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Abstract : To enhance phytoremediation, which removes heavy metal from soil, transgenic plants were applied to contaminated soil. We constructed a transformation vector expressing both TgMTP₁ (T. goesingense metal tolerance protein):HA and TgMTP:GFP genes. Transgenic plants were generated using an Agrobacterium-mediated transformation system that expressed the two vectors. Screening and analysis confirmed the incorporation of foreign genes into the Arabidopsis thaliana genome. Callus was induced in the 116 T3 line. These transgenic plants and calli were used for further analyses on the accumulation of Ni. The 116 T3-line plants and calli from selected lines were resistant to heavy metals and accumulated Ni in their leaves. The expression level of TgMTP RNA was equal in all leaves, but protein stability increased in the leaves with Ni treatment. According to these results, we suggest that TgMTP₁-overexpressing plants may be useful for phytoremediation of soil.

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Abstract : Horticultural traits and genetic relationship were evaluated for 83 melon (Cucumis melo L.) cultivars. Survey of a total of 36 characteristics for seedling, leaf, stem, flower, fruit, and seed and subsequent multiple analysis of variance (MANOVA) were conducted. Principal component analysis (PCA) showed that 8 principle components including fruit weight, fruit length, fruit diameter, cotyledon length, seed diameter, and seed length accounted for 76.3% of the total variance. Cluster analysis of the 83 melon cultivars using average linkage method resulted in 5 clusters at coefficient of 0.7. Cluster I consisted of cultivars with high values for fruit-related traits, Cluster II for soluble solid content, and Cluster V for high ripening rate. Genotyping of the 83 cultivars was conducted using 15 expressed-sequence tagged-simple sequence repeat (EST-SSR) from the Cucurbit Genomics Initiative (ICuGI) database. Analysis of genetic relatedness by UPGMA resulted in 6 clusters. Mantel test indicated that correlation between morphological and genetic distance was very low (r = -0.11).

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Abstract : To investigate the optimal conditions for shoot organogenesis in Astragalus sinicus L., hypocotyl explants were cultured in Murashige & Skoog’s (MS) medium supplemented with 0.1, 1.0, 2.0, or 4.0 mg/L 2,4-dichlorophenoxy acetic acid (2,4-D) for 6 weeks. 2,4-D concentration significantly effected morphogenesis: some produced calli with adventitious shoots and roots, some produced calli with adventitious roots, some produced only calli, and some produced deep-brownish calli with roots. The formation of calli with shoots and/or roots was observed at lower levels of 2,4-D, whereas calli without shoots or with deep-brownish roots were formed after treatment with higher levels of 2,4-D. Also, a shoot organogenesis ability of callus clones was observed after treatment with medium with 0.1 or 1.0 mg/L 2,4-D grown in MS medium with combinations of benzyl adenine (BA) and 2,4-D for 4 weeks. Medium with a combination of BA and 2,4-D was effective for shoot formation, whereas root organogenesis from calli decreased. The greatest amount of shoot formation was obtained when calli were cultured in MS medium containing 1.0 mg/L 2,4-D and 0.5 mg/L BA. Upon shoot transfer into ½ MS basal medium, plantlets developed, and the plantlets grew well in soil in a greenhouse.

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Abstract : Somatic embryogenesis is as an excellent technology for potential use in plant mass production, germplasm conservation, or genetic engineering. We examined the effect of cold storage using 3 embryogenic callus lines with different levels of embryogenesis competence derived from immature zygotic embryo cultures of Kalopanax setemlobus. Somatic embryo induction, germination and plant conversion were evaluated after 1, 3 and 6 months storage at 4°C in the dark. Most cold-stored embryogenic calli formed somatic embryos normally even after 6 months; however, the induction rate was gradually decreased by increasing the storage period. The most competent line tended to show a slight decline in somatic embryo induction rate, as compared with other lines after cold storage. In general, cold storage resulted in reduced somatic embryo germination and plant regeneration, although 93% somatic embryo germination and 91% plant conversion were achieved regardless of the storage period. Cold storage led to cell browning and degradation. Additionally, the cell structures were confirmed by the aceto-carmine and evans blue dye evaluation. Collectively, our results showed that embryogenic callus of K. septemlobus could be preserved at 4°C without subculture for 6 months, and suggested the need for storage of relatively more competent embryogenic calli lines to support somatic embryo induction.

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Abstract : Aronia (Aronia melanocarpa, Black chokeberry) is an important cash crop in domestic agriculture. We investigated the effects of plant growth regulators on shoot proliferation and rooting using in vitro tissue culture. The most effective shoot multiplication was observed on WPM (woody plant medium) supplemented with 1.0 mg/L zeatin (8.3±1.0 shoots/explant), while the highest rooting rate was obtained from half-strength WPM with 3.0 mg/L IBA (8.8 roots/explant). The rooted plantlets all survived in the artificial soil mixture (with a mixture of peat moss : perlite : vermiculite, 1:1:1, v/v/v) and grew up relatively uniform, ranging from 14 to 16 leaves, 8 to 10 cm in stem height, and 2.3 to 2.8 mm in stem diameter. While experimenting with 5 different varieties of Aronia, we found out that each variety had different characteristics of shoot proliferation and rooting. The total numbers of proliferated shoots per variety is as follows: 17.4±0.8 for Nero, 14 to 15 for Purple and Mackenzie, and 10 for both Viking and Odamamachiko. Rooting rates were also various depending on the variety: 88% of Odamamachiko, 80% of Viking and Purple, and 76% of Nero and 60% of Mackenzie shoots rooted. The survival rate of the rooted plantlets was from 92% to 100%, varying by type. Further growth appeared to be better in auxin-treated plantlets, compared to untreated ones. Our results showed the possibility of establishing an effective in vitro micropropagation system for Aronia melanocarpa.

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Abstract : This study was conducted to evaluate the effects of different types and concentrations of organic nitrogen sources (L-Glutamine and casein hydrolysate, CH) and plant growth regulators (auxins and cytokinins) on embryogenic tissue proliferation and somatic embryo production in L. kaempferi. Overall, the highest tissue fresh weight was obtained at either 2 or 4 weeks in culture when 1,000 mg/L L-Glutamine was added to the culture medium, which showed similar results with other treatments. In experiments with different types and concentrations of plant growth regulators on somatic embryo production, the highest production (426.3/90 mg tissue) was found when 0.2 mg/L IBA was added; however, no somatic embryos were induced following treatment with 0.2 mg/L BA or Kinetin. The effect of various concentrations of IBA on somatic embryo production was also tested. The best result (303/90 mg tissue) was obtained when plants were treated with 0.2 mg/L IBA; 1.0 mg/L IBA was also effective (281/90 mg tissue). The lowest result (109.3/90 mg tissue) was obtained with 5.0 mg/L IBA.

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Abstract : This experiment compared the regeneration conditions of the radiation mutant spray chrysanthemum ‘purple ND’. The four different flower blooming stages (S1: 10% opened flower, S2: 30% opened flower, S3: 50% opened flower, and S4: 70% opened flower) and different petal parts (TBOP: the basal of petal and TEOP: the end of petal) were used to compare regeneration conditions between plants grown in MS medium supplemented with IAA and BAP. The highest adventitious shooting rate was identified in plants grown on the IAA 1.0mg·L^-1 and BAP 2.0mg·L^-1 when using the end of petal at the S2 stage. It displayed 79.2% regeneration and produced 33.4 shoots. Rooted plantlets were successfully established in the greenhouse, showing the same morphological characteristics of vegetative and reproductive organs with those of the mother plant. Flow cytometry analysis revealed no ploidy variation between the regenerated plants and the mother plant grown under greenhouse conditions.

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Abstract : Safflower (Carthamus tinctorius) seeds are well-known traditional oriental medicines that have long been used for the remedies of blood stasis and bone formation in east Asia. In this study, ethyl acetate (EtOAc) was used for extraction of the main chemical compounds from C. tinctorius seeds. Four major compounds were identified, acacetin, cosmosiin, N-feruloyl serotonin and N-(p-coumaroyl) serotonin. Each compound was evaluated for its inhibitory activity against the inflammatory process of macrophages. All compounds significantly inhibited production of lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and pro-inflammatory cytokines. The protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were dramatically decreased by serotonins in a dose-dependent manner in LPS-stimulated RAW 264.7 macrophages. These results suggest that serotonin derivatives from safflower seeds may reduce inflammation-related diseases.

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Abstract : In order to study genetic engineering in trees, the characterization of genes and promoters from trees is necessary. We isolated the promoter region (867 bp) of Pagns-LTP from poplar (P. alba × P. glandulosa) and characterized its activity in transgenic poplar plants using a β-glucuronidase (GUS) reporter gene. High-level expression of the Pagns-LTP transcript was found in poplar roots, while comparatively low-level expression was found in the young leaves. Pagns-LTP mRNA was not detected in other poplar tissues. Additionally, transgenic poplar plants that contained a Pagns-LTP promoter fused to a GUS reporter gene, displayed tissue-specific GUS enzyme activity localized in root tissue. In silico analysis of the Pagns-LTP promoter sequence reveals the presence of several cis-regulatory elements responsive to phytohormones, biotic and abiotic stresses, as well as those regulating tissue-specific expression. These results demonstrate that the Pagns-LTP promoter has tissue-specific expression activity in poplar roots and leaves that may be involved in organ development and plant resistance to various stresses. Therefore, we anticipate that the Pagns-LTP promoter would be a useful tool to genetically optimize woody plants for functional genomics.

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Abstract : Clubroot disease is one of the most wide-spread and devastating diseases in the cultivation of Chinese cabbage. To develop a protein marker for resistance to clubroot disease in Chinese cabbage, a comparative proteome analysis was performed between a sensitive line, 94SK, and a resistant line, CR Shinki DH. Three proteins of two fold or higher accumulation that are specific to each line were found 3 days after innoculation of the Plasmodiphora brassicae. They are glutamine synthetase, malate dehydrogenase/oxidoreductase and fructose-bisphosphate aldolase in the 94SK and actin, phosphoglycerate kinase, and Cu/Zn superoxide dismutase in the CR Shinki line. From the comparison of the synthesized proteins in the 94SK and the CR Shinki, CR Shinki was found to produce more ATP-binding protein for the ABC transporter while 94SK showed a higher level of pathogenesis-related protein 1 production. All of these proteomic variations may lead to the development of molecular markers to accelerate the breeding process.