Sugar metabolism, photosynthesis, and growth of in vitro plantlets of Doritaenopsis under controlled microenvironmental conditions

Abstract

Suboptimal environmental conditions inside closed culture vessels can be detrimental to in vitro growth and survival of plantlets during the acclimatization process. In this study, the environmental factors that affected Doritaenopsis plantlet growth and the relationship between growth and sugar metabolism were investigated. Cultures were maintained under heterotrophic, photoautotrophic, or photomixotrophic conditions under different light intensities and CO₂ concentrations. Photoautotrophic growth of Doritaenopsis hybrid plantlets could be promoted significantly by increasing the light intensity and CO₂ concentration in the culture vessel. The concentration of different sugars in the leaves of in vitro-grown plantlets varied with different cultural treatments through a 10-wk culture period. Starch, reducing sugars, and nonreducing sugar contents were higher in plantlets grown under photoautotrophic and photomixotrophic conditions than in heterotrophically grown plantlets. Net photosynthesis rates were also higher in photoautotrophically and photomixotrophically grown plantlets. These results support the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high photosynthetic photon flux density. Growth was greatest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high photosynthetic photon flux density. The physiological status of in vitro-grown Crassulacean acid metabolism (CAM)-type Doritaenopsis showed a transition from C3 to CAM prior to acclimatization.

Introduction

Micropropagation is routinely used for rapid multiplication of stock plant material to produce a large number of genetically and physiologically uniform plants. However, the low survival rate of regenerated plants can limit large-scale micropropagation of some plant species (Hazarika 2006). The culture vessel environment is characterized by high relative humidity, poor gas exchange between the culture vessel and the microclimate chamber, low CO₂ concentration, low photosynthetic photon flux density (PPFD), high sugar concentration in the culture medium, and high ethylene. These conditions may affect tissue vigor and shoot multiplication and can impede the successful transfer of micropropagated plants to ex vitro conditions (Kozai et al. 1997; Kozai et al. 2005; Park et al. 2011). Two of the most common problems with in vitro propagation are that the leaves of micropropagated plants may be chlorophyll deficient and contain more water than ex vitro-grown leaves, a condition termed “hyperhydricity.” Hyperhydricity shows altered leaf anatomy and can generate aeration stress that depletes oxygen levels in the cells. Several types of abnormal structures have been observed in in vitro-grown leaves, such as a reduced number of palisade cell layers, irregular stomata, chloroplast degeneration, and the cuticle is either very thin or absent (Chakrabarty et al. 2006; Dewir et al. 2006; Ivanova and Van Staden 2010).

Light intensity and quality are important factors for plant growth and development in vitro (Macedo et al. 2011). Low light conditions inside culture vessels are comparable to shade conditions. Shade-acclimated leaves undergo physiological changes that enable efficient use of limited light resources, but they are predisposed to photodamage by sudden exposure to high light conditions. This scenario can occur when culture-adapted tissues are transferred to soil under greenhouse conditions. The result of this damage is reduced net CO₂ assimilation after transfer to high light conditions, possibly for the entire life of the leaf (Van Huylenbroeck 1994; Van Huylenbroeck et al. 2000).

Conventional in vitro-grown heterotrophic plants have a low photosynthetic rate, and require the addition of sugar to the culture medium to serve as a carbon source. This affects their acclimatization during conversion of the plant tissue from heterotrophic to autotrophic growth. The low photosynthetic rate of plants in vitro is not due to low photosynthetic efficiency but results from the low CO₂ concentration in culture vessels (Park et al. 2011; Xiao et al. 2011).

Many microclimate environmental factors can be controlled to enhance micropropagation efficiency. Since photoautotrophic micropropagation was developed more than two decades ago, numerous studies have been conducted to investigate the effects of the culture vessel microenvironment on plant growth and development and to devise strategies for improving these conditions (Zobayed et al. 2004; Yoon et al. 2009; Kozai 2010). Plant transpiration and nutrient uptake are critically important for a successful photoautotrophic micropropagation. Although physiological changes in some photoautotrophic plants grown in vitro have been studied (Norikane et al. 2010; Badr et al. 2011), there are few reports on plant growth and physiological changes during micropropagation of orchid species.

The purpose of this study was to investigate environmental factors during in vitro culture of Doritaenopsis, an important ornamental orchid in the floriculture industry. The effects of light intensity and CO₂ enrichment on the growth of Doritaenopsis plantlets were explored for possible correlations with sugar metabolism. In addition, the physiological parameters of different culture systems that are currently used for Doritaenopsis were studied to understand their relationships to growth.

Materials and Methods

Plant material and culture medium.

Seeds of Doritaenopsis ‘New Candy’ and Doritaenopsis ‘Mary Anes × Ever Spring’ hybrids were germinated on Hyponex medium (Kano 1965; 6.5 N–4.5 P–19 K at 1 g L⁻¹ and 20 N–20 P–20 K at 1 g L⁻¹) with 0.5 g L⁻¹ activated charcoal (Sigma, St. Louis, MO) and 7 g L⁻¹ phytoagar (Duchefa, Haarlem, Netherlands). After 4 mo, five seedlings (about 1.5 cm in shoot length and 0.2 g fresh weight) were cultured in each of several 250 ml Erlenmeyer flasks. For all treatments, at least five flasks were used for each treatment within a replicate, with three replicates. All cultures were maintained for 10 wk in a growth chamber (Dasol Scientific Co. Ltd., Gyeonggi, Republic of Korea) at 24 ± 1°C with 16-h light/8-h dark photoperiod. After 10 wk of culture, leaf area, leaf length, root length, and number of roots were recorded from each plantlet grown under different environmental conditions.

Light intensity, sucrose, and CO₂ concentration.

Three different growth conditions (heterotrophic, photomixotrophic, and photoautotrophic) were applied by varying sucrose, light, and CO₂ (Table 1). For heterotrophic cultures, 30 g L⁻¹ sucrose was added to the basal Hyponex medium, and the culture vessels were covered with sterile aluminum foil and sealed with a wrap to block air exchange. The conditions for heterotrophic growth included low heterotrophic conditions (40 ± 2 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂) and high heterotrophic conditions (120 ± 5 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂).

Table 1. Description of the treatments used in this study

For photomixotrophic cultures, 30 g L⁻¹ sucrose was added to the basal Hyponex medium which was used for seed germination and the culture vessel (250 ml Erlenmeyer flask) was sealed as before, except a small hole (1 cm diameter) was cut in the top and covered with a membrane filter (0.2 μm pore size; Micropore™, Seoul, Republic of Korea) to allow gas exchange to occur. The rate of gas exchange was calculated as the hourly ventilation rate divided by the air volume of the culture vessel according to Zobayed et al. (2000). The gas exchange for the photomixotrophic experiments was calculated to be 2.83 times/h. The conditions for photomixotrophic growth included low photomixotrophic conditions (40 ± 2 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂), high photomixotrophic conditions (120 ± 5 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂), low photomixotrophic conditions enriched with CO₂ (40 ± 2 μmol m⁻² s⁻¹ PPFD and 1,500 ± 100 μmol mol⁻¹ CO₂), and high photomixotrophic conditions enriched with CO₂ (120 ± 5 μmol m⁻² s⁻¹ PPFD and 1,500 ± 100 μmol mol⁻¹ CO₂).

For photoautotrophic cultures, no sugar was added to the basal Hyponex medium. The culture vessels were closed with aluminum foil containing a small hole that was covered with a membrane filter (1 cm diameter), the same as for the photomixotrophic cultures. The conditions for photoautotrophic growth included low photoautotrophic conditions (40 ± 2 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂), high photoautotrophic conditions (120 ± 5 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂), low photoautotrophic conditions enriched with CO₂ (40 ± 2 μmol m⁻² s⁻¹ PPFD and 1,500 ± 100 μmol mol⁻¹ CO₂), and high photoautotrophic conditions enriched with CO₂ (120 ± 5 μmol m⁻² s⁻¹ PPFD and 350 ± 20 μmol mol⁻¹ CO₂).

CO₂ concentrations in the culture chamber were monitored and controlled using an infrared-type CO₂ controller (Dasol Scientific Co. Ltd., Republic of Korea). Liquid CO₂ was supplied by Praxair Korea Co. Ltd. (Seoul, Republic of Korea). Metal halide and fluorescent lamps were used as light sources.

Acclimatization.

Following the 10-wk culture period, plantlets were transplanted to round pots (9 × 10 cm) that contained pine tree bark (average particle size, >5 mm) as culture soil. During the acclimatization period, the PPFD in the day was adjusted to 100 ± 20 μmol m⁻² s⁻¹, and the day/night temperature was 25 ± 2/22 ± 2°C.

Chlorophyll fluorescence.

Plantlets were kept in the dark for 30 min before chlorophyll measurement. The chlorophyll fluorescence of the abaxial side of fresh leaves was measured (Chakrabarty et al. 2006). Modulated fluorescence was measured using a PAM-200 chlorophyll fluorometer fitted with a DA-200 leaf clip holder (Heinz Walz, Effeltrich, Germany). Data were analyzed by a software provided by the manufacturer. Minimal fluorescence (F ₀) was measured in dark-adapted leaves using incident light of <0.1 μmol m⁻² s⁻¹; maximal fluorescence (F _m) was measured after a 1-s saturating pulse (>3,500 μmol m⁻² s⁻¹) in the same leaves. Maximal variable fluorescence (F _v = F _m − F ₀) and photochemical efficiency of photosystem II (PSII) (F _v/F _m) were calculated for dark-adapted leaves.

Net photosynthetic rate.

The amount of CO₂ absorbed by in vitro-grown plantlets was calculated by measuring the CO₂ concentration in the culture container halfway through the light and dark photoperiods. The concentration of CO₂ was measured by using a gas chromatograph (HP 6890) equipped with a 25 m × 0.53 mm PoraPlot Q capillary column and a thermal conductivity detector (Hewlett Packard, Wilmington, DE). Helium was used as carrier gas and set to a flow speed of 13 mL min⁻¹. Other parameters included an oven temperature of 90°C and an injector temperature of 200°C. The net photosynthetic rate (NPR) of plantlets was calculated according to Kozai and Iwanami (1988).

Malic acid and citric acid contents.

Malic acid and citric acid were analyzed using high-performance liquid chromatography (HPLC). One gram of leaf tissue was finely ground, 2 ml of distilled water was added, soluble materials were extracted for 30 min at 60°C, and then the solution was filtered through a 0.2 μm membrane filter. The organic acids in the filtrate were measured using HPLC (Waters 600S controller, Waters 626 pump, Waters Co., Milford, MA) (Gomis et al. 1992) with a Nova-Pack C18 column (3.9 × 300 mm, Waters Co.), with a mobile phase of 0.2 M KH₂PO₄/H₃PO₄ (pH 2.4) at a flow rate of 0.5 mL min⁻¹, and measurements were performed using ultraviolet light at 215 nm. Totally the tests were repeated three times for each experimental group. Data was expressed with mean and standard errors in the figures to show statistical significance between the treatments.

Carbohydrate extraction and analysis.

Leaves were frozen in liquid nitrogen, transferred to mortars containing liquid nitrogen and ground with a pestle. The liquid nitrogen was allowed to evaporate and the ground tissues were extracted in 3 volumes of 80% ethanol to separate the ethanol-soluble fraction (containing sugars) and the ethanol-insoluble fraction (containing starch). The ethanol-soluble supernatant was removed after centrifugation for 15 min at 8,000×g. The supernatant was passed sequentially through a C₁₈ Sep-Pak cartridge (Waters, Milford, MA) and a 0.2 μm membrane filter. Samples were injected into a Waters HPLC (Waters 626 pump and 600 s controller) using a Rheodyne 7725I injector equipped with a 20 μl sample loop. The isocratic system was operated at 1 mL min⁻¹ using degassed 80% acetonitrile as the mobile phase. Soluble sugar separation was achieved using a carbohydrate WATO44355 (250 × 4:6 mm²) stainless steel column (Waters). Soluble sugars were measured using a refractive index detector (Waters 410) (Ding et al. 1998). For starch determination, the precipitate was washed three times with 2–3 volumes of 40% ethanol to remove soluble sugar residues. Starch was solubilized using HCl–DMSO pretreatment (Lambrechts et al. 1994) and hydrolyzed by incubation with amyloglucosidase (50 units mL⁻¹ in 0.1 M Na-acetate buffer pH 4.2) for 48 h at 55°C, with occasional agitation. Starch was then determined as glucose using the dinitrosalicylic acid assay (Chaplin 1986).

Results and Discussion

Growth and morphological features of plantlets.

For plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions, the leaf area, leaf length, and root length were greater than for plantlets grown in heterotrophic conditions (Fig. 1A, B ). In general, all growth parameters were highest in plantlets grown under CO₂-enriched photoautotrophic and photomixotrophic conditions with high PPFD. The leaves were smaller in L and H treatments (no CO₂ enrichment) compared with the leaves from LC and HC treatments (with CO₂ enrichment). Plantlets grown on medium containing sugar showed more and longer roots than those from the treatments that had no sugar. Statistically, the greatest root growth was observed in HC conditions for both photoautotrophic and photomixotrophic treatments.

Figure 1.

Leaf area, leaf length, root length, and number of roots per plantlet (A) and growth of Doritaenopsis hybrids cultured for 10 wk under different culture conditions. Plantlets were cultured under photoautotrophic (0% sucrose), photomixotrophic (3% sucrose), and heterotrophic (3% sucrose) conditions. Scale bar = 2 cm. L low PPF (40 ± 2 μmol m⁻² s⁻¹), H high PPF (120 ± 5 μmol m⁻² s⁻¹), LC low PPF + CO₂ enrichment (1,500 ± 100 μmol mol⁻¹), HC high PPF + CO₂ enrichment (1,500 ± 100 μmol mol⁻¹).

The heterotrophic cultures, where no gas exchange occurred between the inside and outside of the culture vessel, showed the poorest growth and leaf area decreased to approximately 56% that of the plantlets grown in photoautotrophic and photomixotrophic conditions. Conventional micropropagation techniques are typically performed using small culture vessels under low PPFD conditions without ventilation (Xiao et al. 2011). Many studies indicate that photoautotrophic growth in cultured plantlets can be significantly promoted by increasing the CO₂ concentration and the light intensity, and by removing growth retardant gas such as ethylene from the vessel (Lian et al. 2002; Yoon et al. 2009; Xiao et al. 2011).

Physiological and biochemical features of tissue samples.

Measurement of the photosynthetic efficiency with chlorophyll fluorescence. Chlorophyll fluorescence was used to determine the photosynthetic efficiency. F _o , which is an indicator of the antenna size (number of pigment molecules) of PSII, was lower under heterotrophic treatment conditions after 10 wk of culture. The F _v /F _m ratio, which provides an estimate of the basal efficiency of the PSII photochemical process, was also lower under heterotrophic treatment conditions. The AHC and MHC cultures maintained values of 0.75–0.8 F_v/F_m, and showed the highest photosynthetic efficiency and the highest amount of photosynthesis. However, for the treatments exposed to high light intensity but without CO₂ enrichment, the value of F _v /F _m was lower (Fig. 2). The tissues subjected to high light intensity in the heterotrophic condition showed a significant decrease of the early value of F _v /F _m . Chakrabarty et al. (2006) studied hyperhydricity in apple and suggested that the substantial collapse of photosynthesis that occurred in heterotrophic treatments largely resulted from a severe disruption of the photosystem (or at least of PSII), and was probably not the result of any major effects on other aspects of the photosynthesis.

Figure 2.

Fluorescence parameters (F _v /F _m , F ₀ , and F _m ) during in vitro culture of Doritaenopsis hybrid plantlets. Vertical bars represent mean ± standard error of plantlets from three replicate vessels.

Net photosynthetic rate. The photosynthetic rate was affected by different culture conditions. For plantlets grown in the photoautotrophic and photomixotrophic treatments, depending on the time within the photoperiod, CO₂ absorption decreased during the light cycle and CO₂ absorption increased during the dark cycle (Fig. 3). The plant materials that were cultured in CO₂-enriched conditions (such as AHC and MHC) showed a high CO₂ absorption throughout the culture compared to the other treatments, and CO₂ absorption was increased in plantlets grown in photoautotrophic treatments, especially at Week 8 (Fig. 3). Plantlets grown under strong light with CO₂-enriched condition (HC) seemed to exhibit the Crassulacean acid metabolism (CAM)-type photosynthetic pattern. Doritaenopsis is CAM type plant in nature. In general, CAM plants photosynthesize by absorbing CO₂ during the night (Osmond 1978). However, activation of CAM or C3 metabolism depends on the microenvironment, so in vitro-grown plantlets can show C3-type photosynthesis patterns under certain conditions (Malda et al. 1999) and transition to a CAM-type photosynthetic pattern upon exposure to bright light and CO₂ supplementation.

Figure 3.

Changes in CO₂ uptake during light (A–C) and dark (D–F) periods in Doritaenopsis hybrid plantlets during in vitro culture. Vertical bars represent mean ± standard error of plantlets from three replicate vessels.

Photoautotrophic micropropagation is defined as micropropagation without sugar in the culture medium, where carbohydrate accumulation of tissue cultures and subsequent growth is dependent fully on photosynthesis and inorganic nutrient uptake (Zobayed et al. 2004; Arigita et al. 2010; Kozai 2010). Thus, it also can be called photosynthetic micropropagation in sugar-free medium (Xiao et al. 2011).

In AHC and MHC treatments, malic acid and citric acid content remained high after 6 wk culture (Fig. 4). An increased CO₂ concentration increased the rate of malic acid synthesis and its concentration in the cytoplasm (Zobayed et al. 2000). Our result supports the hypothesis that pyruvate, produced by the decarboxylation of malate, is required for optimal photoautotrophy under high PPFD. Apparently, the pyruvate produced by other metabolic pathways is insufficient to support optimal growth under high PPFD conditions. During heterotrophic (and presumably photomixotrophic) growth, the principal mode of glucose catabolism is via the pentose phosphate pathway. Malate was the dominant fixation product in photoautotrophic cultures of soybean grown under elevated CO₂ concentrations (Horn and Widholm 1994).

Figure 4.

Changes in malic acid and citric acid contents in leaves of Doritaenopsis hybrid plantlets during the in vitro culture period. Vertical bars represent mean ± standard error of plantlets from three replicate vessels.

Changes in carbohydrate content during culture period. The sugar concentration in leaves of in vitro-grown plantlets through 10-wk culture varied with treatments (Figs. 5 and 6). Starch, reducing sugar, and non-reducing sugar contents were higher in plantlets grown under AHC and MHC conditions, with increases after 6 wk culture. Starch, reducing soluble sugar, nonreducing sugar, and total sugar content were higher in photoautotrophic and photomixotrophic conditions than in the leaves and roots of plantlets grown under heterotrophic conditions (Fig. 6). These variations were obvious because NPR rates were very high in photoautotrophically and photomixotrophically grown plants, as previously reported (Lian et al. 2002).

Figure 5.

Changes in sucrose, glucose, and fructose contents in leaves of Doritaenopsis hybrid plantlets under photoautotrophic, photomixotrophic, and heterotrophic conditions over 10 wk culture. Vertical bars represent mean ± standard error of plantlets from three replicate vessels.

Figure 6.

Changes in total sugar (a) and starch (b) of leaves (A–C) and roots (D–F) of Doritaenopsis hybrid plantlets under photoautotrophic, photomixotrophic, and heterotrophic conditions over 10 wk culture. Vertical bars represent mean ± standard error of plantlets from three replicate vessels.

A very good correlation was observed between starch content and high light and CO₂ (AHC and MHC) culture conditions. The photomixotrophic and photoautotrophic groups under high light intensity showed that the starch contents increased after net photosynthesis was increased during the night (Figs. 2 and 6). Photosynthetically active cells and tissues in vitro have two major carboxylation pathways for CO₂ fixation. The first is autotrophic fixation of CO₂ mediated by Rubisco; the second is non-autotrophic carbon fixation mediated by the enzyme phosphoenolpyruvate (PEP) carboxylase, which catalyses the irreversible β-carboxylation of PEP by HCO₃ ⁻ (Bender 1987; Kumar et al. 1988). Under low light, CO₂ enrichment caused the highest activity of PEP carboxylase in the photoautotrophic and photomixotrophic plantlets (data not shown). A similar observation was reported for Platycerium coronarium callus culture (Kwa et al. 1997). The nonphotosynthetic fixation of CO₂ through PEP carboxylase has been previously observed in shoot cultures of eastern white cedar (Nour and Thorpe 1994).

The accumulation of sugars may be attributed to the specific activation of sucrose synthesis by up-regulation of sucrose phosphate synthase (SPS) in the MHC treatment group. Sucrose synthesis in higher plants occurs through the action of SPS and sucrose synthase (SS). SPS activity is influenced by different culture conditions, whereas SS activity increases with increased metabolic carbohydrate demand. The activity of SPS was enhanced by CO₂, which accelerates the biosynthesis of sucrose (Signora et al. 1998). The in vitro-grown plantlets of Doritaenopsis showed accumulation of starch and an increase in sucrose particularly in the AHC and MHC (CO₂ enriched) treatments. However, monosaccharide content decreased over time. The trend for sucrose content increase correlated with the length of the cultivation period, whereas glucose and fructose contents decreased gradually during culture.

Plantlets grown under various conditions were planted in the pots and acclimatized for 10 wk. Most plants were successfully acclimatized, however the plantlets grown in AHC and MHC showed the most vigorous and rapid growth in greenhouse (data not shown)

In conclusion, the results of the studies on growth parameters, photosynthesis, and carbohydrate analyses suggest that photoautotrophic growth in vitro could be promoted significantly by increasing the CO₂ concentration and light intensity in culture vessels. Plant tissues that were cultured in CO₂-enriched conditions, such as AHC and MHC showed higher CO₂ absorption throughout the culture period compared with other treatments, and CO₂ absorption was remarkably high in the photoautotrophic treatments. The findings of the present study will be helpful to enhance acclimatization of Doritaenopsis plantlets to ex vitro conditions.