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Zoysia japonica Steud. is a warm-season lawn grass popular in Korea and elsewhere. They are cultivated in many places such as river banks, roadside, and play grounds. However, there still is a disadvantage of frequent mowing, and the grass grows poorly under the chilly conditions. To develop a grass variety that circumvents these drawbacks, we cloned the chitin-inducible gibberellins-responsive1 gene (CIGR1) from zoysiagrass. The full length of the ZjCIGR1 (Zj; Zoysia japonica Steud.) gene was obtained by 5′/3′ RACE PCR and the phylogenetic tree showed that it belonged to the CIGR1-subgroup in the PAT1-group of GRAS protein family. Expression of the ZjCIGR1 in wild-type plants was confirmed in roots, meristems, leaves, and flowers, especially high in the flowers. The transgenic zoysiagrass was confirmed by PCR using gene-specific primers, phosphinothricin-acetyl-transferase (PAT) strip test, and Southern blot analysis. ZjCIGR1-overexpressing plants acquired tolerance to cold stress displaying morphological phenotypes characteristic of stress resistance. In addition, in the transformants, expression of the ZjCIGR1 as well as cold-regulated (COR) gene was increased compared to the wild-type plants under cold stress condition. These results suggest that ZjCIGR1 gene is an important candidate for regulating cold stress resistance.

Numerous genes are known to regulate the cold response in plants. The ICE1 gene is a regulator of coldinduced transcriptome playing an important role in cold tolerance in Arabidopsis thaliana. Here, the Arabidopsis ICE1 gene was cloned and transformed into A. thaliana and Korean lawn grass (Zoysia japonica) respectively, under the control of a maize ubiquitin promoter. The transgenic Arabidopsis plants showed higher survival rate, less MDA content, and higher levels of proline, SOD, and POD after freezing treatment. In the case of Z. japonica, 43 transgenic plants were produced by Agrobacterium-mediated transformation. Southern blot analysis showed that 9 transgenic lines selected by phosphinothricin (PPT) were transformed with ICE1 independently. Cold treatment significantly induced the transcripts abundant in the up-regulated ZjCBF and ZjDREB1 in transgenic lines OE-18 and OE-19. Meanwhile, transgenic plants showed increased proline level but decreased MDA content and with higher activities of SOD and POD compared with wild type under cold stress. These results suggest that overexpression of the ICE1 gene could improve the antioxidative capacity in conferring cold tolerance to the transgenic zoysiagrass plants.

Zoysiagrass (Zoysia japonica Steud.) is an important turfgrass species used in golf courses and athletic fields. However, zoysiagrass is susceptible to large patch disease caused by Rhizoctonia solani AG2-2 (IV). Chitinases are pathogen-related (PR) proteins induced by viruses, bacteria, and fungi that hydrolyze chitin. Recently, we isolated a class II chitinase gene (Zjchi2) from zoysiagrass. The purified recombinant Zjchi2 showed broad-spectrum activity against various fungi, including R. solani AG2-2 (IV). In the current study, we generated transgenic zoysiagrass overexpressing Zjchi2 and then verified the resistance of transgenic plants to R. solani AG2-2 (IV). Polymerase chain reaction and Southern blot hybridization showed the integration of transgenes in zoysiagrass genomes and constitutive expression of Zjchi2, respectively. Antifungal activity was enhanced significantly in the transgenic zoysiagrass compared with wild-type plants. To our knowledge, this report is the first on the antifungal activity of a class II chitinase in transgenic zoysiagrass.

한국형 잔디에서는 다른 병에 비해 진전 속도가 빠르고 주로 뿌리에서부터 발병하여 잔디를 고사시키고 발병 후 구제하기 매우 어려운 라이족토니아잎마름병(라지패취)이 큰 문제로 대두되고 있다. 라이족토니아잎마름병(라지패취)은 Rhizoctonia solani AG2-2 (IV)병원균에 의해 발생하는데, 이 병원균에 강한 내병성 들잔...

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.

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.

본 연구는 종자로부터 유도된 캘러스를 아그로박테리움을 이용해 감염하여 고체배지에서 배양하는 기존의 방법과 달리, 형질전환에 소모되는 노동력과 시간, 비용을 단축하고자 감염과 공동배양, 균제거와 캘러스 선발까지 액체배양을 시도하였다. 성숙 종자로부터 유도된 캘러스를 바로 아그로박테리움으로 감염함으로써 조직배양으로 인한 체세포 변이의 발생을 최소화하고 감염부터 그 후 캘러스선발까지 총 4단계의 시간과 비용을 최소화하였으며, 감염된 캘러스로부터 재분화 시킴으로써 형질전환 식물체를 얻는 방법을 새롭게 수립하였다. 배양과정 중 감염과 공동배양 기간을 3일로 단축시킴으로써 캘러스의 스트레스를 최소화 하였고, PCR 분석을 통해 원하는 목표 유전자가 형질전환체에 안정적으로 도입이 되는 것도 확인할 수 있었다. 이러한 결과를 종합해 볼 때, 본 실험을 통해 얻어진 새로운 액체배양 방법은 우수한 농업적 형질을 가진 벼 품종 개발시 효율적으로 이용할 수 있을 것으로 생각된다. 【Rice is one of the most important cereal crops as a model plant for functional genomics of monocotyledons and usually transformed using Agrobacterium tumefaciens. However, the transformation's process using previous method is still time consuming and uneconomical, low efficiency. In this study, we established a new method by modifying the general Agrobacterium protocol especially in the infection and co-cultivation, Agrobacterium elimination, infected calli's selection steps using liquid media. We directly inoculated Agrobacterium containing a ZjLsL gene under the control of constitutive promoter into the 1- to 3-week-old rice calli derived from mature seeds. After 3 days of co-cultivation, the infected calli were transferred onto liquid media of Agrobacterium elimination and calli's selection for 3 days. The calli were transferred to calli's growth solid media for 14 days and then the calli transferred to shoot induction and root induction media. Putative transformants were initially selected on the medium containing phosphinothricin, and the PAT protein verified by PAT strip test. This method in this study would lead to reduction of substantial labor and time to generate transgenic plants.】

Zoysia grass and creeping bentgrass are important turf grasses used in parks, gardens and playing fields. Development of grasses with increased tiller formation will enhance their commercial cultivation. To investigate the regulatory mechanism of tiller formation, we cloned the Zoysia japonica Lateral suppressor-like (ZjLsL) gene. The Lateral suppressor (Ls) gene encodes a transcriptional regulator belonging to the plant-specific GRAS protein family of putative transcription factors, and regulates axillary meristem initiation. A full-length DNA of the ZjLsL gene was isolated by 5'/3' DNA walking. Phylogenetic analysis showed that ZjLsL is closely related to Ls genes. Southern blot analysis revealed that zoysia grass has two copies of the ZjLsL gene. ZjLsL expression was detected in all organs of zoysia grass but was most highly expressed in culms. Overexpression of ZjLsL in creeping bentgrass and Arabidopsis plants promoted axillary bud formation. These results suggest that ZjLsL plays an important role in axillary meristem initiation and tiller formation.

With the purpose of improving ginsenoside content in adventitious root cultures of Korean wild ginseng (Panax ginseng Meyer), the roots were treated with different dosages of γ-ray (5, 10, 25, 50, 75, 100, and 200 Gy). The growth of adventitious roots was inhibited at over 100 Gy. The irradiated adventitious roots showed significant variation in the morphological parameters and crude saponin content at 50 to100 Gy. Therefore, four mutant cell lines out of the propagation of 35 cell lines treated with 50 Gy and 100 Gy were selected on the basis of phenotypic morphology and crude saponin contents relative to the wild type control. The contents of 7 major ginsenosides (Rg 1 , Re, Rb 1 , Rb 2 , Rc, Rf, and Rd) were determined for cell lines 1 and 3 from 100 Gy and lines 2 and 4 from 50 Gy treatments. Cell line 2 showed more secondary roots, longer length and superior growth rate than the root controls in flasks and bioreactors. Cell line 1 showed larger average diameter and the growth rate in the bioreactor was comparable with that of the control but greater in the flask cultured roots. Cell lines 1 and 2, especially the former, showed much more ginsenoside contents than the control in flasks and bioreactors. Therefore, we chose cell line 1 for further study of ginsenoside contents. The crude saponin content of line 1 in flask and bioreactor cultures increased by 1.4 and 1.8-fold, respectively, compared to the control. Total contents of 7 ginsenoside types (Rg 1 , Re, Rb 1 , Rb 2 , Rc, Rf, and Rd) increased by 1.8 and 2.3-fold, respectively compared to the control. Crude saponin and ginsenoside contents in the bioreactor culture increased by about 1.4-fold compared to that the flask culture.

Ferulate-5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde, and coniferyl alcohol in the pathways leading to sinapic acid and the syringyl unit of lignin. We have isolated twoF5H genes,OsF5HL andOsF5HL2, from a japonica-type rice (Oryza sativa L. cv. llpoombyeo). They are the firstF5H genes experimentally identified in monocotyledonous plants. Phylogenetic analysis indicated that both genes are closely related to dicotF5H genes fromArabidopsis thaliana andBrassica napus. Southern blot analysis showed that these genes exist as single copies in the rice genome. Alignments of theOsF5HL andOsF5HL2 cDNAs to their genomic DNAs revealed thatOsF5HL has an open reading frame (ORF) of 1590 b from four exons, whileOsF5HL2 has an ORF of 1560 b from two exons. Expression ofOsFSHL is highest in young leaves, whereas that ofOsF5HL2 is greatest in mature leaves. In the roots and stems, transcription levels for both genes are markedly low. These data suggest that theOsF5HL andOsF5HL2 genes belong to theCYP84 subfamily and that their expressions are tissue-specific.

Conventionally, the lateral shoots of chrysanthemum are removed manually, which is time consuming and uneconomical. The development of branchless chrysanthemum will economize its commercial cultivation. To investigate the regulatory mechanism of branchlessness, we undertook cloning of Dendranthema grandiflorum Kitamura Lateral suppressor-like (DgLsL) gene for development of lateral shoot in chrysanthemum. A full-length cDNA of DgLsL gene was isolated by screening cDNA library and performing Rapid Amplification of cDNA Ends (RACE) PCR. Phylogenetic analysis showed that the DgLsL gene is closely related to Lateral suppressor that encodes transcriptional regulator proteins belonging to GRAS family of known transcription factor. Southern blot analysis revealed that DgLsL gene in chrysanthemum genome has one copy. DgLsL expression was apparently up-regulated by ethephon treatment. The expression patterns revealed that DgLsL transcripts were detected in all organs, but showed their highest level in stems.

Plant cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of phenylpropanoids, terpenes, lipids, and alkaloids. We isolated a cDNA clone for cinnamate-4-hydroxylase (C4H) from a Japonica type rice(Oryza sativa L. cv. llpumbyeo). This C4H has a deduced amino acid sequence that is 85% identical tothat ofSorghum bicolor. Our phylogenetic analysis also showed that theOsC4HL gene is closely related toC4H fromS. bicolor. A putative genomic DNA sequence corresponding toOsC4HL contained cis-elements (boxes P, A, L, and TCA motifs), AT-rich elements, and wound-response elements that control gene expression in its promoter region.OsC4HL expression was detected in all the tissue types, with the highest level being measured in the roots. It was also apparently up-regulated by wounding stress. These data suggest that theOsC4HL gene isC4H member in theCYP73 subfamily.

We examined ultrastructural changes of the cell organelles ofArabidopsis stems in response to gamma irradiation. Seedlings treated with 0 to 5 Gy developed normally, while height growth in plants exposed to 50 Gy was significantly inhibited. Based on TEM observations, the chloroplasts were extremely sensitive to such irradiation. In particular, the thylakoids were heavily swollen, some portions of the mitochondria and endoplasmic reticulum were structurally altered, and the plasmalemma had pulled away from the cell wall in places. However, no ultrastructural changes in cell organelles occurred at doses of 0 to 5 Gy.