Gelling agent influences the detrimental effect of kanamycin on adventitious budding in flax

Flax (Linum usitatissimum L.) hypocotyls were cultivated on regeneration media containing various concentrations of kanamycin (an aminoglycoside antibiotic commonly used to select transgenic plant material) solidified with three different gelling agents: gellan gum, agar and a mixture of both. The inhibitory effect of kanamycin on bud regeneration was analyzed. A significant interaction was observed between the nature of the gelling agent and the kanamycin concentration. The antibiotic concentration needed to strongly inhibit bud production varied greatly with the nature of the gelling agent. Gellan gum lowered the inhibitory effect of kanamycin. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of gelling agent on growth and development ofCeratozamia hildae somatic embryos

Individual somatic proembryos ofCeratozamia hildae were exposed to media that differed only in gelling agent utilized. Five different gelling agents were compared in the first experiment: Bacto agar, Agargel, Gel-Gro, Phytagar, and TC agar. In addition, the effect of agar was examined at two levels. Growth, proliferation, and development were assessed. The lower level of agar did not support good somatic proembryo growth. Agargel and the high agar concentration produced cultures with good proliferation. Proembryos exposed to Phytagar, Gel-Gro, and TC agar had the highest proliferation rates. Overall, Gel-Gro was considered the best gelling agent tested. The three concentrations of Gel-Gro used in the second experiment were 2, 4, and 6 g·1⁻, with the lowest concentration representing the control, the recommended concentration. As gelling agent concentration increased, so did mortality; however, the highest Gel-Gro concentration also produced the highest numbers of good-quality, mature somatic embryos. Proliferation rate was greatest at the lowest concentration. These results suggest thatCeratozamia cultures should be exposed to different gelling agents or concentrations of gelling agents at different developmental stages in order to produce the greatest number and highest quality of somatic embryos.     

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow‐growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet‐to‐be cultured microorganisms.     

Control of hyperhydricity in micropropagated apricot cultivars

Summary The effects of different factors on the control and reversion of hyperhydricity during the in vitro propagation of Prunus armeniaca were studied. Treatments that decreased the hyperhydricity but did not affect micropropagation rates were the use of the bottom cooling system for 1 or 2 wk and agargel as gelling agent in ‘Helena’, whereas the best results were obtained with the bottom cooling system for 2 wk and the use of 0.8% agar as gelling agent in ‘Lorna’. Hyperhydric shoots reverted to normal after keeping them for 3 wk in the bottom cooling system.     

Effect of gelling agents and antibiotics on adventitious bud regeneration from In vitro leaves of pear

Summary The effect of the type of gelling agent and of several antibiotics on the adventitious bud regeneration from in vitro leaves was tested on eight pear genotypes. The use of gellan gum (Phytagel™) in the medium instead of agar had a very strong positive effect on the rate of adventitious bud regeneration for all pear genotypes tested in this study. This gelling agent induced faster cell divisions than agar, thus more callus was produced on wound sites and subsequently more buds regenerated. Incubation on gellan gum medium during the first 20 d of bud induction was sufficient to induce a stimulatory effect on regeneration and limited the production of hyperhydric buds. In the prospect of Agrobacterium transformation, the effect of several antibiotics was tested. Cefotaxime (200 mg/l) plus ticarcillin/clavulanic acid (100 mg/l) could be used in the culture medium without affecting the frequency of bud regeneration. The inhibition of bud regeneration was obtained with different kanamycin concentrations according to the gelling agent in the medium. On gellan gum medium, a concentration of 100 mg/l of kanamycin was suitable. These conditions can be recommended for experiments on Agrobacterium-mediated transformation of pear, where bacterial inoculation and presence of antibiotics generally reduce and delay bud regeneration.     

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.     

Low-cost media for <Emphasis Type="Italic">in vitro</Emphasis> conservation of turmeric (<Emphasis Type="Italic">Curcuma longa L.</Emphasis>) and genetic stability assessment using RAPD markers

Up to 73% decrease in cost of media for plant regeneration and in vitro conservation was achieved in Curcuma longa cv Prathibha by using inexpensive carbon source and gelling agent. Laboratory reagent-grade sucrose was replaced by locally available commercial sugar (market sugar or sugar cubes) as carbon source and bacteriological grade agar by isabgol (also named isubgol) as gelling agent. No adverse effects on shoot regeneration and conservation on isabgol-gelled low-cost media were observed as compared to that on agar-gelled control medium (CM). Some 33–56% cultures of C. longa survived up to 12 mo. on isabgol-gelled medium in comparison to only 16% on CM. Genetic stability of 12-month-old in vitro-conserved plants was assessed using 25 random amplified polymorphic DNA (RAPD) primers; no significant variation was observed in RAPD profiles of mother plants and in vitro-conserved plantlets on CM and low-cost media.     

Sago: An Alternative Cheap Gelling Agent for Potato In Vitro Culture

Sago, a processed (gelatinized) edible starch, was successfully used as a gelling agent in culture medium. The efficacy of sago-gelled (80 g dm^–3) medium was studied in ten potato (Solanum tuberosum L.) genotypes during micropropagation and minimal growth conservation. Sago starch provided a firm gelling surface throughout the entire culture period, and fostered optimum plantlet growth in terms of shoot height, number of nodes per plant, number of leaves and fresh mass. No softening of the sago-gelled medium occurred over prolonged (six months) storage. The study showed that sago starch could be used as a substitute to agar in culture medium to substantially reduce the medium cost.     

Use of low-cost gelling agents and support matrices for industrial scale plant tissue culture

The efficacies of sago (from Metroxylon sagu Rottb.) and isubgol (from Plantago ovata Forsk.) as gelling agents and those of filter paper, nylon cloth, polystyrene foam and glass wool cloth as support matrices have been tested for the propagation of plantlets of chrysanthemum (Dendranthema grandiflora Tzvelev). The performances of these low-cost gelling agents and matrices were found satisfactory and could compare well with that of agar. Glass wool cloth was, however, found to be the best matrix. Comparative cost estimations of the matrices and gelling agents have been presented and their merits and/or demerits have been discussed. For a given quantity of a medium, sago and isubgol cost about 1/18th and 1/10th respectively, compared to agar (e.g. Sigma, purified agar, No. A. 7921). The corresponding costs of the matrices are also less than that of agar. The results showed the potential of the substitutes for economic commercial application, replacing the costliest, though not indispensable, gelling agent agar.Abbreviations MS Murashige and Skoog medium (1962) - IAA indole-3-acetic acid - BA 6-benzyladenine - SEM scanning electron microscopy - _m matric potential - osmotic potential     

Micropropagation of ‘Durondeau’ pear in modified-gelled medium

Summary The influence of partial substitution of agar by galactomannans (GMs) in culture media was studied in pear (Pyrus communis L. cv. ‘Durondeau’) micropropagation. GMs. extracted from seeds of Cassia fastuosa (cassia) or Cyamopsis tetragonolobus (guar gum, a commercial GM), were mixed in equal proportions with agar to a final concentration of 0.3% (w/v) for each type of gelling agent. The production of multiple shoots and the formation of roots from shoots were compared with the control solidified with agar alone at a concentration of 0.6% (w/v). In the media solidified with the mixtures of agar/guar and agar/cassia GMs, an, increase of 32 and 17%, respectively, was obtained in the number of regenerated shoots. The modified media promoted a higher number of roots and increased the rooting percentage. A maximum of 91% rooting was obtained in the medium solidified with the agar/cassia GM and containing 9.80 μM indole-3-butyric acid. Less callus formation at the base of the shoot was also observed on this medium. The improved in vitro performance of shoot formation and rooting, combined with a significantly lower cost, suggests a potential use of agar/GM gels in plant tissue culture.     

Guar gum as a gelling agent for plant tissue culture media

Summary Guar gum, a galactomannan derived from the endosperms of Cyamposis tetragonoloba, has been successfully used as a sole gelling agent for plant tissue culture media. Its suitability as a gelling agent was demonstrated by using guar gumgelled media for in vitro seed germination of Linum usitatissimum and Brassica juncea, in vitro axillary shoot proliferation in nodal explants of Crataeva nurvala, rooting of regenerated shoots of the same, in vitro androgenesis in anther cultures of Nicotiana tabacum, and somatic embryogenesis in callus cultures of Calliandra tweedii. The media used for these were gelled with either guar gum (2, 3, or 4%) or agar (0.9%). Guar gum-gelled media, like agar media, supported all these morphogenic responses. Rather, axillary shoot proliferation, rhizogenic and embryogenic responses were better on guar gum-gelled media than on agar media.     

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.     

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.     

Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents

The process of hyperhydricity in tissue cultured plants of Aloe polyphylla is affected by both applied cytokinins (CKs) and the type of gelling agent used to solidify the medium. Shoots were grown on media with agar or gelrite and supplemented with different concentrations of N⁶-benzyladenine (BA) or zeatin (0, 5 and 15 μM). Endogenous CKs were measured in in vitro regenerants after an 8-weeks cycle to examine whether the hyperhydricity-inducing effect of exogenous CKs and gelling agents is associated with changes in the endogenous CK content. On media with agar a reduction in hyperhydricity occurred, while the gelrite treatment produced both normal and hyperhydric shoots (HS). The content of endogenous CKs, determined by HPLC-mass spectrometry, in the shoots grown on CK-free media comprised isopentenyladenine-, trans-zeatin- and cis-zeatin-type CKs. The application of exogenous CKs resulted in an increase in the CK content of the shoots. Following application of zeatin, dihydrozeatin-type CKs were also detected in the newly-formed shoots. Application of BA to the media led to a transition from isoprenoid CKs to aromatic CKs in the shoots. Shoots grown on gelrite media contained higher levels of endogenous CKs compared to those on agar media. Total CK content of HS was higher than that of normal shoots grown on the same medium. We suggest that the ability of exogenous CKs and gelrite to induce hyperhydricity in shoots of Aloe polyphylla is at least partially due to up-regulation of endogenous CK levels. However, hyperhydricity is a multifactor process in which different factors intervene.     

Sodium polyacrylamide polyacrylate, a gelling agent in diets for cabbage looper, omnivorous looper and western avocado leafroller

Ingredient substitutions for agar or carrageenan in artificial diets for lepidopterous larvae were evaluated. A sodium polyacrylamide polyacrylate gelling agent, Water-Lock G-400, was compared with agar for solidifying diets of the cabbage looper, Trichoplusia ni (Hübner), omnivorous looper, Sabulodes aegrotata (Gueneé), and the western avocado leafroller, Amorbia cuneana Walsingham. Water-Lock G-400 is considerably less expensive than agar or carrageenan, it does not require heating to gel, and it is made from a dependable supply of agricultural starch. Sabulodes aegrotata pupae from larvae reared on agar were significantly heavier than those reared on Water-Lock G-400. However, the remainder of the variables observed (larval survival, developmental times, fecundity, sex ratio and pupal weights for A. cuneana and T. ni) were all independent of diet. Decreasing the total amount of ingredients in a Water-Lock-G-400 diet produced fewer and smaller T. ni pupae than did an agar diet when the larvae were reared using a mass production protocol. However, these lower values were still above acceptable levels for mass production and reduced the cost of diet production by 65%.Further ingredient substitutions impaired larval development (i.e., replacing pinto beans with soy protein or combinations of soy protein and -cellulose), while others did not (i.e., excluding pinto beans and reducing water content and replacing pinto beans with -cellulose or corn cob grits). Moisture control by adding corn cob grits to diets increased the yield of pupae, while removing water from diets increased survivorship and the yield of pupae.     

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。     

Gelrite as an alternative to agar for micropropagation and microtuberization of Solanum tuberosum L. cv. Baraka

Summary Phytagel™ allowed the production of longer internodes, faster in vitro tuberization, and larger tubers in Solanum tuberosum L. cv. Baraka as compared to Difco Bacto-agar during both an 8-h photoperiod or in darkness. It also allowed a higher tuberization percentage in the dark. Only a 0.2% (wt/vol) Phytagel allowed optimal micropropagation and microtuberization under the photoperiod regime used. Water availability does not account for the observed differences in growth and tuberization between media containing the above gelling agents. In consequence, Phytagel appears as an advantageous alternative to agar for micropropagation and microtuberization.     

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.     

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     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence