Difco Bacto Agar Research Articles

PM 7/60 (2) Pantoea stewartii subsp. stewartii

Specific scopeThis Standard describes a diagnostic protocol for Pantoea stewartii subsp. stewartii.Specific approval and amendmentApproved in 2005‐09.Revised in 2016‐04.

Cryopreservation of in vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn.

In vitro-grown shoot tips of Alnus glutinosa (L.) Gaertn. were successfully cryopreserved by vitrification. Shoot tips (0.5–1 mm) excised from 6-week-old shoots were precultured in hormone-free Woody Plant Medium (WPM) supplemented with 0.2 M sucrose, for 2 days at 4 °C in the dark, and then treated with a mixture of 2 M glycerol plus 0.4 M sucrose, for 20 min at 25 °C. Osmoprotected shoot tips were first dehydrated with 50 % vitrification solution (PVS2), for 30 min at 0 °C, and then placed in 100 % PVS2, for 30 min at 0 °C. The solution was replaced with fresh 100 % PVS2, and the shoot tips were plunged directly into liquid nitrogen. The shoot tips were rewarmed in a water bath at 40 °C for 2 min, and then washed twice, for 10 min at 25 °C, with 1.2 M sucrose solution, before being transferred onto WPM supplemented with 0.5 mg l−1N6-benzyladenine, 0.5 mg l−1 indole-3-acetic acid, 0.2 mg l−1 zeatin, 20 g l−1 glucose and 6 g l−1 Difco Bacto agar. The shoot tips were kept in darkness for 1 week and under dim lighting for another week, before being exposed to standard culture conditions (16 h photoperiod). This protocol was successfully applied to three alder genotypes, with recovery rates higher than 50 %.

Biotin Sensing in Saccharomyces cerevisiae Is Mediated by a Conserved DNA Element and Requires the Activity of Biotin-Protein Ligase

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

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

To determine the possibility of using guar gum, a colloidal polysaccharide, as a cheap alternative to agar for gelling microbial culture media. 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. 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. 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.

Effect of gelling agents onin vitro tuberization of six potato cultivars

The gelling agent Phytagel™ was compared with Difco Bacto-agar forin vitro tuberization of six potato cultivars (Jaerla, Blanka, Claustar, Kennebec, Desiree, and Baraka). On a culture medium with 6% sucrose but lacking growth regulators, tuberization was higher when Phytagel™ was used rather than Difco Bacto-agar, regardless of the photoperiodic regime used. Chemical analyses of the gelling agents revealed a higher mineral content and organic impurities in Bacto-agar than in Phytagel™, which is therefore recommended for microtuber production. The same gelling agent should be used in all treatments of microtuberization experiments in order to draw meaningful physiological conclusions.

Factors affecting the shoot multiplication of Persian walnut ( Juglans regia L.)

A number of experiments were conducted to identify suitable procedure for in vitro shoot multiplication of Persian walnut ( Juglans regia L.). Three different nutrient media (DKW, MS and WPM) and three different gelling agents (Phytagel, Difco Bacto agar and a mixture of Phytagel and Difco Bacto agar) were studied in the first experiment. Driver and Kuniyuki walnut (DKW) medium solidified with 2.2 g l −1 Phytagel was found optimum. Performance of explants was better on DKW medium than on MS and WPM. The DKW and MS media were not significantly different from each other, but both of them were significantly better than WPM, which was a very poor medium for this species. Phytagel alone was significantly better than Difco Bacto agar or Phytagel combined with Difco Bacto agar. In another experiment different concentrations of BA were studied. Medium containing 1.0 mg l −1 BA and 0.01 mg l −1 IBA was the best, although medium containing 0.6 and 0.8 mg l −1 BA were also successful, and utilisation of 0.4 mg l −1 BA and 0.01 mg l −1 IBA was optimum for shoot elongation. Application of different kinds of auxins (IAA, IBA and NAA at 0.01 or 0.1 mg l −1) with 1.0 mg l −1 BA were also studied. Media containing IBA were significantly better than media containing IAA for shoot fresh weight, but neither of them was significantly different from media containing NAA. Application of 0.01 mg l −1 or 0.1 mg l −1 auxin, with 1.0 mg l −1 BA, was not significantly different for shoot multiplication of Persian walnut. The morphology of shoots on media containing 0.01 mg l −1 IBA was the best.

Organogenesis from megagametophyte and zygotic embryo explants of the gymnosperm Dioon edule Lindley (Zamiaceae, Cycadales)

Organogenic cultures were induced from zygotic embryo and megagametophyte explants of the Central American cycad species, Dioon edule. Plant growth medium consisted of B5 major salts, Murashige and Skoog minor salts and organics, 400 mg l−1 glutamine, 100 mg l−1 arginine, 100 mg l−1 asparagine, 60 g l−1 sucrose, 8 g l−1 Difco Bacto agar and was supplemented with kinetin (0 – 13.94 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (0 – 9.05 μM) arranged as a 5×4 factorial in a randomized block design. Callus initiation occurred on a wide range of medium formulations from megagametophyte explants; however, shoot formation occurred only on medium supplemented with 2.26 μM 2,4-D. In comparison, callus initiation from explanted zygotic embryos occurred on fewer medium formulations, and adventitious shoot induction occurred from callus on formulations with 9.29–13.94 μM kinetin + 0.45–9.05 μM 2,4-D. Rooted shoots, derived from megagametophyte and zygotic embryo cultures, have been regenerated.

Selection of agar for use inSalmonella typhimurium andEscherichia coli mutation assays

The spontaneous and induced revertant frequency of four Salmonella typhimurium strains (TA1535, TA1537, TA98, and TA100) and Escherichia coli [WP2 uvrA (pKM101)] was evaluated using Vogel Bonner minimal plates prepared with ten different agars. In addition to the Difco Bacto agar originally recommended by Ames, Difco Noble, granulated and Bitek agars; BD grade A, BBL granulated and purified agars; Oxoid purified and No. 1 agars; and GIBCO select agar were tested. Several of these agars have been reported as acceptable alternatives for these Salmonella strains, but comparable studies with E. coli have not been done. The bacteria were treated with DMSO or an appropriate positive control in the presence or absence of an Aroclor 1254-induced rat liver activation system. With the exception of Noble agar in the presence of S9, there was little difference among the responses of the Salmonella strains on any of the agars. However, with E. coli the responses include either a reduction or an increase in spontaneous revertants numbers as well as a reduction in absolute and relative induced revertant frequency. Difco Bacto agar appears to be the most consistent agar for use with these strains. As an alternative, only BBL purified agar resulted in consistent results for all of these strains under all testing conditions. These results emphasize the need to evaluate the components of the standard mutation assay when incorporating additional bacterial strains. Suboptimal responses related to the agar or other components could compromise the detection of weak mutagens.

Regeneration of plants from leaflet explants of tissue culture raised safed siris (Albizia procera)

Shoot bud regeneration was obtained from isolated leaflets of Albizia procera cultured on MS medium with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA). The highest numbers of adventitious buds were obtained on MS medium supplemented with 10 μM BA and 1 μM NAA. The replacement of 7 g l-1 Difco bacto agar with 2.6 g l-1 Phytagel in the medium enhanced adventitious bud regeneration. Further, addition of 15 μM silver nitrate promoted callus-free shoot regeneration from leaf explants. The regenerated shoot buds were elongated on MS medium containing 0.01 μM BA and 1 μM NAA. Rooting was obtained on modified MS medium supplemented with 2 μM IBA. To our knowledge this is the first report of direct regeneration of shoots from leaflet explants in A. procera, and should help facilitate genetic transformation in this species.

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

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.

Effects of Nutrient Media on the In Vitro Establishment of Shoots of Pistacia Atlantica Desf. Seedling.

Shoot cultures of Pistacia atlantica Desf. seedlings were established in vitro using apical and nodal segments from six-month-old plants. The most satisfactory results were obtained using a modified Quirin and Lepoivre (1977) macroeement solution identified as QL. 4 which was enriched with 10 mEq/I of NH4NO_ plus 5 mEq/l of NaNO3 and supplemented with MS (1962) micronutrients plus 0.4 mg/l thiamine-HCl, 0.4 mg/l pyridoxine-HCl, 100 mg/l m-inositol, 10-6 M BAP, 5x10-8 M NAA, 30 g/l sucrose and 6 g/l Difco-Bacto agar. All cultures were incubated 24ºC under a 16 h photoperiod (30 \alphaE/m2xs).

Micropropagation of juvenile and mature american beech

The purpose of this study was to micropropagate juvenile and mature American beech (Fagus grandifoliaEhrh.) resistant to beech bark disease. Shoot tips (from juvenile seedlings and root sprouts of mature trees) and buds from branches of mature trees, were cultured and multiplied on aspen culture medium supplemented with 0.89M 6-benzyladenine, 0.27M-naphthaleneacetic acid, 20 g l 1 sucrose, and 7 g l 1 Difco Bacto-agar. Rooting of shoots was achieved using a 1 minute dip in 12.3 mm indole-3-butyric acid, followed by culture in a Horticube saturated with half-strength aspen culture medium containing 20 g l 1 sucrose. Thirteen of 33 mature genotypes were established from shoot tips taken from root sprouts, and six of 41 were established from mature buds. Plantlets of six genotypes were successfully acclimatized to a growth room for three months. Abbreviations:AC ‐ aspen culture medium; BBD ‐ beech bark disease; WS ‐ Wolter Skoog medium

Influence of growth regulators and carbon sources on callus induction, growth and morphogenesis from leaf tissues of peach (Prunus persicaL. Batsch)

SummaryLeaf segments of Prunus persica (L.) Batsch ‘Elberta Queen’ were collected from in vitro-grown proliferating shoots and cultured onto a basal medium containing half-strength Murashige and Skoog (MS) salts, Staba vitamins, 30 g l−1 sucrose and 6.5 g l−1 Difco-bacto agar. The influences of six growth regulators supplemented at three levels (5, 10 and 15 µM) on callus induction were investigated under light conditions (16 h photoperiods). For all growth regulator treatments, caulogenesis was observed along cut margins of leaf explants. Among cytokinins tested, thidiazuron (TDZ) induced compact green calli, 6-benzyladenine (BA) and zeatin induced small calli, and kinetin failed to induce callus. For auxin treatments, both dicamba and 2,4-D induced friable white to yellowish calli. In another experiment, leaf explants collected from greenhouse-grown ‘Bellaire’ and in vitro-grown ‘Elberta Queen’ were cultured onto the basal medium supplemented with a wide range of TDZ concentrations (3 to 23 µM). Cauloge...

In vitro shoot proliferation of Myrtus communis L. from field-grown plants

In vitro vegetative propagation of native Mediterranean myrtle ( Myrtus communis L.) has been obtained from mature field-grown plants. Single node explants were cultivated on a standard basal medium supplemented with 3% sucrose, 10 mg l −1 l-ascorbic acid and 0.7% Difco bacto agar. For the proliferation stage various 6-benzylaminopurine treatments (0–2.0 mg l −1) and explant orientations were tested. A high proliferation rate was obtained from explants placed horizontally on a medium containing 2.0 mg l −1 and 0.05 mg l −1 1-naphthaleneacetic acid (NAA) and subcultured at 6-week intervals. Shoot proliferation medium composition and NAA concentration strongly influenced root induction on propagules.

Effects of source of macronutrients and plant growth regulator concentrations on shoot proliferation of Cornus florida

Nodal stem segments of flowering dogwood (Cornus florida L.) were cultured on media containing seven different sources of macronutrients including full- and half-strength Murashige & Skoog (MS) macrosalts, N6, Anderson's (AND), Quoirin & Lepoivre's (LP), Nitsch & Nitsch (NIT), and Woody Plant Medium (WPM). All media contained MS micronutrients, Staba vitamins, 20 g l-1 sucrose, and 6.5 g l-1 Difco Bacto-agar, and were supplemented with 2.2 μM 6-benzyladenine (BA) and 0.49 μM indole-3-butyric acid (IBA). Following three subcultures, the best shoot proliferation was supported on media containing WPM macronutrients. To optimize the proliferation rate, shoots were cultured on WPM macronutrients supplemented with eight combinations of BA and IBA, and 3.3 μM BA without IBA was determined to be the best.

Enhanced development and differentiation of protoplasts and spores of green and red seaweeds by a Pterocladia agar from New Zealand

Enhanced growth of macroalgae was observed on High Purity Agar, a product of Coast Biologicals Limited of Auckland New Zealand. Increases in rates of cell divisions and tissue differentiation occurred in green and red seaweeds. Protoplasts, spores, and vegetative thalli were all affected. The enhancement of growth and differentiation was concentration dependent, with high concentrations (> 1 %) appearing toxic. Few cells survived on 1 % and 1.5 % agar; over 8 weeks of culture these developed into plantlets six fold larger than those grown on an 0.3 % gel of the same agar, and as much as 11 fold larger than control plants grown on Difco Bacto agar at the same gel concentration. The activity was heat stable, water soluble, and largely bound to the carbohydrate matrix. The chemical nature of the active compound is under investigation.

INFLUENCE OF AGAR AND AMMONIUM NITRATE ON TISSUE NITROGEN, CULTURE GROWTH, AND VITRIFICATION

Shoot tips of Amelanchier were grown on Murashige & Skoog (MS) or Woody Plant (WP) medium containing 4.4 μM benzyladenine and 4, 6 or 8% Difco Bactoagar. Increasing agar concentration reduced culture fresh weight, dry weight, and vitrification, but increased tissue nitrate concentrations. The number of shoots produced per culture was unaffected by agar concentration. Overall, response to agar additions was similar for shoots on both WP and MS salts, although the magnitude of the response may have differed. Amelanchier shoots cultured on WP medium containing 4% agar and 10.5, 15.5, 20.9 or 25.5 mM nitrate showed a linear increase in culture fresh weight, culture dry weight, number of shoots per culture and number of vitrified shoots per culture as medium nitrate concentration increased. Nitrate and total nitrogen content of explants also increased linearly with increasing medium nitrate concentration. Although vitrification has been previously ascribed to elevated ammonium concentrations in culture media, it is possible that elevated medium and tissue nitrate levels may also play a role in vitrification.

Micropropagation and Field Establishment of Tiarella cordifolia

Abstract Tiarella cordifolia L. (foamflower) was proliferated on an MS-based nutrient medium. Proliferation was maximal on medium gelled with 0.4% Difco-Bacto agar containing 1.0 mg benzyladenine (BA)/1 and 0 or 0.025 mg naphthaleneacetic acid (NAA)/1. Proliferation of T. cordifolia var. collina was maximal on medium gelled with Gelrite® containing 0.25, 0.5 or 1.0 mg BA/1 plus NAA (0, 0.025, 0.05, 0.1, 0.5 mg/l). Greenhouse rooting and survival was directly related to an increase in microcutting length, whereas leaf number (two to six leaves per microcutting) had no effect. At least 48% of the microcuttings of T. cordifolia inserted directly in the field rooted and survived.

Regeneration of peppermint and orange mint from leaf disks

Leaf disks from peppermint, spearmint, orange mint, lavender mint and Scotch spearmint were cultured on various Murashige-Skoog-based media in order to regenerate shoots. A significantly larger average number of orange mint leaf disks regenerated shoots on basal medium containing 44.4 μM benzyladenine (BA) and 250 ml l-1 coconut water (CW). Shoots regenerated from peppermint leaf disks cultured on basal medium containing 44.4 μM BA and 250 ml or 450 ml l-1 CW. The most shoots regenerated from orange mint leaf disks cultured on medium containing 10 g l-1 washed Difco Bacto-agar. Disks excised from the bases of the first expanding pair of orange mint leaves cultured under dark conditions regenerated a significantly larger average number of shoots. Histological studies suggested that shoots regenerated from the palisade parenchyma cells associated with vascular tissue.

Carbohydrate requirements for the development of black spruce (Picea mariana (Mill.) B.S.P.) and red spruce (P. rubens Sarg.) somatic embryos

Different carbohydrates were investigated for somatic embryo development of black spruce and red spruce. They were tested in a basal maturation medium consisting of Litvay's salts at half-strength containing 1 g l-1 glutamine, 1 g l-1 casein hydrolysate, 7.5 μM abscisic acid, and 0.9% Difco Bacto-agar. A comparison of different sucrose concentrations showed that 6% was optimal for embryo development. Among the nine carbohydrates tested, sucrose, fructose, glucose, maltose, and cellobiose supported embryo development while arabinose, mannitol, myo-inositol, and sorbitol did not. A comparison of sucrose, glucose, and fructose at three concentrations showed that the general pattern of response for both species followed concentration expressed as a percentage, independent of the molarity of carbohydrate in the medium. Interspecific differences were observed concerning carbohydrate requirements. For red spruce, 6% fructose was found best for embryo development, while no such preference was observed for black spruce. No significant difference was observed in the number of embryos produced with 6% sucrose or 3% sucrose plus an equimolar concentration of either mannitol, sorbitol, or myo-inositol in the maturation medium, suggesting that the effect of the carbohydrate on the maturation was partly osmotic.