Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

Plant regeneration from mesophyll protoplasts ofDianthus superbus

Leaf mesophyll protoplasts ofDianthus superbus were cultured at a density of 5 × 10⁴ protoplasts/ml and divided at about 18% plating efficiency in MS liquid medium supplemented with 0.5 mg/L BAP, 2.0 mg/L NAA and 9% mannitol after 2 weeks. Protocolonies formed after 3 to 4 weeks of culture in the dark at 27°C. These colonies were transferred to continuous illumination (21.5 E m^–2 sec^–1) for 2 weeks where most of the colonies divided to form microcalli, about 2 mm in diameter. Subsequently, green microcalli were transferred to MS solidified medium with 2.0 mg/L 2,4-D that induced shoot-forming calli after 4 weeks. These calli were transferred onto N₆-2 medium containing 0.1 mg/L 2,4-D, 0.1 mg/L NAA, 2.0 mg/L kinetin and 2.0 g/L casein hydrolysate and were cultured under light. After 5 weeks the calli gave rise to multiple shoots (10 to 15 per callus). Upon transfer to MS medium containing 2.0 mg/L NAA, individual shoots were rooted in 4 weeks. The regenerants were successfully transplanted into potting soil.Abbreviations MS Murashige and Skoog - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - N₆ Chu basal salt mixture - MES 2-N-morpholinoethanesulfonic acid     

Rapid plant regeneration from Nicotiana mesophyll protoplasts

A procedure is described for rapid plant regeneration from tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Six to seven days after protoplast isolation, colonies are placed on double filter feeder plates that consist of a strong regeneration medium containing 7.5 mg/l 6-(γ,γ-dimethylallylamino)-purine (2iP) and 0.1 mg/l p-chlorophenoxyacetic acid (pCPA). Complete plants are regenerated in about 5 weeks after transfer to a rooting medium (hormone-free Murashige and Skoog (MS) medium). However, upon remaining on shoot regeneration medium, 50–75 shoots are regenerated from single colonies derived from individual protoplasts. This procedure may reduce the amount of somaclonal variation (as measured by ploidy level) which is usually expressed in plants obtained by conventional regeneration techniques.     

Plant regeneration from mesophyll protoplasts of Moricandia arvensis

Moricandia arvensis is of interest as it is a dicotyledonous species which has C₃ — C₄ intermediate photosynthesis, a mechanism which results in enhanced recapture of photorespired CO₂. Leaves from cultured shoot tips were used as a source for mesophyll cell protoplasts. Approximately 1% of the protoplasts which survived the first few days of culture produced calli. On a suitable regeneration medium, 30–60% of the calli regenerated one or more shoots. From among the regenerating shoots eight were selected, transferred to soil and grown to flowering in the glasshouse; all were fertile. The development of a protoplast regeneration system provides the opportunity to use transformation and somaclonal variation as tools in the genetic analysis of the C₃–C₄ character in this species.Abbreviations GDC glycine decarboxylase - 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzyl aminopurine - NAA naphthalene acetic acid - ABA abscisic acid     

Plant regeneration from mesophyll protoplasts of Lactuca perennis

Cultured protoplasts of young, unexpanded leaves of the wild lettuce, Lactuca perennis, divided to produce cell colonies in an agarose-solidified, modified MS medium with reduced levels of inorganic salts, together with 2,4-d, NAA and zeatin at 0.2, 0.1 and 0.5 mg 1^-1 respectively. Organogenesis followed the initial transfer of protoplastderived colonies to modified MS medium with 2,4-d, NAA and zeatin (0.1, 1.0 and 0.2 mg 1^-1 respectively) and then to full-strength MS medium with 6-BA and NAA (0.4 and 0.05 mg 1^-1). Shoots were rooted on agar-solidified MS medium lacking growth regulators. Regenerated shoots were established ex vitro, 21 weeks after protoplast isolation.Abbreviations 6-BA 6-benzyladenine - BSA bovine serum albumin - d days - 2.4-d 2,4-dichlorophenoxyacetic acid - f. wt. fresh weight - IAA indoleacetic acid - MES 2 [N-morpholino]ethane sulphonic acid - MS Murashige & Skoog (1962)     

Plants regenerated from mesophyll protoplasts of white mulberry

INTRODUCTIONProtoplastcultureis0neofthen1ostrapidlydevel0pingareasinp1anttissueculture,becauseofitsimportancei11plantgeneticmanipulation.However,sofar,thereareonlyafewforesttreespeciesinwhichplantregenerationfr0mprotoplastshaJsbeensuccessful,namelyLiriode…     

Somatic embryogenesis from Lycopersicon peruvianum leaf mesophyll protoplasts

Summary One to five percent of Lycopersicon peruvianum (L.) Mill. leaf mesophyll protoplasts undergo cell division and concomitant organization to form embryogenic-like structures when cultured in Murashige and Skoog medium (1962) containing 3% sucrose, 9% mannitol, 1.0 mg/l kinetin (K) and 1.0 mg/l naphthalene acetic acid (NAA) at pH 5.6–5.8 (medium A). These embryogenic structures, after passing through developmental stages similar to those observed in zygotic embryogeny, are capable of forming shoots on hormone-free medium A. In medium B, wherein 0.5 mg/l of 2,4-dichlorophenoxyacetic (2,4-D) replaced the hormones (K and NAA), embryogenic structures did not develop. However, callus originating in medium B retained morphogenetic capacity as was evidenced by subsequent shoot regeneration when they were transferred to medium A with K and NAA replaced by 1.0 mg/l zeatin (Z). The potential value of incorporating this regeneration trait into Lycopersicon species and cultivated lines for use in tissue culture programs is discussed.Michigan Agricultural Experiment Station Journal No. 9676     

Plant regeneration from mesophyll protoplasts in Isatis indigotica

Protoplasts of an accession of Isatis indigotica Fort. were isolated from mesophyll tissue by enzymatic digestion and cultured using a feeder cell system. Shoot regeneration efficiency was 100% via organogenesis among 627 isolated calluses within 30–37 days. Among these shoot initiating calluses, 162 (22.6%) developed normal shoots with multiple (2–5) shoots per callus. The remaining calluses developed only vitreous shoots. High concentration (5 μM) of indole-3-butyric acid had a positive effect on rooting compared to low concentration (0.5 μM) of indole-3-butyric acid and α-naphthalene-acetic acid. The average rooting efficiency of regenerated shoots of two experiments was higher on LS medium with 5 μM indole-3-butyric acid than on LS medium without growth regulators. Twenty-nine plantlets, with 2–3 expanded leaves and roots were potted in soil and 22 developed normally to maturity in the glasshouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

Isolation and culture of mesophyll protoplasts from Rosa hybrida

After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0 (w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt. 10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise to roots after transfer to SH medium containing 14 μM 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light-affected ca fluxes in protoplasts from vallisneria mesophyll cells

In Vallisneria gigantea Graebner mesophyll cells, red light irradiation induces cytoplasmic streaming by decreasing the Ca²⁺ concentration in the cytoplasm, while far-red light irradiation inhibits it by increasing the concentration (S Takagi, R Nagai 1985 Plant Cell Physiol 26: 941-951). To examine the effects of light irradiation on Ca²⁺ fluxes across the cell membrane, protoplasts are isolated from the mesophyll cells. Changes in Ca²⁺ concentration in a solution bathing the protoplasts are monitored by spectrophotometry, using the Ca²⁺ -sensitive dye murexide. Red light irradiation induces an increase in Ca²⁺ concentration, which means an efflux of Ca²⁺ from the protoplasts. Subsequent far-red light irradiation produces a rapid decrease in Ca²⁺ concentration down to the dark control level; however, this is not observed in the presence of the Ca²⁺ -channel blocker nifedipine. Vanadate inhibits both the streaming and the Ca²⁺ efflux induced by red light irradiation. The results suggest that red light and far-red light control Ca²⁺ movements across the cell membrane, which in turn regulate the streaming.     

Proteases of Melilotus alba mesophyll protoplasts

Proteases from mesophyll protoplasts of Melilotus alba were identified by standard proteolytic assays and separated using different chromatographic techniques. Their characterization also included their subcellular location. Besides the evidence for the multiplicity of the proteolytic enzymes, two protease sets were distinguished endopeptidases, which are exclusively vacuolar, and aminopeptidases, which are widely distributed throughout the cell. Cytosol-located enzymes were tested as substrates of the two sets of proteases, by studying comparatively the time-course changes of enzyme activities during incubation in total protoplast extracts, or in cytosol fractions devoid of vacuolar proteases. The degradation of phosphoenolpyruvate-carboxylase protein, a typical cytosolic enzyme, in the presence of purified amino-and endopeptidases, was also estimated by immunoprecipitation studies. Only the vacuolar endopeptidases are effective in the degradation of cytosolic enzymes. Hydrolytic enzyme activities mostly of vacuolar origin were very stable during incubation in total protoplast extracts. These proteins therefore appear to be particularly resistant to proteolytic attack. The results indicate that, in plants, the effective proteolytic system acting on cytosolic enzymes seems to be vacuole-located, and that the selectivity in protein degradation may be imposed by the susceptibility of the protein being degraded and by its transfer into the vacuoles.Abbreviations Leu-pNA leucine-p-nitroanilide - lys-p-NA lysine-p-nitroanilide - pCMB p-chloromercuribenzoic acid - PEPCase phosphoenolpyruvate carboxylase - PMSF phenylmethylsulfonylfluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Induced morphogenesis in tissue cultures of Solanum xanthocarpum

Summary Differentiation of shoots and roots or unorganized proliferation could be induced in tissue cultures of Solanum xanthocarpum when callus tissues habituated to a medium containing 2.4-D and meso-inositol were transferred to an auxinfree medium or to a medium in which the ratio of auxin and meso-inositol had been altered.     

Sucrose transport into vacuoles isolated from barley mesophyll protoplasts

Sucrose transport has been investigated in vacuoles isolated from barley mesophyll protoplasts. Rates of sucrose transfer across the tonoplast were even higher in vitro than in vivo indicating that the sucrose transport system had not suffered damage during isolation of the vacuoles. Sucrose transport is carrier-mediated as shown by substrate saturation of transport and sensitivity to a metabolic inhibitor and to competitive substrates. A number of sugars, in particular maltose and raffinose, decreased uptake of sucrose. Sorbitol was slowly taken up but had no effect on sucrose transport. The SH-reagent p-chloromercuribenzene sulfonate inhibited sucrose uptake completely. The apparent K_m of the carrier for sucrose uptake was 21 mM. Transport was neither influenced by ATP and pyrophosphate, with or without Mg²⁺ present, nor by protonophores and valinomycin (with K⁺ present). Apparently uptake was not energy dependent. Efflux experiments with preloaded vacuoles indicated that sucrose unloading from the isolated vavuoles is mediated by the same carrier which catalyses uptake. The vacuole of mesophyll cells appears to represent an intermediary storage compartment. Uptake of photosynthetic products into the vacuole during the light apparently minimizes osmotic swelling of the small cytosolic compartment of vacuolated leaf cells when photosynthetic productivity exceeds the capacity of the phloem for translocation of sugars.Abbreviations Hepes 4-(2-hydroxyethyl)-1-piperazincethane-sulfonic acid - pCMBS p-chloromercuribenzene sulfonate Dedicated to Professor Dr. W. Simonis on the occasion of his 75th birthday     

Stimulation of direct embryogenesis from mesophyll protoplasts of Medicago sativa

Mesophyll protoplasts of Medicago sativa were exposed to low voltage electrical fields immediately following isolation. Several exposure times and voltages were utilized. At the lower doses, protoplast aggregation and subsequent embryogenesis were stimulated. A clone of Rangelander, which was directly-embryogenic (i.e. embryos were derived from single mesophyll protoplasts without an intervening callus phase), was induced to form embryos in all samples exposed to the lowest level electrical fields, while unexposed controls formed few or no embryos. A clone of Regen S, which was previously not directly-embryogenic, was induced to follow the Rangelander pattern of development and to produce early globular embryos.Plant Research Centre Contribution No. 1013     

Callus formation from mesophyll protoplasts of Fagus sylvatica L.

Viable protoplasts of European beech (Fagus sylvatica L.) were isolated from sterilized young leaves of juvenile (3–5 years) and mature (40 years) trees. Isolation in a saline solution containing 0.5% (w/v) Pectinol and 2% (w/v) Cellulase R-10 yielded 3×10⁷ protoplasts per gram fresh weight. Protoplast culture in modified Kao and Michayluk (1975) medium resulted in cell wall regeneration and sustained cell divisions with the formation of colonies and microcalli.Abbreviations KM Kao and Michayluk (1975) - LS Linsmaier and Skoog (1965) - MES 2(N-morpholino)-ethane-sulfonic acid - NAA 1-naphthalene acetic acid - PVP Polyvinylpyrrolidone - Tween 80 Polyoxy-ethylene-sorbitan-monooleate Dedicated to Landesforstmeister Prof. Dr. Hans-Joachim Fröhlich on the occasion of his 65th birthday.