Protoplast Yield Research Articles

Plant regeneration from protoplasts isolated from embryogenic suspension cultured cells of Cinnamomum camphora L.

An efficient and reproducible protocol is described for the regeneration of Cinnamomum camphora protoplasts isolated from cultured embryogenic suspension cells. Maximum protoplast yield (13.1+/-2.1x10(6)/g FW) and viability (91.8+/-3.8%) were achieved using a mixture of 3% (w/v) cellulase Onozuka R10 and 3% (w/v) macerozyme Onozuka R10 in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl(2).2H(2)O, and 0.011% (w/v) NaH(2)PO(4).2H(2)O. First divisions occurred 7-10 days following culture initiation. The highest division frequency (24.6+/-2.9%) and plating efficiency (6.88+/-0.8%) were obtained in liquid medium (MS) supplemented with 30 g l(-1) sucrose, 0.7M glucose, 0.1 mg l(-1) NAA, 1.0 mg l(-1) BA, and 1.0 mg l(-1) GA(3). After somatic embryo induction and then shoot induction, the protoplast-derived embryos produced plantlets at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg l(-1) 3-indole butyric acid (IBA). Regenerated plants that transferred to soil have normal morphology.

Production and seeding of protoplasts of Porphyra okhaensis (Bangiales, Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 °C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 °C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 °C, 23.2± 0.24× 106 protoplasts g−1 fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.

Isolation of protoplasts from tissue fragments of Philippine cultivars of Kappaphycus alvarezii (Solieriaceae, Rhodophyta)

Protoplasts were isolated from tissue fragments (<1 mm2) of three Philippine cultivars of Kappaphycus alvarezii: the giant cultivar, cultivar L and Bohol wild type, by enzymatic dissolution of cell walls. Yields of viable protoplasts from young and old thalli (apical, middle, basal segments) were compared at various temperatures, duration of treatment and pH using eight combinations of commercial enzymes (abalone acetone powder and cellulase), and prepared extracts from fresh viscera of abalone (Haliotis asinina) and a terrestrial garden snail. Isolated protoplasts were grown in various culture media, temperatures, photoperiods and irradiance values to determine the conditions that favor germination and growth. Protoplast yields in tissues treated with commercial enzymes and the garden snail extract were lower than those obtained in tissues treated with fresh abalone extracts. Generally, the number of viable protoplasts increased with duration of enzyme treatment at 25 °C with a maximum yield of 8.2 × 103 g−1 tissue at 48 h. Yields were consistently higher in all cultivars at pH 6.1. The yields were also high from the middle segments of the giant cultivar (3.7 × 103 g−1 tissue) and Bohol wild type (4.5 × 103 g−1 tissue) treated with fresh abalone extract, and from basal segments of cultivar L and tissues treated with garden snail extract. The germination rate of protoplasts was highest (39.8%) at 25 °C and 20 μmol photon m−2 s−1, using a 12:12 light dark photoperiod. The filament was 3.7 mm long by Day 5. These findings are relevant to developing cultures from protoplasts for genetic or strain improvement of K. alvarezii cultivars.

Isolation and transformation ofTrichoderma virideprotoplasts

AbstractThe conditions for protoplast isolation and regeneration fromTrichoderma viridewere studied. Protoplasts were optimally isolated when mycelia ofT. viridethat had been cultured for 24 h were digested with 4 mg/ml Glucanex in phosphate buffer (pH 6.98) for 4 h at 30°C, resulting in a protoplast yield of 4.7×107cfu/mg. The maximum regeneration ratio (14.5%) was obtained in mycelia culture medium containing 0.3 mol/l KCl and 0.3 mol/l inositol. In addition, a plasmid pCSSNCC1 carrying a hygromycin resistance gene and an elicitor-producing gene was transformed intoT. virideprotoplasts, with an efficiency of 1–2 transformants/μg DNA. The hygromycin-resistant transformants were determined by PCR and the elicitor protein was detected by ELISA. The results indicate that the elicitor protein was expressed stably inT. viride.

Development and Characterization of a Nonmorphogenetic Cell Line of Wheat (Triticum aestivum)

This study was conducted to compare characteristics of a wheat (Triticum aestivum L.) cell line to those of the maize (Zea mays L.) black Mexican sweet (BMS) cell line and to compare protoplasts isolated from suspension cells of these cell lines. The wheat cell line was established from immature-embryo derived callus of the experimental line ‘ND7532’ and was conditioned for growth in suspension culture. For both cell lines, measurements of packed cell volume (PCV), fresh weight (FW), and dry weight (DW) were taken at 3 day intervals from suspension cultures. Measurements of FW of calluses cultured from suspension cells of both cell lines were taken at 6 day intervals. The morphogenetic potential of the wheat ND7532 cell line was tested in both callus and suspension cultures using media promoting regeneration and/or organogenesis. Growth rates of ND7532 cells in suspension culture were comparable to those of BMS cells. However, relative growth rates of calluses recovered from ND7532 suspension cells were slower than those of calluses recovered from BMS suspension cells. The ND7532 cell line has very limited morphogenetic potential and has been maintained as rapidly growing callus tissue for 11 years. Yields of protoplasts from suspension cells of the two cell lines were comparable, though ND7532 protoplasts were typically smaller. The wheat cell line has is now designated ND7532-NM (nonmorphogenetic) and is available for cellular and molecular biology research.

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume Astragalus melilotoides Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74 +/- 0.6x10(5)/g FW) and viability (87.07 +/- 2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3-7 days following culture initiation. The highest division frequency (9.86 +/- 0.68%) and plating efficiency (1.68 +/- 0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l alpha-naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3 +/- 4.1%) and organogenesis (21.6 +/- 0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.

Tissue degradation and enzymatic activity observed during protoplast isolation in two ornamental Grevillea species

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.

Aspects of isolation underpinning mitotic behaviour in lupin protoplasts

This study reports on the influence of critical isolation factors on the subsequent culture of protoplasts of Lupinus albus L. Protoplasts were isolated from in vitro seedling cotyledons of five early maturing accessions in which protoplast yields and division frequencies appeared to be correlated as a high protoplast yield corresponded with a high division frequency. The overall difference among the accessions for mitosis was non-significant, although the highest yield and division frequency were observed in accession LA132, with Alban giving a significantly lower level. Accession Lucrop produced the lowest number of protoplasts, all of which collapsed during culture. Of the enzyme types used for tissue maceration, Pectolyase Y23, was significantly inferior to Macerase in terms of giving way to mitosis. The extent of division in Macerase-isolated protoplast population was 266% higher than that in the Pectolyase Y23-isolated one. The physiological maturity level of the explant, expressed in terms of developmental age, was optimal when 14–18-day-old seedling cotyledons were used for protoplast production and culture, rather than more mature ones, despite higher protoplast yields in the latter. On K8p medium, the protoplast division frequency was 129% greater when 18-day-old seedling cotyledons were used, than that with any other treatment. This work on protoplast culture of the potentially important lupin species, which is a pulse rich in dietary protein, oil and fibre, allows a further understanding of the biology, with an aim to advance lupin biotechnology.

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126.

Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye ( Secale cereale L.).

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3-5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.

Optimisation of Protoplast Production in White Lupin

The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, -2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.

Plant regeneration from mesophyll protoplasts of the Egyptian medicinal plants Artemisia judaica L. and Echinops spinosissimus Turra

Protoplast-to-plant systems for the Egyptian medicinal plants Artemisia judaica L. and Echinops spinosissimus Turra have been developed. Micropropagated shoots of A. judaica and E. spinosissimus were selected as plant materials suitable for protoplast isolation. The yield and viability of protoplasts in both species were greatly influenced by osmotic potential and enzyme combinations. Sustained cell division and colony formation from the protoplasts of A. judaica were best supported by a medium containing 0.6 mol l −1 glucose, 2.5 μmol l −1 6-benzylaminopurine (BAP) and 5.0 μmol l −1 1-naphthaleneacetic acid (NAA) in 0.6% Agarose (SeaPrep®) block/liquid culture. The highest frequency of colony formation in E. spinosissimus was observed when the protoplasts were embedded in 0.6% Na-alginate block and cultured in a medium containing 1/2 strength Murashige and Skoog, 1962 (MS salts), Gamborg et al., 1968 (B5 vitamin), 0.5 mol l −1 glucose, 2.5 μmol l −1 BAP and 5.0 μmol l −1 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calli when transferred onto 0.25% gellan gum-solidified MSO medium supplemented with 2.5 μmol l −1 BAP and 2.5 μmol l −1 NAA for A. judaica, and 2.5 μmol l −1 BAP and 2.5 μmol l −1 2,4-D for E. spinosissimus. Shoot organogenesis from the protoplast-derived callus was induced on MS medium supplemented with 5.0 μmol l −1 BAP for A. judaica and 2.5 μmol l −1 BAP for E. spinosissimus. The protoplast to plant regeneration protocols developed in this study may provide the basis to investigate cell physiology and biochemistry of desert medicinal plants.

Isolation of protoplasts from undaria pinnatifida by alginate lyase digestion

The aim of this study is to isolate protoplasts from Undaria pinnatifida. Protoplasts of the alga were isolated enzymatically by using alginate lyase, which was prepared by fermenting culture of a strain Vibrio sp. 510. Monofacterial method was applied for optimizing digestion condition. The optimum condition for protoplast preparation is enzymatic digestion at 28°C for 2h using alginate lyase at the concentration of 213.36 U (8 mL) every 0.5g fresh thalline with NaCl 50 and at the shaking speed of 150 r min−1 during digestion. The protoplast yield can reach 2.62±0.09 million per 0.5 g fresh leave under the optimum condition. The enzyme activity is inhibited by Ca2+ and slightly enhanced by Fe2+ and Mn2+ at concentrations of 0.05, 0.08 and 0.10 mol L−1.

Protoplasts from Agrobacterium rhizogenes-transformed cell line of Medicago sativa L. regenerated to hairy roots

Protoplasts were isolated from Agrobacterium rhizogenes A4-transformed cell line of Medicago sativa L. The highest yield of protoplasts (4.2×106 per g fresh weight) was obtained from 12-d-old calluses after being subeultured on fresh medium. The viability of protoplasts reached over 80%. Protoplasts were induced to undergo sustained divisions when cultured in Durand et al. (DPD) medium supplemented with 2 mgl−1 (9.05 μM) 2,4-dichlorophenoxyacetic acid, 0,2mgl−1 (0.93 μM) kinetin, 0.3 M mannitol, 2% (w/v) sucrose, and 500 mgl−1 casein hydrolyzate at a plating density of 1.0×105 per ml. An agarose-beads culture method was appropriate for protoplast division of transformed alfalfa. The division frequency was about 30%. Numerous hairy roots were induced from protocalluses on Murashige and Skoog medium without growth regulators. Paper electrophoresis revealed that all of the regenerated hairy roots tested synthesized the corresponding opines. This protoplast culture system would be valuable for further somatic hybridization in forage legumes.

Effect of biotic factors on the isolation of Lupinus albus protoplasts

Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.

Efficient paclitaxel production using protoplasts isolated from cultured cells of Taxus cuspidata.

Efficient isolation of protoplasts from Taxus cuspidata cultured cells, localization of paclitaxel in the cultured cells, and efficient production of paclitaxel by protoplasts were studied. Hemicellulase, potassium citric acid solution, and degassing treatments were effective in increasing the yield of protoplasts isolated from T. cuspidata cultured cells. Protoplasts yields (3.2 - 6.4 x 10(6) number/g-fresh weight cells) were achieved by combining the various treatments with specific culture and cell phases. It was found that about 30% and 35% of paclitaxel in the cells was located in cell walls and/or between the cell wall and cell membrane (CW) of suspension cells in the growth phase and in the stationary phase, respectively. About 30% and 43% of paclitaxel in the cells was located in CW of the cells grown in solid culture in growth phase and in the stationary phase, respectively. In comparison with cell suspension culture, protoplasts in a static culture and the protoplasts immobilized in agarose gel in shaking culture resulted to about 6 times increase in the extracellular paclitaxel accumulation.

Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts

One prerequisite for plant functional genomic projects is the development of a high-throughput transformation platform. As the moss Physcomitrella patens can be transformed via PEG-mediated DNA-uptake into protoplasts, a semi-continuous bioreactor culture of this plant was optimised regarding protoplast isolation efficiencies. Under standard conditions protoplast yields were 2.8×10 4/mg dry weight. This yield was increased sixfold by supplementation of the medium with 460 mg/l ammonium tartrate. The same effect was achieved by controlling the pH-value in the bioreactor culture with a setpoint of 4.5. In contrast, pH control with a setpoint of 7.5 reduced the protoplast yield compared to a culture without pH control to 11%. A semi-continuous culture of Physcomitrella in a 5-l bioreactor grown at pH 4.5 yielded sufficient cell material for more than 100 transformations per day.

Efficient plant regeneration from protoplasts isolated from long-term, shoot primordia-derived calluses of garlic ( Allium sativum)

We developed an efficient method for the regeneration of plants from garlic (Allium sativum) protoplasts. Protoplasts were isolated from six-year-old cultures of shoot primordia-derived calluses. At the isolation condition applied, high yield of protoplasts was obtained from the calluses compared with that from the initial shoot primordia. Throughout long-term culturing, highly regenerative calluses that propagated rapidly and appeared genetically stable were selected. The frequency of colony formation from protoplasts was clearly enhanced by increasing cell density. Adventitious buds were induced from protoplast-derived calluses at ca. 90 % efficiency, and the regeneration efficiency from adventitious buds averaged nearly 70 %. Plants that regenerated from adventitious shoots showed normal phenotypes, suggesting that long-term culturing of these garlic calluses does not induce dramatic genetic alterations.

Influence of donor material and genotype on protoplast regeneration in banana and plantain cultivars ( Musa spp.)

Among the various strategies for genetic improvement in banana, somatic hybridisation appears to be a promising complement to classical breeding since protoplasts are amenable to plant regeneration. Therefore, the present investigation was undertaken to improve protoplast regeneration in banana. Protoplasts were isolated from young leaves, calli and embryogenic cell suspensions. The highest protoplast yield was obtained with cell suspensions. Mesophyll protoplasts and callus-derived protoplasts did not divide. However, protoplasts isolated from cell suspension developed into plants. Feeder cells and protoplast culture at high density were required for plant regeneration from protoplasts. Plant regeneration through somatic embryogenesis was achieved in Grande Naine and Gros Michel ( Musa spp. Sub-Group cavendish AAA), Currare Enano and Dominico (Sub-Group plantain AAB), SF265, IRFA903 and Col49 (Sub-Group AA). The frequency of embryo formation and plant regeneration was genotype-dependent. Large number of somatic embryos and regenerated plants were produced in SF265 (AA) followed by Dominico (AAB) and Grande Naine (AAA).

Protoplast preparation and transient transformation of Rhizoctonia solani

Optimization of protoplast release (202 × 10−4 ml−1) from a strain of Rhizoctonia solani anastomosis group three was achieved by treating 3 d old mycelia from broth cultures with Novozyme 234 for 3 h at 30°g; C. The only other treatment, of many tested, that yielded any protoplasts (15.6 × 10−4 ml−1) was commercial lysing enzymes from Trichoderma harzianum. Various combinations of commercial enzyme preparations, variations of osmotic stabilisers, and mycelial age, were ineffective in raising the protoplast yield. Regeneration of colonies from protoplasts was maximal (83-87%) when protoplasts were incorporated in cooled agar containing 5% glucose, malt extract and yeast extract, supplemented with 0.8 or 1.0 M mannitol as osmotic stabiliser. Liquid culture broth and other potential osmotic protectants at various concentrations yielded fewer regenerating colonies (5% in broth and 13-75% in agar). The Rhizoctonia strain was highly sensitive to hygromycin B, so transformation of protoplasts was achieved with a polyethylene glycol-based procedure, using plasmid pES200 containing the bacterial hygromycin B phosphotransferase gene driven by a trpC promoter from Aspergillus nidulans. Transformation was achieved using the DNase inhibitor aurintricarboxylic acid and incubation temperatures below 10-12°. It was confirmed by high stringency hybridization of DNA extracted from the transformants, using pES200 as a radiolabelled probe. The transformants grew slowly (25-35 mm diam after 14 d) then ceased growth, and never produced fast-growing sectors indicative of integrated plasmid DNA. Transformation was also achieved with pAXHY2 from Cryphonectria parasitica, and colonies reached 55-65 mm diam before they stopped growing. Southern blots confirmed the presence of non-integrated pAXHY2, but no fast-growing sectors were produced.