• Title/Summary/Keyword: K-L transformation

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Agrobacterium-mediated Transformation of Eleutherococcus sessiliflorus using Embryogenic Calli and the Regeneration of Plants (오갈피(Eleutherococcus sessiliflorus)의 배형성 세포를 이용한 고빈도 형질전환 및 재분화)

  • Jeong, Jae-Hun;Han, Seong-Soo;Choi, Yong-Eui
    • Journal of Plant Biotechnology
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    • v.30 no.3
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    • pp.233-239
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    • 2003
  • We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

Transformation of Potato using the Phosphinothricin Acetyltransferase Gene as the Selectable Marker Gene (감자의 형질전환을 위한 표지유전자로서 Phosphinothricin Acetyltransferase 유전자의 이용)

  • Jeong, J.H.;Yang, D.C.;Bang, K.S.;Han, S.S.
    • Korean Journal of Weed Science
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    • v.18 no.3
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    • pp.205-213
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    • 1998
  • This experiment was carried out to produce herbicide resistant potatoes hawing only chimeric phosphinothricin acetyltransferase (PAT) genes without using antibiotic selectable marker. The pDY502 vector having only PAT gene was reconstructed for transformation of potato. The reconstructed vector was introduced to Agrobacterium tumefaciens MP90 disarmed, and they were used for potato transformation. Hormonal requirement for plant regeneration from leaves and stem explants of potato was investigated. From this experiment, MS medium treated with IBA 0.1 mg/L + BA 0.5 mg/L was the best for potato regeneration, and the ratio of shoot regeneration was 54% for leaf and 46% for stem in that condition. For transformation, explants of potato leaves and stems were cocultured with A. tumefaciens MP90 containing reconstructed vector harvoring only PAT gene. When the potato explants were placed on various concentrations of bialaphos and all the potato explants were dead on medium with over 5.0mg/L bialaphos. By this selection methods, the explants cocultured with Agrobacterium produced the putative transgenic shoots on medium with 5mg/L bialaphos treatment after 3-4 weeks. Second selection was performed by transferring the shoot tips of putative transgenic to medium containing 20mg/L of bialaphos. The shoot tips grew well on the second selection medium, indicating the production of successful transgenic plants. But normal shoots were dead in same cytotoxic medium. Incorporation of the PAT gene into transgenic potatos were confirmed by PCR analysis of DNA and Southern hybridization. These results show that the PAT gene can serve as a selectable marker and herbicide resistant genes for transformation of potato.

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Vacuum infiltration transformation of non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) with the pinII gene and bioassay for diamondback moth resistance

  • Zhang, Junjie;Liu, Fan;Yao, Lei;Luo, Chen;Zhao, Qing;Huang, Yubi
    • Plant Biotechnology Reports
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    • v.5 no.3
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    • pp.217-224
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    • 2011
  • Non-heading Chinese cabbage (Brassica rapa L. ssp. chinensis) is a popular vegetable in Asian countries. The diamondback moth (DBM), Plutella xylostella (L.), an insect with worldwide distribution, is a main pest of Brassicaceae crops and causes enormous crop losses. Transfer of the anti-insect gene into the plant genome by transgenic technology and subsequent breeding of insect-resistant varieties will be an effective approach to reducing the damage caused by this pest. We have produced transgenic non-heading Chinese cabbage plants expressing the potato proteinase inhibitor II gene (pinII) and tested the pest resistance of these transgenic plants. Non-heading Chinese cabbages grown for 45 days on which buds had formed were used as experimental materials for Agrobacterium-mediated vacuum infiltration transformation. Forty-one resistant plants were selected from 1166 g of seed harvested from the infiltrated plants based on the resistance of the young seedlings to the herbicide Basta. The transgenic traits were further confirmed by the Chlorophenol red test, PCR, and genomic Southern blotting. The results showed that the bar and pinII genes were co-integrated into the resistant plant genome. A bioassay of insect resistance in the second generation of individual lines of the transgenic plants showed that DBM larvae fed on transgenic leaves were severely stunted and had a higher mortality than those fed on the wild-type leaves.

Factors Affecting Agrobacterium tumefaciens-mediated Transformation of Panax ginseng C.A. Meyer

  • Kim, Ok-Tae;Jung, Su-Jin;Bang, Kyong-Hwan;Kim, Young-Chang;Shin, Yu-Su;Sung, Jung-Sook;Park, Chun-Geon;Seong, Nak-Sul;Cha, Seon-Woo;Park, Hee-Woon
    • Korean Journal of Medicinal Crop Science
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    • v.15 no.2
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    • pp.100-104
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    • 2007
  • A protocol for the production of transgenic Panax ginseng C.A. Meyer was established via Agrobacterium tumefaciens-mediated genetic transformation of direct somatic embryos. A number of conditions related to the co-cultivation were tested with respect to maximizing transformation efficiency. The results showed that pH of the co-cultivation medium (5.7), the bacterial growth phase (optical density; $OD_{600}$ = 0.8), co-cultivation period (3 days), and acetosyringone concentration $(100\;{\mu}M)$ had positive effects on transformation. Selected plantlets were cultured on the medium at an elevated hygromycin level(30 mg/l). Integration of the transgenes into the P. ginseng nuclear genome was confirmed by PCR analysis using hpt primers and by Southern hybridization using hpt-specific probe. The transgenic plantlets were obtained after 3-month cultivation and did not show any detectable variation in morphology or growth characteristics compared to wild-type plants.

Immunohistochemical study on the expression of matrix metalloproteinase 2 and high-risk human papilloma virus in the malignant progression of papillomas

  • Lee, Ho-Jin;Kim, Jin-Wook
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • v.39 no.5
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    • pp.224-230
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    • 2013
  • Objectives: Papilloma frequently develops as a benign tumor of the head and neck area, but its potential for malignant transformation has yet to be studied. This study aims to provide basic information for papillomas using the immunohistochemical staining of matrix metalloproteinase 2 (MMP-2) and human papilloma virus (HPV) 16 and 18. Materials and Methods: To evaluate the malignant transformation of papillomas, the selected tissue samples were serially diagnosed with pre-cancerous papilloma (with epithelial dysplasia, pseudo-epitheliomatous hyperplasia) or malignant lesion (squamous cell carcinoma, SCC) after the first diagnosis (squamous papilloma, inverted papilloma). The selected tissues were stained with an antibody to MMP-2 and HPV 16-E7, HPV 18-L1. A statistical analysis was performed according to each transformation step. Results: The epithelial layer of papilloma and pre-cancerous papilloma lesions had a similar MMP-2 expression, but that of the malignant lesion had a significantly increased MMP-2 expression. HPV 16 and 18 infection rates were 28.6%, 33.3% and 63.6% in papillomas, pre-cancerous papilloma lesions, and SCC. Conclusions: A relatively high MMP-2 expression and HPV 16 or 18 infection of papillomas may be associated with early events in the multistep processes of malignant transformation of papillomas.