Agrobacterium tumefaciens is a soil-borne bacterium that, in nature, is capable of inserting a discrete portion of its plasmid DNA into the genome of a wide range of dicot species (Valentine, 2003). Grasses were initially considered to be recalcitrant to Agrobacterium infection; however, a breakthrough in the mid 1990s showed that Agrobacterium could be used effectively for producing transgenic rice (Oryza sativa L.) plants (Hiei et al., 1994). Following this success, Agrobacterium-mediated transformation was developed for the other major cereal crops, such as maize (Zea mays L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare L.) (Cheng et al., 2004). Agrobacterium-mediated transformation has the advantage of allowing for the production of transgenics with lower copy number and fewer rearrangements than the free DNA delivery methods discussed above (Hu et al., 2003).

Spurred by the progress in cereal crops, protocols have been developed for Agrobacterium-mediated transformation of forage and turf grasses, such as tall fescue (Dong and Qu, 2005; Wang and Ge, 2005), perennial ryegrass (Lolium perenne L.) (Altpeter et al., 2004; Bajaj et al., 2006; Sato and Takamizo, 2006), Italian ryegrass (Lolium multiflorum Lam.) (Bettany et al., 2003), and creeping bentgrass (Agrostis stolonifera L.) (Han et al., 2005; Luo et al., 2004). Typically, embryogenic calluses first were induced and used to incubate with Agrobacterium. Resistant calluses were obtained after several weeks of antibiotic selection. Transgenic plants finally were regenerated from the resistant calluses. The use of highly embryogenic calluses has been shown to be critically important for the successful generation of transgenic plants. A procedure for Agrobacterium-mediated transformation of tall fescue (Wang and Ge, 2005) is briefly described here and illustrated in Fig. 22-2. Embryogenic calluses induced from seeds (caryopses) of tall fescue (Fig. 22-2A) were fragmented into small pieces and infected with A. tumefaciens strain EHA105 (Hood et al., 1993) (Fig. 22-2B). The hph gene was used as selectable marker and hygromycin as selection agent. Calluses resistant to hygromycin were obtained after 4 to 6 wk of selection in medium containing hygromycin (Fig. 22-2C). Shoots and rooted plantlets were obtained after transferring the hygromycin resistant calluses onto regeneration medium (Fig. 22-2D,E). Soil-grown tall fescue plants were regenerated 4 to 5 mo after Agrobacterium-mediated transformation (Fig. 22-2F). Transformation frequency was in the range of 1.9 to 8.7% when calculated based on the number of transgenic plants recovered and the number of original intact calluses infected. The transgenic nature of the recovered plants was demonstrated by Southern and Northern hybridization analyses. Progeny analysis after vernalization and crossing revealed Mendelian inheritance of transgenes (Wang and Ge, 2005).

 

Fig. 22-2. Transgenic tall fescue plants obtained by Agrobacterium-mediated transformation. (A) Embryogenic callus induced from caryopses of tall fescue. (B) Callus pieces infected with Agrobacterium tumefaciens. (C) Hygromycin resistant calluses obtained after selection of infected callus pieces on medium containing hygromycin. (D) Shoot differentiation of hygromycin resistant calluses after transfer onto regeneration medium. (E) Rooted transgenic tall fescue plantlets obtained after transferring the differentiated shoots to rooting medium. (F) Transgenic tall fescue plants growing in the greenhouse.

 

 

 

<--Previous       Back to Top      Next-->