Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Photoautotrophic cell suspension cultures of periwinkle (Catharanthus roseus (L.) G. Don): Transition from heterotrophic to photoautotrophic growth

Chlorophyllous, heterotrophic periwinkle (Catharanthus roseus (L.) G. Don) cells were capable of sustained photoautotrophic growth in sugar-free B5 medium containing naphthaleneacetic acid and kinetin when provided with a CO₂-enriched atmosphere. An increase in cell fresh weight, first observed approximately 2 weeks after transfer from heterotrophic to photoautotrophic conditions, coincided with the development of maximum chlorophyll content and photosynthetic activity. Electron micrographs revealed that chloroplasts of cells cultured photoautotrophically in continuous light contained large starch granules and exhibited a less extensive thylakoid system than did periwinkle mesophyll chloroplasts. Photoautotrophic cells did not accumulate vindoline or dimeric alkaloids.Abbreviations Chl chlorophyll - dry wt dry weight - fr wt fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida Benth.

Photoheterotrophic and photoautotrophic cell suspension cultures were raised from a callus tissue derived from a Morinda lucida Benth. plant (Rubiaceae). The cultures were characterized with regard to fresh weight, dry weight, cell number, pH, chlorophyll and quinoid natural products. The amount of lipoquinones (phylloquinone, -tocopherol, plastoquinone, ubiquinone) isolated from the photoautotrophic cultures matched the amount detected in an intact leaf. Anthraquinone glycosides which are found in the roots of Morinda plants were not present in the photoautotrophic culture. The photoheterotrophic culture contained only trace amounts of these pigments. Abundant anthraquinone synthesis was observed when photoautotrophic and photoheterotrophic suspension cultures were transferred into darkness, provided sucrose was present in the medium. Induction of synthesis of anthraquinone pigments coincided with a rapid disappearance of lipoquinones from the culture. Thus, in the suspension culture, photoautotrophy correlates with lipoquinone synthesis and heterotrophy correlates with anthraquinone synthesis. This reflects the situation in the intact plants where lipoquinones are chloroplast-associated whereas anthraquinones occur in the roots.Abbreviation HPLC high-performance liquid chromatography     

Photoautotrophic potato cells: Transition from heterotrophic to autotrophic growth

Cells of potato (Solanum tuberosum L.) were obtained which were capable of photoautotrophic growth in liquid suspension culture under a photon flux density of 90–110 μmol m^?2 s^?1 PAR and in an atmosphere enriched with 2% CO₂. These photoautotrophic cells contained between 100 to 200 μg Chl (g fresh weight)^?1 and fixed CO₂ at a maximum rate of 16 μmol CO₂ (g fresh weight)^?1h^?1. In order to obtain cells capable of photoautotrophic growth it was necessary to adapt highly chlorophyllous heterotrophic cells (>50 μg Chl (g fresh weight)^?1) for growth in medium with 2.5 g sucrose 1^?1 (photomixotrophic cells). The photomixotropic cells had a Chl content of ca 100 μg Chl (g fresh weight)^?1 and were capable of photosynthetic activity which allowed them to survive after sugars had been depleted from the medium. It was from the photomixotrophic cells that cells capable of photoautotrophic growth were obtained. Heterotrophic cells initially established in liquid medium with 25 g sucrose I^?1 from chlorophyllous callus contained about 50 to 150 μg Chl (g fresh weight)^?1. However, after 5 to 10 passages the Chl content decreased to a maximum of 15 μg Chl (g fresh weight)^?1. These cells could not be adapted to photomixotrophic or photoautotrophic growth. These cells also were not able to regain Chl or initiate high rates of CO₂ fixation during the stationary phase of growth as did photomixotrophic cells or chlorophyllous heterotrophic cells. The loss of Chl exhibited by the cells during adaption to heterotrophic growth could be attributed at least in part to unbalanced growth (when cell division and growth exceeds Chl accumulation). Sucrose appeared to have an inhibitory effect directly on photosynthesis independent of Chl accumulation.     

Photoautotrophic growth of soybean cells in suspension culture III. Characterization of carbon fixation products under high and low CO2 levels

A photoautotrophic soybean suspension culture (SB-P) was used to study CO₂ assimilation while exposed to elevated or ambient CO₂ levels. These studies showed that under elevated CO₂ (5% v/v) malate is the dominant fixation product, strongly suggesting that phosphoenolpyruvate carboxylase (PEPCase) is the primary enzyme involved in carbon fixation in these cells under their normal growth conditions. Citrate and [aspartate + glutamate] were also significant fixation products during fifteen minutes of exposure to ¹⁴CO₂. During the ten minute unlabeled CO₂ chase however, ¹⁴C-malate continued to increase while citrate and [aspartate + glutamate] declined. Fixation of ¹⁴CO₂ under ambient CO₂ levels (0.037%) showed a very different product pattern as 3-phosphoglycerate was very high in the first one to two minutes followed by increases in [serine + glycine] and [aspartate + glutamate]. Hexose phosphates were also quite high initially but then declined relatively rapidly. Thus, the carbon fixation pattern at ambient CO₂ levels resembles somewhat that seen in C3 leaf cells while that seen at elevated CO₂ levels more closely resembles that of a C₄ plant. The initial fixation product of C₃ plants, 3-PGA, was never detectable under high CO₂ conditions. These data suggest that an in vitro photoautotrophic system would be suitable for studying carbon fixation physiology during photosynthetic and non-photosynthetic growth.Abbreviations SB-P photoautotrophic soybean cells - PEPCase phosphoenol-pyruvate carboxylase - RuBPCase ribulose bisphosphate carboxylase/oxygenase - 3-PGA 3-phosphoglycerate     

Regulating the ratio of photoautotrophic to heterotrophic metabolic activities in photoheterotrophic culture of Euglena gracilis and its application to α-tocopherol production

Methods of regulating the ratio of photoautotrophic to heterotrophic growth rates in photoheterotrophic culture of Euglena gracilis were investigated. In normal photoheterotrophic culture (in the presence of excess organic carbon), the cells grew mainly by organic carbon assimilation (heterotrophic metabolism). The relative contribution of photoautotrophic metabolism increased with the increase in the light supply coefficient, the increase in the CO₂ concentration in the aeration gas and the decrease in the feed rate of organic carbon source. However, limiting the organic carbon supply was the most effective method of shifting the metabolic balance to the photoautotrophic side. In the presence of excess organic carbon source, the -tocopherol contents of the cells in photoheterotrophic culture were low even when the light supply coefficient and CO₂ concentration in the aeration gas were high. By limiting the organic carbon supply to the photoheterotrophic culture, the intracellular content of -tocopherol increased to the same level as those obtained in photoautotrophic cultures.     

Photoautotrophic growth of soybean cells in suspension culture: I. Establishment of photoautotrophic cultures

Highly chlorophyllous photomixotrophic callus was visually selected from callus originating from soybean (Glycine max (L.) Merr. var. Corsoy) cotyledon. Suspension cultures initiated from this callus became photoautotrophic under continuous light with an atmosphere of 5% CO₂ (balance air). Dry weight increases of 1000 to 1400% in the 2-week subculture period have been observed. The cellular Chl content ranged from 4.4 to 5.9 micrograms per milligram dry weight which is about 75 to 90% of the Chl content in soybean leaves under equivalent illumination (300 micro-Einsteins per square meter per second).

No growth can be observed in the dark in sucrose-lacking medium or in the presence of 0.5 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, a concentration which does not inhibit heterotrophic growth (on sucrose). Photoautotrophic growth has an absolute requirement for elevated CO₂ concentrations (>1%). During the 14-day subculture period, growth (fresh weight and dry weight) is logarithmic. Photosynthesis quickly increases after day 4, reaching a peak of 83 micromoles CO₂ incorporated per milligram Chl per hour while dark respiration decreases 90% from day 2 to day 6. The pH of the growth medium quickly drops from 7.0 to 4.5 before slowly increasing to 5.0 by day 14. At this pH range and light intensity (200-300 microEinsteins per square meter per second), no O₂ evolution could be detected although at high pH and light intensity O₂ evolution was recorded.

     

Regulation of Chloroplast Development by Nitrogen Source and Growth Conditions in a Chlorella protothecoides Strain

Chlorella strain (UTEX 27) maintains optimal photosynthetic capacity when growing photoautotrophically in the presence of ammonium. Nitrate-grown photoautotrophic cells, however, show a drastic loss of chlorophyll content and ribulose-1,6-bisphosphate carboxylase/oxygenase activity, resulting in a greater than 10-fold decrease in photosynthetic capacity and growth rate. Nitrate-grown cells are not deficient in protein content, and under mixotrophic and heterotrophic conditions, the alga can utilize nitrate as well as it does ammonium. The alga metabolizes both glucose and acetate in the dark with a doubling time of 5 to 6 hours. However, its growth on acetate is inhibited by light. Ribulose-1,6-biphosphate carboxylase/oxygenase activity correlates well with photosynthetic capacity, and glucose 6-phosphate dehydrogenase and hexokinase activities are altered in a manner consistent with the availability of glucose in growing cells. The alga appears to assimilate ammonium under photoautotrophic conditions primarily via the glutamine synthetase pathway, and shows an induction of both NADH and NADPH dependent glutamate dehydrogenase pathways under mixotrophic and heterotrophic conditions. Multiple isoforms are present only for hexokinase and glucose 6-phosphate dehydrogenase. Etiolated nitrate-grown cells resume greening and increase their photosynthetic capacity after about 6 hours of incubation in the presence of ammonium under photoautotrophic conditions. Similarly, the loss of photosynthetic capacity in ammonium-grown photoautotrophic cells commence about 9 hours after their transfer to heterotrophic nitrate containing media.     

Physiological Responses of Phytoflagellates to Dissolved Organic Substrate Additions. 1. Dominant Role of Heterotrophic Nutrition in Poterioochromonas malhamensis (Chrysophyceae)

Algal heterotrophy is a potentially important considerationin the flow of carbon through aquatic food webs. The physiologicalresponses to organic compound additions under various lightintensities were examined with Poterioochromonas malhamensis,a freshwater chrysophyte with an exceptionally high heterotrophiccapability. P. malhamensis demonstrated a much greater potentialfor heterotrophic growth than for photoautotrophic growth. Whenorganic substrates (glucose, glycerol, or ethanol) were addedto the culture medium, the growth rate of P. malhamensis significantlyincreased while the chlorophyll content cell ^–1 decreased,even at light intensities saturating for photoautotrophic growth.After an initial decline in chlorophyll production caused byorganic substrate uptake, chlorophyll cell¹ increased and theuptake rate of organic substrates decreased, despite the persistenceof a relatively high substrate concentration in the medium.The results are consistent with the production of substance(s)by P. malhamensis that conditioned the culture medium, leadingto a relief of the inhibitory effect of organic substrates onchlorophyll production. (Received October 29, 1990; Accepted May 8, 1991)     

Establishment and growth characterization of suspension culture of cells from the moss, Sphagnum imbrication

A green-pigmented callus of the moss, Sphagnum imbricatum Hornsch. ex. Russ., was induced and a chlorophyllous cell suspension culture was established using a modified Murashige and Skoog's medium without plant hormones. Cell growth in the light in the presence of glucose started after a short lag and was exponential for 12 days. The chlorophyll level was about 15 μg (mg cell dry weight)⁻¹ and photosynthetic activity ca 20 to 50 μmol O₂ (mg chlorophyll)⁻¹h⁻¹. Cell growth in the light was negligible in the absence of glucose under ordinary air, but photoautotrophic growth was possible under elevated CO₂ concentrations. In the dark, the moss cells grew heterotrophically and continued to synthesize chlorophyll, although at a much reduced rate. The suspension-cultured cells redifferentiated protonemata and shoots when transferred to solid Knop's medium. In contrast to the callus cells, which could not assimilate nitrate, redifferentiated plantlets could use nitrate as the sole nitrogen source.     

Photoautotrophic growth of Marchantia polymorpha L. cells in suspension culture

A cell line of M. polymorpha was grown photoautotrophically in liquid suspension culture using 1% CO₂ in air as sole carbon source. The growth rate in terms of cell dry-weight during the exponential phase was 0.171 and the doubling time was 1.76 d. The rate of increase in chlorophyll was 1.6 times higher than the growth rate. The highest content of chlorophyll was 24 mg g^-1 dry weight, and the photosynthetic activity of the cells in the exponential phase, as calculated from the growth rate, was at least 60 mol mg^-1 chlorophyll h^-1.     

Photoautotrophic growth in suspension culture of cells from the moss, Barbula unguiculata

Chlorophyllous cells in suspension culture from the moss, Barbula unguculata Hedw., grown under photoheterotrophic conditions were transferred to photoautotrophic conditions. The cells started to grow photoautotrophically without selection. Maximum growth was observed under irradiances of more than 5 W m² and in an atmosphere enriched with 1% (v/v) CO₂. Under optimum growth conditions, dry weight and chlorophyll content in the culture had increased 20-fold after 20 days of cell growth. High concentration of chlorophyll [10–20 μg (mg dry weight)⁻¹] and high photosynthetic actively [30–70 μmol O₂ evolved (mg chlorophyll)⁻¹ h⁻¹] were observed throughout the culture period. In sugar-free culture medium, cell growth did not occur in the dark or in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) under light, although cell growth was observed in glucose-containing medium under those conditions. When cells that were grown photoautotrophically for one year were transferred to glucose-containing medium under ordinary air, they started to grow heterotrophically both in the light and in the dark.     

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.     

AUTO-, HETERO-, AND MIXOTROPHIC GROWTH OF CHLAMYDOMONAS HUMICOLA (CMLOROIMIYCKAK) ON ACETATE1

Relative growth rate, isocitrate lyase activity, chlorophyll, protein, lipid, and soluble carbohydrate contents were investigated in Chlamydomonas humicola Lucksch during auto-, mixo-, and heterotrnphic growth. Mixotrophic cells have a relative growth rate of 1.66 d ^–1as compared to 0.78 d ^–1 and 0.21 d ^–1 for hetero- and autotrophic cells, respectively. Addition of acetate to autotrophic cells resulted in an increase in cell dry weight during the first day, followed by a rapid decrease and stabilization at 40 pg·cell ^–1. Cellular yield of mixotrophu cells, on a dry weight basis, was 6.6 times that of heterotrophic cells and 21.9 limes that of autotrophic ones. After 4 d, mixotrophic cells were characterized by higher chlorophyll (3.6% dry weight [d.w.]) and protein (58.6% d.w.) contents and lower lipid (4.8% d.w.) and soluble carbohydrate (1.3% d.w.) contents than those of autotrophic (2.6% d.w. chlorophyll, 31.0% d.w. protein, 10.2% d.w. lipid, and 6.5% d.w. soluble carbohydrate) and heterotrophic (1.5% d.w. chlorophyll, 36.9% d.w. protein, 5.6% d.w. lipid, and 6.0% d.w. soluble carbohydrate) cells. The ratio of chlorophyll a/b was highest in heterotrophic cells due to lower chlorophyll b content. Isocitrate lyase activity, a key enzyme in ecetate assimitation, could not be detected in autotrophic cells. Addition of 10 mM acetate to the culture medium of hetero- and mixotrophic cells resulted in increased isocitrate lyase activity with a maximum after 24 h, followed by a decline in activity over a 7-d period. After 7 d of growth, only 0.01 mM acetate was found in the culture medium of mixotrophic cells as compared to 3.2 mM in the medium of heterotrophic ones, from an initial concentration of 10 mM.     

Photosynthetic characterization of photoautotrophic cells cultured in a minimal medium

Photosynthetic properties of photoautotrophic suspensions cultured in a minimal growth medium have been evaluated to determine whether changes have occurred in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, phosphoenol-pyruvate (PEP) carboxylase activity, chlorophyll content, or culture growth. Five photoautotrophic lines Amaranthus powellii, Datura innoxia, Glycine max, Gossypium hirsutum, and a Nicotiana tabacum-Nicotiana glutinosa fusion hybrid were grown in a medium without organic carbon other than phytohormones, and without vitamins. These photoautotrophic lines had total Rubisco activities ranging from 85 to 266 micromoles CO₂ fixed per milligram chlorophyll hour⁻¹, with percent activation of Rubisco ranging from 16 to 53%. Inclusion of protease inhibitors in the homogenization buffer did not result in higher Rubisco activity. PEP carboxylase activity for cells cultured in minimal medium was found to range from 16 to 146 micromoles CO₂ per milligram chlorophyll hour⁻¹, with no higher activity in the C₄Amaranthus cells compared with PEP carboxylase activity in the C₃ species assayed. Rubisco-to-PEP carboxylase ratios ranged from 2.2 to 1 up to 9.4 to 1. Chlorophyll contents increased in all but the Nicotiana cell line, and all of the photoautotrophic culture lines were capable of growth in vitamin-free medium with the exception of SB-P, which requires thiamine.     

Comparison of secondary product accumulation in photoautotrophic,photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures

Summary Photoautotrophic, photomixotrophic and heterotrophic Nicotiana tabacum cell suspension cultures were compared for the constitutive accumulation of secondary metabolites and the elicitor-induced formation of the phytoalexin capsidiol. Nicotine and chlorogenic acid were found in high amounts in the heterotrophic cultures and in moderate concentrations in photomixotrophic but not in photoautotrophic cells. Nicotinic acid-N-glucoside occured in all culture types; in photoautotrophic and photomixotrophic cells the formation of N-methylnicotinic acid (trigonelline) was also observed. Treatment with a fungal elicitor led to substantial accumulation of capsidiol in heterotrophic and photomixotrophic cells and in only low levels in photoautotrophic cultures. Elicitor-treated photomixotrophic cells showed a pronounced increase in cell wall-bound phenolics. The levels of nicotine, nicotinic acid-N-glucoside and trigonelline were not affected by elicitation.Abbreviations hcc heterotrophic cell culture - mcc photomixotrophic cell culture - pcc photoautotrophic cell culture - fr.wt. freshweight - nic-N-glc nicotinic acid-N-glucoside - PMG Phytophthora megasperma f. sp. glycínea - HPLC high performance liquid chromatography - GC gas chromatography - TLC thin layer chromatography - 2,4D 2,4-dichlorophenoxyacetic acid - Kin kinetin - BAP 6-benzylaminopurine - NAA -naphthylacetic acid     

A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum

Chenopodium rubrum photoautotrophic cell suspensions were grown in plastic tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed cell volume, chlorophyll content and oxygen production. Such microtiter dishes are suitable systems for the serial assay of growth inhibition and various physiological effects (i.e. chlorophyll fluorescence, cell viability, oxygen production) of photoautotrophic cells as caused by herbicides and fungal phytotoxins. The applicability of the test system is discussed.Abbreviations pcv packed cell volume - fr.w. fresh weight - rpm revol. per minute - DMSO dimethyl sulfoxide - PMS phenazine methosulfate - NBT nitro-blue tetrazolium chloride     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Long term culture and caffeine production of immobilized coffee (Coffea arabica) L. cells in polyurethane foam

Coffee (Coffea arabica L.) cells could be immobilized in polyurethane foam and subcultured repeatedly for a long time. Four phases were observed for cell growth and caffeine production, I; immobilization, II; growth, III; caffeine production, IV; regrowth. Their periods were influenced by the number of foam particles. Especially in the phase III, the immobilized cells produced a relatively large amount of caffeine in the subculture numbers 5–8 (34 cubes) when the fresh weight of the immobilized cells decreased despite culture in growth medium (DK medium). Caffeine production appeared to have a negative correlation with the growth of the immobilized cells throughout the subcultures.This paper is Part 71 in the series of Studies on Plant Tissue Cultures. For Part 70, see Orihara Y, Furuya T. submitted for publication.