Introduction

Silicon (Si) is the second most abundant mineral element in the soil after oxygen and comprises about 31% of the earth’s crust (Epstein 1999). Si is present as a silicic acid in the soil solution at concentrations normally ranging from 0.1 to 0.6 mM, roughly two orders of magnitude higher than the concentrations of phosphorus in soil solutions (Epstein 1994). Although Si has not been considered as an essential element for higher plants, it has been proved to be beneficial for the healthy growth and development of many plant species, particularly graminaceous plants such as rice and sugarcane, and some cyperaceous plants (Epstein 1994). Si is effective in preventing lodging in rice by increasing the thickness of the cell wall and the size of the vascular bundles (Shimoyama 1958), thereby enhancing the strength of the stems.

The characteristic function of Si is to help plants to overcome multiple stresses including biotic and abiotic stresses (Ma and Yamaji 2006). Si plays an important role in increasing the resistance of plants to pathogens such as blast on rice (Datnoff et al. 1997) and powdery mildew on cucumber (Miyake and Takahashi 1893). Si also alleviates the effects of abiotic stresses including salt stress, metal toxicity, drought stress, radiation damage, nutrient imbalance, high temperature, and freezing (Epstein 1999). Most of all, salt stress has been a major obstacle to the successful use of salt-affected soils for plant cultivation and production. Salt stress has been shown in some investigations to be mitigated by Si. Matoh et al. (1986) reported that silicate at 0.89 mM to reduce the translocation of Na⁺ to the shoots and to increase dry matter production of salt-stressed rice plants as compared to the control. For wheat (Ahmad et al. 1992) and barley (Liang et al. 1996) similar findings have been reported, namely a repression of Na⁺ transport in plants growing in salinized solutions supplied with Si, with concomitant improvement in their growth. Liang (1998) reported that added Si enhanced the growth of salt-treated barley and improved the chlorophyll content and photosynthetic activity of leaf cell organelles of barley. In addition to the impact of Si on plant protection, various other beneficial effects of Si have been reported, such as amelioration of the adverse effects of Al and Mn toxicity to plants (Hodson and Evans 1995), improvement of water use efficiency (Gao et al. 2004; Hu and Schmidhalter 2005), as well as enhancement of the salt tolerance (Liang et al. 1996; Matoh et al. 1986; Schmidhalter and Oertli 1993).

More recently, benefits of Si on salt tolerance of barley and cucumber have been related to antioxidant enzyme activity (Al-aghabary et al. 2004). Many researchs addressed the role of Si to alleviate salt stress in wheat (Ahmad et al. 1992; Liang et al. 2003), tomato (Romero-Aranda et al. 2006), rice (Yeo et al. 1999), maize (Shu and Liu 2001), and alfalfa (Wang and Han 2007). It has been reported that addition of Si to salt-treated barely, significantly increased SOD activity in plant leaves (Liang 1999), and increased SOD, POD, and CAT activity in barely roots (Liang et al. 2003). Liang (1999) examined possible interactions between Si and NaCl in barley with respect to the uptake of Na and the activity of some enzymes related to plant protection against stress conditions. The results indicated that Si was capable of reducing the accumulation of Na in the plant tissue, but was rather not conclusive with respect to the responses of enzyme activity to Si. Yet, there have been only limited number of reports on horticultural crops related to stress aspects.

The genus torenia (Scrophulariaceae) includes 40 species of herbaceous perennial, and almost all of them are found in tropical and subtropical Asia and Africa (Yamazaki 1985). Torenia species are grown as garden plants; flower colors range from white with yellow throats to violet, blue, cobalt, lavender, and purple (Nhut et al. 2013). Torenia fournieri L., known as torenia or wishbone flower, is a common bedding ornamental plant (Tao and Li 2006). It has become a model plant for biological research, and its culture requirements in vitro are well established (Tanimoto and Harada 1981). Residents living within close proximity to beach front areas need to select bedding plants that are more tolerant of high winds, salt spray, and irrigation water containing high levels of salt (Black 2006). Black (2006), and Tjia and Rose (1987) reported that torenia exhibited poor salt tolerance. Although the application of Si to plant species increases tolerance to many abiotic stresses including salt stress, no reports are available concerning the effect of Si on growth and development of torenia. Thus, in this study, the effect of Si on growth and antioxidant activity of torenia (Torenia fournieri Linden ex Foum) ‘Duchess Blue and White’ cultured in vitro under salt stress was examined.

Materials and Methods

Plant materials

Torenia (Torenia fournieri Linden ex Foum ‘Duchess Blue and White’) seeds were initially surface sterilized by washing with sterile distilled water (6 times), then immersed in 70% (v/v) ethanol for 1 min and then washing with sterile distilled water. Then they were further treated with 1.5% (v/v) sodium hypochlorite solution for 10 min. After rinsing 5 times with sterile distilled water, the seeds were cultured on the half strength Murashige and Skoog (1962) medium (MS) containing 3 % (w/v) sucrose and 0.8 % (w/v) agar. The cultures were maintained for 10 days at 25 ± 1°C under darkness and then exposed to 95 μmol·m^-2·s^-1 photosynthetic photon flux density (PPFD) for a 16-h per day photoperiod.

Effect of NaCl concentrations on torenia

The pH of the medium was adjusted to 5.80 using 0.1 N NaOH or 0.1 N HCl before autoclaving at 121°C for 15 min. The 16 days old seedlings removed of roots were cultured on the MS medium containing 0, 25, 50, 100, or 200 mM NaCl for 45 days to test their tolerance to NaCl. All cultures were kept at 25 ± 1°C and 70 ~ 80% relative humidity (RH) under a 16 h photoperiod with 95 μmol·m^-2·s^-1 PPFD provided by cool white fluorescent light in the growth chamber. Those explants which continued to grow and were able to induce growth of healthy shoots was considered as NaCl-tolerant; while those which failed to induce growth of shoots or shoots with bleached or wilted leaves were considered to be NaCl-sensitive.

Effect of Si on survival and growth of shoot buds cultured under salt stress

Shoot buds isolated from 16 days old seedlings were cultured on the MS medium containing 0, 50 or 100 mM NaCl alone or in combination with 1.8 or 3.6 mM Si from K₂SiO₃. Additional K introduced by K₂SiO₃ was subtracted from KNO₃ and loss of NO₃ was subsequently reintegrated by addition of HNO₃. After 45 days, the number of surviving plantlets in each treatment was recorded and length of the shoot and root, stem diameter, number of nodes, number of leaves, number of wilted leaves, chlorophyll content, and fresh and dry weights of surviving plantlets were measured. To estimate chlorophyll content, 10 mg of fresh leaf was ground in 1 mL of 80% ice cold acetone and filtered using filter paper, and volume was brought up to 10 mL using the same solution. The absorbance of the final solution was recorded at 663 and 645 nm and chlorophyll a, b, and total chlorophyll content was calculated as mentioned by Dere et al. (1998). Dry weight was measured after 48 h of drying at 70°C in a dry oven (JSOF-150, JSR Micro, Korea).

Effect of Si on antioxidant activity of plants cultured under salt stress enzyme extraction

Fresh leaves were collected from 35 days old plantlets cultured on the MS medium containing 50 mM NaCl alone or in combination with 1.8 or 3.6 mM Si. Plantlets developed in the 100 mM NaCl treatment were not used for enzyme analysis due to their small sizes. For estimation of enzyme activity and total protein content, fresh leaf tissue (0.1 g) was homogenized in 50 mM phosphate buffer (pH 7.0) containing 1.0 mM EDTA, 0.05% Triton X-100, 1.0 mM polyvinylpyrrolidone (PVP), and 1.0 mM ascorbate. After centrifugation of homogenate at 10,000×g for 20 min at 4°C, the supernatant was used to measure the activities of antioxidant enzymes (Shekhawat et al. 2010). Protein content was estimated by the method of Lowry et al. (1951).

Superoxidase dismutase (SOD; E.C.1.15.1.1)

The SOD activity was assayed by measuring its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT) according to the method of Beauchamp and Fridovich (1971) with 3 mL reaction mixture containing 50 mM phosphate buffer (pH 7.8), 13 mM methionine, 75 μM NBT, 2 mM riboflavin, 0.1 mM EDTA, and a suitable aliquot of enzyme extract. This reaction mixture was incubated for 30 min under fluorescent lamp. A tube containing enzyme was kept in darkness and served as the blank, while the control tube without enzyme was kept in the light. The absorbance was taken at 560 nm and calculations were made by using an extinction coefficient of 100 mM^-1 cm^-1.

Peroxidase (POD; E.C.1.11.1.7)

The enzyme assay was comprising of 0.1 M phosphate buffer (pH 7.0), 0.05 mL guaiacol solution, 0.1 mL enzyme extract, and 0.03 mL hydrogen peroxide. An increase in the absorbance was recorded at 436 nm. Time in minutes has been recorded until the decrease becomes constant. The extinction coefficient was 6.39 per micromole and a protocol was described in Sadasivam and Manickam (1996).

Ascorbate peroxidase activity (APX; E.C.1.11.1.7)

Total APX activity was measured in a reaction mixture that contained a 50 mM phosphate buffer (pH 7.0), 0.6 mM ascorbic acid, and enzyme extract (Chen and Asada 1989). The reaction was initiated by addition of 1 μL substrate H₂O₂ (10%), and the oxidation rate of ascorbic acid was estimated by following the decrease in absorbance at 290 nm for 3 min (molar extinction coefficient 2.8 mM^-1 cm^-1).

Catalase (CAT; E.C.1.11.1.6)

Total CAT activity was measured by the method of Aebi (1984). The assay system was comprised of 50 mM phosphate buffer (pH 7.0), 20 mM H₂O₂, and a suitable aliquot of enzyme i n the final volume o f 3 mL. Decrease in t he absorbance was recorded at 240 nm. The molar extinction coefficient of H₂O₂ at 240 nm was 0.04 μmoL^-1 cm^-1.

Statistical data analysis

Data 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. Graphing was performed with Sigma Plot 10.0 (Systat Software, Inc., San Jose, CA, USA).

Results and discussion

Effect of NaCl concentrations on survival and growth of torenia

A preliminary experiment was carried out for salt sensitivity on growth of torenia ‘Duchess Blue and White’. Shoot growth and root induction were observed within 15 days of culture when shoot buds were cultured on the MS medium with or without NaCl (Fig. 1A-E). However, after six weeks shoot development was affected and differences in growth and injury were observed among the NaCl treatments a nd control (Fig. 1F-J). P lants that w ere salt-stressed often developed visual injury presumably due to excessive salt u ptake. Shoot g rowth was completely inhibited at 200 mM of NaCl (Fig. 1). At concentrations of 25, 50 and 100 mM NaCl the percentage survival were 67.4, 32.7 and 19.4%, respectively (Fig. 1). Torenia exhibited poor salt tolerance.

Effect of Si on survival and growth of torenia cultured under salt stress

Effect of NaCl and Si on survival and growth of torenia ‘Duchess Blue and White’ cultured in vitro is shown in Table 1. Concentrations of NaCl, Si and their interactions had a significant effect on survival (%) plantlets (p = 0.001). Plant survival ratio was significantly reduced by NaCl supplementation as compared to the control. It was 100% in the control, 32.7% in the 50 mM NaCl, and 19.4% in the 100 mM NaCl. The survival ratio (100%) significantly increased when 1.8 or 3.6 mM Si was added to the MS medium containing 50 mM NaCl. However, only 31% of plantlets survived when Si was added to the culture medium containing 100 mM NaCl (Table 1). Thus, at the 100 mM NaCl, the Si supplementation did not alleviate the effect completely, suggesting a possible negative effect of excessive concentration of NaCl.

One of the initial effects of salt stress on plant is the reduction of growth. Shoot and root length of plantlets decreased significantly (p = 0.001) with increasing NaCl concentration in the culture medium. The average length of shoot and root were 3.4 and 3.2 cm in the control, 1.8 and 0.8 cm in the 50 mM NaCl, and 1.9 and 0.3 cm in the 100 mM NaCl treatments, respectively. Decreases in shoot and root development may be due to toxic effects of the NaCl used as well as unbalanced nutrient uptake by the plants (De pascale et al. 2005). Shoot length significantly increased when Si was added to the MS medium containing 50 mM N aCl. This result i s in c oncomitance with t hat of Lee et al. (2010), who reported that addition of Si to soybean plants resulted in increased shoot length. However, no significant difference in shoot length was observed when Si was added to the medium containing 100 mM NaCl. In the present study, root length increased when Si was added to the culture medium containing 50 mM NaCl, but it decreased when Si was added to the medium containing 100 mM NaCl. Stem diameter increased at 50 mM NaCl as compared with the control. The greatest stem diameter was recorded when shoot buds were cultured on the MS medium containing 50 mM NaCl and 3.6 mM Si.

Salinity stress significantly decreased the mean number of nodes and leaves. However, Si supplementation increased number of nodes and leaves in the 50 mM NaCl treatment as compared with the control. Sodium is the primary toxic ion, because it interferes with K uptake as well as and disturbs stomatal regulation which ultimately causes water loss and necrosis. On the other hand, Cl induces chlorotic toxicity symptoms due to impaired production of chlorophyll (Jeong and Lee 1992). In the present study, NaCl had a significant effect on the number of wilted leaves. The number of wilted leaves was 0.0 in the control, and 6.0 in the 50 mM NaCl. However, supplementation of Si e ither at 1 .8 or 3. 6 mM a lleviated i t to have to 3 . 0 and 4.0, respectively as compared to the 50 mM NaCl. Similarly, chlorophyll (Chl) content significantly decreased when the culture medium was supplemented with NaCl. A reduction in Chl levels due to salt stress has been reported in pea (Ahmad and Jhon 2005), rice (Anuradha and Rao 2003), tomato (Al-aghabary et al. 2004), and wheat (Ashraf et al. 2002). In the present study, Chl content increased when 1.8 mM Si was added to the MS medium containing 50 mM NaCl. This result is in concomitance with Al-aghabary et al. (2004) who reported that Si supplementation increased Chl a and Chl b contents in tomato plants under salt stress.

The addition of NaCl to the MS medium significantly reduced fresh and dry weights. However, Si supplementation increased fresh and dry weights in the 50 mM NaCl treatment as compared with the control. An increase in biomass of salt-stressed plants under the influence of Si may be due to the improved photosynthesis and increased Chl content. The greatest fresh and dry weights were recorded when shoot buds were cultured on the MS medium containing 50 mM NaCl and 3.6 mM Si. Our results are in accordance to Savvas et al. (2009) who reported that the combination of salinity with enhanced Si ameliorated the impact of salinity on the fresh and dry weights of both shoot and root. The results suggested the Si supplementation was effectively enhanced tolerance of torenia plants to 50 mM NaCl stress.

Effect of Si on antioxidant activity of torenia cultured under salt stress

Any stressful environment enhances the accumulation of reactive oxygen species (ROS). The excessive production of ROS under salt stress occurred due to impaired electron transport processes in chloroplasts and mitochondria as well as from pathways such as photorespiration causing membrane damage and chlorophyll degradation leading to the development of leaf chlorosis and necrosis (Choi et al. 2002). In the present study, leaf chlorosis and necrosis were observed when the culture medium was supplemented with NaCl alone or in combination with Si treatments as compared t o the control (Fig. 2). The defensive system of plants enables them to ameliorate salt stress via production of enzymatic antioxidants such as SOD, POD, APX, and CAT (Ali et al. 2012). The differences in activities of antioxidant enzymes such as SOD, POD, APX, and CAT are given in Figs. 3A-D. In present study, the activities of SOD, APX, and CAT, but not the POD, were markedly higher in presence of 50 mM N aCl as c ompared with t he c ontrol. The activity of antioxidant enzymes has been reported to increase under saline conditions in cotton (Meloni et al. 2003), cucumber (Lechno et al. 1997), pea (Hernandez et al. 1999), rice (Fadzilla et al. 1997) and wheat (Meneguzzo et al. 1999). Effect of salt stress on the antioxidant enzymes are very complex and depends on the treatment time, plant species, and genotypes. In the present study, when Si was added to the medium containing 50 mM NaCl, activities of SOD, POD, APX, and CAT decreased as compared with there in the 50 mM NaCl treatment. These results were not consistent with previous studies (Liang 1999; Liang et al. 2003) where addition of Si to the salt treatment significantly increased antioxidant enzyme activities as compared to the corresponding salt treatment with no Si.

In conclusions, NaCl treatments significantly decreased plant survival and growth traits of torenia. When Si was added to the MS medium containing 50 mM NaCl, significantly increased length of shoot and root, stem diameter, number of nodes, total Chl content, and biomass were resulted. However, the addition of S i to the medium containing 100 mM NaCl did not increase plant survival nor growth as compared with the control. These results suggest the Si supplementation was effectively enhanced tolerance to the 50 mM NaCl stress. The activities of SOD, APX, and CAT were markedly higher in presence of 50 mM NaCl, while they decreased when Si was added to the medium containing 50 mM NaCl. Thus, Si-mediated resistance was not due to antioxidant enzymes. A number of possible other mechanisms are reported through which Si may increase plant tolerance to salinity including increased plant water status (Romero-Aranda et al. 2006), enhanced photosynthetic activity and maintenance of ultra structure of leaf organelles (Shu and Liu 2001), immobilization of toxic Na⁺ ion (Liang et al. 2003), and reduced Na⁺ uptake and enhanced K⁺ uptake (Yeo et al. 1999). Thus further investigations are needed to reveal the underlying mechanisms of Si-mediated tolerance of torenia to salt stress.