Characterization of plant suspension cultures using the focused beam reflectance technique

Development of bioreactor systems utilizing plant suspension cultures has been hindered by the lack of on-line sensors for monitoring important process variables such as biomass concentration and aggregate size. An optical technique, the focused beam reflectance method (FBRM developed by Lasentec Inc., Redmond, WA), was used to characterize several plant suspension cultures: rice (Oryza sativa), tobacco (Nicotiana benthamiana) and wild Chinese cucumber (Trichosanthes kirilowii). These cultures differ in a number of respects such as individual cell size and morphology, aggregate shape and size distribution, initial culture density, and color. For plant suspensions comprised of relatively spherical aggregates (rice and cucumber), the area under the cube-weighted FBRM chord length distribution was linearly correlated to biomass concentration (R ²>0.99) while the mean of the cube-weighted FBRM chord length distribution was nonlinearly related to aggregate size.     

Analysis of aggregate size as a process variable affecting paclitaxel accumulation in Taxus suspension cultures

Plant cell aggregates have long been implicated in affecting cellular metabolism in suspension culture, yet the rigorous characterization of aggregate size as a process variable and its effect on bioprocess performance has not been demonstrated. Aggregate fractionation and analysis of biomass-associated product is commonly used to assess the effect of aggregation, but we establish that this method is flawed under certain conditions and does not necessarily agree with comprehensive studies of total culture performance. Leveraging recent advances to routinely measure aggregate size distributions, we developed a simple method to manipulate aggregate size and evaluate its effect on the culture as a whole, and found that Taxus suspension cultures with smaller aggregates produced significantly more paclitaxel than cultures with larger aggregates in two cell lines over a range of aggregate sizes, and where biomass accumulation was equivalent before elicitation with methyl jasmonate. Taxus cuspidata (T. cuspidata) P93AF cultures with mean aggregate sizes of 690 and 1,100 μm produced 22 and 11 mg/L paclitaxel, respectively, a twofold increase for smaller aggregates, and T. cuspidata P991 cultures with mean aggregate sizes of 400 and 840 μm produced 6 and 0.3 mg/L paclitaxel, respectively, an increase of 20-fold for smaller aggregates. These results demonstrate the importance of validating experiments aimed at a specific phenomenon with total process studies, and provide a basis for treating aggregate size as a targeted process variable for rational control strategies.     

Selective uptake of pyrrolizidine N-oxides by cell suspension cultures from pyrrolizidine alkaloid producing plants

The N-oxides of pyrrolizidine alkaloids such as senecionine or monocrotaline are rapidly taken up and accumulated by cell suspension cultures obtained from plants known to produce pyrrolizidines, i.e. Senecio vernalis, vulgaris, viscosus (Asteraceae) and Symphytum officinale (Boraginaceae). The transport of the N-oxides into the cells is a specific and selective process. Other alkaloid N-oxides such as sparteine N-oxide are not taken up. Cell cultures from plant species which do not synthesize pyrrolizidine alkaloids are unable to accumulate pyrrolizidine N-oxides. The suitability of the pyrrolizidine N-oxides in alkaloid storage and accumulation is emphasized.     

Epchrosine — a new indole alkaloid isolated from plant cell cultures of Ochrosia elliptica Labill

From plant cell suspension cultures of Ochrosia elliptica Labill. an indole alkaloid of the apparicine type has been isolated, which is not known to occur in differentiated plants. The structure of the new compound, named epchrosine, was established by UV, MS and high resolution two-dimensional ¹H NMR (COSY and NOESY) as (19R,20R)-epoxyapparicine.     

Characterization of aggregate size in Taxus suspension cell culture

Plant cells grow as aggregates in suspension culture, but little is known about the dynamics of aggregation, and no routine methodology exists to measure aggregate size. In this study, we evaluate several different methods to characterize aggregate size in Taxus suspension cultures, in which aggregate diameters range from 50 to 2,000 μm, including filtration and image analysis, and develop a novel method using a specially equipped Coulter counter system. We demonstrate the suitability of this technology to measure plant cell culture aggregates, and show that it can be reliably used to measure total biomass accumulation compared to standard methods such as dry weight. Furthermore, we demonstrate that all three methods can be used to measure an aggregate size distribution, but that the Coulter counter is more reliable and much faster, and also provides far better resolution. While absolute measurements of aggregate size differ based on the three evaluation techniques, we show that linear correlations are sufficient to account for these differences (R ² > 0.99). We then demonstrate the utility of the novel Coulter counter methodology by monitoring the dynamics of a batch process and find that the mean aggregate size increases by 55% during the exponential growth phase, but decreases during stationary phase. The results indicate that the Coulter counter method can be routinely used for advanced process characterization, particularly to study the relationship between aggregate size and secondary metabolite production, as well as a source of reliable experimental data for modeling aggregation dynamics in plant cell culture.     

Induction of pistil-like structures in suspension-derived callus cultures of wheat (Triticum aestivum)

In the following a method for the induction of pistil-like structures in wheat suspension cultures (Triticum aestivum L.) is described. In young influorescences of plants, which were artificially infected with the wheat bunt fungi (Tilletia controversa), organogenic calli with pistil-like structures could be induced on loblolly pine medium + 3 mg/l 3,6-dichloro-2-methoxy benzoic acid. The yield of these structures in calli from a five-month-old suspension culture was up to 100 per gram of callus fresh mass.Abbreviations LM Loblolly pine medium - Dicamba 3,6-dichloro-2-methoxy benzoic acid     

Isochorismate hydroxymutase from Rubiaceae cell suspension cultures

The enzyme catalysing the isomerisation of chorismic to isochorismic acid (isochorismate hydroxymutase E.C. 5.4.99.6) has been detected in protein preparations of various cell suspension cultures derived from plants of Rubiaceae species.Abbreviations OSB o-Succinylbenzoic acid - Tris Tris(hydroxymethyl)aminomethane - DEAE Diethylaminoethyl cellulose     

UDP-glucose:digitoxin 16′-O-glucosyltransferase from suspension-cultured Digitalis lanata cells

Suspension cultures from several cell lines of Digitalis lanata, as well as cultures from 6 other plant species were checked for their ability to form purpurea-glycoside A from digitoxin. An in-vitro assay for the UDP-glucose:digitoxin 16-O-glucosyltransferase (DGT, EC 2.4.1.-) has been established based on an HPLC method. The enzyme is located in the soluble fraction. Its pH optimum is at 7.4. No enzyme activity was found in either purified vacuole preparations or lysed vacuoles. Ascorbate (10 mM) increased the transferase activity about 4-fold. Of the sugar nucleotides tested, only UDP-glucose served as a glucosyl donor. Digitoxin, digoxin, -acetyldigitoxin, and -acetyldigoxin are substrates for the glucosyltransferase. The role of the DGT during the biotransformation of cardenolides in Digitalis lanata cell suspension cultures is discussed.Abbreviation DGT UDP-glucose:digitoxin 16-C-glucosyltransferase     

Induction and in vitro culture of soybean crown gall tumors

Induction of crown galls on 4–6 week old soybean (Glycine max (L.) Merr.) plants cultured in growth chambers was obtained with Agrobacterium tumefaciens strains C58, T37 and ACH5. The crown galls were isolated and cultured in vitro as sterile callus and liquid suspension cultures. Transformation was tested by opine tests and by molecular hybridization of restricted cell DNA with T-DNA fragments. Protoplasts were isolated from suspension cultures. Transformed protoplasts regenerate cell walls, divide and form calli without an exogenous supply of hormones.     

Effects ofVerticillium albo-atrum culture filtrate on somatic embryogenesis in alfalfa

Cell suspensions derived from young petioles of alfalfa (Medicago sativa L.) were cultured in the presence and absence of aVerticillium albo-atrum culture filtrate (20% v/v) for 6 cycles. The frequency of somatic embryogenesis and the growth rate of the suspension cultures were investigated at each cycle. Somatic embryogenesis in the filtrate-treated cultures declined but was still at a relatively high level after 6 subcultures, compared to controls cultures which virtually lost the capacity for embryo formation in the same period. The decline in the embryogenic capacity of filtrate treated-cultures was accompanied by a six-fold increase in the rate of growth of the cultures.     

The effect of physical stress on plant cells in suspension cultures

Summary The effect of the physical (hydrodynamic) stress generated by aeration and agitation in suspension cultures onCatharanthus roseus cells was studied. The intensity of the physical stress was closely related to the growth rate of the cells, the maximum cell mass, the size distribution of cell aggregates, the contents of the cell wall, and each component of the cell wall.     

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.Abbreviations FW fresh weight - I₅₀ inhibitor concentration at which cell growth is inhibited by 50% - MS Murashige and Skoog (1962) culture medium - ZM Black Mexican Zea mays suspension culture of Chourey and Zurawski (1981)     

Directed electrofusion between protoplasts with different responses in a mass fusion system

Protoplasts of Solanum brevidens (leaves) and Nicotiana rustica (suspensions) have been aligned and fused electrically between widely spaced electrodes, and the yield of 1:1 (binary) fusion products in chains of aligned protoplasts has been determined by light microscopy. Leaf protoplasts fuse more easily than protoplasts from suspension cultures (Tempelaar and Jones, 1985), thus electrical parameters and the ratio of leaf: suspension protoplasts can be varied to control the yield of binary and multifusion products. In experiments to determine optimum ratios for electrofusion, up to 60–70% of S. brevidens — N. rustica fusion products were binary at overall fusion frequencies of 40–50%.Fusions in samples of protoplasts with the same characteristics can also be controlled to direct the fusion process towards binary products. However, in this case, at least half of the binary products may be derived from self-fusions.     

Phenylalanine ammonia-lyase activity in suspension cultures of Ulmus pumila and U. campestris treated with spores of Ceratocystis ulmi

Summary Cell suspension cultures of a Ceratocystis ulmi-resistant (Ulmus pumila) and a -susceptible elm (U.campestris) were established from leaf callus tissue. Treatment of cultures with spores of C.ulmi induced a large increase in the activity of phenylalanine ammonialyase, only in the cells of the resistant species U.pumila with a maximum after 24 h. Inoculated U.pumila cells also excreted a red unidentified chemical into the culture medium. Neither responses were induced in inoculated U.campestris cultures. The results are discussed in relation to the development of the elm cell culture system as a model for studying the differential biochemical mechanisms of disease resistance in elms.     

Plant regeneration from cell suspension cultures ofArabidopsis thaliana heynh

A technique is described for the establishment, maintenance, and regeneration of plants from cell suspension cultures ofArabidopsis thaliana (L) Heynh. Friable, rapidly growing cell suspension cultures were initiated from leaf or hypocotyl callus cultures and these have been maintained in liquid culture for 24 months. The cells grown in liquid culture were used to study the effects of growth regulators, medium salts composition, culture temperature, sucrose concentration and medium solidifying agents on morphogenesis. The most important parameters for plant regeneration were culture temperatures lower than 25°C, the medium solidification agent gelrite at 0.2% (w/v) and zeatin or thidiazuron as the choice of cytokinin. These cell suspensions continue to regenerate fertile plants with a total of over 200 plants having been rooted to date and they also serve as convenient sources of cells for protoplast isolation, biochemical, and molecular assays.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indoleacetic acid - BAP 6-benzylaminopurine     

Stable endosperm cell suspension cultures from wild-type and opaque-2 maize

Stable cell suspension cultures have been established from immature endosperms of A69Y wild-type and opaque-2 maize (Zea mays L.). Cultured cells are capable of storage protein (zein) synthesis and accumulation throughout the growth period. Electrophoretic patterns of zeins show, for opaque-2 cells, the preferential inhibition of the accumulation of 22 kDa peptides typical of the mutation. Viable protoplasts, able to regenerate cell walls, as well as to divide and to express foreign DNA in transient expression experiments, can be obtained with high yields from cultures of both genotypes.Abbreviations 02 opaque-2 - wt wild-type - DAP days after pollination - PCV packed cell volume - f.w. fresh weight - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - CAMV cauliflower mosaic virus - CAT chloramphenicol-acetyl-transferase     

Regeneration of haploid and dihaploid plants from protoplasts of supersweet (sh2sh2) corn

Summary Plants were regenerated from maize (Zea mays L.) protoplasts isolated from embryogenic cell suspensions. The donor maize suspension cultures were established from friable callus initiated from microspores of a commercial supersweet hybrid (sh2sh2). The frequency of cell colony formation was higher when protoplasts were cultured on feeder layers of maize cells as compared with a liquid thin layer method. It was demonstrated that haploid and dihaploid soil-grown plants can be regenerated from maize protoplasts isolated from haploid cell cultures.     

Steroidal alkaloids in tissue cultures and regenerated plants of Solanum dulcamara

Callus and cell suspension cultures were established with shoots of the soladulcidine variety of the bittersweet Solanum dulcamara L. Plantlets were regenerated from undifferentiated callus. From mixotrophic callus as well as mixotrophic suspension cultures soladulicidine, solasodine and the corresponding neutral spirostanes tigogenin and diosgenin were isolated and identified by thin layer chromatography and mass spectrometry. Total alkaloid concentrations were about 0.2 mg/g dry weight (callus) and 0.1 mg/g dry weight (green suspension cultures). In the heterotrophic cell line only the neutral sapogenins could be detected. Alkaloid accumulation in callus of Solanum dulcamara could be enhanced by the induction of organogenesis. The shoots of the regenerated plants from the mixotrophic callus contained soladulcidine (1.6 mg/g dry weight) and tigogenin. Thus, in concentration and composition the regenerated plants equalled the source plant.Abbreviations 2.4-D 2.4-dichlorophenoxyacetic acid - NAA naphthylacetic acid - HPLC high performance liquid chromatography - TLC thin layer chromatography     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt