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    3 views6 pages

    X 030020

    Uploaded by

    Bvinosobi

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    Attribution Non-Commercial (BY-NC)
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    hoy eR Acta Botanica Siniea 2003, 45(9): 1103-1108 Generating Marker-Free Transgenic Tobacco Plants by Agrobacterium- mediated Transformation with Double T-DNA Binary Vector ‘ZHIOU Hong: Yan, CHEN Song-Biao!”, LL Xu-Gang!, XIAO Gui-Fang!, WELXiao-Lit, ZHU Zhen!” (1, Institue of Goneties and Developmental Biology, The Chinese Academy of Sclences, Beijing 100101, Chins 2. Cell strwe, Beijing Normal University. Bejing 100878, China) Abstract We have developed a “double T-DNA” binary vector system for generating selectable marker- ‘free transgenic plants by Agrobacteriummediated transformation. The “double T-DNA" binary vector PDLBRBbarm which carried two independent T-DNAs, one containing a selectable marker neomycin Phosphotransferase (npt/T) gene and the other a bar gene, was constructed. Transgenic tobacco (Nicotiana tabacum L) plants were then produced by Agrobacterium-mediated transformation with this vector, Fre- ‘quency of the primary transformants co-integrated with not IT gene and bar gene was 59.2%, Segregation ‘of two T-DNA regions was found in 3 out of 4 T, lines from co-traneformed To plants with nptil and bar. PPT-resistant and kanamycin-sensitive plants were in approximate 19.5% of the T, plants, The result indicated that this “double T-DNA” vector system could be a workable approach to generate transgenic plants free from selectable marker genes. Co-transformation of nptil gene and bar gene to plants with mixtures of Agrobacterium tumefaciens strains containing single T-DNA vectors was also tested, Fre- ‘quency of co-transformed plants was 20.04-47.74 and relatively low as compared with that of “double T- hitp/wiw chineseplantacience.com DNA" vector system. Key words: Asa patt ofthe process of plant transformation, select- able markers are used to select transgenic cells, from which intact transgenic plants can be regenerated (Gleave e¢ al, 1999), However, these selectable marker genes in transgenic plants are usually useless once transgenic plants are produced, The maintenance of marker genes in transgenic plants is not only unnecessary, but also can generate envi- ronmental and consumer concerns (Zechendorf, 1994), Moreover, because there are only a limited number of se lectable marker genes available for plant transformation, the combination of multiple transgenic traits into the same cultivar through repeated transformation is constrained by the presence of marker genes (Yoder etal, 1994), To date, the production of maker-fie transgenic plants has become ‘a major objective for plant biologist and the plant biotech- nological industry (Zuo et al, 2002). Several strategies have so far been proposed to pro- duce transgenic plants free from selectable marker genes, including use of transposable element system fo eliminate marker genes (Goldsbrough ef a., 1993), use of site-spe~ cific recombination systems such as ere/lox (Dale et al, 1991; Russell er ab, 1992; Gleave et at, 1999; Zuo eral, 2001) and RRS (Sugita etal, 2000)t0 excise marker genes, Reveived: 200 28 Accepted: 2002 plant transformation; marker-free: double T-DNA vector, tobacco and the generation of co-transformants, in which the marker gene can be eliminated subsequently in later generations. ‘Among these, the latter appears to be the simplest system and can be suitable for most economically important crops. ‘Several different methods have been employed inthe devel- ‘opment of eo-transformation system for producing marker- free transgenic plants. These include use of a mixture of Agrobacterium tumefaciens strains (Depicker etal, 1985; McKnight ef al, 1989; Komari et a., 1996), use of one Agrobacterium tumefaciens strain containing two binary vectors (Daley et al, 1998) and use of one binary vector containing two T-DNAs (Komari ef al., 1996; Matthews et al, 2001), Of costansformation methods, which is effective for obtaining marker-fre transgenic plants, efficiency of ¢o- transformation and unlinked T-DNAs integration must be high (Yoder et al, 1994), Reports of co-transformation fre- quencies among different methods varied. Study of Komari etal, (1996) showed that using “super binary” vectors which contained two T-DNAs was higher in co-transformation ef- iciency than using the mixture method, and thus more ef fective in generating marker-free transgenic plants, Here we reporta “double TSDNA™ binary veetor system for developing selectable marker-free transgenic plants by ‘Supported by the Hi-Tech Research and Development (863) Program of China (2001AA212041, 2002AA212031) and the State Key Basie Research and Development Plan of China (G2001C8109001). * Coordinate first authors wit the same contribution > Autor for corespondnce, Tel (Fan): +86 (0)10 64852890; E-mail:

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