Isolation and cell regeneration of protoplasts from sugar pine (Pinus lambertiana)

Protoplasts were isolated at high yields from actively growing callus and cell suspensions of cotyledons and needles of mature trees. The best protoplast growth response was obtained from cell suspensions of cotyledon and needle callus. Lower protoplast yields were obtained directly from young needles of flushing buds on explants from mature shoots (30-year-old trees) growing in vitro. In all cases, the first divisions, promoted by dimethyl sulfoxide, were observed in 10–45% of the protoplasts by 7–10 days. After 25–30 days, colonies of 8–10 cells were established. Browning of protoplast-derived cell cultures was observed within 40–45 days (cotyledons) and 20–25 days (mature tree sources).Abbreviations BA N⁶-Benzyladenine - DCR Douglas-fir cotyledon revised medium - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - FDA Fluorescein diacetate - Mes 2-(N-morpholino) ethanesulfonic acid - NAA -naphthaleneacetic acid     

In vitro differentiation of plantlets from embryonic explants of lodgepole pine (Pinus contorta Dougl. ex Loud.)

A protocol is described for plantlet formation in juvenile tissues of Pinus contorta. Shoots were induced on embryonic, cotyledonary and hypocotyl explants cultured on a defined medium supplemented with cytokinin. The concentration of salts, vitamins and cytokinin (benzylamino purine) in the medium, as well as different temperature regimes, strongly influenced the frequency of bud formation. Differentiation of shoot primordia and their subsequent development was also markedly affected by cytokinin exposure times. Bud development and elongation were enhanced by elimination of the phytohormone, reducing the strength of mineral salts, vitamins and sucrose in the medium, as well as by the inclusion of charcoal. Rooting was induced by treating the shoots with a sterilized rooting powder containing indole-butyric acid and culturing them in agar-solidified medium containing reduced mineral salts, vitamins, sucrose and charcoal. The number of chromosomes and their structure were found to be normal in the regenerated plantlets.     

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5–12 months old cell suspensions showing visible colony formation in the range of 180–1050 colonies/10⁶ protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10⁶ protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - NAA naphthaleneacetic acid     

Isolation and culture of gymnosperm root protoplasts (Pinus pinaster)

Various factors affecting the yield and isolation of axenic protoplast cultures originating from Pinus pinaster root segments (in which most cells are differentiating) were studied. In spite of the use of plant material collected from germinating seeds under aseptic conditions, an additional sterilization with 0.1 % w/v mercuric chloride in 50% ethanol was a prerequisite for obtaining an axenic protoplast culture. A pretreatment with 30 mM cysteine in 0.7 M sorbitol for I h tripled the yield. Cen-trifugation at lOOg instead of 40 g further increased the yield to 6 × 103 protoplasts per cm of root segment. Viability ranged from 80 to 91%. Cell divisions occurred after a minimum of 7 days of culture.     

Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea

Summary Embryogenic cell suspension cultures of Pinus caribaea var. hondurensis have been cryopreserved in liquid nitrogen for up to four months, using sucrose and dimethylsulfoxide as cryoprotectants. Post-thaw growth was obtained after a short lag phase. Removal of the remaining liquid around the cells using a filter disc favoured subsequent regrowth of the cells. These reestablished cultures maintained an embryogenic potential similar to non-frozen cultures. The embryos produced were able to regenerate into plants, which are now growing in a greenhouse.Abbreviations BA 6-benzyladenine - DMSO dimethylsulfoxide - FDA fluorescein diacetate - MS Murashige and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - PAR photosynthetically active radiation     

Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth.

In order to study the accumulation and transport of rosmarinic acid in suspension cells of Coleus blumei we established an efficient method to isolate protoplasts and vacuoles. Protoplasts were disrupted by an osmotic shock in a medium with basic pH containing ethylenediamine tetraacetic acid. The resulting vacuoles were purified on a two-step Ficoll gradient. The comparison of the rosmarinic acid contents of cells, protoplasts and vacuoles showed that the depside is localized in the vacuole. Data concerning the yield and purity of the vacuoles are presented. In addition we show that at the physiological pH of the cytoplasm rosmarinic acid is present almost exclusively as an anion and cannot pass a membrane by simple diffusion. We therefore propose a carrier system for the transport of rosmarinic acid into the vacuole.Abbreviations EDTA ethylenediamine tetraacetic acid - HEPES 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethane sulfonic acid - HPLC high performance liquid chromatography - MES morpholinoethane sulfonic acid - NADP⁺ ß-nicotinamide adenine dinucleotide phosphate - PEG polyethylene glycol - RA rosmarinic acid - Tris Tris(hydroxymethyl)aminomethane     

Isolation and culture of protoplasts from immature leaves and embryogenic cell suspensions of Dioscorea yams: tools for transient gene expression studies

Leaf mesophyll protoplasts from immature leaves of in vitro shoot cultures of a range of cultivars of three species of food yam (Dioscorea alata, D. bulbifera and D. cayenensis-rotundata) were isolated and their responses to culture in agarose-solidified media compared. Leaves at early stages of development (< 1.0 cm in length) proved most suitable for production of active yam protoplasts capable of cell division. Formation of cell colonies to the 50-cell stage was observed in protoplast cultures in five of ten cultivars of D. alata and to the 30-cell stage in two cultivars of D. cayenensis-rotundata but not in cultures of D. bulbifera. Embryogenic cell suspension protoplasts of D. alata cv. Oriental Lisbon were successfully transformed with plasmids pBI 221.2, pBI 221.54, pBSGUS1 and pJT137 using a standard polyethylene glycol-mediated uptake method. Levels of transient expression of the uidA gene varied according to the plasmid used and the cell lines from which yam protoplasts were derived. This is the first report of yam protoplast culture leading to cell regeneration and direct gene transfer into protoplasts of this monocotyledonous genus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Callus formation from cotyledon protoplasts of Pinus oocarpa and Pinus patula

Protoplasts were isolated from cotyledons of 11-day-old seedlings of Pinus oocarpa and P. patula ssp. tecunumanii . The best enzyme combination was Cellulase R10 + Pectolyase Y-23, associated with bovine serum albumin. When cultured at a low density [1.25 × 10³ to 5 × 10³ protoplast (ml)⁻¹] in a liquid medium, the cells divided. The medium contained glutamine and casein hydrolysate as nitrogen sources, and glucose as osmoticum. Rate of division was increased by supplementing the medium with l -ornithine, putrescine and spermidine. However, the rate remained low, with an absolute division frequency of ca 1%. Dilution allowed colony proliferation and fragmentation, leading to the formation of numerous microcalli that could be transferred to various solid media for further growth.     

Electrostimulated regeneration of plantlets from protoplasts derived from cell suspensions of barley (Hordeum vulgare)

For transformation and somatic hybridisation of barley ( Hordeum vulgare L.), it is necessary to develop an efficient and reliable system for routne plant regeneration from protoplasts. Freshly-isolated cell suspension-derived protoplasts were treated with both rectangular and exponential electric pulses with the aim of increasing plating efficiency as well as to stimulate regenerative capacity. Suspensions were initiated from callus from immature embryos of barley (cv. Dissa). Increasing field strength, capicitance, or number of applied pulses resulted in a decreased protoplast viability and plating efficency. However, the regeneration of albino leaves and albino plantlets from electro-treated protoplasts was stimulated in comparison with controls.     

A rapid,efficient method for the mass production of pollen protoplasts from Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast.

An optimized protocol was established to isolate large numbers of mature living pollen protoplasts of Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast. Intact pollen grains of P. bungeana or pollen with short tubes were incubated with gentle agitation in a solution of 2% cellulase R-10, 1.5% macerozyme R-10, 15% sucrose, 0.01% H₃BO₃, and 0.01% CaCl₂. Intact pollen protoplasts with diameters of 40 μm were liberated, with an isolation rate of up to 70% after 6 h of enzymatic incubation. The optimal pH and temperature for the reaction were 5.8 and 24 °C, respectively, and the optimal enzymatic digestion conditions were 6 h of incubation in the above solution. The method for isolating pollen protoplasts from P. wilsonii was similar to that for P. bungeana, except that the incubation medium contained 12% rather than 15% sucrose and the optimal enzyme concentrations were 3% cellulase and 2% macerozyme. The isolated pollen protoplasts were demonstrated to be living by microscopy in a fluorochromatic reaction with fluorescein diacetate (FDA).     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

Isolation of protoplasts from edible fungi

Summary Mycelium from the periphery of actively growing colonies on cellophane and from shake flask cultures was used for the isolation of protoplasts from strains ofAgaricus bisporus,Auricularia auricula, Lentinus edodes, Pleurotus sajor-caju, Volvariella bombycina andV. volvacea. The mycelial cells were treated with two mycolytic enzymes, Novozym 234 or lywallzyme. Protoplasts were produced from all the edible fungi tested.Pleurotus sajor-caju gave the highest yield (3.84×10⁷/ml), followed byAu. auricula (7.46×10⁶/ml),Ag. bisporus (2.16×10⁶/ml) andV. volvacea (1.92×10⁶/ml), when treated with lywallzyme.Agaricus bisporus gave the smallest yield of protoplasts when Novozym 234 was used. The effects of different molarities of osmotic stabilizers were also studied. The cellophane method is simple and quick and can be used as a screening procedure. The yields of protoplasts obtained from liquid cultures were usually higher than those from cellophane cultures.

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Aislamiento de protoplastos a partir de setas comestibles

Resumen Para el aislamiento de protoplastos de cepas deAgaricus bisporus, Auricularia auricula,Lentinus edodes, Pleurotus sajor-caju, Volvariella bombycina, y V. volvacea se utilizo micelio periferico de colonias que estaban en crecimiento activo en celofan y en frascos de agitación. Las celulas del micelio se tratarón con dos enzymas mycoliticos: Novozym 234 y lywallzyme. Todas las setas comestibles ensayadas produjerón protoplastos. Mediante tratamiento con lywallzyme la mejor cosecha de protoplastos la proporcionoP. sajor-caju (3.84×10⁷/ml) seguido porAu. auricula (7.46×10⁶/ml),Ag. bisporus (2.16×10⁶/ml) yV. volvacea (1.92×10⁶/ml). Con Novozym 234Ag. bisporus fué la de menor rendimiento. También se estudiarón los efectos de distintos estabilizadores osmoticos a diferentes molaridades.

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Isolement de protoplastes à partir de champignons comestibles

Résumé Du mycélium prélevé à la périphérie de colonies dévoloppées sur cellophane ou à partir de cultures liquides agitées a été utilizé pour la préparation de protoplastes d'Agaricusbisporus,Auricularia auricula, Lentinus edodes, Pleurotus sajor-caju, Volvariella bombycina etV. volvacea. Les cellules mycéliales ont été traitées par deux enzymes mycolytiques, Novozym 234 et lywallzyme. Par traitement avec le lywallzyme, des protoplastes ont été obtenus avec tous les champignons comestibles étudiés,P. sajor-caju donnant le rendement le plus élevé (3.84×10⁷/ml), suivi parAu. auricula (7.46×10⁶/ml,Ag. bisporus (2.16×10⁶/ml etV. volvacea 1.92×10⁶/ml). Avec le Novozym 234,Ag. bisporus a donné le rendement en protoplastes le moins élevé. Les effets de différentes molarités de stabilisateurs osmotiques ont également été étudiés. La méthode de la cellophane est simple et rapide et peut être employée comme procédé de degrossissage. Les rendements en protoplastes obtenus à partir des cultures liquides sont habituellement plus élevés que ceux à partir des cultures sur cellophane.

     

Isolation of protoplasts from edible seaweeds

Protoplasts were isolated enzymatically from three species of Chlorophyta (Enteromorpha linza, Monostroma zostericola andUlva pertusa) with high yield and viability. An enzyme solution appropriate for protoplast isolation from the marine green algae was the following: 2% Cellulase Onozuka R-10, 1.0.M mannitol, pH 6.0. Protoplasts could not be obtained from members of Phaeophyta or Rhodophyta.     

Competition Within Stands of Picea sitchensis and Pinus contorta

Competition was analysed in plots of Picea sitchensis and Pinuscontorta grown in 50 x 50 hexagonal arrays at 14 cm spacingto ages 7 and 5 years, respectively. Relative growth rates in height (RHGR) became positively relatedto tree heights during the year before harvest. Frequency distributionsof tree heights became negatively kurtotic with tendencies towardsleft-skew ness. By the time of harvest, dead trees were evenlydispersed over the plots. Trees with many taller neighbourshad lower RHGRs than trees with few taller neighbours, and theRHGRs of intermediate-sized trees were correlated with their‘competitive status’. Competition was confined mostlyto first-order neighbours. Large trees depressed the RHGRs ofsmaller neighbours and not vice versa; a simple test for this‘one-sided’ competition is described. Neighboursdid not need to greatly overtop a tree to depress its RHGR,they needed only to be at least as tall. Systematic trends inRHGR across the plots, attributed to site heterogeneity, decreasedwith time, and accounted for only about 10 per cent of the variationin RHGR at harvest. Competitive status accounted for 25 and38 per cent of the variation in RHGR in the Picea sitchensisand Pinus contorta plots, respectively. Picea sitchensis, Pinus contorta, competition, monoculture, self-thinning, relative growth rate, growth model     

Plant regeneration from embryogenic cell suspensions and protoplasts in sugarcane (Saccharum spp.hybrid cv. CoL-54)

Embryogenic callus was developed from young leaves of sugarcane (Saccharum spp.hybrid, cv. CoL-54). A good embryogenic callus response was achieved using MS basal medium containing 2.0 mol (0.5 mg l^-1) picloram under dark conditions at 27±1°C. Initiation of fast growing homogeneous cell suspension cultures was achieved in MS and AA media, both supplemented with g mol (2 mg l^-1) 2,4-d and 500 mg l^-1 CH. Embryogenic callus was reinitiated from embryogenic cell suspension cultures using MS medium containing 30 g l^-1 sucrose, 500 mg l^-1 CH and 2.26 mol (0.5 mg l^-1) 2,4-d after 4–6 weeks of culture under 16-h photoperiod conditions. Plant regeneration was achieved after about 4 weeks in MS medium lacking growth regulators but containing CH (500 mg l^-1) and sucrose (60 g l^-1). Rooting was enhanced by transferring regenerated plantlets to half strength MS basal medium.Totipotent protoplasts with an average yield of 2.0×10⁷ to 1.0×10⁸ ml^-1 were obtained from embryogenic cell suspension cultures at log phase, i.e., 4–5 days after transfer to fresh media. The best growth response was achieved when protoplasts were cultured in a modifed KM8P medium at the density of 2.0×10⁵ m l^-1. Protoplasts were mainly embedded in 0.8% sea plaque agarose. Division efficiency of 22.2% was achieved after 20 days of culture and 0.26% of microcolonies continued growth and formed microcalluses after 30 days of culture under dark conditions. Microcalluses were proliferated in MS medium having 2,4-d (2 mg l^-1) under 16-h photoperiod. Transferring these embryogenic calluses in MS medium +9.29 mol kinetin (2 mg l^-1) +5.37 mol NAA (1.0 mg l^-1) + activated charcoal (200 mg l^-1) for 5 weeks favoured plant regeneration. Shoots and roots were further proliferated in half strength MS basal medium for 2–4 weeks. Regenerated plants were transferred to autoclaved sand for 2 weeks under 16-h photoperiod in growth room and transferred to soil in a greenhouse to raise to maturity.Abbreviations MS salts of Murashige & Skoog (1962) basal medium - AA salts of Muller & Grafe (1978) basal medium - N6 saits of Chuet al. (1975) basal medium - 2,4-d 2,4-dichlorophenoxyacetic acid - CH casein hydrolysate - KM8P protoplast culture medium of Kao & Michayluk (1975) - KPR protoplast culture medium of Kao (1977) - P9 protoplast culture medium (Chen & Shih, 1983) - BA Benzyladenine - Picloram 4-amino-3,5,6-trichloropicolinic acid - NAA Naphthalene acetic acid