Internal air current patterns depend on the ventilation method in a scaled-up culture vessel for micropropagation

Air current patterns were visualized inside a scaled-up culture vessel under natural or forced ventilation. Metaldehyde particles were used as tracers, and their patterns were recorded as video images by a high-resolution-and-contrast camera. Under natural conditions, the air currents were mainly influenced by natural convection that developed due to the lighting scheme, which caused differences in temperature among various articles in the chamber, including a sweet potato plantlet, supporting material, a multi-cell tray, and the culture vessel. Under forced ventilation, the air current pattern and air speed were affected by ventilation rates and by air-supply methods that were either parallel downward or circular upward. Uniformity of air movement could be achieved with air distribution pipes inside a modified vessel. Under forced ventilation, growth, photosynthetic rate, and transpiration of the micropropagated plantlets were enhanced around the air outlet as well as the inlet in the large-scale vessel. Those plant responses were probably induced by uniform spatial distribution of air current and gas concentrations.     

Manipulation of the culture environment on <Emphasis Type="Italic">in vitro</Emphasis> air movement and its impact on plantlets photosynthesis

Two-dimensional air current speeds in the culture vessel were measured using a tracer-based visualization technique and the effect of the air movement in the culture vessel on the photosynthesis of in vitro potato plantlets was assessed under a photoautotrophic culture condition. The air current speeds inside the vessel were varied by controlling free convection induced by spatial variations of temperatures in the culture vessel. For all conditions examined, upward air currents were observed around the plantlets in the central part of the culture vessel and downward air currents were observed near inside walls in the culture vessel. The upward and downward air currents were restricted by the presence of the plantlet. The upward air current speeds were affected by plantlet size inside the vessel and it was 24, 8 and 4 mm s⁻¹ in culture vessels with no plantlets, a 10-mm-tall plantlet and a 60-mm-tall plantlet cultured inside the vessel, respectively. The upward air current speed was increased by 2 times by increasing wind velocity above the culture vessel from 0.1 to 1.0 m s⁻¹. Placing the black plate on the medium also increased the air current speeds by 1.5 times. The net photosynthetic rates of the plantlets increased from 2.0 to 2.5 μmol m⁻² s⁻¹ as the upward air current speed in the culture vessel increased from 2.4 to 8.0 mm s⁻¹. The air current speeds in the culture vessel were significantly slow. Enhancement of the air movement in the culture vessel is important to promote photosynthesis of the in vitro plantlets.     

Physical environment in non-ventilated culture vessels affects <Emphasis Type="Italic">in vitro</Emphasis> growth and morphogenesis of several cultivars of <Emphasis Type="Italic">Dianthus Caryophyllus</Emphasis> L.

Summary Differences were shown in the environmental conditions inside four types of non-ventilated culture vessels (glass baby-food jars capped with metal steel or Magenta B-caps, jam glass jars capped with metal steel caps, and Magenta GA7 culture vessels) that were incubated in the same external growth room conditions. Vessel light transmittance varied from 83% to 53% and determined the availability of photosynthetic photon flux density (PPFD) for explants. The mean of medium desiccation in the culture period (30 d) was from 2.8 g in vessels with the lowest rate of gas exchange (0.02 h⁻¹) to 10.4 g in vessels with the highest (1.1 h⁻¹). In all cases, the temperature inside the culture vessel increased during the light period and decreased during the dark period, and relative humidity was close to 100%. Results of in vitro growth and morphogenesis of Dianthus caryophyllus L. evs. Scania, White Sim, Angeline, and Pink Calypso provided evidence that the environmental differences detected inside these four types of non-ventilated culture vessels was sufficient to affect micropropagation, mainly related with the specific sensitivity of each cultivar to the gas exchange and medium desiccation determined by the vessel type.     

Spatial heterogeneity of photosynthetic photon flux density in the canopy ofMiscanthus sinensis

Spatial variation in photosynthetic photon flux density (PPFD) was investigated in detail at different heights within the canopy of aMiscanthus sinensis grassland to evaluate the light environment of microsites for establishment of heliophilic tree seedlings. Highly heterogeneous patterns of light distribution were revealed within the apparently uniform grass canopies, especially under direct light. The frequency distribution patterns of relative PPFD (RPFD) were compared among different solar and sky conditions. With increasing height in the canopy, the mean RPFD value and standard deviation (SD) increased, while the skewness and kurtosis of the distribution decreased. The mean RPFD and SD were higher, especially at higher solar elevation angles, under direct light than those under diffuse light conditions. The frequency distribution of RPFD was more platykurtic under direct light and at higher solar elevation angles.     

Evaluating the usefulness of hemispherical photographs as a means to estimate photosynthetic photon flux density during a growing season in the understorey of Nothofagus pumilio forests

Abstract

Old-growth Nothofagus pumilio forests in Chile are managed employing a shelterwood system. A wide range of canopy openings can be found in old-growth and managed forests. Plant survival and growth in the understorey are influenced by the light available. There are limitations (practical and economic) to monitoring the light in the understorey. The aim of this study was to assess the options to estimate the forest understorey photosynthetic photon flux density (PPFD) measured during the growing season (GS) using canopy openness (CO) estimated by means of hemispherical photographs (HP). PPFD was measured using 31 sensors (Li-190SA quantum sensor) over the course of three GSs (October to March). The sensors were installed in an old-growth stand and another subjected to a regeneration felling under a shelterwood system. One HP was taken above each sensor (during the final GS) and the CO estimated. A comparison of the three seasons revealed that the sum of the PPFD during the GSs did not differ significantly. The CO could be used to effectively predict the sum of the PPFD during a GS (R ² = 0.959). These results demonstrate the usefulness of HPs as a means to estimate the sum of the PPFD during a GS.     

High photosynthetic photon flux and high CO2 concentration under increased number of air exchanges promote growth and photosynthesis of four kinds of orchid plantlets in vitro

Summary In vitro plantlets of Phalaenopsis ‘Happy Valentine’, Neofinetia falcate Hu, Cymbidium kanran Makino, and Cymbidium goeringii Reichb. f. were grown under photoautotrophic [high photosynthetic photon flux (PPF), high CO₂ concentration, and increased number of air exchanges] and heterotrophic (low PPF, low CO₂ concentration, no air exchanges) culture conditions. After 40 d of culture, a significant difference in plantlet growth was observed between the two cultures. Total fresh and dry mass were on average 1.5 times greater in photoautotrophic culture than in heterotrophic culture. Higher net photosynthetic rates were also observed for Phalaenopsis in photoautotrophic culture. In photoautotrophic culture, little difference was observed in air temperature between the inside and outside of the culture vessel, whereas in heterotrophic culture, air temperature inside the culture vessel was 1–2°C higher than that outside the culture vessel. Relative humidity inside the culture vessel was remarkably different between the two cultures: 83–85% in photoautotrophic culture and 97–99% in heterotrophic culture. These results indicated that growth and net photosynthetic rate of in vitro orchid plantlets were susceptible to the culture environments such as PPF, CO₂ concentration, relative humidity (RH), and the number of air exchanges, which would allow a more efficient micropropagation system for these orchid plants.     

Simple light guide for measuring irradiance in an aqueous oxygen electrode chamber

The light-dependent reactions of photosynthesis are often measured with Clark-type oxygen electrodes yet the irradiance level inside aqueous oxygen electrode reaction vessels is seldom reported due to the difficulty of measuring light inside a small volume chamber. We describe a simple light guide terminating in a 90° prism that can be inserted into a reaction vessel. Incoming irradiation is directed to a commercially available quantum sensor positioned at the other end of the light guide. Both materials for and construction of the device are inexpensive. This revised version was published online in June 2006 with corrections to the Cover Date.     

A light distribution model for an internally radiating photobioreactor

Analysis of light energy distribution in culture is important for maximizing the growth efficiency of photosynthetic cells and the productivity of a photobioreactor. To characterize the irradiance conditions in a photobioreactor, we developed a light distribution model for a single-radiator system and then extended the model to multiple radiators using the concept of parallel translation. Mathematical expressions for the local light intensity and the average light intensity were derived for a cylindrical photobioreactor with multiple internal radiators. The proposed model was used to predict the irradiance levels inside an internally radiating photobioreactor using Synechococcus sp. PCC 6301 as a model photosynthetic microorganism. The effects of cell density and radiator number were interpreted through photographic and model simulation studies. The predicted light intensity values were found to be very close to those obtained experimentally, which suggests that the proposed model is capable of accurately interpreting the local light energy profiles inside the photobioreactor system. Due to the simplicity and flexibility of the proposed model, it was also possible to predict the light conditions in other complex photobioreactors, including optical-fiber and pond-type photobioreactors.     

Application of a novel disposable film culture system to photoautotrophic micropropagation of <Emphasis Type="Italic">Eucalyptus uro-grandis (Urophylia x grandis)</Emphasis>

Summary To overcome various disadvantages of conventional culture vessls for plant micropropagation, we previously developed the photoautotrophic micropropagation technique, with special mention for the first practical film culture system, the ‘Miracle Pack’ (MP), which was made of fluorocarbon polymer film (Neoflo^? PFA film) and supported by a polycarbonate frame. While the PFA film has superior thermal stability, high light transmittance and high gas permeability, making the MP system (MP-PFA) superior to conventional culture vessels for the micropropagation of various plant species, its high cost is a disadvantage. In this study, a possible alternative of lower-cost OTP^? film made of TPX (4-methyl-1-pentane polymer) and CPP (a polypropylene), which possesses similar characteristics to PFA film, is evaluated to develop a novel disposable film culture vesel, termed ‘Vitron’, for culturing Eucalyptus (urophylla x grandis), plantlets. The three film culture systems, MP-PFA, MP-OTP (MP with OTP film), and Vitron, were placed under CO₂ enrichment, low photosynthetic photon flux density (PPFD; 45 μmol m⁻² s⁻¹), and sugar-free medium, using phenol resin foam (Oasis^?) as a substrate. In vitro and ex vitro growth and development of Eucalyptus shoots from the four-leaf stage to the rooting stage were compared for all three culture systems. The effects of the duration and concentration of CO₂ enrichments on in vitro growth of Eucalyptus cultured in the Vitron film system were also examined. The best growth and quality of Eucalyptus plantlets was obtained for the Vitron vessel placed in 3000 ppm CO₂ enrichment for 24 hours per day at low PPFD with sugar-free liquid medium and Oasis as substate. Results of this study suggest that the novel Vitron culture system is suitable for the photoautotrophic micropropagation of Eucalyptus. These authors contributed equally to the research results.     

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

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.     

Monitoring and controlling the dissolved oxygen (DO) concentration within the high aspect ratio vessel (HARV)

A probe-type oxygen sensor was developed utilizing a radioluminescent (RL)-based light source and a ruthenium-based sensing chemistry for monitoring the dissolved oxygen (DO) concentration in a modified version of the NASA-designed high aspect ratio vessel (HARV), a batch rotating wall vessel. This sensor provided the means to monitor the DO concentration in the HARV without influencing the flow pattern, thereby retaining the low shear HARV environment conducive to the formation of 3-dimensional cell aggregates. This sensor lost significant signal as a result of exposure to the first three autoclave cycles, but only minimal change in signal was observed following exposure to subsequent autoclave cycles. A new calibration model requiring only one fitted parameter was developed that accurately fit data over the entire range from 0% to 100% oxygen saturation. The ability for DO concentration control within the vessel was demonstrated by using this sensor to monitor the DO concentration inside the HARV.     

Low-cost alternatives for the micropropagation of banana

A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m⁻² s⁻¹ and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m⁻² s⁻¹ and photoperiods ranged from 8–16 hours. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microsite variation in light availability and photosynthesis in a cool-temperate deciduous broadleaf forest in central Japan

An empirical light simulation model was applied to estimate stand scale photosynthesis in a deciduous broadleaved forest in central Japan. Based on diurnal courses of photosynthetically active photon flux density (PPFD), we characterized the components of incoming light within the forest canopy, and found that the instantaneous relative PPFD (PPFD under the canopy relative to that above the canopy) under diffuse light condition was a reliable estimator of the intensity and duration of PPFD. We calculated the daily photosynthesis (A_day) for each PPFD class using photosynthesis–light response curves. Model simulated A_day were corroborated with the estimates obtained from the nearby CO₂ flux tower. The result demonstrated the potential of the light simulation model. The light use efficiency of two dominant species, Betula ermanii as overstory and Sasa senanensis as understory species, were then evaluated. At the forest understory, PPFD under 50 mol m^–2 s^–1 contributed to 77% of the sunshine duration on a completely clear day. Therefore, a higher apparent quantum yield for S. senanensis enhanced the utilization of low PPFD for photosynthesis. On the other hand, at the upper forest canopies, B. ermanii with a higher light-saturated photosynthetic rate used high PPFD efficiently. Consequently, potential of daily net photosynthesis for both B. ermanii and S. senanensis was high under each light condition. Such interspecific difference in the patterns of light utilization was suggested as one of factors allowing coexistence of the two species in the study forest.     

Spatial and seasonal heterogeneity in understory light conditions caused by differential leaf flushing of deciduous overstory trees

In a deciduous broad-leaved forest, we investigated the seasonality and heterogeneity of understory light conditions in relation to the leaf phenology of overstory trees. Fisheye photographs were taken from spring to autumn to estimate direct and indirect light conditions above the understory. Spatial variation in daily direct photosynthetic photon flux density (PPFD) was highest in late May, when the early and the intermediate-flushing trees had finished flushing and the late-flushing trees had just started flushing. After whole canopy closure, spatial variation in direct PPFD became low. Thus, asynchronous overstory leaf flushing in spring resulted in spatial heterogeneity in understory light conditions. These results suggest that the leaf phenological patterns of overstory trees are an important factor in the formation of the understory community.     

Fluctuation of photosynthetic photon flux density within aMiscanthus sinensis canopy

Detailed measurements of diurnal variations in photosynthetic photon flux density (PPFD) were made at seven locations within the canopy of aMiscanthus sinensis grassland to evaluate the light conditions of microsites for heliophilic tree seedlings. Multiple regression analysis revealed that the short-term light fluctuation on a clear day was highly dependent on the wind speed and solar elevation angle, whereas on a cloudy day it was mainly determined by the PPFD incident from above the canopy. The relative PPFD at 40 cm aboveground varied from 0.065 to 0.252, depending on sky conditions and the sensor's position in relation to clumped patches ofM. sinensis. On a clear day, the proportion of PPFD readings above 100 μmol·m⁻²·s⁻¹ contributed by sunflecks ranged between 25.4% and 82.0%. Computer simulation showed that the contribution of sunflecks to the daily carbon gain ofQuercus serrata seedlings may range from 11% to 65%. The mean relative PPFD measured under diffuse light conditions was linearly related to the daily total PPFD and the daily carbon gain by single leaves ofQ. serrata seedlings. This suggests that the relative PPFD under diffuse light conditions provides an appropriate measure of site-specific light availability within a grass canopy.     

In Vitro Determination of Drug Transfer from Drug-Coated Balloons

Drug-coated balloons are medical devices designed to locally deliver drug to diseased segments of the vessel wall. For these dosage forms, drug transfer to the vessel wall needs to be examined in detail, since drug released into the blood is cleared from the site. In order to examine drug transfer, a new in vitro setup was developed combining the estimation of drug loss during advancement to the site of application in a model coronary artery pathway with a hydrogel compartment representing, as a very simplified model, the vessel wall. The transfer of fluorescent model substances as well as the drug paclitaxel from coated balloons to the simulated vessel wall was evaluated using this method. The model was suitable to quantify the fractions transferred to the hydrogel and also to qualitatively assess distribution patterns in the hydrogel film. In the case of fluorescein sodium, rhodamin b and paclitaxel, vast amounts of the coated substance were lost during the simulated passage and only very small fractions of about 1% of the total load were transferred to the gel. This must be attributed to good water solubility of the fluorescent substances and the mechanical instability of the paclitaxel coating. Transfer of the hydrophobic model substance triamterene was however nearly unaffected by the preliminary tracking procedure with transferred fractions ranging from 8% to 14%. Analysis of model substance distribution yielded inhomogeneous distributions indicating that the coating was not evenly distributed on the balloon surface and that a great fraction of the coating liquid did not penetrate the folds of the balloon. This finding is contradictory to the generally accepted assumption of a drug depot inside the folds and emphasizes the necessity to thoroughly characterize in vitro performance of drug-coated balloons to support the very promising clinical data.     

Microsite variation in light availability and seedling growth ofQuercus serrata in a temperate pine forest

Spatial heterogeneity in light availability for tree seedlings under the canopy of a temperate pine forest was studied. Six-day measurements at 10-s intervals revealed a great variety in the temporal patterns of photosynthetic photon flux density (PPFD) and histograms among observation days and microsites; mean daily total PPFD relative to full sun varied from 1.5% to 10.4% depending on the microsites. The occurrence and duration of PPFD above 80 μmol m⁻² s⁻¹, which might reflect sunfleck activity, varied greatly among the microsites. However, several simple empirical relationships were found between some parameters characterizing microsite light availability and sunfleck activity; the diffuse site factor was correlated well with other parameters, including daily total PPFD, daily totals and daily summed durations of high PPFD above any examined threshold level, and its contribution to daily total PPFD. Diffuse site factors which were obtained for 700 microsites within an area of 28 m² on three different occasions during the growing season showed high correlations within the microsite. Based on the regressed relationship between the relative growth rate of current-year seedlings ofQuercus serrata and the microsite diffuse site factor and the results of area-survey measurement of the diffuse site factor, an estimation was made of the abundance of potential ‘safe-sites’ for seedling growth of the species; the ‘safe sites’ were estimated to cover 40% and 0% of the total area of the sunny and shady sites of the forest, respectively.     

Development of a forced ventilation micropropagation system for large-scale photoautotrophic culture and its utilization in sweet potato

Summary A forced ventilation system has been developed for large-scale photoautotrophic micropropagation of chlorophyllous plants. The major goal of the system is to provide a uniform supply of CO₂ inside a large culture vessel (volume 3480 ml) to achieve uniform growth of the plantlets. The system has been designed such that sterile nutrient solution can be supplied throughout the culture period, which is essential for long-term culture. Sweet potato (Ipomoea batatas L. Lam., cv. ‘Beniazuma’) was used as a model plant for photoautotrophic culture with stagnant and nonstagnant nutrient solution in large vessels. Growth and net photosynthetic rates of the plantlets were compared with those of the plantlets grown in a small vessel under photoautotrophic conditions (with natural ventilation) and conventional photomixotrophic conditions. The results indicated that the large vessel with the forced ventilation system was effective for improving growth and uniformity of the plantlets and the rate of net photosynthesis. The stagnant nutrient solution condition under photoautotrophic forced ventilation treatment significantly increased the fresh mass of the plantlets; however, percent dry mass was highest in the treatment with nonstagnant nutrient solution condition. The results demonstrated that the conventional photomixotrophic culture system can cause seriously inhibited growth and development.     

A feedback control system for the precise and continuous regulation of photosynthetic photon flux density

A simple and inexpensive feedback control system that provides continuous and precise control of photosynthetic photon flux density (PPFD) in a whole plant cuvette is described. A ‘Plexiglass’ tank is interposed between a light source and cuvette and PPFD changed by varying the level of dyed liquid in the tank. The amount of liquid pumped into or drained from the tank is a function of the difference (error) between a defined set point value of PPFD and that measured in the cuvette. The set point can be varied as a function of time, can follow the output of a quantum sensor measuring ambient PPFD or can be driven by values of PPFD read from a data file. Within the 0.4 to 0.64 μm waveband, the dye acts as a neutral density filter so that there is no change in spectral distribution with PPFD. Photosynthetic photon flux density in the cuvette was controlled to better than 20 μmol m⁻²s⁻¹ when the set point was varied from 200 to 1100 μmol m⁻²s⁻¹ over 3 min. When the set point was held constant or changed less rapidly, errors did not exceed 5 μmol m⁻²s⁻¹. Net photosynthesis of Western redcedar (Thuja plicata Donn.) seedlings held at 18 °C closely followed rapid changes in PPFD.     

LIGHT EFFECTS ON LEAF MORPHOLOGY IN WATER HYACINTH (EICHHORNIA CRASSIPES)

Water hyacinth leaves in natural populations vary from being long and thin-petioled to being short with inflated petioles. A variety of factors has been used experimentally to alter water hyacinth leaf shape, but what controls the development of leaf morphology in the field has not been established. We measured photosynthetic photon flux density (PPFD) and spectral distribution of radiation in a natural water hyacinth population. PPFD in the center of the water hyacinth mat was reduced to 2.7% of full sunlight, and the red to far red (R:FR) ratio was reduced to 0.28. When shoot tips of plants were exposed to artificial light environments, only plants in the treatment with a R:FR ratio comparable to that in the natural population produced leaves with long, thin petioles. Shoot tips in full sun or covered with clear plastic bags or bags that reduced light quantity without greatly altering light quality produced shorter leaves with inflated petioles. We hypothesize that the altered light quality inside a mat is a major environmental control of water hyacinth leaf morphology.