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.     

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.     

Mass Propagation of Eucalyptus camaldulensis in a Scaled-up Vessel Under In Vitro Photoautotrophic Condition

A scaled-up culture vessel was designed for the large-scalephotoautotrophic micropropagation of chlorophyllous plants.The culture vessel (volume 20 l) contained a plug cell traywith 448 plantlets, and had a forced ventilation system to supplyCO₂-enriched air. A nutrient-reservoir was connected to theculture vessel from which nutrient solution was circulated tothe culture vessel every 24 h. Nodal leafy cuttings of Eucalyptuscamaldulensis L. were cultured photoautotrophically in thissystem without sugar in the nutrient medium, but with an enrichedCO₂concentration and a high photosynthetic photon flux. Thegrowth and the net photosynthetic rate of the in vitro grownplantlets and the survival percentage of the plantlets aftertransplanting to ex vitro conditions were compared with thoseof plantlets grown photoautotrophically under natural ventilationin conventional small culture vessels (Magenta-type vessels;volume 0.4 l). Fresh and dry masses and net photosynthetic ratewere significantly higher in plantlets grown in the scaled-upvessel compared to plantlets grown in the conventional smallvessels (control). The environmental conditions created in thisscaled-up vessel (with forced ventilation) also facilitatedacclimatizationin vitro . Importantly, after transplanting tothe ex vitro condition, plantlets grew well without any specializedexvitro acclimatization treatment. Copyright 2000 Annals of BotanyCompany CO₂enrichment, Eucalyptus camaldulensis L., ex vitro, forced ventilation, natural ventilation, photoautotrophic, scaled-up vessel, survival percentage     

Multiple shoot induction and leaf and flower bud abscission of Annonacultures as affected by types of ventilation

Nodal explants of Annona squamosa L. and Annona muricata L. were cultured in vitro under various types of ventilation: airtight vessel (sealed condition; number of air exchange 0.1 h^–1), natural ventilation (via a polypropylene membrane; number of air exchange 1.5 h^–1), and forced ventilation (5.0 cm³ min^–1 in a 60 cm³ vessel; number of air exchange 5.0 h^–1). In both species, numbers of leaves, leaf areas and numbers of nodes per shoot increased with improving standards of ventilation, while leaf abscissions were substantially reduced; all the leaves had abscised in the airtight vessels after 12–15 days, but none had done so with forced ventilation. Flower-bud abscission in A. muricatashowed a similar trend after 21 days. These effects were associated with reductions in the accumulation of ethylene within the culture vessels, produced by increasing the efficiency of ventilation; ethylene was not detected in those fitted with a forced ventilation system. CO₂ concentrations in culture headspaces and the net photosynthetic rates of the plantlets were also evaluated. CO₂ concentrations decreased well below the ambient in the natural and airtight vessels; however, under forced ventilation, CO₂ concentrations were significantly higher during the photoperiod, compared to those of the natural ventilation and airtight vessel treatments. In general, net photosynthetic rates per unit leaf area increased with increasing photosynthetic photon flux (PPF) and rates were highest in plantlets grown under forced ventilation, intermediate under natural ventilation and lowest in the airtight vessels.Eighteen different media were investigated for their effects on multiple shoot induction in both species. The best medium for multiple shoot induction and growth in A. squamosa was Murashige and Skoog medium (MS) + 6-benzylaminopurine (BA; 1.5 mg l^–1) + casein hydrolysate (1.0 g l^–1) and for A. muricata MS + BA (1.0 mg l^–1) + naphthaleneacetic acid (NAA; 0.1 mg l^–1).     

Developing a Photoautotrophic Micropropagation System for Woody Plants

in vitro including cotyledonary stage somatic embryos have the ability to grow photoautotrophically (without sugar in the culture medium), and that the low or negative net photosynthetic rate of plants in vitro is due not to poor photosynthetic ability, but to the low CO₂ concentration in the air-tight culture vessel during the photoperiod. Furthermore, we have shown that the photoautotrophic growth of several woody plants in vitro can be significantly promoted by increasing the CO₂ concentration and light intensity in the vessel, by decreasing the relative humidity in the vessel, and by using a fibrous or porous supporting material with high air porosity instead of gelling agents such as agar. In this paper, the advantages of photoautotrophic micropropagation in a conventional, small culture vessel with a microporous gas filter for enhancing natural ventilation and in a large culture vessel with a forced ventilation unit are described for woody plants such as acacia (Acacia mangium), coffee (Coffea arabusta), eucalyptus (Eucalyptus camaldlensis), mangosteen (Garcinia mangostana), neem (Azadirachta indica), paulownia (Paulownia fortunei), and pine (Pinus radiata). Received 30 August 2001/ Accepted in revised form 27 September 2001     

Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition

Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m⁻² s⁻¹) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h⁻¹ for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity, emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included 40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation method in photoautotrophic micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Estimation of light intensity distribution in a culture vessel

Distribution of photosynthetic photon flux density (PPFD) in a culture vessel was estimated using a sensor film for measuring the integrated solar radiation. A plantlet model whose leaves were constructed from sensor film was used to simulate a potato plantlet. The plantlet model was put into a culture vessel and, after exposure to culture conditions for 20 days, PPFD was estimated for each individual model leaf based on the degree of fading of the sensor film. The method was able to demonstrate the light intensity distribution patterns inside a small test tube under downward lighting conditions. This technique allows for the estimation of light intensity distribution patterns inside a small culture vessel, which was previously difficult to measure by conventional methods.     

Measurement of gas exchange rates in plant tissue culture vessels

The aerial microenvironment in culture vessels has a significant effect on the growth and development of plantlets in vitro. Since the gas exchange between outside air and inner air can influence the microenvironment of culture vessel, it is necessary to measure the air exchange rate for various vessels. In this study, water vapor was used as the tracer gas, and the change of absolute humidity inside the vessel was calculated continuously by the measured values of a relative humidity sensing element. The outside environment was maintained at constant humidity level by a saturated salt solution. The RH data were transformed into absolute humidity and the specific humidity ratio. The air exchange rates of several tissue culture vessels were then calculated. The exchange rate was between 0.0145 h^–1 to 0.0376 h^–1. This technique provides an inexpensive, rapid and simple way to determine the air exchange rate of a culture vessel within a short period. The effects of the air current velocities on the exchange rates of vessels were also determined.     

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.     

Micropropagation of Potato: Evaluation of Closed, Diffusive and Forced Ventilation on Growth and Tuberization

Different types of ventilation of the culture vessel headspace,each with and without the ethylene inhibitor AgNO₃(3.0 µM)or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid(ACC) (2.0 µM) in the culture medium, were investigatedin terms of their effects on the growth of potato cuttings (Solanumtuberosum L. ‘cara’). Concentrations of CO₂ , O₂andethylene in the culture vessel headspaces were monitored duringthe study. Growth was substantially enhanced and vitrification(stunting and epinasty of leaves and hooking of stem apices)was reduced by increasing the efficiency of ventilation, theeffects being greatest with forced ventilation. In the conventionaldiffusive treatment (via a polypropylene membrane), leaf epinastyoccurred but the leaves were not stunted unless ACC had beenadded. AgNO₃prevented vitrification in the latter case and reducedit in the sealed treatment. On the other hand, with all forcedventilation treatments, even with the addition of ACC, the plantletsgrew well and some of the growth parameters exceeded those inthe diffusive + AgNO₃treatment. Ethylene removal was clearlyan important factor contributing to the better growth foundwith diffusive and especially with the forced ventilation treatment;with the latter, ethylene concentrations in the culture vesselswere virtually zero. In addition, enhanced CO₂supply probablycontributed to the better performance under forced ventilationcompared to diffusive ventilation. Callus developed on the stembases in all sealed (airtight) and diffusive treatments exceptwhere AgNO₃was used. No callus was observed in any treatmentwhere forced ventilation was applied and in vitro tuberization(tuber size) was considerably improved by this treatment. Copyright2001 Annals of Botany Company Callus, ethylene, potato, tuberization, vitrification     

Comparison of responses to CO2 under the conditions of natural respiration and controlled artificial pulmonary ventilation

The relationship between the sensation of lack of air caused by the addition of carbon dioxide to inhaled air and minute pulmonary ventilation was studied in eight healthy male volunteers. Under the conditions of both natural respiration and controlled artificial ventilation of the lungs, ventilation increased in proportion to the increase in the partial pressure of CO₂ in the end-tidal exhaled air. The individual differences in the ventilation responses to CO₂ under the conditions of controlled ventilation and natural ventilation are regarded as a property of voluntary control.     

Photoautotrophic culture of Coffea arabusta somatic embryos: development of a bioreactor for large-scale plantlet conversion from cotyledonary embryos

Somatic embryos were developed from in vitro-grown leaf discs of Coffea arabusta in modified Murashige and Skoog medium under 30 micromol m(-2) s(-1) photosynthetic photon flux (PPF). Cotyledonary stage embryos were selected from the 14-week-old cultures and were placed under a high (100 micromol m(-2) s(-1) PPF for 14 d. These pretreated embryos were grown photoautotrophically in three different types of culture systems: Magenta vessel; RITA-bioreactor (modified to improve air exchange); and a specially designed temporary root zone immersion bioreactor system (TRI-bioreactor) with forced ventilation. The aims of the study were to achieve large-scale embryo-to-plantlet conversion, and to optimize growth of plantlets under photoautotrophic conditions. The plantlet conversion percentage was highest (84 %) in the TRI-bioreactor and lowest in the modified RITA-bioreactor (20 %). Growth and survival of converted plantlets following 45 d of photoautotrophic culture in each of the three culture systems were studied. Fresh and dry masses of leaves and roots of plantlets developed in the TRI-bioreactor were significantly greater than those of plantlets developed in the modified RITA-bioreactor or Magenta vessel. The net photosynthetic rate, chlorophyll fluorescence and chlorophyll contents were also highest in plantlets grown in the TRI-bioreactor. Normal stomata were observed in leaves of plantlets grown in the TRI-bioreactor, whereas they could be abnormal in plantlets from the modified RITA-bioreactor. Survival of the plants after transfer from culture followed a similar pattern and was highest in the group grown in the TRI-bioreactor, followed by plants grown in the modified RITA-bioreactor and Magenta vessel. In addition, ex vitro growth of plants transferred from the TRI-bioreactor was faster than that of plants from the other culture systems.     

Housing and health. What quality of air?

Summary The renewal of air is essential for good hygiene in housing. The list of polluting elements in dwellings is very long: particles, smells, acarus, allergens, formaldehyde, radon. All these polluting elements influence negatively our health. Apart from the complete elimination of the generating cources of polluting elements, an important factor for the pollution treatment is a good-working ventilation. Mechanical ventilation is the only system allowing an acceptable permanent ventilation during all seasons. It can be simple flow by mechanical exhaust and natural air income, or double flow by both mechanical exhaust and air supply. Double flow ventilation allows the filtering of fresh air and the heat transfer from exhaust to fresh air. It enables the elimination of certain allergens and the lowering, to a reasonable value, of the concentration of other polluting agents present in the dwellings inside air.     

Influence of in vitro environment on somatic embryogenesis of Coffea arabica L. cv. Caturra rojo: the effects of carbon dioxide on embryogenic cell suspensions

The influence of environment in the culture vessel is a factor that has very little study in the process of somatic embryogenesis. The present research was carried out with the objective to determine the effects of carbon dioxide on somatic embryogenesis of Coffea arabica cv. Caturra rojo. Embryogenic cell suspensions were cultured under different carbon dioxide concentrations (2.5%, 5.0%, and 10.0%) in the gases mixture and two control treatments, one with passive exchange and the other with forced ventilation. The results demonstrated that there were a larger number of somatic embryos formed with a concentration of 2.5% CO_2. The differentiation of these somatic embryos of coffee in embryogenic cell suspensions (130 × 10³ SE l⁻¹) was also stimulated. The effects of CO₂ on somatic embryogenesis were demonstrated when the control with passive exchange was compared with forced ventilation control, because in the former, where there was an accumulation of CO₂, the production of somatic embryos was greater. CO₂ could stimulate the formation and differentiation of somatic embryos directly, which led to a modification of the pH patterns of the culture medium or indirectly when producing changes in the pH that favored the somatic embryogenesis process.     

Physiology of Eucalyptus plantlets grown photoautotrophically in a scaled-up vessel

Summary Nodal cuttings of Eucalyptus camaldulensis L. plantlets were cultured photoautotrophically (sugar-free nutrient medium and with enriched CO₂ and high photosynthetic photon flux) in a scaled-up vessel (volume 4.0 liters) under forced ventilation (SV-treatment). After 28 d of culture, physiological aspects of the plantlets were compared with plantlets grown photomixotrophically (20 g l⁻¹ sucrose in the medium) in a Magenta vessel (volume 0.4 liters) under natural ventilation (control). In the SV-treatment net photosynthetic rates were enhanced, normal stomatal closing and opening were observed, and the epicuticular leaf-wax content was significantly higher than the control. The anatomical study showed well-organized palisade and spongy mesophyll layers of SV leaves. The SV-treatment also allowed in vitro acclimatization, and after transplanting ex vitro, the transpiration rate and the percent water loss was lower than those of the control and thus the SV plantlets acclimatized easily ex vitro.     

Effect of the application of benzyladenine pulse on organogenesis, acclimatisation and endogenous phytohormone content in kiwi explants cultured under autotrophic conditions.

In traditional in vitro culture, explants grow enclosed in a non-ventilated vessel at high relative humidity with phytohormones continuously present and sucrose as the main energy source. Under such conditions explant growth is far from normal. In this paper, explants of Actinidia deliciosa were cultured in MS medium supplemented with sucrose, benzyladenine and gibberellic acid under autotrophic conditions in glass boxes flushed with air enriched with 600 microl l(-1) CO(2) for the first 20 days and then transferred to MS medium until the end of the culture period. The effect of benzyladenine was assayed in two regimes of application: in cultures for 20 days in the medium or only 24 h in the presence of benzyladenine with the aim of improving shoot proliferation and acclimatisation. The longest explants were those grown under ventilation and pulsed for 24 h with benzyladenine. These explants also rooted spontaneously, whereas those grown with continuous benzyladenine under ventilation or without ventilation grew and rooted poorly. The highest amount of endogenous isoprenoid cytokinins were found in the longest explants grown under ventilation and pulsed for 24 h with benzyladenine; under these conditions zeatin riboside represented two thirds of the entire cytokinin pool. These explants presented the highest amount of indole-3-acetic acid, while abscisic acid content was high in explants cultured under non-ventilated conditions. No differences were observed between explants cultured under ventilation regardless of their exposure to benzyladenine. The longest explants, which also performed best in acclimatisation, also presented a high indole-3-acetic to abscisic acid ratio.     

Voluntary and Involuntary Control of Breathing with Imposed Ventilation Parameters

To clarify the mechanisms of interaction between voluntary and involuntary control of respiratory movements in a waking human, respiratory patterns were studied during self-controlled artificial ventilation used in place of natural breathing. Seven subjects controlled both the duration of artificial inhalations and the flow rate of air at excess pressure, continuously adjusting their actions to obtain the sensation of comfortable breathing. At rest, pulmonary ventilation was higher during self-controlled artificial breathing than during natural breathing. This trend was also noted during exercise. A correlation was observed between the velocity of the movement that started air flow and the artificial ventilation volume (r = 0.91). During self-controlled artificial breathing, the subjects sometimes took natural breaths. Natural inhalations did not influence the beginning or end of an artificial inhalation. Information received from respiratory receptors was assumed to play a certain role in the self-control of artificial breathing.     

Social control of air ventilation in colonies of honey bees,Apis mellifera

Fanning behaviour inside the nest of honey bees is an effective mechanism of ventilation. The following results are reported: (1) With only a single small entrance, the fanning is controlled so as to induce tidal ventilation of the nest as in a typical breathing pattern. (2) Periodic active fanning moves an air current out followed by a passive influx of air. (3) Fanning bees show negative phototaxis. (4) The colonial respiratory activity decreases at night following a pronounced day-night cycle.     

Indoor versus outdoor climate

Investigations in this field have been carried out before. Some of them will be mentioned in conjunction with this investigation. The previous investigations were about pollutants that infiltrate a building or come in with the ventilation air. This was one of the reasons that so-called natural ventilation began to be abandoned in the 1950s. Prior to that, ventilation air was taken directly from the street. In fact, the conditions that will be investigated here received attention long ago. One of the reasons that the outdoor conditions have been considered is because of the transition from older ventilation systems to current ones, where air may be taken from roof level and subjected to some treatment and cleaning. Although the subject is understood at least qualitatively, it can be interesting to look at it again. Modern calculation methods make it easier to study the problem quantitively, i.e., to calculate the indoor concentrations that can arise as a result of pollutants in the outdoor air. Such calculations can also give an idea of how long high levels of pollutants can be maintained under various circumstances.     

木本植物导管长度分布规律研究新方法

从理论上推导出以着色导管数计算导管长度的关系式,为注入颜料法测定植物导管长度分布规律了理论基础,并推导了导管长度分布规律,以及着色导管数与样品长度关系。以香梓、梧桐为试材,研究了导管长主工分布规律,实验结果与理论结果完全一致,表明理论推导和实验结果是可靠的。