Callus and suspension cultures fromGeranium robertianum L (Geraniaceae)

Summary Frieble calli, obtained from petioles ofGeranium roberlianum were used for the production of cell suspension cultures in liquid MS modified medium supplemented with BAP and NAA. Casamino acids were shown to be necessary for suspension cultures establishment With a 15.9×10⁴ cell. ml^–1 concentration a td=38.2h was achieved.     

Protease from callus and cell suspension cultures of Onopordum turcicum (Compositae)

Summary Germinated seeds of Onopordum turcicum, which is a kind of thistle, were induced to develop callus on Murashige-Skoog medium. Both callus and suspension cultures of the plant synthesized extracellular and intracellular protease as a product of primary metabolism. The proteolytic activities of the enzyme complex from cell suspension and callus cultures were higher than those from the leaves and seeds of the plant.     

Callus concentration regulates somatic embryo production in orchardgrass suspension cultures

Summary The effects of callus inoculation concentration and culture duration on somatic embryogenesis of orchardgrass,Dactylis glomerata L., were evaluated in suspension cultures of an embryogenic genotype Embryogen-P. Somatic embryo formation was induced in liquid SH medium containing 30 μM dicamba (SH-30 and 1.5% casein hydrolysate; embryo development was in liquid SH medium without plant growth regulators (SH-0); and embryo maturation and germination occurred on solid SH-0 medium. Callus proliferation in SH-30 suspension cultures was greatest when callus was inoculated into the liquid medium at a relatively high concentration of 4% (4 g callus/100 ml medium), but the induction of somatic embryos was highest in this medium if the callus was inoculated at a lower concentration (<2%). In a second experiment, somatic embryo yield was highest when SH-0 development medium was inoculated with suspension culture callus at 0.1% concentration and declined markedly as inoculation concentration increased. Cell concentration is a critical factor in regulating the somatic embryogenesis response in orchardgrass suspension cultures.     

Alkaloid production in Vernonia cinerea: Callus, cell suspension and root cultures

Fast-growing callus, cell suspension and root cultures of Vernonia cinerea, a medicinal plant, were analyzed for the presence of alkaloids. Callus and root cultures were established from young leaf explants in Murashige and Skoog (MS) basal media supplemented with combinations of auxins and cytokinins, whereas cell suspension cultures were established from callus cultures. Maximum biomass of callus, cell suspension and root cultures were obtained in the medium supplemented with 1 mg/L alpha-naphthaleneacetic acid (NAA) and 5 mg/L benzylaminopurine (BA), 1.0 mg/L NAA and 0.1 mg/L BA and 1.5 mg/L NAA, respectively. The 5-week-old callus cultures resulted in maximum biomass and alkaloid contents (750 microg/g). Cell suspension growth and alkaloid contents were maximal in 20-day-old cultures and alkaloid contents were 1.15 mg/g. A 0.2-g sample of root tissue regenerated in semi-solid medium upon transfer to liquid MS medium containing 1.5 mg/L NAA regenerated a maximum increase in biomass of 6.3-fold over a period of 5 weeks. The highest root growth and alkaloid contents of 2 mg/g dry weight were obtained in 5-week-old cultures. Maximum alkaloid contents were obtained in root cultures in vitro compared to all others including the alkaloid content of in vivo obtained with aerial parts and roots (800 microg/g and 1.2 mg/g dry weight, respectively) of V. cinerea.     

Calli and suspension cultures for biomass production ofEuphorbia characias L. subsp.characias

Summary Friable calli and cell suspension cultures were obtained from leaf segments ofEuphorbia characias L. subsp.characias, in Murashige and Skoog (MS) basal medium supplemented with 1g.l^–1 casein hydrolyzate (CH), 5mg.l^–1 ascorbic acid, 1.0mg.l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.75mg.l^–1 benzyl adenine (BA). The highest callus specific growth rate (=0.085.day^–1), calculated for 1 year old calli cultures, was obtained with 0.25 mg.l^–1 2,4-D and 0.50mg.l^–1 BA. Suspension cultures started with an inoculum of 8.0×10⁴ cells.ml^–1 in supplemented liquid MS medium, gave a specific growth rate =0.256.day^–1.     

Callus formation from protoplasys of Sorghum cell suspension cultures

Cell suspension cultures have been obtained from three cultivars of Sorghum bicolor L. Moench. Protoplasts readily obtained from these cultures underwent sustained cell division and callus formation.     

A structured growth model for Cynara cardunculus cell suspension

In this study a model was developed to describe the growth of Cynara cardunculus L. suspended cells as a function of the availability of two substrates, sucrose as the carbon and energy source and phosphate. It was assumed that the maintenance energy need was fulfilled by the consumption of extracellular carbohydrates, in non-limiting conditions, or by the consumption of structural biomass when sucrose is depleted. A production of secondary metabolites was also assumed. This model was developed based on a structured model previously described by Van Gulik et al. (1993). The model was applied to the experimental results of C. cardunculus suspended cells grown in a Gamborg B₅ medium supplemented with 2% sucrose, using a non-linear regression program.     

Callus and cell suspension cultures of Rudgea jasminoides,a tropical woody Rubiaceae

Rudgea jasminoides is a woody Rubiaceae that produces phytoalexins in response to fungal inoculation, the response being dependent of the seasonal conditions. With the aim of studying phytoalexin induction under controlled conditions, callus cultures were established from petiole explants of R. jasminoides on a modified basal MS medium supplemented with picloram alone or in combination with kinetin. The highest frequency of callus formation was observed in solid medium containing 2.22 M kinetin and 2.07 M picloram. Development of fast-growing friable white callus was achieved in the absence of kinetin, in cultures supplemented only with 8.28 M picloram. Cell suspension cultures were established from this friable callus by transferring pieces directly to the same medium without agar. Preliminary experiments revealed that cell suspension cultures of R. jasminoides represent a useful system to analyse induced defensive metabolites produced by this Rubiaceae species.     

Salvia suspension cultures as production systems for oleanolic and ursolic acid

Oleanolic acid (OA) and ursolic acid (UA) are triterpenic acids with diverse biological activities that are of interest to the pharmaceutical industry. To investigate the scope for producing these compound using cell suspension cultures of Salvia species, calli from Salvia officinalis, S. virgata and S. fruticosa were induced using several plant growth regulator combinations. Eleven lines were selected for suspension induction from a pool of calli. Six suspension cultures were established successfully and cultivated in the respiration activity monitoring system (RAMOS^®) to obtain online data on their growth kinetics and to establish appropriate sampling schedules for the determination of their OA and UA production. Based on their observed growth behaviour, OA and UA contents, and aggregation properties, one suspension culture from each studied Salvia species was selected for further optimisation. The µ_max values for these suspension cultures ranged from 0.20 to 0.37 day^?1, their OA and UA contents were greater than 1.3 and 1.2 mg g^?1, respectively, and they afforded maximum volumetric yields of 21.0 mg l^?1 for OA and 32.8 mg l^?1 for UA. These results will be useful in the development of a refined Salvia suspension-based process for OA and UA production.     

Immobilization of Rubia tinctorum L. suspension cultures and its effects on alizarin and purpurin accumulation and biomass production

The present study is investigating the immobilization of Rubia tinctorum L. suspension cultures. The effects of three inoculation volumes and three immobilization materials (loofa, sisal and jute) on fresh and dry weights of biomass as well as on alizarin and purpurin production were determined in this study. Two grams of four-week old callus tissue were transferred to liquid medium to establish suspension cultures. After four weeks, suspension cultures of R. tinctorum at concentration of 8?×?10⁵?living cells/ml were immobilized with lignocellulosic materials and the cells were attached to all immobilization materials at the end of the first week and started to form aggregates on them. At the fourth week of these batch systems, biomass was measured approximately three times higher than the starting suspension cultures. The highest fresh weight was obtained (339.40?g/l) from sisal with ? inoculation ratio. Immobilization materials and inoculation volumes had an effect on dry weights, and accordingly, the most effective combinations were jute with ? (J3) and ? (J1) inoculation volumes with 7.86 and 7.82?g/l dry weights, respectively. Alizarin and purpurin contents of immobilized cells, analyzed with U-HPLC method, were 6.05 and 22.91 times higher than inoculated cells. All immobilization materials used in this study had no negative effect on to cells and biomass accumulation was enhanced. Concomitantly with rapid biomass increase, alizarin and purpurin production was ascended.     

New disposable tubes for rapid and precise biomass assessment for suspension cultures of mammalian cells

We present a new approach for biomass assessment in cell culture using a disposable microcentrifuge tube. The specially designed tube is fitted with an upper chamber for sample loading and a lower 5 microL capillary for cell collection during centrifugation. The resulting packed cell volume (PCV) can be quantitatively expressed as the percentage of the total volume of the sample. The present study focused on the validation of the method with mammalian cell lines that are widely used in bioprocessing. Using several examples, the PCV method was shown to be more precise, rapid, and reproducible than manual cell counting.     

Callus formation from protoplasts of cell suspension cultures of Rosa 'Paul's Scarlet'

Sustained divisions of protoplast-derived cells obtained from cell suspension cultures of Rosa‘Paul's Scarlet’ leading to colony and callus formation was achieved. The rather low plating efficiencies observed for agar-plated protoplasts were partly due to early arrests in further development of dividing protoplast-derived cells and cell clusters. Successful cultivation of dividing protoplast-derived cells was particularly dependent upon frequent subculturing of the precultures and on an efficient procedure for viable protoplast isolation. Colony formation was largely independent of medium composition.     

Correlation between biomass and medium conductivity in suspension cultures of rice cells

Summary An intercept in the linear relationship between the biomass increase and decrease in medium conductivity was found in suspension cultures of rice cells. It was due to the. consumption of medium salts by the cells during the lag phase. An equation was established for accurate monitoring of the cell growth which aids understanding of the cell physiology especially during the initial stage of cell growth.     

Increased shikonin production inLithospermum erythrorhizon suspension cultures within situ extraction and fungal cell treatment (elicitor)

Summary The effect ofin situ extraction and elicitor treatment on shikonin production was studied with the suspension cultures ofLithospermum erythrorhizon. Shikonin concentration of 60 mg/L was achieved by the use of both techniques which was 24 times higher than that of control culture, and 65 times higher in terms of shikonin productivity. The host-pathogen effect of elicitor treatment andin situ extraction for product removal were effective for shikonin production.     

Taxol production in suspension cultures of Taxus baccata

The response of Taxus baccata (PC2) to basic manipulations of culture conditions is described. Suspension cultures of Taxus baccata (PC2) were maintained at 25°C on a modified B5 medium with two-week transfers. Under these conditions, no taxol^® is formed. However, if the cells are left in the same medium for 7 or more additional days, taxol is produced and released (ca. 90%) into the extracellular medium. Levels as high as 13 mg 1^–1 extracellular taxol were achieved in shake flask cultures and taxol was the primary taxane formed representing between 50 and 80% of total taxane in the medium. The cells are sensitive to changes in culture conditions and cultures cycle through periods of high (13 mg 1^–1) and low (<0.1 mg 1^–1) levels of taxol production during extended culture. Picloram was the most effective of the auxins tested with respect to cell growth but it suppressed taxol production. Addition of fructose to moderately-productive cultures (ca. 4 mg 1^–1) improved taxol production, but cultures in a high producing state did not respond. Glucose suppressed taxane production. Two isoprenoids (geraniol and pinene) had a modest effect on taxol production when added to cultures at 10 mg 1^–1.®|Taxol is a registered trademark of Bristol Meyer Squibb for paclitaxel     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Physical stress for overproduction of biomass and flavonoids in cell suspension cultures of Boesenbergia rotunda

Flavonoid, a bioactive compound isolated from the rhizomes of Boesenbergia rotunda, exhibited antibiotic and anti-inflammatory properties and has also shown high inhibitory activity of dengue-2 virus protease. Several factors are responsible for the production of flavonoid in cell cultures. In the present study, the effects of initial inoculation volume, temperature and speed of agitation on cell growth, total and selected flavonoid in suspension cultures of B. rotunda were determined. High performance liquid chromatography analysis showed that a 2 % inoculation volume induced a significantly high accumulation of biomass and of flavonoid in the cells. The cells cultured at 25 °C showed significantly high biomass and selected flavonoid accumulation while differences in medium agitation significantly affected the yield of selected flavonoid.     

Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

Callus production,suspension culture and in vitro alkaloid yields of Ephedra

The effects of a range of plant growth regulators on callus production in various Ephedra species were examined. Species examined were E. andina, E. distachya, E. equisitina, E. fragilis var, camplyopoda, E. gerardiana, E. intermedia, E. major ssp procera, E. minima and E. saxatilis. All species produced callus on modified MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Neither indole-3-acetic acid nor 3-indolebutyric acid induced significant callus formation but the latter maintained growth of established callus cultures in several species. Suspension cultures of several species were established in MS medium supplemented with 0.25 M kinetin and 5.0 M 2,4-dichlorophenoxyacetic acid or 1-naphthaleneacetic acid. Sustained fresh weight doubling times of 70±7h were recorded for cell suspension cultures of E. andina grown in a semi-continous air-lift bubble bioreactor and a minimum doubling time of 56 h was recorded for E. andina in batch culture. It also proved possible to immobilise E. andina batch cultures in sodium alginate beads.Neither parent plants or in vitro cultures of E. distachya, E. fragilis or E. saxatilis produced alkaloids. Trace quantities of 1-ephedrine and trace-0.14% dwt d-pseudoephedrine were produced by in vitro cultures of other species. The ability to produce alkaloid diminished to zero with successive subcultures.Abbreviations Eph 1-ephedrine - Peph d-pseudoephedrine - RGR relative growth rate - KIN kinetin - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IBA 3-in-dolebutyric acid - IAA indole-3-acetic acid     

Bacoside production by suspension cultures of Bacopa monnieri (L.) Pennell

Cell suspension cultures of Bacopa monnieri (L.) Pennell, grown in modified MS medium, grew some 5–6 fold over 40 days. Selected cell lines produced the important saponin, bacoside A, up to 1 g/100 g dry wt after this time.