Biolistic transformation (particle bombardment) is based on the delivery of DNA into plant cells by high velocity gold or tungsten particles (Sanford, 1988). Because DNA delivery by particle bombardment is a physical process that does not depend on bacteria, it has become a versatile and effective transformation method for many species, particularly monocot plants (Wang and Ge, 2006). Biolistic transformation of embryogenic cultures has led to the production of transgenic tall fescue plants (Chen et al., 2003, 2004; Cho et al., 2000; Hu et al., 2005; Spangenberg et al., 1995; Wang et al., 2003a, 2001). Morphogenic calli or embryogenic suspension cells have been used as ideal targets for biolistic transformation. The chimeric hygromycin phosphotransferase gene (hph) and phosphinothricin acetyltransferase gene (bar) have been used as selectable markers. The integration and expression of an hph gene in transformed cells results in the ability to phosphorylate hygromycin, thus rendering the cells resistant to this antibiotic. Similarly, the expression of a bar gene in transformed cells results in the acetylation of phosphinothricin (PPT) and renders the cells resistant to this herbicide. The use of antibiotic selection provides an advantage for the transformed cells to proliferate and regenerate into plants, because most of the target cells are not transformed. The construction of a chimeric gene requires a promoter, which directs when and where the gene should be expressed. The commonly used promoters include cauliflower mosaic virus (CaMV) 35S promoter, rice actin promoter, and maize ubiquitin promoter.
The procedure for biolistic transformation of tall fescue using the hph gene as the selectable marker and hygromycin as the selection agent is illustrated in Fig. 22-1. Embryogenic cultures were bombarded with tiny gold particles coated with DNA (Fig. 22-1A,B). Hygromycin resistant calluses were obtained after selection in medium containing the antibiotic (Fig. 22-1C). Transgenic plants were recovered from the hygromycin-resistant calluses (Fig. 22-1D,E) and established in the greenhouse (Fig. 22-1F). To demonstrate that the regenerated plants are true transgenics, the plants need to be analyzed using molecular techniques, such as polymerase chain reaction (PCR), and Southern and Northern hybridization analyses. Polymerase chain reaction is an effective method for rapid screening of the transgenics. Southern hybridization analysis is used to confirm stable integration of a transgene in the plant genome. Expression levels of the transgene in individual plants can be detected by Northern hybridization analysis (Spangenberg et al., 1998).
Fig. 22-1. Transgenic tall fescue plants obtained by biolistic transformation. (A) PDS/1000 biolistic device used for biolistic transformation. (B) Embryogenic cells of tall fescue plated on filter paper for microprojectile bombardment. (C) Hygromycin resistant calluses obtained after bombardment and hygromycin selection. (D, E) Transgenic shoots and plantlets regenerated from the hygromycin resistant calluses. (F) Transgenic tall fescue plants growing in the greenhouse.
An analysis of progenies showed stable meiotic transmission of transgenes following Mendelian rules in transgenic tall fescue plants obtained from biolistic transformation (Wang et al., 2003a). Although transgenics with a single copy transgene were obtained by biolistic transformation, the majority of the transformants had complex transgene integration patterns with multiple or rearranged copies of the transgene (Chen et al., 2003; Spangenberg et al., 1995; Wang et al., 2003a). This problem associated with biolistic transformation led to the development of transformation technology based on the use of Agrobacterium tumefaciens.
|<--Previous||Back to Top||Next-->|