A study of cell wall regeneration of protoplasts inMarchantia polymorpha L.

Protoplasts ofMarchantia polymorpha L. were isolated from suspension cells. Regeneration of cell walls on the surface of the protoplasts began within a few hr of cultivation. New cell walls completely covered the surface of the protoplasts within 48 hr. Coumarin and 2,6-dichlorobenzonitrile treatment inhibited the formation of the new cell wall. In the initial stage of cell wall regeneration, endoplasmic reticula developed remarkably close to the plasma membrane in the protoplasts, but no development of Golgi bodies was observed at the same locus. This may suggest that the Golgi bodies do not play an active role in the cell wall formation, at least not in very early periods of cell wall regeneration. The development of endoplasmic reticula and an ultrastructural change of plasma membrane from smooth to rough may be important in the cell wall formation of protoplasts.     

Studies on Chlorella protoplasts

Protoplasts of Chlorella saccharophila (Krüger) Nadson were obtained by cellulase digestion of the microfibrillar inner compount of the cell wall after the resistant outermost layer had been scratched with sea sand. The absence of the cell wall was demonstrated immunologically, electron microscopically and by staining, thus confirming the protoplastic nature of the treated cells. After transfer to an enzyme-free medium regeneration of a thin cell wall was observed. The regeneration of the cell wall obviously followed the same steps as does the cell wall development of the autospores. At least 50% of the protoplasts were able to form colonies when plated on a suitable agar medium.     

The role of microtubules and cell-wall deposition in elongation of regenerating protoplasts of Mougeotia

Protoplasts of the filamentous green alga Mougeotia sp. are spherical when isolated and revert to their normal cylindrical cell shape during regeneration of a cell wall. Sections of protoplasts show that cortical microtubules are present at all times but examination of osmotically ruptured protoplasts by negative staining shows that the microtubules are initially free and become progressively cross-bridged to the plasma membrane during the first 3 h of protoplast culture. Cell-wall microfibrils areoobserved within 60 min when protoplasts are returned to growth medium; deposition of microfibrils that is predominantly transverse to the future axis of elongation is detectable after about 6 h of culture. When regenerating protoplasts are treated with either colchicine or isopropyl-N-phenyl carbamate, drugs which interfere with microtubule polymerization, they remain spherical and develop cell walls in which the microfibrils are randomly oriented.     

Plant regeneration from protoplasts of a commercial Asian long-grain javanica rice

A reproducible plant regeneration system has been developed for protoplasts from embryogenic cell suspension cultures of the commercial Asian long-grain javanica rice, Oryza sativa cv. Azucena. Protoplasts were isolated routinely from cell suspensions with yields of 5.5–12.0 × 10⁶ g^-1 fresh weight. A membrane filter nurse-culture method was adopted and was essential to support sustained mitotic division of protoplast-derived cells, leading to cell colony formation. The protoplast plating efficiency was higher when suspension cells of Lolium multiflorum, rather than those of the japonica rice O. sativa L. cv. Taipei 309, were employed as nurse cells. A two-step shoot regeneration procedure, in which protoplast-derived calli were cultured initially on medium semi-solidified with 1% (w/v) agarose followed by culture on medium containing 0.4% (w/v) agarose, induced plant regeneration from protoplast-derived calli. Fifteen percent of protoplast-derived tissues regenerated shoots; tissues not subjected to this treatment failed to develop shoots.     

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Summary Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.     

Physiological properties and cytological features of protoplasts prepared fromChlorella saccharophila

Summary Protoplasts were prepared from cells ofChlorella saccharophila by treatment with a mixture of pectinase and cellulase. The yield of protoplasts is dependent upon the culture conditions prior to cell wall digestion. In thin section chemically-fixed protoplasts were without wall remnants at the surface of the plasma membrane. Of particular interest is the relationship between the Golgi apparatus and a nuclear envelope-endoplasmic reticulum continuum. Protoplasts have a photosynthetic capacity lying between 70 and 80% of that of normal cells, but show the same response towards CO₂ concentration and DCMU inhibition. Protoplasts also respond to changes in the osmolarity of the surrounding medium in accordance with the Boylevan't Hoff equation as if they are an osmometer. The nonosmotic volume (NOV) was calculated.Abbreviations GA Golgi apparatus - ER endoplasmic reticulum - NE nuclear envelope - PM plasma membrane - N nucleus - S starch - M mitochondria - V vacuole     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Involvement of arabinogalactan proteins in the regeneration process of cultured protoplasts of Marchantia polymorpha

Protoplasts of Marchantia polymorpha L. (liverwort) regenerated new cell walls in initial culture. However, the survival rate of regenerated cells decreased rapidly after this stage. The decrease in survival rate was suppressed by the β-glucosyl Yariv reagent (βglcY), which binds to arabinogalactan proteins (AGPs), only when it was added to culture medium during the period of incipient cell wall regeneration. The addition of βglcY after the period of incipient cell wall regeneration had no effect on the survival rate. These results suggested the involvement of AGPs in the cell wall regeneration process. After cell wall regeneration, the regenerated cells started to divide actively after being transferred to a medium with 1% activated charcoal (AC). Protoplasts that had been cultured with βglcY during the period of incipient cell wall regeneration and then transferred to the AC medium divided vigorously, and the cell division rate was remarkably increased (>80%). However, without transfer to the AC medium, βglcY at concentrations higher than 20 μg ml⁻¹ inhibited cell division. No effect on cell survival nor cell division was observed with the α-galactosyl Yariv reagent. Staining of β-1,3-glucan (callose) with aniline blue (AB) showed that a large amount of β-1,3-glucan was deposited in the regenerated cell walls of the protoplasts cultured without βglcY, while little or no β-1,3-glucan was stained by AB in protoplasts cultured with βglcY. These results suggest that AGPs and β-1,3-glucan play important roles in the survival and subsequent cell division of regenerated cells of M. polymorpha protoplast cultures.     

Mating reaction in yeast protoplasts

Protoplasts prepared from complementary haploid strains ofSaccharomyces cerevisiae were studied with regard to their ability of conjugating. Neither fresh protoplasts nor the growing protoplasts possessing fibrillar walls exhibited sex specific agglutination or fusion. However, they were capable of inducing sexual activation in normal cells of opposite mating type. After completing the regeneration of cell walls the protoplasts could conjugate either with each other or with cells of opposite sex. The frequency of conjugations was low, about 1%, and was largely dependent on the degree of completition of the wall during regeneration. From the results the following conclusions may be drawn: 1. The initiation of mating is dependent on the integrity of the cell wall. 2. The sex specific morphogenetic changes do not occur in wall-less protoplasts but may happen after the protoplasts have regenerated their cell walls. 3. The lysis of cell walls does not occur until the walls come into close contact. 4. The fusion of plasma membranes in sex-activated protoplasts cannot be induced by artefucial agglutination.     

Morphological and ultrastructural studies on protoplast production and reversion of the higher basidiomyceteAgaricus bisporus

Protoplasts have been isolated from young vegetative mycelia ofAgaricus bisporus by an enzyme mixture of novozym and chitinase. Protoplasts were released through ruptures in the wall, initially at the apices, but also later from older parts of the hyphae, indicating that they may lack the cell wall. The process of regeneration of these protoplasts has been investigated in liquid medium in which the protoplasts produced short chains of convoluted cells that finally produced a hypha. Electron microscopy has shown that at the start of regeneration two different kinds of fibrils were produced at the external surface of the protoplasts. Later, the thickness of the cell wall increased, and there was a deposit of amorphous material giving rise to a complete new wall.     

Asymmetric protoplast fusion between sweet potato and its relatives,and plant regeneration

Experiments were conducted to asymmetrically fuse protoplasts from sweet potato (Ipomoea batatas L. Lam.) and its wild relativesI. trifida Don. andI. lacunosa L. Protoplasts of sweet potato were treated with iodoacetamide, whereas those ofI. trifida Don. andI. lacunosa L. were irradiated with X-rays. The asymmetric protoplast fusion was carried out by the electrofusion method and by polyethylene glycol treatment. Electrically-fused protoplasts initiated cell division, and then formed calli earlier than the polyethylene glycol-fused protoplasts. Plant regeneration occurred only in electrofused calli, suggesting that polyethylene glycol had some toxic effect on plant regeneration ability. Analysis of peroxidase isozymes confirmed the interspecific hybrid characteristics of both the fusion-derived calli and regenerated plants.     

Effekte der Phospholipase D auf den Elektronentransport und die Lipidzusammensetzung isolierter Spinatchloroplasten

Summary The process of cell wall regeneration around two species of higher plant protoplasts has been studied using reflection scanning electron microscopy. The first stage in the process is the formation of short fibres from randomly spaced centres. With protoplasts of tobacco leaf (Nicotiana tabacum L., cv White Burley) these fibres then elongate and interlace apparently at random to give rise to a matted continuous layer of wall. Protoplasts of a suspension culture of grapevine cells (Vitis vinifera L. cv Müller Thurgau) produce short fibres but these fail to elongate. Budding is observed during wall regeneration around vine protoplasts. The results are discussed in terms of the mechanical properties of the wall and its relationship to changes in plasmalemma morphology which are observed during wall formation.Abbreviation SEM scanning electron microscopy     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

The junction between the plasma membrane and the cell wall in fern protonemal cells,as visualized after plasmolysis,and its dependence on arrays of cortical microtubules

Summary The junction between the plasma membrane and the cell wall in the subapical region of tip-growing protonemata of the fernAdiantum capillus-veneris was visualized by plasmolyzing the cells with a 1 M solution of NaCl. When the protonemata were treated with this solution, cells were rapidly plasmolyzed and the plasma membrane became detached from the cell wall around the entire periphery of the cell, with the exception of the subapex. In the subapical region, the connection between the cell wall and the plasma membrane remained undisturbed, whereas the membrane in other regions, as well as at the apex, was detached from the cell wall. As a result, the protoplasm appeared to adhere to the wall by a ringlike band of plasma membrane at the subapex. The location of the junction coincided with that of a circular array of microtubules (MTs) and microfilaments (MFs) at the cell cortex. The subapical junction disappeared when protonemata were treated with colchicine, cytochalasin B (CB), and blue-light irradiation, all of which are known to disrupt circular arrays of MTs. CB and blue light also disrupt the array of MFs but colchicine does not. Thus, the junction depends on the cortical MTs and not on the MFs. This finding indicates that the junction between the plasma membrane and the cell wall is sustained by a cortical array of MTs and suggests the presence of a specific and localized transmembrane structure.Abbreviations CB cytochalasin B - MF microfilament - MT microtubule     

Protoplasts from the cyanobacterium,Spirulina platensis

Protoplasts were obtained from the filamentous blue-green algaSpirulina platensis by treating the filaments with 0.05% (w/v) lysozyme in 0.03m phosphate buffer. The protoplasts regenerated cell walls and formed colonies when plated on a regeneration medium. The highest percentage of regeneration, 40% was obtained after 21 days.     

Efficient plant regeneration from protoplasts isolated from embryogenic suspension cultures of Cyclamen persicum Mill.

A protocol for plant regeneration from protoplasts has been developed, and then successfully applied to different genotypes of Cyclamen persicum Mill. Protoplasts were isolated from embryogenic suspension cultures by enzymatic digestion in 2% cellulase R10 and 0.5% macerozyme R10. Yields obtained varied between 1 and 5 × 10⁵ protoplasts per gram fresh mass depending on the genotype. Protoplasts were immobilized in alginate films, which promoted proper cell wall regeneration. The highest cell division frequencies were found in modified Kao and Michayluk (1975, Planta 126:105–110) medium containing the same types and concentrations of plant growth regulators that were applied for suspension culture (2.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid and 0.8 mg l⁻¹ 6-(γ,γ-dimethylallylamino)purine). Cell division was recorded for all 11 tested genotypes in frequencies of up to 12% and 18% after 7 and 14 days, respectively. However, cell division frequency varied strongly between different genotypes. After 4–6 weeks calluses were released from the alginate films and further cultured on hormone-containing medium for continued growth or transferred to hormone-free medium for regeneration of somatic embryos. Plant regeneration via somatic embryogenesis succeeded in 9 out of the 11 genotypes under investigation. Up to now protoplast-derived plants from four genotypes have been successfully transferred to soil.     

Genotype-dependent whole plant regeneration from protoplasts of red clover (Trifolium pratense L.)

Protoplasts are useful for subcellular studies, in vitro selection, somatic hybridization and transformation. Whole plant regeneration from protoplasts is a prerequisite to producing altered crop plants using these methods. Whole plant regeneration was achieved from leaf- and suspension culture-derived protoplasts of T. pratense. Regeneration was most dependent upon identifying genotypes with genetic capacity to regenerate. Additional factors that were used to select genotypes, but which proved to be less important, were a high rate of cell growth in culture and a high plating efficiency of protoplasts. One genotype was identified which had a regeneration response equivalent to that of T. rubens and which regenerated from both leaf- and suspension culture-derived protoplasts.Research supported by USDA/CRGO Grant No. 81 CRCR-1-0613     

Formation of cytoplasts from giant protoplasts in culture

Summary Protoplasts isolated from calli ofNicotiana plumbaginifolia can dramatically increase in volume without showing indications of cell wall synthesis. After reaching a critical size, the plasma-rich giant protoplasts show multiple formation of cytoplasts, which are released from the mother cells. The anucleate cytoplasts display the same increase in size as the nucleate protoplasts. Both cell types retain a spherical shape for several months, indicating that no major synthesis of cell wall occurred.     

Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers

Protoplasts ofCunninghamella elegans, showing 11-, and 11-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.     

Plant regeneration from protoplasts of Laminaria japonica Areschoug (Laminariales, Phaeophyceae) in a continuous-flow culture system

A continuous-flow culture system was developed for culturing Laminaria japonica protoplasts. Protoplasts were settled on 5-μm pore size nylon mesh fixed inside a 50-ml plastic syringe, and cultured in Provasoli's enriched seawater with iodine medium with a gentle upward flow generated by a peristaltic pump. In the culture system, 50% of the protoplasts regenerated their cell wall within 24 hours and almost all protoplasts regenerated a cell wall after 3 days culture. After cell wall regeneration, a number of cells divided and regenerated into sheet-shaped thalli. The thalli transferred to a tissue culture flask developed into sporophyte-like plantlets within 1 month. Plantlets then differentiated into blade, stipe, and holdfast, with a proper mucilage canal. Received: 21 April 1997 / Revision received: 27 June 1997 / Accepted: 5 July 1997