Gamborg's B5 Medium Research Articles

Indole Alkaloid Production in Callus Cultures of Uncaria rhyncophylla (MIQ) MIQUEL.

The effects of growth hormones and nutrients on growth and alkaloid production in callus cultures of Uncaria rhynchophylla (MIQ.) MIQUEL (Rubiaceae) were investigated. Gamborg B5 (B5) medium supplemented with indoleacetic acid (10-4M) and 6-benzylaminopurine (3×10-5M) was optimized both for the growth (1.8g fresh weitht/5 weeks) and alkaloid production (1.73 mg/g dry weight/5 weeks). The influence of sucrose concentration was examined, and 2% sucrose was found to be the most appropriate for the growth and alkaloid production. The effect of the composition of nitrogen sources was examined. Maximal growth and alkaloid production were obtained when 6 mM ammonium chloride and 25mM potassium nitrate were added to the B5 medium. Hirsuteine, hirsutine, 3α-dihydrocadamibine and ursolic acid were isolated from the callus. The 3α-dihydrocadambine concentration of callus was ca. 50-fold higher than that of the hooks and stems of U. rhynchophylla.

Fast ion bombardment mass spectrometry of sanguinarine and coptisine from Chelidonium majus L. callus cells

AbstractThis paper reports the identification of sanguinarine and coptisine isolated from the crude extract of callus culture of Chelidonium majus by positive fast ion bombardment (FIB+) mass spectrometry (MS) and FIB+ tandem mass spectrometry (MS‐MS). Callus was cultivated on Gamborg B5 medium for three weeks at 25°C and extracted with 70% ethanol. The alkaloids were extracted into chloroform, following ion pair formation, and then separated on a silica gel column. The presence of sanguinarine and coptisine was initially determined from chromatographic analysis, and the structures of the compounds were confirmed by MS. MS‐MS analysis was not suitable for coptisine since this compound did not produce daughter ions.

Growth and steroidal saponin production in hairy root cultures of Solanum aculeatissimum

Hairy root cultures of Solanum aculeatissimum were established by trans-formation using Agrobacterium rhizogenes strain 15834. Root growth and production of steroidal saponin were investigated under various culture conditions. Transformed roots grew better in Gamborg's B5 medium containing 3 % sucrose under continuous light than in the dark. Also, the roots turned light green when cultured under continuous light. Green hairy roots produced aculeatiside A (6.71mg ·) L(-1) and aculeatiside B (6.39mg · L(-1)) after 8 weeks of culture, while no steroidal saponin was detected in hairy roots cultured in the dark. Of the three culture media tested, Gamborg's B5 medium was superior for growth and steroidal saponin production. Growth and steroidal saponin production were enhanced when 100μg · L(-1) auxin except for 2,4-D was added to the medium. The addition of 2,4-D inhibited growth. Production of steroidal saponin was highest with NAA. Transformed roots used in this experiment were confirmed that hairy roots examined contain both TL-DNA and TR-DNA region of Ri plasmid by PCR amplification analysis of DNA.

Cell Wall Pigment Formation of in vitro Cultures of the Liverwort Ricciocarpos natans

Abstract The liverwort Ricciocarpos natans has been cultivated on Gamborg B5 medium with different levels of ammonium, nitrate, phosphate and sucrose. The formation of the cell wall pigments riccionidin A and B was shown to be dependent on the level of these nutrients as well as on the intensity of light. A decrease in nitrogen supply (mainly nitrate) induced the formation of both wall pigments, whereas phosphate and high levels of sucrose inhibited the pigment synthesis. Decreasing the illumination rate led to lower anthocyanidin contents. Dedifferentiated cultures also produced wall pigments, but their amount was much lower compared to differentiated cultures.

Growth kinetics and stoichiometry of Mentha citrata shooty teratomas transformed by Agrobacterium tumefaciens

Culture characteristics of genetically transformed Mentha citrata shooty teratomas were studied in liquid Murashige and Skoog and Gamborg's B5 media. Based on calculated kinetic and yield parameters, a stoichiometric equation was developed to describe growth in media containing both nitrate and ammonia. Measurements of oxygen uptake rate showed that delivery of adequate oxygen to completely submerged shoots depends on the elimination of hydrodynamic boundary layers at high external liquid velocity.

Production of valepotriates by hairy root cultures of Centranthus ruber DC

Hairy root cultures of Centranthus ruber DC. were established by infection of sterile plantlets with Agrobacterium rhizogenes, strain R1601. The transformed roots were grown in 12 different, hormone-free liquid media, and valtrate, isovaltrate, 7-desisovaleroyl-7-acetylvaltrate, 7-homovaltrate, didrovaltrate and isovaleroxyhydroxydidrovaltrate were quantified by high performance liquid chromatography. The highest overall valepotriate content (3.0% dry wt) was observed in half-strength Gamborg B5 medium supplemented with 3% sucrose. This concentration is very similar to that found in the roots of parent plants grown in the field. The use of N,N-dimethylmorpholinium iodide, a plant bioregulator, was very detrimental to the hairy root growth and to the valepotriate production. The hairy roots cultured in half strength Gamborg B5 liquid medium supplemented with 3% sucrose for 45 days produced over 31 mg/g dry wt valepotriates.

Regeneration of whole plants from protoplasts isolated from tissue-cultured shoot primordia of garlic (Allium sativum L.)

Protoplasts derived from tissue-cultured shoot primordia of garlic (Allium sativum L.) initiated successive cell divisions within 4 days and formed small individual calli (0.2mm in diameter) after 5 weeks of culture on Gamborg's B5 medium supplemented with 0.1% casein hydrolysate, 1mg/1 1-naphthaleneacetic acid and 1mg/1 6-benzylaminopurine. Plating efficiency was roughly 5% at the density of 1x10(4) protoplasts/ml of medium. Adventitious buds developed from the calli during subsequent subculture on Gamborg's B5 medium supplemented with 40mg/l adenine and 10% coconut milk. When transferred to the same medium without supplements, these buds grew into shoots and rooted. The regenerated garlic plantlets were successfully transferred to the greenhouse and grew into whole plants.

Dauricine production in cultured roots of Menispermum dauricum

Root Cultures of Menispermum dauricum were established from adventitious roots formed on the leaf segments. These roots produce dauricine as a major constituent and the optimum conditions for dauricine production in cultured roots were investigated. A dauricine content of more than 0.5% dry weight was achieved in Gamborg's B5 medium containing 3% sucrose and 7.5 μM NAA. Tyrosine and tyramine, possible precursors of dauricine, increased dauricine formation. There was a positive correlation between dauricine content and root growth.

Vessel Volume, Gelling Agent, and Basal Salts Affect pH and Gel Strength of Autoclave Tissue Culture Media

Effects of autoclaving volume, gelling agent (Bactoagar, Gel-gro, Phytagar), and basal salts [Murashige and Skoog (MS); Woody Plant Medium (WPM); Gamborg B5 (GB)] on gel strength and pH of tissue culture media were tested. Gel strength was significantly affected by gelling agent and basal medium. MS media were generally softer than comparable WPM or GB media. As the vessel volume during autoclaving decreased, gel strength significantly decreased with Phytagar and Bactoagar gelling agents; Gel-gro had greater gel strength at the intermediate volume of medium autoclave. In all cases, autoclaving resulted in a pH decrease of 0.2 to 0.5 pH units. Lower pH values were associated with softer gels. The type of gelling agent did not greatly affect the postautoclave pH; mean values among gelling agents were within 0.05 pH units. Postautoclave pH of MS medium was lower than that of WPM or GB. This study verifies the need to observe uniform sterilization protocols to maintain consistency in the chemical and physical properties of media.

Tropane Alkaloids in Transformed Roots ofDatura quercifolia

Hairy root cultures of DATURA QUERCIFOLIA were established following infection with AGROBACTERIUM RHIZOGENES strain LBA 9402. Eight tropane alkaloids were identified in the hairy roots, hyoscyamine being the major constituent. The growth and the hyoscyamine content of transformed roots were investigated under various conditions. Gamborg B5, Murashige and Skoog, and Woody Plant media were tested. Gamborg B5 medium was the best for growth as well as for hyoscyamine accumulation. The influence of sucrose concentration was examined and a 5% concentration was found to be the most appropriate for growth and for alkaloid production. After 35 days of incubation in this medium, the hyoscyamine content of the roots was 1.24% based on dry weight. The influence of gibberellic acid and of Amberlite XAD-4 resin on hyoscyamine production was tested.

Acridone Alkaloids from Cell Suspension Cultures ofThamnosma montana

Cell Suspension cultures of THAMNOSMA MONTANA were grown in Gamborg B5 medium which was best suited for acridone alkaloid formation. From the lyophilized cell material nine acridones and three acridone-monoglucosides have been isolated which were found for the first time in the genus THAMNOSMA. Moreover, N-methylacridone which is known from the intact plant was found. Gravacridonol monoglucoside was identified as a new alkaloid. Interestingly, most isolated alkaloids belong to the dihydrofuroacridones.

Involvement of Phenolic Esters in Cell Aggregation of Suspension-Cultured Rice Cells.

Fluorescence microscopy of rice (Oryza sativa L.) callus sections showed that all of the walls fluoresced blue in water (pH 5.8) and green in ammonia (pH 10.0), both characteristics of feruloyl esters. Such fluorescence in the walls of cells cultured in Gamborg's B5 medium was much stronger than that in amino acid (AA) medium. Laser scanning microscopy showed that the level of fluorescence was higher in the intercellular layer, especially at corner junctions between cells, suggesting that ferulic acid ester derivatives are located in the middle lamella as well as in the wall. Extracellular polysaccharides appearing during cultivation in AA medium were more highly feruloylated than those in B5 medium during cultivation. Both the levels of ferulic and diferulic acid and the relative proportion of diferulic acid in the walls of cells increased on transfer of the cells cultured in AA medium to B5 medium. The walls of cells cultured in B5 medium maintained constant levels and proportions of the phenolic acids. Removal of phenolic acids from wall preparations by carboxylesterase facilitated the solubilization of noncellulosic polysaccharides. Treatment of the cell aggregates grown in AA medium with an enzyme that hydrolyzes feruloyl esters decreased the size of the aggregates to between 20 and 500 [mu]m, compared with an original size between 200 and 1000 [mu]m. These findings suggest that feruloyl and diferuloyl esters between polysaccharides are involved in the aggregation of cultured rice cells.

Embryo Rescue Culture Medium for Improved Synthesis of 4x Wheat × Rye Hybrids

The cross-incompatibility barrier between 4x wheat and rye has limited the genetic base for triticale breeding. Experiments designed to improve the synthesis of wheat-rye amphihaploids were conducted. The effects of 2,4-D on crossability and 3x hybrid embryo differentiation, and the influence of one-step and two-step media on the culture of immature 3x embryos in vitro, were investigated. Application of 10 mg L-1 2,4-D slightly improved seed set but significantly reduced the frequency of normal embryos. In contrast to the reported favourable effect of 2,4-D on haploid embryo formation in wheat × maize crosses, the application of 2,4-D in the present study offers no real advantage on amphihaploid embryo formation from 4x wheat × rye crosses. For small and immature wheat-rye hybrid (3x) embryos, optimum recovery in vitro was obtained via a two-step procedure consisting of a semi-solid MN medium followed by MS medium supplemented with IAA (1 mg L-1) and BAP (1 mg L-1). For bigger and well-differentiated embryos, the use of a one-step Gamborg's B5 medium was sufficient.

In vitro cultures of Solanum malacoxylon Sendt.: nutritional requirements and sterol production

Callus cultures were established from hypocotyl, root and leaf explants of Solanum malacoxylon. The growth rate of calli was evaluated on Murashige and Skoog medium as well as Gamborg's B5 medium. Sterols were isolated from calli grown on both media however the B5 proved to be more effective in promoting the production of these metabolites. No calcitriol was found in the cultures. Feeding experiments with vitamin D3 were scarcely effective in promoting the production of vitamin D3 hydroxylated metabolites.

Cell suspension cultures from Tropaeolum majus L. establishment and growth conditions

Friable calli, obtained from Tropaeolum majus leaves, on Gamborg B5 medium supplemented with 2,4-D, kinetin and casein amino acids, were used for the establishment of cell suspension cultures in the same liquid medium. Different basal media, inoculum densities, sucrose concentrations, nitrogen sources and cysteine levels were assayed for biomass production. The best growth rates were achieved in Gamborg B5 medium supplemented with 1·5 mg litre −1 2,4-dichlorophenoxyacetic acid, 0·1 mg litre −1 kinetin, 2 g litre −1 casein amino acids, 100 mg litre −1 cysteine and with an initial inoculum of 50 g (fresh weight) litre −1. The doubling time obtained under these conditions was about 55 h.

Effect of gibberellin biosynthesis inhibitors on shoot regeneration from hypocotyl explants of Albizzia julibrissin

Hypocotyl explants of Albizzia julibrissin were placed on Gamborg's B5 medium supplemented with various levels of paclobutrazol, uniconazole, prohexadione calcium, or GA3. Callus formation was evident within one week after placement of the explants on the culture media. Green nodule-like structures protruded from the distal end of the explants within 10 days and developed into shoots within a month. These shoots readily formed adventitious roots when placed on fresh culture medium. All three of the gibberellin biosynthesis inhibitors increased shoot formation compared to the control. The number of shoots per explants was increased 107, 79, and 168% by 0.3-0.4 μM paclobutrazol, uniconazole, and prohexadione calcium, respectively. In contrast to the gibberellin biosynthesis inhibitors, GA3 decreased shoot formation. These results indicate that modification of gibberellin status can have a strong impact on the number of shoots formed.

Perfusion strategy for rosmarinic acid production by Anchusa officinalis

The production of an intracellular secondary metabolite rosmarinic acid (RA) by plant cell suspensions of Anchusa officinalis cultivated with intermittent medium exchange is investigated. Initially, a two-stage perfusion culture method was employed. After being cultured in the batch mode for ca. 6 days in B5 medium plus 3% sucrose, 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and 0.1 mg/L kinetin (2,4-D B5 medium), Anchusa culture was cultivated to high cell density by perfusion during the growth stage using a hormone-free Gamborg B5 medium supplemented with 6% sucrose. This was followed by a production stage, in which a complete medium exchange into B5 medium plus 3% sucrose and 0.25 mg/L naphthleneacetic acid (NAA) was conducted. The two-stage perfusion culture had a higher maximum culture RA concentration but a lower RA content per cell than the batch stock culture maintained in the 2,4-D B5 medium. Higher culture RA concentration was due primarily to high cell density. The high packed cell volume, however, seemed to reduce the synergistic effect of NAA on RA synthesis. Subsequently, a single-stage perfusion culture method was investigated. The best result was obtained by growing the culture in the batch mode for ca. 10 days using B5 medium supplemented with 3% sucrose and 0.25 mg/L NAA, followed by perfusing the culture with B5 medium plus 6% sucrose and 0.25 mg/L NAA at a constant perfusion rate of 0.1/day. A maximum cell dry weight of 35 g/L and a RA concentration of almost 4 g/L were achieved. This is the highest RA concentration ever reported in the Anchusa culture.

In vitro-Selection and Regeneration of Hydroxyproline-Resistant Lines of Winter Wheat with Increased Proline Content and Increased Frost Tolerance

An embryogenic callus obtained from immature embryos of a Finnish winter wheat ( Triticum aestivum 1. cv. Jo 3063) was used for in vitro -selection of hydroxyproline (Hyp) resistant calli. Calli were plated on solid Gamborg B5 medium containing 10–20mM Hyp and 2mgL −1 2,4-D. From 6018 embryogenic calli exposed to Hyp in the course of three subcultures nine calli proved to be Hyp-resistant and remained viable and embryogenic. These were transferred to a regeneration medium and finally to Gamborg B5 medium before being transplanted to soil. The regenerated plants were grown at 18°C for 6 weeks and then cold hardened at 2°C for 18 weeks. The mean osmotic potential of the Hyp-resistant cold hardened regenerates was significantly lower than that of hardened controls. At the same time their mean proline content and their mean frost tolerance were significantly higher compared with regenerated controls.

Growth characteristics of Glycyrrhiza glabra cell suspension cultures

Callus and cell suspension cultures of licorice (Glycyrrhiza glabra) have been established in Gamborg's B5 medium supplemented with 2% sucrose, 1.0 mg 1-1 2,4-dichlorophenoxyacetic acid and 0.1 mg 1-1 kinetin. Biomass accumulation was affected by the density and age of inoculum, vessel size and sucrose level. 8% sucrose medium in 2-1 flasks induced biphasic growth, with fructose as the only source of carbon and energy in the second phase.

Initiation and growth of cell lines of Taxus brevifolia (Pacific yew)

Conditions have been established for the induction and maintenance of callus cultures of Taxus brevifolia (Pacific yew) from bark, stem, and needle tissues. Cultures were established on a modified Gamborg's B5 medium, 1% sucrose, 0.2% casamino acids and 1 mg/L 2,4-D. There was no apparent inhibition of callus induction as a result of taxol concentration in the explant material. Cell lines derived from explants of individual trees were used to investigate growth characteristics. Although none of the cell lines contained taxol, some contained low levels of related taxanes. Variability was observed with each cell line in response to light, and auxin type and concentration. Growth index was most affected by cell line, followed by auxin type and concentration. These culturing methods may be useful for the goal of developing a highproducing cell line applicable for large-scale taxol production.