Introduction

Carnation is one of the most important species in the worldwide market of cut flowers (Birlanga et al. 2015). Recently domestic production and cultivation area of carnation has been decreasing in South Korea, while the amount of import and the royalty paid to foreign carnation cultivars have continued to increase from the past decade (Ministry of Agriculture, Food and Rural Affairs Database 2015). In an effort to solve these problems, the Rural Development Administration has bred many domestic cultivars. Although growers are not quite satisfied with the performance of the new domestic cultivars in terms of flower quality and productivity, there have not been studies to obtain high quality propagated plants of these cultivars. Carnation is mainly propagated by cutting and production of propagated plants by cutting method in Korea is estimated to occupy about 20% of the cultivated area (Lee et al. 2006). However, it is desirable to use micropropagated virus-free plants, especially for those to be used as mother plants.

Tissue culture or micropropagation is powerful technique for rapid propagation of physiologically uniform and healthy plantlets which can’t be done by seed propagation or other vegetative propagation method of low efficiency in limited period of time (Jeong et al. 1995; Murch et al. 2000; Pretto and Santarem 2000; Zobayed and Saxena 2004). However, the widespread use of micropropagation is limited in some situations due to high production costs. These costs are mostly attributed to a low growth rate, a significant loss of plantlets during in vitro tissue culture process due to microbial contamination, physiological and morphological disorders, poor rooting, low survival rate at the ex vitro acclimatization stage, and high labor costs (Afreen-Zobayed et al. 2000; Jeong et al. 1996; Kozai 1991; Xiao et al. 2011). In order to overcome these problems, the photo-autotrophic system has shown to be worthy to consider a suitable technique due to its advantages (Kozai 1991).

Plantlets grown in the photo-autotrophic system can synthesize carbohydrates by utilizing CO₂ in the atmosphere, defused into the tissue culture vessel through air membrane, so that the system enables the plantlets to be ready for the acclimatization step ex vitro. The photo-autotrophic system has many advantages in terms of fast growth rate of plantlets, few physiological disorders, high survival rate of plantlets, and low cost even though there are differences among plant species (Hwang and Jeong 2012; Ticha et al. 1998). The growth rates of plantlets of potato (Kim et al. 1999; Kozai et al. 1988a), strawberry (Fujiwara et al. 1988; Kozai and Sekimoto 1988; Kozai et al. 1988b), tobacco (Kozai et al. 1990b; Mousseau 1986), Phalaenopsis (Doi et al. 1989), and gerbera (Jeong et al. 1999) were cultured on the medium without supplementation of sucrose under high level of CO₂ and/or high light intensities were greater than those were cultured on the medium with supplementation of sucrose under low levels of CO₂ and low light intensities. Maintaining properly the in vitro environmental condition is critical to promote plantlet growth by increasing metabolic processes. In this study the effect of condition with different levels of medium sucrose, light intensity, and air CO₂ has been investigated in carnation which are very prone to hyperhydricity to see how plantlet growth under photo-mixotrophic and photo-autotrophic culture conditions are affected.

Materials and Methods

Plant materials and culture conditions

Shoot explants of carnation (Dianthus caryophyllus L.) obtained from in vitro-grown plantlets were stuck into and cultured on the MS medium (Murashige and Skoog 1962). The pH of all media was adjusted to 5.70 prior to autoclaving at 121℃ for 20 min. Media in the quantity of 50 mL per container were supplemented with one of the two levels of sucrose, 0 and 30 g·L^-1, and were solidified with 0.8% agar (w/v) in 85 mm (diameter) x 110 mm (height) round plastic containers (Gaooze 0811C, Korea Scientific Technique Industry Co., Ltd., Suwon, Korea).

Each container was capped with a lid which had a 10 mm diameter round hole made and sealed by a sheet of HEPA filter (Nihon Millipore Ltd., Yonezawa, Japan), with an estimated number of air exchanges per hour of 2.8. All the cultures were maintained in culture rooms at 24℃/22℃ (day/night) temperatures and 70-80% relative humidity (RH). Cultures were subjected to a light intensity of 50 or 200 μmol·m^-2·s^-1 photosynthetic photon flux density (PPFD), measured at the top of culture containers, for a 16 h photoperiod provided by white light emitting diodes (LED, 400-700 nm, PSLED-1203-50A, Force Lighting Co., Ltd., Hwaseong, Korea), and CO₂ concentration of 350 or 1,000 μmol·mol^-1 in culture rooms (Fig. 1). The experiment was set up in a completely randomized design and each treatment consisted of 3 replicates of 4 explants in each of two containers. All the measurements were taken in triplicates, totally 24 shoot explants per treatment, to verify the reproducibility of the results.

Measurements of growth and morphological parameters

After four weeks of culture, shoot length, number of nodes, root length, number of roots, fresh and dry weights of shoot, root and whole plantlet, tissue water content, leaf length, and leaf width were measured. Shoot, root, and total dry weights were measured a fter 72 h of d rying in a 70°C oven (FO-450M, Jeio Technology Co., Ltd., Daejeon, Korea).

Measurements of total chlorophyll content

For total chlorophyll content measurement, fresh leaf tissues (the same 20 mg) taken from the third leaf from the top in each treatment were extracted with 80% (v/v) acetone for 24 h. Total chlorophyll content was determined by measuring the absorbance at 645 and 663 nm by a spectrophotometer (Uvikon 992, Kotron Instrumentals, Milano, Italy) according to the method of Arnon (1949).

Statistical analysis

Data collected were analyzed for statistical significance by the SAS (Statistical Analysis System, V. 9.1, Cary, NC, USA) program. The experimental results were subjected to an analysis of variance (ANOVA) and Duncan’s multiple range tests at a p ≤ 0.05 probability level. Graphs were plotted with the SigmaPlot 10.0 (Systat Software Inc., San Jose, CA, USA).

Results and Discussion

The supplementation of sucrose to the medium significantly influenced the shoot length, number of nodes, root length, shoot and total fresh weights, dry weights of shoot, root and whole plantlet, and tissue water content (Table 1). Light intensity significantly affected shoot length, number of nodes, number of roots, root and total fresh weights, shoot and total dry weights, tissue water content, leaf length, leaf width, and total chlorophyll content. The CO₂ supplemented to the culture room also affected shoot length, number of nodes, total chlorophyll content, root length, shoot and total fresh weights, shoot and total dry weights, leaf length, and leaf width.

Figure 2A shows the effect of level of medium sucrose, light intensity, and air CO₂ on shoot length of carnation plantlets cultured in vitro for four weeks. Shoot length increased in the treatment without supplementation of sucrose as compared to those with supplementation of sucrose. Generally, a photo-autotrophic condition with CO₂ supplementation to the culture room and high light intensity promoted the growth and development of the shoot, while it had little influence on the shoot length, thus resulting in more compact shoot growth. Similar results were obtained with number of nodes as the shoot length. The treatments without supplementation of sucrose to the medium showed decrease in the number of nodes when the cultures were supplied with higher light intensity along with a higher concentration of CO₂. In contrast, in the photo-mixotrophic condition, number of node increased by a higher level of CO₂ when the light intensity was increased (Fig. 2B).

Root length also showed a similar trend as the shoot length. The treatment without supplementation of sucrose showed root length to increase, especially in the treatment of a low light intensity. Root length increased in the treatment of a lower light intensity in the photo-autotrophic condition. On the other hand, in the photo-mixotrophic condition, root length increased with increased light intensity (Fig. 3A). Deng and Donnelly (1993) obtained results that the photo-autotrophic condition producing plantlets with longer length of the longest root as compared to the photo-mixotrophic condition. The number of roots increased with increasing light intensity. The photo-autotrophic condition with a higher light intensity showed unexpected decrease in the number of roots with the increased CO₂ concentration, which implied that 1,000 μmol·mol^-1 CO₂ was excessively too high for the young cultures (Fig. 3B).

As for the fresh weight of whole plantlet, the photo-autotrophic condition gave better results than the photo-mixotrophic condition. Kozai et al. (1988a) also reported that the total fresh weight of carnation plantlets was greater under the photo-autotrophic than photo-mixotrophic condition. Total fresh weight increased with an increase in CO₂ concentration and light intensity (Fig. 4A). Similar results were reported by Deng and Donnelly (1993), Nguyen and Kozai (2001), Xiao et al. (2007), and Zhang et al. (2009) who described that high light intensity and/or high CO₂ concentration increased total fresh weight. Total dry weight showed very similar trend as the total fresh weight. However, the photo-mixotrophic condition gave better results than the photo-autotrophic condition (Fig. 4B). Similar results have been obtained for tobacco (Tichá et al. 1998). The tissue water content of the plantlets grown in the photo-autotrophic condition was significantly higher than that of the plantlets grown in the photo-mixotrophic condition. The tissue water content was slightly lower in plantlets cultured under a high than a low light intensity, indicating less succulent growth in this condition (Fig. 5A). Total chlorophyll content was significantly higher in the photo-autotrophic than in the photo-mixotrophic condition. Increasing CO₂ concentration and light intensity increased the total chlorophyll content (Fig. 5B). Studies on increasing the total chlorophyll content of plantlets by increasing the concentration of CO₂ in the culture room have been reported in tobacco (Mosseau 1986), cymbidium (Kozai et al. 1990a), potatoes (Cournac et al. 1992), and Limonium spp. ‘Ocean Blue’ (Jeong and Jeong 2002).

Leaf l ength and leaf w idth s howed v ery similar trend each other. In the photo-autotrophic condition, leaf length and width were significantly increased. However, in the photo-mixotrophic condition, they significantly increased only under the high light intensity and high CO₂ concentration (Fig. 6A and B).

Overall, supplemented sucrose to the medium negatively affected the growth and development of carnation plantlets cultured in vitro. However, light intensity and CO₂ concentration positively affected total fresh and dry weights, and total chlorophyll content, while slightly suppressing the shoot length. In conclusions, better growth and development of the plantlets were observed under the photo-autotrophic than photo-mixotrophic condition. The finding is in agreement with previous reports that the photo-autotrophic condition promotes the growth of various plant species as compared to the photo-mixotrophic condition (Kozai et al. 1991; Oh et al. 2009; Xiao and Kozai 2004; Zobayed et al. 1999).