A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions of Musa (Banana and plantain)

A tissue culture technique for rapid clonal propagation and storage under minimal growth conditions is presented in this paper. Shoot-tip cultures of Musa cultivars (both banana and plantain) are induced by culturing small excised shoot apices on modified MS semisolid medium supplemented with various concentrations and combinations of auxins and cytokinins. The effects of cytokinin concentration in the medium as well as the genotypic configuration of the cultivars on the rate of shoot-bud proliferation have been tested. The established shoot-tip cultures grown on modified MS semisolid medium supplemented with IAA (0.18 mg/l) and BA (2.30 mg/l) have been successfully stored at 15°C with 1000 lux light intensity up to 13–17 months depending on the cultivar. The cultivars tested in the present investigation seem to vary in their ability to withstand minimal growth temperature.Abbreviations BA Benzyladenine - IAA Indoleacetic acid - IBA Indolebutyric acid - MS Murashige and Skoog     

Effect of glycine betaine on the sucrose catabolism of an l-lysine producing mutant of Brevibacterium lactofermentum

Summary Effect of exogenous betaine on the growth of an l-lysine-producing mutant of Brevibacterium lactofermentum was examined in a medium containing different carbon sources such as glucose, fructose, or sucrose. The growth rate decreased significantly with a rise in temperature when sucrose was the carbon source. Both the specific sucrose consumption rate and the invertase activity of the mutant decreased with the culture period when the cultivation temperature was 35°C. The addition of betaine restored both growth and invertase activity on medium containing sucrose as the carbon source at 35°C. Betaine protected the invertase activity against the inactivating effects of high temperature in vitro. Furthermore, the addition of exogenous invertase into production medium at 35°C restored the growth rate to that at 32°C. These results indicated that growth decreased on medium containing sucrose at 35°C due to a decrease in invertase activity, and that addition of betaine was an effective way to enhance growth on this medium at a higher temperature. Offprint requests to: Y. Kawahara     

Isolation of thermotolerant,fermentative yeasts growing at 52°C and producing ethanol at 45°C and 50°C

Five thermotolerant, alcohol-producing yeast cultures were isolated from samples obtained from India. Two were identified as ofKluyveromyces marxianus. All five grew on plate-cultures up to 52°C, with maximum growth rates in liquid culture at 40°C. All produced relatively high alcohol concentrations: 5.7 to 7.0% (w/v) at 45°C and 5.0 to 5.5% (w/v) at 50°C when growing on 14.0% (w/v) glucose. All five isolates fermented diluted molasses containing 16.0% (w/v) total sugars, producing 5.6 to 6.0% (w/v) alcohol concentrations. Supplementing the molasses with P, K, Mg and Mn resulted in a 13 to 20% increase in alcohol production at 40°C. The maximum amounts of alcohol produced on supplemented molasses were 7.5 to 8.0 and 6.5 to 7.0% (w/v) at 37°C and 40°C, respectively.     

High frequency of shoot multiplication and bulblet formation of garlic in liquid cultures

In vitro shoot proliferation and bulblet production of garlic (Allium sativum L.) was studied in liquid cultures. Shoots grown in vitro were used as explants and were cultured in MS medium supplemented with 2% (w/v) sucrose and 0.5 mg l^–1 2-iP. Three culture methods (semi-solid, liquid-immersion and raft) were compared for shoot proliferation. Explants in liquid (immersion) culture exhibited an increased multiplication rate and fresh weight of shoots after 3 weeks of culture as compared with the other treatments. Bulblet formation and growth were studied in liquid medium with different concentrations of sucrose (2–13%). MS medium containing 11% (w/v) sucrose was optimal for bulblet development and bulblets developed in this medium within 9 weeks in culture. The highest multiplication rate was (135 bulblets/explant) found when explants were cultured in bulbing medium (MS medium containing 0.1 mg l^–1 NAA+11% (w/v) sucrose) supplemented with 10 M JA. Growth retardants CCC, B-9, ABA also promoted induction and growth of bulblets. Darkness promoted the bulblet induction and growth compared to light conditions (16-h photoperiod of 50 mol m^–2 s^–1). The dormancy of bulblets was broken by cold treatment at 4 °C for 8 weeks.     

Production of extracellular alkaline proteases by <Emphasis Type="Italic">Aspergillus clavatus</Emphasis>

Summary The production of extracellular alkaline proteases from Aspergillus clavatus was evaluated in a culture filtrate medium, with different carbon and nitrogen sources. The fungus was cultivated at three different temperatures during 10 days. The proteolytic activity was determined on casein pH 9.5 at 37 °C. The highest alkaline proteolytic activity (38 U/ml) was verified for culture medium containing glucose and casein at 1% (w/v) as substrates, obtained from cultures developed at 25 °C for 6 days. Cultures developed in Vogel medium with glucose at 2% (w/v) and 0.2% (w/v) NH₄NO₃ showed higher proteolytic activity (27 U/ml) when compared to the cultures with 1% of the same sugar. Optimum temperature was 40 °C and the half-lives at 40, 45 and 50 °C were 90, 25 and 18 min, respectively. Optimum pH of enzymatic activity was 9.5 and the enzyme was stable from pH 6.0 to 12.0.     

Cryopreservation of in vitro-grown apical meristems of lily by vitrification

Apical meristems from adventitious buds induced by culturing of bulb-scale segments of Japanese Pink Lily (Lilium japonicum Thunb.) were successfully cryopreserved by a vitrification. The excised apical meristems were precultured on a solidified Murashige & Skoog medium, containing 0.3 M sucrose, for 1 day at 25°C and then loaded in a mixture of 2 M glycerol plus 0.4 M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) at 25°C for 20 min or at 0°C for 110 min prior to a plunge into liquid nitrogen. After rapid warming in a water bath at 40°C, the meristems were placed in 1.8 ml of 1.2 M sucrose for 20 min and then, placed on filter papers over gellan gum-solidified MS medium. The revived meristems resumed growth within 5 days and directly produced shoots. The rate of shoot formation was approximately 80% after 4 weeks. When bulb-scale segments with adventitious buds were cold-hardened at 0°C for more than 7 days before the procedure, the rates of shoot formation were significantly increased. This vitrification method was successfully applied to five other lily cultivars. Thus, this vitrification procedure for cryopreservation appears promising as a routine method for cryopreserving meristems of lily.Abbreviations DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige & Skoog (1962) medium - PVS2 vitrification solution     

Cryopreservation of Grapevine (Vitis spp.) Embryogenic Cell Suspensions by Encapsulation–Vitrification

Embryogenic cell suspensions of two grapevine rootstocks: 110 Ritcher (V. berlandieri × V. rupestris), 41B (V. vinifera × V. berlandieri) and several table grape and wine cultivars (Vitis vinifera) were successfully cryopreserved by the encapsulation–vitrification method. Embryogenic cell suspensions were precultured for 3 days in liquid MGN medium supplemented with daily increasing sucrose concentrations of 0.25, 0.5, 0.75 M. Precultured cells were encapsulated and directly dehydrated with a highly concentrated vitrification solution prior to immersion in liquid nitrogen for 1 h. After rewarming at 40 °C for 3 min, cryopreserved cells were post-cultured on solid MGN medium supplemented with 2.5 g l^–1 activated charcoal. Surviving cells were transferred to solid MGN medium for regrowth or solid MG medium for embryo development and then to solid WPM for plant regeneration. Optimal viability was 42–76% of cryopreserved cells when cell suspensions were precultured with a final sucrose concentration of 0.75 M and dehydrated with PVS2 at 0 °C for 270 min. Biochemical analysis showed that sucrose preculture caused changes in levels of total soluble protein and sugars in cell suspensions. Although the increase in fresh weight was significantly lower in cryopreserved cells than in control cells, the growth pattern of the cryopreserved cells and control cells was the same after two subcultures, following re-establishment in cell suspensions. Protocol developed in this study suggests a universal and highly efficient cryopreservation system suitable for several genetically diversed Vitis species.     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Effect of sugar type on the efficiency of plant regeneration from protoplasts isolated from shoot tip-derived meristematic nodular cell clumps of Lilium x formolongi hort.

Summary Suspension cultures composed of meristematic nodular cell clumps of Lilium x formolongi hort were established from shoot tips placed on MS medium supplemented with 1 mg/l picloram and 30 g/l sucrose, glucose, fructose or sorbitol. Protoplasts isolated from these cultures were embedded in 1 g/l gellan gumsolidified 1/2MS medium with 1 mg/l picloram and the different kinds of sugars at 0.5 M, and cultured at 25 °C in the dark. The highest plating efficiency (13.7%) was obtained when the protoplasts were isolated from the cell clumps which had been subcultured in MS medium containing glucose and were likewise cultured in MS medium supplemented with 0.5 M glucose. Plants were regenerated from the protoplast-derived calli on 1/2MS medium containing 2.5–10 g/l sucrose or 5–10 g/l glucose. These results suggest that the kinds of sugar and concentration are important parameters affecting protoplast isolation, proliferation and plant regeneration in L. x formolomgi hort.Abbreviations FW fresh weight - MS Murashige and Skoog (1962) - 1/2MS medium MS medium containing half strength mineral salts - 1/2MS-0 1/2MS medium containing no growth regulators - NAA 1-naphthaleneacetic acid - p-calli protoplast-derived calli - PE plating efficiency - picloram 4-amino-3,5,6-trichloro-picolinic acid     

Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration

Summary In vitro-grown apical meristems of wasabi (Wasabia japonica Matsumura) were successfully cryopreserved by vitrification. Excised apical meristems precultured on solidified M S medium containing 0.3M sucrose at 20°C for 1 day were loaded with a mixture of 2M glycerol and 0.4M sucrose for 20 min at 25°C. Cryoprotected meristems were then sufficiently dehydrated with a highly concentrated vitrification solution (designated PVS2) for 10 min at 25°C prior to a plunge into liquid nitrogen. After rapid warming, the meristems were expelled into 2 ml of 1.2M sucrose for 20 min and then plated on solidified culture medium. Successfully vitrified and warmed meristems remained green after plating, resumed growth within 3 days, and directly developed shoots within two weeks. The average rate of normal shoot formation amounted to about 80 to 90% in the cryopreserved meristems. This method was successfully applied to three other cultivars of wasabi. This vitrification procedure promises to become a routine method for cryopreserving meristems of wasabi.Abbreviations BA 6-benzylaminopurine - DMSO dimethylsulfoxide - EG ethylene glycol - LN liquid nitrogen - MS medium Murashige and Skoog medium (1962) - PVS2 vitrification solution     

Optimisation of culture conditions for production of eicosapentaenoic acid byMortierella elongate NRRL 5513

Summary WhenMortierella elongata NRRL 5513 was cultured in shake flasks at 25°C, mycelial growth reached a stationary phase at 48 h but maximum eicosapentaenoic acid (EPA) production was observed at 6 days. When incubated at 11°C, EPA production also continued to rise during the stationary phase of growth, reaching a maximum after 10 days. An initial culture pH of 6.1 was found to be optimum for EPA production. The effect of temperature on EPA production was dependent on medium constituents. In glucose and linseed oil supplemented media, optimum temperature for EPA production was 11 and 15°C respectively. A maximum EPA yield of 0.61 g/l was obtained in linseed oil (2%), yeast extract (0.5%) supplemented basal medium. Maximum EPA content as a percentage of lipids (15.12%) was observed when the latter medium was supplemented with 0.25% urea.     

Characteristic symptoms of photosynthesis inhibition by herbicides are expressed in photomixotrophic tissue cultures of Nicotiana

A photomixotrophic tissue culture system for Nicotiana plumbaginifolia and N. tabacum has been developed in which a primary symptom (bleching) of the inhibition of photosynthetic electron transport by herbicides can be observed. Photomixotrophic cultures were initiated and maintained in the light on medium containing 0.2–0.3% sucrose or glucose (low-sugar medium) as sole source of respirable carbohydrate. The usual medium for growing heterotrophic cultures contains 2–3% sucrose or glucose (high-sugar medium). Callus grown on low-sugar medium achieved a fresh weight three to four times greater in the light than in the dark and reached about half that of callus grown on high-sugar medium. Carbon-dioxide fixation rates were an order of magnitude higher in cultures grown on low-sugar medium in the light than in those grown on high-sugar medium or in any of the dark-grown cultures. The lightdependent growth and CO₂-fixation rates of cultures grown on low-sugar medium indicated that a major proportion of the weight increase resulted from photosynthesis. Under these photomixotrophic conditions it was found that a number of photosystem-II herbicides, at concentrations which inhibit photosynthetic electron transport, also inhibited the light-dependent component of callus growth, and caused bleaching. These effects could not be demonstrated on high-sugar medium.Abbreviations PSII photosystem II For common names of the herbicides the reader is referred to Weed Res. 19, 401–406 (1979)     

Low-temperature storage of Rauvolfia serpentina Benth. ex Kurz.: An endangered,endemic medicinal plant

On a standard shoot culture medium, nodal cultures of Sarpagandha (Rauvolfia serpentina) could be maintained for nine months at 25° C by replacing cotton plugs with polypropylene caps as enclosures for culture tubes. Low temperature incubation of in vitro cultures appeared highly promising because cultures exhibited normal health even after 15 months of storage at 15° C; while 10°C and 5°C were found deleterious to growth of the cultures of R. serpentina.     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to a _w 0.93 were more rapid than in unsupplemented media ( a _w 0.99). Although growth in unsupplemented medium was lower at 35°C, incubation at 21°C or 35°C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 μg potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35°C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35°C than in cultures incubated at 21° in media both with and without 300 μg potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at - 18°C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 μg potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21°C. Sensitivity to freezing increased when cultures were incubated at 21°C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Isolation and characterization of a thermophilic acetotrophic strain of Methanothrix

An enrichment culture which converted acetate to methane at 60°C was obtained from a thermophilic anaerobic bioreactor. The predominant morphotype in the enrichment was a sheathed gas-vacuolated rod with marked resemblence to the mesophile Methanothrix soehngenii. This organism was isolated using vancomycin treatments and serial dilutions and was named Methanothrix sp. strain CALS-1. Strain CALS-1 grew as filaments typically 2–5 cells long, and cultures showed opalescent turbidity rather than macroscopic clumps. The cells were enclosed in a striated subunit-type sheath and there were distinct cross-walls between the cells, similar to M. soehngenii. The gas vesicles in cells were typically 70 nm in diameter and up to 0.5 m long, and were collapsed by pressures over 3 atm (ca. 300 kPa). Stationary-phase cells tended to have a higher vesicle content than did growing cells, and occasionally bands of cells were seen floating at the top of the liquid in stationary-phase cultures. Acetate was the only substrate of those tested which was used for methanogenesis by strain CALS-1, and acetate was decarboxylated by the aceticlastic reaction. The optimum temperature for growth of strain CALS-1 was near 60°C (doubling time=24–26 h), with no growth occurring at 70°C and 37°C. The optimum pH value for growth was near 6.5 in bicarbonate/CO₂ buffered medium and no growth occurred at pH 5.5 or pH 8.4. No growth was obtained below pH 7 when the medium was buffered with 20 mM phosphate. Strain CALS-1 grew in a chemically defined medium and required biotin. Sulfide concentrations over 1 mM were inhibitory to the culture, and growth was more rapid with 1 mM 2-mercaptoethane sulfonate (coenzyme M) or 1 mM titanium citrate as an accessory reductant than with 1 mM cysteine. It is likely that strain CALS-1 represents a new species in the genus Methanothrix.     

Complex responses to culture conditions in Pseudomonas syringae pv. tomato DC3000 continuous cultures: The role of iron in cell growth and virulence factor induction

The growth of a model plant pathogen, Pseudomonas syringae pv. tomato DC3000, was investigated using a chemostat culture system to examine environmentally regulated responses. Using minimal medium with iron as the limiting nutrient, four different types of responses were obtained in a customized continuous culture system: (1) stable steady state, (2) damped oscillation, (3) normal washout due to high dilution rates exceeding the maximum growth rate, and (4) washout at low dilution rates due to negative growth rates. The type of response was determined by a combination of initial cell mass and dilution rate. Stable steady states were obtained with dilution rates ranging from 0.059 to 0.086 h^?1 with an initial cell mass of less than 0.6 OD₆₀₀. Damped oscillations and negative growth rates are unusual observations for bacterial systems. We have observed these responses at values of initial cell mass of 0.9 OD₆₀₀ or higher, or at low dilution rates (<0.05 h^?1) irrespectively of initial cell mass. This response suggests complex dynamics including the possibility of multiple steady states. Iron, which was reported earlier as a growth limiting nutrient in a widely used minimal medium, enhances both growth and virulence factor induction in iron‐supplemented cultures compared to unsupplemented controls. Intracellular iron concentration is correlated to the early induction (6 h) of virulence factors in both batch and chemostat cultures. A reduction in aconitase activity (a TCA cycle enzyme) and ATP levels in iron‐limited chemostat cultures was observed compared to iron‐supplemented chemostat cultures, indicating that iron affects central metabolic pathways. We conclude that DC3000 cultures are particularly dependent on the environment and iron is likely a key nutrient in determining physiology. Biotechnol. Bioeng. 2010;105: 955–964. © 2009 Wiley Periodicals, Inc.     

Establishment and Growth of Embryogenic Suspension Cultures of Ocotea catharinensis Mez. (Lauraceae)

The focus of this study was to test the effects of 2,4-D, sucrose, culture media and initial inocula on the development of embryogenic suspension cultures of Ocotea catharinensis Mez. (Lauraceae). Suspension cultures were established in half-strength MS medium supplemented with 2% (w/v) sucrose either in the absence or in the presence of 2.2 μM 2,4-D, when higher cell viability was achieved. Under this culture condition the maximum fresh weight increase occurred in the fourth week. The cultures were yellow and consisted of a mixture of highly cytoplasmic single cells and small cell aggregates (<0.25 mm). The best proportion of inoculum per volume of medium for suspension culture development was 5% (w/w). Suspension cultures consisting of somatic embryos at the globular and cotyledonary stages (structures ranging from 1 to 3 mm) were successfully established on half-strength MS supplemented with 2% (w/w) sucrose through repetitive embryogenesis from the desiccated mature somatic embryos used as initial inoculum. The failure to initiate liquid cultures from non-desiccated mature somatic embryos was overcome by pre-treatment with air desiccation and reduction of the water content to 6.1 g H₂O g⁻¹ dry weight.     

Cryopreservation in liquid nitrogen of cultured navel organe (Citrus sinensis Osb.) nucellar cells and subsequent plant regeneration

Suspension cultured cells of nucellar callus of navel orange (Citrus sinensis Osb. var. brasiliensis Tanaka) were successfully cryopreserved. The nucellar cells were cryoprotected in Murashige-Tucker basal medium supplemented with 5% DMSO+1.2 M sucrose in an ice bath for 1 h, and then were frozen in this solution at a cooling rate of 0.5°C/min to –40°C prior to immersion in LN₂. After rapid thawing in a +40°C water bath, regrowth was achieved by transferring the treated cells, without washing, onto filter paper discs over nutrient media solidified with agar. The viability after thawing, as evaluated by FDA and phenosafranine double staining, was about 70% of controls. The revived cells resumed growth within 3 days and produced cotyledonary embryos that developed into plants within 2 to 6 months of culture. Plants regenerated from cryopreserved cells were morphologically uniform and had the characteristics typical of navel orange.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - LN₂ liquid nitrogen - NAA -naphthaleneacetic acid - SE standard error     

Interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii.

The interactive effects of solutes, potassium sorbate and incubation temperature on growth, heat resistance and tolerance to freezing of Zygosaccharomyces rouxii were investigated. Growth rates in media supplemented with glucose, sucrose or NaCl to aw 0.93 were more rapid than in unsupplemented media (aw 0.99). Although growth in unsupplemented medium was lower at 35 degrees C, incubation at 21 degrees C or 35 degrees C had little effect on growth in media supplemented with glucose and sucrose. The addition of 300 micrograms potassium sorbate/ml to media resulted in reduced growth rates, particularly at 35 degrees C. Heat resistance of Z. rouxii was substantially greater in cultures previously incubated at 35 degrees C than in cultures incubated at 21 degrees C in media both with and without 300 micrograms potassium sorbate/ml. Zygosaccharomyces rouxii was tolerant to freezing at -18 degrees C for up to 120 d in all test media supplemented with glucose, sucrose or NaCl. The addition of 300 micrograms potassium sorbate/ml to sucrose-supplemented media resulted in increased resistance to freezing in cultures previously incubated at 21 degrees C. Sensitivity to freezing increased when cultures were incubated at 21 degrees C in media not supplemented with solutes. Glucose and sucrose provided the best protection against inactivation by heating and freezing, regardless of the presence of potassium sorbate in growth media.     

Chlorella vulgaris (Chlorellaceae) does not secrete autoinhibitors at high cell densities

Filtrates (conditioned medium) from high-density Chlorella vulgaris cultures in photobioreactors were obtained and tested for autoinhibitory activity under different conditions. Exponentially growing cells were inoculated at low initial cell concentration (2 × 10⁵ cells/ml) in 90% conditioned medium (CM) supplemented with 10% fresh medium (FM) at low (atmospheric) CO₂ levels. The time sequence of DNA histograms of cells in CM cultures showed that there is an accumulation of cells with two and four DNA equivalents in the culture over a period of time, signifying a blockage of cells at the division stage of the cell cycle. Examination of the chemical composition of CM showed the presence of high concentrations (> 10 mM) of bicarbonate. Adding similar bicarbonate concentrations to FM were found to have similar effects as CM cultures, causing blockage of cell division, though the intensity of the blocking effect was lower. The bicarbonate-free CM did not show any cell cycle modulating or inhibitory activity. The growth of cells cultivated at high (5%) CO₂ levels in 90% CM supplemented with 10% FM was comparable to 10% FM cultures, indicating nutrient limitation in 90% CM culture. When the 90% CM culture was supplemented with 100% nutrients, the growth rate and final cell concentration was similar to 100% FM culture. Based on these results we conclude that C. vulgaris does not secrete any autoinhibitor(s) or cell cycle modulating compound(s) under the conditions from which the CM was obtained.