<Emphasis Type="Italic">In vitro</Emphasis> culture studies on <Emphasis Type="Italic">Stevia rebaudiana</Emphasis>

Summary Shoot apex, nodal, and leaf explants of Stevia rebaudiana Bertoni can regenerate shoots when cultured on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA; 8.87 μM) and indole-3-acetic acid (5.71 μM). Rooting of the in vitro-derived shoots could be achieved following subculture onto auxin-containing medium. A survival rate of 70% was recorded at the hardening phase on the substrate cocopeat. The presence of the sweet diterpene glycosides, viz. stevioside and rebaudioside, was confirmed in the in vitro-derived tissues of Stevia using HPTLC techniques. Callus cultured on agar-solidified MS medium supplemented with BA (8.87 μM) and indole-3-butyric acid (9.80 μM) showed the highest sweetener content.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

In vitro propagation of Alstroemeria ‘Alsaan’

Plantlets were regenerated from Alstroemeria Alsaan rhizome tips cultured in vitro on solid and liquid media based on Murashige and Skoog salt formulation. The quality of the cultures was superior when intact rather than longitudinally sliced rhizome tips were used as explants and when a temperature of 8°C rather than 22°C was used at the initiation stage. More roots were produced on rhizome tips containing a rhizome apical meristem than on rhizome sections lacking such a meristem. Most (90%) of the rooted plantlets were successfully acclimatized and developed into true-to-type flowering plants.     

Combined Effects of Algal (<Emphasis Type="Italic">Chlorella vulgaris</Emphasis>) Food Level and Temperature on the Demography of <Emphasis Type="Italic">Brachionus havanaensis</Emphasis> (Rotifera): a Life Table Study

We evaluated the combined effects of food (0.5 × 10⁶, 1.0 × 10⁶ and 2.0 × 10⁶ cells ml⁻¹ of Chlorella vulgaris) and temperature (15, 20 and 25 °C) on life history variables of B. havanaensis. Regardless of Chlorella density there was a steep fall in the survivorship of B. havanaensis at 25 °C. Both food level and temperature affected the fecundity of B. havanaensis. At any given food level, rotifers cultured at 15 °C showed extended but low offspring production. At 25 °C, offspring production was elevated, the duration of egg laying reduced and the fecundity was higher during the latter part of the reproductive period. The effect of food level was generally additive, at any given temperature, and higher densities of Chlorella resulted in higher offspring production. Average lifespan, life expectancy at birth and generation time were 2–3 times longer at 15 °C than at 25 °C. At 20 °C, these remained at intermediate levels. The shortest generation time (about 4 days) was observed at 25 °C. Gross and net reproductive rates and the rate of population increase (r) increased with increasing temperature and generally, at any given temperature, higher algal food levels contributed to higher values in these variables. The r varied from 0.11 to 0.66. The survival patterns and lower rates of reproduction at 15 °C suggest that the winter temperatures (10–15 °C) prevailing in many waterbodies in Mexico City allow this species to sustain throughout the year under natural conditions.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

A preliminary evaluation of the use of microbial culture filtrates for the control of contaminants in plant tissue culture systems

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from 1/4 to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strenth (1/4 B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1/4 B5) was 40 mg g^-1 and 22 mg g^-1 fresh weight respectively but decreased to less than 1 mg g^-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4–0.6 mg g^-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

Thidiazuron-induced regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.: Micropropagation in solid and liquid culture systems

The goals of this study were to investigate thidiazuron (TDZ)-induced morphogenesis of Echinacea purpurea L. and to assess the possibility of developing a liquid-based protocol for rapid micropropagation. Callus development and root organogenesis were observed on leaf explants cultured on media containing 2,4-dicholorophenoxyacetic acid or dicamba, but no plantlets were regenerated. Addition of TDZ to the culture medium as the sole growth regulator resulted in the production of regenerable callus cultures. The highest rate of regeneration was observed for explants cultured on medium with TDZ at 2.5 μM or higher. Tissue derived from 1.0 μM TDZ treatments was used to initiate liquid cultures. All liquid treatments produced a similar number of regenerants but significantly more healthy plants were obtained from cultures grown in the presence of 0.1 and 1.0 μM TDZ. This TDZ-based micropropagation system is the first liquid, large-scale propagation protocol developed for the mass production of E. purpurea plants.     

Plantlet regeneration of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> var. adik-adik by tissue culture

Three color morphotypes of Kappaphycus alvarezii var. adik-adik (brown, green and red) collected from a farming area in Tictauan Is., Zamboanga City, Philippines were used as explants in the study in order to micropropagate ‘new’ plants. Individual sections of sterile Kappaphycus alvarezii var. adik-adik, initially cultured in a 48-well culture plate containing ESS/2 + E3 + PGR, released callus cells after 4–5 days of incubation at 23–25°C, 13:11H LD cycle and 10–15 μmol photons m⁻² s⁻¹ light intensity. True calli were formed after 29–35 days following dense formation of filaments or undifferentiated round cells at the medullary and inner cortical layers of the section. Plantlets (2–3 mm long) of Kappaphycus alvarezii var. adik-adik were able to regenerate after 98, 150 and 177 days in-vitro among the reds, greens, and browns, respectively. This study established successful methods for the production and regeneration of tissue explants of Kappaphycus alvarezii var. adik-adik which can possibly be used to mass produce ‘new’ cultivars for land- and sea-based nurseries as sources for commercial farming. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

<Emphasis Type="Italic">In vitro</Emphasis> organogenesis of <Emphasis Type="Italic">Pelecyphora aselliformis</Emphasis> erhenberg (cactaceae)

Summary In vitro propagation of Pelecyphora aselliformis, a Mexican cactus which is considered rare and is highly valued in the commercial market, was initiated using seeds as explants. The longitudinal explants from seedlings germinated in vitro were cultivated on Murashige and Skoog medium containing 8.8 μM benzyladenine (BA) or 4.6 μM kinetin at pH 7.0. After 120 d, each explant gave rise to five shoots and this number of shoots increased 20–25% after subculture. The hyperhydricity was similar in both media, but callus formation was lower on the medium with BA. The shoot development, in terms of epicotyl length, and fresh and dry weight after 6 wk, was also recorded. The epicotyl length was similar on shoot-forming media but the quality of shoots was better on media containing BA. In about 1 yr, 500–600 well-defined shoots were obtained. The rooting of shoots was very slow and a vigorous radical system was observed after 1 yr of culture.     

Combined Effects of Food Concentration and Temperature on Competition Among Four Species of <Emphasis Type="Italic">Brachionus </Emphasis>(Rotifera)

We evaluated the effect of algal food density (1.5 × 10⁶, 3.0 × 10⁶ and 4.5 × 10⁶ cells ml⁻¹ of Chlorella) and temperature (22° and 28 °C) on competition among the rotifers Brachionus calyciflorus, Brachionus havanaensis, Brachionus patulus and Brachionus rubens, based on population growth experiments for 24 days. The growth experiments were conducted seperately for each individual rotifer species (i.e., controls), and in mixtures of all four species in equal initial proportions (i.e., under competition). The population growth of B. calyciflorus, B. havanaensis, B. patulus and B. rubens grown separately at two temperatures and at three algal food densities showed typical patterns of lag, exponential and retardation phases in the controls. This pattern differed considerably under competition. In general, we observed that in all of the test species, the highest growth rates were observed at higher food levels and in the absence of congenerics. At 22 °C, under the lowest food level, the differences in the population abundances of B. havanaensis, B. patulus and B. rubens grown alone and in the presence of competition were large. However, these differences reduced as food density was increased from 0.5 × 10⁶ to 4.5 × 10⁶ cells ml⁻¹. At 28 °C and at the lowest food level, all of the other rotifer species eliminated B. havanaensis in mixed cultures. Each brachionid species had a higher rate when grown alone than when cultured with other species. The highest r (mean ± standard error: 0.54 ± 0.01 day⁻¹) was recorded for B. havanaensis at 28 °C under 4.5 × 10⁶ cells ml⁻¹ of algal food density. At 28 °C at low algal food density, the presence of competitors resulted in negative population growth rates for three of the four rotifer species tested.     

Life-history traits of the acarophagous lady beetle,<Emphasis Type="Italic">Stethorus japonicus</Emphasis> at three constant temperatures

Stethorus japonicusKamiya (Coleoptera: Coccinellidae) is an indigenous ladybird beetle in Japan, which feeds on many spider mite species. We evaluated the development, survivorship and life-history parameters of this lady beetle on a diet of eggs of the two-spotted spider mite, Tetranychus urticae Koch (red form) (Acari: Tetranychidae). In addition, the effect of short photoperiod on its reproduction was assessed. Survival rates from egg to adult were more than 71% at temperatures between 17.5 and 30 °C. The highest immature mortality was 100% at 35 °C followed by 76% at 15 °C and 52% at 32.5 °C. The lower threshold temperature for development from egg to egg-laying adult was 13.0 °C and the thermal constant was calculated as 238.7° days. Based on these data, the maximum number of generations that could complete development in a year under field conditions in Ibaraki, central Japan, would be between five and seven. The intrinsic rates of natural increase (r_m) were 0.093 at 20 °C, 0.156 at 25 °C and 0.241 at 30 °C. Reproductive diapause was induced at photoperiods with light phases shorter than 13 h at 18 °C.     

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling

Photosynthetic activity, in leaf slices and isolated thylakoids, was examined at 25° C after preincubation of the slices at either 25° C or 4° C at a moderate photon flux density (PFD) of 450 mol·m^–2·s^–1, or at 4° C in the dark. The plants used wereSpinacia oleracea L.,Cucumis sativus L. andNerium oleander L. which was acclimated to growth at 20° C or 45° C. The plants were grown at a PFD of 550 mol·m^–2·s^–1. Photosynthesis, measured as CO₂-dependent O₂ evolution, was not inhibited in leaf slices from any plant after preincubation at 25° C at a moderate PFD or at 4° C in the dark. However, exposure to 4° C at a moderate PFD induced an inhibition of CO₂-dependent O₂ evolution within 1 h inC. sativus, a chilling-sensitive plant, and in 45° C-grownN. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO₂ concentration but was reduced at O₂ concentrations of less than 3%. Methyl-viologen-dependent O₂ exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition inS. oleracea, a chilling-insensitive plant, or in 20° C-grownN. oleander. The CO₂-dependent photosynthesis, measured at 5° C, was reduced to about 3% of that recorded at 25° C in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent O₂ exchange, measured at 5° C, was greater than 25% of the activity at 25° C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chilling-sensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures.Abbreviations Chl chlorophyll - DPIPH reduced form of 2,6-dichlorophenol-indophenol - DMQ 2,5-dimethyl-p-benzoquinone - MV methyl viologen - 20°-oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - PFD photon flux density (photon fluence rate) - PSI and PSII photosystem I and II, respectively     

<Emphasis Type="Italic">Arthrobacter ardleyensis</Emphasis> sp. nov., isolated from Antarctic lake sediment and deep-sea sediment

Three psychrotrophic Arthrobacter strains, isolated from Antarctic lake sediment (An24, An25^T) and deep-sea sediment (ZX6) were studied. Their 16S rRNA gene sequences showed highest similarities (97.0–97.9%) with those of A. nicotianae and A. protophormiae. All three strains underwent rod–coccus morphological change, had high mol% G+C content, were aerobic to slightly anaerobic, and grew between 0°C and 30°C, with optimal growth temperature around 25°C. The cell wall peptidoglycan was A4 variant. DNA–DNA hybridization, physiological and chemotaxonomic studies indicated that these three strains constituted a new homogeneous genomic species within the genus Arthrobacter, for which the name Arthrobacter ardleyensis, with the type strain An25^T (CGMCC 1.3685, JCM 12921) was proposed.M. Chen and X. Xiao contributed equally to this paper.     

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.     

Biosynthesis of Dibenzyl Trisulfide (DTS) from somatic embryos and rhizogenous/embryogenic callus derived from Guinea hen weed (<Emphasis Type="Italic">Petiveria alliacea</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) leaf explants

A procedure for producing somatic embryos enriched with dibenzyl trisulfide (DTS) using a hormone-dependent culture system is reported for Petiveria alliacea L. (Guinea hen weed). Leaf explants were cultured on a Murashige and Skoog medium supplemented with a range of naphthaleneacetic acid (NAA) concentrations and a fixed concentration of benzyladenine (BAP) at 11.0 μM and sucrose or glucose at 30 g l⁻¹. Leaf explants cultured on all media types started to form callus at the cut surfaces of the discs 10–14 d after initiation. The type of sugar used influenced average fresh weight, the propensity to form roots, as well as the embryogenic response. The highest mean fresh weight (337.7 ± 26.18 mg) and mean root number (23.7 ± 1.69) was produced on media enriched with sucrose and supplemented with 53.7 μM NAA and 11.0 μM BAP. An ethanol extract of rhizogenic/embryogenic callus or somatic embryos was subjected to high-performance liquid chromatography analysis, which revealed the presence of DTS in both extracts. UV spectral analysis and the use of standard quantitation procedures showed that the quantity of DTS in the somatic embryo extract, at 0.16% (w/v), was approximately 30-fold higher than in rhizogenic/embryogenic callus (0.0055% w/v) of similar fresh weight. These results indicate that it is possible to biosynthesize approximately 6 mg of natural DTS from 3,808 mg of fresh somatic embryos within 10 wk from less than three leaf explants.     

Development of the starfish <Emphasis Type="Italic">Asterias amurensis</Emphasis> under laboratory conditions

Under laboratory conditions the development of the starfish Asterias amurensis Lütken from Vostok Bay (Sea of Japan) was studied at 14 and 17°C. At 14°C and a salinity of 31.6–32.6, ciliated coeloblastulae hatched from egg envelopes 19 h after fertilization. At this temperature the development proceeded slowly and stopped at the stage of bipinnaria. At 17°C and normal salinity of seawater, the development of A. amurensis was successful. The swimming blastula appeared in 14 h. It took 30.5 h for the embryos to reach the gastrula stage. The larvae began swimming in a horizontal position with the apical tip ahead. The dipleurula appeared at 60 h. These larvae began feeding. At 71 h after the beginning of development, the early bipinnaria has developed. In the larva, the edged ciliated band, the preoral plate, and the anal plate were already formed. At the age of 4.2 days, the larvae reached the stage of bipinnaria and the brachiolaria stage developed by 26–28 days after fertilization. The larvae had three identical brachiolar arms with attachment papillae on their tips and an attachment disk. In 37–44 days (at 17°C) the pelagic phase of A. amurensis development was completed by the attachment of larvae to the bottom plates and termination of metamorphosis. Most likely, the specificity to a substrate is not expressed in the brachiolaria of A. amurensis. They can settle on almost any hard substrate which is coated with a bacterial film. The newly settled juvenile starfish had five well-developed arms and moved using their ambulacral podia.Original Russian Text Copyright © 2005 by Biologiya Morya, Kashenko.     

Development, survival and reproduction of Euseius finlandicus (Acari: Phytoseiidae) at different constant temperatures

Development, survival and reproduction of Euseius finlandicus Oudemans were studied at seven constant temperatures (15, 20, 25, 27, 30, 32 and 34°C) in the laboratory. Within the temperature range tested, developmental period from egg to adult varied from 148 to 360.5h and 133.7 to 336.5h for females and males, respectively. The lower thermal threshold for immature development for females and males was 8.9 and 6.4°C, respectively. Survival during immature development exceeded 90% at all the temperatures from 15 to 32°C, but at 34°C an abrupt decline was recorded. Female longevity decreased gradually from 82.7d at 15°C to 12.2 d at 34°C. The mean generation time ranged from 44.3d at 15°C to 15.9d at 32°C. The highest r _m value (0.2817) was obtained at 30°C and the lowest at 15°C (0.0976). Temperatures above 30°C had an adverse effect on population increase.     

Tissue culture propagation of Mongolian cherry (<Emphasis Type="Italic">Prunus fruticosa</Emphasis>) and Nanking cherry (<Emphasis Type="Italic">Prunus tomentosa</Emphasis>)

In vitro culture establishment, shoot proliferation and ex vitro rooting responses of Mongolian cherry (Prunus fruticosa L.), and Nanking cherry (Prunus tomentosa L.), were examined using various combinations of growth regulators. Dormant buds, taken during winter months, were used as explants. In both species, Murashige and Skoog Minimal Organic (MSMO) solid medium supplemented with 0.49 M indole-3-butyric acid (IBA) and either 4.44 or 8.88 M 6-benzylaminopurine (BA), was the best for culture initiation, and with 8.88–15.16 M BA for shoot proliferation. Good rooting responses were also obtained with shoots produced on media containing 0.91 M thidiazuron (TDZ). Auxin treatments were required for ex vitro rooting of approximately 20 mm long shoots in peat/perlite (1:1 v/v) mixture, at 25 °C, under mist. The best rooting (79%) was obtained with IBA/NAA (naphthaleneacetic acid) (9.80/2.69 M) combination. A commercial rooting powder, Rootone F, containing IBA/NAA (0.057/0.067%), was also effective (73%). The ex vitro rooted plantlets did not require any additional acclimatization prior to transplanting to the regular greenhouse conditions.