Studies on the Hill reaction of membrane fragments of blue-green algae III. Fluorescence characteristics of membrane fragments of Anabaena variabilis and Anabaena cylindrica

Fluorescence spectra of the pigment system at –196°Cin membrane fragments of Anabaena variabilis and A. cylindricawere investigated. The fluorescence spectra of membrane fragments having four emissionbands at 645–655, 685, 695 and 725 nm were basically similarto those reported for intact cells of blue-green algae, thoughthe emission from phycocyanin (645–655 nm) was far strongerwith membrane fragments than with intact algal cells. Incubation of membrane fragments of A. variabilis in a dilutebuffer (10^–2M, pH 7.5) caused an increase in the 645 nmfluorescence and slight decreases in the 685 and 695 nm fluorescences,but had no influence on the 725 nm fluorescence. The decreasein the 685 and 695 nm fluorescences of A. cylindrica was moremarked and had the same kinetics as the inactivation of photosystemII reaction measured by DPIP-photoreduction. When membrane fragments of A. cylindrica were incubated in thebuffer solution at room temperature or in the presence of MgCl₂(10^–3M) at 0°C; phycobilin aggregates, which emittedthe 655 and 685 nm fluorescence, were solubilized. This solubilizationwas not observed with membrane fragments of A. variabilis. (Received August 31, 1972; )     

Comparison of main emissions at--196?C from phycobilisomes of two blue-green algae Anabaena cylindrica and Anacystis nidulans

Main emissions at—196?C from phycobilisomes of two blue-greenalgae Anabaena cylindrica and Anacystis nidulans were studiedwith special reference to allophycocyanin (APC) B content. Supplementaryexperiments were done with Anabaena variabilis (M-2 and M-3).The main emission from phycobilisome of Anacystis nidulans richin APC B was located at 681 nm. The location was identical tothat of the main emission from APC B but at a shorter wavelengththan that of in vivo emission (685 nm). Results indicate thatAPC B acts as the energy output of phycobilisomes, but thatthe in vivo 685 nm emission is not attributed to APC B. The main emission of the phycobilisome of Anabaena cylindricawas always located at 685 nm irrespective of the preparationmethod; 0.75 M phosphate buffer [Plant Physiol., 63: 615–620(1979)] or 30% polyethylene glycol [Special Issue of Plant &Cell Physiol., No. 3, p. 23–31 (1977)]. This alga alsocontained a special form of APC, but its content was very low.The location of its emission band (681 nm) was identical tothat of APC B, but shorter than that of the main band of phycobilisomes(685 nm). The 685 nm emitter in phycobilisomes showed a charactersimilar to chlorophyll a but not phycobiliproteins in treatmentsfor aqueous extraction or methanol extraction. Results indicatethat the pigment is probably chlorophyll a as we assumed previously.The 685 nm emission from phycobilisomes of Anabaena variabilis(M-2 and M-3) showed the same character. Results were interpreted as indicating that (i) the contentof the special form of APC varies with the species or strainof blue-green algae and (ii) the energy at the phycobilin levelis transferred directly from APC to pigment system II chlorophylla when the amount of the special form of APC is low. (Received October 24, 1979; )     

Light Inhibits Flagellation in a Chlamydomonas Mutant

We have isolated a new flagellar mutant in Chlamydomonas reinhardtii.When the mutant was cultured under the white fluorescent lamp({small tilde}4,800 lux), most cells had no flagella. However,when the cultures were put in the dark, flagellation occurred.Greater than 70% of the cells had flagella within 12–16h after the transfer. The flagellar morphology varied from "rod-shape"(same as the wild-type flagella) to "disk-shape". The disk-shapedflagella had the axonemes which were curved into a loop withinthe swollen membrane. Hence, this mutant is called loop-1. Light-inhibitionof flagellation was restored in the presence of 10^–5 MDCMU. The spectral dependency of the photo-inhibition of flagellation,determined using the Okazaki Large Spectrograph, showed maximaleffectiveness at 400–420 nm and 600–680 nm. Theseresults suggest that photosynthesis inhibits flagellation ofloop-1 cells. (Received July 27, 1989; Accepted January 29, 1990)     

Induction of Oligosporogeneous and Asporogeneous Mutants of Blue-green Alga, Anabaena doliolum by Acriflavine and Acridine Orange

Acriflavine and acridine orange were highly effective in producingmutants defective in spore differentiation in the blue-greenalga, Anabaena doliolum. Acridine orange produced both oligosporogeneous(Osp) and asporogeneous (Sp^–) mutants in high yield withthe frequency ranging from 2.4 to 21 per 10⁴ survivors, whereaswith acriflavine no oligosporogeneous mutants were detected,although it produced non-sporulating (Sp^–) mutants withthe frequency of 1.4 per 10⁴ survivors. Mutants isolated throughthese dyes were stable and showed no tendency towards spontaneousor induced reversion. Behaviour of various mutants indicatedtheir being blocked at different stages of sporulation withdifferent mutagenic sites. Induction of high numbers of Ospand Sp^– mutants after acridine dye treatment indicatesthe involvement of an extrachromosomal determinant in sporulationin Anabaena doliolum. Ohgosporogeneous mutants, asporogeneous mutants, Anabaena doliolum, blue-green algae, mutagenesis, acriflavine, acridine orange     

A probable lack of function of c-type cytochrome in the photosynthetic system of the blue-green alga Anabaena variabilis

Quantitative study of the cytochrome c acting in the photosyntheticsystem of the blue-green alga Anabaena variabilis (M-2) wasdone with membrane fragments and intact cells. Membrane fragments highly active in the NADP⁺-Hill reaction(above 200 µmoles/mg chl.a;-hr) retained photoresponsivecytochrome c equal only one-tenth that of P700, while the plastocyanincontent was almost equal to that of P700. The cytochrome contentin intact cells was a little larger than that in membrane fragmentson the chlorophyll a basis. However, the values relative toP700 (1/9) and plastocyanin (1/10) were identical with thosein membrane fragments. The content was also far smaller thanthat of reaction center II's (1/6). If the cytochrome mediatesall electrons from reaction center II, the cytochrome oxidation-reductionshould have a rate constant of 2.4?102 sec^–1 which isone order above of the rate constant of the cytochrome reduction(2.3 to 3.5?10¹sec^–1). These quantitative relationshipsindicate that in Anabaena variabilis (M-2), c-type cytochrome,either cytochrome f or algal cytochrome c, cannot function inthe main electron flow between two reaction centers. (Received September 8, 1978; )     

Effect of Indoleacetic Acid on Growth and Dinitrogen Fixation in Cyanobacteria

The effect of IAA on growth, dinitrogen fixation, and heterocystsfrequency of Anabaena PCC 7119 and Nodularia sp. have been investigated.Concentrations of IAA ranging from 10^–10 to 10^–4M did not change the growth of Anabaena PCC 7119. Concentrationshigher than 10^–4 M were inhibitory. Similar results werefound in Nodularia sp. although in this case the inhibitoryeffect appeared with 10^–5M of IAA. Neither the nitrogenaseactivity nor the heterocysts frequency were enhanced by IAAtreatment. (Received June 17, 1986; Accepted January 22, 1987)     

Influence of Environmental Stress on the Germination of Anabaena vaginicola Akinetes

Germination of akinetes of Anabeana vaginicola v. fertilissimaPrasad in response to environmental stress was studied. Additionof nitrate to the medium induced early and maximum germination(96 per cent), whereas less than half of the akinetes germinatedwhen either nitrate or phosphate was omitted from the medium.The pH range over which germination occurred was 7.0–9.0.The desiccated akinetes after rehydration germinated after acertain lag period, depending upon the dehydration state. Thetemperature optimum for germination and vegetative growth wasthe same (25 °C) and germination did not occur at 5 °Cor above 35 °C. The limit of heat shock tolerated was 55°C for 4 min. In addition to white light, only the red partof the visible spectrum induced germination. Ultraviolet radiationreduced germination rate presumably by inducing thymine dimersin DNA. The photoreactivating system (s) in akinetes is certainlynon-photosynthetic. LD₅₀ photon flux densities were 300 Jm^–2for akinetes and 240 Jm^–2 for vegetative cells. Anabaena vaginicola, blue-green alga, akinete, germination, environmental stress     

A study of feeding in predacious ciliates using prey ciliates labeled with fluorescent microspheres

Feeding in predacious estuarine ciliates was investigated ina series of laboratory experiments using a new method of preylabeling which facilitates microscopic indentification of ingestedprey items. Ingestion rates of Mesodinium pulex, Euplotes vannusand E.woodruffi were estimated using the appearance, insidethe predator, of bacteriovorous ciliates (Metanophrys sp., Cyclidiumsp.and Pleuronema sp ) labeled with fluorescent microspheres. Preyremain motile and have presumably unaltered surface characteristics.Ingestion rates of log-growth phase predators increased withprey density. Mesodinium pulex ingested 0 15–0.32 cellsh^–1 over a prey concentration of 60–2300 ml^–1.Maximum ingestion rates of E. woodruffi and E. vannus were 4.5and 3.4 cells h^–1 respectively, estimated at prey abundancesof 75 and 172 cells ml^–1 respectively. Comparisons offeeding rates on prey of different sizes, and the effects ofstarvation, indicated that ingestion is likely limited by differentfactors in ‘raptorial’ (M pulex) and ‘filterfeeding’ (Euplotes spp.) predators.     

The Use of Organic and Inorganic Compounds to Increase the Accumulation of Indole Alkaloids in Catharanthus roseus (L.) G. Don Cell Suspension Cultures

Smith, J. I, Smart, N. J., Kurz, W. G. W. and Misawa, M. 1987.The use of organic and inorganic compounds to increase the accumulationof indole alkaloids in Catharanthus roseus (L.) G. Don cellsuspension cultures.—J. exp. Bot. 38: 1501–1506. The addition of sodium chloride, potassium chloride or sorbitolto 5–d–old cell suspension cultures of Catharanthusroseus stimulated an increase in the intracellular accumulationof catharanthine and other indole alkaloids within 48–72h. The magnitude of the response depended upon the concentrationof the compound added. The use of such inexpensive and readilyavailable compounds to increase the yields and reduce the requiredculture times has considerable potential for the productionof useful secondary metabolites from cell cultures of C. roseusand other plant species.     

Regulation of Free Calcium Concentration in the Pitchers of the Carnivorous Plant Sarracenia purpurea: A Model for Calcium in the Higher Plant Apoplast?

The pitcher of the carnivorous plant Sarracenia purpurea L.contains an entrapped body of liquid within which its prey isdigested. Free calcium in the pitcher is derived from eitherthe pitcher walls or from prey falling into the pitcher; inthe absence of exogenous (prey-derived) calcium it will dependon the active and passive calcium regulatory properties of thepitcher walls and may to some extent therefore mimic calciumin the apoplast of plant cells. Using a calcium-specific electrode,the free calcium concentration of the pitchers of Sarraceniaplants was investigated and the effect of adding a variety ofconcentrations of calcium in water determined. The mean pitcherfree calcium concentration in vivo was 2.3 x 10^–5 M±2.5x 10^–5 M; when pitchers were washed and filled with watercontaining lower calcium concentrations, the concentration inthe pitcher water rose to 1–5 x 10^–5 M. When highercalcium concentrations (up to 1 x 10^–4 M) were added,the pitcher calcium concentration declined to 1–7 x 10^–5M. Concentrations of calcium above 1 x 10^–4 M were alsoreduced, but to a lesser extent. Metabolic inhibition of activeion transport, while inhibiting pitcher acidification, did notinhibit regulation of pitcher free calcium, suggested that itoccurs as a result of calcium exchange sites in the pitcherwalls. The data are discussed in relation to the physiologyof Sarracenia pitchers and to the usefulness of the pitcheras a model for free calcium in the higher plant apoplast. Sarracenia purpurea L., carnivorous plant, pitcher, free calcium, plant apoplast     

Cell-based imaging of sodium iodide symporter activity with the yellow fluorescent protein variant YFP-H148Q/I152L

The sodium iodide symporter (NIS) mediates iodide (I^–) transport in the thyroid gland and other tissues and is of increasing importance as a therapeutic target and nuclear imaging reporter. NIS activity in vitro is currently measured with radiotracers and electrophysiological techniques. We report on the development of a novel live cell imaging assay of NIS activity using the I^–-sensitive and genetically encodable yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In FRTL-5 thyrocytes stably expressing YFP-H148Q/I152L, I^– induced a rapid and reversible decrease in cellular fluorescence characterized by 1) high affinity for extracellular I^– (35 µM), 2) inhibition by the NIS inhibitor perchlorate, 3) extracellular Na⁺ dependence, and 4) TSH dependence, suggesting that fluorescence changes are due to I^– influx via NIS. Individual cells within a population of FRTL-5 cells exhibited a 3.5-fold variation in the rate of NIS-mediated I^– influx, illustrating the utility of YFP-H148Q/I152L to detect cell-to-cell difference in NIS activity. I^– also caused a perchlorate-sensitive decrease in YFP-H148Q/I152L fluorescence in COS-7 cells expressing NIS but not in cells lacking NIS. These results demonstrate that YFP-H148Q/I152L is a sensitive biosensor of NIS-mediated I^– uptake in thyroid cells and in nonthyroidal cells following gene transfer and suggest that fluorescence detection of cellular I^– may be a useful tool by which to study the pathophysiology and pharmacology of NIS. thyroid; fluorescence microscopy; FRTL-5 cells     

Characterization of regulatory mechanisms and states of human organic cation transporter 2

Polyspecific organic cation transporters (OCTs) have a large substrate binding pocket with different interaction domains. To determine whether OCT regulation is substrate specific, suitable fluorescent organic cations were selected by comparing their uptake in wild-type (WT) human embryonic kidney (HEK)-293 cells and in HEK-293 cells stably transfected with hOCT2. N-amidino-3,5-diamino-6-chloropyrazine-carboxamide (amiloride) and 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP) showed concentration-dependent uptake in hOCT2 at 37°C. After subtraction of unspecific uptake determined in WT at 37°C or in hOCT2 at 8°C saturable specific uptake of both substrates was measured. K_m values of hOCT2-mediated uptake of 95 µM amiloride and 24 µM ASP were calculated. Inhibition of amiloride and ASP uptake by several organic cations was also measured [IC₅₀ (in µM) for amiloride and ASP, respectively, tetraethylammonium (TEA) 98 and 30, cimetidine 14 and 26, and tetrapentylammonium (TPA) 7 and 2]. Amiloride and ASP uptake were significantly reduced by inhibition of Ca²⁺/CaM complex (–55 ± 5%, n = 10 and –63 ± 2%, n = 15, for amiloride and ASP, respectively) and stimulation of PKC (–54 ± 5%, n = 14, and –31 ± 6%, n = 26) and PKA (–16 ± 5%, n = 16, and –18 ± 4%, n = 40), and they were increased by inhibition of phosphatidylinositol 3-kinase (+28 ± 6%, n = 8, and +55 ± 17%, n = 16). Inhibition of Ca²⁺/CaM complex resulted in a significant decrease of V_max (160–99 photons/s) that can be explained in part by a reduction of the membrane-associated hOCT2 (–22 ± 6%, n = 9) as determined using FACScan flow cytometry. The data indicate that saturable transport by hOCT2 can be measured by the fluorescent substrates amiloride and ASP and that transport activity for both substrates is regulated similarly. Inhibition of the Ca²⁺/CaM complex causes changes in transport capacity via hOCT2 trafficking. organic cation transport; fluorescence measurement; 4-[4-(dimethylamino)-styryl]-n-methylpyridinium; amiloride     

Bulbing in Long-day Onion (Allium cepa L.) Cultured In Vitro: Comparison Between Sugar Feeding and Light Induction

Bulbs were obtained on onion plants cultured in vitro. No bulbinghappened under long days with fluorescent light and 30–40g l^–1 sucrose. Bulb formation was mainly dependent eitheron sucrose concentration in the culture medium, or on lightspectral composition. Raising the sucrose concentration from40 to 120 g l^–1 increased plant basal swelling and stoppedfurther vegetative development. These plants were not dormant.When fluorescent light was enriched in incandescence duringa long day period, bulbs were obtained in two months and underwenta consecutive dormancy. Bulb, dormancy, light spectral quality, photoperiod, R: FR ratio, sugar, tissue culture     

Photochemical Apparatus Organization in the Diatom Cylindrotheca fusiformis: Photosystem Stoichiometry and Excitation Distribution in Cells Grown under High and Low Irradiance

The photochemical apparatus organization in the thylakoid membraneof the diatom Cylindrotheca fusiformis was investigated in cellsgrown under high and low irradiance. High light (HL, 200µE.m^–2.s^–1)grown cells displayed a relatively low fucoxanthin to chlorophyll(Chl) ratio, a low photosystem (PS) stoichiometry (PSII/PS I=1.3/1.0)and a smaller photosynthetic unit size in both PS I and PS II.Low light (LL, 30µE.m^–2.s^–1) grown cells displayeda 30% elevated fucoxanthin content, elevated PS II/PS I=3.9/1.0and larger photosynthetic unit size for PS II (a change of about100%) and for PS I (by about 30%). In agreement, SDS polyacrylamidegel electrophoresis of thylakoid membrane polypeptides showedgreater abundance of PS I, RuBP carboxylase and ATP synthasepolypeptides in HL cells. In contrast, LL grown cells exhibitedgreater abundance of light-harvesting complex polypeptides.Assuming an efficiency of red (670 nm) light utilization of1.0, the measured efficiency of blue (481 nm) light utilizationwas 0.64 (HL cells) and 0.72 (LL cells). The lower efficiencyof blue versus red light utilization is attributed to the quenchingof absorbed energy by non-fucoxanthin carotenoids. Differencesin the efficiency of blue light utilization between HL and LLgrown cells are attributed to the variable content of fucoxanthin.The results support the hypothesis of a variable Chl a-Chl c-fucoxanthinlight-harvesting antenna associated with PS II and PS I in Cylindrotheca. (Received February 10, 1988; Accepted April 6, 1988)     

Isolation, Purification and Some Properties of Cytochrome c-551 from Chromatium vinosum

Cytochrome c-551 was isolated and purified from a photosyntheticbacterium Chromatium vinosum by ammonium sulfate fractionation,ion-exchange chromatography and gel filtration. The cytochromehad absorption maxima at 280, 407 and 523–524 nm in theoxidized form, and 416, 521 and 549.5 nm in the reduced form.The reduced-minusoxidized difference millimolar absorption coefficientwas 9.90 mM^–1cm^–1 for the wavelength pair, 550.5minus 540 nm. The molecular weight of the cytochrome was 16,000by gel filtration on Sephadex G-100 and 15,500 by sodium dodecylsulfate-polyacrylamidegel electrophoresis. The midpoint redox potential was +240 mVat pH 8.0. Cytochrome c-551 was released from bacterial cells when spheroplastswere produced but EDTA and lysozyme treatments. The releasedcytochrome had the same properties as those of the cytochromepreparation obtained by disruption of cells through a Frenchpressure cell. This confirms the earlier suggestion that cytochromec-551 is located in the periplasmic space of cells. (Received August 21, 1982; Accepted October 28, 1982)     

Occurrence of mycosporine-like amino acids in the red-tide dinoflagellate Alexandrium excavatum: UV-photoprotective compounds?

Water extracts of the red-tide dinoflagellate Alexandrium excavatumgrown at ‘high’ light intensity (200 µE m^–2s^–1) show a broad absorbance maximum in the UV regionof the spectrum (310–360 nm). Using TLC and reverse-phaseHPLC a series of mycosporine-like amino acids have been characterized:mycosporine-glycine (_max = 310 nm), palythine (_max = 320 nm),asterina-330 (_max = 330 nm), shinorine (_max = 334 nm), porphyra-334(_max= 334 nm), palythenic acid (_max = 337 nm) and the isomericmixture of usujirene and palythene (_max = 359 nm). From theobserved spectral changes during transference from ‘low’(20 µE m^–2 s^–1) to ‘high’ (200µE m^–2 s^–1) light intensities and vice versa,the series of compounds are supposed to be biogenically relatedto one another. The presence of these compounds in A.excavatumis discussed in relation to their possible role in the photoprotectionto deleterious UV radiation.     

Effect of cell flotation on growth of Anabaena circinalis under diurnally stratified conditions

We tested the hypothesis that the growth rate of Anabaena circinalis,under diurnally stratified conditions, would increase as flotationvelocity increased owing to higher light availability. An insitu experiment compared the growth of diurnally stratifiedpopulations of A. circinalis with flotation velocities of 0.5and 1.0 m h^–1, with neutrally buoyant populations thatwere exposed to either mixed or persistently stratified conditions.The experiment was conducted in the turbid lower Murray Riverin South Australia (vertical attenuation coefficient = 4.52± 0.36 m^–1). To represent the mixing patterns,A. circinalis was contained in diffusion chambers that weremoved to different positions in the water column throughoutthe day. Diurnal populations with flotation velocities of 1.0and 0.5 m h^–1 grew at 0.23 ± 0.01 and 0.15 ±0.01 day^–1, respectively. Mixed populations grew at 0.19± 0.01 day^–1, whereas persistently stratified populationsgrew at 0.43 ± 0.01 day^–1. Results were used toextend a model that predicts growth of A. circinalis when exposedto the different mixing patterns. The model showed that bloomsare unlikely to be formed when the period of diurnal stratificationis <1 week, regardless of flotation velocity. When the diurnallystratified period is >1 week, flotation velocity is importantand a bloom may form depending on values assigned to the growthperiod and maximum mixed depth (Z_m).     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Xylem fluxes of fixed N through nodules of the legume Acacia littorea and haustoria of an associated N-dependent root hemiparasite Olax phyllanthi

Nodulated 1-1.5-year-old plants of Acacia littorea grown inminus nitrogen culture were each partnered with a single seedlingof the root hemiparasite Olax phyllanthi. Partitioning of fixedN between plant organs of the host and parasite was studiedfor the period 4–8 months after introducing the parasite.N fluxes through nodules of Acacia and xylem-tapping haustoriaof Olax were compared using measured xylem flows of fixed Nand anatomical information for the two organs. N₂ fixation duringthe study interval (635 µg N g FW nodules^–1 d^–1)corresponded to a xylem loading flux of 0.20 µg N mm^–2d^–1 across the secretory membranes of the pencycle parenchymaof the nodule vascular strands. A much higher flux of N (4891µg mm^–2 d^–1) exited through xylem at the junctionof nodule and root. The corresponding flux of N from host xylemacross absorptive membranes of the endophyte parenchyma of Olaxhaustorium was 1.15 µg N mm^–1 d^–1, six timesthe loading flux in nodules. The exit flux from haustorium toparasite rootlet was 20.0 pg N mm^–1 d^–1, 200-foldless than that passing through xylem elements of the nodule.Fluxes of individual amino compounds in xylem of nodule andhaustorium were assessed on a molar and N basis. N flux valuesare related to data for transpiration and partitioning of Cand N of the association recorded in a companion paper. Key words: Olax phyllanthi, host-parasite relationships, N flux, Acacia, N₂ fixation     

Somatic Embryogenesis and Its Hormonal Regulation in Tissue Cultures of Freesia refracta

Somatic embryogenesis can be induced in tissue cultures of Freesiarefracta either directly from the epidermal cells of explants,or indirectly via intervening callus. These two pathways ofsomatic embryogenesis can be controlled and regulated by varyingthe combinations and levels of exogenous hormones. When younginflorescence segments were cultured in vitro on modified N₄(MN₄) medium supplemented with 2 mg l^–1 indoleacetic acid(IAA) and 3 mg l^–1 6-benzylaminopurine (BAP), some ofthe epidermal cells began to exhibit the features of embryogeniccells. These cells produced embryoids and developed into newplants through direct somatic embryogenesis. If the same explantswere placed on Murashige and Skoog's (MS) medium containing2 mg l^–1 IAA, 05 mg l^–1 BAP and 05 mg l^–1naphthaleneacetic acid (NAA), pale-yellow translucent nodularcalluses appeared on the surface of the explants. When thiskind of callus was transferred to MN6 medium with 2 mg l^–1IAA and 3 mg l^–1 BAP, embryoids formed which further developedinto plantlets. The regenerated plants were morphologicallynormal and possessed the normal diploid chromosome number of2n = 22. A similar result has also been obtained with youngleaf explants of this plant. The early segmentations of embryogeniccells and the development of embryoids were studied using histologicaland scanning electron microscopic techniques, and the resultshave been discussed in association with the ontogeny and originof the embryoids. Freesia refracta Klatt, somatic embryogenesis, plant regeneration, exogenous hormones