- Cytological Study of the Introduction of Agrobacterium tumefaciens Spheroplasts into Nicotiana tabacum Protoplasts.
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Jung Hye Kim, Yong Bum Koo, Ki Yung Lee
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Yeungnam Univ J Med. 1985;2(1):175-181. Published online December 31, 1985
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DOI: https://doi.org/10.12701/yujm.1985.2.1.175
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 Abstract
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- Agrobacterium tumefaciens induces cancerous growths called crown galls at wound sites on dicotyledonous plants. A large plasmid called T1 plasmid is responsible for virulence. Upon tumor induction, part of the plasmid, termed T-DNA, becomes integrated into plant genome and its genetic sequences are expressed. These properties allow T1 plasmids to be used as gene vectors in plants. Several in vitro methods for the transfer of T1 plasmid into plant cell have been developed. One of them is the treatment of bacterial spheroplasts and plant protoplasts mixture with polyethylene glycol that is generally used as fusogen in cell-to-cell fusion. Several workers investigated the interaction of bacterial spheroplasts with plant protoplasts in the presence of polyethylene glycol and suggested that the interaction is not fusion but endocytosis. In this report we observed the interaction of Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts by electron microscope. Agrobacterium tumefaciens spheroplasts with Nicotiana tabacum protoplasts were prepared and mixed in the presence of polyethylene glycol and high pH-high Ca²⁺ buffer. Then the interaction of the spheroplasts with the protoplasts was examined by transmission electron microscope. After the treatment of polyethylene glycol the spheroplasts adhered to the surface of the protoplasts and then they were engulfed by the protoplasts. After the high pH-high Ca²⁺ buffer treatment the engulfed spheroplasts lost their cell integrity. No fusion process was observed. Thus all these observation suggest that the introduction process of Agrobacterium tumefaciens spheroplasts into Nicotiana tabacum protoplasts with the aid of polyethylene glycol is endocytosis.
- Comparative Genetic Characterization of Plasmids of Agrobacterium Species Isolated in Korea.
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Jung Hye Kim, Yong Bum Koo, Ki Young Lee, Jae Kyu Chung
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Yeungnam Univ J Med. 1984;1(1):41-48. Published online December 31, 1984
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DOI: https://doi.org/10.12701/yujm.1984.1.1.41
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Abstract
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- The soil bacterium Agrobacterium tumefaciens is a plant pathogen that causes crown gall tumors by infecting the wounded dicotyledonous plants and subsequent integration of bacterial DNA into plant nuclear DNA. Virulent A. tumefaciens strains harbor a large Ti (tumor–inducing) plasmid that carries genes essential for tumorigenesis. In the present study, 13 strains (Malus pumila Mill; A₁₋₃, Populus monilifera; W₁₋₆, Populus tomentiglandlosa; P₁₋₃ and Rosa species; R₁) of Agrobacterium isolated in korean crown gall tumors and plasmids were observed in 6 strains (W₂, W₃, W₆, P₁, P₃ and A₂). The test for crown gall tumor formation was resulted only in ATCC15955 and KW2 strains inoculated into the stem of sun flower and the development was observed for 4 and 6 weeks after inoculation. Above two Ti plasmids (pTi) were purified by cesium chloride-ethidium bromide density gradient centrifugation and digested with restriction enzyme and fragments of pTiATCC15955 and pTiKW₂ observed by EcoR I ; 25&27, Hind III ; 23&21, BamH I ; each 20 and Hpa I ; 12&27. And sizes of pTiATCC15955 and and pTiKW₂ calculated as 200 and 87 kbases. Octopine was isolated from tumor tissue (W₁₋₆ and P₁₋₃) and these strains confirmed as octopine type.
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