Photoautotrophic in vitro Multiplication of the Orchid Dendrobium under CO2 Enrichment

An attempt to reduce the production cost on tissue cultured plants, photoautotrophic culture of a high value orchid Dendrobium was established under CO2-enriched conditions. The shoot length and the number of leaves were almost equal in plantlets grown on medium with 2 % sucrose or without sucrose and under normal or enhanced (40 g m-3) CO2 concentration, whereas the fresh and dry masses were higher in cultures grown in sucrose containing media or under CO2 enrichment. Development of roots was observed only on media without sucrose, but CO2 enrichment did not have significant effects on in vitro rootings. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hardening by abscisic acid of tobacco plantlets grownin vitro

Tobacco plantlets were grownin vitro on Murashige and Skoog’s medium with2 % of saccharose. Addition of 0.01 mM abscisic acid (ABA) into the medium decreased stomatal conductance of the adaxial epidermis and especially the abaxial epidermis without negative effects on growth parameters. As a result the rate of water loss from ABA-treated plantlets taken out of cultivation vessels was slower than that of control plantlets. This could help their acclimation after transplantation toex vitro conditions.     

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.     

Protein as a sole source of nitrogen for <Emphasis Type="Italic">in vitro</Emphasis> grown tobacco plantlets

We tested the capability of plants to utilize protein as the exclusive source of nitrogen. The aim of this study was to find out how such a nutrition affected plantlet growth, photosynthetic performance, and N assimilation metabolism in tobacco (Nicotiana tabacum L., cv. Petit Havana SR1) grown in vitro. Plantlets grown in a casein-supplemented (CA) medium were compared to plantlets grown in a complete Murashige-Skoog (MS) medium, plantlets grown in an ammonium-deficient medium (N1), or plantlets grown in a nitrate-reduced medium (N2). In addition, the plantlets were grown in the presence or absence of 1.5 % (m/v) saccharose as an additional carbon source. Casein, similarly as inorganic N limitation, reduced generally plantlet growth, whereas no significant effects were observed on photosynthetic parameters evaluated by chlorophyll a fluorescence. Although addition of saccharose stimulated the plantlet growth particularly in the MS, it showed a rather negative influence both on the growth and on the photochemical efficiency of photosystem II in the plantlets grown in the CA and N1. The activities of enzymes involved in N assimilation, such as nitrate reductase (NR) and glutamine synthetase (GS), were lower in the plantlets grown in the CA, N1, and N2, both in leaves and in roots. On the other hand, glutamate synthase and glutamate dehydrogenase were employed by the plantlets grown in the CA. The presence of saccharose in the growth medium stimulated mainly NR and GS activities in the MS grown plantlets, whereas enzyme activities of the plantlets grown on the N1, N2, and CA were not significantly influenced. We proved that the tobacco plantlets can utilize casein as the sole source of N particularly during their photoautotrophic cultivation. Contrary to positive effects of photomixotrophic nutrition for the MS grown plantlets, exogenous sugar seemed to diminish the ability of the casein-supplemented plantlets to utilize efficiently the additional C source.     

Photosynthesis and photoprotection in Nicotiana tabacum L. in vitro-grown plantlets

Nicotiana tabacum L. plantlets were cultured in vitro photoautotrophically (0% sucrose) and photomixotrophically (3% or 5% sucrose) at two irradiances (80 or 380 mumol m-2 s-1) with the aim of investigating the effect of these culture conditions on photosynthetic parameters and on protective systems against excess excitation energy. In plantlets grown photoautotrophically under higher irradiance photoinhibition was demonstrated. These plantlets had a decreased chlorophyll (Chl) a + b content and Chl a/b ratio, an increased content of xanthrophyll cycle pigments and a higher deepoxidation state, a decreased maximum photochemical efficiency of photosystem II (PS II) and actual photochemical efficiency of PS II, and an increased non-photochemical quenching. In the photoautotrophically grown plantlets and those photomixotrophically grown with 3% sucrose, the increase of growth irradiance from 80 to 380 mumol m-2 s-1 stimulated the activities of ascorbate-glutathione cycle enzymes with the exception of ascorbate peroxidase. Ascorbate peroxidase activity was not affected by the increase in growth irradiance but a significant decrease with increasing sucrose concentration was evident. The higher concentration of sucrose in the medium (5%) in combination with the higher irradiance inhibited photosynthesis (decrease in Chl a + b content and net photosynthetic rate) but no significant changes in activities of ascorbate-glutathione cycle enzymes were found. These results suggest that exogenous sucrose added to the medium improved high irradiance and oxidative stress resistance of the plantlets but the effect of sucrose is concentration dependent.     

Leaf Structure of Tobacco In Vitro Grown Plantlets as Affected by Saccharose and Irradiance

Tobacco plantlets were cultured in vitro under high (200 µmol m^–2 s^–1) or low (60 µmol m^–2 s^–1) irradiance with or without saccharose in the medium. Light microscopy and image analysis were used to evaluate the effect of these culture conditions on leaf anatomy. Addition of saccharose resulted in thicker leaves (all leaf layers) and larger mesophyll cells under both growth irradiances. Various irradiance affected leaf anatomy differently when plantlets had been cultivated in presence or absence of saccharose in the medium. While under high irradiance in presence of saccharose leaf thickness and number of chloroplasts per cell section were increased, plantlets grown under high irradiance in absence of saccharose had thinner leaves and less chloroplasts per cell section. The changes were more pronounced in palisade parenchyma layer.     

In vitro sucrose concentration affects growth and acclimatization of <Emphasis Type="Italic">Alocasia amazonica</Emphasis> plantlets

Plantlets of Alocasia amazonica were regenerated on the MS medium supplemented with different concentrations (0–9%) of sucrose. An absence of sucrose in the growth medium induced generation of leaves, however, it decreased multiplication. On contrary, sucrose supply of 6% or 9% enhanced multiplication but hampered photoautotrophic growth (generation of leaves). Increasing sucrose supply also increased sugars and starch content and number of stomata and decreased water potential and size of stomata during in vitro growth period. During ex vitro acclimatization, shoot length, root length, leaf number and root number of Alocasia plantlets grown with 3% sucrose, were found to be better among the other studied sucrose concentrations. Under ex vitro acclimatization, number of stomata, contents of various carbohydrates in the leaves were increased but size of stomata decreased with increasing sucrose supply during in vitro growth period. Moreover, water potential of leaves of plantlets, which have been grown with a sucrose concentration other than 3%, was decreased. During in vitro growth, net CO₂ assimilation rate (P_N), transpiration (E), stomatal conductance (g_s) and variable fluorescence to maximum fluorescence ratio (Fv/Fm) were unaffected, however, during acclimatization these were changed and maximum P_N, E, and g_s were observed in the plantlets micropropagated with 3% sucrose. Fv/Fm was decreased severely in the plantlets micropropagated with 6% sucrose during acclimatization. Thus a sucrose concentration of 3% in the medium is appeared to be better among studied concentrations for both in vitro growth and ex vitro acclimatization of A. amazonica plantlets.     

巴戟天种质离体保存研究

以巴戟天试管苗为材料,研究不同浓度的无机盐、蔗糖和四种植物生长调节剂(CCC、PP333、ABA、MH)对巴戟天试管苗保存的影响。结果表明:巴戟天试管苗在无生长调节剂的MS、1/2MS、1/4MS三种无机盐浓度培养基上均能保存培养360d,存活率达90%;蔗糖浓度为20~40g/L时,植株生长健壮,能延长保存时间;四种生长调节剂均能诱导试管苗侧芽的生成,抑制顶芽和叶片的生长。其中以PP333促进壮苗效果最好,能提高试管苗素质,在1/2MS+PP3330.5~1.0mg/L+蔗糖30g/L培养基上,试管苗能保存480d,存活率达100%。     

Effects of CO2 and photosynthetic photon flux on yield, gas exchange and growth rate of Lactuca sativa L. 'Waldmann's Green.'

Enrichment of CO2 to 46 mmol m-3 (1000 mm3 dm-3) at a moderate photosynthetic photon flux (PPF) of 450 micromoles m-2 s-1 stimulated fresh and dry weight gain of lettuce leaves 39% to 75% relative to plants at 16 mmol m-3 CO2 (350 mm3 dm-3). Relative growth rate (RGR) was stimulated only during the first several days of exponential growth. Elevating CO2 above 46 mmol m-3 at moderate PPF had no further benefit. However, high PPF of 880-900 micromoles m-2 s-1 gave further, substantial increases in growth, RGR, net assimilation rate (NAR) and photosynthetic rate (Pn), but a decrease in leaf area ratio (LAR), at 46 or 69 mmol m-3 (1000 or 1500 mm3 dm-3) CO2, the differences being greater at the higher CO2 level. Enrichment of CO2 to a supraoptimal level of 92 mmol m-3 (2000 mm3 dm-3) at high PPF increased leaf area and LAR, decreased specific leaf weight, NAR and Pn and had no effect on leaf, stem and root dry weight or RGR relative to plants grown at 69 mmol m-3 CO2 after 8 d of treatment. The results of the study indicate that leaf lettuce growth is most responsive to a combination of high PPF and CO2 enrichment to 69 mmol m-3 for several days at the onset of exponential growth, after which optimizing resources might be conserved.     

Phenol content, acidic peroxidase and IAA-oxidase during somatic embryogenesis in Theobroma cacao L.

Calli were induced in cacao cotyledon explants on a half-strength Murashige and Skoog medium containing 6 × 10-2 g m-3 saccharose and various combinations of 2,4-dichlorophenoxyacetic acid (2,4-D) with kinetin (kin), benzylaminopurine (BAP) or 2-isopentenylphosphate (2-iP). Experiments were carried out on two clones of cacao differing in their susceptibility to black pod disease. The highest percentage of explants forming callus and the most rapid callus development were obtained with 10-6 g m-3 2,4-D and 0.5× 10-6 g m-3 kin. Somatic embryogenesis and rhizogenesis were induced by transferring 3-week-old callus in a half strength Murashige and Skoog medium containing 3 × 10-2 g m-3 saccharose and NAA or IBA in the 0 to 5 × 10-6 g m-3 concentration range. No differentiation could be observed when the medium was supplemented with kin or BAP. The conversion of callus into somatic embryos and roots was accompanied by a drop in phenol content and an increase in peroxidase and IAA-oxidase activities. Moreover, cell differentiation was characterized by the persistence in the callus of one acidic soluble isoperoxidase which was not detected in nondifferentiating callus. Although some differences were noticed between the clones, alterations responsible for cell differentiation were the same in both genotypes. This revised version was published online in July 2006 with corrections to the Cover Date.     

Photosynthetic characteristics of Cymbidium plantlet in vitro

The photosynthetic characteristics of the Cymbidium plantlet in vitro cultured on Hyponex-agar medium with 2% sucrose were determined based on the measurements of CO₂ concentration inside and outside of the culture vessels. The CO₂ measurements were made with a gas chromatograph at a PPF (photosynthetic photon flux) of 35, 102 and 226 mol m^-2 s^-1, a chamber air temperature of 15, 25 and 35°C and a CO₂ concentration outside the vessel of approximately 350, 1100 and 3000 ppm. The net photosynthetic rates were determined on individual plantlets and were expressed on a dry weight basis. The steady-state CO₂ concentration during the photoperiod was lower inside the vessel than outside the vessel at any PPF greater than 35 mol m^-2s^-1 and at any chamber air temperature. The photosynthetic response curves relating the net photosynthetic rate, PPF, and CO₂ concentration in the vessel and chamber air temperature were similar to those for Cymbidium plants grown outside and other C₃ plants grown outside under shade. The results indicate that CO₂ enrichment for the plantlets in vitro at a relatively high PPF would promote photosynthesis and hence the growth of chlorophyllous shoots/plantlets in vitro and that the plantlets in vitro would make photoautotrophic growth under environmental conditions favorable for photosynthesis.Abbreviations C_in CO₂ concentration in the culture vessel - C_out CO₂ concentration outside the vessel (in the culture room) - PPF photosynthetic photon flux     

Nutrient-encapsulation of potato nodal segments for germplasm exchange and distribution

Nutrient-encapsulation technique using in vitro grown nodal segments was developed as an alternative method for distribution of potato germplasm. The nodal cuttings of two potato genotypes, Kufri Jyoti and Kufri Lauvkar, were encapsulated in calcium-free Murashige and Skoog's (MS) medium containing either 2 or 3 % sodium alginate and 0, 1, 2 or 3 % saccharose. The encapsulated segments were stored in tubes with or without semisolid MS medium, and incubated in the dark at 25 ± °C for 3 to 6 weeks. Presence of saccharose in the beads was found detrimental for regrowth of new shoots. In absence of saccharose, about 82 % and 53 % encapsulated segments initiated regrowth after 3 and 6 weeks of dark storage, respectively, in tubes containing MS medium. Storage in empty tubes deteriorated the encapsulated segments, and depressed shoot formation. For potato germplasm distribution, the encapsulated segments can be transported in small tubes containing semisolid MS medium. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of elevated CO2 concentration on acclimation of tobacco plantlets to ex vitro conditions

Nicotiana tabacum L. plants grown in vitro were transferred to ex vitro conditions and grown for 28 d in a greenhouse under normal CO2 concentration (C, 330 mol mol^-1) or elevated CO2 concentration (E, 1000 mol mol^-1). Stomatal conductances of abaxial and adaxial epidermes measured under optimal conditions were not significantly affected by growth under E, but the stomatal regulation of gas exchange was better. Leaf photosynthetic rate (A) of elevated CO2 plants was similar to that of control plants when both were measured under normal CO2, but higher when both were measured under elevated CO2. The A of elevated CO2 plants was much higher than the A of control plants when measured under their respective growth CO2 concentration, which resulted in their higher growth rate. Chlorophyll a and b contents, and activities of whole electron transport chain and of photosystem (PS) II were not markedly affected by growth under E, and the maximum efficiency of PSII measured as the ratio of variable to maximum fluorescence was even slightly increased. Hence no down-regulation of photosynthesis occurred in transplanted plants grown for 4 weeks under E. The contents of -carotene and of xanthophyll cycle pigments (violaxanthin + antheraxanthin + zeaxanthin) were lower in E plants. The degree of de-epoxidation of xanthophyll cycle pigments was not changed or was even lower after transfer to ex vitro conditions, which indicated that no photoinhibition occurred. Therefore, CO2 enrichment can improve acclimation of in vitro-grown plantlets to ex vitro conditions.Keywords: Carotenoids, chlorophyll content and fluorescence, Nicotiana tabacum stomatal conductance      

Long-term growth of soybean at elevated [CO2] does not cause acclimation of stomatal conductance under fully open-air conditions

Accurately predicting plant function and global biogeochemical cycles later in this century will be complicated if stomatal conductance (g(s)) acclimates to growth at elevated [CO(2)], in the sense of a long-term alteration of the response of g(s) to [CO(2)], humidity (h) and/or photosynthetic rate (A). If so, photosynthetic and stomatal models will require parameterization at each growth [CO(2)] of interest. Photosynthetic acclimation to long-term growth at elevated [CO(2)] occurs frequently. Acclimation of g(s) has rarely been examined, even though stomatal density commonly changes with growth [CO(2)]. Soybean was grown under field conditions at ambient [CO(2)] (378 micromol mol(-1)) and elevated [CO(2)] (552 micromol mol(-1)) using free-air [CO(2)] enrichment (FACE). This study tested for stomatal acclimation by parameterizing and validating the widely used Ball et al. model (1987, Progress in Photosynthesis Research, vol IV, 221-224) with measurements of leaf gas exchange. The dependence of g(s) on A, h and [CO(2)] at the leaf surface was unaltered by long-term growth at elevated [CO(2)]. This suggests that the commonly observed decrease in g(s) under elevated [CO(2)] is due entirely to the direct instantaneous effect of [CO(2)] on g(s) and that there is no longer-term acclimation of g(s) independent of photosynthetic acclimation. The model accurately predicted g(s) for soybean growing under ambient and elevated [CO(2)] in the field. Model parameters under ambient and elevated [CO(2)] were indistinguishable, demonstrating that stomatal function under ambient and elevated [CO(2)] could be modelled without the need for parameterization at each growth [CO(2)].     

Changes in carbohydrates and freezing tolerance during cold acclimation of red raspberry cultivars grown in vitro and in vivo

Changes in LT50 and carbohydrate levels in response to cold acclimation were monitored in vitro and in vivo in red raspberry ( Rubus idaeus L.) cultivars with different levels of cold hardiness. Entire micropropagated plantlets or shoot tips from 3 cultivars were harvested before, during and after cold acclimation. Cane samples from container-grown plants of 4 cultivars were harvested before and during cold acclimation and deacclimation. Samples were evaluated for cold hardiness (LT50) by controlled freezing, then analyzed for carbohydrates, including starch, sucrose, glucose, fructose and raffinose. Hardiness of cold-acclimated 'Muskoka' and 'Festival' was superior to that of 'Titan' or 'Willamette'. In vitro plantlets had higher levels of soluble carbohydrates on a dry weight basis and higher ratios of sucrose:(glucose+fructose) than the container-grown plants. Total soluble carbohydrates, primarily sucrose, accumulated during cold acclimation in both plantlets (33–56% relative increase) and plants (143–191% relative increase). Sucrose increased 124–165% in plantlets and 253–582% in container-grown plants during acclimation and declined rapidly to the level of control plants during deacclimation. Glucose and fructose also accumulated, but to a lesser extent than sucrose. Raffinose concentrations were very low, but increased significantly during cold acclimation. In vitro, genotype hardiness was related to the high concentrations of total soluble carbohydrates, sucrose and raffinose. In vivo, hardier genotypes had lower concentrations of starch than the less hardy genotypes. These results demonstrated the importance of soluble carbohydrates, especially sucrose, in cold hardening of red raspberry and that the in vitro conditions or controlled acclimation conditions do not necessarily reflect the phenomena observed in vivo.     

Organogenesis andin vitro flowering ofEchinochloa colona. Effect of growth regulators and explant types

Echinochloa colona regeneration via organogenesis in callus cultures derived from leaf base and mesocotyl expiants andin vitro flowering were achived. Shoot bud regeneration was achieved on Murashige and Skoog’s (MS) basal medium supplemented with 6.66 μM 6-benzylaminopurine (BAP), 2.68 μM 1-naphthalene acetic acid (NAA) and 3 % (m/v) saccharose. Regenerated shoots were rooted on half strength basal MS medium with 2 % (m/v) saccharose devoid of growth regulators. About 90 -95 % of rooted plantlets survived in the greenhouse.In vitro flowering was induced in the regenerated shoots derived from callus on half strength MS medium supplemented with 4.4 μM BAP, 74.07 μM adeninesulphate, 0.72 μM gibberellic acid, and 3 % (m/v) saccharose. The frequency ofin vitro flowering was 80 – 90 % in three repeated experiments. Fertile seeds were recovered fromin vitro grown plantlets which were subsequently germinated into plants. Acknowledgement: The authors wish to thank to the Department of Environment and Forests, Government of India for financial assistance to undertake this investigation.     

Factors influencing the production of hardened glaucous carnation plantlets in vitro

Medium type, its water status and the relative humidity in the culture vessel modified carnation leaf development in vitro. Carnation shoot apices cultured on liquid or on 0.8% agar solidified media developed into plantlets having succulent and translucent leaves which are not transplantable to non-aseptic conditions. Increasing the agar and/or sucrose concentration in the medium as well as decreasing the relative humidity in the culture vessel by a desiccant promoted glaucous leaf production. Increased water status (H₂O and relative humidity) increased shoot proliferation and translucency of leaves. Decreased water status reduced shoot proliferation but induced the formation of glaucous leaves. The culture of apices for 5–6 days on liquid medium prior to their sub-culture to 1.5% agar medium improved shoot proliferation and normal leaf development. An agar slant prevented the submergence of apices in water accumulating on the medium and thus reduced leaf translucency. Survival was further increased by the transfer of plantlets in uncapped culture vessels to a desiccator for 1–2 weeks prior to transplanting to soil.     

Pear plantlets cultured ‘in vitro’ under lime-induced chlorosis display a better adaptive strategy than quince plantlets

In many fruit trees species lime-induced chlorosis causes serious economic damage. In the last few years, the in vitro culture technique has been applied to test rootstocks for susceptibility to iron chlorosis and to study biochemical and molecular aspects of the syndrome. In this study in vitro shoot cultivation of quince rootstocks MA, BA29 and pear cv. ‘Conference’ was used to unravel the relationship between iron deficiency, presence of bicarbonate, and growth development and chloroplast pigment behaviour. Stresses were obtained by using MS medium supplemented with various concentrations of FeNaEDTA and/or KHCO₃. Substrate pH, shoot growth and development parameters, total leaf iron and photosynthetic pigment contents of plantlets were independently determined after 24 days of culture. The medium was acidified by the species under all stress conditions. Iron deficiency and bicarbonate condition led to different growth patterns and modular development among the genotypes. This reflects a different sensitivity and plastic adaptation to the elements of this stress. Iron concentration in leaves and the shoot apex of plantlets decreased in all genotypes. However, chloroplast pigments only decreased in quince plantlets under iron deficiency and bicarbonate condition while they did not change in pear plantlets. Our results demonstrate firstly that a plantlet without a root system, which is cultivated in vitro, is able to sense iron deficiency and bicarbonate enriched conditions and, consequently, activates biochemical and physiological responses. Secondly, acidification appears to be related to iron concentration in tissues of quince rootstocks and pear cultivar. Finally, chloroplast biochemical elements are strongly and differently regulated under both stress conditions and between genotypes.     

High frequency somatic embryogenesis and plant regeneration in root explant cultures of carnation

Root explants excised from carnation plants maintained in vitro formed off-white, friable calluses after three weeks of culture on Murashige and Skoog (MS) medium supplemented with 1 mg l⁻¹ thidiazuron (TDZ) and 1 mg l⁻¹ α-naphthalaneacetic acid (NAA). These calluses were subsequently transferred to MS basal medium where, after an additional four weeks of culture, approximately 50% of the calluses formed somatic embryos. However, calluses formed on root explants that had been cultured on MS medium supplemented with 2,4-dichlorophenoxyacetic acid did not produce somatic embryos upon transfer to MS basal medium. Somatic embryos developed into plantlets and subsequently were grown to maturity. These results indicate that root explants have a high competence for somatic embryogenesis in carnation. J. Seo and S.W. Kim contributed equally to this work.     

Photosynthetic Acclimation to Elevated CO2 Occurs in Transformed Tobacco with Decreased Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Content

Inhibition of net carbon assimilation rates during growth at elevated CO2 was studied in transgenic tobacco (Nicotiana tabacum L.) plants containing zero to two copies of antisense DNA sequences to the small subunit polypeptide (rbcS) gene of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). High- and low-Rubisco tobacco plants were obtained from the selfed progeny of the original line 3 transformant (S.R. Rodermel, M.S. Abbott, L. Bogorad [1988] Cell 55: 673-681). Assimilation rates of high- and low-Rubisco tobacco plants increased 22 and 71%, respectively, when transferred from 35- to 70-Pa CO2 chamber air at 900 [mu]mol m-2 s-1 photon flux density. However, CO2-dependent increases of net carbon assimilation rates of high- and low-Rubisco plants virtually disappeared after 9 d of growth in elevated CO2 chamber air. Total above-ground dry matter production of high- and low-Rubisco plants was 28 and 53% greater, respectively, after 9 d of growth at 70 Pa compared with 35 Pa CO2. Most of this dry weight gain was due to increased specific leaf weight. Rubisco activity, Rubisco protein, and total chlorophyll were lower in both high- and low-Rubisco plants grown in enriched compared with ambient CO2 chamber air. Soluble leaf protein also decreased in response to CO2 enrichment in high- but not in low-Rubisco tobacco plants. Decreased Rubisco activities in CO2-adapted high- and low-Rubisco plants were not attributable to changes in activation state of the enzyme. Carbonic anhydrase activities and subunit levels measured with specific antibodies were similar in high- and low-Rubisco tobacco plants and were unchanged by CO2 enrichment. Collectively, these findings suggested that photosynthetic acclimation to enriched CO2 occurred in tobacco plants either with or without transgenically decreased Rubisco levels and also indicated that the down-regulation of Rubisco in CO2-adapted tobacco plants was related to decreased specific activity of this enzyme.