Development of marker-free transgenic sorghum ... - Plant Sciences

98 Plant Cell Tiss Organ Cult (2009) 99:97–108sorghum events from 300 immature embryos using animproved protocol employing a standard binary vector(with hpt as the selectable marker), cold pre-treatedimmature embryos and activated charcoal. Four additionalstudies (Gurel et al. 2009; Jeoung et al. 2002; Gao et al.2005a, b) used positive selectable markers (i.e. the Escherichiacoli phosphomannose isomerase (pmi) gene and thegreen fluorescent protein gene (gfp). Recently, heat treatmentof immature embryos prior to infection with A.tumefaciens has been used (Gurel et al. 2009).Although each of these studies has contributed to sorghumtransformation, none have enabled production oflarge numbers of marker-free transgenic sorghum plantsusing standard binary vectors. In the present study, wedescribe the use of standard binary vectors with bar as aselectable marker and the sorghum public line P898012 toestablish a rapid and reproducible A. tumefaciens-mediatedtransformation system that generates large numbers oftransgenic sorghum events and marker-free progeny insegregating generations.Materials and methodsVectors and bacterial strainsThe binary vector pZY102 with the GUS-intron gene(Zeng et al. 2004) (http://plantsci.missouri.edu/muptcf/pzy101.html) was used to evaluate transformation parameters.Two vectors containing an altered tRNA lys and asorghum lysyl tRNA synthetase (Wu et al. unpublished)were also constructed and employed: the pBluescript vectorwas digested with KpnI and Ecl136II to remove theKpnI and Ecl136II (SacI) sites. Each fragment was ligatedwith an adaptor incorporating two ScaI sites and a singleBglII site, respectively, to generate the shuttle vector, SBSpBS.An intermediate binary vector, Bin19(-), was firstgenerated by removing a 2,047-bp fragment between Pme Iand ClaI sites from Bin19 vector (Frisch et al. 1995). Thetwo restriction sites were then filled in with Klenowpolymerase fragment and ligated and inserted into the BglIIsite of SBS-pBS, generating pSBS19(-). The altered tRNAgene (TC2) or sorghum lysyl tRNA synthetase (SKRS)gene fragments were inserted into the HindIII-KpnI siteof SBS19(-), generating SBS-TC2 and SBS19-SKRS,respectively. The ScaI fragment from SBS-TC2 or SBS19-SKRS containing T-DNA left and right border region wasthen inserted into the Sca I site of the standard binaryvector pZY101(Vega et al. 2008) that contains the bar geneas a selectable marker to generate vectors pZY101-TC2(Fig. 1a) and pZY101-SKRS (Fig. 1b), respectively. Thethree vectors, pZY102, pZY101-TC2 and pZY101-SKRSwere mobilized into A. tumefaciens strain EHA101(Hood et al. 1986) and their integrity within A. tumefacienswas confirmed by restriction enzyme digestion.Plant materialsThe public sorghum line P898012 was used for all transformationexperiments. All plants (except for those used inexperiments L73-L79, which were grown in the field duringthe summer) were grown in greenhouses at the Universityof Missouri, Columbia, Missouri, with day/nighttemperatures of 26/21°C, a photoperiod of 16 h light/8 hdark, in 3-gal pots containing Promix soil mixed with 2 oz.of Osmocote (14-14-14). About 32–35 days after planting,2 oz. of iron sulfate and 2 oz. of Osmocote (18-6-12) weremixed with the top portion of the soil of each pot; the soilmoisture and plants were checked daily and watered asneeded. Embryos were isolated from immature panicles,11–14 days after pollination.TransformationThe procedure reported here is based on the work of Zhaoet al. (2000) and Cai et al. (2002). Table 1 summarizesthe general procedure, cultivation periods and formulationof the media. A key modification was the use of lowlevels of glufosinate-ammonium (DL-phosphinothrithin,PPT, Aventis CropScience) at a concentration of2.5 mg l -1 in combination with a shortened selectionperiod (4–8 weeks). Other modifications included: (1) cocultivationmedium (TM) was overlaid with a piece ofsterile filter paper, (2) reduction of MS salts to 2.15 g l -1in the infection medium (IM) and TM, (3) addition ofPVPP to final concentrations of 1 or 0.5% in all mediumpreparation, with the exception of IM, (4) Trans-Zeatinribosidesubstitution for zeatin in shooting medium (SM),(5) elimination of selection agent in SM, and (6) no useof PHI-V (extended selection medium, CM with PPT10 mg l -1 , instead) or PHI-W (regeneration medium, CMsupplemented with kinetin 0.5 mg l -1 ; Zhao et al. 2000).Briefly, A. tumefaciens strain EHA101 harboring standardbinary vector was clonally isolated from a -80°C glycerolstock onto an AB minimal medium (Chilton et al.1974) plate containing appropriate antibiotics. The platewas incubated at 28°C for 3 days until single coloniesdeveloped. This master plate was used on a weekly basisfor up to a month. For the inoculation, a single colonywas streaked out onto YEP (5 g l -1 yeast extract,10 g l -1 peptone, 5 g l -1 NaCl, and pH 7.0) containingthe same antibiotics as the AB plate. The YEP plate wasthen incubated at 20°C for 2–3 days until bacterial coloniesdeveloped fully. A. tumefaciens colonies were thentaken from the YEP plate, suspended in 5 ml of IM123