• Applied Biological Chemistry
• /
• v.47 no.2
• /
• pp.192-196
• /
• 2004

In order to measure cellular physiological activity including ion channel activity, protoplasts were isolated from the root tissue of tomato plant. The general methods recommended were not efficient enough to make protoplasts from the root tissue. Among various conditions tested, we found that a two-step treatment of osmosis is very efficient for the isolation of protoplasts. In this procedure, root tissues were preincubated in a solution containing 300 mM sorbitol for 30 min. Then, they moved to the reaction solution containing 700 mM sorbitol as well as cell wall-digesting enzymes. The formation of protoplast was greatly increased by this method. In order to find the optimal condition of the two-step method, various conditions of pH, osmotic pressure, incubation time, and the concentrations of cell wall-digesting enzymes were tested. The yield of protoplast isolation was maximal at pH 5.0 after 2 hr incubation. Mixed enzymes of 3% cellulase, 1 % macerozyme, and 0.1 % pectolyase showed maximal protoplast isolation. The physiological activity of isolated protoplast evaluated by measuring the cellular ATPase activity was as high as that measured from the preparation of root tissue. The protoplasts isolated by this method were remained healthy up to 4 hrs which is enough time to measure the cellular physiological activity. These results show that the two-step treatment of osmotic pressure was successful to obtain high yield of healthy protoplast from tomato root tissue.

• The Korean Journal of Mycology
• /
• v.14 no.4
• /
• pp.273-280
• /
• 1986

The degree of autolysis and lytic enzyme production in the culture filtrate of Rhizopus oryzae was investigated. The formation of protoplast by using autolytic enzymes from Rh. oryzae was also attempted. Protoplasts were liberated from Rh. oryzae mycelium by lytic enzymes present in autolytic-phase culture filtrate. Maximum release of chitosanase and proteolytic enzyme into culture filtrate during autolysis was corresponded to maximum protoplast-liberating activity. High yields of protoplasts were obtained from 10 hr-age of Rh. oryzae mycelium with 0.5 M mannitol as osmotic stabilizer. The optimum temperature and pH for mycelium digestion were $25{\sim}30^{\circ}C$ and $6.0{\sim}6.5$ respectively. The mycelium of the 18 hours cultures were treated with autolytic enzyme in same volume of osmotic stabilizer at $30^{\circ}C$ for 5 hours and then it was confirmed by scanning electoron microscope that protoplast were produced beside the digesting cell wall of the fungi.

• Microbiology and Biotechnology Letters
• /
• v.14 no.2
• /
• pp.175-179
• /
• 1986

In order to develope a protoplast fusion of the genus Cellulomonas having high assimilibility of cellulose, the optimum conditions for the protoplast formation of Cellulomonas flavigena NCIB 12901 was investigated and observed by means of Scanning Electron Microscope. The results suggested that the susceptibility of the cell wall by lysozyme treatment on protoplast formation was considerably depend on the cultural periods of the cells. Cells of C. flavigena at mid exponential phase could more efficiently convert to protoplast cells than those at late exponential phase did. The rate of the protoplast formation was 95%, even though the rate was over 99.9% on counting by indirect method after osmotic shock treatment, when cells of the organism at mid exponential phase were treated with lysozyme (400$\mu\textrm{g}$/$m{\ell}$) for 6 hours and observed by SEM. In the evaluation of protoplast formation of the genus Cellulomonas, direct method of the observation under Scanning Electron Microscope was much more reliable than the counting method of protplasts after osmotic shock treatment. Because defferences between the number of spheroplast and protoplast were not able to be figured out on counting the number of protoplast after osmotic shock treatment.

• Journal of Mushroom
• /
• v.2 no.1
• /
• pp.33-37
• /
• 2004

To develop neohaplonts for Ganoderma breeding, protoplasts were isolated from dikaryotic mycelium and regenerated. Selection rate of neohaplonts varied between ASI7074, ASI7091, ASI7094, ASI7100 and ASI7115, showing 5.24% on the average. Auxotropic mutants from Ganoderma monokarions were recovered by UV irradiation on protoplasts. Protoplast survival rates were 1.9% ASI 7074, 0.17% ASI 7091, and zero percent ASI 7100 using 300 second irradiation. Four auxotrophic strains were recovered from 1,536 colonies screened that will be further utilized for protoplast fusion and transformation.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.32 no.1
• /
• pp.117-120
• /
• 1999

High yields of protoplasts were obtained following enzymatic digestion of the vagetative thalli of marine green alga Monostroma nitidum. The enzyme mixtures containing $4\%$ Cellulase R-10+$3\%$ Macerozyme R-10+$3\%$ Abalone acetone power produced $4.41\times10^6$ protoplasts per 300 mg of fresh tissue. The highest yield of protoplasts was obtained by 270 minutes treatment of the thalli in enzyme solution. Freshly isolated protoplasts were spherical in shape and ranged between $13\~33\mu$m in diameter. The high efficiency of differentiation were obtained by incubating freshly isolated protoplasts in 0.4 M mannitol f/2 medium for 7 days and then transferring to 0.2 M mannitol f/2 medium. Protoplasts began to form new cell walls three days after initial culture and began to germinate after 10 days, and then form a leafy thallus after further culture in f/2 medium. The addition of antibiotics in media inhibited the differentiation of protoplasts in culture.

• Korean Journal of Microbiology
• /
• v.22 no.1
• /
• pp.49-56
• /
• 1984

Formation and regeneration of protoplasts by Novozym 234 from Kluyveromyces fragilis N100 and Candida pseudotropicalis CBS607 were studied. This enzyme was more effective on cells grown at exponential phase than those at stationary one to convert intact cells into protoplasts. As osmotic stabilizer, ammonium sulphate was suitable for not only protoplast formation but also regeneration in K. fragilis as well as in C. pseudotropicalis. Optimal enzyme concentration was 3mg per ml in K. fragilis and 1~3mg per ml in C. pseudotropicalis, respectively. After the exposure of K. fragilis cells to 3mg per ml of enzyme for 3hr at 30$^{\circ}C$ , approximately 95% of protoplast formation of all observed cells was obgained, while about 100% from C. pseudotropicalis under the same condition was produced. The regeneration frequency of protoplasts by this enzyme was much lower than that by snail enzyme(Glusulase) although Novozym 234 converted cells from above two species into protoplasts free of cell debris effectively, compared with Glusulase. Novozym 234 appears to be suitable for subcellular fractionation to obtain nuclei or other organelles rather than protoplast regeneration.

• Korean Journal of Microbiology
• /
• v.27 no.3
• /
• pp.216-220
• /
• 1989

As the bsic study about protoplast fusion of amylolytic fungus Aspergillus oryze and nonamyloytic sugar fermenter, Saccaromyces cerevsisae, the intraspecific protoplast fusion of A. oryzae was carried out and the properties of the obtained fusants were investigated. For protoplast fomation from mycellia of auxotrophs, Novozyme 234 as lytic enzyme was the most effective and optimal pH was determined to be pH 5.5-6.0. When the two types of protoplasts were treated with a fusogen including 30% PEG4000, they fused effectively and most of fusants were heterokaryons. Protoplasts aggregated with 30% PEG4000 after fusion treatment were observed by the microscope. Protoplast regeneration frequency was 1.46 to 13.8% and complementation frequency of fusion was 0.12 to 0.16. Fusant strains had a 1.5-fold DNA content compared to that of parent strain. And amylase activity was intermediate between those of parent strains.

• Korean Journal of Microbiology
• /
• v.31 no.4
• /
• pp.318-325
• /
• 1993

The optima] conditions for the formation, the regeneration. and the spheroplast fusion of Flavimonas aryz/habitans spheroplasts were investigated. Cells were transformed to spherop]asts effectively by treatment of 0.5% volume (v/v) of 0.] M EDTA and ]00 flg/ml lysozyme at $37^{\circ}C$ for 30 min without shaking. Magnesium chloride and calcium chloride were effective on the stabilization of spheroplasts. and 20 mM calcium chloride in the rich regeneration medium improve the yield of regenerants as much as 3.5-fo]d. Addition of 0.8% bovine serium albumine (BSA) in dilution buffer for spheroplast formation improved the stabilization of spheroplasts over extended periods (4-6 hr) at room temperature. and thus increased the yield of recombinants to 4.5-fold. The spheroplast formation frequency and regeneration frequency of F aryzihabitans strain was 90.10% and 3.800/." respectively. The first regenerated cell of F. aryzihabitans spheroplasts were appeared 6 hours after plating. By I I hours after plating, 80% of spheroplasts were regenerated on thc rich regeneration medium containing 0.5 M sucrose. The intraspeci11c spheroplast fusion of F urvz/habitans was carried out and the properties of obtained fusants were investigated. Formation of fusion products was effective when the Flav/munas spheroplast mixture was treated with 40%(w/v) PEG6000 and 20 mM CaCl, for 10 min at room temperature. and thc formation of frequency of recombinants were $2.0{\times}10^{-5}~3.6{\times}10^{-5}$. All tested recombinant clones were very stable on further propagation.

• Korean Journal of Microbiology
• /
• v.24 no.2
• /
• pp.154-160
• /
• 1986

In order to develope interspecific fusion of the genus Cellulomonas capable of assimilation cellulose, the optimun conditions for the protoplast formation was investigated to examine the susceptibility of cell wall, between different species of the same genus using scanning electron microscope. The variation in the susceptibilities of Cellulomonas sp. CS 1-1 and C. flavigena to lysozyme treatment were considerably remarkable, although they belong to the same genus. The rate of protoplast formation of CS1-1 was 99.9% being treated with lysozyme $(100{\mu}g/ml)$ for 30 minute and that of C. flavigena was about 80% being treated at the concentration of $600{\mu}g/ml$ of lysozyme for 6 hours. The susceptibility of cell wall to the lysozyme treatment on protoplast formation of the strain, CS1-1 seems not to be depend on the cultural periods of cells. On the contrary, that of C. flavigena was considerably depend on the periods. Cells of C. flavigena at mid exponential phase could be more efficiently converted to protoplast cells than those at late exponential phase be done. The rate of the protoplast formation was 95%, when cells of C. flavigena at mid exponential phase were treated with lysozyme $600{\mu}g/ml$ for 6 hours and observed by SEM. In the evalution of protoplast formation of the CS1-1 results of counting method in plate after osmotic shock treatment were similar to the results of the direct observation method by means of SEM. But in the case of C. flavigena the latter method was much more reliable than the former, because the differences between the number of spheroplasts and protoplasts were not able to figure out on conuting the number of protoplast after osmotic shock tretment.

• Korean Journal of Microbiology
• /
• v.22 no.1
• /
• pp.35-40
• /
• 1984

Attempts were made to optimise protoplast formation and regeneration methods to improve the protoplast fusion frequencies of Streptomyces coelicolor. The yields of protoplast formation and regeneration were varied with different growth phase of the culture. Maximum yields were obtained when cells were taken from the late logarithmic phase. Protoplast formation reached almost its maximum with lysozyme treatment at a concentration of 2mg/ml without any other lytic enzyme. A high frequency of protoplast regeneration was accomplished by overlay method: the method gave 14% recovery of regenerated protoplast versus 1.8% recovery for monolay method. A recombinant frequency of 1.8X10^-2 was obtained by protoplast fusion using PEG 1000(50% w/v).