Improved growth and acclimatization of somatic embryo-derived Oplopanax elatus plantlets by ventilated photoautotrophic culture

To improve large-scale in vitro production of Oplopanax elatus Nakai, we cultured somatic embryo-derived plantlets under a heterotrophic condition (semi-solid culture with sucrose), photoautotrophic condition (semi-solid culture without sucrose), or modified photoautotrophic condition (liquid culture with forced ventilation). The plantlets grown under the modified photoautotrophic condition had more leaves as well as higher chlorophyll content, and higher net photosynthetic rate than those grown under the conventional conditions. Further, the photoautotrophically grown plantlets acclimatized better and sooner upon ex vitro transplantation than did the conventionally cultured plantlets. Consequently, a photoautotrophic culture method with forced ventilation is effective for enhancing the growth and acclimatization of O. elatus.     

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.     

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.     

Acclimation of tobacco plantlets to ex vitro conditions as affected by application of abscisic acid

Plantlets of Nicotiana tabacum L. cv. Petit Havana SR1 were grown in vitro on Murashige and Skoog medium containing 2% saccharose, and then transplanted ex vitro into pots with coarse sand and Hewitt nutrient solution. In the first day after transplantation, the anti-transpirant abscisic acid (ABA; 0.01, 0.05 or 0.10 mM) was added to the substrate. Leaf stomatal conductance (gs), which was high in plants during the first days after transplantation similarly as in plantlets grown in vitro, was considerably decreased by ABA-treatment. However, in the further days gs decreased more quickly in control than in ABA-treated plants, and after 2 or 3 weeks gs was significantly lower than that of plantlets grown in vitro but similar in control and ABA-treated plants. Two weeks after transplantation, net photosynthetic rate, chlorophyll a + b content, maximal photochemical efficiency, and actual quantum yield of photosystem II in plant leaves were higher in comparison with those in plantlets grown in vitro. ABA-treatment had slight positive or insignificant effect on photosynthetic parameters and enhanced plant growth. Thus ABA application can alleviate 'transplant shock' and speed up acclimation of plantlets to ex vitro conditions.     

Photoautotrophic micropropagation

Summary Sugar in the medium is considered to be an essential cause for the high production costs of plantlets in conventional, heterotrophic micropropagation. Chlorophyllous explants, shoots, and plantlets in vitro have high photosynthetic ability to develop photoautotrophy, but their photosynthetic activity is restricted largely by the low CO₂ concentration in the vessel during the photoperiod and in part by the presence of sugar in the medium. The growth of plantlets in vitro is often greater under photoautotrophic conditions than under heterotrophic conditions, provided that the in vitro environment is properly controlled for promoting photosynthesis. The advantages and disadvantages of photoautotrophic micropropagation are discussed.     

Physiological changes and growth of micropropagated chile ancho pepper plantlets during acclimatization and post-acclimatization

Little is known about physiological changes that occur with micropropagated chile ancho pepper (Capsicum annuum L. cv. San Luis) plantlets during acclimatization. Plantlets were transferred to ex vitro conditions to study selected physiological changes and growth performance during acclimatization and post-acclimatization. The physiology of the plantlets was characterized by measuring leaf gas exchange and water status. Plant growth was determined by assessing plant height, leaf number, total leaf area, relative growth rate (RGR), and leaf, root, and stem dry matter (DM). Chile pepper plantlets became acclimatized within 6 days after transplantation. During this period, physiological adjustments occurred, which were critical for plantlet survival. After initial ex vitro transplanting, plantlets experienced water deficit [leaf wilting and reduced relative water content (RWC)], which corresponded with reduced stomatal conductance (g _s) and transpiration (E), and an increase in stomatal resistance (r _s). Thus, leaf stomata that developed in vitro were functional ex vitro. Because of this stomatal control, plantlets minimized transplant shock, recovered and survived. Prior to transplanting, plantlets were photomixotrophic, as indicated by low photosynthetic rates (A). During acclimatization, RWC, g _s, E, and A were significantly lower two days after transplanting. However, within 6 days after transplanting, plantlets recovered and became photoautotrophic – attaining high A, g _s, and E. Water use efficiency was initially low during the first days after transplanting, but increased dramatically at the end of the acclimatization period in part due to increased A. The stabilization and improvement of plantlet water status and gas exchange during acclimatization and post-acclimatization closely correlated with increased plantlet growth. This revised version was published online in June 2006 with corrections to the Cover Date.     

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     

Photoautotrophic culture conditions and photosynthetic photon flux influence growth of <Emphasis Type="Italic">Lilium</Emphasis> bulblets <Emphasis Type="Italic">in vitro</Emphasis>

Summary Lilium Asiatic hybrid ‘Mona’ bulblets were cultured in vitro for 100 d under photoautotrophic (CO₂-enriched conditions and without sucrose in the medium) and heterotrophic (non-enriched CO₂ conditions and sucrose-supplemented medium) methods and under various levels of photosynthetic photon flux (PPF). Bulblet growth and net photosynthetic rate (NPR) were analyzed. CO₂₋ and PPF-enriched conditions enhanced the overall growth of bulblets, scale leaves, and roots. Heterotrophic conditions enhanced bulblet growth but higher PPF levels were inhibitory to the development of scale leaves. These results indicate the CO₂₋ and PPF-enriched conditions (photoautotrophic conditions) are beneficial for the production of high-quality bulblets of Asiatic hybrid lilies in vitro     

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.     

Improvement of <Emphasis Type="Italic">ex vitro</Emphasis> transfer of tobacco plantlets by addition of abscisic acid to the last subculture

Tobacco (Nicotiana tabacum L.) plantlets were grown on Murashige and Skoog medium in ventilated Magenta boxes and for the last subculture 10 μM ABA was added to the medium. After three weeks plantlets were transferred into pots with Perlite moistened with water and grown in controlled conditions (16-h photoperiod, day/night temperature 25/20 °C, air humidity about 45 %) either under low or high irradiance of 150 (LI) and 700 (HI) μmol m⁻² s⁻¹, respectively. Content of endogenous ABA was 271.7 pmol g⁻¹(f.m.) in ABA treated plantlets, while in control plantlets it was only 53.3 pmol g⁻¹(f.m.). After ex vitro transfer, stomatal conductance and transpiration rate decreased considerably in comparison with in vitro grown plantlets and remained lower also 7 d after ex vitro transfer, especially in ABA-treated plants and so wilting of plants was practically eliminated. Net photosynthetic rate also decreased 1 d after ex vitro transfer but after 7 d it was mostly higher than that of in vitro grown plantlets. Water use efficiency significantly increased in ABA-treated plants. Chlorophyll a+b content did not change immediately after ex vitro transfer, nevertheless, after 7 d chlorophyll content was higher in ABA-treated plants. Pool of xanthophyll cycle pigments (XCP) and the degree of their deepoxidation (DEPS), which are connected with harmless dissipation of light energy, increased under high irradiance. Contents of XCP and ABA precursors (neoxanthin and violaxanthin) were lower in ABA-treated plants than in control plants indicating less stress in these plants. Most chlorophyll a fluorescence parameters did not change considerably after ex vitro transfer and so the photoinhibition was not observed even under HI. Slight increase in non-photochemical quenching under HI in ABA-treated plants suggested their better photoprotection. Thus application of ABA to the last subculture can improve acclimatization of in vitro grown plants to ex vitro conditions     

High irradiance can minimize the negative effect of exogenous sucrose on the photosynthetic capacity of <Emphasis Type="Italic">in vitro</Emphasis> grown coconut plantlets

There is increasing evidence that the sucrose normally added to the culture medium affects negatively the photosynthetic capacity of plantlets. At the same time, however, sucrose cannot be eliminated from the medium, as it is required for normal in vitro growth. We argue that this is true only under the conventional light conditions of growth-rooms. In the present paper irradiance of growth-rooms was increased 10 times and although the sucrose-inhibitory effect was found at high sucrose concentrations, it was possible to grow coconut (Cocos nucifera L.) plantlets without sucrose. Those plantlets showed both high photosynthetic capacity and comparable in vitro growth to those grown with sucrose in the medium under conventional growth-room irradiance. Nevertheless, the best growth was achieved under mixotrophic conditions where at high irradiance and moderate sucrose concentrations plantlets accumulated 27 % more biomass than plantlets grown without sucrose under high irradiance and 43 and 73 % more biomass than their counterparts at low irradiance with or without sucrose, respectively.     

Effect of abscisic acid on photosynthetic parameters during <Emphasis Type="Italic">ex vitro</Emphasis> transfer of micropropagated tobacco plantlets

The aim of this research was to determine whether exogenous abscisic acid (ABA) applied immediately after ex vitro transfer of in vitro grown plants can improve their acclimatization. Tobacco (Nicotiana tabacum L.) plantlets were transferred into pots with Perlite initially moistened either by water or 50 μM ABA solution and they were grown under low (LI) or high (HI) irradiance of 150 and 700 μmol m⁻² s⁻¹, respectively. Endogenous content of ABA in tobacco leaves increased considerably after ABA application and even more in plants grown under HI. Stomatal conductance, transpiration rate and net photosynthetic rate decreased considerably 1 d after ex vitro transfer and increased thereafter. The gas exchange parameters were further decreased by ABA application and so wilting of these plants was limited. Chlorophyll (a+b) and β-carotene contents were higher in ABA-treated plants, but the content of xanthophyll cycle pigments was not increased. However, the degree of xanthophyll cycle pigments deepoxidation was decreased what also suggested less stress in ABA-treated plants. No dramatic changes in most chlorophyll a fluorescence parameters after ex vitro transfer suggested that the plants did not suffer from restriction of electron transport or photosystem damage.     

Responses of Tobacco Plantlets to Change of Irradiance During Transfer from in vitro to ex vitro Conditions

Chlorophyll a fluorescence kinetics, net photosynthetic rate (P _N), water relations, and photosynthetic pigment contents were studied during acclimation of in vitro grown tobacco to higher irradiance (HL; 700 mol m^–2 s^–1). Plantlets were grown on medium containing sucrose in glass vessels (G-plants) or in Magenta boxes (M-plants) with better CO₂ supply in the latter ones. The effect of HL was studied either (1) in plantlets grown under original in vitro conditions (closed vessels), (2) in in vitro plantlets exposed to ambient CO₂ concentration (covers removed), or (3) in plantlets transplanted to ex vitro into pots with sand and nutrient solution. Higher P _N, and fraction of closed photosystem 2 (PS2) centres (1 – q_P), and lower content of xanthophyll cycle pigments were found in M-plants compared to G-plants. HL treatment caused photoinhibition particularly in plants kept in closed vessels. This was indicated by the decrease in the ratio of F_v/F_m and by the increase in non-photochemical quenching, 1 – qp, and content of xanthophyll cycle pigments. Better CO₂ supply ensured by the removal of closure lead to the moderate reduction of symptoms of photoinhibition, although stomatal conductance (g _s), transpiration rate (E), and P _N were negatively affected. The main reason was the decrease in relative air humidity, which caused similar reduction of P _N, E, and g _s after the transfer of plantlets to ex vitro. Nevertheless, plant response to HL seemed not to be affected by any possible root injury caused by transfer to ex vitro. The differences in contents of xanthophyll cycle pigments, degree of de-epoxidation, P _N, and quenching parameters between M- and G-plantlets were still significant 7 d after ex vitro transfer and HL acclimation.     

Stomatal characteristics and leaf anatomy of potato plantlets cultured in vitro under photoautotrophic and photomixotrophic conditions

Summary Potato plantlets (Solanum tuberosum L. cv. Benimaru) were cultured under photoautotrophic (without any sucrose in the nutrient medium and with enriched CO₂ and high photosynthetic photon flux) and photomixotrophic conditions (20 g 1⁻¹ sucrose in the medium). Leaf anatomy and stomatal characteristics of the leaves were studied in relation to stomatal size and density. Leaf diffusive resistance, transpiration rate, and wax content of the leaves were also investigated. In the photoautotrophic treatment, stomata behaved normally by closing in the dark and opening in the light. The stomatal density increased twofold compared to that of the photomixotrophic treatment. Relatively thick leaves and an organized palisade layer were observed and the epicuticulal wax content was remarkably higher in this treatment, i.e., seven times greater than that of photomixotrophic treatment. In general, higher diffusive resistance of the leaves was observed than under photomixotrophic conditions; also the resistance increased in darkness and decreased in the light. All these characteristics led the plantlets to have a normal and controlled transpiration rate, which was exceptionally high in the photomixotrophic treatment throughout the light and the dark period.     

Environmental control for the large-scale production of plants through in vitro techniques

Leafy or chlorophyllous explants of a number of plant species currently micropropagated have been found to have high photosynthetic ability. Their growth and development have been promoted on sugar-free medium rather than on sugar-containing medium, provided that the environmental factors, such as CO₂ concentration, light intensity and relative humidity, are controlled for promoting photosynthesis and transpiration of explants/shoots/plantlets in vitro. Thus, environmental control is essential for promoting photosynthetic growth and development of in vitro plantlets. Several types of sugar-free (photoautotrophic) culture systems for large-scale micropropagation of plants have been developed. Advantages of sugar-free over conventional (heterotrophic or photomixotrophic) micropropagation systems are as follows: growth and development of plantlets in vitro are faster and more uniform, plantlets in vitro have less physiological and morphological disorders, biological contamination in vitro is less, plantlets have a higher percentage of survival during acclimatization ex vitro, and larger culture vessels could be used because of less biological contamination. Hence, production costs could be reduced and plant quality could be improved significantly with photoautotrophic micropropagation. Methods for the measurement and control of in vitro environments and the beneficial effects of environmental control on photosynthetic growth, development, and morphogenesis in large-scale production of micropropagated plantlets are presented. This revised version was published online in June 2006 with corrections to the Cover Date.     

Influence of in vitro growth conditions on in vitro and ex vitro photosynthetic rates of easy- and difficult-to-acclimatize sea oats (Uniola paniculata L.) genotypes

Net photosynthetic rates (P _n) of easy (EK 16-3) and difficult-to-acclimatize (EK 11-1) sea oats genotypes were examined under the following culture conditions: (1) photoautotrophic [sugar-free medium, high photosynthetic photon flux (PPF), high vessel ventilation rates and CO₂ enrichment, (PA)]; (2) modified photomixotrophic [sugar-containing medium diluted with sugar-free medium over time, high PPF, and high vessel ventilation rates (PM)]; (3) modified photomixotrophic enriched [same as PM with CO₂ enrichment, (PME)]; or (4) conventional photomixotrophic [sugar-containing medium, low PPF, and low vessel ventilation rates (control)]. Regardless of genotype, plantlets cultured under PA conditions died within 2 wk, whereas under PM and PME conditions, plantlets increased their P _n. After 6 wk, P _n per gram dry weight was 1.7 times greater in EK 16-3 than EK 11-1 plantlets cultured under PME conditions. In vitro-produced leaves of EK 16-3 plantlets were elongated with expanded blades, whereas EK 11-1 produced short leaves without expanded blades, especially under control conditions. After in vitro culture, EK 16-3 PME plantlets exhibited the highest dry weights among treatments. EK 16-3 PME and EK 16-3 PM had similarly high survivability, shoot and root dry weights and leaf lengths ex vitro compared to EK 16-3 control and EK 11-1 PM and PME plantlets. Ex vitro growth, survivability and P _n per leaf area of either genotype were not affected by CO₂ enrichment under modified photomixotrophic conditions. These results suggest that growth and survivability of sea oats genotypes with different acclimatization capacities can be enhanced by optimizing culture conditions.     

Promoting root induction and growth of in vitro macadamia (<Emphasis Type="Italic">Macadamia tetraphylla</Emphasis> L. ‘Keaau’) plantlets using CO<Subscript>2</Subscript>-enriched photoautotrophic conditions

In this study, a rooting protocol was developed for macadamia plantlets with healthy roots and enhanced growth performance, along with enhanced photosynthetic capability. In vitro-grown shoots rooted in vented vessels containing vermiculite as the supporting material exhibited 100% frequency of root induction, whereas when shoots were grown in non-vented vessels containing a solidified Murashige and Skoog (MS) medium, the frequency of root induction was less than 30%. The formation of root with callus, hyperhydricity, and leaf necrosis was observed in this photomixotrophic closed system. The modification of the vented photoautotrophic system with different concentrations of CO₂ and sucrose were investigated using vermiculite as the supporter. The number of roots, root length, root surface area, fresh weight, and dry weight were significantly higher in plantlets grown in CO₂-enriched (1,000 μmol CO₂ mol⁻¹) photoautotrophic conditions. The water content in both root and shoot tissues of plantlets cultured under photoautotrophic conditions was maximized. In addition, shoot and leaf performances were enhanced in plantlets cultured under CO₂-enriched photoautotrophic conditions. The supplementation of sucrose (29–88 mM) to culture media in both ambient and elevated CO₂ conditions affected a reduction in the shoot and root performance of in vitro plantlets. Chlorophyll a, chlorophyll b, and total carotenoids in the leaf tissues of plantlets acclimatized in CO₂-enriched photoautotrophic conditions were enriched, leading to increasing photosynthetic abilities, including chlorophyll fluorescence and net photosynthetic rate. From this investigation, a root induction protocol was established and the production of healthy macadamia plantlets was successfully implemented using CO₂-enriched photoautotrophic conditions.     

Changes in photosynthetic activity,pigment composition,electrolyte leakage,lipid peroxidation,and antioxidant enzymes during ex vitro establishment of micropropagated <Emphasis Type="Italic">Rauvolfia tetraphylla</Emphasis> plantlets

The effects of photosynthetic photon flux density (PPFD) on antioxidant metabolism and photosynthetic properties in leaves during ex vitro establishment of micropropagated Rauvolfia tetraphylla plantlets were investigated. In vitro-propagated plantlets were acclimatized at either 50 (Low-light = LL) or 300 (High-light = HL) μmol m⁻²s⁻¹ photosynthetic PPFD for 4 weeks under controlled conditions. Increases in chlorophyll (Chl) a, b and carotenoid levels were observed in plantlets acclimatized at both light intensities. At transplantation, micropropagated plantlets were not photosynthetically active, but the net photosynthetic rate increased in newly formed leaves over time during acclimatization. The observed differences in pigment contents and photosynthetic rates suggested adaptation of plantlets from heterotrophic to autotrophic mode of nutrition during acclimatization. Changes in activities of antioxidant enzymes were also observed during acclimatization. Superoxide dismutase activity increased in plantlets acclimatized at HL intensities. Likewise, changes in activity of catalase and ascorbate peroxidase were also detected. These observed changes reflected the ability of plants in developing an antioxidant enzymatic defense system aiding in survival against oxidative stress and in reducing release of free radicals.     

姜试管苗移栽过程中的蒸腾及光合性能变化

用TPS-1型光合测定仪测定了移栽驯化过程中姜试管苗的光合速率、蒸腾速率等生理指标变化,结果表明：姜试管苗在移栽过程中叶片表观量子产额、羧化效率、净光合速率逐渐增加,而蒸腾速率和气孔导度则呈下降趋势,这有利于试管苗适应外界环境和自养。     

Growth and Photosynthetic Characteristics of Solanum tuberosum Plantlets Cultivated in Vitro in Different Conditions of Aeration, Sucrose Supply, and CO(2) Enrichment

Growth characteristics, oxygen exchange, and carbohydrate and chlorophyll contents were determined 30 days after subculturing of single node-derived plantlets of Solanum tuberosum cv Haig cultivated in vitro. Cultivation conditions were: (a) photomixotrophy in closed vessel, (b) photomixotrophy in closed vessel on medium supplemented with silver thiosulfate, (c) photomixotrophy in aerated vessel, (d) photoautotrophy in air, (e) photoautotrophy in CO₂-enriched air. In photomixotrophic conditions, aeration of the vessel enhanced sucrose utilization and had a positive effect on plantlet growth. In photoautotrophic conditions, growth of the plantlets was slow in air and was strongly enhanced by CO₂ enrichment of the atmosphere. Starch to sucrose ratios were higher in plants grown photoautotrophically than in plants grown with sucrose in the medium. Oxygen exchange characteristics on a chlorophyll basis were similar between the plantlets when measured under moderate light, and resembled those of greenhouse plant leaves. In high light, however, plantlets grown photoautotrophically in a CO₂-enriched atmosphere had higher oxygen exchange rates. We concluded from these results that potato plantlets in vitro in conditions (c), (d), and (e) developed C3-plant photosynthetic characteristics, which were in photoautotrophically grown plantlets comparable to those of field-grown plants.