• ALGAE
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• v.37 no.2
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• pp.105-121
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• 2022

Modern seaweed farming relies heavily on seedlings from natural beds or vegetative cuttings from previous harvests. However, this farming method has some disadvantages, such as physiological variation in the seed stock and decreased genetic variability, which reduces the growth rate, carrageenan yield, and gel strength of the seaweeds. A new method of seedling production that is sustainable, scalable, and produces a large number of high-quality plantlets is needed to support the seaweed farming industry. Recent use of tissue culture and micropropagation techniques in eucheumatoid seaweed production has yielded promising results in increasing seed supply and growing uniform seedlings in large numbers in a shorter time. Several seaweed species have been successfully cultured and regenerated into new plantlets in laboratories using direct regeneration, callus culture, and protoplast culture. The use of biostimulants and plant growth regulators in culture media increases the seedling quality even further. Seedlings produced by micropropagation grew faster and had better biochemical properties than conventionally cultivated seedlings. Before being transferred to a land-based grow-out system or ocean nets for farming, tissue-cultured seedlings were recommended to undergo an acclimatization process to increase their survival rate. Regular monitoring is needed to prevent disease and pest infestations and grazing by herbivorous fish and turtles during the farming process. The current review discusses recent techniques for producing eucheumatoid and other valuable seaweed farming materials, emphasizing the efficiency of micropropagation and the transition from laboratory culture to cultivation in land-based or open-sea grow-out systems to elucidate optimal conditions for sustainable seaweed production.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.30 no.2
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• pp.195-200
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• 1985

This experiment was carried out to know the processes of protoplast isolation, culture and plant regeneration in aims of introducing foreign genes into plant cells through plant gene vector, and cellular selection for plant improvement. The main results indicated that 2% cellulase plus 0.5% macerozyme is proper for isolation of protoplasts from leaf mesophyll cells of N. plumbaginifolia, plating efficiency was higher in 1.4-2.0 x 10$^4$ cells/ml, complete cell wall was regenerated after 2 days culture, cell division and cell mass were observed after 4 days and 2 weeks, respectively, colony was developed after 3 weeks culture, addition of 1-2mg/l BA promoted shoot differentiation while root differentiation did not required hormone and seeds were harvested from more than 100 cell lines for further investigation and study.

• ALGAE
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• v.38 no.1
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• pp.1-22
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• 2023

An enormous body of research is focused on finding ways to commercialize carotenoids produced by the unicellular green alga, Haematococcus, often without the benefit of a sound phylogenetic assessment. Evidence of cryptic diversity in the genus means that comparing results of pigment studies may be confounded by the absence of a phylogenetic framework. Moreover, previous work has identified unnamed strains that are likely candidates for species status. We reconstructed the phylogeny of an expanded sampling of Haematococcus isolates utilizing data from nuclear ribosomal markers (18S rRNA gene, 26S rRNA gene, internal transcribed spacer [ITS]-1, 5.8S rRNA gene, and ITS-2) and the rbcL gene. In addition, we gathered morphological, ultrastructural and pigment data from key isolates of Haematococcus. Our expanded data and taxon sampling support the concept of a new species, H. privus, found exclusively in North America. Despite overlap in numerous morphological traits, results indicate that ratios of protoplast length to width and akinete diameter may be useful for discriminating Haematococcus lineages. High growth rate and robust astaxanthin yield indicate that H. rubicundus (SAG 34-1c) is worthy of additional scrutiny as a pigment source. With the description of H. privus, the evidence supports the existence of at least five, species-level lineages in the genus. Our phylogenetic assessment provides the tools to frame future pigment investigations of Haematococcus in an updated evolutionary context. In addition, our investigation highlighted open questions regarding polyploidy and sexuality in Haematococcus which demonstrate that much remains to be discovered about this green flagellate.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.234-234
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• 2022

Gene-editing is currently one of the most popular technologies in recent years. Development of the new crop using the gene editing have advantage of improved accuracy and efficiency compared with conventional breeding. Soybean (Glycine max L.) is one of the most important crops worldwide used as food and forage. We tried to establish a system for breeding improvement of soybean through gene-editing technology. For the gene-editing system of soybean, i) selection of efficiency gRNA of targeted gene, ii) efficient genetic transformation of the selected gRNA, iii) selection of trans-clean mutant is essential. First of all, we investigated the selection conditions of gRNA with high editing efficiency of targeted gene using isolated protoplast of soybean. Furthermore, we performed the Agrobacterium-mediated genetic transformation of various soybean cultivars. We identified the tissue culture ability in 23 soybean cultivars for genetic transformation of soybean. The six cultivars with high tissue culture ability were selected and confirmed the transgenic plants in four cultivars. Finally, we established a speed-breeding system as a powerful tool for the fast selection of trans-clean mutants from transgenic plants. Our laboratory will provide the valuable system for improvement of soybean by the gene-editing technology.

• Journal of Plant Biotechnology
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• v.50
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• pp.11-18
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• 2023

Manihot esculenta Crantz, commonly known as cassava, is a staple aliment that is a significant source of revenue for farmers. The embryogenic callus is crucial in the genetic engineering of various crop species, including cassava. Four cultivar cassava landraces from East Java were assessed for their ability to produce friable embryogenic callus (FEC) for protoplast isolation. In this study, four cassava cultivars; (Kaspro, Kuning, Gajah, and Gendruwo); were used to obtain FEC, which involved the culture of immature leaf lobes (ILLs) and apical buds (ABs) media containing MS supplemented with 33 μM picloram and 2 μM of CuSO₄ (M1) or MS supplemented with 50 μM 2,4-D and 2 μM CuSO₄ (M2). The highest FEC induction efficiency ranged from 72% to 57%, and the highest FEC number ranged from 4.7 to 3.7 with AB explants in media containing MS + 33 μM pilocram and 2 μM CuSO₄ (M1). On the other hand, the efficiency of somatic embryogenesis induction ranged from 67% to 53%, and the number ranged from 4.4 to 3.4. The efficiencies of FEC induction ranged from 48% to 42%, and the number ranged from 3.1 to 2.6 with AB explants in media containing MS + 50 μM 2,4-D and 2 μM CuSO₄ (M2); the efficiency of FEC induction ranged from 56% to 50%, and the value ranged from 3.6 to 2.4 with ILL explants. The FEC induction of the Gendruwo cultivar, which was examined using AB and ILL explants, demonstrated the lowest efficiency. Nevertheless, all four cultivars showed the ability to generate FEC, even though their effectiveness differed depending on the explant genotype and the applied media.

• Journal of Korean Society of Forest Science
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• v.81 no.3
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• pp.273-279
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• 1992

Protoplasts isolated from leaf mesophyll tissues of Populus koreana ${\times}$ P. nigra var. italica were fused with those of P. euramericana cv. Guardi. Well expended healthy leaves of 5 to 7 week-old-plantlet grown in vitro were used as source materials. Leaves from P. koreana ${\times}$ P. nigra var. italica and P. euramericana cv. Guardi were digested in enzyme solution I (2.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v) and enzyme solution II (1.0% Cellulase, 1.2% Hemicellulase, 0.4% Macrozyme, 2.0% Driselase, 0.05% Pectolyase ; w/v), respectively, The highest frequency of fusion among the protoplasts originated from the two source materials was approximately 21% using 40% PEG or 15% dextran. In addition, fusion frequency was enhanced by incorporating 30mM of $Ca^{2+}$ in eluting solution at pH 10.5. Dividing cells and/or mint-calli were obtained by culturing the fusion products in a liquid 8p-KM medium supplemented with 0.6M sucrose, $0.45{\mu}M$ 2, 4-D, and $0.5{\mu}M$ BA. Shoots were regenerated from the fusion product-derived calli after culture on MS medium containing $5.0{\mu}M$ zeatin. To verify the putative hybrid or cybrid, SDS-PAGE was carried out. From the 24 regenerants, just two plants showed intermediate protein band patterns compared with those of the original source plants.

• Korean Journal of Food Science and Technology
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• v.41 no.5
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• pp.541-546
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• 2009

The objectives of this study were to evaluate the physico-chemical and sensory characteristics of a Korean traditional alcoholic beverage (yakju) prepared using different nuruk (Korean-style koji) concentrations and yeasts such as the fusant FA776 and Saccharomyces cerevisiae KOY-1, respectively. The fusant FA776, which has alcohol-fermenting and starch-utilizing properties, was formed by Saccharomyces cerevisiae KOY-1 and Saccharomyces diastaticus KCTC1804. The fermentation trial was conducted in a 5 L lab-scale jar at $25^{\circ}C$. The maximum alcohol production of the K-100 and F-50 reached levels of 135.0 mg/mL and 119.4 mg/mL, respectively. The pH values were in a range of 4.3-4.5. Total acidity was in a range of 0.47-0.60%. Organic acids and amino acids were analyzed in order to evaluate variations in its composition and content via HPLC analysis. Organic acids including lactic acid, citric acid, malic acid, and pyruvic acid, and 16 kinds of amino acids, including aspartic acid, were detected in all treatments. K-100 showed the highest amino acid contents, whereas F-50 exhibited the lowest amino acid contents. Volatile flavor components such as phenylethyl alcohol, isoamyl alcohol, 2-methylthiophane, isobutyl alcohol, and ethyl succinate were detected as a major component in all treatments, as determined via gas chromatography. The results of our sensory evaluation demonstrated that Yakju fermented by the FA776 fusant yielded more favorable results than S. cerevisiae KOY-1.

• Korean Chemical Engineering Research
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• v.47 no.2
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• pp.220-229
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• 2009

Strain improvement and morphology investigation in bioreactor cultures were undertaken in suspended cultures of Phellinus linteus mycelia for mass production of protein-bound polysaccharides(soluble ${\beta}$-D-glucan), a powerful immuno-stimulating agent. Phellineus sp. screened for this research was identified as Phellinus linteues through ITS rDNA sequencing method and blast search, demonstrating 99.7% similarity to other Phellinus linteus strains. Intensive strain improvement program was carried out by obtaining large amounts of protoplasts for the isolation of single cell colonies. Rapid and large screening of high-yielding producers was possible because large numbers of protoplasts ($1{\times}10^5{\sim}10^6\;protoplasts/ml$) formed using the banding filtration method with the cell wall-disrupting enzymes could be regenerated in relatively high regeneration frequency($10^{-2}{\sim}10^{-3}$) in the newly developed regeneration medium. It was demonstrated that the strains showing high performances in the protoplast regeneration and solid growth medium were able to produce 5.8~6.4%(w/w) of ${\beta}$-D-glucan and 13~15 g/L of biomass in stable manners in suspended shake-flask cultures of P. linteus mycelia. In addition, cell mass increase was observed to be the most important in order to enhance ${\beta}$-D-glucan productivity during the course of strain improvement program, since the amount of ${\beta}$-D-glucan extracted from the cell wall of P. linteus mycelia was almost constant on the unit biomass basis. Therefore we fully investigated the fungal cell morphology, generally known as one of the key factors affecting cell growth extent in the bioreactor cultures of mycelial fungal cells. It was found that, in order to obtain as high cell mass as possible in the final production bioreactor cultures, the producing cells should be proliferated in condensed filamentous forms in the growth cultures, and optimum amounts of these filamentous cells should be transferred as active inoculums to the production bioreactor. In this case, ideal morphologies consisting of compacted pellets less than 0.5mm in diameter were successfully induced in the production cultures, resulting in shorter period of lag phase, 1.5 fold higher specific cell growth rate and 3.3 fold increase in the final biomass production as compared to the parallel bioreactor cultures of different morphological forms. It was concluded that not only the high-yielding but also the good morphological characteristics led to the significantly higher biomass production and ${\beta}$-D-glucan productivity in the final production cultures.

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.

• The Korean Journal of Mycology
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• v.25 no.2 s.81
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• pp.143-151
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• 1997

Interspecific hybrids between Aspergillus niger and Penicillium notatum (Tyr-), hyperlipolytic enzyme-producing fungi, were obtained by nuclear transfer technique. Optimal conditions for formation of intergeneric hybrids were investigated. Maximum production of protoplasts was obtained by 1% Novozyme 234 at $30^{\circ}C$ for 3 hrs and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6 M KCl. Frequencies of hybrid formation by nuclear transfer were $3.8{\times}10^{-3}{\sim}1.3{\times}10^{-3}$. From the observation of genetic stability, conidial size, DNA content, and nuclear stain, it was suggested that their karyotypes are aneuploid. The hybrids showed $1.2{\sim}1.7$ fold higher lipase activities than parental strains. It was strongly supported by results of this study that nuclear transfer technique is much more efficient in the formation of intergeneric hybrids than protoplast fusion and is very useful for the improvement of strains.