Studies on the Growth in Culture of Plant Cells: VIII. THE PRODUCTION OF ETHYLENE BY SUSPENSION CULTURES OF ACER PSEUDOPLATANUS, L.

Sycamore cell suspension cultures in a synthetic medium releaseethylene; during a 24-day incubation period a single culture(initial volume 70 ml) produces c. 4 µ moles. There isa very sharp peak of ethylene production between day 10 andday 14 of culture; at the peak of production c. 2 nmoles ethyleneare released per million cells in 24 h. Evidence is presentedthat 2,4-D enhances ethylene production independently of itseffects on culture growth. Under the standard conditions of culture (250-ml Erlenmeyerflasks closed with aluminium foil and containing 70 ml cellsuspension) the concentration of ethylene in the gas phase ofthe cultures rises above 10 ppm. No evidence was obtained thatthis ethylene is inhibitory to culture growth or that a criticallevel of ethylene is necessary to initiate cell division incultures at a critically low cell density. The low rate of ethylene release by stationary phase culturesis temporarily enhanced by the addition of various solutes andfurther depressed by dilution with water.     

Nitrogen Utilization in N-limited Barley During Vegetative and Generative Growth: III. POST-ANTHESIS KINETICS OF NET NITRATE UPTAKE AND THE ROLE OF THE RELATIVE ROOT SIZE IN DETERMINING THE CAPACITY FOR NITRATE ACQUISITION

Barley (Hordeum vulgare L., cvs Golf, Mette, and Laevigatum)was grown under nitrogen limitation in solution culture untilnear maturity. Three different nitrogen addition regimes wereused: in the ‘HN’ culture the relative rate of nitrate-Naddition (RA) was 0·08 d^–1 until day 48 and thendecreased stepwise to, finally, 0·005 d^–1 duringgrain-filling; the ‘LN’ culture received 45% ofthe nitrogen added in HN; the ‘CN’ culture was maintainedat RA 0·0375 d^–1 throughout. Kinetics of net nitrateuptake were measured during ontogeny at 30 to 150 mmol m^–3external nitrate. V_max (which is argued to reflect the maximuminflux rate in these plants) declined with age in both HN andLN cultures. A pronounced transient drop was observed just beforeanthesis, which correlated in time with a peak in root nitrateconcentration. Similar, but less pronounced, trends were observedin CN. The relative V_max (unit nitrogen taken up per unit nitrogenin plants and day) in all three cultures declined from 1·3–2·3d^–1 during vegetative growth to 0·1–0·7d^–1 during generative growth. These values are in HN andLN cultures 15- to more than 100-fold in excess of the demandset by growth rates throughout ontogeny. Predicted balancingnitrate concentrations (defined as the nitrate concentrationrequired to support the observed rate of growth) were below6·0 mmol m^–3 in HN and LN cultures before anthesisand then decreased during ontogeny. In CN cultures the balancingnitrate concentration increased during grain-filling. Apartfrom the transient decline during anthesis, most of the effectof ageing on relative V_max can be explained in terms of reducedcontribution of roots to total biomass (R:T). The loss in uptakeper unit root weight is largely compensated for by the declinewith time in average tissue nitrogen concentrations. The quantitativerelationships between relative V_max and R:T in ageing plantsare similar to those observed for vegetative plants culturedat different RAs. The data support the contention that the capacity for nitrateacquisition in N-limited plants is under general growth control,rather than controlled by specific regulation of the biochemicalpathway of nitrate assimilation. Key words: Barley, nitrogen concentration, root: total plant biomass ratio, V_max     

DNA methylation as a key process in regulation of organogenic totipotency and plant neoplastic progression?

Summary Progressive loss of organogenic totipotency appears to be a common event in long-term plant tissue culture. This loss of totipotency, which has been proposed to be a typical trait of plant neoplastic progression, is compared to some mechanisms that occur during the establishment of animal differentiation-resistant cancer lines in vitro. Evidence is presented that alteration in DNA methylation patterns and expression of genes occur during long-term callus culture. An effect of the auxin, 2,4-dichlorophenoxyacetic acid, in the progressive methylation, is moreover suggested. Methylation of genes relevant to cell differentiation and progressive elimination of cells capable of differentiation is proposed as being responsible for this progressive loss of organogenic potential. Finally, the epigenetic alteration (DNA methylation) that occurs during prolonged periods of culture may induce other irreversible genetic alterations that ultimately make the loss of totipotency irreversible.     

Analysis of the expression and function of the σB-dependent general stress regulon of Bacillus subtilis during slow growth

Glucose-limited continuous cultures were used to analyze σ^B activity at decreasing growth rates. Expression of the σ^B-dependent genes gsiB and ctc started to increase at a growth rate of 0.2 h^–1, and both genes were induced approximately fivefold at a growth rate of 0.1 h^–1 as compared to expression at the maximal growth rate. However, maximal σ^B activity was only reached when the growth stopped as a result of the exhaustion of the carbon and energy source glucose. During glucose-limited growth, increased expression of the general stress regulon at growth rates below 0.2 h^–1 did not provide wild-type cells with a growth advantage over sigB mutants. Instead, expression of the stress regulon seems to constitute a significant burden during glucose-limited growth, resulting in a selective growth advantage of the sigB mutant as compared to the wild-type at a growth rate of 0.08 h^–1. Received: 7 January 1999 / Accepted: 22 March 1999     

Mechanisms of somatic embryogenesis in cell cultures: Physiology,biochemistry, and molecular biology

Summary One of the most characteristic cell functions in plants is totipotency. Somatic embryogenesis can be regarded as a model system for the investigation of mechanisms of totipotency, because a high frequency and synchronous embryogenic system from single somatic cells has been established in carrot suspension cultures. Four phases are recognized in this process, and several molecular markers, viz. polypeptides, mRNAs, antigens against monoclonal antibodies, can be detected during the expression of totipotency, but they disappear during its loss. Four organ-specific genes have been isolated from hypocotyls and roots by differential screening. They were expressed preferentially after the globular-heart stages of embryogenesis, and were strongly suppressed by auxin. A CEM 1 gene was isolated by differential screening of embryogenic cell clusters. This gene was expressed strongly and transiently during the proglobular and globular stages. The sequence of CEM 1 was found to encode a polypeptide showing high homology to the elongation factor isolated from eucaryotic cells. Thus good progress is being made in understanding the basic mechanisms of somatic embryogenesis. Presented in the Session-in-Depth Developmental Biology of Embryogenesis at the 1991 World Congress on Cell and Tissue Culture, Anaheim, California, June 16–20, 1991.     

A Possible Role for Urease as a Storage Protein in Aspergillus tamarii

A marked decrease in mycelial urease activity during the endogenousphase of undifferentiated Aspergillus tamariicultures was foundto be independent of preparative procedures but related to thedepletion of external nutrients. The enzyme, which was synthesizedduring the active growth stage, was produced in similar quantitieswith ammonium or urea as sole nitrogen source and at its peakrepresented c. 8·5 per cent of the total soluble proteinpool of the mycelium. It was found to show maximum activityat pH 8·20–8·65 when measured in cell-free,phosphate-buffered extracts. Isolation of urease from differentstages of the endogenous phase by affinity chromatography hasshown that the observed decrease in activity was due to breakdownof the enzyme protein in mature cultures, followed by the progressivedeactivation of residual enzyme during the autolytic stage.Since selective inhibition of 80–90 per cent of activityby acetohydroxamic acid in media containing urea as the onlynitrogen source or total repression of urease synthesis by L-histidinein ammonium-grown cultures did not interfere with normal growth,it was concluded that in A. tamarii urease fulfils the functionof a storage protein with a measure of catalytic activity. Aspergillus tamarii, urease, storage protein, nitrogen metabolism     

Aerobic Growth on Nitroglycerin as the Sole Carbon, Nitrogen, and Energy Source by a Mixed Bacterial Culture

Nitroglycerin (glycerol trinitrate [GTN]), an explosive and vasodilatory compound, was metabolized by mixed microbial cultures from aeration tank sludge previously exposed to GTN. Aerobic enrichment cultures removed GTN rapidly in the absence of a supplemental carbon source. Complete denitration of GTN, provided as the sole C and N source, was observed in aerobic batch cultures and proceeded stepwise via the dinitrate and mononitrate isomers, with successive steps occurring at lower rates. The denitration of all glycerol nitrate esters was found to be concomitant, and 1,2-glycerol dinitrate (1,2-GDN) and 2-glycerol mononitrate (2-GMN) were the primary GDN and GMN isomers observed. Denitration of GTN resulted in release of primarily nitrite-N, indicating a reductive denitration mechanism. Biomass growth at the expense of GTN was verified by optical density and plate count measurements. The kinetics of GTN biotransformation were 10-fold faster than reported for complete GTN denitration under anaerobic conditions. A maximum specific growth rate of 0.048 ± 0.005 h⁻¹ (mean ± standard deviation) was estimated for the mixed culture at 25°C. Evidence of GTN toxicity was observed at GTN concentrations above 0.3 mM. To our knowledge, this is the first report of complete denitration of GTN used as a primary growth substrate by a bacterial culture under aerobic conditions.     

Determination in plant cells

The possibility that some of the variation in callus cultures involves epigenetic changes is examined in cultures established from the hypoootyls and roots ofEuphorbia heterophylla. It is shown that the responses of the cultures are affected by the light regimes under which they are grown and that in the dark and under short photoperiods, there are differences between the two types of culture with respect to pigmentation, auxin requirement, capacity to regenerate buds and roots and in certain isozyme patterns, whereas the two cultures are similar from the first passage under continuous light. However, these differences are only maintained for 2–3 passages, after which the root callus becomes similar to the hypoc otyl callus. Evidence is presented that these differences between cultures are epigenetic. Callus cultures established from the apical meristems of shoots and roots ofE. hetero phylla show similar differences to those observed between hypocotyl and root cultures and these differences are also lost after 3 passages. These results indicate that the cells of apical meristems are not totally uncommitted, but are determined as ‘shoot’ and ‘root’ meristem colls, respectively. The practical importance of a better understanding of epigenetic effects in plant cells is strassed.     

In vitro selection and characterization of Ni-tolerant callus lines of Setaria italica L

Nickel tolerant callus lines of Setaria italica L. were developed from callus cultures grown on MS medium supplemented with 0.5 mg·dm⁻³ kinetin+2.0 mg·dm⁻³ 2,4-D+2.0 mg·dm⁻³ Ni⁺². Standard growth parameters such as callus fresh and dry weight, growth tolerance index were used as indicators of nickel toxicity. Measurements as early as 2 weeks after the beginning of the treatments did not yield consistent results. However, growth tolerance index at 4, and 8 weeks after the beginning of treatments yielded significant differences among the non-tolerant and tolerant calli. The tolerant calli has enhanced growth at 2.0 mg·dm⁻³ Ni⁺² while non-tolerant calli showed a reverse trend in growth in the presence of 2.0–2.5 mg·dm⁻³ of nickel. The tolerant calli differentiated into mass of embryogenic calli within 4 weeks of culture which could be maintained for prolonged period without loss of regenerative capacity.     

The flavonoid rutin induces astrocyte and microglia activation and regulates TNF-alpha and NO release in primary glial cell cultures

Astrocyte and microglia cells play an important role in the central nervous system (CNS). They react to various external aggressions by becoming reactive and releasing neurotrophic and/or neurotoxic factors. Rutin is a flavonoid found in many plants and has been shown to have some biological activities, but its direct effects on cells of the CNS have not been well studied. To investigate its potential effects on CNS glial cells, we used both astrocyte primary cultures and astrocyte/microglia mixed primary cell cultures derived from newborn rat cortical brain. The cultures were treated for 24 h with rutin (50 or 100 μmol/L) or vehicle (0.5% dimethyl sulfoxide). Mitochondrial function on glial cells was not evidenced by the MTT test. However, an increased lactate dehydrogenase activity was detected in the culture medium of both culture systems when treated with 100 μmol/L rutin, suggesting loss of cell membrane integrity. Astrocytes exposed to 50 μmol/L rutin became reactive as revealed by glial fibrillary acidic protein (GFAP) overexpression and showed a star-like phenotype revealed by Rosenfeld’s staining. The number of activated microglia expressing OX-42 increased in the presence of rutin. A significant increase of nitric oxide (NO) was observed only in mixed cultures exposed to 100 μmol/L rutin. Enhanced TNFα release was observed in astrocyte primary cultures treated with 100 μmol/L rutin and in mixed primary cultures treated with 50 and 100 μmol/L, suggesting different sensitivity of both activated cell types. These results demonstrated that rutin affects astrocytes and microglial cells in culture and has the capacity to induce NO and TNFα production in these cells. Hence, the impact of these effects on neurons in vitro and in vivo needs to be studied.     

Spatial integration of the partners and heteromorphism of the cyanobacteriumNostoc muscorum CALU 304 in a mixed culture with theRauwolfia tissue

A comparative morphological study was conducted ofNostoc muscorum CALU 304 grown either as a pure culture on standard media or as a mixed culture withRauwolfia callus tissue on a medium for plant tissue cultivation. The interaction of the cyanobacterial and plant partners results in their spatial integration into aggregates of specific anatomy, which arise periodically during the mixed culture growth. The morphology of the cyanobacterial cells varies depending on their localization in the mixed aggregate. The degree of cyanobacterial heteromorphism increases with the time of growth of the association. Evidence of the plant origin of the factors inducing heteromorphic changes inN. muscorum was obtained, as well as evidence indicating that these factors can rapidly diffuse in agarized medium. A conclusion is inferred that the heteromorphic cells correspond to bacterial forms that appear during unbalanced growth as an adaptation to altered environmental conditions.     

Impact of dust exposure on mixed bacterial cultures and during eukaryotic cell co-culture infections

On a daily basis, humans, and their colonizing microbiome, are exposed to both indoor and outdoor dust, containing both deleterious organic and inorganic contaminants, through dermal contact, inhalation, and ingestion. Recent studies evaluating the dust exposure responses of opportunistic pathogens, such as Escherichia coli and Pseudomonas aeruginosa, revealed significant increases in biofilm formation following dust exposure. In this study, the effects of dust exposure on mixed bacterial cultures as well as HT-29 co-cultures were evaluated. As it was observed in pure, single bacterial cultures earlier, neither indoor nor outdoor dust exposure (at concentrations of 100 μg/mL) influenced the growth of mixed bacterial liquid cultures. However, when in paired mixed cultures, dust exposure increased sensitivity to oxidative stress and significantly enhanced biofilm formation (outdoor dust). More specifically, mixed cultures (E. coli-Klebsiella pneumoniae, K. pneumoniae-P. aeruginosa, and E. coli-P. aeruginosa) exhibited increased sensitivity to 20 and 50 mM of H2O2 in comparison to their pure, single bacterial culture counterparts and significantly enhanced biofilm production for each mixed culture. Finally, bacterial proliferation during a eukaryotic gut cell (HT29) co-culture was significantly more robust for both K. pneumoniae and P. aeruginosa when exposed to both house and road dust; however, E. coli only experienced significantly enhanced proliferation, in HT29 co-culture, when exposed to road dust. Taken together, our findings demonstrate that bacteria respond to dust exposure differently when in the presence of multiple bacterial species or when in the presence of human gut epithelial cells, than when grown in isolation.     

Investigations on the Growth and Metabolism of Plant Cells: II. Variables affecting the Growth of Tissue Explants and the Development of a Quantitative Method using Carrot Root

This paper describes the technique of growing small discs ofcarrot phloem in pure culture and with the precautions necessaryto standardize the rate of growth at the highest practicablelevel. It deals with the procedure necessary to remove replicatesamples of the material, with variability along the radial andlongitudinal axes of the carrot, and with the regulation ofthe culture conditions. Evidence is adduced of the existence of a ‘staling’factor in crowed cultures.     

Totipotency and embryogenesis in plant cell and tissue cultures

Summary An important development in the field of plant cell and tissue culture has been the demonstration in the past decade of the totipotency of higher plant cells. Isolated single cells were first successfully grown on a nurse tissue separated by a filter paper and gave rise to a callus tissue. Later, completely isolated single cells of tobacco were grown in microchambers to form small clumps of cells which then could be differentiated to form adult tobacco plants. Indirect evidence of the totipotency of higher plant cells has also been provided in a number of other plants. Embryo-like structures (or embryoids) or whole plants, or both, have been obtained from such highly differentiated cells as the pollen grains (gametic and haploid), photosynthetic palisade cells in leaves, epidermal cells from the hypocytyl, and the triploid endosperm cells; all of these cell types perform very highly specialized functions in the plant. Plant protoplasts (cell wall is digested with enzymes) have also been cultured to give rise to normal adult plants. In many instances embryoids have been produced in vitro from several species of flowering plants which do not show such asexual activity in nature. These embryoids are normally indistinguishable morphologically from embryos produced by gametic fusion, often follow the same pattern of cell divisions and differentiation as the developing zygote, and are economically important as they provide clonal populations. Early work in this area emphasized the necessity of dissociating tissues into single cells and providing a nutritional environment identical to that of the zygote in the embryo sac (usually by supplementing the medium with liquid endosperm from coconuts), before the cells could be released morphogenetically to express their totipotency by forming embryoids. Much of the recent work, however, has shown that perfect development of embryoids can be obtained in completely synthetic media in callus tissues as well as in suspension cultures. This paper is dedicated to the memory of the late Professor Philip R. White, a dear friend who provided much counsel and inspiration to us both. for his pioneering work, valuable contributions and untiring efforts in developing the science of plant cell, tissue, and organ culture. Florida Agricultural Experiment Stations Journal Series No. 4699. Presented in the Symposium on Functional Differentiated Systems at the 23rd Annual Meeting of the Tissue Culture Association, Los Angeles, California, June 5–8, 1972.     

Biodegradation kinetics of 4-fluorocinnamic acid by a consortium of Arthrobacter and Ralstonia strains

Arthrobacter sp. strain G1 is able to grow on 4-fluorocinnamic acid (4-FCA) as sole carbon source. The organism converts 4-FCA into 4-fluorobenzoic acid (4-FBA) and utilizes the two-carbon side-chain for growth with some formation of 4-fluoroacetophenone as a dead-end side product. We also have isolated Ralstonia sp. strain H1, an organism that degrades 4-FBA. A consortium of strains G1 and H1 degraded 4-FCA with Monod kinetics during growth in batch and continuous cultures. Specific growth rates of strain G1 and specific degradation rates of 4-FCA were observed to follow substrate inhibition kinetics, which could be modeled using the kinetic models of Haldane–Andrew and Luong–Levenspiel. The mixed culture showed complete mineralization of 4-FCA with quantitative release of fluoride, both in batch and continuous cultures. Steady-state chemostat cultures that were exposed to shock loadings of substrate responded with rapid degradation and returned to steady-state in 10–15 h, indicating that the mixed culture provided a robust system for continuous 4-FCA degradation.     

The Heterotrophic Nutrition of Chlorella vulgaris (Brannon No. 1 Strain): With two Figures In the Text

A range of sugars, sugar alcohols, sugar phosphates, organicacids, and monohydric alcohols have been tested as carbon sourcesfor growth and as respiratory substrates using Chlorella vulgaris,Brannon I, grown in darkness. Much higher rates of growth and respiration were obtained withd-glucose than with any other substance tested. Ethanol (at0·005 M.) sustained both growth and respiration at c.50 per cent, of the level with glucose (0·028 M. or higher).Evidence was obtained that the organism can become ‘adapted’to utilize d-galactose and sucrose as effective carbon sources.Sustained growth was not obtained with any of the other substancestested. The glucose monophosphates, methanol and certain organic acids(oxalacetate, -ketoglutarate, cis-aconitate, and pyruvate) clearlystimulated oxygen uptake but to a less extent than ethanol.The other substances tested were either inhibitory to respirationor inactive or of very low activity as substrates. The growth in darkness and in liquid culture of Chlorella whensupplied with d-glucose was insensitive to pH over the range4·5 to 7·0 and was markedly enhanced by a highlevel of aeration. Gains in cellular dry weight ranging from45 to 90 per cent, of the weight of d-glucose disappearing fromthe culture medium were recorded in growth experiments; measurementsof CO₂ evolution in the Warburg indicated retention of up totwo-thirds of the glucose-C in cell material.     

Growth characteristics of the cyanobacterium Nostoc flagelliforme in photoautotrophic, mixotrophic and heterotrophic cultivation

Nostoc flagelliforme is a terrestrial cyanobacterium with high economic value. Dissociated cells separated from a natural colony of N. flagelliforme were cultivated for 7 days under either phototrophic, mixotrophic or heterotrophic culture conditions. The highest biomass, 1.67 g L⁻¹ cell concentration, was obtained under mixotrophic culture, representing 4.98 and 2.28 times the biomass obtained in phototrophic and heterotrophic cultures, respectively. The biomass in mixotrophic culture was not the sum as that in photoautotrophic and heterotrophic cultures. During the first 4 days of culture, the cell concentration in mixotrophic culture was lower than the sum of those in photoautotrophic and heterotrophic cultures. However, from the 5th day, the cell concentration in mixotrophic culture surpassed the sum of those obtained from the other two trophic modes. Although the inhibitor of photosynthetic electron transport DCMU [3-(3,4-dichlorophenyl)-1,1-dimethylurea] efficiently inhibited autotrophic growth of N. flagelliforme cells, under mixotrophic culture they could grow by using glucose. The addition of glucose changed the response of N.flagelliforme cells to light. The maximal photosynthetic rate, dark respiration rate and light compensation point in mixotrophic culture were higher than those in photoautotrophic cultures. These results suggest that photoautotrophic (photosynthesis) and heterotrophic (oxidative metabolism of glucose) growth interact in mixotrophic growth of N. flagelliforme cells.     

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

Comparative Physiology of Apple Rootstocks: II. The Effect of pH and Aeration on the Growth of Malling Apple Rootstocks in Water Cultures

Three Malling apple rootstocks, M.IX, M.II, and M.XVI, weregrown in aerated and non-aerated nutrient solutions at pH 3·6,4·0, and 5·5, and their growth and survival comparedwith rootstocks grown in sand and in soil. No plants survivedin the non-aerated cultures due to severe bacterial and fungalattack on the roots. In the aerated series a marked differencein susceptibility of rootstocks to root infection was found,M.XVI being considerably more resistant than M.II or M.IX. Morerootstocks survived at the lower pH's (4·0 and 3·6)but among surviving plants of M.XVI growth was much less thanat pH 5·5. It would appear therefore that factors makingfor survival do not of necessity make for optimum growth. Insand and soil cultures all plants survived.     

The Effect of the Female Gametophyte on the Growth of Cultured Douglas-fir Embryos

Several clones of Douglas-fir, Pseudotsuga menziesii (Mirb.)Franco, embryos grown in aseptic culture in the presence orabsence of the female gametophyte or a crushed aqueous extractof it, have shown striking differences in overall length, sizeand substance(s) responsible for this colour of the cotyledonsand in root length. The growth-promoting effect is diffusiblein agar, heat-stable and has detectable activity at a concentrationof about 0·1 to 1·0 per cent (v/v) in a basalnutrient medium. Evidence is provided that extracts of gametophytemay have synergistic effects with coconut water in promotingmorphogenesis of Douglas-fir cells in suspension culture. Pseudotsuga menziesii, Douglas-fir, female gametophyte, einbryo culture