Evaluation of guar gum derivatives as gelling agents for microbial culture media

Guar gum, a galactomannan, has been reported to be an inexpensive substitute of agar for microbial culture media. However, its use is restricted probably because of (1) its highly viscous nature even at high temperatures, making dispensing of the media to Petri plates difficult and (2) lesser clarity of the guar gum gelled media than agar media due to impurities present in guar gum. To overcome these problems, three guar gum derivatives, carboxymethyl guar, carboxymethyl hydroxypropyl guar and hydroxypropyl guar, were tested as gelling agents for microbial growth and differentiation. These were also evaluated for their suitability for other routine microbiological methods, such as, enumeration, use of selective and differential media, and antibiotic sensitivity test. For evaluation purpose, growth and differentiation of eight fungi and eight bacteria grown on the media gelled with agar (1.5%), guar gum (4%) or one of the guar gum derivatives (4%), were compared. All fungi and bacteria exhibited normal growth and differentiation on all these media. Generally, growth of most of the fungi was better on guar gum derivatives gelled medium than on agar medium. The enumeration carried out for Serratia sp. and Pseudomonas aeruginosa by serial dilution and pour plate method yielded similar counts in all the treatments. Likewise, the selective succinate medium, specific for P. aeruginosa, did not allow growth of co-inoculated Bacillus sp. even if gelled with guar gum derivatives. The differential medium, Congo red mannitol agar could not differentiate between Agrobacterium tumefaciens and Rhizobium meliloti on color basis, if gelled with guar gum or any of its derivatives However, for antibiotic sensitivity tests for both Gram-positive and -negative bacteria, guar gum and its derivatives were as effective as agar.     

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.     

Guar gum: a cheap substitute for agar in microbial culture media

AIMS: To determine the possibility of using guar gum, a colloidal polysaccharide, as a cheap alternative to agar for gelling microbial culture media. METHODS AND RESULTS: As illustrative examples, 12 fungi and 11 bacteria were cultured on media solidified with either guar gum or agar. All fungi and bacteria exhibited normal growth and differentiation on the media gelled with guar gum. Microscopic examination of the fungi and bacteria grown on agar or guar gum gelled media did not reveal any structural differences. However, growth of most of the fungi was better on guar gum media than agar, and correspondingly, sporulation was also more advanced on the former. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method yielded similar counts on both agar and guar gum. Likewise, a selective medium, succinate medium used for growth of Pseudomonas sp. did not support growth of Bacillus sp. when inoculated along with Pseudomonas on both agar or guar gum supplemented medium. CONCLUSIONS: Guar gum, a galactomannan, which is 50 times cheaper than Difco-bacto agar, can be used as a gelling agent in place of agar in microbial culture media. SIGNIFICANCE AND IMPACT OF THE STUDY: As the media gelled with guar gum do not melt at temperature as high as 70 degrees C, these can be used for isolation and maintenance of thermophiles.     

Xanthan gum: an economical substitute for agar in plant tissue culture media

Xanthan gum, a microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used as a solidifying agent for plant tissue culture media. Its suitability as a substitute to agar was demonstrated for in vitro seed germination, caulogenesis and rhizogenesis of Albizzia lebbeck, androgenesis in anther cultures of Datura innoxia, and somatic embryogenesis in callus cultures of Calliandra tweedii. Culture media used for eliciting these morphogenic responses were gelled with either 1% xanthan gum or 0.9% agar. Xanthan gum, like agar, supported all these responses.     

Gum katira – a cheap gelling agent for plant tissue culture media

Gum katira, an insoluble gum derived from the bark of Cochlospermum religiosum, has been successfully used as a gelling agent in tissue culture media for in vitro shoot formation and rooting in Syzygium cuminii and somatic embryogenesis in Albizzia lebbeck. The epicotyl segments, excised from in vitro grown seedlings of S. cuminii, developed shoots when cultured on MS medium (Murashige and Skoog, 1962), supplemented with 4% sucrose and 1 mg l^–1 BA. The so-developed shoots were rooted on Knop's medium, augmented with 2% sucrose and 1 mg l^–1 IAA. For somatic embryogenesis, hypocotyl segments derived from in vitro developed seedlings of A. lebbeck were cultured on B5 medium containing 2% sucrose. Media were gelled with either 3% gum or 0.9% agar. The quantitative response obtained on media fortified with either of the gelling agents was not significantly different. The media gelled with gum katira were almost as transparent as the liquid medium. However, viscosity of gum katira gelled medium was less than one-sixth of the viscosity of agar-gelled media, and therefore, shaking ofthe culture vessel often resulted in submersion of the explants. Nevertheless, even these submerged explants responded positively. To increase the firmness of the gum katira-gelled medium, various combinations of agar (0.2–0.6%) and gum (1–3%) were used. However, the viscosities of the media gelled with 3% gum katira as well as different concentrations of agar (0.2–0.6%) were lower than that of the medium containing only gum katira (3%). Moreover, the explant productivity obtained in neither of these combinations was more than those recorded on the control media, which were gelled either with 0.9% agar or 3% gum alone.     

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.     

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.     

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.     

Effects of different gelling agents on the different stages of rice regeneration in two rice cultivars

Plant tissue culture technology offers a solution for meeting the increasing commercial demand on economically important plants such as rice, a widespread dietary staple. However, significant genotype-specific morphogenetic responses constitute a considerable on rice regeneration in plant biotechnology contexts. Aside from genotype dependency, the components of the nutrient media including gelling agents have an important impact on regeneration efficiency. The current study explores the effect of different gelling agents on various stages of rice regeneration in two Egyptian rice cultivars-Sakha104 and Giza178. Media solidified with varying concentrations of a variety of gelling agents (agar, bacto agar, gelrite and phytagel) were tested for their impact on the frequency of callus induction, shoot regeneration and rooting. The results indicated gellan gum (gelrite and phytagel) was superior to agar products (agar and bacto agar) for callus induction. By contrast, no significant differences were found between different gelling agents for shoot regeneration. Gellan gum and media solidified with bacto agar were found to lead to significantly higher root regeneration than agar. The Sakha104 cultivar showed better responses than Giza 178 for callus induction and similar performance to the Giza 178 cultivar for root regeneration irrespective of the gelling agent. This work provides insights into the impact of different gelling agents on the morphogenetic response of two rice cultivars and can be used to help maximize the frequency of rice regeneration.     

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。     

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.     

Factors affecting adventitious regeneration from in vitro leaf explants of 'Improved French' plum, the most important dried plum cultivar in the USA

An adventitious shoot regeneration protocol from in vitro leaves of the most important dried plum cultivar in the USA, ‘Improved French’, has been established. Factors affecting regeneration were studied in order to optimise regeneration. The proliferation medium in which the shoots, used as the source of leaf explants, were cultured had a strong influence on subsequent regeneration. Shoot regeneration was observed at a mean frequency of 52% when a Murashige-based and Skoog-based shoot culture medium with 3 μM N⁶-benzylaminopurine and 0.25 μM indole-3-butyric acid (IBA) was employed compared with shoot regeneration frequencies of less than 5% for a Quoirin-based and Lepoivre-based shoot culture medium, with 8.9 μM N⁶-benzylaminopurine and 0.49 μM IBA. The shoot regeneration medium contained α-naphthaleneacetic acid at 2.0–6.0 μM and thidiazuron at 4.5–15.0 μM. 2,4 Dichlorophenoxy-acetic acid at 9.0 μM was included in the medium but only for the first 4 days of culture. Shoot regeneration frequencies were positively related to thidiazuron concentration and significantly greater (P < 0.05) for 9–15 μM thidiazuron than for the media with 4.5 μM thidiazuron. Leaf explants, incubated in a 16-h-light/8-h-dark photoperiod or in the dark for 1 week followed by exposure to light, showed significantly more organogenic activity (P < 0.01) than was observed for leaves cultured in the dark for 2 or 3 weeks before they were transferred to the light. The utilisation of Bacto agar (0.7%) as the gelling agent increased organogenesis compared with media gelled with TC Agar (0.7%), or an agar–gellan gum blend (Agargel™) (0.45%). The addition of the ethylene inhibitor silver thiosulphate at 60–120 μM also improved organogenesis. When all the studied factors were optimised, a regeneration rate of 65% was achieved. Rooting frequency of regenerated shoots was significantly increased (P < 0.05) by the use of full-strength Murashige and Skoog salts (40%) or 100 mg L⁻¹ phloroglucinol (53%) to the rooting medium.     

Improved micropropagation in Polygala myrtifolia

Summary Stem segments from apical shoot tips of Polygala myrtifolia were used as primary explants to establish in vitro cultures. Axillary shoots produced on non-contaminated explants were excised and recultured in the same medium to increase the stock of shoot cultures. Equal molar concentrations of five cytokinins [2-isopentenyladenine, kinetin, zeatin, N ⁶-benzyladenine (BA), and adenine] were tested for ability to induce axillary shoot development from double-node stem segments. The highest rate of axillary shoot proliferation was induced on Murashige and Skoog agar medium supplemented with 1.8 μM BA. Seven indole-3-acetic acid (IAA) concentrations (0, 2.9, 5.7, 8.6, 11.4, 14.3, 17.1 μM) were tested to determine the optimum conditions for in vitro rooting of microshoots. Up to 72% of the microshoots rooted with 14.3 μM IAA. Other auxins tested, α-naphthaleneacetic acid and indole-3-butyric acid, were less effective than IAA in inducing adventitious root formation. All rooted plantlets having more than three roots were successfully established in soil.     

Agar/galactomannan gels applied to shoot regeneration from tobacco leaves

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum — a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm⁻³ each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm⁻³). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.     

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 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.     

Influence of gelling agents on culture medium gel strength, water availability, tissue water potential, and maturation response in embryogenic cultures of Pinus strobus L.

Summary Maturation of somatic embryos of Pinus strobus L. was evaluated on media containing various types (agars and gellan gum), brands and concentrations of gelling agents in the presence of 80 μM ABA and 0.09 M sucrose. The media were characterized with respect to gel strength, water potential and water availability. Embryogenic tissue and somatic embryos cultured on medium with various concentrations of gellan gum were used to determine their water potential (Ψ). Regardless of the type of gelling agent used, gel strength increased with gelling agent concentration and was critical to the maturation response. High gel strength was associated with reduced water availability from the medium to the cultures. The water potential of gelled maturation medium remained constant between 0.4 and 1.0% gellan gum. It is concluded that the embryogenic tissue was exposed to varying amounts of water at the onset of and during the culture period, and that the amount of water in the culture environment in turn influenced the maturation response. Cotyledonary somatic embryos derived from gellan gum medium of high gel strength had a lower Ψ than somatic embryos matured on medium of lower gel strength. Once somatic embryos developed to the cotyledonary stage on the maturation medium, they were transferred to the germination medium. The germination frequency and the number of morphologically normal germinants were higher for somatic embryos matured on medium of high gel strength. Raising the concentration of the gelling agent in the maturation medium may be an alternative to the use of solutes to restrict water available to the embryogenic cultures.     

Isubgol as an Alternative Gelling Agent for Microbial Culture Media

Isubgol, the mucilaginous husk derived from the seeds of Plantago ovata, has been successfully used as a gelling agent for microbial culture media. As illustrative examples, fast growing symbiotic bacterium, Rhizobium meliloti and saprophytic fungi, Aspergillus flavus and Penicillium chrysogenum were cultured on media gelled with either Isubgol or agar. All the three microbes employed in the study exhibited normal growth when cultured on their respective media gelled with Isubgol. Rather, Isubgol gelled medium appears to promote the growth of bacterial cultures as the colonies on this medium were denser than the corresponding ones on the medium gelled with agar. Likewise, on Isubgol gelled medium, sporulation in both the fungi took place earlier than on the medium gelled with agar, thus indicating the promotive influence of the former gelling agent.     

Direct measurement of water availability in gelled plant tissue culture media

Summary Water constitutes nearly 100% of the volume and 95% of the mass of gelled plant tissue culture media. Even so, plant growth and development responses observed to occur with relatively small changes in gelling agent concentration (0.1% of media total mass) have been attributed to changes in media water availability. Measurements with three alternative direct techniques, specific for measuring physiochemical water availability indicated the effects of a change of this magnitude in gelling agent concentration negligibly affected the media water potential and water conductivity. Sensitive pressure membrane measurements indicated that incremental gelling agent concentration increases of 0.1% (of media total mass) within the range normally used for plant tissue culture media, depressed water matric potential only 1–2cm H₂O (1–2×10⁻⁴ MPa (mega pascal.)); these values were confirmed with equally sensitive tensiometer measurements. Moreover, no effect of concentration on water movement could be detected with a precise constant-head permeameter over a broader range of gelling agent concentrations. These results indicate that eitherin vitro plants are extremely sensitive to subtle shifts in water status, or other physiochemical factors that also change with gelling agent concentration are contributing to the reported effects on plant growth and development.     

`Isubgol' as an alternative gelling agent in plant tissue culture media

`Isubgol', the mucilaginous husk derived from the seeds of Plantago ovata, was successfully used as a gelling agent in tissue culture media for in vitro seed germination, shoot formation and rooting in Syzygium cuminii and anther culture in Datura innoxia. For seed germination, Knop's basal medium supplemented with 1% sucrose was employed, whereas for the development of shoots the epicotyl segments excised from in vitro-developed seedlings were cultured on MS basal medium supplemented with 4% sucrose and 1 mg/l 6-benzyladenine. Shoots that developed from the epicotyl segments were rooted on Knop's medium enriched with 2% sucrose and 1 mg/l indole-3-acetic acid. The anthers of D. innoxia excised at the late uninucleate to early binucleate stages of microspore development were cultured on Nitsch's basal medium containing 2% sucrose. Media were either gelled with 0.9% agar or 3% `Isubgol'. The response on media gelled with `Isubgol' in each of the cases was similar to that on media solidified with agar. Received: 9 October 1996 / Revision received: 22 July 1996 / Accepted: 30 July 1997