Induction of efficient cell division in alfalfa protoplasts

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.Contribution No. 774 Ottawa Research Station     

Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Plant regeneration through somatic embryogenesis from suspension culture-derived protoplasts of Paspalum scrobiculatum L.

Protoplasts were released from embryogenic suspension culture of Paspalum scrobiculatum and cultured in either liquid or semisolid KM medium supplemented with 2,4-D in the dark at 24°C with or without a feeder layer. Cell wall formation was observed in 75% of the plated protoplasts. Microcolonies developed after 10 d of culture, which in turn formed callus upon transfer to M-2 medium (Nayak and Sen, 1989). The highest plating effeciency (ca 7%) was obtained in thin-layer liquid culture. The macrocalli formed somatic embryos which regenerated to plantlets. The plantlets were grown to flowering plants upon transfer to soil.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA Fluoresceine diacetate - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige & Skoog medium (1962)     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

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.     

Effects of different feeder layers on culture of bovine embryonic stem cell-like cells in vitro

To find a suitable feeder layer is important for successful culture conditions of bovine embryonic stem cell-like cells. In this study, expression of pluripotency-related genes OCT4, SOX2 and NANOG in bovine embryonic stem cell-like cells on mouse embryonic fibroblast feeder layers at 1–5 passages were monitored in order to identify the possible reason that bovine embryonic stem cell-like cells could not continue growth and passage. Here, we developed two novel feeder layers, mixed embryonic fibroblast feeder layers of mouse and bovine embryonic fibroblast at different ratios and sources including mouse fibroblast cell lines. The bovine embryonic stem cell-like cells generated in our study displayed typical stem cell morphology and expressed specific markers such as OCT4, stage-specific embryonic antigen 1 and 4, alkaline phosphatase, SOX2, and NANOG mRNA levels. When feeder layers and cell growth factors were removed, the bovine embryonic stem cell-like cells formed embryoid bodies in a suspension culture. Furthermore, we compared the expression of the pluripotent markers during bovine embryonic stem cell-like cell in culture on mixed embryonic fibroblast feeder layers, including mouse fibroblast cell lines feeder layers and mouse embryonic fibroblast feeder layers by real-time quantitative polymerase chain reaction. Results suggested that mixed embryonic fibroblast and sources including mouse fibroblast cell lines feeder layers were more suitable for long-term culture and growth of bovine embryonic stem cell-like cells than mouse embryonic fibroblast feeder layers. The findings may provide useful experimental data for the establishment of an appropriate culture system for bovine embryonic stem cell lines.     

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.     

An embryogenic cell suspension culture of Picea glauca (White spruce)

A cell suspension culture of Picea glauca (White spruce) which continuously produces somatic embryos has been established. Embryogenic callus derived from cultured zygotic embryos was used to initiate the culture. Numerous embryos at various early stages of development were recognized; they exhibited a meristematic embryonic region and suspensor consisting of elongate, vacuolated cells. The culture also contained clumps of meristematic cells and large irregular — shaped cells. The culture could be readily re-established on solid medium.     

Protoplast-to-plant regeneration in cotton (Gossypium hirsutum L. cv. Coker 312) using feeder layers

Summary We report the regeneration of protoplasts isolated from two embryogenic cell lines of Gossypium hirsutum L. cv. Coker 312 initiated from hypocotylderived callus. Protoplasts plated on cellulose nitrate filters and placed over feeder layers formed embryogenic callus from which plants were regenerated. Plating efficiency up to 12.8% depended upon the cell line. Addition of phytohormones to the protoplast medium had no stimulating effect on plating efficiency. The influence of feeder cells and conditioned medium on plating efficiency was significantly different for the two cell lines.Abbreviations ACM autoclaved conditioned medium - AFC autoclaved feeder cells - BM basic medium - BM+ basic medium with phytohormones - CM non-autoclaved conditioned medium - FC non-autoclaved feeder cells - FDA fluorescein diacetate - MM maturation medium - NAA 1-naphtaleneacetic acid - PCM protoplast culture medium - PCM+ protoplast culture medium with phytohormones - SC settled cells - 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylamino purine     

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H⁺ extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H⁺ extrusion. These observations can be explained on the basis of a plasmamembrane located, H⁺-translocating redox system. The electrochemical technique is a useful method of studying this system.     

Production of L-DOPA from cell suspension culture of Mucuna pruriens f. pruriens

Summary Production of L-DOPA was studied in cell suspension culture of Mucuna pruriens f. pruriens. Suspension culture was established in MSI medium composed of half concentration of Murashige and Skoog's salts and 2% sucrose. A two-stage cell suspension culture was developed for enhanced accumulation of L-DOPA. In the first stage, the culture system was composed of MSI medium without CaCl, which was suitable for cell growth and in the second stage MSI medium containing 42.5 mg.l^–1 KH₂PO₄ and 4% sucrose favoured L-DOPA production. A discernible higher production of L-DOPA was obtained in this two-stage cell suspension culture in comparison to single stage culture.     

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.     

Plant regeneration from indica rice (Oryza sativa L.) protoplasts

Rice (Oryza sativa L.) plants of the indica cultivar IR54 were regenerated from protoplasts. Conditions were developed for isolating and purifying protoplasts from suspension cultures with protoplast yields ranging from 1·10⁶ to 15·10⁶ viable protoplasts/1 g fresh weight. Protoplast viability after purification was generally over 90%. Protoplasts were cultured in a slightly modified Kao medium in a Petri plate by placing them onto a Millipore filter positioned on top of a feeder (nurse) culture containing cells from a suspension culture of the japonica rice, Calrose 76. Plating efficiencies of protoplasts ranged from 0.5 to 3.0%; it was zero in the absence of the nurse culture. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the protoplasts. After three weeks the Millipore filter with callus colonies were transferred off feeder cells and onto a Linsmaier and Skoog-type medium for an additional three weeks. Selected callus colonies that had embryo-like structures were then transferred to regeneration medium containing cytokinins, and regeneration frequencies up to 80% were obtained. Small shoots emerged and were transferred to jars for root development prior to transferring to pots of soil and growing the plants to maturity in growth chambers. Of the cytokinins evaluated, N⁶-benzylaminopurine was the most effective in promoting shoot formation; however, kinetin was also somewhat effective. Regeneration medium could be either an N₆ or Murashige and Skoog basal medium. Of 76 plants grown to maturity, 62 were fertile, and the plant heights averaged about three-fourths the height of seed-grown plants.Two other suspension cultures of IR54, one developed from the protoplast callus of the initial IR54 line, and the other developed from callus produced by mature seeds, have yielded protoplasts capable of regenerating plants when using cells of the Calrose 76 suspension as a nurse culture. In addition, protoplasts obtained from three-week-old primary callus of immature embryos of IR54 were capable of regenerating plants when using the same culture conditions.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - pcy packed cell volume - BAP N⁶-benzylaminopurine - FDA fluorescein diacetate - FW fresh weight - IAA indole-3-acetic acid Media AA Muller and Grafe (1978) - CPW Frearson et al. (1973) - Kao^* Kao (1977) - LS Linsmaier and Skoog (1965) - MS Murashige and Skoog (1962) - N₆ Chu et al. (1975) - PCM Ludwig et al. (1985)     

Plant regeneration through somatic embryogenesis from suspension cultures of a minor millet,Paspalum scrobiculatum

Compact, friable and embryogenic calli were initiated from immature inflorescences and young leaf bases of one week old seedlings of Paspalum scrobiculatum cultured on MS medium supplemented with 2,4-D. A stable, embryogenic suspension culture was initiated from these calli and maintained in a liquid version of the same MS medium. Embryogenic calli and somatic embryos were obtained by plating suspension culture cells onto semi-solid medium containing 2,4-D. Complete, normal plantlets developed on 2,4-D free medium at a high frequency from somatic embryos. NAA and BAP in the medium promoted plant development.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA Abscisic acid - MS Murashige and Skoog (1962) - CM Coconut milk     

Alkaloid production in cell suspension cultures of Thalictrum flavum and T. dipterocarpum

Both cell suspension cultures of Thalictrum flavum and T. dipterocarpum were found to produce berberine (0.3 and 0.4 g/l, respectively) as a main alkaloid. Berberine production in the latter was markedly stimulated by 1-naphthaleneacetic acid in combination with 6-benzylaminopurine, whereas it was rather suppressed by the same auxin in the former. T. flavum cultures accumulated berberine and columbamine in the cells without releasing them into medium. On the other hand, T. dipterocarpum cultures released berberine into medium during the logarithmic growth phase, but thereafter accumulated all the berberine synthesized in the cells.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - BA 6-benzylaminopurine - TFG a culture strain of T. flavum ssp. glaucum - TDP a culture strain of T. dipterocarpum     

Cyclic adenosine 3':5'-monophosphate in axenic rye grass endosperm cell cultures

Cyclic adenosine 3′:5′-monophosphate (cAMP) was extensively purified from rye grass (Lolium multiflorum) endosperm cells grown in axenic suspension culture. The cAMP was purified by neutral alumina and anion and cation exchange chromatography. The cAMP was quantitated by means of a radiochemical saturation assay using a beef heart cAMP-binding protein and also by an assay involving activation of beef heart protein kinase. The cAMP levels found (corrected for recovery of tracer cyclic 3′,5′-[8-³H]AMP included from the point of sample extraction) ranged from 2 to 12 pmol/g fresh weight. The material purified from rye grass cultures was indistinguishable from authentic cAMP with respect to chromatography in two cellulose thin layer systems, behavior on dilution in both the saturation and protein kinase activation assays, and rates of degradation by a mammalian cAMP phosphodiesterase. The cAMP from rye grass cultures was completely degraded by a mammalian cAMP phosphodiesterase, and 1-methyl-3-isobutylxanthine inhibited such degradation. The protein kinase activation and saturation assays gave essentially the same values for the cAMP content of axenic rye grass culture extracts. Material satisfying the above criteria for identity with cAMP was also isolated from the culture medium. The increase observed in medium cAMP levels during culture growth provides evidence for the synthesis and secretion of cAMP by rye grass endosperm cells in suspension culture.     

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)     

Isoquinoline alkaloids from cell suspension cultures of Fumaria capreolata

Fumaria capreolata was taken into cell suspension culture. The culture yielded a biomass of about 12 g dry weight per liter of medium; the dried cells contained ca. 0.1% of alkaloids. Besides choline, the following ten known isoquinoline alkaloids were isolated from the cell extract in crystalline form: coptisine, dehydrocheilanthifoline; (+)-isoboldine; magnoflorine; N-methylcoclaurine; (+)-reticuline; (–)-pallidine; protopine; sanguinarine; (–)-scoulerine. This is the most diverse isoquinoline alkaloid spectrum thus far published for a cell suspension culture.     

High frequency callus formation from maize protoplasts

Summary A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10³–10⁵ protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

A two-layer culture method was established that uses an organic solvent to remove shikonin derivatives produced on cell surfaces during the culture of suspension cells of Lithospermum erythrorhizon. Some paraffins and a fatty acid ester made suitable solvents, whereas olefins and aromatic solvents extensively inhibited the production of shikonin derivatives. The yield of derivatives increased with an increase in the carbon chain length of the n-paraffin used as the solvent and when the oxygen supply was sufficient it reached the value found for the ordinary culture method.