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     

Protoplast production in Chondrus crispus gametophytes (gigartinales,rhodophyta)

Protoplasts were isolated from female gametophytes of Chondrus crispus (Stackh.) using commercial cellulase and various carrageenases prepared from marine bacteria. Depending on the nature of the donor tissue (apices or whole thallus, wild or cultivated strains), yields ranged from 1.0–8.5×10⁸ protoplasts per gram of fresh tissue. Preincubating the tissue with a potassium chelator, Kryptofix 222, enhanced protoplast yields by 30–50 %. Based on staining with fluorescein diacetate most protoplasts were viable. A few protoplasts regenerated a cell wall and divided.     

Asymmetric somatic hybridization between wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) and <Emphasis Type="Italic">Avena sativa</Emphasis> L.

Protoplasts from cell suspensions of young-embryo-derived calli, whichwere non- regenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of300 μW/cm2 for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Common wheat is one of the most important cereal crops in the world. The improvement of its yield and quality by the introduction of heterologous gene(s) is very significant. Avena sativa L. (2n = 42), belonging to the Avena tribe, possesses resistance to drought, coldness and many dis-eases. Its contents of proteins and fat in seed, especially lysine and unsaturated fatty acid are highest in crops, therefore it is regarded as healthy food. Sexual hybridization between wheat and Avena sativa…     

The isolation, culture and regeneration of Petunia leaf protoplasts

Methods are described for the enzymatic release of protoplasts from leaves of Petunia hybrida and for the utilization of protoplasts in studies in plant developmental biology. As a result of spontaneous fusion during cell wall degradation of leaf material, fresh preparations can contain a high proportion of multinucleate protoplasts. This level can be dramatically reduced by a gradual plasmolysis of the material prior to enzyme incubation.Leaf protoplasts maintained in liquid media are seen to undergo cell wall synthesis, “budding,” and limited regenerated cell division sometimes associated with anthocyanin production. Under such conditions, multinucleate cells are formed as a result of mitosis without cytokinesis.Protoplasts, plated out in a fully defined medium, undergo cell wall synthesis followed by sustained progeny cell division with eventual cell colony production. Cell colonies, derived from individual mesophyll protoplasts, grow rapidly upon subculture, to produce callus capable of shoot differentiation and ultimately whole plant formation. Protoplasts isolated from varieties of P. hybrida were found to differ in their cultural requirements.     

Asymmetric somatic hybridization between wheat (Triticum aestivum) and Avena sativa L.

Protoplasts from cell suspensions of young-embryo-derived calli, which were nonregenerable for long-term subculture and protoplasts from embryogenic calli with the regeneration capacity of 75% of the same wheat Jinan 177, were mixed as recipient. Protoplasts from embryogenic calli of Avena sativa (with the regeneration capacity of less than 10%) irradiated with UV at an intensity of 300 μW/cm² for 30 s, 1 min, 2 min, 3 min, 5 min were used as the donor. Protoplasts of the recipient and the donor were fused by PEG method. Many calli and normal green plants were regenerated at high frequency, and were verified as somatic hybrids by chromosome counting, isozyme, 5S rDNA spacer sequence analysis and GISH (genomic in situ hybridization). Fusion combination between protoplasts either from the cell suspensions or from the calli and UV-treated Avena sativa protoplasts could not regenerate green plants.     

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.     

Isolation, purification and regeneration of protoplasts from Sporotrichum thermophile conidiospores

Protoplasts of uniform size were prepared from mononucleated conidiospores of Sporotrichum thermophile. Conidia were preincubated in glucose yeast extract medium at 45 C for 4 h. The conidia were collected resuspended in buffer containing 0.6 M KCl (as stabilizer), and incubated with Novozyme SP249 and Cellulase CP at 37 C for 6 h. The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 50% sucrose. The purified protoplasts were regenerated in glucose yeast extract broth after 7 h of incubation at 45 C.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA|Benzyladenine - BA Benzyladenine     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

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.     

Protoplast formation from submerged mycelium and from spore germinants of Streptomyces coelicolor

Stages in the formation of protoplasts from S. coelicolor strain A3(2) have been studied by transmission electron microscopy. Protoplasts liberated from submerged mycelial growth were variable in size and were released when digestion of the cell wall by lysozyme had completely or almost completely taken place. Protoplasts did not fully adopt the typical rounded shape until after release. A single region of cytoplasm gave rise to more than one protoplast unit. Protoplasts released from spore germinants escaped from the tip of the germ tube, which was the region of the cell wall most susceptible to digestion. Protoplasts derived from spore germinants were more consistent in size and rounded up more rapidly. If a cross-wall had formed in a germinant then it gave rise to separate protoplasts from each cellular compartment. Protoplasts of either type contained a single DNA region. These studies give an indication of the cellular organization of a streptomycete colony, which can be visualized as a multinucleated assemblage of cellular units in a common cytoplasm. The assembly of units separates into a number of protoplasts on digestion of the cell wall.     

DEVELOPMENT OF PROTOPLASTS FROM HOLDFASTS AND VEGETATIVE THALLI OF MONOSTROMA LATISSIMUM (CHLOROPHYTA, MONOSTROMATACAE) FOR ALGAL SEED STOCK

The aim of this study was to isolate and cultivate the protoplasts of the green alga Monostroma latissimum Wittrock and subsequently induce them to form algal filaments to act as an algal "seed" stock. Protoplasts of the alga were isolated enzymatically with 4% cellulase Onozuka R-10 and 2% Macerozyme R-10. The highest number of protoplasts was obtained on a 50-rpm shaker with 1.2 M of sorbitol after 6 h of incubation, with a yield of 9 × 106 protoplasts·g−1 of fresh thallus (including holdfast). Protoplasts from both holdfasts and erect thalli usually began to form new cell walls within 5 h after isolation and began to divide from day 6 to day 9 in PES medium; cell clusters, filaments, and/or tubular thalli were formed from day 14 to day 18. For algae collected in March, about 60% of protoplasts isolated from vegetative thalli regenerated to form tubular thalli, and about 45% of protoplasts isolated from holdfasts regenerated to form filaments. However, for algae collected in May, about 1% of protoplasts isolated from vegetative thalli developed directly to form tubular thalli, and 59% of protoplasts regenerated to form cell clusters without the ability to differentiate, whereas protoplasts isolated from holdfasts failed to develop. Regenerated filaments were kept in an incubator for more than 3 years at 24° C under the low irradiance of 66μmol photons·m−2·s−1. After this time, they retained the ability to develop to form tubular thalli under irradiance of 166 and 300 μmol photons·m−2·s−1 at 18°–30° C. Subsequently, these tubular thalli can develop to form leafy thalli after being cultivated at high irradiance of 300 μmol photons·m−2·s−1 and at 18°–22° C. Therefore, the filaments could serve as"seed" stock for algal mass culture.     

Protoplasts of Pyricularia oryzae P2 : Preparation and Regeneration into Hyphal Form

Protoplasts of Pyricularia oryzae P₂, a rice blast mold, were prepared in high yield from the young mycelium of the fungus using lytic enzymes from Bacillus circulans WL 12. The majority of the protoplasts had one nucleus per cell. The protoplasts formed a cell wall and eventually reverted to normal mycelial form in liquid medium. The process of regeneration was studied under phase-contrast and electron microscopes. The protoplast built a very thick wall prior to the protrusion of a germ-tube like hypha. Golgi apparatus-like structures appeared in the early stage of regeneration and disappeared later. Electron-transparent amorphous structures accumulated during regeneration. Lomasomes were observed in the regenerated cell walls.     

Somatic hybridization in Citrus: navel orange (C. sinensis Osb.) and grapefruit (C. paradisi Macf.)

Summary Protoplasts of navel orange, isolated from embryogenic nucellar cell suspension culture, were fused with protoplasts of grapefruit isolated from leaf tissue. The fusion products were cultured in the hormone-free medium containing 0.6 M sucrose. Under the culture conditions, somatic embryogenesis of navel orange protoplasts was suppressed, while cell division of grapefruit mesophyll protoplasts was not induced. Six embryoids were obtained and three lines regenerated to complete plants through embryogenesis. Two of the regenerated lines exhibited intermediate morphological characteristics of the parents in the leaf shape. Chromosome counts showed that these regenerated plants had expected 36 chromosomes (2n=2x=18 for each parent). The rDNA analysis using biotin-labeled rRNA probes confirmed the presence of genomes from both parents in these plants. This somatic hybridization system would be useful for the practical Citrus breeding.     

3,4-Dehydroproline inhibits cell wall assembly and cell division in tobacco protoplasts.

We investigated the function of cell wall hydroxyproline-rich glycoproteins by observing the effects of a selective inhibitor of prolyl hydroxylase, 3,4-dehydro-L-proline (Dhp), on wall regeneration by Nicotiana tabacum mesophyll cell protoplasts. Protoplasts treated with micromolar concentrations of Dhp do not develop osmotic stability and do not initiate mitosis. The architecture of regenerated cell walls was examined using deep-etch, freeze-fracture electron microscopy of rapidly frozen tobacco cells. Untreated protoplasts assemble a dense fibrillar cell wall consisting of laterally associating subelementary fibrils. In contrast, treatment of protoplasts with Dhp alters the structure of the regenerated wall fibrils in several ways: first, the microfibrils are coated with globular knobs; second, some larger fiber bundles have an open ribbon-like appearance; and third, the smallest subelementary fibrils were not visible. Tobacco cells develop an abnormal morphology as a consequence of this abnormal cell wall structure. Thus, inhibition of prolyl hydroxylase results in the regeneration of a cell wall with abnormal structural and functional properties. These data provide experimental evidence that hydroxyproline-rich glycoproteins are important for the structural integrity of primary cell walls and for the correct assembly of other wall polymers, and that wall structure is an important regulator of cell division and cell morphology.     

ELECTRON MICROSCOPIC OBSERVATIONS OF CULTURED CELLS AND PROTOPLASTS OF AMMI VISNAGA

Protoplasts were prepared from cultured cells of Ammi visnaga (Umbelliferae) by enzymatic digestion of the cell walls and examined microscopically. Staining of fresh protoplasts with Calcofluor and silver hexamine demonstrated the apparent absence of wall material. Protoplasts contained more cell organelles than the whole cells, particularly endoplasmic reticulum and associated polysomes. The plasmalemma of most protoplasts appeared smooth; some protoplasts were connected by structures resembling plasmodesmata. Multinucleates resulting from fusion were frequently observed.     

Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta)

Protoplasts were isolated enzymatically from the red alga Gracilariaverrucosa using only two enzymes: agarase prepared from marinebacteria and commercial cellulase. Yields of protoplasts weredependent on the donor material and by choosing young bladesor algae in a state of higher growth rate, the production ofprotoplasts reached a maximum of 10⁷ protoplasts per gram offresh tissue. Cell viability was better with NaCI used as osmoticumthan with sorbitol in the culture medium and on reducing culturemedia to normal osmolarity in 4 d. 25% of the cultured protoplastswere able to regenerate a cell wall (i.e. cellulose) within7 d as confirmed by staining with calcofluor white althoughonly a few protoplasts were able to divide. During the first24 h of culture, the synthesized agar contained higher amountsof L-galactose-6-sulphate than the cell wall of thalli. Theamount of agar in the protoplasts, however, did not increase,indicating that the protoplasts synthesize a qualitatively differentcellwall. Key words: Agarase, agar, cell wall regeneration, Gracilaria verrucosa, protoplasts     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts