The role of <Emphasis Type="Italic">meta</Emphasis>-topolins in alleviating micropropagation problems

Low shoot multiplication, morphological abnormalities, poor rooting frequency and high cost of production are among the factors challenging the micropropagation of ornamental perennials and garden plants. Most of these problems can be alleviated by using the appropriate type and concentration of plant growth regulator(s) (especially cytokinins) in developing efficient micropropagation protocols. In this study, we investigated the effects of five different aromatic cytokinins (BA, Kin, mT, mTR and MemTR) on adventitious shoot production from shoot-tip explants of B. greenii, a critically endangered plant with horticultural potential. Of all the cytokinin concentrations evaluated, the highest adventitious shoot production (5.88 ± 0.73 shoots/explant) was observed in cultures containing 7 μM MemTR. Low adventitious shoot production, which was not significantly different from that of the control, was observed at all the concentrations of kinetin (Kin), suggesting that it is a weak cytokinin for shoot production in this species. All the treatments with BA alone showed higher adventitious shoot production when compared to the BA treatments supplemented with NAA concentrations. At equimolar concentrations, however, all the BA concentrations had a higher abnormality index than the other cytokinins. It is noteworthy that the abnormality index in all the topolin treatments was much lower than that recorded at the lowest BA concentration. Almost all the abnormality indices recorded with mTR and MemTR concentrations were lower than that of the control. Given that the explants used were from BA-containing cultures, it is likely that the abnormalities recorded using mTR and MemTR were carry-over effects of BA. Culturing under 16 h light/8 h dark conditions resulted in a higher production of adventitious shoots with lengths greater than 10 mm compared to culturing under continuous light. This measure could help reduce the cost of production. Regenerated shoots were successfully rooted and acclimatized with a 65% survival frequency and no observable morphological variation. The developed micropropagation protocol has the potential for producing more than 60,000 transplantable shoots per year from a single shoot-tip explant of this critically endangered species.     

Influence of Different Cytokinins on the Transpiration and Senescence of Excised Oat Leaves

In order to investigate the possibility that cytokinins control transpiration indirectly through affecting leaf senescence, a direct comparison was made of the effect of different cytokinins on transpiration and senescence of oat leaves (Avena sativa L. cv. Forward). Senescence was assessed by measuring chlorophyll loss. The synthetic cytokinins N⁶ benzyladenine (BA) and kinetin delayed senescence and increased transpiration of oat leaves to a greater extent than did the naturally occurring compounds zeatin, N^b-Δ² isopentenyladenine (i⁶ Ade) and 6-ø-hydroxybenzyladenosine (hyd-BA riboside). During the early stages of the transpiration experiment zeatin showed similar or greater activity than BA. This period was longest when freshly excised leaves were used, was reduced when leaves were used after incubation in distilled water in the dark for 20 h and was eliminated by incubation in cytokinin solution in the dark. After this period the activity of zeatin declined relative to BA. The effect of cytokinins in increasing transpiration occurred only in the light; no effect was observed in the dark. BA showed higher activity than zeatin in senescence tests but both cytokinins were less effective as the tests progressed, this decrease in activity being more rapid when older leaves were used. The results are discussed in relation to the mechanisms by which endogenous cytokinins might control sensecence and transpiration in oat leaves and to the value of the oat leaf senscence and transpiration bioassays as tests for cytokinin activity of plant extracts.     

Shoot organogenesis and plant regeneration in <Emphasis Type="Italic">Metabriggsia ovalifolia</Emphasis>

An efficient propagation and regeneration system via direct shoot organogenesis for an endangered species, Metabriggsia ovalifolia, was established. High activity cytokinins [6-benzyladeneine (BA) and thidiazuron (TDZ)] and low activity auxins [α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA)] could directly induce adventitious shoots from leaf or petiole explants within 5 weeks. Cytokinins (TDZ or BA) combined with auxin (NAA) in the induction media induced more adventitious shoots than when auxins or cytokinins were used alone. Adventitious shoots could be induced and also mass-propagated on media containing 2.5–5.0 μM TDZ (or BA) and 0.25–0.5 μM NAA. Adventitious roots differentiated at the proximal end of shoots on rooting media containing half-strength MS salts and 0.5 μM IBA, 0.5 μM NAA, 0.1% activated charcoal or no plant growth regulators. Over 90% of plantlets survived following acclimatization and transfer to a potting mixture (1:1, sand:vermiculite) in basins.     

Cytokinin and explant types influence in vitro plant regeneration of Leopard Orchid (<Emphasis Type="Italic">Ansellia africana</Emphasis> Lindl.)

The effects of explant and cytokinin types on in vitro plant regeneration of Ansellia africana were investigated. The exogenous addition of cytokinins is not required for the proliferation of new protocorms from Trimmed protocorm cluster (TPC) explants. To the contrary, nodal and shoot-tip explants produced a single shoot in response to the addition of cytokinins. Overall plant growth in terms of shoot length, leaf number, frequency of root organogenesis, root length, and fresh weight/plant were significantly higher in media containing meta-Topolin Riboside (mTR) in both nodal and shoot-tip explants. Thidiazuron (TDZ) and 6-benzyladenine (BA) induced stunted and hypertrophied shoots at their highest level (15 μM). In addition root differentiation and root growth were significantly lower on P668 media with TDZ and BA. Zeatin was capable of inducing a significantly higher root organogenesis frequency and root length in TPC explants as compared to other cytokinins. However, TPC explants produced a significantly greater number of longer shoots (>3 cm) on P668 media with mTR. Hyperhydric shoots were produced from TPC explants. The occurrence of hyperhydricity is discussed with respect to the culture vessel used in this study.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Protein,fatty acid,and pigment content of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under different light regimes

The effects of irradiance and photoperiod on growth rates, chlorophyll a, β-carotene, total protein, and fatty acid content of Chlorella vulgaris were determined. The maximum growth rate (1.13 day⁻¹) was at 100 μmol photons m⁻² s⁻¹ and 16:8-h light/dark photoperiod. Chlorophyll a and β-carotene contents significantly differed under different light regimes with chlorophyll a content lower at high irradiance and longer light duration, while β-carotene showed the inverse trend. The total protein and fatty acid content also significantly differed in different light regimes; the maximum percentage of protein (46%) was at 100 μmol photons m⁻² s⁻¹ and 16:8 h photoperiod, and minimum (33%) was at 37.5 μmol photons m⁻² s⁻¹ and 8:16 h photoperiod; the total saturated fatty acids increased, while monounsaturated and polyunsaturated fatty acids decreased with increasing irradiance and light duration.     

Changes in endogenous cytokinin profiles in micropropagated <Emphasis Type="Italic">Harpagophytum procumbens</Emphasis> in relation to shoot-tip necrosis and cytokinin treatments

Changes in cytokinin (CK) profiles and their physiological implications in micropropagated Harpagophytum procumbens [(Burch.) DC. ex Meisn.] tissues in relation to shoot-tip necrosis (STN) and CK treatments were studied. Total CK content was quantified in benzyladenine (BA)-treated necrotic and normal plantlets and in plantlets treated with the CKs BA, meta-topolin (mT) and meta-topolin riboside (mTR) with and without the auxin indole-3-acetic acid (IAA). Generally necrotic shoots yielded more total CK compared to normal shoots. Cytokinin accumulation was higher at the basal section (basal > middle > top). Further analysis of the CKs based on structural and functional forms revealed excessive accumulation of 9-glucosides (deactivation products—toxic metabolites) and limited amounts of O-glucosides (storage forms—re-utilizable) in necrotic and BA-treated shoots compared to normal and topolin-treated cultures. The addition of IAA enhanced the formation of 9-glucosides in BA-treated cultures but reduced it in topolin-treated cultures. The symptom of STN could therefore be attributed to conversion of active cytokinins to other forms such as 9-glucosides which are neither active nor reversibly sequestrated to active forms. Literature shows that metabolites like 9-glucosides of BA have a detrimental effect in plant tissue culture.     

Responses of Two Coastal Algae (Skeletonema costatum and Chlorella vulgaris) to Changes in Light and Iron Levels1

Iron (Fe) is essential for phytoplankton growth and photosynthesis, and is proposed to be an important factor regulating algal blooms under replete major nutrients in coastal environments. Here, Skeletonema costatum, a typical red-tide diatom species, and Chlorella vulgaris, a widely distributed Chlorella, were chosen to examine carbon fixation and Fe uptake by coastal algae under dark and light conditions with different Fe levels. The cellular carbon fixation and intracellular Fe uptake were measured via ¹⁴C and ⁵⁵Fe tracer assay, respectively. Cell growth, cell size, and chlorophyll-α concentration were measured to investigate the algal physiological variation in different treatments. Our results showed that cellular Fe uptake proceeds under dark and the uptake rates were comparable to or even higher than those in the light for both algal species. Fe requirements per unit carbon fixation were also higher in the dark resulting in higher Fe: C ratios. During the experimental period, high Fe addition significantly enhanced cellular carbon fixation and Fe uptake. Compared to C. vulgaris, S. costatum was the common dominant bloom species because of its lower Fe demand but higher Fe uptake rate. This study provides some of the first measurements of Fe quotas in coastal phytoplankton cells, and implies that light and Fe concentrations may influence the phytoplankton community succession when blooms occur in coastal ecosystems.     

Somatic embryogenesis and plant regeneration of <Emphasis Type="Italic">Lilium ledebourii</Emphasis> (Baker) Boiss., an endangered species

A somatic embryogenesis (SE) protocol was established for the regeneration of Lilium ledebourii (Baker) Boiss. whole plants using new vegetative bulblet microscales and transverse thin cell layers (tTCLs) of young bulblet roots as the explant sources. Bulblets were induced from bulb scale explants cultured for at least 3 months in the dark on Murashige and Skoog (MS) medium containing 3% sucrose, 0.8% agar, and different concentrations of α-naphthaleneacetic acid (NAA), 6-benzyladenine (BA), and thidiazuron. Embryo-like structures were obtained from tTCL explants of 3-month-old bulblets (excised from bulb scale explants) following culture on solid MS medium containing 3% sucrose and various concentrations of NAA and BA for 3 months in the dark. Both the explant source and the type of plant growth regulators affected the differentiation of somatic embryos. The highest percentage (65.55%) of embryogenesis was obtained from bulblet microscale tTCLs cultured on solid MS medium containing 0.54 μM NAA and 0.44 μM BA. Plants with normal shoots and roots were obtained following a 3-month culture of embryos on growth regulator-free MS medium at 25 ± 1°C under a 16/8-h light/dark photoperiod (light intensity 40 μmol m⁻² s⁻¹, cool-white fluorescent light). The plants were successfully acclimatized in the growth chamber.     

Biomass and lipid productivities of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> under autotrophic,heterotrophic and mixotrophic growth conditions

Biomass and lipid productivities of Chlorella vulgaris under different growth conditions were investigated. While autotrophic growth did provide higher cellular lipid content (38%), the lipid productivity was much lower compared with those from heterotrophic growth with acetate, glucose, or glycerol. Optimal cell growth (2 g l⁻¹) and lipid productivity (54 mg l⁻¹ day⁻¹) were attained using glucose at 1% (w/v) whereas higher concentrations were inhibitory. Growth of C. vulgaris on glycerol had a similar dose effects as those from glucose. Overall, C. vulgaris is mixotrophic.     

Physiological responses of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> to different nitrogen forms

The responses of carbon and nitrogen metabolisms in the medical plant Catharanthus roseus to the nitrogen solutions (N1, N2 and N3) containing different ratio of nitrate to ammonium (1:0, N1; 1:1, N2; 1:3, N3) were investigated here. The plants in N3 nitrogen solution were strongly inhibited in photosynthetic gas exchange and carbohydrate accumulation, reflecting the toxicity symptom of excess ammonium continuously accumulated in plants. The treatment with N2 nitrogen solution, however, displayed an obviously synergistic effect on plant growth and metabolisms in contrast to nitrate as the sole source. The short-term (7 days) exposure of plants to N2 nitrogen solution resulted in an increased shoot/root ratio, leaf mass ratio, and Pn, as well as the elevated levels of sucrose, glutamate, aspartate, proline and threonine. The plants in N2 nitrogen solution accumulated twofold catharanthine and vinblastine than did the plants in N1 or N3 nitrogen solution after the long-term incubation. Internal nitrate had an increased accumulation in the plants in N2 nitrogen solution compared to the counterparts. The supply of N2 or N3 nitrogen solution to plants for 7 days induced an over tenfold increase of ammonium in leaves as compared to the case using N1 nitrogen solution. The increased ammonium ion promoted the activities of NADH-dependent glutamate dehydrogenase (NADH-GDH) both in the leaf and root of plants. Under the ammonium-containing solution (N2 and N3 nitrogen solutions), there was a significantly increased activity for glutamine synthase (GS) in the root during experiment and for nitrate reductase (NR) in the leaf and root only after 21 days of treatment. The performed correlation analysis revealed a negative relation between soluble sugars and internal ammonium, whereas a positive correlation of alkaloid production with glutamate and aspartate.     

Arabinogalactan proteins are involved in cell aggregation of cell suspension cultures of <Emphasis Type="Italic">Beta vulgaris</Emphasis> L.

Arabinogalactan proteins (AGPs) are glycoproteins present at cell surfaces. Although exact functions of AGPs remain elusive, they are implicated in plant growth and development. The aim of this study was to evaluate the role of AGPs in the process of cell aggregation of Beta vulgaris L. suspension cultures. It was observed that B. vulgaris suspension cultures accumulated AGPs in parallel form to its cell growth. The AGPs maximum content in the stationary phase was 0.330 mg g⁻¹ dry weight (DW) in the cell wall (CW) and 1.534 mg g⁻¹ DW in the culture medium (CM), generating cell aggregates >500 μm (93.21% DW). The addition of tunicamycin (TM) caused a reduction of AGPs content in CW and CM of 46 and 64%, respectively. These changes were associated with inhibition of growth and the reduction of the cell aggregates >500 μm (50.0% DW). When TM was removed from the CM, cell growth, aggregation, and AGPs content on CW and CM were recovered. Precipitation of AGPs with Yariv reagent generated a reduction of 61.14% of AGPs content in CW and a total inhibition of AGPs secretion in CM. This Yariv treatment generated a reduction in the cell aggregates >500 μm of 51.31% of DW. When the Yariv reagent was removed from the culture, cells did not recover their AGPs accumulation. In addition, cell cultures did not recover their ability to grow and aggregate. These results indicate that AGPs are molecules required in the cellular aggregation process of B. vulgaris L. suspension cultures.     

Influence of auxins,cytokinins, and nitrogen on production of rutin from callus and adventitious roots of the white mulberry tree (<Emphasis Type="Italic">Morus</Emphasis><Emphasis Type="Italic">alba</Emphasis> L.)

Rutin is an economically valuable flavone compound with anticancer activity, dietary effects, and anti-aging activity. In this study, callus and adventitious roots were induced from three Morus (mulberry) species. Among the three mulberry species tested for rutin production, roots of the Sugye (M. alba L.) had the highest levels (242.2 μg/g fresh tissue) of rutin. In addition, the mature leaves of this type of tree promoted higher levels of rutin compared to those of young leaves or those undergoing senescence. Adding auxins such as indole-3-acetic acid (IAA), 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) not only enhanced the development of callus and adventitious roots but also increased the protein and rutin contents. In contrast, adding cytokinins such as 6-benzyladenine (BA) and kinetin (KN) retarded callus and adventitious root development as well as the protein and rutin contents. Callus in suspension culture in the presence of IAA produced more rutin than that in the absence of IAA. However, rutin secretion into a medium was greater in the absence of IAA. Different ammonium/nitrate (AM/NI) ratios in a root suspension culture also greatly affected rutin production and its secretion into a liquid medium. As a result, the highest level of rutin was produced when adventitious roots were grown in a 34/66 AM/NI full-strength standard MS medium containing 5 mg/l IAA.     

Adsorption of turbid materials by the cyanobacterium <Emphasis Type="Italic">Phormidium parchydematicum</Emphasis>

The present study investigated the adsorption of turbid materials such as clays, by microalgae. Among six tested microalgae, including Chlorophyceae and Cyanophyceae, a cyanobacterium, Phormidium parchydematicum strain KCTC 10851BP, and unicellular alga, Chlorella vulgaris strain UTEX 265, showed a higher turbidity-removal efficiency (TRE) of 99% and 93%, respectively, for clay-containing water after 24 h, which was much higher than the 36% for the control. The TREs of all the treatments were >95% after 24 h, except for the treatment with a lower algal density and optical density (OD) = 0.1. Phormidium parchydematicum demonstrated a slightly higher TRE than a polyaluminum chloride coagulant (Al₁₃(OH)₂₈Cl₉SO₄) for a turbid field water. Scanning electron microscope (SEM) observations revealed a dense adsorption of clay particles to the surface of P. parchydematicum. Thus, it would appear that P. parchydematicum and C. vulgaris can be used for clay removal in turbid water by sedimentation through microalgae–clay flocculation.     

Micropropagation of calla lily (<Emphasis Type="Italic">Zantedeschia albomaculata</Emphasis>) via <Emphasis Type="Italic">in vitro</Emphasis> shoot tip proliferation

Summary A method for the micropropagation of Zantedeschia albomaculata is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. Of the four cytokinins tested, 6-benzyladenine (BA) and thidiazuron (TDZ) were found to be more effective. An optimal concentration of BA (8.87 μM) or TDZ (4.54 μM) developed an average of 3.8 and 3.2 shoots per explant, respectively, but increasing concentrations of cytokinins often led to lower proliferation rate and stunted growth. Addition of auxins to the MS medium supplemented with 8.87 μM BA slightly enhanced multiple shoot formation in the explants. Multiplication of six cultivars of Zantedeschia genus comprising different flower types and colors were tested and achieved using only one regeneration medium (MS+8.87 μMBA+2.46 μM IBA). Different MS medium strength, air temperature (15, 20, 25, and 30°C) and light quality [fluorescent, red + blue, red and blue light provided by a LED (light-emitting diode) system] were used (without phytohormone) with the aim of stimulating in vitro shoot and root development. Half-strength MS or MS and cultures maintained at 25°C were found to be equally suitable for shoot tip culture of Z. albomaculata. Shoot elongation as well as fresh and dry weight were significantly increased when cultures were kept under red or blue light.     

In vitro propagation of the endangered plant <Emphasis Type="Italic">Centaurea ultreiae</Emphasis>: assessment of genetic stability by cytological studies,flow cytometry and RAPD analysis

The effect of different cytokinins on multiple shoot regeneration from shoots of Centaurea ultreiae was studied. The culture system consisted of solid basal half-strength Murashige and Skoog medium supplemented with one of four cytokinins [6-benzyladenine (BA), zeatin, kinetin, or N⁶-(2-isopentyl) adenine (2-iP)] at each of five different concentrations. The highest multiplication rate (5.52 shoots per explant) was obtained in the medium supplemented with 4.44 μM BA. Shoots were successfully rooted (91% success) by dipping the basal end into a solution containing 10 M 1-naphthaleneacetic acid for 30 s. Genetic stability of the regenerated plants was assessed by random amplified polymorphic DNA (RAPD) analysis and flow cytometry. In the initial randomly selected plant material (control) and 20 of its regenerants, 2,688 bands were generated by RAPD with 12 different primers, and the same banding profiles were exhibited. Molecular and cytological analyses did not reveal genomic alterations in any of the regenerated plants obtained on medium containing 4.44 μM BA. The success of acclimatization to environmental conditions—100% of plants were successfully acclimatized—suggests that the micropropagation system described is a reliable method for propagation of C. ultreiae.     

Cyanobactericidal effect of <Emphasis Type="Italic">Rhodococcus</Emphasis> sp. isolated from eutrophic lake on <Emphasis Type="Italic">Microcystis</Emphasis> sp

A bacterium, which was observed in all cultivations of Microcystis sp., was isolated and designated as Rhodococcus sp. KWR2. The growth of bloom-forming cyanobacteria, including four strains of Microcystis aeruginosa and Anabaena variabilis, was suppressed by up to 75–88% by 2% (v/v) culture broth of KWR2 after 5 days. But KWR2 did not inhibit eukaryotic algae, Chlorella vulgaris and Scenedesmus sp. An extracellular algicidal substance produced by KWR2 showed a cyanobactericidal activity of 94% and was water-soluble with a molecular weight of lower than 8 kDa.