Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (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.     

Optimization of Parameters for Isolation of Protoplasts from the Antarctic Sea Ice Alga Chlamydomonas Sp. ICE-L

The present study was initiated to provide a systematic protocol for producing protoplasts from the Antarctic sea ice alga Chlamydomonas sp. ICE-L suitable for physiological studies. The results showed that the mixtures of 3.0% Cellulase R-10 and 2.0% Macerozyme R-10 were most effective for isolating protoplasts from this alga. Optimum pH and temperature for hydrolytic enzyme reaction were pH 6.0 and 15^∘C, respectively. Mannitol and sorbitol were found to be the excellent osmotic stabilizers. Growth conditions of the algae prior to enzyme treatment also influenced the yield of protoplasts greatly. At the optimized condition, protoplast production was 47.8%, and the viability of isolated protoplasts was more than 97.6% as confirmed by Evan's blue staining.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

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.     

Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml^-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×10⁶ viable protoplasts per g fresh weight and their size ranged from 12 to 44 m. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×10⁵ protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [¹⁴C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN₃, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.     

玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

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.     

Isolation of Allium pollen protoplasts

Studies on protoplasts isolation were carried out with mature pollen grains of 29 samples of species of Allium aflatunense, A. cepa, A. fistulosum, A. karataviense, A. longicuspis, A. nutans, A. odorum, A. sativum and A. schoenoprasum. Surface sterilized pollen grains drifted from crushed anthers were incubated in an enzyme solution containing 1% (w/v) cellulase Onozuka R-10, 1% (w/v) Macerozyme R-10, 0,5 mol l^-1 sucrose and the basal salts of Nitsch medium. Protoplasts were released within 3 to 120 min, either from the pollen grain, through a slightly disturbed germination pore (narrow aperture), or through a wider aperture, when the exine surrounding the germination pore was disturbed. For the first time, protoplasts were obtained from 13 genotypes of 6 Allium species, at a rate of 1 to 30% of the digested intact pollen grains, depending on the genotype.     

Influence of pH and sucrose concentration on nonenzymatic and enzymatic isolation of protoplasts from mature pollen of Tulbaghia violacea

Studies on protoplast isolation were carried out with mature pollen grains of Tulbaghia violacea Harv. (Liliaceae). Pollen grains drifted from surface sterilized crushed anthers were incubated either in a nonenzymatic solution composed of Nitsch medium and sucrose, or in the same solution supplemented with 1% cellulase Onozuka R-10 and 1% Macerozyme R-10. The process of protoplast release was studied as a function of pH and sucrose concentration of nonenzymatic and enzymatic solutions. For nonenzymatic isolation, the tested range of pH and sucrose concentration was from 3.3 to 13.1 and from 0.015 to 1.12 M (final solution osmolality from 200 to 1,300 mOs kg^-1 H₂O), respectively. In the former case, the release of protoplasts occurred only at nonphysiological pH (12.2 to 13.1) and could be observed after several seconds to 120 min, depending on pH and sucrose concentration of medium. Under enzymatic incubation, viable protoplasts were released more rapidly (3 to 35 min) and in more physiological conditions, the optimum being pH 5.8 and final medium osmolality 652 mOs kg^-1 H₂O. Speed, manner of protoplast release, number and quality of protoplasts were dependent on interactions of pH and sucrose concentration.     

An Efficient Protocol for Ulmus minor Mill. Protoplast Isolation and Culture in Agarose Droplets

We describe here an efficient and reproducible protocol for isolation and culture of protoplasts from Ulmus minor. Different sources of donor tissues were tested for protoplast isolation: callus and juvenile leaves from in vitro and greenhouse plants. Several combinations and concentrations of hydrolytic enzymes were used. Comparative tests between Cellulase Onozuka R10 and Cellulase Onozuka RS were made and the last one proved to be more efficient. Both the pectinases used, Macerozyme Onozuka R10 and Pectinase (Sigma^®), were efficient in protoplast isolation and there was no need for a more active pectinase. In vitro leaves proved to be the best source for protoplast isolation and produced an average of 3.96 × 10⁷ protoplasts per gram of fresh weigh. Elm mesophyll protoplasts were cultured using the advantageous method of agarose droplets and a modification of the Kao and Michayluk culture medium, using two plating densities (1 × 10⁵ and 2 × 10⁵ protoplasts ml^?1). Protoplast division and evolution into colonies and microcalli was promoted in the agarose droplets plated at 2 × 10⁵ protoplasts ml^?1. Ten weeks after protoplast culture initiation a plating efficiency of 2.7% was attained and the bigger microcalli, with at least 0.5 mm diameter, were transferred to a solid medium previously used for the production of embryogenic callus.     

A simple method for mass isolation of protoplasts from species of Monostroma, Enteromorpha and Ulva (Chlorophyta, Ulvales)

A simple enzyme mixture containing 2% Cellulase Onozuka R–10 and1% Macerozyme R–10 prepared in deionised water supplemented with 3% NaCland 1 mM CaCl₂ was developed for isolating rapidlyprotoplasts from different species of Monostroma,Enteromorpha and Ulva. The yield fordifferent species of Monostroma ranged from 9.6 ×10⁶ to 10.2 × 10⁶ cells g^–1f. wt thallus, and forEnteromorpha from 3.48 × 10⁶ to 11.7× 10⁶ cells g^–1 f. wt and forUlva from 4.58 × 10⁶ to 26.8 ×10⁶ cells g^–1 f. wt. The overallregeneration rate of the protoplasts isolated was usually > 90% and showednormal morphogenesis. The method yields rapid mass production of viableprotoplasts with high regeneration rates.     

Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 M 6-benzyladenine, 4.5 M 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid monohydrate - PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate     

Isolation of megabase-size DNA from sorghum and applications for physical mapping and bacterial and yeast artificial chromosome library construction

A method was developed for the isolation of megabase-size DNA fromSorghum bicolor. Sorghum protoplasts were isolated from young leaf tissue, embedded in an agarose matrix as microbeads or plugs, followed by cell lysis and protein degradation. The DNA prepared by this method was larger than 1 Mb in size and readily digestible with restriction enzymes. The DNA was shown to be suitable for physical mapping, and was successfully used for the construction of BAC and YAC libraries.     

三倍体‘银中杨’叶肉原生质体制备的优化

以三倍体杨树品种‘银中杨’（Populus alba×P.berolinensis Yinzhong）无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明：酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BAS,酶解时间为8 h,原生质体产量和活力分别为2.13×10⁷个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高（1.06×10⁷个/g）,可满足进一步的原生质体培养等技术的要求。     

In vitro culture of Cucumis sativus L. XVIII. Plants from protoplasts through direct somatic embryogenesis

A procedure is described for the isolation and culture of protoplasts from embryogenic callus (gel-like callus — GLC) and embryogenic suspension cultures (ESC) of Cucumis sativus c.v. Borszczagowski. Maximal protoplast yields from GLC and ESC were 5×10⁶ and 1×10⁷ protoplasts/g tissue respectively. They were obtained following 14–16 h digestion with 1.2% Cellulase Onozuka R-10, 1.2% Macerozyme R-10 and 0.3% Driselase. At a plating density of 2×10⁵ / ml, first divisions occurred in 4–5 days and 7–8 days in ESC-and GLC-derived protoplasts respectively. The highest percentage of direct embryogenesis (over 80%) was observed with ESC. It was possible to obtain approximately 5000 embryo structures / g tissue. Some embryos converted into plants after 6 weeks, but most of them after 2 months of culture. ESC-derived plants, when transferred into the glasshouse, bloomed normally, and set seeds.Abbreviations CMS Murashige & Skoog (1962) medium for cucumber - GLC gel-like callus - ESC established embryogenic suspension culture - 2,4-d 2,4-dichlorophenoxyacetic acid     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

An evaluation of methods for quantifying the enzymatic degradation of red seaweed Grateloupia turuturu

For better exploitation of the red seaweed Grateloupia, enzymatic digestion of the thallus may be a way to increase access to metabolites of industrial interest. With this aim, we have tried to find a method to quantify the efficiency of enzymatic digestion. Vegetative algal material was treated with polysaccharidases (Onozuka R-10 cellulase, agarase, and Ultraflo L mixture). The proportion of degraded surface area was determined by microscopic measurement of the residue surface using imaging software and compared with the analysis of carbohydrates and R-phycoerythrin released in the incubation solution. Both the reducing carbohydrate concentration and percentage of degraded surface area appeared the most reliable methods to study enzymatic efficiency. The amount of solubilized total carbohydrates, and particularly that of R-phycoerythrin, showed non-specific variations, so no conclusions could be drawn. The application of this procedure to the screening of the efficient digestion of Grateloupia material demonstrated that cell walls were only partially digested by polysaccharidase enzymes alone. The Ultraflo L mixture and Onozuka R-10 cellulase produced a greater degradation of Grateloupia tissues and a higher release of reducing carbohydrates, whereas agarase did not display any specific action. Thus, the proposed procedure based on the quantification of residue surface area seems to be an accurate method to analyze enzymatic digestion. Other tests using different concentrations and combinations of enzymes are now required.     

Callus proliferation from leaf protoplasts using phenylurea type cytokinin, 4-PU,inBetula platyphylla VAR.Japonica

Summary Protoplasts were isolated from leaves ofBetula platyphylla var.japonica using a 0.6M mannitol solution containing 1% Cellulase Onozuka R-10 and 1% Driselase. The cell division and colony formation were largely enhanced using Murashige and Skoog (1962) liquid medium at half strength (1/2 MS), containing 0.6M mannitol, 0.09M sucrose, and factorial combinations of 0.1–30 μM N-(2-chloro-4-pyridyl)-N′-phenylurea (4-pu) and 0.1–10 μM 1-naphthaleneacetic acid (NAA) or 0.1–30 μM 2,4-dichlorophenoxyacetic acid (2,4-D). The optimal protoplast density was 5–7 × 10⁴/ml. Continuous callus proliferation from protoplasts was achieved by transferring colonies to fresh 1/2 MS agar medium containing 1 μM NAA and 1 μM 4-pu with no mannitol. It appeared that supplementation of the medium with phenylurea type cytokinin, 4-pu gave the successful callus proliferation from the protoplasts ofB. platyphylla.     

Studies on a drought resistant legume: The moth bean,Vigna aconitifolia (Jacq) marechal. I. Protoplast culture and organogenesis

High yields of viable protoplasts were obtained from callus cultures derived from shoot apices of Vigna aconitifolia (JACQ) Marechal. The protoplasts divided and formed cell clusters on modified MS medium. The protoplast-derived callus formed multiple shoot buds on MS and B5 basal media without supplements, on MS medium containing supplements and on B5 medium containing charcoal (0.25%). Shoot formation occurred.Abbreviations MS Murashige and Skoog (1962) - B5 B5 basal medium (Gamborg et al 1968) - CM coconut milk - NAA 1-napthaleneacetic acid - BA 6-benzyladenine - IAA indole-3-acetic acid - 2,4 D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - CB Cellulase from Penicillium funiculosum - C Cellulase from Trichoderma reesei - CRIO Cellulase R10 - MR10 Macerozyme R10 - PDS Potassium dextran sulphate NCL communication No. 3376