Genetic Transformation of Iris germanica Mediated by Agrobacterium tumefaciens

A protocol was developed for production of transgenic iris plants ( Iris germanica L. 'Skating Party') from regenerable suspension cultures via Agrobacterium-mediated transformation. We tested a series of selection agents, and identified hygromycin and geneticin as the most suitable for selecting transformed iris cells. Suspension cultures of iris were cocultured for 3 days with A. tumefaciens LBA 4404(pTOK233) carrying an intron-interrupted uidA (GUS) gene encoding β-glucuronidase, and hpt (hygromycin) and nptII (geneticin) selectable marker genes. Hygromycin- or geneticin-resistant calli having GUS enzyme activity were identified and used to induce plant regeneration. More than 300 morphologically normal transgenic iris plants were obtained in ≈6 months. About 80% of the transformants were GUS-positive and NPTII- positive (paromomycin-resistant). Integration of transgenes into the nuclear genome of iris plants was confirmed by Southern blot analysis. We have, therefore, developed an efficient A. tumefaciens-mediated transformation system for Iris germanica, which will allow future improvement of this horticulturally important ornamental monocot via genetic engineering. mation). Microprojectile bombardment and direct gene transfer to protoplasts are used commonly to transform a variety of monocoty- ledonous plants (Vain et al., 1995). However, stable (integrative) transformation of only two horticulturally important ornamental monocots, Dendrobium Swartz orchid (Kuehnle and Sugii, 1992) and Gladiolus L. (Kamo et al., 1995), by microprojectile bombard- ment have been reported. Agrobacterium-mediated transformation has significant advan- tages over other approaches such as integrating a few copies of T- DNA with defined border sequences and minimal rearrangement in the plant genome, preferential integration into transcriptionally active regions of the chromosome, high quality and fertility of resultant transgenic plants, and easy manipulation (Komari et al., 1998; Tingay et al., 1997). Methods for transforming dicotyledonous species with Agro- bacterium are well established. In contrast, until recently monocoty- ledons were considered beyond the range of A. tumefaciens transfor- mation methods. Various attempts to infect monocots with Agro- bacterium were made in the 1970s and 1980s, but no conclusive evidence of integrative transformation was reported (Conner and Dommisse, 1992; Smith and Hood, 1995). Successful A. tumefa- ciens-mediated transformation, however, is now possible in several agronomically important monocots including corn (Zea mays L.), wheat (Triticum aestivum L.), rice (Oryza sativa L.), barley ( Hor- deum vulgare L.), and sugar cane ( Saccharum spp. L.) (Arencibia et al., 1998; Cheng et al., 1997; Hiei et al., 1994; Ishida et al., 1996; Tingay et al., 1997). The utility of A. tumefaciens for stable (integrative) transformation of ornamental monocots was demon- strated only in Anthurium scherzerianum Schott 'Rudolph' and 'UH1060' (Chen and Kuehnle, 1996). We report here on the development of an efficient A. tumefaciens-mediated transforma- tion method for Iris.