• The Korean Journal of Mycology
• /
• v.15 no.2
• /
• pp.80-86
• /
• 1987

The author isolated high cellulolytic fungi from natural sources and determined optimal condition of protoplast formation and fusion as fundamental step for improvement of the isolated it's cellulolytic ability. Three different cellulolytic fungi, such as Aspergillus sp., Penicillium sp. and Trichoderma sp., were isolated from soil. Their cellulolytic activities were compared with that of Aspergillus niger which was useful industrially and had cellulase activity. It was Aspergillus sp. that showed the highest activity of all these four fungi. And then it was followed by Penicillium sp., Trichoderma sp., and Aspergillus niger in order. An auxotrophic mutant of Aspergillus sp. was obtained by UV mutagenesis method. Having try to produce protoplast from mycelia, the author found that ${\beta}-glucuronidase$, at pH 6.0, was effective cell-wall lytic enzyme. And the optimal concentration of this enzyme was 5,000 unit/ml. Regeneration rates of wild type, met. auxotroph and arg. auxotroph, in presence of osmotic stabilizer, were 7. 0%, 7. 5% and 5.2%, respectively. PEG with M.W. 6,000 was effective stimulator for protoplast fusion in the concentration of 30% (W IV). In such a condition, we obtained 1.2% cell fusion rate.

• Microbiology and Biotechnology Letters
• /
• v.20 no.4
• /
• pp.384-389
• /
• 1992

Bacillus sp. YBL-7 which had been isolated from ginseng root-rot suppressive soil was able to antagonize Fusarium solani causing ginseng root-rot by their antibiotic substance. In order to develop multifunctional antagonist on Bacillus sp. YBL-7 as a biocontrol agent against Fusarium salam', optimal conditions for protoplast transformation system of Bacillus sp.YBL-7 by the vector plasmid pE194 were investigated. The protoplasts of Bacillus sp. YBL-7 were obtained at best efficiency by treatment with 200${\mu}g$/ml of lysozyme in the pH 7.0 of SMM buffer for 90 minutes at $40^{\circ}C$. The cell wall of the protoplast was regenerated on the agar plate containing 1.2% agar and 0.7 M mannitol. Under the best condition for protoplast formation and regeneration, the optimal transformation was achieved with 40% polyethylene glycol (M.W. 4000) treatment for 10minutes. The vector plasmid pE194 showed the best transformation frequency at 5$\mu$g/ml of final concentration. The pE194 was very stable over 80% in the transformants.

• Korean Journal of Food Science and Technology
• /
• v.22 no.6
• /
• pp.611-617
• /
• 1990

This study was conducted to breed a high vitamin $B_{12}$ producer by the fusion of protoplasts between Bacillus natto and Bacillus megaterium. Auxotrophic mutants of Bacillus natto SH-34 ($thr^-try^-rif^r$) and Bacillus megaterium BK-13 ($arg^-ade^-lys^-str^r$) which showed high protease activity and production of vitamin $B_{12}$, respectively, were isolated for the fusion experiment. Protoplasts were induced by incubating the cells with lysis solution containing $500{\mu}/ml$ lysozyme, and the ratio of protoplast and regeneration formation were ranged from 99% and 67%, respectively. Fusion frequencies of fusants between Bacillus natto SH-34 and Bacillus megaterium BK-13 were appeared in the ranges of $1.0{\times}10^{-5}$ under the treatment of 30% PEG 6000 containing 3% PVP. The fusant, MNF-72 showed the highest product yield of $7.85{\mu}g/g-cell\;vitamin\;B_{12}$ in production medium. For the improvement of productivity, the immobilization of fusants with sodium alginate was carried out. In batch and continuous fermentation systems, the productivity were determined to be $0.58{\mu}g/ml.hr\;and\;0.80{\mu}g/ml.hr\;vitamin\;B_{12}$ under optimum condition, respectivity.

• Applied Biological Chemistry
• /
• v.42 no.1
• /
• pp.12-19
• /
• 1999

To improve biopolymer productivity and properties of Bacillus strains, protoplast fusion was performed between biopolymer producing Bacillus subtilis K-1 and lactose utilizing Bacillus coagulans. The results were as follows; Protoplasts mixed in fusion fluid containing 33% PEG 6000, 1% PVP and 10 mM $CaCl_2$ were reacted for 5 min at $37^{\circ}C$ and then centrifused protoplasts were directly overlaid on the selective media containing $100\;{\mu}g/ml$ antibiotics and incubated for 3 days. At this conditions, the frequency of protoplast fusion was generally in the range of $4.6{\times}10^{-5}\;to\;1.8{\times}10^{-7}$ in ratio. Segregation ratio was observed between 1 to 6% indicating genetic stability of all the fusants. Fusants growth were also observed on the media contained amino acid and antibiotics as required marked materials. DNA contents of the selected fusants were 1.6 to 2.7 times more than that of parent strains. With observation by TEM microscopy, spherical protoplasts were first released from the swollen parental cells and then contracted to fuse in the process of fusion. And fused cells were observed representative vesicle. Originally, the parental cells were observed as in the morphology of thick-walled and double membrane-surrounded rod shape with TEM microscopy.

• Korean Journal of Plant Resources
• /
• v.33 no.5
• /
• pp.536-549
• /
• 2020

Soybean (Glycine max (L.) Merrill) is one of the most important crops of the world. With the completion of the soybean genome sequence, the Korean soybean core collection consisted of 430 accessions with genetic and phenotypic diversity was constructed in recent year. The availability of genome sequences and core collection will result in the crop improvement by molecular breeding using the various accessions and genome editing approaches. Efficient tissue culture techniques, such as haploid production, protoplast culture and plant regeneration from various organs are essential for the successful molecular biological approach and crop improvement. However, soybean is still considered to be recalcitrant in tissue culture because of the low frequency of regeneration and limitation of available responsive cultivars. In this study, we discuss the recent studies of tissue culture technology and methodology for efficient tissue culture to genetic improvement and application of molecular biotechnology in soybean.

• Journal of The Korean Society of Grassland and Forage Science
• /
• v.13 no.3
• /
• pp.170-176
• /
• 1993

The yield, viability and continuous culture of isolated Italian ryegrass protoplasts were investigated. The effects of cold treatment (4^{\circ}C.$) for 7 days and basic LS medium supplemented with 5mg/l $AgNO_3$ showed effectively on embryogenic callus induction and regeneration responses of immature and mature embryos or young inflorescences subcultured every 4 weeks on basic medium. The optimum combinations of growth regulator on the regeneration responses was 0.2mg/l BAP and 2mg/l 2, 4-D. Calli induced inflorescences were suspended in its liquid medium for 5 days before enzyme treatment. Maximum protoplast yield and viability were obtained after digestion in enzyme solution contained 4% cellulase R10. 2% macerozyme and 2% pectinase in 0.6M mannitol. Cell division and microcalli development were observed in isolated protoplasts cultured in agarose culture of KM8P medium.

• Journal of Plant Biotechnology
• /
• v.37 no.1
• /
• pp.77-83
• /
• 2010

Eighteen cultivars of tomato were tested for their regeneration response. Lycopersicon esculentum cv. 2001-58 showed a very high frequency of regeneration (93%). We evaluated the effect of two compounds with known antioxidant activity (ascorbic acid and cystein). The use of ascorbic acid ($200\;-\;300\;{\mu}M/L$) has a positive effect on shoot regeneration. To develope a system for plastid transformation in tomato via homologous recombination, we constructed the tomato plastid expression vector (pKRT22-AG) harboring 2.2 kb flanking sequences cloned from intact plastid genome and gfp gene. To investigate the factors affecting the delivery system of the pKRT22-AG into chloroplast using bombardment, We assessed the optimal DNA concentration, gold particle volume and target distance. Expression of the GFP protein was observed within chloroplast on protoplast of cotyledon explant by confocal laser scanning microscopy, which indicates that the protocol developed in this study be useful for the production of plastid transgenic plants in tomato.

• Plant Biotechnology Reports
• /
• v.2 no.3
• /
• pp.219-226
• /
• 2008

Transgenic plants with the herbicide-resistance gene (bar gene) were obtained via organogenesis from isolated mesophyll protoplasts of Nierembergia repens after applying electroporation. Transient ${\beta}-glucuronidase$ (GUS) activity of electroporated protoplasts assayed 2 days after applying an electric pulse showed that optimum condition (transient GUS activity 319 pmol 4 MU/mg per min and plating efficiency 2.43%) for electroporation was 0.5 kV/cm in field strength and $100{\mu}F$ in capacitance. The protoplasts electroporated with the bar gene at this condition initiated formation of microcolonies on medium after 2 weeks. After 4 weeks of culture, equal volume of fresh 1/2-strength Murashige and Skoog (MS) medium containing 0.2 mg/l bialaphos was added for selection of transformed colonies. After 6 weeks of culture, growing colonies were transferred onto regeneration medium containing 1.0 mg/l bialaphos, on which they formed adventitious shoots 1-2 months after electroporation. The adventitious shoots rooted easily after transfer onto MS medium with bialaphos lacking plant-growth regulators. Transformation of these regenerants with the bar gene was confirmed by Southern analysis. Some of the transformants showed strong resistance to the application of bialaphos solution at 10.0 mg/l.

• Proceedings of the Botanical Society of Korea Conference
• /
• 1995.06a
• /
• pp.40-56
• /
• 1995

During the past two decades, China has seen her great progress in plant biotechnology. Since the Chinese market of herb medicine is huge, while the plant resources are shrinking, particular emphasis has been placed in plant tissue and cell cultures of medicinal plants, this includes fast propagation, protoplast isolation and regeneration, cell suspension cultures and large scale fermentation. To optimize culture conditions for producing secondary compounds in vitro, various media, additives and elicitors have been tested. Successful examples of large scale culture for the secondary metabolite biosynthesis are quite limited : Lithospermum ery throrhizon and Arnebia euchroma for shikonin derivatives, Panax ginseng, P. notoginseng, P. quinquefolium for saponins, and a few other medicinal plants. Recent development of genetic transformation systems of plant cells offered a new approach to in vitro production of secondary compounds. Hairy root induction and cultures, by using Ri-plasmid, have been reported from a number of medicinal plant species, such as Artemisia annua that produces little artemisinin in normal cultured cells, and from Glycyrrhiza uralensis. In the coming five years, Chinese scientists will continue their work on large scale cell cultures of a few of selected plant species, including Taxus spp. and A. annua, for the production of secondary metabolites with medicinal interests, one or two groups of scientists will be engaged in molecular cloning of the key enzymes in plant secondary metabolism.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.1
• /
• pp.72-78
• /
• 2001

It is possible ot prepare protoplasts of the zygomycete fungus, Phycomyces blakesleeanus, by digesting the cell wall of spore germlings with commercially available chitinase and chitosanase. However, the cells without any cell walls immediately form large aggregates, and thus, it is difficult to isolate the individually separated protoplasts. Inherent problem with the formation of aggregates in preparing protoplasts could be solved by the use of bovine serum albumin (BSA). As a result, we were able to prepare a large number of single protoplsts quickly and easily. We took time-lapse photomicrographs of the formation of protoplasts, and found that there were certain regions of the cell wall of spore germlings that were sensitive to chitinase and chitosanase, although the cell wall of the original spores is known to be insensitive to these enzymes. There are two kinds of cell walls on a spore germling; one with a bound wheat germ agglutinin (WGA), and the other a bound concanavalin A (ConA). Furthermore, only cells with walls which had bound WGA were able to regenerate, while those with walls with bound ConA were not able to regenerate.