Protoplast Yield Research Articles

Isolation and cell wall regeneration of protoplasts from Posidoniaoceanica and Cymodoceanodosa

A method for the isolation of protoplasts from the seagrasses Posidonia oceanica and Cymodocea nodosa is described. Isolation of protoplasts was achieved using a combination of cellulase Onozuka R-10, hemicellulase and pectinase. Purification was carried out by Ficoll gradient centrifugation. A yield of 1.87 (± 0.16 SD)×10 6 protoplasts per gram of fresh tissue was obtained from mesophyll cells of P. oceanica. The viability of isolated protoplasts was 82.5% (± 10.6) as confirmed by fluorescein diacetate staining. A high percentage of protoplasts of P. oceanica (61.5±14.8%) regenerated the cell wall within 7 days as confirmed by staining with calcofluor white, but only a few protoplasts were able to divide. Callus-like structures were noticed after 20–30 days in culture. A lower yield of protoplasts, 6.9 (± 3.8)×10 5 protoplasts per gram of fresh tissue, was obtained from mesophyll of C. nodosa. Viability of these protoplasts was 67.9% (±12.6) after isolation. Some possible applications of the method are discussed.

Production and regeneration of protoplasts from gremmeniella abietina and ascocalyx abietis

This work was undertaken to develop a system of protoplast isolation and regeneration for G. abietina and A. abietis that could be of use for the genetic manipulation of both species. Nuclear staining was performed to assess the nuclear conditions of the protoplasts. Of the 19 enzyme complexes studied, only 10 were found to have some lytic effect on either Gremmeniella, A. abietis, or both. Only snail enzyme (from Szeged University), Novozym 234, lytic enzyme L1, or mixtures of snail enzyme and Novozym 234 produced satisfactory yields of protoplasts. Regeneration of protoplasts was observed on complete and on minimal medium, and occurred from protoplasts plated out directly onto the surface and from those embedded in the agarose. In most cases, embedding increased the frequency of regeneration. Protoplasts formed after incubation at 20 degrees C regenerated at a frequency of approximately 5%, as opposed to 2% for those produced at 30 degrees C. As roughly 40% of the protoplasts were anucleate, the percentage of regeneration can be estimated as about 12.5% at 20 degrees C and 5% at 30 degrees C. Protoplasting appears to be a satisfactory method of obtaining material for genetic experiments with G. abietina and A. abietis when other methods are not directly applicable.

Optimum conditions for yeast protoplast release and regeneration in Saccharomyces cerevisiae and Candida tropicalis using gut enzymes of the giant African snail Achatina achatina.

Release of viable protoplasts of Saccharomyces cerevisiae and Candida tropicalis was achieved using fresh crude enzyme extracts of the giant African snail Achatina achatina. Optimum results of 2.8 x 10(6) protoplast ml(-1) were obtained when 1 g (wet wt) of cell slurry from the yeast strains was first treated with 1% beta-mercaptoethanol for 10 min and incubated with the undiluted crude enzyme for 120 min using 1.0 mol l(-1) sorbitol as osmotic stabilizer. Protoplast yield was enhanced with higher enzyme concentrations, longer digestion times and treatment of cells with beta-mercaptoethanol. Percentage regeneration of protoplast to viable cells in isotonic medium containing 0.01 mol l(-1) CaCl2 was in the range of 52-77%. These findings could be useful in the genetic manipulation of yeast of industrial importance.

Can protoplast production from in vitro cultured shoots of Tanacetum vary during the season?

Two different experiments were carried out to study the production of protoplasts and the variation of protoplast yield from in vitro cultured shoot tips of tansy (Tanacetum vulgare L.) and pyrethrum (Tanacetum cinerariifolium (Trevir.) Schiltz-Bip). In the first experiment, light had more pronouced effect for tansy than for pyrethrum. When the donor tissues of tansy were cultured under high light intensity the leaves contained anthocyanin and became brown during enzyme maceration. In contrast, donor tissues cultured under low light intensity produced leaves without anthocyanin. Depending on the light intensity of donor tissues, on average 5.8 - 6.8 x 106 and 3.4 - 4.3 x 106 protoplasts were isolated from one gram of mesophyll leaves of tansy and pyrethrum, respectively. In the second experiment, the production of protoplasts from tansy and pyrethrum varied seasonally. The most successful season for the production of protoplasts from in vitro cultured shoot tips was between December and April, when also the highest number of protoplasts could be isolated. It was not possible to state whether Tanacetum species have rhythms, which could cause physiological or chemical changes for the in vitro grown shoot tips. However, some external or internal, possible seasonal-dependent stimuli may have caused variation in the number of protoplasts isolated from tansy and pyrethrum and favoured protoplast production during winter and spring.

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 106protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.

Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. Lepideus, the mycelia were grown in ten different media at 28 °C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and β-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO 4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28 %.

Effect of Osmotic Stabilizers on Protoplast Generation of Chlorella ellipsoidea Yellow/White Color Mutants.

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 μE/m2/s and no reversion to normal cells at 0–26.8 μE/m2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

Effect of osmotic stabilizers on protoplast generation of Chlorella ellipsoidea yellow/white color mutants

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 μE/m 2/s and no reversion to normal cells at 0–26.8 μE/m 2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

Cryopreserved callus, a source of protoplasts for citrus improvement

SummaryThe availability and maintenance of embryogenic callus is a major limitation for large-scale application of somatic hybridization for citrus breeding. The suitability of cryopreserved callus as source of protoplasts was evaluated. Sweet orange callus were frozen by slow cooling and stored for two years in liquid nitrogen. Cryopreserved callus were fast thawed and used as source of material for protoplast isolation, protoplast fusion and plant regeneration, in comparison with control non-cryopreserved callus. No differences were found in protoplast yield, quality, growth and regeneration capacity between both callus types. Protoplasts isolated from cryopreserved callus were also successfully used in somatic fusion assays. Plants regenerated from protoplasts of the two sources had the same phenotypic characters and no differences were detected by microsatellite analysis. Availability of cryopreserved callus facilitates the development of breeding programmes based on somatic hybridization, avoiding the risks and high labour needs associated with standard maintenance by periodical subcultures.

Effect of sorbitol pretreatment on yield and viability of protoplasts isolated from etiolated shoots of three cultivars of<i> Sorghum bicolor</i>.

Effect of sorbitol pretreatment on yield and viability of protoplasts isolated from etiolated shoots of three cultivars of<i> Sorghum bicolor</i>.

Somatic embryogenesis and plant regeneration from litchi protoplasts isolated from embryogenic suspensions

High yields of protoplasts were isolated from litchi embryogenic suspensions, which were maintained by alternative culture in liquid and on solid media containing silver thiosulfate. Protoplasts in liquid culture and agarose beads were unable to divide sustainedly, whereas embedding of protoplasts in Ca-alginate supported cell division to microcalli and the direct formation of somatic embryos from protoplasts. Nurse cells of litchi further enhanced the culture efficiency when protoplasts were cultured in Ca-alginate beads. White non-hyperhydric somatic embryos were developed from protoplast-derived microcalli or proembryos, and 33.1% of white somatic embryos regenerated into plantlets.

Regeneration of the thallus of Monostroma oxyspermum (Chlorophyta) from protoplasts in axenic culture

High yields of protoplasts were isolated from Monostroma oxyspermum (Kützing) Doty by enzymatic degradation of the cell wall. Optimal conditions for the isolation of the maximum numbers of viable protoplasts were found to be treatment with 3% cellulase Onozuka with 0.6 M sorbitol or mannitol for an incubation period of 2 h. The regeneration frequency of isolated protoplasts was more than 90%. Even when regenerating under uniform conditions, protoplasts followed two different developmental patterns. Usually, protoplasts formed a cell wall and then developed into sporangia. These liberated biflagellated motile swarmers that settled and grew into thalli similar to the parent thallus in successive cultures. In the other type of development, some protoplasts underwent repeated cell divisions and developed a monostromatic thallus directly. Supplementation of the culture medium with sorbitol as an osmoticum inhibited cell divisions and further development of protoplasts of Monostroma.

Culture and regeneration of mesophyll-derived protoplasts of sorghum [ Sorghum bicolor (L.) Moench

A protocol for plant regeneration from mesophyll/protoplasts of sorghum [Sorghum bicolor (L.) Moench] was developed. The yield of intact protoplasts, their subsequent divisions and regeneration were genotype-dependent. The genotype 296B was always more responsive than IS 32266. For 296B, the sixth leaf from 18-day-old plants kept in dark for 2 days before harvesting was found to be the most suitable source of viable protoplasts. The first division was observed 10–12 days after plating, and the second division after 12–14 days. The maximum plating efficiency was 4.8% in 296 B, followed by 2.48% in IS 32266. Microcolonies were visible after 25–30 days, and microcalli after 60–75 days. Whole plants were obtained after 6–8 weeks of culture of microcalli on MS medium containing 0.2 mg l–1 kinetin and 2 mg l–1 BAP. The frequency of regeneration in 296B and IS 32266 was 12.80% and 10.58%, respectively. Ten plants transferred to pots in the glasshouse established well. The seeds collected from glasshouse-grown plants were sown in the field where plants were grown to maturity.

Isolation of protoplasts from in vitro-grown quince BA29 leaves

The aim of this research was to isolate protoplasts from in vitro-grown quince BA 29 leaves. Effects of various concentrations of cellulase and pectinase and different incubation periods were evaluated. The most effective treatment included (per ml) 50 units cellulase and 10 units pectinase, in the incubation medium and incubation for 21 h at 50 rpm in the dark at 27°C. This treatment produced a protoplast yield of 8.2×107 gfw−1 of which 90% were viable. Protoplast viability was influenced by length of the incubation period and ranged from about 40 to 100% among the various experiments.

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase.

; Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 µg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 µl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10(7) cells per gram of fresh weight alga.

An Optimized Method for the Isolation of Protoplasts from Penicillium simplicissimum to Produce Sealed Plasma Membrane Vesicles

As a first step to the production of plasma membrane vesicles, protoplast production from hyphae of Penicillium simplicissimum was optimized with reference to the following criteria: (i) protoplast yield; (ii) percentage of viable protoplasts judged via fluorescent dyes; (iii) percentage of protoplasts which were able to regenerate a cell wall, and (iv) the degree of damage to the plasma membrane estimated from the rate of glucose-stimulated proton excretion. Four mixtures of cell wall lytic enzymes were tested. The effect of different pretreatments (homogenization of mycelial pellets, addition of proteases, and addition of dithiothreitol) was also examined. Protoplast production was further optimized with respect to the choice of osmotic stabilizer, the pH during cell wall digestion, the age of the mycelium and the duration of cell wall digestion. The optimal conditions for the production of protoplasts from hyphae of P. simplicissimum were determined. In general, conditions which increased the protoplast yield, also damaged the plasma membrane (manifested as a decrease in the rate of glucose-induced proton excretion). The highest yield was 2 × 109 protoplasts per gram of mycelium (dry weight). A membrane fraction was obtained by mechanical homogenization of the protoplasts. Orientation and membrane integrity of the vesicles in the membrane fraction were characterized by the activity of the plasma membrane H+-ATPase (vanadate-sensitive ATP-hydrolyzing activity and proton pumping activity). The main part of vesicles (80%) were right-side-out.

An optimized method for the isolation of protoplasts from Penicillium simplicissimum to produce sealed plasma membrane vesicles

As a first step to the production of plasma membrane vesicles, protoplast production from hyphae of Penicillium simplicissimum was optimized with reference to the following criteria: (i) protoplast yield; (ii) percentage of viable protoplasts judged via fluorescent dyes; (iii) percentage of protoplasts which were able to regenerate a cell wall, and (iv) the degree of damage to the plasma membrane estimated from the rate of glucose-stimulated proton excretion. Four mixtures of cell wall lytic enzymes were tested. The effect of different pretreatments (homogenization of mycelial pellets, addition of proteases, and addition of dithiothreitol) was also examined. Protoplast production was further optimized with respect to the choice of osmotic stabilizer, the pH during cell wall digestion, the age of the mycelium and the duration of cell wall digestion. The optimal conditions for the production of protoplasts from hyphae of P. simplicissimum were determined. In general, conditions which increased the protoplast yield, also damaged the plasma membrane (manifested as a decrease in the rate of glucose-induced proton excretion). The highest yield was 2 × 109 protoplasts per gram of mycelium (dry weight). A membrane fraction was obtained by mechanical homogenization of the protoplasts. Orientation and membrane integrity of the vesicles in the membrane fraction were characterized by the activity of the plasma membrane H+-ATPase (vanadate-sensitive ATP-hydrolyzing activity and proton pumping activity). The main part of vesicles (80%) were right-side-out.

Protoplast culture and paclitaxel production by Taxus yunnanensis

Viable protoplasts of Taxus yunnanensis were isolated from friable, light yellow callus. Protoplast yield was dependent on callus age, with a maximum from 20-day-old callus. Protoplasts were induced to undergo sustained divisions and to form cell colonies when cultured in medium consisting of B5 salts, KM vitamin and organic components, 0.45 M fructose, 3.0 mg l-1 2,4-dichlorophenoxyacetic acid and 0.1 mg l-1 kinetin. The planting density was 2.5–3.0×105 protoplasts per ml of culture medium. Cell-free extract from callus enhanced protoplast division and the highest plating efficiency was about 7%. Protoplast-derived colonies showed significant variations in both growth and paclitaxel content. A negative correlation existed between paclitaxel accumulation in colonies and their growth to some extent (r = −0.4485). Among 70 colonies isolated from the heterogeneous protoplast cultures, colony TY-7 accumulated the highest paclitaxel content. Paclitaxel accumulation in colony TY-7 was not great enough to produce paclitaxel for commercial purposes, however, success in inducing colony formation from T. yunnanensis protoplasts provides an opportunity to obtain cell lines with high paclitaxel productivity from mutagenized protoplast cultures.

Plant regeneration from protoplasts of Triticum aestivum L. cv. Nakasoushu

A fast-growing, small, granular, embryogenic callus was selected from primary calli induced from the Japanese wheat cultivar Nakasoushu and the Australian wheat cultivar Bodallin. Regenerable and fine suspension cultures were induced three to six months after liquid culture was initiated and were characterized by dense cytoplasm and active division. These suspension cultures routinely provided high yields of protoplasts with about 90% viability when incubated in a modified KMP (Kao and Michayluk, 1975) medium containing 1 mg l-1 2,4-D (2,4-dichlorophenoxyacetic acid), and 1 mg l-1 zeatin. Nakasoushu and Bodallin protoplasts divided at frequencies of 8.6% and 11.1%, respectively, in agarose-solidified media. When Nakasoushu protoplasts were cultured with effective nurse cells of sorghum and wheat, protoplast division increased to 16.9% and 12.6%, respectively. Plating efficiencies varied from 0.03% to 2.5%. After subculture, protocolonies yielded embryogenic calli and somatic embryos, from which green plants were eventually regenerated. Whole plants obtained from Nakasoushu protoplasts were fertile, demonstrating the first report of Japanese cultivars in wheat protoplast cultures.

ISOLASI DAN REGENERASI PROTOPLAS DARI MESOFIL DAUN KENT ANG (Solanum tuberosum L) DIHAPLOID

The isolation and regeneration of potato (Solanum tuberosum L) protoplasts have been carried out. Mesophyl cell protoplast were isolated from two dihaploid cultivars of potato BF 15 and SVP 10 leaves used four different enzymes solution. Protoplast were cultured onto four different cultures media to increase plating efficiency. Calli were then transferred to ten different regeneration media. Using cellulose RS 0.5 % and pectolyase Y-23 0.05 % protoplast yield of both cultivars were improved. Medium VKM supplemented with 0.2 mg/l 2, 4-D, 1.0 mg/l NAA and 0.5 mg/l zeatin or 2iP were increase recovery of colonies from protoplast up to 5.9%. Regeneration medium containing zeatin did always produce more shoots than those of 2iP. Genotypes dependant regeneration frequencies have also been showed in this experiments