Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum)
Introduction
The warm-season grass Paspalum notatum (bahiagrass) is a widely grown forage crop, particularly in the tropical and subtropical regions. Its mode of reproduction is either diploid-sexual or obligate-apomictic in tetraploid genotypes (Burton and Forbes, 1960). The grass performs well under severe drought and grazing stress. However, one of the major limitations for cattle production on bahiagrass and some other forage grasses, especially warm-season grasses, is the low nutritive value and poor digestibility in comparison to most temperate grasses or non-grass forage (Akin and Burdick, 1975; Reid et al., 1990). It is believed that the low digestibility of tropical pasture grasses is due to its high lignin content (Akin et al., 1983; Jung and Vogel, 1986). Improved tetraploid cultivars of bahiagrass with increased yield and enhanced forage performance have been developed (Burton, 1982), but diploid-sexual cultivars that would allow a combined breeding approach of conventional and transgenic techniques are lacking behind. Therefore, genetic transformation could add to quality improvement and enhancement of alternative uses in diploid bahiagrass.
Plant regeneration from in vitro-cultures of bahiagrass has been demonstrated previously via organogenesis from inflorescence-derived callus (Bove and Mroginski, 1986), via somatic embryogenesis from seed-derived callus (Marosky and West, 1990; Akashi et al., 1993), from callus derived from the apical meristems of seedlings (Grando et al., 2002) and from seed-derived embryogenic cell suspension cultures (Akashi et al., 1993). Recently, successful transformation of tetraploid (apomictic) bahiagrass for herbicide (l-phosphinothricin, l-PPT) resistance using microprojectile bombardment of embryogenic calluses was reported (Smith et al., 2002). According to this report, selection of transformants was greatly hampered by the spontaneous development of resistant non-transgenic cells. In a preliminary report, we have demonstrated particle inflow gun-mediated transformation for bialaphos (active ingredient l-PPT) resistance of diploid bahiagrass (Gondo et al., 2003). In these experiments, we used an efficient regeneration system that regularly yields green plants. However, after particle bombardment and subsequent growth on bialaphos-containing selection medium only albino transgenics could be recovered. Recovery of green transgenic plants in diploid bahiagrass has not been described.
Here we report the transformation of callus cultures of diploid bahiagrass and the recovery of green plants after the application of a modified callus induction and regeneration protocol. Despite the presence of the herbicide in the selection medium albinos did not develop. The plasmid pDB I (Becker et al., 1994), containing the GUS reporter gene under control of the rice actin1 promoter (Act1) and the bialaphos resistance gene (bar) under control of the CaMV 35S promoter, was used for transformation experiments. Under optimized conditions, 360 target pieces yielded 22 transgenic plants, 8 of which are independent lines.
Section snippets
Formation of embryogenic callus and microcallus culture
Seeds of diploid bahiagrass (Paspalum notatum Flügge cv. Pensacola) were surface-sterilized in 70% (v/v) ethanol for 1 min and 2% (v/v) sodium hypochlorite for 15 min, followed by three washings with sterile water. Seeds were cultured at 80–90 seeds per 90 mm Petri dish on filter paper (Advantec Toyo Co., Japan) and 5 mL liquid MS medium (Murashige and Skoog, 1962) containing 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 3% sucrose as described previously (Gondo et al., 2003). After 14 d of
Embryogenic callus formation and selection of microcallus
Embryogenic callus formed faster on filter paper with liquid MS medium than on solid medium. Embryogenic mirocalli were subcultured, and after 5 subcultures a single seed-derived fast growing callus culture, showing shiny, compact and nodular clusters and a homogeneous appearance of the subcallus population, was selected as target tissue for particle bombardment.
Plant regeneration from microcalli under non-selective conditions
Since the initial transformation experiments with microcalli cultured and bombarded on MS medium with 2,4-D (MS-D) yielded nothing but
Discussion
We have established an efficient particle bombardment transformation protocol for the warm-season forage grass P. notatum (bahiagrass). A key factor for the success was likely the specific quality of the target tissue used, a condition also observed by others (e.g., Sivamani et al., 1996; Bec et al., 1998; Zhang et al., 2002; Goldman et al., 2003). Callus formation in heterozygous bahiagrass (Burton, 1982) is normally low (Bove and Mroginski, 1986; Marosky and West, 1990; Akashi et al., 1993;
Acknowledgements
We are grateful to Professor Horst Lörz (Hamburg University, Germany) for providing the plasmid pDB I.
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