Agrobacterium-Mediated Genetic Transformation of Switchgrass

and the bar gene was transmitted through both male and female gametes and expressed in T1 progeny. Although Agrobacterium tumefaciens has been successfully used Microprojectile bombardment and gene transfer by to transfer genes to a wide range of plant species, it has received litA. tumefaciens are currently the two most common tle attention for transformation of forage grasses. Therefore, the obmethods for achieving genetic transformation in higher jective of the present study was to demonstrate Agrobacterium-mediated transformation of switchgrass (Panicum virgatum L.). The plants. Although not universally accepted (Smith et al., A. tumefaciens strain AGL 1 carrying the binary vector pDM805, 2001), it has been reported that Agrobacterium-mediated coding for the phosphinothricin acetyltransferase (bar) and -gluctransformation leads to clean, discrete, low copy, welluronidase (gus) genes, was utilized in these experiments. Somatic emdefined, unrearranged DNA insertions into the plant bryos, embryogenic calluses, mature caryopses, and plantlet segments genome (Chilton, 1993; Repellin et al., 2001; Upadhyaya served as target tissues for infection. Treated cultures were selected et al., 2000). There are now several publications describin the presence of 10 mg L 1 bialaphos and the resultant plantlets ing genetic transformation in various cereal species uswere treated with the herbicide Basta [monoammonium 2-aminoing Agrobacterium (Repellin et al., 2001). The only pre4(hydroxymethylphosphinyl)butanoate]. T-DNA delivery efficiency vious report of gene transfer in a forage or turf grass by was affected by genotype, explant used and the presence or absence this method was for GFP in creeping bentgrass {Agrostis of acetosyringone (3 ,5 -dimethoxy-4 -hydroxyacetophenone) during inoculation and cocultivation. Approximately 600 transgenic plants palustris Huds. [ A. stolonifera var. palustris (Huds.) were produced, and transformation efficiencies ranged from 0 to Farw.]; Yu et al., 2000}. nearly 100%. Stable integration, expression, and inheritance of both The objectives of the present study were to demontransgenes were confirmed by molecular and genetic analyses. Apstrate high efficiency Agrobacterium-mediated transforproximately 90% of the tested plants appeared to have only one or mation in switchgrass, and to show sexual transmission two copies of the T-DNA inserts. Controlled crosses between T0 of the transgenes and their expression in T1 progeny. and nontransgenic ‘Alamo’ plants indicated the expected ratio of 1:1 The accomplishment of such provides an alternative to (transgenic:nontransgenic) in T1 plants for both transgenes according microprojectile bombardment for genetic manipulation to a 2 test at P 0.05. These results indicate that the Agrobacterium of this species. method is effective for transferring foreign genes into switchgrass. MATERIALS AND METHODS S is a warm season (C4) perennial grass Plant Material and Culture Conditions that is native to the tall grass prairies of North Embryogenic calluses were initiated from various explants America (Moser and Vogel, 1995). Although switchof different genotypes of cv. Alamo according to established grass is an important forage crop, it has also recently procedures (Table 1). Callus induction medium consisted of received interest for its potential as a bioenergy crop Murashige and Skoog (MS) salts and vitamins (Murashige and (Sanderson et al., 1996; McLaughlin et al., 1999). ImporSkoog, 1962) supplemented with 22.5 M 2,4-dichlorophentant to the improvement of this species is the developoxyacetic acid (2,4-D) and 5 M 6-benzylaminopurine (BAP). ment of cellular and molecular approaches, including Maltose (30 g L ) was substituted for sucrose. Cultures were gene transfer, that can be used to supplement convenmaintained at 27 C in the dark. Resulting calluses were subcultional breeding programs. tured every 4 wk. For plant regeneration, calluses were transThe current status of forage and turf grass biotechnolferred to MS medium supplemented with 1.4 M gibberellic acid (GA3) and incubated at 27 C with a 16-h photoperiod ogy has been recently reviewed (Forster and Spangen(cool white fluorescent bulbs at 80 mol m 2 s ). berg, 1999; Spangenberg et al., 2001; Wang et al., 2001). Transgenic plants have been reported for only 6 genera Agrobacterium Strain, Plasmid, and Culture and 11 species (Wang et al., 2001); the only warm season grass listed is switchgrass. This was achieved in our laboThe transformation experiments were conducted using ratory by microprojectile bombardment of embryogenic A. tumefaciens strain AGL1 (Lazo et al., 1991) containing the calluses with a GFP-BAR plasmid (Richards et al., 18.15-kilobase (kb) transformation vector pDM805 (Tingay 2001). Integration of both the green fluorescent protein et al., 1997). This plasmid contains the bar gene under the control of the maize ubiquitin 1 (Ubi1) promoter and the (gfp) and bar genes was shown by Southern blot hybriduidA (gus) gene under the control of the rice actin 1 (Act1) ization. Fluorescing pollen was observed in T0 plants promoter. The Agrobacterium was grown from a single colony in MG/L medium (Garfinkel and Nester, 1980) supplemented M.N. Somleva, USDA-ARS-PWA, Western Regional Research Center, 800 Buchanan St., Albany, CA 94710-1105; Z. Tomaszewski, Zabinskiego 18, Apt. 30, 02-793 Warsaw, Poland; B.V. Conger, Dep. of Abbreviations: Act1, actin 1 promoter; bar, phosphinothricin acetylPlant Sciences and Landscape Systems, Univ. of Tennessee, 2431 transferase gene; GA3, gibberellic acid; gfp, green fluorescent protein Center Drive, Knoxville, TN 37996-4561. Received 28 Jan. 2001. *Corgene; gus, -glucuronidase gene; kb, kilobase; MS, Murashige and responding author ([email protected]). Skoog medium; PCR, polymerase chain reaction; Ubi1, ubiquitin 1 promoter; X-Gluc, 5-bromo-4-chloro-3-indolyl-3-glucuronic acid. Published in Crop Sci. 42:2080–2087 (2002).