Screening of new resistance gene to rice stripe virus (RSV) in Rice (Oryza sativa L.)

Tackmin Kwon,Jong-Hee Lee,Sais-Beul Lee,Soo-Kwon Park,Un-Ha Hwang,Dong-Jin Shin,Jun-Hyun Cho,You-Chun Song,Min-Hee Nam,Dong-Soo Park 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Rice stripe disease, caused by rice stripe virus (RSV), is one of the major virus diseases in east Asia. The objective of this study was conducted to identify new resistance genetic source to rice stripe virus (RSV) disease. Genetic diversity of 155 rice cultivars was evaluated using 9 co-dominant InDel markers and STS marker ST10. These cultivars were classified into two groups by cluster analysis based on Nei`s genetic distances. The marker showed different band pattern among RSV resistance or susceptible cultivar. In comparison with bioassay for RSV resistance and genotyping using SSR markers showed that Stv-bi and InDel 7 marker observed recombination value within 3.8% and RSV resistance gene was closely related to InDel 7. Also InDel 7 divided as resistance type alleles and susceptible type alleles except for some varieties. Interestingly, 02428, Daw dam, Erguailai, Padi Adongdumarat, PERVOMAJSZKIJ, and Tung Ting Wan Hien 1 showed Japonica type in InDel 7 marker. However, these cultivars revealed resistant to RSV bioassay. These results indicate that those cultivar can be able to get the different gene resistance with Stv-bi gene. Newly identified resistance gene is considered useful for improving RSV resistance in japonica rice. Therefore, we will progress the allelism test and genetic analysis for identification of new gene source.

Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

Tackmin Kwon 한국분자세포생물학회 2016 Molecules and cells Vol.39 No.9

The efficiency of Agrobacterium-mediated transfor-mation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

Mitochondrial Porin Isoform AtVDAC1 Regulates the Competence of Arabidopsis thaliana to Agrobacterium-Mediated Genetic Transformation

Kwon, Tackmin Korean Society for Molecular and Cellular Biology 2016 Molecules and cells Vol.39 No.9

The efficiency of Agrobacterium-mediated transformation in plants depends on the virulence of Agrobacterium strains, the plant tissue culture conditions, and the susceptibility of host plants. Understanding the molecular interactions between Agrobacterium and host plant cells is crucial when manipulating the susceptibility of recalcitrant crop plants and protecting orchard trees from crown gall disease. It was discovered that Arabidopsis voltage-dependent anion channel 1 (atvdac1) mutant has drastic effects on Agrobacterium-mediated tumorigenesis and growth developmental phenotypes, and that these effects are dependent on a Ws-0 genetic background. Genetic complementation of Arabidopsis vdac1 mutants and yeast porin1-deficient strain with members of the AtVDAC gene family revealed that AtVDAC1 is required for Agrobacterium-mediated transformation, and there is weak functional redundancy between AtVDAC1 and AtVDAC3, which is independent of porin activity. Furthermore, atvdac1 mutants were deficient in transient and stable transformation by Agrobacterium, suggesting that AtVDAC1 is involved in the early stages of Agrobacterium infection prior to transferred-DNA (T-DNA) integration. Transgenic plants overexpressing AtVDAC1 not only complemented the phenotypes of the atvdac1 mutant, but also showed high efficiency of transient T-DNA gene expression; however, the efficiency of stable transformation was not affected. Moreover, the effect of phytohormone treatment on competence to Agrobacterium was compromised in atvdac1 mutants. These data indicate that AtVDAC1 regulates the competence of Arabidopsis to Agrobacterium infection.

Fine mapping and identification of candidate rice genes associated with qSTV11 SG , a major QTL for rice stripe disease resistance

Kwon, Tackmin,Lee, Jong-Hee,Park, Soo-Kwon,Hwang, Un-Ha,Cho, Jun-Hyun,Kwak, Do-Yeon,Youn, Yeong-Nam,Yeo, Un-Sang,Song, You-Chun,Nam, Jaesung,Kang, Hang-Won,Nam, Min-Hee,Park, Dong-Soo Springer-Verlag 2012 TAG. Theoretical and applied genetics. Theoretisch Vol.125 No.5

Genetic transformation of a high-molecular-weight glutenin Dx5 to Donjin rice cultivar

Soo-Kwon Park,Tackmin Kwon,Woon-Ha Hwang,Donjin Shin,Se Yun Oh,Seong-Hwan Oh,You-Chun Song,Min-Hee Nam,Dong-Soo Park 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

In order to improve rice dough functionality, we cloned 4 kinds of high-molecular-weight glutenin subunit (HMW-GS) genes from bread wheat, ‘Jokyeong’. Among them, we first examined Dx5 gene to generate marker-free transgenic rice for advanced quality processing of bread and noodles. The GluB1 promoter was inserted into binary vector for seed specific expression of the Dx5 gene. Two expression cassettes comprised of separate DNA fragments containing only the high-molecular-weight glutein subunit (HMW-GS) protein (Dx5) and hygromycin phosphotransferase II (HPTII) resistance genes were introduced separately to tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected to rice calli at 3: 1 ratio of Dx5 and HPTII, respectively. Then among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted both with Dx5 and HPTII gene to rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcriptsin rice seeds was examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only Dx5 gene were successfully screened at T1 generation. This result also provides that co-infection system with two expression cassettes could be efficient strategy to generate marker-free transgenic rice plants.

A large-scale screening analysis for the evaluation of Bakanae disease in rice

Myung-Hee Kim,Saet-Byeol Lee,Tackmin Kwon,Un-Ha Hwang,Soo-Kwon Park,Yeong-Nam Youn,Jong-Hee Lee,Jun-Hyun Cho,Dongjin Shin,Sang-Ik Han,Un-Sang Yeo,You-Chun Song,Min-Hee Nam,Dong-Soo Park 한국육종학회 2013 한국육종학회 심포지엄 Vol.2013 No.07

Bakanae disease of rice, caused by Fusarium moniliforme Sheldon, the imperfect stage of Gibberella fujikuroi, is one of the most important rice diseases worldwide, but no rice variety has been found to be completely resistant to this fungus. Cultivation of resistant cultivars is the most beneficial way of reducing quantitative or qualitative losses to for bakanae disease in rice. To facilitate the study of this disease, accurate and large scale screening methods were developed for the inoculation and evaluation of Bakanae disease. Even and large scale infection was achieved by using F. moniliforme spore in tissue embedding cassette and seedling tray. The efficiency of F. moniliforme infection with the concentration of 1×106 spore/ml caused better distribution (F-value=33.96) than 1×102 (F-value=10.69), and 1×104 spore/ml (F-value=2.63). We established new criteria of healthy and non-healthy plant, and introduced calculation of proportion of healthy plants to meet fast evaluation of resistance level of each variety. The effect of F. moniliforme strains containing different genetic background was also evaluated with rice varieties to figure out the stability of resistance level. GA3 response of rice variety was significantly correlated with bakanae disease, but it did not adequate for direct indicator of bakanae disease resistance. These results indicated that a large scale infection method developed in this study is fast and reproducible, as well as a disease evaluation system provides an accurate measurement of bakanae disease resistance of rice.

Phosphate uptake and growth characteristics of transgenic rice under high phosphate soils

Woon-Ha Hwang,Soo-Kwon Park,Tackmin Kwon,Sais-Beul Lee,Min-Hee Nam,Doh-Hoon Kim,Dong-Soo Park 한국육종학회 2012 한국육종학회 심포지엄 Vol.2012 No.07

Farmers have use phosphate fertilizer to provide sufficient yields. However, overuse of phosphorus accumulate in soil and causes soil and water pollution. We evaluated the phosphate acquisition and growth characteristics of OsPT1 transgenic rice (OsPT1-OX, over-expressing the high affinity phosphate transporter 1) in high phosphate soils with different level of nitrogen fertilizer treatment to investigate removing ability of excessive phosphate from soil. OsPT1-OX had shorter culm length but more tillers than those of wild-type plants in each soil conditions. Phosphate content per dry weight of OsPT1-OX was 1.8 times higher than that of wild-type under control fertilizer treated conditions. Although the dry weight of OsPT1-OX was not different from that of wild-type plants, whole plant phosphate content was 1.7 times higher than that of wild-type plants under control fertilizer conditions. Tiller number and phosphate content per dry weight of wild-type plants increased following high levels of phosphate application but did not change by following additional nitrogen application. Tiller number and phosphate content per dry weight of OsPT1-OX did not change under the high phosphate condition, but increased following nitrogen application under similar conditions. Whole plant phosphate content was highest under high nitrogen and high phosphate application conditions. These results suggest that OsPT1-OX may reduce phosphate content in soils containing excess phosphate and may be further effective under high nitrogen condition.

Cytokinin Response Factor 2 positively regulates salicylic acid-mediated plant immunity in <i>Arabidopsis thaliana</i>

Japanese Society for Plant Cell and Molecular Biol 2016 Plant biotechnology Vol.33 No.3

Generation of Transgenic Rice without Antibiotic Selection Marker through Agrobacterium-mediated Co-transformation System

Soo-Kwon Park(박수권),Tackmin Kwon(권택민),Jong-Hee Lee(이종희),DongJin Shin(신동진),Woon-Ha Hwang(황운하),You-Chun Song(송유천),Jun-Hyun Cho(조준현),Min-Hee Nam(남민희),Seung-Ho Jeon(전승호),Sang-Yeol Lee(이상열),Dong-Soo Park(박동 한국생명과학회 2012 생명과학회지 Vol.22 No.9

작물의 수확량이나 병 저항성을 증가시키는 형질전환 식물체 개발은 세계 식량 부족을 해결하는 좋은 방법이다. 하지만 항생제나 제초제의 사용은 형질전환 작물의 안전에 대해서 일반 사람들의 심각한 우려를 초래한다. 본 연구에서는, 아그로박테리움을 이용한 동시 형질전환 방법을 이용하여 한국의 밀 재배종인 ‘조경밀’의 유전자인, 고분자 글루테닌 서브유닛[high molecular-weight glutenin subunit (HMW-GS)] Dx5가 삽입된 마커프리 형질전환벼를 개발하였다. 각각 Dx5 유전자와 하이그로마이신(HPTII) 저항성 유전자만으로 구성된 두 종류의 발현 카셋트(Two expression cassettes)를 독립적으로 아그로박테리움 EHA105에 도입하였고, Dx5와 HPTII가 도입된 각각의 EHA105 아그로박테리움을 3:1 비율로 혼합하여 벼 캘러스에 접종하였다. 66개의 HPTII 저항성 형질전환체 중에서 벼 게놈에 Dx5와 HPTII가 모두 삽입된 2개의 형질전환 라인을 획득하였다. Dx5와 HPTII가 벼 게놈에 도입된 것을 Southern blot을 통해서 다시 확인하였다. 또한, semi-quantitative RT-PCR을 통해 형질전환벼 T1 세대 종자의 밀 Dx5 전사여부를 확인하였고 결국, Dx5 유전자만을 가지는 마커프리 형질전환벼를 T1 세대에서 선발할 수 있었다. 본 연구 결과는 두 종류의 발현 카셋트를 사용한 아그로박테리움 동시 접종 시스템이 마커프리 형질전환벼를 생산하기 위한 효과적인 전략이 될 수 있음을 보여준다. Development of transgenic plant increasing crop yield or disease resistance is good way to solve the world food shortage. However, the persistence of marker genes in crops leads to serious public concerns about the safety of transgenic crops. In the present paper, we developed marker-free transgenic rice inserted high molecular-weight glutenin subunit (HMW-GS) gene (Dx5) from the Korean wheat cultivar ‘Jokyeong’ using Agrobacterium-mediated co-transformation method. Two expression cassettes comprised of separate DNA fragments containing only the Dx5 and hygromycin resistance (HPTII) genes were introduced separately into Agrobacterium tumefaciens EHA105 strain for co-infection. Each EHA105 strain harboring Dx5 or HPTII was infected into rice calli at a 3: 1 ratio of EHA105 with Dx5 gene and EHA105 with HPTII gene expressing cassette. Then, among 66 hygromycin-resistant transformants, we obtained two transgenic lines inserted with both the Dx5 and HPTII genes into the rice genome. We reconfirmed integration of the Dx5 and HPTII genes into the rice genome by Southern blot analysis. Wheat Dx5 transcripts in T1 rice seeds were examined with semi-quantitative RT-PCR. Finally, the marker-free plants containing only the Dx5 gene were successfully screened at the T1 generation. These results show that a co-infection system with two expression cassettes could be an efficient strategy to generate marker-free transgenic rice plants.

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean

Kim, Hye Jeong,Cho, Hyun Suk,Pak, Jung Hun,Kwon, Tackmin,Lee, Jai-Heon,Kim, Doh-Hoon,Lee, Dong Hee,Kim, Chang-Gi,Chung, Young-Soo Korean Society for Molecular and Cellular Biology 2018 Molecules and cells Vol.41 No.5

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from $T_2$ seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than ${\underline{n}}on-{\underline{t}}ransgenic$ (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants ($T_2$) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.