Identification of an agar constituent responsible for hydric control in micropropagation of radiata pine

Tissue cultured Pinus radiata grown on media containing agar as the gelling agent display toxic symptoms and poor long-term shoot survival, however it does have the attribute of hydric control, through a mechanism which, until now, has not been elucidated. Gelrite as an alternative gelling agent is clearly non-toxic but results in hyperhydric (vitrified) tissues. In an effort to overcome these problems, the controlling mechanism found in agar was examined. Hydric control was shown to be effected by a non-gelling, cold-water soluble constituent of a commercial agar, rather than by physical properties of the gel. It could be separated from low molecular weight components of the agar responsible for the toxic symptoms by dialysis. It was identified as being an agaroid-type xylogalactan bearing pyruvate and sulphate substituents. Improved management of gelling agents in culture medium has contributed substantially to a thirty fold increase in propagation rates.     

Seasonal changes in agar characteristics of two populations ofPterocladia capillacea in Gran Canaria,Spain

Agar characteristics ofPterocladia capillacea were examined seasonally at two intertidal populations exposed to different wave energy on the northern rocky shore of Gran Canaria Island. Plants were collected monthly from August 1991 to July 1992. Agar yield, gel strength, melting and gelling temperature and chemical properties such as sulphate and pyruvate content were measured. Percent epiphytism was determined on both populations, together with the changes in biomass as dry weight. Specimens in the sheltered habitat were larger and more epiphytized than ones in the exposed area. There was a clear seasonal change in agar characteristics in both populations. Agar yields decreased in late spring and early summer, although in the sheltered habitat fluctuations were more erratic. Gel strength increased in winter, reaching a maximum in December–February. No significant differences were found in agar yield, gel strength or melting and gelling temperatures, but there was a difference between fresh to dry weight ratio. The role of the exposure degree as a possible environmental factor responsible for this behavior is discussed. Agars ofPterocladia capillacea from Canary Islands show characteristics for industrial use.Author for correspondence     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

微生物固体培养基凝固剂研究进展

明胶是最早使用的固体培养基凝固剂,已逐渐被琼脂所代替。琼脂由于形成凝胶后透明度高、保水性好、无毒、不被微生物液化等优点,逐渐成为最常用的凝胶剂。后来,又发现无机硅胶、瓜尔胶、卡拉胶在某些情况下可用作凝固剂。近年来,兴起一种基于微生物快速检测的快速测试片,其所用凝固剂发展到黄原胶、刺槐豆角、聚丙烯酸系等。但新型凝固剂在使用过程中仍存在许多弊端,因此,从吸水和保水的机理出发对其研究和改性是一项重要的任务。     

Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta)

The effect of season on yield and physical properties of agars extracted from Gracia gracilis and G. bursa-pastoris were determined. The agar yield from G. gracilis was maximum during spring (30%) and minimum during autumn (19%). In G. bursa-pastoris, the agar yield was greatest in summer (36%) and lowest in winter (23%). Agar yield from G. bursa-pastoris was positively correlated with temperature (r=0.94; P<0.01) and salinity (r=0.97; P<0.01) and negatively with nitrogen content (r=−0.93; P<0.01). Agar gel strengths fluctuated from 229 to 828 g cm⁻² and 23 to 168 g cm⁻² for G. gracilis and G. bursa-pastoris, respectively. The gelling temperature showed significant seasonal variation for both species. Chemical analysis of agar from the two seaweeds indicated variation in 3,6-anhydrogalactose and sulfate content (P<0.01). Furthermore, there was an inverse correlation between the two chemical variables. In general, agar extracted from G. gracilis possessed better qualities than agar extracted from G. bursa-pastoris and can be considered a candidate for industrial use.     

Comparison of the rheological and diffusion properties of some gelling agents and blends and their effects on shoot multiplication

The rheological and diffusion properties of blends of agar/guar gum, agar/Phytagel and Phytagel/guar gum were analysed and compared to those properties of agar or Phytagel applied alone at two different gelling concentrations. Moreover, their effects on the shoot multiplication of the apple scion Galaxy and two black locust clones (SF63, SF82) were studied, and their cost benefits over agar were calculated. Elastic hydrogel formation was demonstrated for each blend by rheological measurements, but the gel strength depended on the types and concentrations of the applied gelling agents and blends. Guar gum was able to speed the diffusion in the different blends, and diffusion was independent of gel strength. The rate of shoot multiplication increased (to 8.9 shoots per explant) and the percent of hyperhydrated shoots decreased (to 12%) when the blend of agar/guar gum was used for the shoot multiplication of apple. Similarly, the highest multiplication rates of black locust clones (between 3.9 and 4.1) were obtained on media solidified by blends containing guar gum. The best shoot performance with the lowest percent of hyperhydrated shoots (11–12% in SF63 and 2–23% in SF82) was achieved using agar alone or the agar/guar gum blend. The shoot multiplication was improved of both species and the production cost was reduced by 42% by using the agar/guar gum blend.     

The agar component of the red seaweed Gelidium purpurascens

Chemical composition and rheological properties of agar isolated from Gelidium purpurascens, the agar after alkaline treatment, and a commercial agar are presented. The agar and alkali-treated agar gave weaker gels, as measured with an Instron 1122, than those of commercial agar. Xylose, glucose and glucuronic acid in the agar were removed together with 86% of the nitrogen content on alkaline treatment, indicating occurrence of these residues as carbohydrate-protein complexes. Sequential extraction of the alga accounted for low yields of agar as losses incurred on ethanol precipitation. Acid treatment of the residue from exhaustive aqueous extraction of the alga liberated a further 10% agar with increased gel strengths despite increased glucose inclusion, suggesting a lack of involvement of these ‘contaminant’ carbohydrate-protein residues in helix coil formation during gelling.     

Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis

The red seaweed, Gracilaria lemaneiformis growing as an aquaculture bioremediator along the coasts of Liaodong Peninsula, China, was investigated for the agar production. An eco-friendly method called agar photobleaching extraction process was developed for the benefit of workers' health and safety of the environment. The native agar (NA), alkali-modified agar (AA), chemical-bleached agar (CA) and photobleached agar (PA), which were extracted using different processes, were evaluated for their physical and chemical properties. The PA showed most desirable performances in terms of gel strength, gelling temperature, sulfate content and 3,6-anhydro-l-galactose content. Among the different processed agars, PA gel strength was 1913 g/cm2, the highest among the different processed agars, which increased 8.6% on the basis of the AA. Further we applied this new technique to extract agars from Gracilaria asiatica, and similar results were obtained with that of G. lemaneiformis. This indicates that the agar photobleaching extraction process is a feasible method for Gracilaria species and has a potential application. During the whole agar photobleaching extraction process the pigment content of G. lemaneiformis declined gradually and the TOC concentration in photobleaching solution increased along with the increase in the irradiation time. The mechanism of agar photobleaching could be elucidated by the photolysis theory.     

Agar from the red seaweed,Laurencia flexilis (Ceramiales,Rhodophyta) from northern Philippines

The worldwide production of the gelling agent agar mainly rely on the red algae of the order Gracilariales and Gelidiales for raw material. We investigate here the potential of a species from another red algal order, Ceramiales as an agar source. The agar from Laurencia flexilis collected in northern Philippines was extracted using native and alkali treatment procedures and the properties of the extracts were determined using chemical, spectroscopic and physical methods. The native agar, 26% dry weight basis, forms a gel with moderate gel strength (200 g cm^?2). Alkali‐treatment did not enhance the gel strength, indicating insignificant amounts of galactose‐6‐sulfate residue, the precursor of the gel‐forming 3,6‐anhydrogalactose (3,6‐AG) moieties. Furthermore, the Fourier transform infrared and chemical analysis showed low sulfate and high 3,6‐AG levels, not affected significantly by the alkali treatment. Nuclear magnetic resonance spectroscopic analysis revealed 3‐linked 6‐O‐methyl‐D‐galactose and 4‐linked 3,6‐anhydro‐L‐galactose as the major repeating unit of the native extract, with minor sulfation at 4‐position of the 3‐linked galactose residues. The native and alkali treated agars have comparably high gelling and melting temperatures, whereas the former exhibits higher gel syneresis. Laurencia flexilis could be a good source of agar that possesses physico‐chemical and rheological qualities appropriate for food applications. Due to the inability of alkali treatment to enhance the key gel qualities of the native extract, it is recommended that commercial agar extraction from this seaweed would be done without pursuing this widely‐used industrial procedure.     

Effects of phosphate and nitrate supply on productivity,agar content and physical properties of agar ofGracilaria strain G-16S

Gracilaria strain G-16S was cultured in various phosphorus (P) supply rates with low or high nitrogen (N) supply to determine the effects of nutrient supply on its productivity, agar content and physical properties of the agar. Productivity was reduced after four weeks of growth in zero P supply as plants reached 0.07% P tissue content (critical level), with fragmentation of these plants by six weeks (0.05% P; minimum viable level). Native agar content was higher in low P and high N, or low N conditions. Agar content appeared to increase with decreasing P under high N supply. This increase was not apparent with alkali treatment prior to extraction. Agar gel strength was greatly increased by alkali treatment. The highest gel strengths were obtained under high N supply at all P supply rates except zero P, and under low N supply at 12 M P week^–1. Native agar gel strengths showed a similar pattern on a lower scale. Melting temperatures were higher in agars with higher gel strengths. Dynamic gelling temperatures were generally high for alkali-treated agar, with agar from plants grown in zero P supply showing a slightly elevated gelling temperature. Melting and gelling temperatures of native agars with the highest gel strengths were in the same range as bacteriological agar. These results show that P and N supply affects productivity, agar content and agar physical properties, but the tradeoffs between a slightly higher agar quantity under nutrient limitation and higher agar quality under nutrient-replete conditions seem to favor the latter.     

Differential performance of marubakaido apple rootstock shoots grown in culture media containing different agar brands: dynamic rheological analysis

Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents. In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921 agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921 and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance, i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars.     

Effects of environmental factors on carrageenan fromGymnogongrus griffithsiae (Gigartinales,Rhodophyta)

The effects of environmental factors on substratum coverage, thallus length, production of iota carrageenan and gelling behavior were investigated in field populations of the red algaGymnogongrus griffithsiae (Turner) Martius. Irradiance and water temperature affected thallus substratum coverage and water gel strength of the carrageenan. The significant monthly differences in thallus length, carrageenan content and carrageenan gelling properties were not attributable to seasonal variations in irradiance and water temperature. The effect of temperature and N loading on biomass productivity, carrageenan yield and gelling characteristics were also investigated using cultures. N-enriched cultures showed decreased gel yields, higher viscosities and higher water gel strengths in the carrageenans extracted from these cultures. Milk reactivity tests were correlated with thallus nitrogen and water dessert gel strength, but neither culture temperature nor N loading showed a significant effect. The field and culture studies indicate how temperature, irradiance and N availability affect growth and hydrocolloid production.Author for correspondence     

The influence of gelling agent purity and ion additions on in vitro tomato pollen germinability and pollen tube growth on semi-solid substrates

The physicochemical properties and inherent ion content of the gelling agents used for the preparation of semi-solid substrates significantly affect the germination of tomato pollen in vitro. The addition of Ca, K and Mg to semi-solid, agar-based, substrates improved the germination of tomato pollen when the inherent Ca content of the agar was low, but was without effect or even inhibitory when the Ca level was high. However, Κ and/or Mg addition was beneficial irrespective of the agar source. When agarose replaced agar and K, Mg and Ca were added individually or in combination, pollen germination and pollen tube growth were affected differently by each ion but were optimal only in the presence of all three ions, reflecting the absence of these ions in agarose. An involvement of Na was also implicated since reduction of the inherently high Na content of agar by washing improved germination, with or without the addition of Κ, Mg and Ca. Since >3 mM Ca in the semi-solid substrate impairs tomato pollen germination, the gelling agent must be of high purity, which in the case of agar may entail washing, followed by the addition of K, Mg and Ca. The adoption of such a medium would permit the standardization of semi-solid substrates for in vitro tomato pollen germination studies.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

Rapid and inexpensive recovery method of DNA fragments from agarose and polyacrylamide gels by a cotton-wool column tube.

We developed a rapid, convenient, simple, and inexpensive method for isolating pure DNA from agarose and polyacrylamide gels using cotton wool tubes. DNA fragments ranging in size from 193-23,130 bp can be easily recovered within 2 hours by centrifugation through cotton wool from gel slices. The recovery rate of this method is 35% to 50%, when estimated for isolation of lambda DNA-HindIII fragments. We have also recovered 700-bp polymerase chain reaction (PCR) products using cotton wool tubes from electrophoresis on both a 0.8% agarose gel and a 6% polyacrylamide gel, in which satisfactory yields of more than 50% were obtained. The DNA thus recovered in this way is biologically active and can be used as a substrate for further experimental procedures without additional purification steps.     

Seasonal changes in the chemical and gelling characteristics of agar from Gracilaria verrucosa collected in Turkey

The chemical and gelling properties of agar from G. verrucosa collected from Izmit bay in Turkey at different months of the year were studied. Purification of agar was performed by using amylase treatment and isopropyl alcohol precipitation. The phycocolloid content was between 24.0–43.0% of the algal dry weight and was maximum in summer collected algae. Relative total sulfate and 3,6-anhydrogalactose content in the agar were determined from the ratios of infrared spectroscopy band intensities at 1250/2920 cm^–1 and 930/2920 cm^–1, respectively. 3,6-anhydrogalactose and sulfate contents were the highest in agar from algae collected from June until November and January until July, respectively. The gel strength of native agar were the highest from in autumn collected algae and increased to about 1250 N m^–2 after alkali treatment. Thus, this study demonstrated that G. verrucosa from Turkey produces an agar with optimal chemical and gelling properties after alkali-treatment in fall and winter collected algae and may be used for industrial agar production.     

Optimization of agar extraction from local seaweed species,Gracilaria salicornia in Tanzania

This study aimed to develop agar extraction protocols for Gracilaria salicornia from Tanzania and investigate its physico‐chemical characteristics. A 3³ factorial experimental design was used in the extraction of agar whereby three independent variables of NaOH concentration (10, 20 and 30% w/v), alkali pre‐treatment duration (0.5, 1 and 2 h) and extraction temperatures (115, 120 and 125°C) were used to determine the optimum conditions for production of high‐quality agar. Agar yield, gel strength, sulfate content, gelling and melting temperatures were evaluated as dependent variables. The optimal condition was observed at 30% NaOH concentration, 2 h alkali pre‐treatment duration and 120°C extraction temperature. The yield, gel strength, sulfate content, gelling and melting temperatures of the agar obtained under these conditions were 26.9 ± 0.7%, 510.3 ± 16.2 g cm^?2, 0.29 ± 0.04%, 39.3°C and 88.4°C, respectively. These properties are very close to that of imported commercial agar. It was concluded that the local agar is capable of replacing imported agar for most general purposes. This offers a new possibility of using quality local agar in place of commercial agar.     

Agar yield and quality of Gracilaria chilensis (Gigartinales,Rhodophyta) in tank culture using fish effluents

Tank cultivation of Gracilaria using fish effluents has permitted a production of 48 kg m^–2 yr^–1 and can reduce the dissolved nitrogen loads in the seawater. We report the yield, gel strength, gelling and melting point of agar from Gracilaria cultivated in tanks with seawater previously utilized in intensive, land-based salmon cultures and compared to a control using directly pumped seawater, over a study period of 22 months. The results show that the highest agar yield (20 to 22%) was obtained when Gracilaria was cultivated with pure seawater as compared to the fish effluents. The gel strength, gelling and melting point were higher in the agar obtained from algae cultured with fish effluents. During the spring, the gel strength, gelling and melting point increased in tanks with fish effluents and decreased in tanks with a supply of pure seawater.     

Evaluation of kappa carrageenan as a substitute for agar in microbiological media

Seventy-one samples of the colloid kappacarrageenan extracted from 12 seaweed species were subjected to a number of standard physical demands of solid bacteriological culture media. All samples had a lower melting temperature (less than 67° C) than agar and a gelling (setting) temperature between 16° C and 51° C, some the same and others lower or higher than agar. Temperature spreads were narrow (ca 10° C) to broad (ca 30° C), depending on the seaweed source, but none were as broad as that of agar (ca 40° C). The majority of commercially prepared samples held a slant when incubated at 37° C, but California seaweed colloids were best at 28° C in this test. The majority of samples released little to no water of syneresis in slant tests as well as in plates. Some plates prepared with the colloid were crystal clear as compared to agar plates. All test microorganisms grew as well on kappa-carrageenan media as on agar media. Some media responses could be attributable to the seaweed species, but others could be traced to chemical extraction methods and modification of the colloid.     

Influence of the medium-solidifying agent, the nutrient, and the genotype on the production of gametangia by Phytophthora ramorum in vitro

The effect of different parameters, including the type of nutrients, the quality of the gelling agent, and the genotype of the strain, were evaluated in the production of gametangia by Phytophthora ramorum in vitro. By comparing different agar sources on a carrot-based medium, a delay or a failure in the production of oospores was observed in pairings carried out on media supplemented with technical agar. In contrast, oospores were produced on other agar types, the production on media supplemented with agarose being slightly higher. The formation of gametangia was also influenced by the genotype of the strains involved in the pairing. A European A1 strain producing very few chlamydospores was found to be a better mating partner than other A1 strains. Using a carrot-agarose medium and selected genotypes, all European isolates were characterized in terms of mating type. A macroscopic experiment highlighted a particular spatial distribution of P. ramorum oospores in vitro. A method using polycarbonate membrane was evaluated to assess the selfing ability of P. ramorum.