In vitro propagation ofMedicago andLotus species by node culture

Summary Nodes ofMedicago sativa, Lotus corniculatus, Lotus tenuis, andLotus pedunculatus were cultured on MS basal media with different growth regulators. InM. sativa each node produced one shoot and the apical dominance was unaffected by high levels of cytokinins, and subsequent cycles of culture. Shoot development was stimulated by the presence ofN ⁶-isopentenyl-adenine in the culture medium and was dependent on the genotype of the explant. Shoot development was not affected by the original position of the node on the plant nor by the plant age. Shoots rooted in MS medium gelled with starch and containing 2 mg·liter⁻¹ indol-3-acetic acid. In the threeLotus species, node culture was a more effective technique than inM. sativa. The number of shoots per node increased with the time of culture and with the presence of 0.05 mg·liter⁻¹ of 6-benzylaminopurine. The highest number of shoots derived from one node was achieved inL. pedunculatus and inL. tenuis by culturing single nodes, whereas inL. corniculatus stem segments had to be totally covered by the medium for success. Rooting was easily achieved in MS medium with or without auxins.     

Influence of ventilation closure, gelling agent and explant type on shoot bud proliferation and hyperhydricity in Scrophularia yoshimurae—A medicinal plant

Summary We report an improved procedure of in vitro propagation of Scrophularia yoshimurae—a medicinally important plant species indigenous to Taiwan. Induction of maximum shoot buds (22.75 per explant) was obtained with shoot tip explant cultured on Murashige and Skoog medium supplemented with 1.0mgl^−1 benzyladenine (BA) and 0.2mgl^−1 α-naphthaleneacetic acid and gelrite using dispense paper (DP) for ventilation closure of culture vessels. The type of gelling agents (agar and Gelrite) affected both quantity and quality of the shoots induced. Using aluminum foil for ventilation closure resulted in a higher number of hyperhydric shoots. Hyperhydricity was reduced by culturing shoots on a medium devoid of plant growth regulators in conjunction with the use of DP. Plantlet growth in vessels using DP was healthier and all plantlets survived after being transplanted to soil.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and bulblet growth from lily bulbscale explants as affected by retardants,sucrose and irradiance

Bulbscales of oriental lily hybrid Star Gazer were used as the explants. Bulblets were formed on the basal portion of the excised bulbscales on MS medium supplemented with growth retardants, different sucrose concentrations and exposed to continuous light or dark. ^{Alar, Cycocel} and Paclobutrazol in concentration 1 mg dm⁻³ produced higher number of bulblets as compared to the control. The number of bulblets, however, decreased with the increase in concentration of the growth retardants. The number of bulblets was higher at 90 than at 60 g dm⁻³ sucrose and when the bulbscales were exposed to continuous light than to darkness. The growth retardants, higher sucrose concentration and continuous dark stimulated fresh mass of bulblets. The number of bulblets having roots and leaves decreased in medium with Alar, Cycocel and Paclobutrazol as compared to the control. A few bulblets produced roots and leaves in medium with 90 g dm⁻³ sucrose and none of the regenerated bulblets produced leaves under continuous dark.     

Growth studies on xenic cultures of Entamoeba gingivalis using established media

Wantland's egg medium, modified Shaffer-Frye (MSF) medium and Tryptose-Trypticase-Yeast Extract-Serum-Blood (TTY-SB) medium were compared with variations of the latter two media for their ability to support xenic growth of Entamoeba gingivalis. Wantland's egg medium was unsuitable for growth of E. gingivalis. Accompanying bacteria became resistant to penicillin and streptomycin, overwhelming the amoeba culture. MSF medium was also unsuitable for the cultivation of E. gingivalis. Bacterial growth was heavy and protozoan growth sparse. MSF medium without mercaptosuccinic acid, but with rice starch, dextran or levan substituted for glucose and with Yersinia enterocolitica added, supported limited growth of the amoeba. Unmodified TTY-SB medium did not sustain growth of E. gingivalis. However, when rice starch suspension was substituted for glucose, l-cysteine HCl was deleted, and a Crithidia sp. was added to the E. gingivalis culture grown xenically, enhanced growth of the oral amoeba resulted in this modified TTY-SB medium. E. gingivalis is very sensitive to changes in incubation temperature. Optimum growth was found to be in the narrow range from 34.5 to 35°C for all media tested.     

A simple method for mass propagation of Spathiphyllum cannifolium using an airlift bioreactor

Summary A method for the micropropagation of Spathiphyllum cannifolium is presented using shoot tip proliferation onto Murashige and Skoog (MS) medium supplemented with different plant growth regulator concentrations and combinations. The proliferation responses were significantly influenced by the cytokinin type and concentrations. Supplementation of the medium with benzyladenine (BA; 4.44–13.32 μM) increased the shoot proliferation rate significantly as compared to other treatments. When cytokinins were used with auxin (indole-3-butyric acid, IBA and naphthalene acetic acid. NAA), the number of shoots per explant increased in comparison with treatments with BA alone. The largest number of shoots, 9.3 per explant, was obtained with 13.32 μM BA and 4.9 μM IBA. Different MS medium strengths and sucrose concentrations were used with the aim to stimulate in vitro shoot proliferation. Full MS medium with 30 gl⁻¹ sucrose was found to be suitable for shoot tip culture of Spathiphyllum. Comparative studies between gelled medium and bioreactor culture [continuous immersion (with or without net) and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were more efficient in continuous immersion (with net) bioreactor with low cytokinin-supplemented media. Plantlets from the bioreactor were cultured hydroponically for 30 d and 100% of plants were rooted and acelimatized successfully. Rapid and efficient multiplication rate in bioreactor and successful transfer to greenhouse makes this protocol suitable for large-scale multiplication of this important foliage plant.     

The effect of phosphate concentration on growth and agar content of Gelidium robustum (Gelidiaceae,Rhodophyta) in culture

Phosphate concentration of the growth medium was found to affect the growth rate and agar yield of a clone of Gelidium robustum grown in the laboratory. To study differences in growth we used phosphate concentrations from 0 to 200 µM. To determine the effect of phosphate on agar yield and its properties we used concentrations from 0 to 20 µM. Growth rates generally increased with increasing phosphate concentration, with the highest growth rate (21% d^–1) obtained at 150 µM. Agar yield as percentage of fresh weight was highest (10%) in the algae grown with low phosphate concentrations, but agar yield as percentage of dry weight was highest(43%) at 20 µM of phosphate. Gel strength increased with phosphate concentration with a maximum of 160 g m^–2 for 0.75% gels for the cultures at 20 µM. Melting and gelling temperatures of the gels were also affected by phosphate concentration of the growth medium. Starch yield was highest in algae grown in low phosphate concentrations.     

Growth yield and energy generation in anaerobically-grown Campylobacter spec.

An anaerobic continuous culture study was made with Campylobacter spec. to determine growth yields under various growth conditions. The growth media contained 0.1% (w/v) yeast extract as carbon source. When grown in an aspartate-limited culture Y _asp ^max was 4.6. Inclusion of formate in the culture medium hardly affected the true growth yield. The number of ATP equivalents generated in the fumaratereductase system was 0.66 and the Y _ATP ^max was 7.0. In the nitrate reduction with formate 1.7 ATP equivalents were generated, and a YNO _3- ^max of 12.2 was observed. The true growth yield obtained with a mixture of lactate and aspartate was lower than that found with aspartate alone.     

Novel thermoactive glucoamylases from the thermoacidophilic Archaea Thermoplasma acidophilum,Picrophilus torridus and Picrophilus oshimae

The thermoacidophilic Archaea Thermoplasma acidophilum (optimal growth at 60 °C and pH 1–2), Picrophilus torridus and Picrophilus oshimae (optimal growth at 60 °C and pH 0.7) were able to utilize starch as sole carbon source. During growth these microorganisms secreted heat and acid-stable glucoamylases into the culture fluid. Applying SDS gel electrophoresis activity bands were detected with appearent molecular mass (Mw) of 141.0, 95.0 kDa for T. acidophilum, 133.0, 90.0 kDa for P. torridus and 140.0, 85.0 kDa for P. oshimae. The purified enzymes were incubated with various polymeric substrates such as starch, pullulan, panose and isomaltose. The product pattern, analyzed by HPLC, showed that in all cases glucose was formed as the sole product of hydrolysis. The purified glucoamylases were optimally active at pH 2.0 and 90 °C and have an isoelectric points (pI) between 4.5 and 4.8. Enzymatic activity was detected even at pH 1.0 and 100 °C. The glucoamylases were thermostable at elevated temperature with a half-life of 24 h at 90 °C for both P. torridus and T. acidophilum, and 20 h at 90 °C for P. oshimae. The enzyme system of T. acidophilum has a lower K _m value for soluble starch (1.06 mg/ml) than the enzymes from P. oshimae and P. torridus (4.35 mg/ml and 2.5 mg/ml), respectively. Enzyme activity was not affected by Na⁺, Mg⁺⁺, Ca⁺⁺, Ni⁺⁺, Zn⁺⁺, Fe⁺⁺, EDTA and DTT. This revised version was published online in August 2006 with corrections to the Cover Date.     

Selective recovery of Zn2+ from waste slag from a metal-processing plant by the microscopic fungus Verticillium marquandii

The successful use of hay extract medium for growth of Verticillium marquandii in the presence of toxic black slag, resulted in selective and efficient Zn²⁺ recovery. An alkaline black waste slag originating from short rotary furnace, contained Zn²⁺ and Pb²⁺ (20% and 3.5%, respectively) in a form of compounds only slightly soluble in water. After 90 h of the culturing in bioreactor on hay extract medium with starch (50 g l^–1) and black waste (5 g l^–1) supplements, 80% of zinc and 0.5% of lead were bound by the mycelium. The pH value of the culture medium played a crucial role for limitation of the waste inhibitory effect on mycelium growth and for heavy metal recovery by the fungus.     

Influence of nitrogen availability on agar-polysaccharides fromGracilaria verrucosa strain G-16: structural analysis by NMR spectroscopy

The influence of nitrogen availability on the chemical structure of agar polymers isolated fromGracilaria verrucosa strain G-16 was ascertained by sequential solvent extraction and carbon 13 nuclear magnetic resonance (¹³C NMR) spectroscopy. Agar isolated fromG. verrucosa strain G-16 cultured under nitrogen limited conditions showed large non-polar components but produced spectra indicative of only minor amounts of methylation. These agars also produced spectra suggesting the presence of floridean starch. The nitrogen supplemented cultures ofG. verrucosa strain G-16 produced agars of a more polar nature (hot-water soluble) and contained little detectable starch. The data suggest that the higher gelling temperatures of agar from nitrogen limited plants is not due to markedly higher methylation. These data also suggest that nitrogen effects on agar content may not be as significant as previously thought.     

Competitive growth of slow growingRhizobium japonicum against fast growingEnterobacter andPseudomonas species at low concentrations of succinate and other substrates in dialysis culture

A cultivation system with simultaneous growth of six bacterial cultures in separate bags in dialysis culture was developed. In a medium with no added carbon source (one half concentrated Hoagland solution, water deionized and distilled), cell number ofRhizobium japonicum increased during a 7 day period by a factor of 35, whereas the number ofEnterobacter aerogenes cells decreased to one half. With a concentration of 100 nM succinate as an additional carbon source in the inflow,Rhizobium japonicum 61-A-101 cell number increased by a factor of 50 during an 8 day period, whereas cell number ofEnterobacter cloacae NCTC 10005 only doubled and ofEnterobacter aerogenes NCTC 10006 decreased. At 10 mM concentration of succinate in the inflow, doubling time the twoEnterobacter strains was about 12 h, compared to about 24 h for theRhizobium japonicum strain. Varying the succinate concentration from 10 mM to 100 nM in the inflow,Rhizobium japonicum 61-A-101 surpassed theEnterobacter aerogenes strains in the growth rate between 1 mM and 100 M succinate in the inflowing medium. Three otherRhizobium japonicum strains (fix⁺ and fix^-) did grow with a similar rate as strain 61-A-101 at very low concentrations of substrate. Growth rates for the strains were confirmed by protein data per culture. Growing in competition with twoPseudomonas strains,Rhizobium japonicum RH 31 Marburg (fix^-) did overgrow alsoPseudomonas fluorescens, was however outgrown byPseudomonas putida. In utilizing low concentrations of a¹⁴C labelled organic acid (malonate), three strains ofRhizobium japonicum left 2–4 times smaller amounts of¹⁴C in the medium than two species ofPseudomonas and two species ofArthrobacter.On sabbatical leave at ANU     

Industrial wastewaters and dewatered sludge: rich nutrient source for production and formulation of biocontrol agent, <Emphasis Type="Italic">Trichoderma viride</Emphasis>

Axenic cultivation of biocontrol fungus Trichoderma viride was conducted on a synthetic medium and different wastewaters and wastewater sludges in shake flasks to search for a suitable raw material resulting in higher biocontrol activity. Soluble starch based synthetic medium, dewatered municipal sludge, cheese industry wastewater sludge, pre-treated and untreated pulp and paper industry wastewater and slaughter house wastewater (SHW) were tested for T. viride conidia and protease enzyme production. The maximum conidia production followed the order, soluble starch medium (>10⁹ c.f.u./mL), untreated pulp and paper industry wastewater (4.9 × 10⁷ c.f.u./mL) > cheese industry wastewater (1.88 × 10⁷ c.f.u./mL) ≈ SHW (1.63 × 10⁷ c.f.u./mL) > dewatered municipal sludge (3.5 × 10⁶ c.f.u./mL) > pre-treated pulp and paper industry wastewater (1.55 × 10⁶ c.f.u./mL). The protease activity of T. viride was particularly higher in slaughterhouse wastewater (2.14 IU/mL) and dewatered municipal sludge (1.94 IU/mL). The entomotoxicity of soluble starch based synthetic medium was lower (≈6090 SBU/μL) in contrast to other raw materials. The entomotoxicity inversely decreased with carbon to nitrogen ratio in the growth medium and the conidia concentration and protease activity also contributed to the entomotoxicity. The residual c.f.u./g formulation of T. viride conidia were up to approximately, 90% after 1 month at 4 ± 1 °C and about 70% after 6 months at 25 ± 1 °C. Thus, production of T. viride conidia would help in marketability of low cost biopesticide from the sludge and safe reduction of pollution load.     

Low-cost alternatives for the micropropagation of banana

A 90% resource cost reduction in tissue culture of banana was achieved by replacing tissue culture grade sucrose and Gelrite in the medium with locally available commercial sugar and a starch/Gelrite mixture and by using sun light instead of artificial light. The micropropagation of Musa `Grande Naine' by shoot tip culture was used as model. Thirteen commercial sugars from different countries were tested. Best results were achieved using white and light brown sugars with low electrical conductivity. Sugars of cane or sugar beet origin were suitable. Starches of corn or potato could partially substitute for Gelrite and agar. In all experiments, micropropagation rates under natural light conditions were equal to or higher than under the controlled conditions of a growth room with PPFD of 65 μmol m⁻² s⁻¹ and a 16-h photoperiod. Plants were exposed to average PPFD levels of 58–96 μmol m⁻² s⁻¹ and photoperiods ranged from 8–16 hours. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of Ptilotus plant tissue on pH of in vitro media

Changes occur in the pH of in vitro nutrient media during preparation and over the culture period. The direction and extent of the changes depend upon the initial pH and the presence or type of gelling agent. Agar-based medium was progressively acidified in the presence of a living Ptilotus exaltatus explant. This was not a response to wounding and the rate of acidification could be maintained by frequent replacement of the medium. The rate appears to be pH dependent with an equilibrium occurring at pH4. The pH effect was not accounted for by K⁺ uptake.     

葡萄糖对小球藻(Chloralla sp. HN08)光合作用和生长的影响

【目的】探讨葡萄糖作为外加碳源对热带海洋小球藻(Chloralla sp.HN08)生物质生产和脂、光合色素、碳水化合物及可溶性蛋白等细胞主要成份含量的影响。【方法】分析比较小球藻HN08在光合自养和兼养(添加10 g/L葡萄糖)2种营养方式下的生长速率、细胞密度、光合放氧速率、油脂相对含量,以及可溶性总糖、淀粉和可溶性蛋白的含量。【结果】结果表明,在光照条件下葡萄糖(10 g/L)能促进小球藻(Chloralla sp.HN08)生长,提高细胞终密度,而异养条件下藻细胞逐渐衰亡。兼养条件下,细胞相对生长速率及细胞终密度分别是自养条件下的6.8倍和1.3倍。兼养藻细胞中可溶性糖、淀粉、油脂含量显著高于(P0.05)光合自养细胞,然而可溶性蛋白质和光合色素含量显著低于(P0.05)光合自养细胞。添加葡萄糖的小球藻液的光饱和点和呼吸速率均高于光自养条件下的细胞,但2种培养条件下藻液的净光合速率无显著差异(P0.05)。【结论】光照条件下,添加葡萄糖可显著提高小球藻HN08相对生长速率和细胞终密度,促进油脂与淀粉的积累。     

Establishment of in vitro plants, cell and tissue cultures from Oldenlandia affinis for the production of cyclic peptides

In vitro cultured plants from Oldenlandia affinis were established from seeds and grown on a hormone-free medium. In vitro plants produced the cyclic peptide kalata B1 in concentrations of 0.67 mg g⁻¹ dry weight after growth of 30 days. This was approximately 50% of the concentration analysed in green house plants (shoot tips), where different concentrations have been determined in leaves (1.82 mg g⁻¹), shoot tips (1.36 mg g⁻¹), stems (0.36 mg g⁻¹), and in flowers (0.16 mg g⁻¹). Callus and cell suspension cultures could be initiated from aseptic root, stem and leaf explants of O. affinis seedlings and plants. Different light intensities were shown to affect culture growth as well as chlorophyll synthesis. The friable callus was then used for the establishment of a cell suspension culture. Fresh and dry weight measurements showed that growth was optimal on MS medium supplemented with 0.4 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-d). Leaf suspensions cultured on this medium showed a 4-fold increase of biomass by the first week of incubation. No quantifiable amounts of kalata B1 were produced under these conditions. Morphological differentiation seems to be essential for cyclic peptide production. Therefore, several undifferentiated as well as organised cell lines of O. affinis have been developed. These cell lines will constitute a worthwhile starting point for the optimisation of kalata B1 synthesis in liquid media to the objective of producing cyclic peptides under controlled and defined conditions in bioreactors.     

Starch processing wastewater as a new medium for production of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Production of Bacillus thuringiensis (Bt) based bioinsecticide was studied by using starch processing wastewater (SPW) as a raw material. Results indicated that the nutrients contained in SPW were sufficient for growth, sporulation and δ-endotoxin production of Bacillus thuringiensis subsp. kurstaki (Btk). The final cell counts and spore counts achieved in SPW medium were 72% and 107% respectively higher than those in the soybean meal based commercial medium. Higher δ-endotoxin yield of 2.67 mg mL⁻¹ and higher entomotoxicity of 1,050 IU μL⁻¹ were also obtained in SPW medium as compared with the commercial medium at the end of fermentation. The morphological observations also revealed that the fermentation cycle of Btk could be shortened in this new medium. This process provides solutions for safe SPW disposal and production of high potency and low cost bioinsecticide.     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

Cassava as a Cheap Source of Carbon for Rhizobial Inoculant Production using an Amylase-producing Fungus and a Glycerol-producing Yeast

Summary The aim of this research was to develop methods to use low-cost carbon compounds for rhizobial inoculant production. Five raw starch materials; steamed cassava, sticky rice, fresh corn, dry corn and sorghum were tested for sugar production by an amylase-producing fungus. Streamed cassava produced the highest amount of reducing sugar after fermentation. Bradyrhizobium japonicum USDA110, Azorhizobium caulinodans IRBG23, Rhizobium phaseoli TAL1383, Sinorhizobium fredii HH103, and Mesorhizobium ciceri USDA2429 were tested on minimal medium supplemented with reducing sugar obtained from cassava fermentation. All strains, except B. japonicum USDA110, could grow in medium containing cassava sugar derived from 100 g steamed cassava per litre, and the growth rates for these strains were similar to those in medium containing 0.5 (w/v) mannitol. The sugar derived from steamed cassava was further used for production of glycerol using yeast. After 1 day of yeast fermentation, the culture containing glycerol and heat-killed yeast cells, was used to formulate media for culturing bradyrhizobia. A formulation medium, FM4, with a glycerol concentration of 0.6 g/l and yeast cells (OD₆₀₀ = 0.1) supported growth of B. japonicum USDA110 up to 3.61 × 10⁹ c.f.u./ml in 7 days. These results demonstrate that steamed cassava could be used to provide cheap and effective carbon sources for rhizobial inoculant production.     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.