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Unformatted text preview: Plant Cell, Tissue and Organ Culture 78: 107111, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands. 107 Agrobacterium tumefaciens-mediated transformation of Rosa hybrida using the green fluorescent protein (GFP) gene C.K. Kim 1 , J.D. Chung 2 , S.H. Park 3 , A.M. Burrell 3 , K.K. Kamo 4 & D.H. Byrne 3 , 1 Department of Horticulture, Sangju National University, Sangju 742-711, South Korea; 2 Department of Horticulture, Kyungpook National University, Daegu 702-701, South Korea; 3 Department of Horticultural Sciences, Texas A&M University, College Station, TX 77843-2133, USA; 4 USDA-ARS, Florist and Nursery Plants Research Unit, Beltsville, MD 20705-2350, USA ( requests for offprints: Fax: + 1-979-845-0626; E-mail: firstname.lastname@example.org) Received 21 February 2003; accepted in revised form 10 November 2003 Key words: genetic engineering, marker gene, rose, transgenic plants, vir gene Abstract Embryogenic calluses of Rosa hybrida cultivar Tineke were transformed with Agrobacterium tumefaciens strain LBA4404 containing the binary vector pBIN m-gfp5-ER into which the vir E/ vir G genes had been inserted. Visual- ization of GFP-expressing cells enabled visual selection of dividing, embryogenic cell clusters that were transgenic. When the Agrobacterium strain with the bifunctional fusion marker containing additional vir E/ vir G genes was used, the number of green fluorescent calluses increased. Transformation of the GFP-expressing rose plants was confirmed by Southern blot analysis. Abbreviations: GFP green fluorescent protein gene; GUS -glucuronidase Rose is one of the most economically important flowers worldwide. Genetic improvement of rose through conventional breeding is limited by several factors such as polyploidy and the highly heterozy- gous nature of existing cultivars. Genetic engineering, based on tissue culture technology, provides an option for overcoming some of these restrictions. There are only a few published reports on Agrobacterium-mediated (Firoozabady et al., 1994; van der Salm et al., 1996) and bombardment- mediated transformation in roses (Marchant et al., 1998). Firoozabady et al. (1994) reported successful transformation of Rosa hybrida cv. Royalty follow- ing cocultivation of friable embryogenic callus with Agrobacterium tumefaciens strain LBA4404. Sub- sequent reports are of transformation of rose by cocul- tivation with A. tumefaciens strain GV3101 containing an npt II gene and rol gene from A. rhizogenes using roots derived from stem sections of the rootstock R. hybrida cv. Moneyway (van der Salm et al., 1996). Grafting onto the transformed rootstock resulted in stimulation of both root development of rootstock and axillary-bud release of the transformed scion. In ad- dition, Marchant et al. (1998) developed transgenic plants from embryogenic callus of R. hybrida through biolistic transformation. All these experiments used the -glucuronidase (GUS) reporter gene to identify transformed tissue.transformed tissue....
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