Mesophyll protoplasts ofAvena sativa: Characterization of the first cell cycles

Summary Cells from a fully habituated nonorganogenic sugarbeet callus line show very large nuclei, that are very irregular in shape, with deep invaginations and many nucleoli. Micronuclei can also be seen. Fluorimetric analyses of the DNA content in the habituated cells show an abnormal distribution indicating polyploidy and aneuploidy. Such features closely resemble those observed in animal cancer cells.     

Patterns of direct and indirect embryogenesis from mesophyll protoplasts of Medicago sativa

Clones of three cultivars of Medicago sativa (Rambler, Regen S and Rangelander) were used as sources of mesophyll protoplasts. Although all three clones readily produced protoplasts, the subsequent development patterns in culture varied greatly among genotypes, with protoplasts from Regen S and Rambler forming calli which could be induced to form embryos, and protoplasts from Rangelander undergoing direct embryogenesis. Protoplasts of Regen S exhibited high rates of division while those of Rangelander tended to aggregate with only a few cells per aggregate surviving. The surviving cells gave rise to proembryos within the aggregates; these proembryos developed into differentiated embryos after 5–7 weeks of culture. Based on the initial protoplast population, the efficiency of embryo formation averaged 0.13% and ranged from 0.001–0.4%. Observations during the early stages of culture indicated that cell aggregation was a prerequisite for direct embryogenesis.     

Donor tissue and culture condition effects on mesophyll protoplasts of Medicago sativa

Mesophyll protoplasts were produced from clones of two cultivars of Medicago sativa, Rangelander and Regen S. Protoplasts from the Regen S clone generally gave rise to calli while those from the Rangelander clone would undergo direct embryogenesis. Effects of plant growth conditions, donor tissue pretreatment and protoplast culture conditions on mesophyll protoplast production and subsequent development patterns were investigated. The major factor determining whether or not mesophyll protoplasts would be produced from either of the clones was the pretreatment in water of shoots excised from the donor plants. Pretreatment in water containing growth regulators did not alter protoplast production or development in the Regen S clone. Pretreatment of the Rangelander clone shoots with abscisic acid or naphthaleneacetic acid was slightly beneficial to embryo production while pretreatment with benzylaminopurine was detrimental. Altered leaf morphology induced by growth condition changes did not affect mesophyll protoplast production or subsequent development patterns when shoots were pretreated in water. Culture of protoplasts in liquid droplets or solid agar medium increased low density protoplast survival and subsequent embryo production in the Rangelander clone.     

Plant regeneration from suspension culture and mesophyll protoplasts of Medicago sativa L.

A system was established for achieving plant regeneration from mesophyll protoplasts and cotyledon-derived cell suspension cultures of alfalfa, Medicago sativa L. Peeled leaflets or cells from 6-day-old cell suspensions were incubated in an enzyme mixture containing 1% Driselase, 1% Rhozyme, 0.1% Cellulase and 72 gl^-1 mannitol at pH 5.8 for 2–16 h to liberate protoplasts. A complex Kao medium supported cell division and colony formation, whereas a high auxin/low cytokinin treatment on Schenk and Hildebrandt medium followed by culture on growth regulator-free Blaydes or Linsmaier and Skoog medium resulted in somatic embryo formation. Of the three varieties tested. Citation, Answer and Regen S, the latter two produced embryos from which plants could be regenerated.     

Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.)

Summary We used a tetraploid clone derived from an anther culture operation of Ladak alfalfa to study the pathway of direct embryogenesis from leaf-mesophyll protoplasts. About 72% of the protoplasts divided, and 7% of those produced proembryos. Approximately 38% of the proembryos developed into green embryos, and 33% initiated calluses. Other proembryos dedifferentiated into calluses which later redifferentiated embryos. Sixteen percent of the embryos developed directly into plants, whereas 81% produced plants indirectly via secondary embryos. The remaining 3% of the primary embryos failed to develop into plants. The lowest plating efficiency for direct embryogenesis was 0.3%. The high percentage of direct embryogenesis observed was related to the genetic nature of the clone, low density of liquid medium, low protoplast culture density, and the composition of culture media.Contribution no. 90-61-J from the Kansas Agricultural Experiment Station.     

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     

Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Avena sativa L.) leaves

The uptake of [1-³H]geranylgeranyl diphosphate (GGPP) into protoplasts and intact etioplasts and the metabolic interconversion therein was studied after a 2 min pulse of white light. The chlorophyll synthetase reaction, Chlide+GGPPChl_GG, was taken as a natural probe for the etioplast compartment. This reaction yields labeled ChL_GG and, by hydrogenation, labeled Chl_P, when [1-³H]GGPP receives access to the etioplast stroma. It was found that penetration across the plastid envelope was rapid and that penetration across the plasma membrane of protoplasts, however, was slow. A cellular pool of soluble GGPP was detected. This pool was lost, in part, during preparation of the protoplasts and almost completely during preparation of the etioplasts. The membrane-bound phytol pool of etioplasts could not be replaced by exogenous [³H]GG. The endogenous GG and phytol pools of protoplasts, which were larger than those of etioplasts, could be replaced in part by exogenous [³H]GGPP. That part of this pool exists as soluble GGPP or as a direct precursor in the cytoplasm is discussed.Abbreviations GGPP geranylgeranyldiphosphate - Chl_GG geranylgeranyl chlorophyllide a - Chl_P phytyl chlorophyllide a - IPP isopentenyl diphosphate - Chlide chlorophyllide a     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

DNA fiber autoradiography of tobacco mesophyll protoplasts

DNA fiber autoradiography was successfully applied to protoplasts isolated from tobacco leaf mesophyll. After an incubation period of 37 h, DNA began to be labeled with ³H-thymidine. These cells, with regenerated thin cell walls, were then effectively disrupted in a lytic solution containing sodium dodecyl sulfate. DNA fiber autoradiography confirmed that the mean replicon size of this plant is approximately 60 in good agreement with the values reported previously in animal cells.     

Plantlets from mesophyll protoplasts of Solanum xanthocarpum

Young leaves of Solanum xanthocarpum from axenic shoot cultures released viable protoplasts when treated with appropriate enzymes. The protoplasts on culture in modified Murashige and Skoog (1962) medium supplemented with 2,4-dichlorophenoxy-acetic acid (0.5 mg/l), naphtha leneacetic acid (1 mg/l), kinetin (1 mg/l) and organic nutrients of KM (Kao and Michayluk 1975) regenerated to form callus tissue as a result of repeated divisions. Protoplast-derived calli differentiated into shoots on MS medium enriched with kinetin (0.5 mg/l) and rooting could be initiated by transferring the shoot-buds to basal medium.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Rhizogenesis fromNigella sativa protoplasts

Summary Protoplasts were isolated for the first time from cell suspensions ofNigella sativa. These were then cultured in media and observed at regular intervals. Different concentrations of auxin and kinetin were tried with success to obtain root from the callus tissues of the protoplasts.     

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     

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     

Fine structure of isolated mesophyll protoplasts of tobacco

Summary Protoplasts of palisade cells isolated enzymatically from mature leaves of tobacco were studied with the electron microscope. A cell wall was completely absent, and the chloroplasts contained large inclusion bodies which were believed to be a crystalline form of fraction I protein. The fine structure of the protoplasts was otherwise that of healthy mesophyll cells, indicating that they are in a good physiological state. Some protoplasts were multinucleate as a result of fusion during the isolation process.     

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)     

Culture of soybean mesophyll protoplasts in alginate beads

Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.     

Plants regenerated from mesophyll protoplasts of white mulberry

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