Yong-Ik Kwon

Molecular cloning and characterization of chitinase genes from zoysiagrass ( Zoysia Japonica Steud.)

Zoysiagrass (Zoysia Japonica Steud.) is used frequently in golf courses and athletic fields. However, Zoysiagrass suffers from large-patch disease caused by Rhizoctonia solani AG2-2 (IV), which results in physical and economic loss. In this study, two full-length chitinase genes encoding pathogen-related proteins were isolated from zoysiagrass. Structural and expression analyses of these genes were carried out. The two isolated chitinases were classified into class Ib (Zjchi1) and class II (Zjchi2). Zjchi1 and, Zjchi2 expression was high in root and stolen and was induced in seedlings by Rhizoctonia solnai AG2-2 (IV) infection. To assess their antifungal activity, the two chitinases were overexpressed in Escherichia coli and purified using Ni2+ and glutathione affinity column chromatography. The purified recombinant chitinases showed broad-spectrum antifungal activity against Rhizoctonia solnai AG2-2 (IV), Rhizoctonia solnai AG-1 (IA), Rhizoctonia cerealis, Botrytis cinerea, Fusarium culmorum, Fusarium graminearum and Trichoderma reesei.

Investigation into effects of transgenic glufosinate-resistant Zoysia grasses with herbicide application on bacterial communities under field conditions

Zoysia grass (Zoysia japonica Steud.) is a commercially valuable turfgrass that is popularly cultivated for use in sports and recreational environments. A field study was conducted in 2013 to evaluate the effects of transgenic herbicide-resistant Zoysia grasses in combination with the herbicide glufosinate on microbial communities. We investigated population changes in cultivable total bacteria and glufosinateresistant bacteria using the traditional plate-count method. The results showed that total and glufosinate-resistant bacteria counts decreased in September. Significant differences in the abundance of cultivable bacteria were observed between Zoysia grass lines as well as between March and September samples in both glufosinate-treated and nontreated plots. However, there was no significant difference in the abundance of total cultivable bacteria between Zoysia grass lines in March or between Basta-treated and untreated plots in September. To assess the possible horizontal gene transfer (HGT) of the bar gene across microorganisms, total soil DNAs and genomic DNAs of glufosinate-resistant bacteria isolated from soils and water were analyzed by PCR. No positive DNA bands were found, indicating that HGT did not occur during this experimental period. We also investigated changes in taxonomic distribution of the bacterial community using 16S rRNA gene clone libraries constructed from soil samples. Although bacterial diversities increased in September, there were no significant differences in species richness between genetically modified (GM) and non-GM Zoysia grasses as well as between glufosinatetreated and untreated soils.

Plant regeneration of Korean wild ginseng (Panax ginseng Meyer) mutant lines induced by γ-irradiation (60Co) of adventitious roots

An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.

Molecular Identification of Zoysia japonica and Zoysia sinica (Zoysia Species) Based on ITS Sequence Analyses and CAPS

Zoysiagrasses are important turf plants used for school playgrounds, parks, golf courses, and sports fields. The two most popular zoysiagrass species are Zoysia japonica and Zoysia sinica. These are widely distributed across different growing zones and are morphologically distinguishable from each other; however, it is phenotypically difficult to differentiate those that grow along the coastal line from those in beach area habitats. A combination of morphological and molecular approaches is desirable to efficiently identify these two plant cultivars. In this study, we used a rapid identification system based on DNA barcoding of the nrDNA-internal transcribed spacer (ITS) regions. The nrDNA-ITS regions of ITS1, 5.8S nrDNA, and ITS2 from Z. japonica, Z. sinica, Agrostis stolonifera, and Poa pratensis were DNA barcoded to classify these grasses according to their molecular identities. The nrDNA-ITS sequences of these species were found at 686 bp, 687 bp, 683 bp, and 681 bp, respectively. The size of ITS1 ranged from 248 to 249 bp, while ITS2 ranged from 270 to 274 bp. The 5.8S coding region ranged from 163 164bp. Between Z. japonica and Z. sinica , nineteen (2.8%) nucleotide sites were variable, and the G+C content of the ITS region ranged from 55.4 to 63.3%. Substitutions and insert/deletion (indel) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted to cleaved amplified polymorphic sequence (CAPS) markers, and applied to the Zoysia grasses sampled 본 연구는 산림청 국립산림품종관리센터 산림생명 자원개발사업, 농림축산식품부 농림수산식품기술 기획평가원 농림축산식품부사업(과제번호: 31502 5-3), 교육부 한국연구재단(과제번호: 2009-0094 059)과 기초연구사업(과제번호: 2016R1A6A1A0 3012862), 농촌진흥청 차세대 바이오그린21사업 (과제번호: PJ011244)의 지원을 받아 연구되었음. HORTICULTURAL SCIENCE and TECHNOLOGY 35(3):344-360, 2017 URL: http://www.kjhst.org

Rapid metabolic discrimination between Zoysia japonica and Zoysia sinica based on multivariate analysis of FT-IR spectroscopy

This study aims to establish a system for the rapid discrimination of Zoysia species using metabolite fingerprinting of FT-IR spectroscopy combined with multivariate analysis. Whole cell extracts from leaves of 19 identified Zoysia japonica, 6 identified Zoysia sinica, and 38 different unidentified Zoysia species were subjected to Fourier transform infrared spectroscopy (FT-IR). PCA (principle component analysis) and PLS-DA (partial least square discriminant analysis) from FT-IR spectral data successfully divided the 25 identified turf grasses into two groups, representing good agreement with species identification using molecular markers. PC (principal component) loading values show that the region of the FT-IR spectra are important for the discrimination of Zoysia species. A dendrogram based on hierarchical clustering analysis (HCA) from the PCA and PLS-DA data of turf grasses showed that turf grass samples were divided into Zoysia japonica and Zoysia sinica in a species-dependent manner. PCA and PLS-DA from FT-IR spectral data of Zoysia species identified and unidentified by molecular markers successfully divided the 49 turf grasses into Z. japonica and Z. sinica. In particular, PLS-DA and the HCA dendrogram could mostly discriminate the 47 Z. japonica grasses into two groups depending on their origins (mountainous areas and island area). Considering these results, we suggest that FT-IR fingerprinting combined with multivariate analysis could be applied to discriminate between Zoysia species as well as their geographical origins of various Zoysia species.

Molecular cloning and characterization of β-1,3-glucanase gene from Zoysia japonica steud

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Rhizoctonia leaf blight (large patch) has become a serious problem in Korean lawn grass, which is extremely hard to treat and develops mostly from the roots of lawn grass to wither it away. Rhizoctonia leaf blight (large patch) is caused by Rhizoctonia solani AG2-2 (IV). To develop zoysia japonica with strong disease tolerance against this pathogenic bacterium, β-1,3-glucanase was cloned from zoysia japonica, which is one of the PR-Proteins known to play a critical role in plant defense reaction. β-1,3-glucanase is known to be generated within the cells when plant tissues have a hypersensitive reaction due to virus or bacterium infection and secreted outside the cells to play mainly the function of resistance against pathogenic bacteria in the space between the cells. This study utilized the commonly preserved part in the sequence of corn, wheat, barley, and rice which had been researched for their disease tolerance among the β-1,3-glucanase monocotyledonous plants. Based on the part, degenerate PCR was performed to find out the sequence and full-length cDNA was cloned. E.coli overexpression was conducted in this study to mass purify target protein and implement in vitro activation measurement and antibacterial test. In addition, to interpret the functions of ZjGlu1 gene, each gene-incorporating plant transformation vectors were produced to make lawn grass transformant. Based on ZjGlu1 protein, antibacterial activity test was conducted on 9 strains. As a result, R. cerealis, F. culmorum, R.solani AG-1 (1B), and T. atroviride were found to have antibacterial activity. The gene-specific expression amount in each organ showed no huge difference in the organs based upon the transformant and against 18s gene expression amount.

An efficient gene targeting system using homologous recombination in plants

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.