Protoplast Density Research Articles

Phytosulfokine alpha enhances regeneration of transformed and untransformed protoplasts of Brassica oleracea.

Phytosulfokine-α (PSK-α) is a disulfated pentapeptide (YIYTQ) acting as an intercellular signal peptide and growth factor. It was originally isolated from conditioned medium of asparagus mesophyll cell cultures in 1996 and later characterized as a hormone-like signal molecule with important roles in numerous processes of in vivo plant growth and development. It is currently becoming a valuable mitogenic factor in plant breeding and biotechnology due to its stimulatory effect on in vitro cell elongation, proliferation and differentiation. The focus of our work was to review current knowledge about the roles of PSK-α in plant biotechnology and to evaluate its influence on the regeneration of protoplasts of four Brassica oleracea cultivars (two cauliflower and two cabbage) cultured under two distinctive protocols and with different protoplast densities. Protoplast regeneration was studied due to its high value for plant genome editing, which is generally limited by the inefficient regeneration of treated protoplasts of numerous important plant genotypes. Our hypothesis was that the stress related to PEG-mediated protoplast transformation and the following decrease in viable protoplast density in culture could be alleviated by the addition of PSK-α to the culture medium. We therefore tested whether PSK-α could increase cell division at the early stages of culture (5 and 15 days after protoplast isolation) and stimulate the formation of microcallus colonies up to the 30st day of culture and to evaluate its influence on callus organogenesis leading to shoot regeneration. The PSK-α showed a strong stimulatory effect on untransformed protoplast regeneration already during the first days of culture, accelerating cell division up to 5.3-fold and the formation of multicellular microcallus colonies up to 37.0-fold. The beneficial influence was retained at later stages of regeneration, when PSK improved shoot organogenesis even if it was present only during the first 10 days of culture. The highest numbers of shoots, however, were regenerated when PSK was present during the first days of culture and later in solid shoot regeneration medium. Finally, the addition of PSK-α to PEG-transformed protoplasts significantly enhanced their division rate and the formation of microcallus colonies in selection media, up to 44.0-fold.

Cotyledon peeling method for passion fruit protoplasts: a versatile cell system for transient gene expression in passion fruit (Passiflora edulis).

Passion fruit (Passiflora edulis) is a perennial evergreen vine that grows mainly in tropical and subtropical regions due to its nutritional, medicinal and ornamental values. However, the molecular biology study of passion fruit is extremely hindered by the lack of an easy and efficient method for transformation. The protoplast transformation system plays a vital role in plant regeneration, gene function analysis and genome editing. Here, we present a new method ('Cotyledon Peeling Method') for simple and efficient passion fruit protoplast isolation using cotyledon as the source tissue. A high yield (2.3 × 107 protoplasts per gram of fresh tissues) and viability (76%) of protoplasts were obtained upon incubation in the enzyme solution [1% (w/v) cellulase R10, 0.25% (w/v) macerozyme R10, 0.4 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] for 2 hours. In addition, we achieved high transfection efficiency of 83% via the polyethylene glycol (PEG)-mediated transformation with a green fluorescent protein (GFP)-tagged plasmid upon optimization. The crucial factors affecting transformation efficiency were optimized as follows: 3 μg of plasmid DNA, 5 min transfection time, PEG concentration at 40% and protoplast density of 100 × 104 cells/ml. Furthermore, the established protoplast system was successfully applied for subcellular localization analysis of multiple fluorescent organelle markers and protein-protein interaction study. Taken together, we report a simple and efficient passion fruit protoplast isolation and transformation system, and demonstrate its usage in transient gene expression for the first time in passion fruit. The protoplast system would provide essential support for various passion fruit biology studies, including genome editing, gene function analysis and whole plant regeneration.

Protoplast isolation and transient gene expression in different petunia cultivars.

The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 105 protoplast density, 40% polyethylene glycol (PEG) concentration, 10µg plasmid DNA, and 15min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.

Optimization of protocol for efficient protoplast isolation and transient gene expression in carnation

The optimal protocols for protoplast isolation and transfection in carnation cv. Scarlet were developed by optimizing the factors involved in protoplast isolation and transfection processes. The best protoplast yield and viability were achieved using 0.5 M mannitol, enzyme concentrations of 1.0% cellulase and 0.1% macerozyme, and digestion time of 8 h. In contrast, the best transfection efficiency was obtained using protoplast density of 2.5 × 105, polyethylene glycol (PEG) concentration of 20%, and transfection time of 15 min. The protocols could produce reasonable protoplast yield and transfection efficiency in other carnations. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all carnations was confirmed using PCR analysis. To our knowledge, this is the first report on transient gene expression in carnation protoplasts. This transient gene expression system could be valuable for genome editing of unwanted genes in carnations using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 technology.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method.

We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

Isolation and culture of strawberry protoplasts and field evaluation of regenerated plants

Protoplasts are an useful biotechnological tool for plant improvement. In strawberry, very few studies on protoplast technology have been carried out. In this investigation, a procedure for the isolation and culture of strawberry protoplasts, cv. ‘Chandler’, has been developed. The effect of several factors affecting the successful isolation of protoplasts and formation of microcalli, e.g. explant source, washing procedure, hormonal composition of the culture medium and protoplast density, were evaluated. For shoot regeneration, microcalli derived from isolated protoplasts were transferred to MS medium supplemented with 0.2 mg l−1 NAA and either 5 mg l−1 BA or 3 mg l−1 TDZ, obtaining a similar regeneration rate, 17%, in both media. Twenty-one independent protoclones were transferred to field conditions for agronomic evaluation. Significant alterations in the growth habit, density of foliage, leaf color and leaf morphology were detected in some lines. Fruit yield was significantly reduced in 15 out of the 21 protoclones evaluated due to a reduction in fruit weight and/or the number of fruits. Ploidy level was unaffected in a sample of 6 lines selected at random; however, a study of genetic stability by using 10 EST-SSR markers showed genetic alterations in all the lines analyzed. Despite the high rate of somaclonal variation detected in the protoclones, some of the lines displayed an agronomical behavior similar to control plants, indicating that this protocol could be useful for genetic improvement in this species.

Pre-isolation, isolation and regeneration protoplasts from leaf mesophyll of in vivo Malus domestica ‘Anna’ cv.

Abstract To isolate protoplast, a pre-treatment was completed with the order to reduce and identify the phenolic contents round the year to encourage the isolation of protoplasts. Protoplasts from in vivo mesophyll leaves of apple cultivar “Anna” was isolated from 15 days old leaves by plasmolying in medium containing 90 g L-1 mannitol for half hour, then 130 g L-1 mannitol for half hour. Then using enzymatic mixture involving (1.5% cellulase + 0.5% pectianase + 1.5% Macrozyme) Prior to isolation. Anyhow, diverse factors, for example, Osmotic pressure, incubation period, sieve pore size, centrifugation period and hormonal balance was estimated using the techniques for isolation. The quantity of cells was computed as the quantity of cells per square on haemocytometer. A considerable higher yield of protoplast formation was noted in the CPW medium using a pore size of 25 µm with using incubation period for 20 hours. Moreover, the best protoplast regeneration with using of protoplast density of 2.0 x 105 in MS medium supplemented by 1.0 mg L-1NAA and 0.3 mg L-1BAP. We believed that our protocol might encourage the plant recovery using in apple somatic hybridization programs.

Protoplast isolation and regeneration from Hecatonema terminale (Ectocarpales, Phaeophyceae) using a simple mixture of commercial enzymes

Protoplast systems are essential for genome-editing and gene silencing technologies. In brown algae, protoplast isolation has been hampered by protocols that use non-commercial enzymes or crude extracts. This study is the first to report the production of protoplasts from cell-filament suspension cultures of the brown alga, Hecatonema terminale (Kutzing) Kylin, using different mixtures of commercial enzymes and chelation pre-treatment. In this study, mixture A (cellulase RS and alginate lyase) with chelation pre-treatment produced the highest number of protoplasts (3.52 ± 0.23 × 105 protoplasts g−1 FW). Chelation pre-treatment showed high effects on all kinds of enzyme mixtures. The effects of these different mixtures were examined by two-way ANOVA. We also investigate the optimal protoplast density and regeneration medium for protoplast regeneration. Of 16 combinations for regeneration media, RM1 with lowest initial protoplast density (2.4 × 103 protoplasts mL−1) showed the highest value (74%) of final plating efficiency (FPE) after 13 days of culture. The well-defined heterotrichous thalli with phaeophycean hairs were clearly distinguished after 22 days of culture.

Evaluation of an Anthocyanin, Cyanidin 3,5-di-O-glucoside, as an Allelochemical in Red Callus of a Mangrove Sonneratia ovata, Using Protoplast Co-Culture Bioassay Method with Digital Image Analysis

protoplasts to examine the allelopathic activities. Protoplasts were isolated with Cellulase R10 and Driselase 20 in 0.6 M mannitol solution and purified by density gradient centrifugation on 0.6 M sucrose. Protoplasts were co-cultured in 50 μL of liquid Murashige and Skoog’s (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid and 0.1 μM benzyladenine and 0.6 M mannitol solution in a 96-well culture plate. Protoplast density ranged from 5 × 103/mL to 105/mL. Cell division of lettuce protoplasts was strongly inhibited by addition of S. ovata protoplasts, and non-spherical cell enlargement was slightly inhibited. By contrast, digital image analysis of scanned 96-well culture platesrevealed no inhibition in accumulation of yellow color in lettuce protoplasts. An anthocyanin, cyanidin 3,5-di-O-glucoside (cyanin), was identified and its content in the red callus was ca. 1 mM of fresh weight. The effects of cyanin on the growth of lettuce protoplasts at three stages were similar to those of red S. ovata protoplasts. From these results, cyanin was most likely the allelochemical contained in red callus of S. ovata. The allelopathic activity of cyanin was compared with that of other putative allelochemicals in several plant materials, using the protoplast co-culture method with digital image analysis.

<i>In Vitro</i> Bioassay of Allelopathy in Four Bamboo Species; <i>Bambusa multiplex, Phyllostachys bambusoides, P. nigra, Sasa kurilensis</i>, Using Sandwich Method and Protoplast Co-Culture Method with Digital Image Analysis

Moderately strong allelopathic activities were found in four bamboo species, Bambusa multiplex cv. Houraichiku; Phyllostachys bambusoides cv. Madake; P. nigra cv. Hachiku; Sasa kurilensis cv. Chishimazasa, which are of different classification or of different ecological distributions, using the “Sandwich Method”, which assays the dried leaves on growth of lettuce seedlings. Only small difference of activity was found among the four bamboo species. In addition, “Protoplast Co-culture Method” for assay of allelopathy in a 50 μL liquid medium using a 96 well culture plate, was applied to the suspension cultures of the four bamboo species. Protoplasts were isolated from two-week cultured suspension cells of four bamboo species using Cellulase RS and Pectolyase Y-23 in 0.6 M mannitol. At low protoplast densities of bamboo, B. multiplex and P. bambusoides stimulated the recipient lettuce growth, i.e., non-spherically cell enlargement and cell divisions observed under an inverted microscope, while protoplasts of P. nigra and S. kurilensis were less stimulatory or inhibitory. Inhibitory effect of S. kurilensis was the strongest among four bamboo species. Furthermore, highly inhibitory effects of S. kurilensis protoplasts on yellow color accumulation of lettuce protoplasts were clearly observed by analysis of a scanned digital image of a 96-well culture plate. Differences and causes of the allelopathic activities were discussed comparing with other plant species studied using the same assay methods.

Dendrobium protoplast co-culture promotes phytochemical assemblage in vitro.

The present study is intended to analyze the occurrence of potent, low produce, naturally occurring stilbenes in protoplasts of wild species and hybrids of Dendrobium. The wild species selected for the study was Dendrobium ovatum, endemic to Western Ghats of India. Protoplasts were isolated from leaves and tepal tissues of all the species and were cultured purely to generate homofusants and cross-cultured to raise heterofusants. Phytochemical composition of protoplast culture with atypical and pure microcolonies was performed using mass spectrometry. Enzyme cocktail of 4% pectinase together with 2% cellulase displayed the highest competence for protoplast isolations. Maximum protoplast density of 30.11×104/ml was obtained from D. ovatum leaves in 2h. Subcellular features such as the presence of partially formed cell wall, the position of the nucleus, chloroplast density, colony existence, and integrity of the plasma membrane were analyzed. Among the pure and cross-cultured protoplasts, the number of heterofusants and homofusants formed were enumerated. The spectral feature extraction of the mass spectrometry indicated the presence of five phenolic marker compounds, viz., tristin, confusarin, gigantol, moscatilin, and resveratrol, some of them in pure and others in assorted protoplast cultures raised from Dendrobium leaves and tepals. The study demonstrated that protoplast fusion technique enabled phytochemical assemblage in vitro as stilbenes tend to get restricted either in a tissue or species specific manner. This is the first report showing the presence of resveratrol, moscatilin, tristin, gigantol, and confusarin in wild and hybrid species from cultured Dendrobium protoplasts in vitro.

Teknik Isolasi dan Kultur Protoplas Tanaman Padi

<p>Protoplast<br />fusion or somatic hybridization technology is an alternative<br />technology for production hybrids of plants that are difficult<br />to be produced by conventional methods due to their sexual<br />incompatibility. An experiment was conducted to develop<br />techniques for isolation, purification, and culture of rice<br />protoplasts of cultivar IR64 and a wild rice species (Oryza<br />officinalis). Optimization of protoplast isolation and purification<br />methods from both rice genotypes were successfully<br />done. The highest protoplast density was obtained by<br />digesting embryonic callus or stems of young seedling in an<br />enzyme solution containing of 2% cellulose, 0.1% pectolyase,<br />0.5% macerozyme, 0.5% driselase, 5 mM ES, and 13% mannitol<br />in CPW solution. The protoplast digestion was done for<br />three hours by soaking in the enzyme solution followed by<br />shaking at 50 rpm under a room temperature. Purification of<br />the protoplasts were done by separating them from plant<br />debris using a 25% sucrose solution. Protoplast regeneration<br />was not successful using although different media compositions<br />and conditions. Growth process from cell division to<br />cell aggregate was only successful on IR64 protoplast culture<br />on a medium that contained AgNO3.</p>

A modified method for preparing meiotic chromosomes based on digesting pollen mother cells in suspension.

BackgroundMeiotic chromosome preparation is a key step in plant meiotic research. Pollen mother cell (PMC) wall elimination is beneficial to cytogenetic experimental procedures. Without wall interference, these procedures are easier and more successful. In existing methods it is difficult to eliminate PMC walls completely and uniformly. In this paper, we present an improved method for digesting PMC walls, and one for providing massive chromosomal spreads on a slide for other cytogenetic experimental procedures.ResultsThree plants were selected to exhibit the modified meiotic chromosome preparation method. PMCs were dispersed as single cells and incubated in a mixed enzyme solution (3 % cellulose + 0.3 % pectinase + 1 % snailase) for 1.5–2.5 h. In total, 28.28 % cells were lost during this process. There were 800–1900 spreads on every slide and no PMC wall interference was found on any of the slides. The spreads were also evenly distributed on the slides. More spreads were obtained when PMC and protoplast densities in the suspension were increased. All three plants’ spreads were successfully used to locate a 5 s rDNA conserved sequence. The Nicotiana hybrid’s spreads were successfully used to identify the hybrid’s parental genome.ConclusionThis is an alternative method for meiotic chromosome preparation. Through this method, PMC walls can be completely and uniformly eliminated, and hundreds of spreads on every slide can be obtained. These spreads can be successfully used for DNA in situ hybridization.

Allelopathy in a Leguminous Mangrove Plant, Derris indica: Protoplast Co-culture Bioassay and Rotenone Effect

To investigate allelopathic activity of a leguminous mangrove plant, Derris indica, the 'Protoplasts Co-culture Method' for bioassay of allelopathy was developed using suspension culture. A suspension culture was induced from immature seed and sub-cultured in Murashige and Skoog's (MS) basal medium containing 10 μM each of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BA). The protoplasts were isolated using the separate wells method with 2% each of Cellulase RS, Driselase 20 and Macerozyme R10 in 0.4 M mannitol solution. Protoplast cultures of D. indica revealed that high concentrations of cytokinins, BA and thidiazuron, were effective for cell divisions. The co-cultures of D. indica protoplasts with recipient lettuce protoplasts using 96 multi-well culture plates were performed in MS basal medium containing 0.4 M mannitol solution and 1 μM 2,4-D and 0.1 μM BA. The protoplast density of D. indica used in co-culturing varied from 6 x 10(3) - 10(5) / mL. Very strong inhibitory allelopathic effects of D. indica protoplasts on lettuce protoplast growth were found. A similar strong inhibitory allelopathic activity of dried young leaves on lettuce seedling growth was also observed by using the sandwich method. Rotenone, which is a component of Derris root, dissolved in DMSO, was highly inhibitory on the growth of lettuce protoplasts in culture and this could be one of the causes of the strong allelopathic activity of D. indica.

Studies on the isolation and culture of protoplasts from Kappaphycus alvarezii

In this study, protoplasts were successfully isolated from Kappaphycus alvarezii using snail enzymes, abalone enzymes and cellulase. The optimum enzymic ratio was fixed to be 20% of abalone enzyme, 12% of cellulase and the osmotic stabilizer was 2.0 mol/L glucose. The optimum enzymic hydrolysis conditions were found to be dark enzymolysis at 30°C continuing for 4.0 h. The resultant density and yield of protoplasts achieved 32.60×104 mL−1, 65.20×104 g−1 tissue for Kappaphycus alvarezii. Finally, under the temperature of 20°C, light intensity of 1 500–2 000 lx and photoperiod of 12 h/d, two developmental pathways were investigated: (1) callus-like cell mass and regenerated plantlet occurred on protoplast; (2) young shoots and callus-like cell mass occurred in tissue blocks after enzymolysis.

An inverse relationship between allelopathic activity and salt tolerance in suspension cultures of three mangrove species, Sonneratia alba, S. caseolaris and S. ovata: development of a bioassay method for allelopathy, the protoplast co-culture method.

A bioassay method for allelopathy, the 'protoplast co-culture method' was developed to study the relationship between salt tolerance and allelopathy of three mangrove species, Sonneratia alba, S. caseolaris, and S. ovata. Plants of S. alba grow in the seaward-side high salinity region and plants of the latter two species grow in upstream-side regions of a mangrove forest, respectively. Effects of five sea salts (NaCl, KCl, MgCl2, MgSO4 and CaCl2) on the growth of the suspension cells of the latter two species were first investigated by a small-scale method using 24-well culture plates. S. ovata cells showed higher tolerance than S. caseolaris cells to NaCl and other salts, but were not as halophilic as S. alba cells. Protoplasts isolated from suspension cells were co-cultured with lettuce protoplasts in Murashige and Skoog's (MS) basal medium containing 1 μM 2,4-dichlorophenoxyacetic acid, 0.1 μM benzyladenine, 3% sucrose and 0.6-0.8 M osmoticum. S. caseolaris protoplasts had a higher inhibitory effect on lettuce protoplast cell divisions than S. alba protoplasts at any lettuce protoplast density, and the effect of S. ovata was intermediate between the two. These results were similar to those obtained from a different in vitro bioassay method for allelopathy, the 'sandwich method' with dried leaves. The inverse relationship between allelopathic activity and salt tolerance in suspension cells of Sonneratia mangroves is discussed.

Preparation of protoplasts from Chlorella protothecoides

This paper describes an enzymatic method for yielding protoplasts from the microalga Chlorella protothecoides. Four kinds of commercially available enzymes were tested. The enzymatic digestion was optimal with 2% cellulase R-10 and 1% snailase prepared in 25 mM Tris buffer (pH 6.0) containing 0.6 M D-mannitol, and the protoplast density could reach the peak after treatment at 30°C for 16 h. Nearly all liberated protoplasts were green in the presence of 0.01% phenosafranin, indicating their high viability. The regeneration rate was about 70% when 0.6 M D-mannitol was used as an osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this algal species.

Interspecific somatic hybrids between Cyclamen persicum and C. coum, two sexually incompatible species

By applying polyethylene glycol (PEG)-mediated protoplast fusion, the first somatic hybrids were obtained between Cyclamen persicum (2n = 2x = 48) and C. coum (2n = 2x = 30)-two species that cannot be combined by cross breeding. Heterofusion was detected by double fluorescent staining with fluorescein diacetate and scopoletin. The highest heterofusion frequencies (of about 5%) resulted from a protocol using a protoplast density of 1 × 10(6)/mL and 40% PEG. The DNA content of C. coum was estimated for the first time by propidium iodide staining to be 14.7 pg/2C and was 4.6 times higher than that of C. persicum. Among 200 in vitro plantlets regenerated from fusion experiments, most resembled the C. coum parent, whereas only 5 plants showed typical C. persicum phenotypes and 46 had a deviating morphology. By flow cytometry, six putative somatic hybrids were identified. A species-specific DNA marker was developed based on the sequence of the 5.8S gene in the ribosomal nuclear DNA and its flanking internal transcribed spacers ITS1 and ITS2. The hybrid status of only one plant could be verified by the species-specific DNA marker as well as sequencing of the amplification product. RAPD markers turned out to be less informative and applicable for hybrid identification, as no clear additivity of the parental marker bands was observed. Chromosome counting in root tips of four hybrids revealed the presence of the 30 C. coum chromosomes and 2-41 additional ones indicating elimination of C. persicum chromosomes.

Plant regeneration from Gossypium davidsonii protoplasts via somatic embryogenesis

Protoplasts isolated from wild cotton Gossypium davidsonii were cultured in KM8P medium supplemented with different phytohormones. The most effective combination was 0.45 µM 2,4-dichlorophenoxyacetic acid, 2.68 µM α-naphthaleneacetic acid and 0.93 µM kinetin and the division percentage at the 8th day was 30.78 ± 3.04 %. The density of protoplasts at 2–10 × 105 cm−3 was suitable for protoplast division and calli formation, with a division percentage of 32.21 ± 3.64 % and a plating efficiency of 9.12 ± 2.61 % at the 40th day. The optimal osmotic potential was achieved using 0.5 M glucose or 0.1 M glucose plus 0.5 M mannitol. Protoplasts were cultured in three ways, a double-layer culture system, with liquid over solid medium was proved to be the best way. Embryo induction was further increased by addition of 0.14 µM gibberellic acid.

Establishment of Protoplast Culture of Solanum sisymbriifolium

The optimal culture conditions were studied for plant regeneration from mesophyll protoplasts of Solanum sisymbrizfolium. Aseptic seedlings ofS. sisymbrizfolium were used as a material for protoplast culture. Many viable protoplasts were isolated by incubating leaf slices in an enzyme solution containing 0.250/0 Meiserase and 0.050/0 Macerozyme for 16 h at 25 'C wlthout shaking. Protoplast density of 5.0 X 10*/ml in Kao medium containing 5.0mg/1 NM, 1.0mgfl 2,4-D and 1.0mg/1 BA was optimal for colony formation. Most colonies formed when protoplasts were cultured at 25 'C after initial culture at 30'C for one week. On the MS agar medium with 1.0mg/1 zeatin, 38.50/0 of protoplast~lerived calli differentiated shoots. These shoots rooted on 1/2 MS medium with 5.0mg/1 sucrose and 2.5g/1 gellan gum, and developed into whole plants.