Adsorption ofCatharanthus roseus cells on various support particles

Summary Adsorption ofC. roseus cells producing indole alkaloids on various support particles were investigated in an attempt to find a suitable support material for surface culture of plant cells. Five different support particles namely gelatin, agar, alginate, polypropylene and glass beads were tested. Gelatin was found to be the most effective adsorbent resulting in nearly 30% adsorption of cells initially present in suspension. Adsorption isotherm of cells on gelatin beads was represented by a three parameter expression due to sigmoidal shape of the isotherm. The constants of the adsorption isotherm were determined using the experimental data.     

Enhancement of producing catharanthine by suspension growth ofCatharanthus roseus

Summary Increased production of catharanthine (about 200 g/mL) was obtained at low light intensity under chemostat cultivation as compared with batch and fed-batch processes. Photo-inhibition was observed at high light intensity. Cells after being frozen with 5% DMSO produced more catharanthine (up to 350 g/L) in batch culture than those from conventional culture techniques.     

Biotransformation of vinblastine to vincristine by cell suspension cultures ofCatharanthus roseus

Summary Cell suspension cultures ofCatharanthus roseus convert vinblastine to vincristine after 2 days incubation.     

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.     

Indole alkaloid production by transformed and non-transformed root cultures ofCatharanthus roseus

Summary Ten transformed and two non-transformed root lines ofCatharanthus roseus were established. A systematic study of the growth kinetics and alkaloid content was performed over a culture cycle and showed significant differences between transformed and non-transformed cultures. Mean doubling times for transformed and normal root lines were 2.8 and 19.5 days, respectively. Alkaloid content in hairy roots was from two- to threefold higher than in the non-transformed tissues. The established transformed root lines produced a wide variety of indole alkaloids as can be observed from their complex thin layer chromatography patterns. A large quantity of serpentine was determined in two of the transformed root cultures. Alkaloid content, both quantitatively and qualitatively, has been stable in the hairy root cultures for more than 2 yr of subculturing.     

Ultrastructure ofCatharanthus roseus cells culturedin vitro and exposed to conditions for alkaloid accumulation

Summary Periwinkle (Catharanthus roseus) cells cultured in 1-B 5 medium display the ultrastructure of parenchyma cells. The parenchyma character remained unchanged when cells were exposed to any one of three different conditions effecting alkaloid accumulation. Transfer of cells to alkaloid production medium for 2 weeks (condition 1) accorded two special features,i.e., unusually big lipid droplets in the cytoplasm and, upon fixation, one or several electron-dense droplets of spongy precipitate in vacuoles. Among hormone-autotrophic cultures (condition 2) some cells showed a fine electron-dense vacuolar precipitate. Addition ofPhythium homogenate (fungal elicitor) to cells cultured in 1-B 5-medium for 10 days (condition 3), cells showed a frequent appearance of singular big lipid droplets in the cytoplasm, whereas vacuoles remained devoid of precipitate. The appearance of big lipid droplets and of vacuolar precipitate is interpreted as progressing cytodifferentiation, but is coincidental with alkaloid accumulation.NRCC no. 24524.     

Effect of aeration rate and influence of pCO2 in large-scale cultures ofCatharanthus roseus cells

Summary Catharanthus roseus cells were grown at various aeration rates using normal or CO₂-enriched air. Kinetic data showed a detrimental effect of the increase of the gassing rate on the growth characteristics due to CO₂ stripping. When the CO₂ partial pressure in the culture was maintained at a constant level of 20 mbar, better growth and enhanced conversion yields were obtained.     

Comparative effects of fusion facilitators on electrofusion attributes of N. tabacum mesophyll protoplasts

Mesophyll protoplasts isolated from in vitro-grown Nicotiana tabacum L. shoots were subjected to electrofusion.Dielectrophoresis was induced by an AC field of 50 V cm^-1 inter-electrode distance and 0.5 MHz oscillation frequency. Fusion was effected by two 0.7 kV cm^-1 DC pulses, each of 50 s duration, applied within one second of each other. Various chemical treatments were tested for their effects on dielectrophoresis efficiencies (percentages of protoplasts that made contact with at least one other protoplast under the AC field), fusion efficiencies (percentages of protoplasts participating in fusion events), cell lysis (percentages of protoplasts bursting during the electrofusion processes), overall viabilities of fusion products 24 h post-fusion and overall plating efficiencies 7 d post-fusion (percentages of fusion-derived cells that had undergone division). The various attributes assessed on the electrofusion of protoplasts in the control treatment, 10% mannitol, differed considerably for experiments carried out on different days. Relative to the control treatment, only the Ca²⁺ treatments, and to a lesser extent lipase treatment reduced dielectrophoresis efficiencies. Polyamines, cytochalasins and Ca²⁺ treatments significantly reduced cell lysis percentages. All electrofusion facilitators tested (except for spermine at 150 mg l^-1, the cytochalasins B and D, and Ca²⁺ treatments) increased fusion efficiencies to more than 1.5 times those obtained with the standard 10% mannitol electrofusion medium. Ca²⁺ treatments increased overall viabilities of fusion products by more than 1.5 times. With the exception of the prostaglandins, lecithin and CaCl₂ treatments, overall plating efficiencies were reduced by treatment of protoplasts with fusion facilitators. Substantial increases in overall plating efficiencies over those observed in the control treatment were obtained using prostaglandin F_2a, lecithin and CaCl₂.2H₂O treatments. The implications of the results are discussed.Abbreviations AC alternating current, approx.-approximately - BA benzylaminopurine, cv.-cultivar - DC direct current, diam.-diameter - FDA fluorescein diacetate - MS Murashige & Skoog (1962) - NAA napthaleneacetic acid - PCM protoplast culture medium - PIM protoplast isolation medium - PPM protoplast purification medium - rpm revolutions per minute - SD(n) standard deviation of a variate - SEM standard error of the mean     

Genetic transformation ofCatharanthus roseus G. Don byAgrobacterium rhizogenes

Catharanthus roseus plantlets were inoculated with differentAgrobacterium rhizogenes strains. This plant species is known to produce secondary metabolites and axenic hairy-root cultures are an alternative to extraction of plant tissue for the compounds which are synthesized in roots. Hairy root lines were established from inoculations with the agropine strain 15834 and transformed plants were obtained after spontaneous regeneration. Phenotypic alterations of both the root system and the aerial parts were observed in transformed plants. All the tissues analyzed contained agropine and mannopine. T-DNA analysis confirmed the presence of TL- and TR-DNAs either as distinct inserts, or as a single and continuous insert including the region between TL and TR on pRi 15834.     

Factors affecting the electrofusion efficiency ofPorphyra protoplasts

The effects of various factors on the electrofusion efficiencies ofPorphyra protoplasts were investigated. These factors were protoplast stabilizing reagents, divalent cations, membrane digestive enzymes and cold storage of the protoplasts. Fusion efficiencies were dependent on the concentrations of reagents used to adjust the osmotic pressure of the medium. With mannitol or sorbitol the maximum fusion efficiency (approximately 16%) was observed at concentrations of 0.6 to 0.7 M; glucose was less effective. Brief treatment of the protoplasts with pronase stimulated electrofusion, whereas treatment with proteinase K, trypsin, phospholipase C or lipase repressed fusion. The addition of Ca²⁺ at 10^-5 to 10^-4 M in the protoplast medium enhanced the fusion efficiency to approximately four times that of the non-treated control. Sr²⁺ and Co²⁺ also stimulated electrofusion, but less effectively than Ca²⁺. The fusion capacity of the protoplasts remained stable for about 3 h when kept on ice, but decreased gradually when left at room temperate.     

Indole alkaloid production by hairy root cultures ofCatharanthus roseus: Growth kinetics and fermentation

Summary Growth kinetics and indole alkaloid production ofCatharanthus roseus hairy root cultures were studied in shake flasks and in a small scale fermenter. A logistic growth model commonly used for microbes described well the growth of hairy roots. Of the several parameters analyzed during the cultivation of hairy roots, a linear relationship between sucrose consumption and dry weight increase was obtained. This suggests the validity of sugar analysis as a means in monitoring the growth of hairy roots in fermenters.     

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

Effect of acetylsalicylic acid on secondary metabolism ofCatharanthus roseus tumor suspension cultures

Summary Addition of various concentrations (0.5–20 mM) of acetylsalicylic acid (ASA) to tumor lines ofCatharanthus roseus cultivatedin vitro and requiring corn starch as carbon source, produced remarkable effects on secondary metabolite production. An increase of 505% total alkaloids per culture (cells plus liquid medium), 1587% total phenolics (liquid medium), 612% total furanocoumarins (liquid medium) and 1476% total anthocyanins (liquid medium) was detected. 1 mM ASA in combination with other elicitors, such as homogenates ofAspergillus fumigatus or trans-cinnamic acid, did not further increase the metabolite content substantially. The results suggest that ASA could act as a new biotic elicitor of metabolite production inC. roseus cell suspension culture.     

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.     

Improvement of the catharanthine productivity in hairy root cultures ofCatharanthus roseus by using monosaccharides as a carbon source

Summary Sucrose, glucose, and fructose as carbon sources in culture medium were assessed in hairy root cultures ofCatharanthus roseus. The cultures preferentially consumed sucrose, resulting in about 40% (dry wt) higher growth rate. However, fructose enhanced the cathranthine yield about two-fold. The elevated yield was not seemingly ascribed to the higher osmolarity per unit weight of fructose than sucrose. A two stage culture using sucrose (1st) and fructose (2nd) improved volumetric yields of catharanthine about two-fold, i.e. 41 mg/l.     

Modulation and direction of the electrofusion response in plant protoplasts

In experiments on electrofusion of protoplasts (from Solanum brevidens, S. tuberosum, Nicotiana plumbaginifolia) the presence of divalent cations (Ca²⁺) at 1 mM in the fusion medium was found to increase the yield of hybrids observed directly after fusion and decrease the duration of pulse needed for fusion. Pretreatment of protoplasts with the polyamine spermine also enhanced fusion yield, and when combined with 1 mM Ca²⁺ the effects were additive. The improvement in fusion yield (2–4 fold) was most marked for protoplast populations (e.g. from suspension cultured cells) that were least responsive to electrofusion in mannitol alone. Short term viability, judged from FDA fluorescence was found to be high at these increased fusion levels. Optimum fusion parameters for electrofusion thus may be determined from short term experiments. Attempts to direct to fusion response between populations of protoplasts of identical properties by pretreatment of one fusion partner with spermine were inconclusive.     

Protoplast regeneration and organogenesis from pea protoplasts

Summary Protoplasts of young Pisum Sativum L. seedlings from 7 different genotypes were isolated and regenerated to the callus stage. Germinating embryos were cultivated with cotyledons removed, thus avoiding intracellular starch accumulation in donor tissue. The first lateral shoots provided a source of homogenous meristematic cells which gave rise to sustained protoplast division and resulted in callus formation within 4, weeks. Root formation occurred on hormone-free medium and shoots developed on medium containing kinetin, 2iP or zeatin in the third subculture, when subcultured in monthly intervals. This work was made possible by a joint GFP/BMFT—project to H.-J. Jacobsen (grant A 24/87-ZF).     

Viable somatic hybrids are obtained by direct current electrofusion of chemically aggregated plant protoplasts

Using low levels of polyethylene glycol as aggregating agent, protoplasts of Nicotiana glauca and N. langsdorffii were electropulsed (square wave direct current electric pulses) and on subsequent culture on a selective medium (without phytohormones), calli developed. The hybrid character of some of the calli was demonstrated by direct comparison of the isoelectrophoretic pattern of the ribulose-1, 5-bisphosphate carboxylase subunits with those of the parent plants. In optimum conditions, the electrofusion technique described is about 30-fold more efficient for hybrid production than the conventional polyethylene glycol method when applied to the same plant systems.     

Protoplast fusion inStreptomyces avermitilis

Summary The power of protoplast fusion as a generally applicable method for obtaining genetic recombination is demonstrated by the recombination of genes involved in avermectin biosynthesis. A backcross ofStreptomyces avermitilis strain MA6202, an improved mutant that had lost the ability to carry out the methylation of the C-5 hydroxyl of the avermectin molecule, with the original soil isolate MA4680 resulted in the recovery of at least one unambiguous recombinant class despite the instability of rifampicin resistance, one of two markers initially used for recombinant selection. Such intrinsic instability is frequently encountered in streptomycete genetics, and this result delineates the utility of protoplast fusion as a genetic tool. Other difficulties addressed include recovery of complementary recombinant classes, differences in recombination frequency due to colony density on regeneration medium, and alteration in plating efficiency on diagnostic media following protoplasting and regeneration. The results of a cross between a nicotinamide auxotroph MRG1003 and a lysine auxotroph MRG 1004 are included to aid in the elucidation of these problems as well as to support the finding of homologous recombination inS. avermitilis.