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     

Isolation Conditions for High Yields of Protoplasts from Laminaria saccharina and L. digitata (Phaeophyceae)

In an investigation of the main factors determining protoplastyield in Laminaria saccharina and L. digitata, protoplasts wereisolated from epidermal, cortical and medullary cells of vegetativethallus by incubation with commercial cellulases, crude andpurified mannuronate lyases and purified guluronate lyases.Treatment of the tissue with the calcium chelator EGTA beforeenzymatic digestion greatly increased the protoplast yield.Preplasmolysis was also necessary to obtain large numbers ofhealthy protoplasts and this was most effective when carriedout during chelation with EGTA. Purification of the mannuronatelyases by ion exchange chromatography reduced the toxicity ofthe crude enzyme preparation. The activities of the wall degradingenzymes were differentially influenced by pH and the optimumfor alginate-lyase activity (8.0) was higher than that for cellulaseactivity (<6.0). Protoplast yield decreased linearly withincreasing pH in the enzyme medium over the range tested (6.0–8.0),and this suggests that cellulases are more critical to walldigestion than alginate-lyases. Ionic osmotica gave improvedyields compared with sugar alcohols or sugars. Increasing thecalcium concentration of the enzyme medium brought about anexponential decrease in protoplast yield and wall digestionwas almost completely inhibited at concentrations exceeding8.0 mol m^–3. However, low levels of calcium (<2.0 molm^–3) were beneficial to protoplast viability. Yields of10⁷ to 10⁸ protoplasts g^–1 fr. wt. were consistently obtainedand 20% to 30% of these regenerated new cell walls within 1–2d of culture. Key words: Laminaria protoplasts, cell wall, alginate-lyases     

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     

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo'Green Delica'

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumismelo‘Green Delica’ were used as explants for protoplastisolation and culture. Protoplasts isolated from cotyledonsand etiolated half-expanded leaves cultured in Durand, Potrykusand Donn (DPD) medium supplemented with 0.9 µMbenzylaminopurine(BAP), 3.6 µM2,4-dichlorophenoxyacetic acid (2,4-D) and1% sucrose, using the agarose bead culture method, were ableto form cell walls and subsequently go through cell division.Pretreatment of half-expanded leaf explants in the dark for14 d provided the best material for protoplast isolation andcell division. Approximately one third of protoplasts from etiolatedhalf-expanded leaves formed microcolonies. For hypocotyl protoplasts,none of the treatments used were suitable to induce cell division.There was no significant difference between sucrose, glucose,and sucrose plus glucose, in culture media on the plating efficiencyof leaf protoplasts ofC. melo‘Green Delica’; however,bigger colonies were formed in media supplemented with 1% sucrose.No shoot or whole plant regeneration was achieved. However,the methods reported here provide further information onC. meloprotoplastculture.Copyright 1998 Annals of Botany Company Cucumis melo,protoplast culture, 2,4-D, BAP, yeast extract, casein hydrolysate.     

The Effects of Cell Cycle Parameters on Cell Wall Regeneration and Cell Division of Cotton Protoplasts (Gossypium hirsutum L.)

Protoplasts of cotton cotyledons were isolated and culturedto undergo cell wall regeneration and cell division. DNA contentand cell cycle parameters of nuclei from cotyledons and/or protoplastswere determined by flow cytometry. The DNA content of cotton,Gossypium hirsutum L., was estimated to be 4·34±0·12pg DNA per nucleus. There was a strong positive correlation between G₂ or Sand G₂,and cell wall regeneration and cell division and a strong negativecorrelation between G₁, and cell wall regeneration and celldivision of cotton cotyledon protoplasts. The cell cycle statusof cotyledons changes during their development; as the cotyledonsenlarge, the proportion of cells in G₀ and G₁ phases of thecell cycle increases. The implication of these results in relationto protoplast growth and development is discussed. Key words: Cell cycle parameters, cell wall regeneration, cell division, flow cytometry, Gossypium     

Inhibiting Effect of Ammonium Ion in Protoplast Culture of Some Asteraceae Plants

Protoplasts with high division activity were obtained reproduciblyfrom mesophyll cells of axenic shoot culture of three wild speciesof Asteraceae, Artemisia vulgaris L., Chrysanthemum indicumL. and Ch. zawadskii Herbich, by means of a suitable combinationof enzymes with a gentle pipetting treatment. Ammonium ion was found to inhibit cell division in the protoplastculture of these Asteraceae species but to accelerate it inthe case of tobacco. The inhibiting effect of ammonium ion wasdrastic on the protoplast of Artemisia but moderate on thatof Ch. indicum and Ch. zawadskii. The total concentration ofthe major inorganic elements had little effect on the divisionfrequency of protoplasts in these species. ³Present address: Kirin Brewery Co., Ltd., Plant Research Center,Kitsuregawa, Tochigi-ken 329-14, Japan. (Received October 12, 1983; Accepted December 20, 1983)     

High Frequency Divisions in Leaf Base Protoplasts of Wheat (Triticum aestivum L.)

Protoplasts were isolated from the basal meristematic region of leaves from 6-day-old seedlings of wheat (Triticum aestivum). Protoplasts divided when cultured on MS medium (as agarose beads) in presence of nurse tissue. Donor seedlings when grown on BAP-supplemented MS medium were found to be considerably superior for protoplast isolation and culture than when grown on MS basal medium, in terms of protoplast viability, cell wall formation and cell division frequency. In addition, reduction of ammonium content of the culture medium, together with a dark Incubation, led to a high protoplast division frequency of about 70%. Microcolonies of 10-to 12-celled stages were obtained in Triticum aestivum, varieties HD 2329, HD 2285, Kalyan Sona, Arjun and CPAN 1676.     

Isolation and Characterization of a Cytokinin Up-Regulated Gene from Tobacco Mesophyll Protoplasts

We have isolated a cytokinin up-regulated cDNA clone, H13, froman early stage of cultured tobacco mesophyll protoplasts bya differential display method. The expression of this gene wasspecifically induced by natural and synthetic cytokinins includingN-(2-chloro-4-pyridyl)-N'-phenylurea (4PU30), a diphenylurea-typecytokinin, although the simultaneous presence of auxin was alsorequired. It seems that the preceding treatment of the tobaccomesophyll protoplasts by auxin is necessary for the gene torespond to cytokinin. The addition of a cytokinin antagonist,compound 182, which suppressed the induction of cell divisionin tobacco mesophyll protoplasts, completely abolished the expressionof this gene. Though the predicted gene product of H13 did notsuggest us any sequences of defined functions, two domains ofthe predicted sequence had significant homology to several reportedsequences in the data base. The gene product of H13 is proposedto have a role in regenerating cell wall in cultured protoplasts,since a cDNA clone E6, from cotton fiber cells, which has themost closely related structure to H13, has been isolated fromcells which showed active cellulose synthesis. This suppositionis supported by the evidence that in the absence of cytokinin,cell wall regeneration was significantly suppressed, resultingin failure of the induction of cell division. Thus, the geneproduct of H13 is supposed to have a role in regenerating cellwalls and facilitating the progression of the cell cycle, resultingin the sustained cell division of tobacco mesophyll protoplasts. ¹These authors are equally contributed to this work.     

Isolation of Protoplasts from Kappaphycus alvarezii var. tambalang (Rhodophyta) and Secretion of i-Carrageenan Fragments by Cultured Cells

A method for generating protoplasts from the carrageenan-producingred alga Kappaphycus alvarezii was developed. Digestions withcellulase and _k-carrageenase produced only a few cortical cellprotoplasts, while digestions with cellulase and i-carrageenaseonly produced epidermal cell protoplasts. When both carrageenaseswere used in the digestion media with cellulase, protoplastswere released from all cell types and yields ranged from 1·0to 1·2x10⁷ cells g^–1 with sizes from 5 to 200 µmdiameter. Protoplasts were subsequently cultured to study cellwall regeneration. Calcofluor-positive material (probably cellulose)was detected within 6 h after removal of protoplasts from thewall digestion media, whereas, i-carrageenan fragments weredetected in all regenerating protoplast cultures 24 h afterremoval from the digestion media. Protoplasts continued to produceCalcofluorpositive material and secrete carrageenan fragmentsinto culture media for several days. However, cells culturedin media augmented with K⁺ ions stopped secreting carrageenanfragments after 24 h. Cells cultured for 48 h in seawater labelledweakly with an i-carrageenan hybridization probe, but not atall with a corresponding _k-probe. Cells cultured for 48 h, blottedto nylon membranes and probed with anti-carrageenan monoclonalantibodies, showed the presence of gelling carrageenan subunitsin the cell walls. Key words: -Carrageenan, Kappaphycus, protoplasts, Rhodophyta     

The Investigation of Stomatal Ionic Relations using Guard Cell Protoplasts: I. METHODOLOGY

Clint, G. M. 1985. The investigation of stomatal ionic relationsusing guard cell protoplasts. 1. Methodology.—J exp. Bot.36: 1726–1738. A study was made of the methodology for the production and useof guard cell protoplasts in ion transport studies, with particularemphasis placed on the effects of the composition of the externalmedium on protoplast survival and performance. Addition of externalKCl to media during the production of guard cell protoplastsfrom Commelina communis L. was found to improve viability andto increase K⁺ content and physiological competence of the isolatedprotoplasts. Addition of low levels (20 x 10^–3 mol m^–3)CaCl₂ increased protoplast yield and the maintenance of viabilityin long-term incubation. Ambiguities and uncertainties werefound in the application of methods commonly used for the assessmentof viability of isolated protoplasts. Poor yields (despite highpercentage recoveries) together with difficulties in the assessmentof viability were considered to pose major potential problemsin the use of guard cell protoplasts in ion transport studies. Key words: Guard cell protoplasts, ion transport, Commelina communis     

Sucrose biosynthesis in Dunaliella

Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.     

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.     

Induction of Cell Division in Leaf Cells of Coconut Palm by Alteration of pH and its Correlation with Glyoxalase-I Activity

Cell division in suspension cultures obtained from leaf cellsof coconut was influenced by pH of the culture media. A 3-foldincrease in cell number was obtained at pH 7.0 compared to suspensionsgrowing at pH 5.0. The pH of both cells and media changed after48 h of growth. Internal cell pH showed a significant increasewhen cultures were grown at pH 7.0 and 8.0 and increased onlyslightly at pH 5.0 and 6.0. Glyoxalase-I activity of cells insuspension culture was found to be pH-depcndent, showing maximumactivity at pH 7.0. Glutathione, a co-enzyme for the substratemethylglyoxaJ for glyoxalase-I, produced a 2-fold increase incell number at a concentration of 5 x 10^–3 mol dm ^–3.The polyamine, spermidine, promoted cell division maximallyat a concentration of 10^–6 mol dm^–3. Methylglyoxal-bis(guanylhydrazone), an inhibitor of spermidine biosynthesis,strongly inhibited cell division giving maximum inhibition ata concentration of 3 x 10^–6 mol dm ^–3. These resultsindicate a positive correlation between cell division and glyoxalase-Iactivity. Key words: Cocos nucifera, glyoxalase-I, pH, spermidine     

A Cryomicroscopic Study of Cylindrocystis brebissonii De Bary and Two Species of Micrasterias Ralfs (Conjugatophyceae, Chiorophyta) during Freezing and Thawing

The changes in morphology of the unicellular algae Cylindrocystisbrebissonii and two species of Micrasterias during freezingand thawing were observed on a light microscope fitted witha temperature controlled stage. At slow rates of cooling extensiveshrinkage of the protoplast was observed. The response of thecell wall varied with cell-type. In C. brebissonii plasmolysiswas not observed and the cell wall and protoplast shrank together.In Micrasterias the cell wall did not contract and a distinctplasmolysis was observed. Following freezing to and thawingfrom –25?C cells of C. brebissonii were non-viable butremained osmotically responsive. Cooling at faster rates inducedintracellular ice formation in all cell-types. The criticalrate of cooling varied with cell-type and was determined bycell volume and suface area. Intracellular gas bubbles wereobserved during thawing following both rapid and slow cooling. Following cooling in dimethylsulphoxide cells of C. brebissoniiwere protected against freezing injury. The recovery on thawingfrom –196?C being determined by the rate of cooling, anoptimum rate of 1?C min^–1 was observed. During slow ratesof cooling (<2?C min^–1) cells remained unshrunken,at faster rates (10?C min^–1) the loss of cell viabilitywas related to osmotic shrinkage during cooling rather thanto nucleation of intracellular ice. Intracellular ice formationwas observed only following significantly faster rates of cooling(>20?C min^–1). Key words: Cylindrocystis, Micrasterias, cryomicroscopy, freezing injury     

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

High Efficiency Plant Regeneration from Protoplasts of the Indica Cultivar IR58

Procedures have been established for embryogenic cell suspension,protoplast culture, and plant regeneration from the Indica ricecultivar, IR58. Embryogenic cell suspension was establishedin modified R2 medium and maintained in amino acid-based AAmedium. Protoplasts were cultured in R2 basal medium containingB5 vitamins. Plants were regenerated in MS basal medium with3% maltose as carbon source. Selection of embryogenic protoplast-derivedcalli is an important step for high-frequency plant regeneration.More than 900 plants were regenerated through this procedure. Key words: Rice (Oryza sativa, Indica, Protoplast, Maltose     

Endogenous Cytokinins in Flower Bud Forming Explants of Tobacco

The accumulation of endogenous cytokinins was studied in pedicelexplants of tobacco (Nicotiana tabacumL.) during regenerationof flower buds in vitro. Maximal bud formation was induced onmedia containing 1.0 mmol m^–3 of benzyladenine or dihydrozeatin.No buds were formed in the absence of cytokinin. The levelsof dihydrozeatin, zeatin, and the corresponding ribosides weredetermined in explants cultured in the presence or absence ofcytokinin by means of a competitive ELISA technique. In explantsincubated without a cytokinin, only the dihydrozeatin concentrationincreased significantly during the first day of incubation anddecreased during the second day. No increase was observed inexplants incubated in the presence of benzyladenine. The concentrationof dihydrozeatin in these bud-forming explants was only 10 to15% of the concentration built up in explants cultured on dihydrozeatininstead of benzyladenine. This suggests that the endogenouscytokinins only play a minor role in the regeneration of flowerbuds in vitro. Key words: cytokinin, flower bud development, tissue culture, tobacco     

The Effects of Extracellular Calmodulin on Cell Wall Regeneration of Protoplasts and Cell Division

The effects of anti-calmodulin (CaM) serum, CaM antagonist W7-agaroseand exogenous pure CaM on cell wall regeneration of protoplastsand cell division for Angelica dahurica and other plants werestudied. Anti-CaM serum inhibited cell wall regeneration ofprotoplasts and the first cell division in dose-dependent manner,while the same amount of preimmune serum had a much less inhibitoryeffect than anti-CaM serum. The first cell division was alsoinhibited by CaM antagonist W7-agarose. The addition of exogenouspure CaM enhanced cell wall regeneration of protoplasts andthe cell division for several species of plants, while the sameamount of bovine serum albumin had no obvious effect. CaM wasdetected in the normal culture medium by means of enzyme-linkedimmunosorbent assay. Its content increased with the culturetime. The results suggest that extracellular CaM plays an importantrole in promoting cell wall regeneration of protoplasts andcell division. The possible mechanisms by which extracellularCaM achieves its effects are discussed. (Received February 24, 1994; Accepted November 14, 1994)