Introduction

Dieffenbachia is one of the most popular ornamental indoor foliage plants cultivated throughout the world. It has simple and alternate leaves bearing white spots and flecks. There are around thirty recognized species (Mayo et al. 1997) and, in addition, more than a hundred cultivars have been developed through interspecific hybridization and from somaclonal variants produced through tissue culture (Henny and Chen 2004). Even though cultivars of Dieffenbachia can be propagated through seeds and vegetative means, tissue culture methods of propagation are followed by commercial horticulturists due to poor seed production and germination (Henny 1988; Henny and Chen 2004). The tissue culture method of propagation can also enhance the commercialization of new cultivars within 2–3 year compared to 7–10 year through traditional breeding (Shen et al. 2008). However, a major limitation of tissue culture propagation is high mortality of plants during acclimatization (Chandra et al. 2010). This is because of the controlled environmental condition of in vitro culture, which results in the formation of plantlets of abnormal morphology, anatomy and physiology (Pospisilova et al. 1999; Dewir et al. 2006, 2014). Micropropagated plants usually reveal several physiological and anatomical abnormalities during in vitro growth such as low photosynthesis, non-proper functioning of stomata, malfunctioning of housekeeping systems mainly due to high humidity inside the culture vessel (Hazarika 2003; Dewir et al. 2014). After ex vitro transfer, these plantlets might easily be impaired by sudden changed environmental conditions, and so need a period of acclimatization to correct abnormalities (Preece and Sutter 1991; Pospisilova et al. 1999). As a consequence, in vitro raised plants are grown in environmentally controlled growth chambers/green houses and factors such as light, temperature and humidity are gradually enhanced by step-by-step acclimatization of plants (Kirdmanee et al. 1995; Pospisilova et al. 1999; Hazarika 2003).

In order to increase growth and reduce mortality in plantlets at the acclimatization stage, research has been focused on the control of environmental factors which facilitate optimal rate of survival of plantlets (Hazarika 2003; Dewir et al. 2005). Photosynthetic photon flux density (PPFD) is one important factor, which can be controlled at the acclimatization stage, because it can lead to the depression of photosynthetic efficiency (photoinhibition) mainly due to oxidative damage to the photosystem II as in the case of natural environment (Powles 1984). At high PPFD, an increase in photosynthetic carbon fixation can occur, which varies depending on growth and PPFD and may lead to different susceptibilities to photoinhibition (Powles 1984). However, above a certain threshold, carbon fixation becomes saturated and photosynthesis is incapable of using all the energy absorbed by the plants. Under conditions of excess light absorption, the chloroplast lumen becomes acidic in nature, which reduces the electron transport chain, and excitation energy accumulates in the chloroplast which can lead to the generation of reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (Asada 1999). Such accumulation of ROS may lead to lipid peroxidation and to a reduced accumulation of enzymatic antioxidants such as superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) as a defense system in the plants (Asada 1999). Some dehydrins (DHN) have been implicated to serve as antioxidants scavenging free radicals (Hara et al. 2003) and may act as protectants of osmoregulation. It is also believed that DHN can accumulate in the acclimatizing plants because it was reported that light induced expression of DHN during seedling de-etiolation in sunflower (Natali et al. 2007). Recently, Dewir et al. (2015) confirmed the accumulation of DHN in acclimatizing African violet plants and that DHN might be playing a role in overcoming lipid peroxidation during ex vitro acclimatization.

Not much work has been carried out on the effect of PPFD during in vitro acclimatization of micropropagated plants and the role of light on antioxidant mechanism. Therefore, in the present study, we carried out ex vitro acclimatization experiments in Dieffenbachia cultivars to study the effect of PPFD on various growth and photosynthetic parameters and the antioxidant enzymes SOD, APX, CAT and DHN in order to clarify their role in overcoming light induced stress during the process of acclimatization.

Materials and methods

Chemicals and reagents

All chemicals were of tissue culture grade, except for enzyme-related analyses, which were of HPLC grade. All reagents and chemicals were obtained from Sigma-Aldrich (Poole, UK), unless otherwise stated.

Plant material and PPFD treatments during acclimatization

Micropropagated plantlets of three Dieffenbachia ‘Vesuvius’, ‘Camille’ and ‘Green Magic’ at the 3–4 leaf stage, were gently removed from in vitro culture medium and the roots were washed under tap water. Plantlets were transplanted into culture pots (300 mL plastic coffee cups with three holes at the bottom) filled with sterilized compost (John Innes No. 1, Westland Ltd., UK) and grown in a cultivation chamber (Sanyo, Japan) for 4 weeks under PPFD of 35, 70 and 100 µmol m−2 s−1 and a 16-h photoperiod using metal-halide lamps (Sanyo, Japan). The environment in the cultivation chamber was adjusted to 25 ± 2 °C air temperature and 40–50 % relative humidity (RH). Plants were irrigated twice a week and a compound fertilizer (19: 19: 19; N: P2O5: K2O) at 1 g L−1 was applied in the irrigation water once after 3 weeks. Growth parameters such as fresh weight, dry weight, number of leaves/plantlet, shoot and root length, and leaf area were determined after 4 weeks in the growth chamber.

Analysis of photosynthetic parameters

Determination of chlorophyll and carotenoid contents

The amount of chlorophyll-a, chlorophyll-b and total carotenoids in the fully expanded young leaves were determined by spectrophotometric analysis after 4 weeks in acclimatization. Chlorophyll was extracted from leaf tissue by grinding in a mortar with liquid nitrogen to a fine powder and adding 100 mg to a 2 mL Eppendorf tube. One mL of 80 % acetone was added and the powder was homogenized by inverting for 10 min in ice using a shaker. The absorbance was measured at 470, 649 and 665 nm using a Heliosepiclon spectrophotometer (Unicam, UK). Chlorophyll and carotenoid concentrations were calculated from the spectrophotometric data using the formulae of Lichtenthaler and Welburn (1983). There were three replicates within each treatment.

Chlorophyll fluorescence

Chlorophyll fluorescence parameters were measured on the abaxial surface of freshly detached leaf discs. Plants were kept for 30 min in the dark prior to measurement. Modulated fluorescence was measured using a portable chlorophyll fluorometer (PEA, Hansatech Instrument Ltd., version 1.21, Norfolk, UK). Minimal fluorescence (F 0) was measured for 30 min in dark-adapted leaves using light of <0.1 μmol m−2 s−1 and maximal fluorescence (F m) was measured after a 1 s saturating pulse (>3500 μmol m−2 s−1) in the same leaves. Maximal variable fluorescence (F v = F m − F 0) and the photochemical efficiency of PSII (F v/F m) were calculated for dark-adapted leaves (Dewir et al. 2005). Four plants were randomly selected and measurements were made on the fully expanded young leaves using a standard leaf chamber. There were four single-leaf replications within each treatment.

Stomatal resistance

Stomatal resistance of plant leaves (resistance to loss of water vapour through the stomata; mol H2O m−2 s−1) was estimated using an AP4 Porometer (Delta-T Devices Ltd., Cambridge, UK) (Monteith et al. 1988). Four plants were randomly selected and measurements were taken from fully expanded young leaves using a standard leaf chamber. There were four single-leaf replications within each treatment.

Photosynthetic rate and transpiration rate

Photosynthetic rate and transpiration rate were measured with a portable LCi photosynthesis system (ADC BioScientific Ltd., UK). Photosynthetic measurements were carried out under the following conditions: CO2 concentration of the in-flow air was at 350 μmol mol−1, RH at 60 % and leaf temperature was 25 °C. Data was either logged on a PCMCIA-type 1 memory card or was sent directly to a dumb terminal via an RS232 serial link connector. Four plants were randomly selected and measurements were made after 4 weeks on the fully expanded young leaves using a standard leaf chamber. There were four single-leaf replications within each treatment.

Analysis of biochemical parameters

Antioxidant enzymes assay

To determine the activities of antioxidant enzyme, 0.5 g FW of fully expanded young leaves was homogenized under liquid nitrogen with 1.5 mL of extraction buffer using pre-chilled mortar and pestle. The homogenate was filtered through four layers of cheesecloth and centrifuged at 22,000×g for 20 min at 4 °C. The supernatant was re-centrifuged at 22,000×g for 20 min at 4 °C and was then used for the assays.

Superoxide dismutase assay

Superoxide dismutase (SOD; EC 1.15.1.1) activity was determined according to Giannopolitis and Ries (1977). The assay mixture consisted of three reagents (xanthine, xanthine oxidase and cytochrome c) dissolved in potassium buffer stored at RT during use and at 4 °C between uses. Xanthine (0.5 mM) was prepared fresh each day and stored at RT during use. SOD anions (\({\text{O}}_{2}^{ \cdot - }\)) are generated by a xanthine/xanthine oxidase (XOD) system and detected by a chromagen solution. Xanthine oxidase (5 µL of Sigma X 4500, diluted with 495 µL of potassium phosphate buffer, final concentration, 0.32 U mL−1) was prepared fresh each day and stored on ice during use. Cytochrome c (12.5 mg in 0.5 mL) was prepared in phosphate buffer and stored on ice during use and at −20 °C between uses. The assay mixture was prepared from 8.85 mL potassium phosphate buffer, 1 mL xanthine and 50 µL cytochrome. SOD was prepared fresh each day by diluting 10 µL of a Sigma-Aldrich product (S8409, from preparation bovine erythrocytes) with 90 µL buffer (960 U mL−1, final concentration) and stored on ice during use. The assay mix (10 mL) was prepared just before use. This was sufficient for at least 32 wells. Aliquots (50 µL) of sample were added to the wells on a 96-well plate (Fisher Scientific, Loughborough, UK). Some wells were also used as controls either by replacing the sample with SOD or with buffer. Xanthine oxidase solution (100 µL) was added immediately before use to the assay mix, the wells were loaded as appropriate and readings started within 1 min after this addition. The assay mixture (250 µL) was pipetted into each well and mixed carefully. The plate was immediately transferred to the VersaMax™ plate reader (Molecular Devices, Sunnyvale, CA, USA), and the increase in absorbance was monitored at 550 nm using a kinetic program for at least 5 min. All data are expressed as unit mL−1.

Catalase assay

Catalase (CAT; EC 1.11.1.6) activity was determined according to Clairbone (1985). Aliquots (50 µL) of the leaf extract were added to each well of a 96-well plate (Fisher Scientific), and then 25 µL of 40 µM H2O2 was added. The plates were then incubated for 30 min at 37 °C. After incubation, 50 µL of reaction buffer containing 100 µM Amplex Red reagent and 0.4 U mL−1 of horseradish peroxidase (Sigma-Aldrich, Poole, UK) was added to each well. The resulting fluorescence was measured using a CytoFluor II fluorescence microplate reader (PerSeptive Biosystems, Framingham, USA) with 540 nm excitation and 590 nm emission. The Amplex Red stock solution was prepared at 10 mM in DMSO and aliquots were stored at −20 °C. CAT activity was calculated from a standard curve prepared using purified catalase (0–2000 U mL−1; Sigma-Aldrich).

Glutathione peroxidase (GPX) assay

GPX activity was determined according to Tappel (1978). The assay mixture was prepared by mixing 9.2 mL of buffer (5 mM potassium HEPES, containing 1 mM dipotassium ethylenediaminetetraacetate (EDTA) with 1 mM NADPH, followed by the addition of 100 µL of 100 U mL−1 glutathione reductase (GR; Sigma G3664 from Saccharomyces cerevisiae), 50 µL of 200 mM glutathione (GSH), and 95 µL of 10 mM potassium cyanide. The assay mixture (290 µL) was added to each well of a 96-well plate, followed by 15 µL of plasma before mixing. The reaction was started by adding 5 µL of 0.042 % (w/w) H2O2 solution to each well. The rate of change of absorbance at 340 nm was measured for at least 10 min in a VersaMax™ plate reader. GPX activity was calculated in pmol mL−1 min−1.

Immunoblots for DHN proteins detection

DHN proteins were detected according to Rihan et al. (2014) with modifications described by Dewir et al. (2015). Total proteins from the fully expanded young leaves were extracted as described previously by Ni et al. (1996). Samples from −80 °C were thawed on ice, placed in a mortar, and 2 mL of extraction buffer (100 mM potassium phosphate, 1 mM dithiothreitol, 1 mM EDTA, 1 % Triton X-100, 10 % glycerol, pH 7.8) was added to 1 g of tissues and ground with a pestle. One mL of the liquid slurry was transferred to a microfuge tube (1.5 mL) on ice. The supernatant was centrifuged twice at 13,000×g for 15 and 10 min, respectively, at 4 °C. Total protein content was evaluated using the Pierce BCA assay kit following the manufacturer’s instructions. The clear supernatant containing proteins was separated using SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) (Laemmli 1970) and visualized using Coomassie blue staining solution. Unstained gels were used for immunoblot and the proteins were transferred to a PVDF membrane (pore size 0.2 µm) (Millipore, Cat. No. ISE000010) by electro-blotting at 100 V for 35 min. 1× Tris glycine transfer buffer was prepared by dissolving 3.05 g L−1 Tris–HCl and 14.4 g L−1 glycine in 20 % (v/v) methanol (Towbin et al. 1979). After transferring the proteins onto the PVDF membrane, the membrane was blocked using PBST (phosphate buffer saline + 0.05 Tween 20 %) + 5 % Marvel (a low fat skimmed milk powder) (Iceland Ltd.) for 1 h at RT with mild shaking. After incubation, the membrane was incubated with a primary antibody raised against the DHN K-segment obtained from Dr. Michael Wisniewski‘s Laboratory, (USDA-ARS, Appalachian Fruit Research Station, USA). Primary antibodies were diluted 1:1000 in a total volume of 20 mL PBST supplemented with 4 % of Marvel semi-skimmed milk and the membrane was incubated at 4 °C overnight with very gentle shaking. The membranes were then washed three times with PBST while agitating to wash off the excess primary antibody, for 5 min each wash, and the membrane was then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (PBS diluted 1:20,000) (Abcam) for 1 h at RT with mild shaking to detect primary antibodies attached to the DHN protein. The membranes were washed three times with PBS for 5 min each and then incubated with ECL detection reagent (Luminata crescendo western HRP substrate; Millipore, WBLUR0100) in the dark for 5 min. The image from the membrane was captured using a UVP gel documentation system.

Experimental design and data analysis

All experiments were set up in a completely randomized design. All data were subjected to ANOVA and Duncan’s multiple range test (DMRT) using SAS program (Version 6.12, SAS Institute Inc., Cary, USA).

Results and discussion

Effect of PPFD on growth parameters

Fresh and dry biomass of plants and root length were highest at 100 µmol m−2 s−1 PPFD in ‘Camille’ and ‘Green Magic’, whereas 70 µmol m−2 s−1 PPFD was optimum for all these growth parameters with ‘Vesuvius’ (Table 1). Low PPFD treatment (35 µmol m−2 s−1) was superior with respect to the number of leaves per plant and leaf area per plant with ‘Camille’ and ‘Green Magic’. ‘Green Magic’ gave the highest values for all growth parameters compared to the other two cultivars. In many plant species, the leaves formed in vitro are unable to develop further under ex vitro conditions and they are replaced by newly formed leaves (Diettrich et al. 1992). However, if the ex vitro transplantation of plantlet is successful, the increase in their growth can be enormous; for example, the ex vitro transplanted plants were taller, had higher dry mass, larger leaf area when compared to in vitro plantlets in Nicotiana tabacum (Pospisilova et al. 1999).

Table 1 Effect of photosynthetic photon flux density (PPFD) on growth parameters of in vitro raised Dieffenbachia cultivars during acclimatization
Full size table

Effect of PPFD on physiological parameters

Both chlorophyll and carotenoid contents were highest with 100 PPFD in all three cultivars (Table 2). Net photosynthesis values increased with each increment in PPFD in ‘Camille’ and ‘Green Magic’, while ‘Vesuvius’, net photosynthesis values decreased slightly. Thus, ex vitro transplantation of plants grown under low irradiance is suggested for successful acclimatization of in vitro raised plants. However, the acclimatization process is a genotype-dependent. An incremental increase in chlorophyll content has been reported in many plant species after ex vitro transplantation (Pospisilova et al. 1988; Kozai and Zobayed 2000). Exposure of Calathea louisae and Spathyphyllum floribundum plantlets to high irradiance immediately after transplantation caused photoinhibition and even chlorophyll photo-bleaching (Van Huylenbroeck 1994; Van Huylenbroeck et al. 1995). The increase in photosynthetic activity recorded here showed that plants are able to respond to increases in PPFD but this represents a risk of ROS formation if the energy captured cannot be fixed chemically.

Table 2 Effect of photosynthetic photon flux density (PPFD) on physiological parameters of in vitro raised Dieffenbachia cultivars during acclimatization
Full size table

The F v/F m was highest with low PPFD (35 µmol m−2 s−1) with all three cultivars and F v/F m values decreased significantly with increased PPFD (Table 2). The results demonstrate that photoinhibition occurs after transplanting the micropropagated plant, even at low PPFD. Similar decreases in F v/F m with increasing PPFD during ex vitro acclimatization were also reported with microcuttings of Rhododendron during acclimatization (Matysiak 2004). Poorly differentiated chloroplast of in vitro established leaves has been suggested to be the reasons for this result (Lee et al. 1985) and together with water stress, arising from non-functional stomata, which plants have to deal with transplanting (Debergh et al. 2000) results in a low resistance against photoinhibition (Matysiak 2004).

Poorly developed stomata, high stomatal densities and a lack of a thick cuticle are characteristics of micropropagated plants (Pospisilova et al. 1999). Decreased stomatal resistance with increased light intensities was observed in Dieffenbachia plantlets (Table 2). In contrast to the current results, low stomatal conductance was reported in Malaus pumila plantlets after transfer to ex vitro conditions (Diaz-Perez et al. 1995). Increments in transpiration rate with an incremental increase in PPFD were observed in all the three Dieffenbachia cultivars (Table 2). This might be a result of a decreased development of epi-cuticular waxes as reported in other species, tobacco seedling leaves (Pospisilova et al. 1988) and Brassica oleracea (Grout and Aston, 1977; Wardle et al. 1979), Leucaena leucocephala (Dhawan and Bhojwani 1987) and Prunus serotina (Drew et al. 1992). The combined effects reported on physiological aspects of photosynthesis lead to the conclusion that for Dieffenbachia the low level of PPFD appears to be the safest option for acclimatization as it will lead to a reduced risk of photoinhibition.

Effect of PPFD on antioxidant enzymes

Differential SOD activity was observed with Dieffenbachia cultivars exposed to the three levels of PPFD (Fig. 1). SOD accumulation was very low with ‘Green Magic’, but a fivefold increment in activity was evident with ‘Camille’. Among the three light treatments, 100 PPFD was responsible for a stimulated activity of SOD enzyme (Fig. 1a).

Fig. 1
figure 1

Activity of antioxidant enzymes in three in vitro raised Dieffenbachia cultivars as affected by three PPFDs (35, 70 and 100 µmol m−2 s−1) 4 weeks after acclimatization. a Superoxide dismutase (SOD); b catalase (CAT); c glutathione peroxidase (GPX). Values are mean ± standard error (n = 3). Different letters above bars indicate significant differences between means (Duncan’s multiple range test; P < 0.05). LSD0.05 values 31.72, 3.85 and 37.11 for SOD, CAT and GPX, respectively

Full size image

The level of CAT activity was similar at all three levels of PPFD treatments (Fig. 1b). ‘Green Magic’ showed slightly higher levels of CAT activity compared to ‘Vesuvius’ and ‘Camille’. ‘Green Magic’ showed comparatively lower GPX activity in comparison to ‘Vesuvius’ and ‘Camille’ (Fig. 1c). ‘Vesuvius’ showed a twofold increment of GPX activity when compared to ‘Green Magic’. Among the three PPFD treatments, plants exposed to low PPFD (35 µmol m−2 s−1) showed comparatively lower GPX activity, whereas plants exposed to higher PPFD (100 µmol m−2 s−1) had a fourfold higher GPX activity in ‘Vesuvius’ and ‘Green Magic’.

It was reported that an inhibition of growth in natural plants is related to physiological and metabolic changes when they are exposed to higher excitation energy, which results in an enhanced accumulation of ROS which can induce oxidative stress (Asada and Takahashi, 1987). The accumulation of ROS in chloroplasts under extreme light stress was reported by Mishra et al. (1995). When ROS are accumulated, a light defensive mechanism will be activated in the plants and various antioxidant enzymes such as SOD, CAT and APX are activated. This was supported in the present study where increased PPFD was responsible for increases of SOD, CAT and GPX levels (Fig. 1). During oxidative stress, SOD destroys superoxide by converting it into hydrogen peroxide, which in turn is detoxified by CAT or GPX reactions (Asada 1999). Similar observations on increased SOD activity during acclimatization phase of plantlets of Cucurbita pepo and Vinca major have been reported (Logon et al. 1998). Also increased CAT activity has been reported during acclimatization of Calthea under increased PPFD (Van Huylenbroeck et al. 2000). It is assumed that the elevated SOD, CAT in combination with GPX in leaves would lower the ROS levels in Dieffenbachia cultivars.

Effect of PPFD on the level of DHN

DHN are a group of proteins that include the late embryogenesis abundant (LEA) proteins which accumulate during seed development and are associated with an acquisition of desiccation tolerance in developing seeds (Dure et al. 1981). DHN-like proteins also accumulate in response to other stresses such as cold and salt stress and one such DHN is COR15 which accumulates in response to exposure to low non-freezing temperatures (Close 1997). In recent years, various studies have reported accumulation of DHN in diverse species and tissues in response to abiotic stresses (Kosova et al. 2007). Accumulation of DHN is thought to offer membrane stability during dehydration and confer freezing, drought and salt stress tolerance in Arabidopsis and rice (Cheng et al. 2001; Puhakainen et al. 2004). We evaluated the effects of light treatments on DHN accumulation during acclimatization of Dieffenbachia cultivars. The immunoblot analysis of DHN protein fraction extracted from leaves of Dieffenbachia cultivars revealed the presence of three major bands with molecular weight of approximately 55, 40 and 22 kDa (Fig. 2). All of these three bands were expressed under exposure to 30, 70 and 100 μmol m−2 s−1 PPFD in all the three cultivars, however, quantitative differential expression showed that their levels were increased under the highest PPFD (100 μmol m−2 s−1). Our results showed the accumulation of various DHN during light induced stress during ex vitro acclimatization and indicate that DHN proteins may be involved in safeguarding cell membranes during light induced stress. Recently, Dewir et al. (2015) reported on accumulation of DHN in relation to a light-induced stress at both low and high PPFD (35 and 100 μmol m−2 s−1) during ex vitro acclimatization of African violet plants. The results of the present study are in accordance with the work of Hara et al. (2003) and Dewir et al. (2015) who have demonstrated the role of DHN in inhibition of lipid peroxidation in transgenic tobacco and acclimatizing African violet plants, respectively. We are of opinion that DHN might play a role in overcoming lipid peroxidation in Dieffenbachia plants during ex vitro acclimatization. However, further investigations are necessary to clarify the role of DHN during ex vitro acclimatization of tissue cultured plant species.

Fig. 2
figure 2

Immunoblot analysis for detection of dehydrins (DHN) in in vitro raised Dieffenbachia ‘Vesuvius’ as affected by three PPFD (35, 70 and 100 µmol m−2 s−1) 4 weeks after acclimatization. Values are mean ± standard error (n = 3). Different letters above bars indicate significant differences between means (Duncan’s multiple range test; P < 0.05; LSD0.05 value = 34.76)

Full size image

Conclusions

In this study, the effect of PPFD (35, 70 and 100 µmol m−2 s−1 PPFD) on ex vitro acclimatization of micropropagated Dieffenbechia plants was evaluated. Biomass of plants, plant height, root length, content of chlorophyll and carotenoids, net photosynthesis were optimal in plant irradiated at 100 µmol m−2 s−1 PPFD. However, this treatment was responsible for high stomatal resistance, enhanced transpiration and it was responsible for accumulation of various antioxidant enzymes as well as DHN proteins. These results clearly depict that enhanced PPFD is responsible for oxidative stress; therefore, low PPFD (35 µmol m−2 s−1) is suggested for ex vitro acclimatization of micropropagated Dieffenbachia plants.