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.     

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     

Techniques for enhanced release of leaf protoplasts in Populus

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.Abbreviations BA benzyladenine - NAA naphthalene acetic acid - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - (NC-XXXX) North Central Forest Experiment Station accession number assigned to Populus hybrid clones     

Plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides

A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA.     

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.     

Plant regeneration from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis,S. macrocephala and S. scabra

Plant regeneration from protoplasts is a prerequisite to the production of modified plants using somatic hybridization and transformation. Whole plant regeneration was achieved from protoplasts isolated from seedling cotyledons of Stylosanthes guianensis, S. macrocephala and S. scabra, three economically important species of this tropical forage legume genus. The effects of both protoplast density and protoplast culture method on cell division and plating efficiency are presented.Abbreviations BAP 6-benzylaminopurine - MES 2-(N-Morpholino) ethanesulfonic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalenacetic acid On leave from: Departamento de Genética, Escola Superior de Agricultura Luiz de Queiroz, Universidade de São Paulo, Brasil     

Seaweed protoplasts: status,biotechnological perspectives and needs

Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research.     

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.     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Studies on plant regeneration from cotyledonary protoplasts in Brassica campestris

Protoplasts isolated from both 7-day-old light-grown and 4-day-old dark/dim light-grown cotyledons of four Brassica campestris varieties (Arlo, Sonja, Bunyip and Wonk Bok) were cultured in three liquid media: modified K8P, modified MS and modified Pelletier's B to compare the capacities for cell division and plant regeneration. Following cell wall regeneration the cultured protoplasts from dark/dim light-grown cotyledons of four varieties showed rapid division and high frequency of cell division compared with those isolated from light-grown cotyledons. The frequencies of cell division were significantly influenced by varieties and culture media but only in cultured protoplasts isolated from dark/dim light-grown cotyledons. The interaction between varieties and media was also significant. Cell colonies formed within 7–14 days in protoplast cultures from dark/dim light-grown cotyledons, and calli subsequently grown on a solid medium developed shoots when transferred onto a regeneration medium. Three of four tested varieties (Arlo, Sonja and Bunyip) showed shoot regeneration within 2–3 months after protoplast isolation, with a high degree of reproducibility in Arlo and Bunyip. Regenerated shoots, which were induced to root on half-strength MS medium with 0.1 mg.l^–1 IBA, survived in soil and grew to produce siliques and set viable seeds in the greenhouse. The present report is the first to document the production of regenerated plants that set seeds in Brassica campestris from cotyledonary protoplasts.Abbreviations BAP benzylaminopurine - CPW Composition of Protoplast Washing-solution - 2,4-D 2,4-dichlorophenoxyacetic acid - EDTA ethylenediamine-tetraacetic acid - GA₃ gibberellic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - KT kinetin - FDA fluorescein diacetate - SDS sodium dodecyl sulfate     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

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

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     

Callus formation from isolated sunflower (Helianthus annuus) mesophyll protoplasts

Mesophyll protoplasts of the cultivated sunflower,Helianthus annuus, have been consistently found not to divide or regenerate calli, despite the efforts of several groups. In the present report, we describe the conditions for donor plant culture, protoplast isolation, and their culture that were suitable for repeated regeneration of green, nodular, vigorously growing calli from isolated sunflower mesophyll protoplasts. The best conditions for protoplast isolation employed the use of both CAYLA cellulase and CAYLA pectinase. Culture conditions were not much different from those established earlier for sunflower hypocotyl protoplasts. The most startling observation was the great variability of division frequencies between experiments even under strictly controlled, identical experimental conditions. This finding points to an important influence of a variable in the physiological state of the donor plant which is difficult to control.     

An Improved Protocol for Microcallus Production and Whole Plant Regeneration from Recalcitrant Banana Protoplasts (Musa spp.)

One important limitation for routine production of somatic hybrids in banana (Musa spp.) is the difficulty in protoplast regeneration. To facilitate protoplast regeneration in banana, the crucial step of microcallus production was optimised for the following parameters: nurse culture medium, duration of microcalli on nurse culture, differing nurse cells, and filter composition. A comparative study between two nurse cell media, Ma₂ and PCM, significantly affected the number of microcalli produced, which was 90 × 10³ per Petri dish on Ma₂ with 0.5 μM zeatin and 9.0 μM 2,4 D, and 30 × 10³ per Petri dish on PCM. Moreover, continuous production of microcalli was achieved on Ma₂ and the frequency of embryogenic cell aggregates was higher among microcalli on Ma₂-medium. However, no cell division was observed in protoplasts cultured on Ma₂ in which nurse cells were maintained for 2 weeks suggesting a requirement of effective presence of nurse cells for cell division of banana protoplasts. Use of a filter in conjugation with nurse cells resulted in greater than 7-fold increase in the number of microcalli. Flow cytometry analysis of 124 protoplast-derived plants showed the presence of hexaploid plants (mother plant is triploid) at the frequency of 4%. Together, these data are indicative of the complex factors involved in the regulation of plant cell division and growth. Each individual aspect must be optimised for efficient protocol development.     

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental <Emphasis Type="Italic">Grevillea</Emphasis> species

Summary Degradative changes in tissue during protoplast isolation were a contributing factor to low protoplast yields in the saltsensitive Grevillea arenaria (R. Brown) and the salt-tolerant Grevillea ilicifolia (R. Brown). Protein and malondialdehyde content decreased significantly during the protoplast isolation procedure. Acid and neutral proteases were identified, and high acid protease activities were correlated to low protoplast yields. Acid phosphatase, catalase, polyphenol oxidase and lipoxygenase activities increased in both Grevillea species with cell wall digestion. High activities of catalase and low levels of polyphenol oxidase were correlated with high protoplast yields. Levels of acid phosphatase and lipoxygenase were not good indicators of final protoplast yields. The addition of the anti-oxidant, reduced glutathione, and the acid protease inhibitor, pepstatin A, significantly increased protoplast yields. Strategies were identified to minimize deleterious degradative effects during the isolation of protoplasts, including strict pH control, testing a number of cell wall digestion enzymes, and the addition of anti-oxidative metabolites and protease inhibitors.     

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.     

Culture and regeneration of Populus leaf protoplasts isolated from non-seedling tissue

Leaf protoplasts of Populus alba L. × P. grandidentata Michx. (NC-5339) were isolted from shoot cultures of non-seedling origin and cultured through plant regeneration. Complete protoplast development was dependent on providing a stress-free culture environment which included eliminating ammonium, agar, exudate build-up, and light during the culture period. Contact with a solid surface appeared to stimulate development and thus the protoplasts were cultured in a liquid floating-disc system in which they adhered to the fibers of a polyester screen. Protoplasts exhibited a slow, staged development which resulted in cell division 6 weeks following protoplast isolation. The resulting colonies proliferated rapidly and rooted spontaneously. Shoot regeneration occurred when the protoplast-derived calli were exposed to thidiazuron, and such shoots could be readily rooted. This is the first report of reproducible plant regeneration from leaf protoplasts of non-seedling origin of a tree species.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Plasmolytic behavior of the donor cell may affect protoplast response

Protoplast donor tissues (leaves of shoots in culture) from a herbaceous plant ( Solanum etuberosum ) and two woody species ( Populus alba × P. grandidentata cv. Crandon and Betula platyphylla szechuanica ) were compared during plasmolysis in a range of osmotic agents and potentials. Cells from both Solanum and Populus , species proven to be amenable to protoplast division and regeneration, plasmolyzed readily at higher osmotic potentials than cells from Betula , a species recalcitrant to prolonged culture after protoplast isolation. Betula leaf mesophyll cells exhibited persistent membrane-to-wall attachments and many failed to plasmolyze even under extreme osmolarity. Although their leaves exhibited similar photosynthetic rates, photosynthetic capacity was lost from Betula protoplasts upon isolation, and retained by Solanum protoplasts. Differential stress after isolation was not detectable through vital staining, but only Solanum and Populus gave both high protoplast yields and high plating efficiencies in continued culture.     

Protoplast isolation and transient gene expression in the single-cell C4 species, Bienertia sinuspersici

Although transient gene expression using reporters such as green fluorescent protein is a versatile tool for examining gene functions and intracellular protein trafficking, the establishment of a highly efficient gene manipulation method remains a challenge in many plant species. A reliable transformation protocol has not yet been established for the three single-cell C₄ species, despite their potential of serving as model systems for their extraordinary C₄ photosynthetic metabolism. We report the first protocol optimized for isolating a large-scale and homogenous population of protoplasts from chlorenchyma cells of the single-cell C₄ species Bienertia sinuspersici. Cytochemical staining confirmed the preservation of the unusual subcellular compartmentation of organelles in chlorenchyma cells after cell wall digestion. Approximately 84% of isolated protoplasts expressed the reporter fluorescent protein following our optimized polyethylene glycol-mediated transfection procedures. Fluorescent fusion protein tagged with various intracellular sorting signals demonstrated potential use of the transient gene expression system in subcellular protein localization and organelle dynamics studies. Further applications of the current protoplast isolation and transfection techniques in understanding the novel single-cell C₄ photosynthetic mechanism are discussed.