Carotenoid accumulation in Haematococcus pluvialis in mixotrophic growth

The microalga Haematococcus pluvialis was cultured with NaNO₃ from 0 to 1 g l^–1 and optimal growth was obtained at 0.15 g l^–1. Sodium acetate and malonate (from 0 to 2% w/v) enhanced the accumulation of astaxanthin three and five times higher, respectively, than in autotrophic control cultures. However, high concentration of those compounds strongly inhibited growth. The ratio chlorophyll a/total carotenoids was a good indicator of the extent of nitrogen deficiency in the cells.     

NaCl stress inhibits maltose fermentation by Saccharomyces cerevisiae

While fermentation of 20 g glucose l^–1 by Saccharomyces cerevisiae was not impaired by high NaCl concentrations, fermentation of 20 g maltose l^–1 was significantly decreased by 0.7 M NaCl, and completely inhibited with 1.4 M NaCl. No glycerol was produced in response to the salt stress when yeast cells were fermenting maltose. Active maltose transport, and not intracellular hydrolysis, was the metabolic step severely impaired by the NaCl stress.     

Exopolysaccharide, pigment and protein production by the marine microalga Chroomonas sp. in semicontinuous cultures

The marine microalga Chroomonas sp. isolated from Venezuela was grown in semicontinuous culture in order to study the effect of renewal rate and nutrient concentration on alloxanthin, chlorophyll a, carotenoid, carbohydrate, exopolysaccharide, protein and cell productivity. Maximal cell productivity of 8.43 ± 1.8 and 8.81 ± 2.3 × 10⁹ cell l^–1 day^–1 were achieved with renewal rates of 30 and 40%. Maximal protein and chlorophyll productivity of 64.64 ± 2.3 and 2.72 ± 0.3 mg l^–1 day^–1 were obtained with renewal rate of 20 and 30%. Biochemical composition of Chroomonas sp. was influenced by renewal rate. Nutrient concentration seems not to affect cell, protein, chlorophyll and carotenoid productivity. However, carbohydrate and exopolysaccharide productivity of 7.56 ± 0.4 and 9.57 ± 1.2 mg l^–1 day^–1 were increased at 12 mM NaNO₃(P < 0.05). Also, alloxanthin and chlorophyll a production analysed by HPLC, were higher between 8 and 12 mM NaNO₃ at a renewal rate of 30%. Results demonstrated that a renewal rate of 30% and nutrient concentration at 8 mM NaNO₃ optimize the cell, protein, carbohydrate, chlorophyll a, and exopolysaccharide productivity in semicontinuous cultures of Chroomonas. This microalga, as biological source of commercially valuable compounds, shows high capacity for changing its productivity and biochemical composition in semicontinuous system on the basis of nutrient concentration and the renewal rate.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressure

Somatic embryogenesis through callus initiation has been quantified under salt stress conditions for 8 wheat cultivars currently cultivated in Morocco. The cultivars were classed according to the mean number of somatic embryos formed per immature embryo half and regenerated plants per 100 explants under saline conditions. Regenerated plants from control callus (R_0–0) and callus initiated on 10 g l^–1 NaCl (R_0–10) did not show significant differences concerning plant height, spike length and grain number per ear but, the R₀ plants remained less developed than parent plants. When watered with a solution containing more than 20 g l^–1 NaCl, the seeds of cultivar Te derived from R_0–10 regenerated plants exhibited the best elongation of roots and coleoptiles. Furthermore, a chlorophyll fluorescence test showed a clear improvement in salt tolerance of R_0–10 plants at four to five-leaf stage, compared to R_0–0 plants. It is concluded that plant regeneration from callus initiated on high NaCl levels may be a valid method of selection for salt tolerance.     

Photoautotrophic micropropagation of Russet Burbank Potato

The photoautotrophic micropropagation of potato cv. Russet Burbank was investigated. Single node microcuttings were grown for four weeks on Murashige and Skoog (MS) medium with or without sucrose (30 g l^–1) in the growth room at 21/19 °C day/night temperature, with 16-h photoperiod at 150 mol m^–2 s^–1, with or without supplemental CO₂ at 1500 l l^–1. A 20% increase in the number of nodes per stem (from 7.5 to 9.4) and a 50% increase in stem dry weight were observed in cultures grown on media with sucrose and in CO₂ enriched atmosphere comparing to the conventionally micropropagated cultures or the cultures grown photoautotrophically on media without sucrose but in air supplemented with 1500 l l^–1CO₂. Stems of these cultures (from media with sucrose in CO₂ enriched air) almost doubled in length the stems of cultures from the other two treatments. No significant differences were observed between Control (MS medium supplemented with sucrose, 30 g l^–1) and photoautotrophic cultures coming from MS medium with no sucrose grown under 1500 l l^–1 of CO₂. Photoautotrophic cultures produced stems averaging 43.3 mm, with 7 nodes and weighing 9.2 mg (dry weight), similar to conventionally grown in vitro cultures (47.9 mm with 7.5 nodes, 9.7 mg dry weight). Growers may consider photoautotrophic culturing of potato in areas where the high sterility levels are difficult to maintain. Supplementing air in the growth room with 1500 l l^–1 of CO₂ could be beneficial for potato plantlet production even on media containing sucrose since it significantly improved quality, size and biomass of produced plantlets, speeding up the multiplication.     

Production of a recombinant, immunogenic protein, P64k, of Neisseria meningitidis in Escherichia coli in fed-batch fermenters

P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, codifying for this protein, was cloned in Escherichia coli and the P64k protein was expressed in Escherichia coli K12 W3110 under the control of the tryptophan promoter. The recombinant bacteria were grown in batch or fed-batch cultures. P64k was expressed as an intracellular soluble form at about 40% of the total cellular protein. A final productivity of 215 mg l^–1 h^–1 and 11 g cell dry wt l^–1 were obtained when the fed-batch culture conditions were optimised, compared to 30% of total protein, and a productivity of 76 mg l^–1 h^–1 and 5.1 g cell dry wt l^–1 in batch cultivation.     

Biomass production in mixotrophic culture of Euglena gracilis under acidic condition and its growth energetics

When Euglena gracilis was grown in the heterotrophic condition with glucose and (NH₄)₂SO₄ as the carbon and nitrogen source, a high cell yield (4.28–4.48 g l^–1) was obtained and the culture pH decreased to 1.6–2. The biomass production in the heterotrophic culture was compared to those in the autotrophic and mixotrophic cultures. Autotrophic growth was 4.7–6.3% of the heterotrophic one, whereas about 15–19% higher growth was obtained in the mixotrophic culture. Moreover, good production of chlorophyll (39.4 mg l^–1) and carotenoids (13.8 mg l^–1) were attained in the mixotrophic culture, giving the highest fermenter productivity with respect to biomass as well as chlorophyll and carotenoids. Through an energetic analysis in the mixotrophic culture, it was estimated about 25–28% of the total ATP requirement is formed in the photochemical reactions. This resulted in an improved biomass production in the mixotrophic culture of E. gracilis.     

Growth of Corynebacterium glutamicum in glucose-limited continuous cultures under high osmotic pressure. Influence of growth rate on the intracellular accumulation of proline,glutamate and trehalose

In order to determine the response of Corynebacterium glutamicum to osmotic stress under different growth conditions, the bacteria were grown in glucose-limited continuous cultures at osmotic pressures of 0.4–2.4 osmol kg^–1 by addition of NaCl to the culture medium. Steady-state continuous cultures were obtained for all investigated osmotic pressures. Increasing the medium osmolality resulted in a higher specific glucose-uptake rate, a lower glucose-to-biomass conversion yield, as well as important changes in the cellular content. A short-term response to the addition of NaCl to a continuous culture was the rapid but transient uptake of Na⁺ ions. At steady state a higher osmotic pressure resulted in a strong increase of the intracellular concentrations of proline, from 5 mg/g to 125 mg/g dry weight, and of trehalose from 20 mg/g to 60 mg/g dry weight. The level of glutamate, which was the dominant intracellular amino acid at low osmotic pressure at 55 mg/g dry weight, was not affected by the addition of NaCl. The influence of the specific growth rate, between 0.1 h^–1 and 0.4 h^–1, on the intracellular metabolite concentration was also determined. The level of proline was found to increase strongly with the growth rate, whereas the trehalose content decreased slightly and the glutamate content did not change. The observed net increase in accumulated metabolites may be related to a requirement of a higher turgor pressure for rapid cell growth.     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

The Molecular Analysis of the Light Adaptation Reactions in the Yellow-green Alga Pleurochloris meiringensis (Xanthophyceae)

The xanthophycean alga Pleurochloris meiringensis was homocontinuously cultured under high light (16 W/m²) and low light (2 W/m²) conditions. In low light cells, the chlorophyll a content and the dry weight on per cell basis is increased, the maximal photosynthetic capacity per chlorophyll is decreased. The content of chlorophyll c, vaucheriaxanthin-ester and heteroxanthin is similar in both cultures, whereas the content of diadinoxanthin and ß-carotene is twice as high in high light cultures. High light cells contain more photosystem I and cytochrome f per chlorophyll than low light cells, whereas the Q_B content is found to be unchanged. Therefore, the ratio reaction center II/reaction center I is twofold higher in low light cells than in high light ones. The regulation of energy distribution between the photosystems is examined by fluorescence emission spectra at 77 K scanned after different preillumination of the cells. No wavelength dependent state I/state II transition can be detected. However, P. meiringensis regulates the energy distribution in response to light intensity: The higher the irradiance of preillumination, the higher the energy transfer to photosystem I. The sensitivity of the regulation to light intensity is increased in low light cells.     

Photosynthetic characteristics of chloroplasts isolated fromMesembryanthemum crystallinum L., a halophilic plant capable of Crassulacean acid metabolism

Photosynthetically highly active chloroplasts were routinely obtained by rupture of leaf protoplasts from the halophyteMesembryanthemum crystallinum which exhibited the photosynthetic characteristics of either a C₃ plant when grown with 20 mmol l^-1 NaCl in the rooting medium, or a Crassulacean-acid-metabolism (CAM) plant when grown with 400 mmol l^-1 NaCl. Photosynthesis rates of C₃ and CAM chloroplasts were 150–250 and 90–150 μmol mg^-1 chlorophyll h^-1, respectively. Because of osmotic adjustment, CAM chloroplasts required higher sorbitol concentrations (0.7–0.8 mol l^-1) in the assay medium than C₃ chloroplasts (0.3–0.4 mol l^-1) for optimum activity. Substitution of sorbitol by NaCl as the osmoticum strongly reduced photosynthesis of CAM chloroplasts. Rates of electron transport (ferricyanide reduction, uncoupled) remained unaffected over a range of sorbitol concentrations (0 to 1 mol l^-1). Sensitivity of electron transport to increasing levels of NaCl was less pronounced than the NaCl-sensitivity of CO₂ fixation by intact chloroplasts. The CAM chloroplasts showed a broad pH optimum of photosynthesis between pH 7.0 and 8.2; photosynthesis of C₃ chloroplasts dropped markedly below pH 7.6. The CAM chloroplasts maintained a higher transenvelope proton gradient than C₃ chloroplasts both in the light and dark. External pyruvate (5 mmol l^-1) inhibited photosynthesis of CAM chloroplasts, but not of C₃ chloroplasts. Inhibition was reduced by increased external concentrations of orthophosphate.     

Selection of atrazine-resistant plants by <Emphasis Type="Italic">in vitro</Emphasis> mutagenesis in pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis>)

The effects of atrazine on cotyledon cultures of Capsicum annuum (L.) cv. G₄ were investigated with a view of establishing a system for in vitro selection of resistant mutants. At low levels of herbicide produced little growth inhibition, some chlorophyll loss occurred associated with the production of albino shoots. At 20 mg l⁻¹ atrazine bleaching was more pronounced and was accompanied by the development of necrotic spots; however, efficient bleaching was associated with severe suppression of growth. Mutagenized cotyledon explants resulted in production of herbicide-resistant plants on medium containing selective levels of sucrose (0.5%) and atrazine (20 mg l⁻¹). Differential morphogenetic responses were observed when the levels of sucrose (0.5–5%) were altered. Shoot regeneration was maximum in 2 sucrose and the regenerating ability decreased with a further increase in sucrose concentration (3%–5%). However, lowering of sucrose concentration from 2 to 0.5% caused complete bleaching of explants and permitted the selection of herbicide-resistant plants. Complete atrazine-resistant plantlets were obtained after rooting of regenerated green shoots on rooting medium containing 10 mg l⁻¹atrazine, 1.0 mg l⁻¹IAA and 0.5% sucrose. Leaf-segment assay of differentiated plants revealed that all regenerants were resistant to the atrazine. Reciprocal crosses between atrazine-resistant and -sensitive plants showed a non-Mendelian transmission of resistance trait.     

Effect of 2,4-Dichlorophenoxyacetic Acid and NaCl on the Establishment of Callus and Plant Regeneration in Durum and Bread Wheat

Optimal callus induction and plant regeneration were obtained in bread and durum wheat by manipulating the NaCl concentration in the induction medium. Immature embryos from a high regeneration line of spring wheat (Triticum aestivum L.), 'MPB-Bobwhite 26', and an elite durum wheat (Triticum turgidum var. durum L.), 'Mexicali', were cultured in E3 induction medium consisting of Murashige and Skoog (MS) medium, 2.5 mg l^–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2% sucrose and 0.9% Bacto agar. The treated embryos were transferred to E3 liquid medium supplemented with various levels of 2,4-D and NaCl. Incubation on medium containing 2.5 mg l^–1 2,4-D for 45 days produced callus and plant regeneration in 'MPB-Bobwhite 26', but lower callus yield and plant regeneration in 'Mexicali', indicating that 2,4-D alone was not sufficient for callus induction and plant regeneration in this durum variety. Callus yield and regeneration frequencies were higher in 'Mexicali' embryos that were incubated in media containing 2 mg l^–1 2,4-D and 2 mg l^–1 NaCl. The presence of NaCl in the medium beyond the initiation phase was detrimental to plant regeneration. The use of NaCl in the callus formation could form the basis for improved transformation of durum wheat varieties.     

Photosynthetic ability in dark-grown Reboulia hemisphaerica and Barbula unguiculata cells in suspension culture

Suspension cultured cells of the liverwort, Reboulia hemisphaerica and of the moss, Barbula unguiculata were independently subcultured in the medium containing 2% glucose in the dark or in the light for more than one year, and the photosynthetic activities of the final cultures were determined. Throughout the culture period light-grown cells of both species contained high amount of chlorophyll (4 to 34 g mg^–1 dry weight) and showed a high photosynthetic activity (10 to 84 mol O₂ mg^–1 chlorophyll h^–1). Dark-grown cells of R. hemisphaerica showed the same level of chlorophyll content and photosynthetic O₂ evolving activity as light-grown cells. Although chlorophyll content in dark-grown B. unguiculata cells was ten-fold lower than that in light-grown cells, the photosynthetic activity of these dark-grown cells was higher than that of light-grown cells based on chlorophyll content.     

Anthraquinones production,hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from intermediary and production medium strategies

The effects of medium strategies [maintenance (M), intermediary (G), and production (P) medium] on cell growth, anthraquinone (AQ) production, hydrogen peroxide (H₂O₂) level, lipid peroxidation, and antioxidant vitamins in Morinda elliptica cell suspension cultures were investigated. These were compared with third-stage leaf and 1-month-old callus culture. With P medium strategy, cell growth at 49 g l^–1, intracellular AQ content at 42 mg g^–1 DW, and H₂O₂ level at 9 mol g^–1 FW medium were the highest as compared to the others. However, the extent of lipid peroxidation at 40.4 nmol g^–1 FW and total carotenoids at 13.3 mg g^–1 FW for cultures in P medium were comparable to that in the leaf, which had registered sevenfold lower AQ and 2.2-fold lower H₂O₂ levels. Vitamin C content at 30–120 g g^–1 FW in all culture systems was almost half the leaf content. On the other hand, vitamin E content was around 400–500 g g^–1 FW in 7-day-old cultures from all medium strategies and reduced to 50–150 g g^–1 FW on day 14 and 21; as compared to 60 g g^–1 FW in callus and 200 g g^–1 FW in the leaf. This study suggests that medium strategies and cell growth phase in cell culture could influence the competition between primary and secondary metabolism, oxidative stresses and antioxidative measures. When compared with the leaf metabolism, these activities are dynamic depending on the types and availability of antioxidants.Abbreviations AQ Anthraquinone - DW Dry cell weight - FW Fresh cell weight - G Intermediary medium - M Maintenance medium - MDA Malondialdehyde - P Production medium - ROS Reactive oxygen species - TBA Thiobarbituric acid - t_d Doubling time     

Photorespiratory toxicity in autotrophic cell cultures of a mutant of Nicotiana sylvestris lacking serine: glyoxylate aminotransferase activity

Procedures were devised for heterotrophic culture and autotrophic establishment of protoplast-derived cell cultures from the sat mutant of Nicotiana sylvestris Speg. et Comes lacking serine: glyoxylate aminotransferase (SGAT; EC 2.6.1.45) activity. Increasing photon flux rates (dark, 40, 80 mol quanta·m^-2·s^-1) enhanced the growth rate of autotrophic (no sucrose) wild-type (WT) cultures in air and 1% CO₂. Mutant cultures showed a similar response to light under conditions suppressing photorespiration (1% CO₂), and maintained 65% of WT chlorophyll levels. In normal air, however, sat cultures developed severe photorespiratory toxicity, displaying a negligible rate of growth and rapid loss of chlorophyll to levels below 1% of WT. Low levels of sucrose (0.3%) completely reversed photorespiratory toxicity of the mutant cells in air. Mutant cultures maintained 75% of WT chlorophyll levels in air, displayed light stimulation of growth, and fixed ¹⁴CO₂ at rates identical to WT. Autotrophic sat cultures accumulated serine to levels nearly nine-fold above that of WT cultures in air. Serine accumulated to similar levels in mixotrophic (0.3% sucrose) sat cultures in air, but had no deleterious effect on fixation of ¹⁴CO₂ or growth, indicating that high levels of serine are not toxic, and that toxicity of the sat mutation probably stems from depletion of intermediates of the Calvin cycle. Autotrophic sat cultures were employed in selection experiments designed to identify spontaneous reversions restoring the capacity for growth in air. From a population of 678 000 sat colonies, 23 plantlets were recovered in which sustained growth in air resulted from reacquisition of SGAT activity. Twenty-two had SGAT levels between 25 and 50% of WT, but one had less than 10% of WT SGAT activity, and eventually developed symptoms typical of the sat mutant. The utility of autotrophic sat cultures for selection of chloroplast mutations diminishing the oxygenase activity of ribulose-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) is discussed.Abbreviations Chl chlorophyll - DW dry weight - FW fresh weight - SGAT Serine:glyoxylate aminotransferase - WT wild-type