Dedifferentiation and microtubule reorganization in the apical cell protoplast ofSphacelaria (Phaeophyceae)

Summary The apical cell ofSphacelaria, a tip-growing filamentous brown alga, and its protoplast constitute a model for the investigation of the consequences of cell wall removal on microtubular cytoskeletal organization and cell polarity. In the apical cell, the microtubular cytoskeleton is strongly polarized and, in most cases, extends from two centrosomes to the cortex where it constitutes a fine meshwork. Observations of microtubule dynamics throughout the cell cycle emphasize the coincidence between orientation of the mitotic axis and cell polarity. Just after protoplast isolation, dramatic alterations of initial polarity are observed, whatever the mitotic stage. In particular, the coincidence between cytoplasmic polarity and polarity of the system nucleus-centrosomes is lost in most cases. 12–24 h after protoplast isolation, the cell shows a more symmetrical organization while a dense cortical microtubular network spreads out concomitantly with wall reformation. Our discussion emphasizes the possible relationship between cell polarity and cell totipotency, and the relevance of such a model for higher plant studies.     

Protoplast regeneration from gametophytes and sporophytes of some species in the order Laminariales (Phaeophyceae)

Summary Protoplasts were isolated from sporophytes and from gametophyte cultures of several species in the order Laminariales. For each example, the isolation and culture procedures were investigated systematically, to identify conditions leading to plant regeneration. After dedifferentiation through a filamentous stage, protoplasts isolated from adultLaminaria saccharina sporophytes regenerated polystichous bladelets. In contrast, cells isolated fromLaminaria digitata sporophytes proved recalcitrant in culture, except when the donor plants were undifferentiated sporelings. The most critical factors for protoplast development were the origin of explants, the osmoticum used for cell isolation, cultivation in plain seawater, and the absence of stress during the first two weeks of culture. We also found that protoplast isolation from the sporophytes of members of the Laminariales results in the release of hydrogen peroxide, up to 5–120 μM final concentration in the macerating medium, a characteristic which may be related to protoplast recalcitrance. Protoplasts isolated from the gametophytic phase readily regenerated into normal gametophytes, capable of gametogenesis and producing sporophytes by fertilization.     

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     

Regeneration of cell suspension derived <Emphasis Type="Italic">Apium graveolens</Emphasis> L. protoplasts

Cytoplasmatic male sterility (CMS), which can be achieved by protoplast fusion and regeneration, has potential to greatly facilitate hybrid breeding of celery (Apium graveolens L.). Therefore as a first step we developed a simple and efficient protoplast isolation and regeneration protocol for three commercial A. graveolens varieties (green and white celery and celeriac). To this end, cell suspensions from independent cell lines of open pollinated cultivars and inbred lines were initiated as a source for protoplast isolation. Comparative analyses showed that culturing was most successful in modified Kao and Michayluk liquid medium supplemented with 0.3 mg l^?1 2,4-D. The cytokinin type (TDZ or zeatin) and concentration had no significant effect on regeneration efficiency. Microcalli were obtained within 15 days to 5 weeks after protoplast isolation. Supplementing the culture medium with 25% conditioned medium increased microcolony formation for some of the cultured lines. Plants were obtained within 2 months of microcallus culturing and these were all diploid, suggesting genetic inheritance consistency. The efficiency of regeneration mainly depended on the specific genotype, with outcrossing genotypes displaying high heterogeneity in regeneration responses whereas inbred lines did not regenerate. The protocol presented here enables to implement protoplast fusion in celery breeding.     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

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.     

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.     

A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Review: Seaweed tissue culture as applied to biotechnology: Problems,achievements and prospects

Advances have been made in cell and tissue culture of seaweeds to define a unique branch of in vitro techniques; however, they are lagging far behind those of land plants and have limited applications. Explants can be cultivated axenically in enriched or artificial seawater culture media, and regeneration and even callus formation are achieved. In this state of the art technique, seaweed tissue culture may be already useful for certain biotechnological applications, such as clonal propagation of seed material for mariculture. Nevertheless, the absolute control of growth and development as it is exerted in higher plant tissue culture is lacking, and it is required for more complex biotechnological applications in seaweeds. Definitively, we need appropriate cells (competent cells) to induce growth with the most effective chemical regulators in culture medium adjusted towards the addition of carbon sources. Still, free cells and protoplast isolation and regeneration in marine seaweeds constitute the most developed topic in seaweed tissue culture. The regulation of growth and development of seaweed free cell and protoplast cultures may sustain a purposeful use of techniques in the era of genomic applications.     

Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

Yeast protoplasts immobilized in alginate: Cell wall regeneration and reversion to cells

Yeast protoplasts may regenerate the cell wall and revert to cells if immobilized in a 2%–5% Ca-alginate gel and cultured in an osmotically stabilized medium. The method of protoplast immobilization and subsequent isolation from the gel is described in detail. The reversion yield is dependent of the actual gel concentration, gel shape (beads vs. sheets) and of a medium molarity, and it may be up to 90%. The morphology of the cell wall regeneration and morphology of reversion to the cell forms correspond to protoplast development in gelatin or agar gels.     

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Optimized Chemodiversity in Protoplast-derived Lines of St. John's Wort (<Emphasis Type="Italic">Hypericum perforatum</Emphasis> L.)

Summary A procedure for protoplast isolation and plant regeneration of St. John's wort has been developed to utilize cell-to-cell variability for optimum production of valuable medicinal compounds. Calluses, induced from hypocotyl segments of St. John's wort seedlings, were used for protoplast isolation, induction of sustained cell division, and ultimately, plant regeneration. Callus-isolated protoplasts at a density of 2.0×10⁵ per ml were embedded in 0.6% Na-alginate blocks and cultured in a medium containing modified Murashige and Skoog (MS) salts, 2.5 μM 6-benzylaminopurine (BA), 5.0 μMα-naphthaleneacetic acid (NAA), and 0.5 moll⁻¹ glucose. Protoplast-derived colonies formed compact calluses when transferred onto 0.35% gellan gum-solidified MS medium supplemented with 2.5 μM BA and 2.5 μM NAA. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5 μM thidiazuron. Complete plantlets were obtained from the regenerated shoots on MS basal medium. A greater than 3-fold variation of antioxidant activity was observed among the protoplast-derived plantets and chemically distinct germplasm lines were selected on the basis of phytochemical profiles. The protoplast to plant regeneration protocol developed in this study provides the foundation for development of novel genotypes with potential expansion of the genetic diversity through somatic hybridization, and organelle transplantation.     

Isolation of cytoplasts from Satsuma mandarin (Citrus unshiu Marc.) and production of alloplasmic hybrid calluses via cytoplast–protoplast fusion

Cytoplasm of Satsuma mandarin (Citrus unshiu Marc.) is known to influence seedlessness. Transfer of cytoplasm to a seedy cultivar could possibly lead to the production of seedless citrus fruits. In the present paper cytoplasts were isolated from cell suspension-derived protoplasts of Satsuma mandarin via ultra-centrifugation in a discontinuous gradient. No nucleus could be detected in the cytoplasts by DAPI (4′, 6-diamidino-2-phenylindole) staining compared with normal protoplasts. The cytoplasts, with high viability and small size, did not divide during solid embedding culture. Cytoplasts of Satsuma mandarin were electrically fused with embryogenic protoplasts of Murcott tangor (C. reticulata × C. sinensis), which led to regeneration of several cell lines. Flow cytometry (FCM) indicated that the cell lines were diploids. Simple sequence repeats (SSR) and cleaved amplified polymorphism sequence (CAPS) showed that the cell lines got their nuclear DNA from the protoplast parent, whereas the cytoplast parent donated the mtDNA, confirming transfer of mtDNA from Satsuma mandarin into Murcott tangor via cytoplast–protoplast fusion though no polymorphism was detected in chloroplast DNA between the fusion partners. This is the first report on isolation and characterization of cytoplasts, together with cytoplast–protoplast fusion in Citrus, which has a potential for citrus cultivar improvement involving cytoplasm transfer via cytoplast–protoplast fusion.     

Efficient regeneration of <Emphasis Type="Italic">Sphagnum fallax</Emphasis> from isolated protoplasts

Summary While the in vitro clonal propagation of peat mosses (Sphagnaceae) in bioreactors has been established since the late 1980s, it has never been possible to regenerate Sphagnum species from isolated protoplasts, which is a key step towards the production of closely defined genetically modified clones. The present study describes an efficient protocol for protoplast isolation and regeneration of Sphagnum fallax. Protoplast survival rates of over 50% and regeneration rates of up to 20% were achieved by using excised capitulum buds as starting material and by co-cultivating Sphagnum protoplasts with protoplasts from a chlorophyll-deficient Solanum hybrid clone. Besides the effects of nutrient components and differential osmotic readjustment of the regenerant cell clusters, the interference of unique Sphagnum phenolics, sphagnum acid and hydroxybutenolide, with protoplast isolation efficiency is demonstrated.     

Protoplasts fromPodospora anserina: Isolation,purification, and transformation

Protoplasts fromPodospora anserina mycelium were produced using the commercially available enzyme Novozym 234. Different parameters involved in protoplast isolation were analyzed in order to establish optimal conditions, and protoplast production was notably increased. For the purification of protoplasts, several techniques based on both centrifugation and filtration were assayed, with filtration yielding the best results. Regeneration of protoplasts was studied on different media and osmostic stabilizers, and about 80% regeneration was obtained. The good physiological condition of the protoplasts produced with this method is demonstrated by the lack of cell wall and high regeneration rate and transformation frequencies.     

Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

Spatial organization of axonal microtubules

Several workers have found that axonal microtubules have a uniform polarity orientation. It is the "+" end of the polymer that is distal to the cell body. The experiments reported here investigate whether this high degree of organization can be accounted for on the basis of structures or mechanisms within the axon. Substantial depolymerization of axonal microtubules was observed in isolated, postganglionic sympathetic nerve fibers of the cat subjected to cold treatment; generally less than 10% of the original number of microtubules/micron 2 remained in cross section. The number of cold stable MTs that remained was not correlated with axonal area and they were also found within Schwann cells. Microtubules were allowed to repolymerize and the polarity orientation of the reassembled microtubules was determined. In fibers from four cats, a majority of reassembled microtubules returned with the original polarity orientation. However, in no case was the polarity orientation as uniform as the original organization. The degree to which the original orientation returned in a fiber was correlated with the number of cold-stable microtubules in the fiber. We suggest that stable microtubule fragments serve as nucleating elements for microtubule assembly and play a role in the spatial organization of neuronal microtubules. The extremely rapid reassembly of microtubules that we observed, returning to near control levels within the first 5 min, supports microtubule elongation from a nucleus. However, in three of four fibers examined this initial assembly was followed by an equally rapid, but transient decline in microtubule number to a value that was significantly different than the initial peak. This observation is difficult to interpret; however, a similar transient peak has been reported upon repolymerization of spindle microtubules after pressure induced depolymerization.     

Protoplast isolation and regeneration from <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariales,Rhodophyta)

The tropical agarophyte Gracilaria changii has been much researched and documented by the Algae Research Laboratory, University of Malaya, especially with regards to its potential as a seaweed bioreactor for valuable compounds. Protoplast regeneration of this seaweed was developed following the optimization of protoplast isolation protocol. Effect of the concentration and combination of isolating enzymes, incubation period, temperature, enzyme solution pH, tissue source on the protoplast yields were used to optimize the isolation protocol. The enzyme mixture with 4% w/v cellulase Onozuka R-10, 2% w/v macerozyme R-10 and 1 unit mL^-1 agarase was found to produce the highest yield of protoplast at 28°C and 3 h incubation period. Thallus tips gave higher yields of protoplasts than middle segments. Freshly isolated G. changii protoplasts were cultured in MES medium. Regeneration of protoplast cell walls after 24 h was confirmed by calcofluor white M2R staining under UV fluorescence microscopy. The protoplasts with regenerated cell walls then underwent a series of cell division to produce callus-like cell masses in MES medium. Following this, juvenile plants of G. changii were obtained.     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.