PATTERNS OF MITOSIS AND DIFFERENTIATION IN CELLS DERIVED FROM PEA ROOT PROTOPLASTS

Mitotic figures of diploid, tetraploid, octaploid and 16-ploid nuclei were observed in cultures of pea root protoplasts whose initial DNA content was apparently 2C and 4C. The distribution of these mitotic figures in the different ploidy levels paralleled the distribution of mitotic figures in the culture of intact root explants and may be related to the hormonal stimulation of mitoses in these cultures. The patterns of the time course of both DNA synthesis and cell division in the protoplast cultures were similar to such patterns observed in the culture of intact root explants, although longer lag periods were observed in the protoplast cultures. Mitotic abnormalities including both chromosome breakage and spindle disfunction were observed in protoplast cultures. A large portion of the cell pairs derived from mitoses (27 % in one experiment) contained Feulgen-positive micronuclei. An accumulation of an as yet unidentified differentiation product termed dense cytoplasmic protoplast derivative was observed. Some of the conditions influencing the development of these derivatives are reported.     

Fusion between interphase and mitotic plant protoplasts : Induction of premature chromosome condensation

Morphological changes in interphase nuclei were cytologically studied in heterophasic dinucleate cells formed by the fusion of mitotic and interphase plant protoplasts. Mitotic protoplasts were isolated from a partially synchronized suspension culture of wheat (Triticum monococcum). The mitotic cells were accumulated by colchicine after release of hydroxyurea block. Treatment of protoplast populations with polyethylene glycol-dimethyl sulphoxide solution resulted in metaphase-interphase fusion. Three hours after fusion, the appearance of chromosomes with single chromatid as well as of fragmented, pulverized chromatin in heterophasic cells indicated the induction of premature chromosome condensation (PCC) in somatic wheat cells. Condensation in interphase nuclei of mitotically inactive rice protoplasts was also detected after fusion with mitotic wheat protoplasts.     

Culture conditions of protoplast-derived cells of nicotiana sylvestris for mutant selection

Large numbers of protoplasts showing reproducible high plating efficiency can be isolated from in vitro propagated, haploid and diploid, plants of Nicotiana sylvestris. Their successful use in the selection of biochemical mutants depends on the establishment of suitable selection parameters: culture medium, cell density, age of cells at selection etc. Plating of protoplasts at low densities as well as simulation and reconstruction experiments of mutant selection were employed to optimize such selection parameters. The results show that some of the principles determined for tobacco protoplast cultures manipulated at low densities or in view of mutant selection are of more general value. However, requirements specific to N. sylvestris protoplast cultures have also been established; they play a decisive part in the successful isolation of resistant mutants in this species.Abbreviations AEC S-aminoethylcysteine - BA benzyl-adenine - NAA napthaleneacetic acid - p-cells or p-colonies protoplast-derived cells or colonies     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Controlled cell wall regeneration for efficient microinjections of Nicotiana tabacum var. Carlson protoplasts

Nicotiana tabacum var. Carlson protoplast culture conditions were modified to contain a cell wall inhibitor, 2,6-dichlorobenzonitrile, in order to delay cell wall regeneration and to allow efficient nuclear and cytoplasmic microinjections. Under modified conditions, the protoplast preparations appeared healthier as compared to the control protoplasts and showed no resistance at all during microinjection. Furthermore, the duration of protoplast microinjection was extended for up to 3–4 days. In order to set up nuclear microinjections, the nuclei of these protoplasts were stained either before or after immobilization without any adverse effect on their mitotic activity. Successful cytoplasmic microinjections were demonstrated by injecting Alfalfa mosaic virus (AMV) RNA, which resulted in viral infection of 14% of the injected protoplasts.Abbreviations AMV Alfalfa Mosaic virus - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - DB 2,6-dichlorobenzonitrile - LR lissamine rhodamine - NAA 1-naphthalene-acetic acid     

Exogenous auxin and cytokinin dependent activation of CDKs and cell division in leaf protoplast-derived cells of alfalfa

Alfalfa leaf protoplast cultures were used to study the role ofexogenously supplied auxin and cytokinin on the level and activity ofCdc2-related protein kinases and progression through the first celldivision cycle after re-activation of cell division. Among the threealfalfa Cdc2-related kinases studied, the Cdc2MsA/B kinase (PSTAIRE)showed only significant activity during the first four days ofprotoplast culture while the Cdc2MsD (PPTALRE) and Cdc2MsF kinases(PPTTLRE) exhibited only low or undetectable activity, respectively,during this period. Although the Cdc2MsA/B protein could be detectedin leaves and freshly isolated protoplasts in variable amounts, thekinase was never active in these cells. The kinase protein disappearedfrom protoplast-derived cells at the beginning (8h) of culture but itssynthesis re-commenced dependent on the presence of exogenous auxin butnot cytokinin. The cytokinin response of alfalfa protoplast-derivedcells varied significantly in different experiments although cytokininwas always required for completion of the first cell division cycle.Frequently both auxin and cytokinin was required for DNA replication asnot more than 5% of cells could incorporate BrdU into their DNAduring three days and significant Cdc2MsA/B activity could not bedetected in the absence of exogenous cytokinin. In other protoplastpopulations, the Cdc2MsA/B kinase was activated by auxin alone andallowed the protoplast-derived cells to enther the S-phase at a similarrate observed in parallel cultures with both auxin and cytokinin. Evenin these cultures, however, ca. 95% of the protoplast-derivedcells were arrested before mitosis without exogenous cytokinin supplywhich could be correlated with decreasing Cdc2MsA/B activity. Theseobservations suggest, that although cytokinin is required for bothG0-G1/S and G2/M cell cycle transitions, in certain cultures theG1/S requirement is overcome by some unknown factors (e.g.conditions of explants; endogenous cytokinins etc.). Furthermore, ourexperiments indicate, that the roles of cytokinin are related to thepost-translational regulation of the Cdc2MsA/B kinase complex atboth cell cycle transition points in alfalfa leaf protoplast-derivedcells. Finally, as a marker for the transition from the differentiated(G0) stage to the activated (G1) stage, we suggest using the parametersof nuclear morphology (size and ratio ofnucleus/nucleolus).     

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.     

Analysis of population cytokinetics of chick myocardial cells in tissue culture

The growth of embryonic chick cardiac myocytes and fibroblasts in tissue culture was evaluated by the kinetics of nuclear labeling during continuous exposure to [3H]thymidine. The fraction of mitotically active cells, the mean intermitotic period and the population doubling times were determined in each cell type during 3 weeks in culture. After 24 hr in culture, 90% of the muscle cells were mitotically active with minimal population doubling times of 65 hr. By 17 days in culture only 5% of the myocytes continued to divide with population doubling times greater than 3000 hr. Primarily, the lengthening of doubling times was due to a withdrawal of cells from the mitotic cycle and much less to a lengthening of the intermitotic period. Growth of cardiac muscle cells from embryonic hearts from 4 to 10 days of development was also compared. Muscle cells from younger hearts displayed greater mitotic activity than those from older hearts at equivalent times in culture.     

Soybean protoplast culture and direct gene uptake and expression by cultured soybean protoplasts

A method was developed for culturing protoplasts freshly isolated from developing soybean (Glycine max L.) cotyledons. First cell divisions were observed within 5 days after protoplast isolation and microcalli, consisting of about 20 cells, were formed within 10 days. Thirty days after protoplast isolation, callus tissues were observed without the aid of a microscope. A 30 to 50% plating efficiency was consistently obtained. Using a polyethylene glycol-electroporation technique, DNA was introduced into these protoplasts. The protoplasts were then cultured to form callus. Chloramphenicol acetyltransferase (CAT) activity was detected in protoplast cultures 6 hours after introduction of a 35S-CAT-nopaline synthase 3′ chimeric gene. The highest CAT activity was detected in 3-day-old electroporated protoplast cultures, indicating transient expression of the introduced gene. Some CAT activity was detected in 40-day-old callus cultures and in geneticin (G418) selected callus tissues which also received a chimeric neomycin phosphotransferase II gene, indicating the presence of stable transformants. A control chimeric gene with an inverted 35S promoter failed to produce any CAT activity in this system.     

Explanation for the lack of division of protoplasts from stems of rubber-tree (Hevea brasiliensis)

Application of in vitro culture techniques to Hevea brasiliensis has encountered serious difficulties. In particular, protoplasts have been found to be recalcitrant to division. The aim of the present work was to compare biochemically non-mitotic protoplasts isolated from young Hevea stems with mitotic protoplasts from Citrus, tobacco and rice cells. Hevea tissue maceration did not promote ethylene production contrary to the mitotic systems, rather aminocyclopropanecarboxylic acid (ACC) synthesis occurred. Large increases in superoxide dismutase (SOD) and peroxidase (PER) activities during maceration indicated the occurrence of a peroxidative phenomenon. During Hevea protoplast culture, a rapid decrease in viability was associated with an increase in ethylene production, reactions common to stress conditions. Activities of enzymes involved in the production and elimination of toxic oxygen forms were not related to protoplast reactivity. Differences in polyamine content and especially the high putresceine/spermididine + spermine ratio could be used, together with ethylene rise during culture, as markers of Hevea protoplast recalcitrance in culture. Thus, a number of physiological phenomena are associated with lack of mitotic division of stem protoplasts of rubber.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

Structural and ultrastructural analysis of Solanum lycopersicoides protoplasts during diploid plant regeneration

Light, fluorescence and electron microscopy were used to analyse the structural properties of protoplasts obtained from established suspension culture of Solanum lycopersicoides Dun, composed of meristematic cell aggregates. Four types of protoplasts were distinguished immediately after isolation: (1) mononuclear; (2) polynuclear, (3) anuclear and (4) homogeneous protoplasts. Only mononuclear protoplasts were capable of complete cell wall regeneration and mitotic division. Other types of protoplasts were eliminated during culture. Three phases were distinguished in the developing protoplast culture: (1) the elimination phase during which protoplasts damaged during isolation underwent complete degradation; (2) a phase of intense division during which both mitotic cell division and amitotic nuclear division took place; and (3) a stabilization phase leading to the formation of suspension culture. The cell suspension culture obtained from protoplasts was capable of regenerating diploid plants.     

Preparation of protoplasts in tissues of <Emphasis Type="Italic">Ficus elastica</Emphasis> callus cultures differing in the endogenous cyrokinin activity. The role of cytokinins in formation of protoplasts

The rate of formation of protoplasts in Ficus elastica callus cultures differing on endogenous cytokinin activity was examined. This rate was shown to be determined by cytokinin synthesis leading to formation of cells whose walls exhibit a changed rate of protoplast formation. In contrast to a culture exhibiting a high cytokinin activity, the culture with a low cytokinin activity produces a greater amount of protoplasts having a smaller intact cell surface. As compared to this culture, the callus culture exhibiting a high cytokinin activity produces a small amount of protoplasts characterized by a high viability. This fact points to the functional role of endogenous cytokinins in plant cells.     

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

The aim of this work was to improve plating efficiency of sugar beet mesophyll protoplast cultures. Preliminary experiments showed that cultures of good quality, viable protoplasts were obtained in rich media based on the Kao and Michayluk formulation and with the calcium alginate as an embedding matrix. Nevertheless, in these cultures cell divisions were either not observed or very seldom confirming earlier reported recalcitrance of sugar beet protoplasts. The recalcitrant status of these cultures was reversed upon application of exogenous phytosulfokine (PSK)—a peptidyl plant growth factor. The highest effectiveness of PSK was observed at 100 nM concentration. Plating efficiencies obtained in the presence of PSK reached approximately 20% of the total cultured cells. The stimulatory effect of phytosulfokine was observed for all tested breeding stocks of sugar beet. Our data indicate that PSK is a powerful agent able to overcome recalcitrance of plant protoplast cultures.     

Somatic hybridization in higher plants.

Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two non-allelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco.     

Formation of protoplasts from cultured tobacco cells and Arabidopsis thaliana by the action of cellulosomes and pectate lyase from Clostridium cellulovorans

The crude culture supernatants from Clostridium cellulovorans were tested for their ability to convert plant cells to protoplasts. The supernatants readily released protoplasts from cultured tobacco cells and Arabidopsis thaliana. The crude culture supernatant from pectin-grown cells was more active than supernatants from glucose-, cellobiose-, xylan-, and locust bean gum-grown cells. After removal of cellulosomes, the crude culture supernatant lost its protoplast formation activity. The protoplast formation activity of the crude culture supernatant from C. cellulovorans was more effective than those of commercial enzymes based on protein content.     

Evaluation of parameters affecting the yield, viability and cell division of Pinus pinaster protoplasts

Various factors affecting the yield and viability of Pinus pinaster Ait. cotyledon protoplasts and the mitotic activity of or regenerated cells are described. A study of the effect of sterilization procedures of the plant material showed that whereas the organs collected from disinfested seedlings allow for good yield and viability of isolated protoplasts, germination under non-sterile conditions favours a greater germinating capacity and stronger mitotic activity. Numerous clusters of from 10 to 15 cells were formed after 20 days of culture when a 5% aqueous solution of calcium hypochlorite was used as a sterilizing agent.
The effects of an additional purification of the enzymes showed that although yield and viability of the protoplasts are only slightly improved, the more highly purified enzymes on the other hand enhanced the mitotic activity markedly. Between the two total enzyme concentrations used (0.2 and 0.4%, and in which the relative ratio of each element was unchanged), only the lowest level supplied a debris-free protoplast suspension; mitotic activity occurred only in that case.
Comparison of the populations of cotyledon protoplasts collected from seedlings at two different growth stages (not fully-developed or fully-expanded cotyledons) did not reveal any appreciable difference in their size distribution. Neither was the extent of cellular viability affected by the degree of cell differentiation at the time of collecting. On the other hand, the yield of protoplasts and the mitotic activity of the regenerated cells were greater when partially-developed organs were used. Moreover a pretreatment of the elongating cotyledons with a mineral (half-strength MS macronutrients and full-strength micronutrients) and hormonal (15 μ M BAP, 0.5 μ M NAA) solution improved cell division frequency.     

Somatic hybridization in higher plants

Summary Somatic hybridization in higher plants has come into focus since methods have been established for protoplast fusion and uptake of foreign DNA and organelles by protoplasts. Polyethylene glycol (PEG) was an effective agent for inducing fusion. Treatment of protoplasts with PEG resulted in 5 to 30% heterospecific fusion products. Protoplasts of different species, genera and even families were compatible when fused. A number of protoplast combinations (soybean + corn, soybean + pea, soybean + tobacco, carrot + barley, etc.) provided fusion products which underwent cell division and callus formation. Fusion products initially were heterokaryocytes. In dividing heterokaryocytes, random distribution of mitotic nuclei was observed to be accompanied by multiple wall formation and to result in chimeral callus. Juxtaposition of mitotic nuclei suggested nuclear fusion and hybrid formation. Fusion of heterospecific interphase nuclei was demonstrated in soybean + pea and carrot + barley heterokaryons. Provided parental protoplasts carry suitable markers, the fusion products can be recognized. For the isolation and cloning of hybrid cells, fusion experiments must be supplemented with a selective system. Complementation of two nonallelic genes that prevent or inhibit growth under special culture conditions appears as the principle on which to base the selection of somatic hybrids. As protoplasts of some species have been induced to regenerate entire plants, the development of hybrid plants from protoplast fusion products is feasible and has already been demonstrated for tobacco. Presented in the formal symposium on Somatic Cell Genetics at the 27th Annual Meeting of the Tissue Culture Association, Philadelphia, Pennsylvania, June 7–10, 1976.     

Isolation of Staphylococcus aureus protoplasts using lysoamidase

The effect of the bacteriolytic enzyme preparation, lysoamidase, on Staphylococcus aureus 209P cells was studied. The protoplast formation was examined by spectrophotometric, biochemical and electron microscopic methods. Optimal conditions for isolation of S. aureus protoplasts were chosen. The susceptibility of S. aureus cells to lysoamidase depended on the culture age: the maximum effect was observed in the logarithmic growth phase. The protoplast yield was 80% when 1 M sucrose was used as an osmotic stabilizer. Lysoamidase caused local disruptures of the staphylococcus cell walls, which resulted in the formation of osmotically fragile spheroplasts and the release of protoplasts into the medium. The protoplasts obtained could retain 85-90% of the respiration activity and were able of cell wall regeneration.     

Factors influencing transient gene expression in electroporated tall fescue protoplasts

For the rapid establishment of optimal conditions for a genetic transformation system for tall fescue, several factors influencing transient gene expression were studied in protoplasts, after the reporter β-glucuronidase gene was introduced by electroporation. In a time-course study of transient gene expression, GUS activity peaked at 24 h after electroporation. Among the different field strength conditions tested, maximum GUS activity was observed at 750 V/cm. Increases in the amount of plasmid DNA to 80 μg/ml led to increased GUS activity. GUS activities increased in linear fashion with increasing protoplast densities up to 2 × 10⁶/ml. Age of suspension cells from which protoplasts were derived influenced transient expression with maximum GUS activity obtained in 3- and 5-day-old suspensions. These results show that monocot and dicot protoplasts respond similarly in electroporation.