophyll content was determined by measuring the absorbance at 645 and 663 nm with a spectrophotometer (USB 2000 Fiber Optic Spectrometer, Ocean Optics Inc., Dunedin, FL, USA).

To study the antioxidant enzyme activities, leaf proteins were extracted by grind ing 0.1 g of the leaf sample with a 1.5 mL protein extraction solution. They were then centrifuged at 13000 rpm for 20 minutes at 4°C. Afterwards, the supernatant was taken out and kept in 1.5 mL Eppendorf tube (3810X, Eppendorf, Hamburg, Germany) at 4°C. The total protein contents of the samples were estimated according to the method of Bradford (1976) based on a bovine serum albumin (BSA) standard curve.

The extracted carnation proteins were also used for antioxidant enzyme analysis. The superoxide dismutase (SOD) activity was measured by applying the nitro blue tetrazolium (NBT) repression method (Giannopolitis and Ries 1977). The guaiacol peroxidase (GPX) activity was estimated based on the number of enzymes expected for the formation of tetraguaiacol per minute (Shah et al. 2001). The catalase (CAT) enzyme activity was determined following Cakmak and Marschner’s method (1992), and the ascorbate peroxidase (APX) activity was assayed as illustrated by Nakano and Asada (1981).

The mineral contents of the in vitro plants were determined by using an inductively coupled plasma (ICP) spectrometer (Optima 4300DV/5300DV, Perkin Elmer, Waltham, MA, USA) following the method developed by Yin et al. (2013). In vitro plants were dried and powdered with a stainless steel mill (Cytclotec, Model 1093, Tector, Hoganas, Sweden) and ashed at 525°C for 4 hours in a Naberthern muffle furnace (Model LV 5/11/B180, Lilienthal, Breman, Germany). These ash particles were resolved with 25% HCl acid solution, then analyzed by ICP.

Statistical analysis

The collected data were analyzed for statistical significance with the SPSS (Statistical Package for the Social Sciences) program (SPSS 13.0, SPSS INC., Chicago, Illinois, USA), using an analysis of variance (ANOVA) of two factors. When the significance was defined, Duncan’s multiple range tests (DMRT) was applied at a p ≤ 0.05 level. Graphs were drawn with the OriginPro 9.0 program (OriginPro 9.0, OriginLab Co., Northampton, MA, USA).

Results and Discussion

The hyperhydricity of micropropagated shoots, formerly known as vitrification, undoubtedly results from culture conditions, and may be influenced by stress factors which include wounding, infiltration of the soft culture medium, a high ionic intensity, a high nitrogen concentration, certain balances of plant growth regulators, and a humid, airtight atmosphere. Stress factors are detrimental to the in vitro plant morphology and physiology. Some studies on propagating in vitro carnations have recorded symptoms of hyperhydricity (Saher et al. 2004;Muneer et al. 2017). Muneer et al. (2017) implemented blue and red LED t reatments to m od erate hyperhydricity in the ‘Green Beauty’, ‘Purple Beauty’, and ‘Inca Magic’ cultivars of carnation. In this study, we observed that red LEDs induced fewer instances of hyperhydricity in carnation ‘Marble Beauty’ grown in unventilated cultures than white LEDs did. Additionally, the ventilation condition also affected the hyperhydricity of carnations (Fig. 2); the average hyperhydricity percentage of carnations in ventilated cultures was 38.4%, much lower than that of carnations in unventilated cultures (62.7%), similar to results reported by Majada et al. (2000). Majada et al. (2000) showed that the ventilation condition resulted in differing anatomical features of the shoots and leaves in carnations. The anatomical differencess of carnation ‘Marble Beauty’ leaves and shoots under ventilated and unventilated conditions in this experiment were in accordance with the results of Majada et al. (2000). When grown in ventilated cultures, the hyperhydricity of potato and eucalyptus leaves was reduced (Zobayed et al. 2001). This may be due to the fact that ventilated containers can enhance CO₂ exchange and reduce the amount of ethylene in the container to help in vitro plants grow well (Jackson et al. 1991). Fig. 1

The combination of different light qualities and ventilation conditions greatly influenced the growth of carnation ‘Marble Beauty’ over the 56 days of culture; the number of shoots, shoot length, total number of nodes, root length, and number of roots were all significantly affected (Fig. 1 and Table 1). As shown in Table 1, the characteristics of carnation ‘Marble Beauty’ shoots cultured under different light qualities present statistically significant differences. Carnations grown under blue LEDs had the highest number of shoots (5.1 shoots/explant), regardless of the ventilation condition. In Dendrobium candidum in vitro cultures, blue LEDs changed the levels of endogenous hormones, especially cytokinins, and significantly promoted shoot induction (Lin et al. 2011). Blue LEDs were also very effective in promoting the production of flavonoids in Cyclocarya paliurus leaves. There were significant positive correlations between the leaf flavonoid contents and the relative gene expression of key enzymes (phenylalanine ammonia lyase, PAL; 4-coumaroyl CoA-ligase, 4CL; and chalcone synthase, CHS).

As Table 1 illustrates, shoot lengths were significantly influenced by not only the ventilation condition but also by the light quality. The shoot length was the greatest when the in vitro carnations were grown under red LEDs in unventilated cultures (8.42 cm). Red + blue LEDs combined with ventilated culture resulted in the lowest shoot length (2.48 cm) (Table 1, Fig. 3). This was similar to the responses of carnation cultivars ‘Purple Beauty’ and ‘Green Beauty’ to different light qualities (Manivannan et al. 2017). Red light increases stem elongation, a means considered to be interfered by high levels of endogenous gibberellin (GA) (Toyomasu et al. 1993). Red LEDs also promoted stem elongation of Oncidium plantlets in vitro (Chung et al. 2010). The total number of carnation ‘Marble Beauty’ nod es w as s ignificantly a ffected by the different light qualities. Nguyen et al. (2016) showed that LEDs also influenced the regeneration of Coffea canephora, especially at the callus stage induced from leaves.

On d ay 56, the root s ystem of c arnation ‘Marble Beauty’ plants displayed significant differences, indicative of responses to the light quality and ventilation condition. The number of roots was high when in vitro carnations were grown in ventilated cultures with white LEDs, blue LEDs, or red + blue LEDs (12.5,11.2,11.3, respectively), whereas it was low for in vitro carnations grown in unventilated cultures und er white LEDs, blue LEDs o r red + b lue LEDs (Table 1). Blue LEDs stimulated the development of the root system and resulted in a higher number of leaves per shoot in Vanilla planifolia in vitro (Ramírez-Mosqueda et al. 2017). White LEDs also increased the root biomass of Codonopsis lanceolata seedlings (Ren et al. 2018). Dianthus caryophyllus ‘Hong Hac’ cultured in erlenmeyer flasks closed with both a cotton plug and/or a plastic bag ventilated with the filter membrane had a greater increase in the number of both leaves and roots (Nguyen et al. 2014). Persian walnut plants cultured in the medium supplemented with 3% sucrose in vessels closed with a clear polypropylene lid with two syringe filters on the lid had the longest roots (3.45 cm) and the highest rooting formation percentage (62.5%) (Amin et al. 2014). Fig. 4

The light quality and ventilation condition both affected the fresh and dry weights of carnation shoots and roots. Carnations had high shoot fresh weights when treated with white LEDs or blue LEDs in unventilated cultures (748 and 674 mg, respectively). Red LEDs on carnations grown in ventilated cultures also resulted in a high shoot fresh weight (648 mg). Manivannan et al. (2017), reported that carnations ‘Green Beauty’ and ‘Purple Beauty’ had the highest fresh and dry weights when they were cultured under blue LEDs. However, the ventilation condition resulted in significant differences of the fresh and dry weights of carnation shoots and roots. The average fresh weight of carnations in unventilated cultures (611.7 mg) was higher than that of carnations i n ventilated cultures (517 mg). In c ontrast, t he root dry weight was higher for carnations in ventilated cultures (61 mg) than that of carnations in unventilated cultures (49 mg). The fresh weight of carnations was higher when grown in unventilated cultures than in ventilated cultures. However, the dry shoot weight of in vitro carnations in ventilated cultures was higher than that of in vitro carnations in unventilated cultures. The explants grown in unventilated cultures have been more dehydrated and had a larger water weight removed when dried. The fresh root weight of carnations in ventilated cultures (77 mg) was higher than that in unventilated cultures (37 mg) (Fig. 5).

Chlorophyll absorbs the light energy that drives photosynthesis. The thylakoid membranes contain the site of the light-dependent, energy-conserving responses of photosynthesis. The total chlorophyll contents (chlorophyll a + chlorophyll b) and the chlorophyll a/b ratio had been understood to vary predictably with shading (Martin and Warner 1984;Chow et al. 1991). In this experiment, the total chlorophyll contents were significantly different between carnation ‘Marble Beauty’ grown in ventilated cultures and those grown in unventilated cultures. The total chlorophyll contents of in vitro plants were higher when grown in ventilated cultures (0.45 μg·mg^-1 FW) than when grown in unventilated cultures (0.21 μg·mg^-1 FW), regardless of the light quality (Table 2). Amin et al. (2014) reported similar findings when micropropagating Persian walnut plants in ventilated and unventilated cultures. In ventilated cultures, the total chlorophyll contents of carnation ‘Marble Beauty’ were the highest when treated with white LEDs (0.6 μg·mg^-1 FW) (Table 2), similar to when carnation ‘Purple Beauty’ was cultured under white light combined with 1000 μmol·mol-1 CO₂ (Park et al. 2018). The combination of light treatments and ventilation conditions resulted in statistically significant differences in the chlorophyll a/b ratio of carnations in vitro. The chlorophyll a/b ratio of carnations grown under white LEDs in unventilated cultures was the highest (5.13) (Table 2). This ratio exceeds the standard ratio, measured between 3.0 and 3.6, and is representative of poor photosynthesis by carnations grown in such conditions (Wild et al. 1973).

The light quality and ventilation conditions also affected the characteristics and performance of the stomata (Fig. 6). Carnations grown under blue LEDs or red + blue LEDs had a higher number of stomata per given leaf area with higher activity exhibition, than carnations grown under red LEDs or white LEDs did. Blue LEDs led carnations to have larger stomata than r ed LEDs d id . This was a lso the case i n the study conducted by Manivannan et al. (2017), where carnations ‘Green Beauty’ and ‘Purple Beauty’ grown under blue LEDs had a higher density of larger stomata than those grown under red LEDs did. The enhancement of the stomatal density by blue LEDs was much more pronounced for in vitro grapevine plants (Poudel et al. 2008). Blue light caused the stomata of Xanthium strumarium L. leaves to open nearly ten times more effectively than red light did (Sharkey and Raschke 1981). Besides, the stomata of carnation plants cultured in vitro in ventilated cultures would relate to the growth of in vitro plants acclimatized in the ex vitro stage in the greenhouse. The lack of ventilation in unventilated cultures limited gaseous transfers, affecting the humidity grades inside the vessel (Majada et al. 2000). In this study, for all light qualities considered, the stomata were higher in d ensity a nd had t hicker cell walls when carnations were grown in unventilated cultures, which are similar to the results in the micropropagation of Persian walnut plants (Amin et al. 2014), and Scrophularia yoshimurae Yamazaki (Chen et al. 2006) in vitro. When carnations were cultured in ventilated vessels, the exchange of CO₂ with the outside resulted in excellent stomatal performance, w here t he s tomata r espond ed until t he C O₂ concentration increased (Morison 1998).

Giannopolitis and Ries (1977) updated that SOD was the initial enzyme associated with the ROS excess in plants, which resolved the serious reactive superoxide radicals into hydrogen peroxide and molecular oxygen. Enzymes, especially H₂O₂ scavengers such as APX, GPX, and CAT, form the antioxidant defense systems to protect plant cells by scavenging ROS (Gill and Tuteja 2010). In this study, the different light qualities combined with ventilation conditions improved the intracellular antioxidant enzyme activities of carnation ‘Marble Beauty’. That is to say, the light quality in conjunction with the ventilation conditions magnified the activities of antioxidant enzymes GPX, APX, and CAT and led to statistically significant differences on this carnation. The effects on the SOD activity of the combination of light treatments and ventilation conditions in this study were not significantly different from the results of Manivannan et al. (2017) on carnations ‘Green Beauty’ and ‘Purple Beauty’. The light quality affected the principal components of the enzymatic antioxidant metabolism (Shohael et al. 2006) and the activity of the antioxidant enzymes of carnations (Manivannan et al. 2017). Carnations grown under blue LEDs in ventilated cultures had the most powerful CAT activities (31 unit min^-1·mg^-1 protein), while carnations grown under red + blue LEDs in ventilated cultures had the greatest APX activities (58 unit min^-1·mg^-1 p rotein). Red LEDs on carnations in unventilated vessels also induced the strongest GPX activities (173 unit min^-1·mg^-1 protein) (Fig. 7). Interestingly, APX and CAT activities were higher for carnations in ventilated cultures than unventilated cultures, while GPX activities displayed the opposite trend. AbdElgawad et al. (2016) illustrated that antioxidant enzyme activities would generally increase in response to stresses, but may not extend further, or even decline, depending the type of enzyme when stresses are combined with elevated CO₂ levels.

Macronutrients are usually significantly correlated to molecular structures and required in large quantities by plants (in excess of 10 mmol kg^-1 of DW) (Hopkins 1999). They play an important role in the growth and development, as well as the breeding processes, of plants. How the contents of K, Ca, Mg, Na, and P are a ffected by the light quality and the ventilation condition in carnation ‘Marble beauty’ are very important. Carnations in unventilated cultures had higher c ontents of K , Ca, Mg, and Na when t reated with red LEDs than with other LEDs. This is similar to the results observed by Manivannan et al. (2017) in carnation ‘Green Beauty’ and ‘Purple Beauty’. However, the highest levels of K, Ca, and Mg (15.19, 4.45, and 1.63 mg·g^-1 DW, respectively) were measured in carnations grown under blue LEDs in ventilated cultures (Table 3). Additionally, K and Ca levels were higher for carnations grown in ventilated cultures than those grown in unventilated cultures. The results of this study were similar to the results of culturing Dianthus caryophyllus ‘Nelken’ tissue in well-ventilated and less-ventilated vessels (Dantas et al. 2001). The physiological processes of plants n eed K t o manage yield s and features (Wang and Wu 2013). Ca plays an important role in the cell membrane (Sharma et al. 2012). Na⁺ ions are necessary for the sustenance of the turgor pressure and the transport channel through the cell membrane (Hopkins 1999). Mg²⁺ ions are related to the gas exchange and affected the photosynthetic system of coffee plants cultured under d ifferent l ight intensities (Kaio et al. 2017). P exists in the form of phosphate esters in plants, creates phospholipid membranes to protect cells, contributes to the formulation of DNA, RNA, ATP, and ADP molecules (Hopkins 1999), and affected the photosynthesis and root systems o f Lotus japonicus (Rochelle et al. 2016). This was also true in this experiment, where carnations had the highest P content when grown in ventilated vessels under white LEDs (8.44 mg·g^-1 DW) ( Table 3), and had the highest number of roots (12.5) (Table 1).

Micronutrients are claimed in small quantities (less than 10 mmol kg^-1 DW) and play an e ssential role in r egulating the enzyme activities (Hopkins 1999). The light quality and ventilation condition resulted in statistically significant differences in the contents of Cu, Zn, Mn, Fe, B, Mo, and Si in carnation 'Marble Beauty' (Table 4). In addition, the ventilation condition significantly influenced the Zn content of the carnations. Carnations grown in unventilated containers had the highest Cu content when grown under blue LEDs, and the highest Mo content when grown under red + blue LEDs. Carnations in ventilated containers had the highest Zn, Mn, and Fe contents when treated with blue LEDs, and the highest B and Si levels when grown with red LEDs. Manivannan et al. (2017) also reported that blue LEDs resulted in increased levels of Zn, Mn and Fe and that red LEDs enhanced B levels in carnations ‘Purple Beauty’. Cu seemed to operate originally as a cofactor for a wide array of oxidative enzymes (Hopkins 1999). Zn was involved in the carbonic anhydrase activity, stomatal aperture, and transpiration in the leaves of cauliflower (Brassica oleracea) (Parma et al. 1995). Mn appeared on the formation of essential secondary metabolites, and was a co-factor element of SOD (Pedas et al. 2014). Mo, like Mn, is a co-factor of enzymes and participated in the nitrogen metabolism (Kaiser et al. 2005). B seemed to appear in the cell wall, and is involved in both cell division and elongation. The Si is stored in the cell walls, prevents fungal infection, and limits stem breakage in heavy rain (Hopkins 1999).

In conclusion, the light quality and ventilation condition noticeably affected the physiology, antioxidant enzyme activities, and nutrient contents in carnation ‘Marble Beauty’. Future studies should examine how the light quality and ventilation condition affect the acclimation, growth and development of carnations in greenhouses.