• Proceedings of the Korean Society of Crop Science Conference
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.20-20
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• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• Proceedings of the Korean Society of Crop Science Conference
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• 한국작물학회 2017년도 9th Asian Crop Science Association conference
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• pp.97-97
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• 2017

Plant genomes include heterochromatic loci that consist of repetitive sequences and transposable elements. LTR retrotransposon is the major class of transposons in advanced plants in terms of proportion in plant genome. The elements contribute not only to genome size but also to genome stability and gene expression. A number of cases have been reported transposon insertions near genic regions affect crop traits such as fruit pigments, stress tolerance, and yields. Functional LTR retrotransposons produce extrachromosomal DNA from genomic RNA by reverse transcription that takes place within virus-like-particles (VLPs). DECREASED DNA METHYLATION 1 (DDM1) plays important roles in maintaining DNA methylation of heterochromatin affecting all sequence contexts, CG, CHG, and CHH. Previous studies showed that ddm1 mutant exhibits massive transcription of retrotransposons in Arabidopsis, but only few of them were able to create new insertions into the genome. RNA-dependent RNA POLYMERASE 6 (RDR6) is known to function in restricting accumulation of transposon RNA by processing the transcripts into 21-22 nt epigenetically activated small interfering RNA (easiRNA). We purified VLPs and sequence cDNA to identify functional LTR retrotransposons in Arabidopsis ddm1 and ddm1rdr6 plants. Over 20 LTR copia and gypsy families were detected in ddm1 and ddm1rdr6 sequencing libraries and most of them were not reported for mobility. In ddm1rdr6, short fragments of ATHILA gypsy elements were detected. It suggests easiRNAs might regulate reverse transcription steps. The highest enriched element among transposon loci was previously characterized EVADE element. It has been reported that active EVADE element is more efficiently silenced through female germline than male germline. By genetic analyses, we found ddm1 and rdr6 mutation affect maternal silencing of active EVADE elements. DDM1-GFP protein accumulated in megaspore mother cell but was not found in mature egg cell. The fusion protein was also found in early embryo and maternal DDM1-GFP allele was more dominantly expressed in the embryo. We observed localization of DDM1-GFP in Arabidopsis and DDM1-YFP in maize and found the proteins accumulated in dividing zone of root tips. Currently we are looking at cell cycle dependency of DDM1 expression using maize system. Among 10 AGO proteins in Arabidopsis, AGO9 is specifically expressed in egg cell and shoot meristematic cells. In addition, mutation of AGO9 and RDR6 caused failure in maternal silencing, implying 21-22 nt easiRNA pathway is important for retrotransposon silencing in female gametophyte or/and early embryo. On the other hand, canonical 24 nt sRNA-directed DNA methylation (RdDM) pathways did not contribute to maternal silencing as confirmed by this study. Heat-activated LTR retrotransposon, ONSEN, was not silenced by DDM1 but the silencing mechanisms require RdDM pathways in somatic cells. We will propose distinct mechanisms of LTR retrotransposons in germline and somatic stages.

• Proceedings of the Plant Resources Society of Korea Conference
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.22-22
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• 2018

실고사리[Lygodium japonicum (Thunb.) Sw.]는 국내 자생하는 덩굴성 양치식물로 유인재배가 용이하여 실내 외 조경 및 관상소재로 활용이 가능하다. 한방에서는 전초를 해금사초, 포자를 해금사, 뿌리와 지하경을 해금사근이라 약재로 사용한다. 본 연구는 관상 및 약재로 이용이 가능한 실고사리의 대량생산을 위한 기내 외 번식방법을 개발하고자 수행되었다. 식물재료는 경상북도 의성군 일대에서 성체를 수집하여 청주의 일반하우스에 식재하여 성숙한 포자를 채취하였다. 포자를 기내 발아시켜 전엽체를 획득한 다음 8주 간격으로 계대하면서 연구의 재료를 확보하였다. 전엽체의 증식과 생육에 적합한 배지를 비교 하고자, 1/4, 1/2, 1, 2MS배지와 Knop배지를 조성하여 배양하였다. 배양방법은 전엽체 300mg을 메스로 균일하게 다져서 배양하는 방법을 이용하였으며, 배양환경은 온도($25{\pm}1.0^{\circ}C$), 광도($30{\pm}1.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$), 광주기(16/8h light/dark)로 조절되었다. 연구결과, 모든 처리구의 생체중은 초기접종량 보다 11배 이상 증가하였고, 그 중 1MS배지에서는 전엽체의 생체중이 7.3g으로 가장 많이 증가하였다. 뿐만 아니라, 형태형성도 우수하여 모두 정상적인 전엽체의 형태인 하트형으로 발달이 유도되었다. 전엽체로부터 포자체 형성을 유도하고자, 원예상토, 피트모스, 펄라이트 및 마사토의 혼합비율을 5종류로 달리하여 사각분($7.5{\times}7.5{\times}7.5cm$)에 혼합토양을 충진하였다. 전엽체 1g과 증류수 25mL를 핸드블랜더로 10초간 분쇄하여 사각분의 토양표면에 균일하게 분주하는 방법을 사용하였다. 이후 온도($25{\pm}1.0^{\circ}C$), 광도($43{\pm}2.0{\mu}mol{\cdot}m^{-2}{\cdot}s^{-1}$), 광주기(16/8h light/dark), 습도($72{\pm}2.0%$)를 유지하면서 10주간 재배되었다. 연구결과, 원예상토 단용, 원예상토와 펄라이트 및 마사토가 2:1(v:v)로 혼합된 토양에서 각 498.0, 402.5, 482.5개의 포자체가 형성되어 사각분 면적대비 7.16개($cm^2$)가 생산되었다. 한편 포자체의 생육은 원예상토와 펄라이트가 2:1(v:v)로 혼합된 토양에서 생체중, 엽장, 엽폭, 근장 및 SPAD value 등이 우수하였다.

• FLOWER RESEARCH JOURNAL
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• 제18권3호
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• pp.179-185
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• 2010

This study was carried out to investigate the optimal culture conditions for gametophytes growth and sporophytes regeneration of Pyrrosia linearifolia in order to provide for the masspropagation system foundation of Pyrrosia linearifolia using their life cycle. Among many different media, 2MS medium was most effective in prothallus proliferation. Prothallus growth was promoted as the total concentration of nitrogen sources increased, and the best result was observed on 120 mM nitrogen. The best concentration of sucrose was 3%. The addition of 5~20 mM IAA, NAA, BA and kinetin promoted the propagation of prothallus. But 2iP demonstrated the most inhibitory effect on prothallus proliferation. Gametophytes shaking-cultured with liquid medium showed similar growth with solid medium and normal formation of reproductive organs. Shoot regeneration was most effective on 1/8MS medium, but growth was promoted on 1/2MS medium. For promotion of shoot regeneration and growth, the suitable concentrations of sucrose and $NaH_2PO_4$ were 1% and $50{\sim}100mg{\cdot}mL^{-1}$ in 1/8MS medium, respectively.

• Proceedings of the Plant Resources Society of Korea Conference
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• 한국자원식물학회 2019년도 춘계학술대회
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• pp.43-43
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• 2019

Deparia pycnosora (Christ) M. Kato is a fern used as ornamental plant. In addition, it is called "Teol-go-sa-ri" in Korean name. The aim of this study was to develop a practical propagation method of D. pycnosora using tissue culture technique. Prothallus obtained from spore germination was the used as experiment materials. The prothalli (300 mg) used in all experiments were sub-cultured for 8-week intervals. The most suitable media for prothallus propagation were identified by culturing 300 mg of prothalli in $1/4{\times}$, $1/2{\times}$, $1{\times}$, $2{\times}$ MS medium and in Knop medium for 8 weeks. Also, the prothalli were cultured by chopping with a scalpel. In addition, sucrose, activated charcoal, and total nitrogen source were added in different concentrations based on the culture medium selected. Cultures were maintained at a temperature of $25{\pm}1^{\circ}C$, light intensity of $30{\times}1.0{\mu}mol-m-2{\cdot}s-1$, and a photoperiod of 16/8 h (light/dark) in in vitro. The results showed that optimum was achieved prothallus fresh weight and development in $1{\times}$ MS medium. When other components were added to the basic $1{\times}$ MS medium, prothallus propagation was maximized in $1{\times}$ MS medium supplemented with 2% sucrose, 0.2% activated charcoal, and 60 mM total nitrogen. To select a suitable soil mixture for sporophyte formation, 1.0 g of prothallus was blended with distilled water, spread on five combinations of different soil substrates (decomposed granite, horticultural substrates, peat moss, and perlite), and cultivated for 12 weeks. The sporophyte cultures were maintained at a temperature of $25{\pm}1^{\circ}C$, light intensity of $43{\pm}2.0{\mu}mol-m-2{\cdot}s-1$, humidity of $84{\pm}1.4%$, and a photoperiod of 16/8 h (light/dark). As a results, horticultural substrate alone, 2:1 (v:v) mixtures of horticultural substrate and perlite, and 2:1 mixtures of horticultural substrate and decomposed granite induced 208.0, 201.3 and 248.8 sporophytes per pot, respectively. Therefore, this result could provide a practical mass propagation method of D. pycnosora

• Korean Journal of Plant Resources
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• 제32권4호
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• pp.290-295
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• 2019

This study was carried out to select the optimal condition for prothallus propagation and sporophyte formation of Leptogramma pozoi (Lag.) Ching subsp. mollissima (Fisch. ex Kunze) Nakaike and to provide basic data for mass production system. In the propagation, 300 mg of prothallus were inoculated in different kinds of medium [1/4, 1/2, Murashige and Skoog (MS), Knop], sucrose (0, 1, 2, 3, 4%), and cultured for 8 weeks. Sporophyte formation studies were carried out by inoculating blended prothallus into artificial soils. The soils were mixed with horticultural substrate, peatmoss, perlite, and decomposed granite at different ratios or only with the horticultural substrate. Then the prothallus was cultivated for 12 weeks to figure out the formation and growth of the sporophytes. The growth and development of prothalli were excellent on MS medium. According to medium components, prothallus growth was favorable in all treatments except 0% sucrose treatment and the highest in 2% sucrose. In the experiment of soil mixtures, sporophyte formation was the highest in the horticultural substrate:perlite 2:1 (v:v) and horticultural substrate:decomposed granite 2:1 (v:v) treatment, and the overall growth was good in the horticultural substrate:decomposed granite 2:1 (v:v) mixed soil.