In Vitro Cell.Dev.Biol.—Plant (2011) 47:658–666 DOI 10.1007/s11627-011-9382-3
BIOTECHNOLOGY/GENETIC TRANSFORMATION/FUNCTIONAL GENOMICS
Comparison of three selectable marker genes for transformation of tall fescue (Festuca arundinacea Schreb.) plants by particle bombardment Danfeng Long & Xueli Wu & Zhimin Yang & Ingo Lenk & Klaus Kristian Nielsen & Caixia Gao
Received: 29 October 2010 / Accepted: 27 June 2011 / Published online: 26 July 2011 / Editor: J. Finer # The Society for In Vitro Biology 2011
Abstract A variety of selection systems have been developed for transformation of forage crops. To compare the most frequently used systems, we tested three selectable marker genes for their selection efficiency under four selection procedures for the production of transgenic tall fescue. Embryogenic calluses initiated from mature embryos were bombarded with three constructs containing either the phosphinothricin acetyltransferase (bar) gene, the hygromycin phosphotransferase (hpt) gene or the neomycin phosphotransferase II (nptII) gene. Transformation efficiency was strongly influenced by the selectable marker gene, selection procedure and genotype. The highest transformation efficiency was observed using the bar gene in combination with bialaphos. Average transformation efficiencies with bialaphos, phosphinothricin (glufosinate), hygromycin and paromomycin selection across the two callus lines used in the experiments were 9.4%, 4.4%,
D. Long School of Life Science, Lanzhou University, Lanzhou, China X. Wu : Z. Yang College of Horticulture, Nanjing Agricultural University, Nanjing, China I. Lenk : K. K. Nielsen Research Division, DLF-Trifolium Ltd., Hoejerupvej 31, Store Heddinge, Denmark C. Gao (*) The State Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China e-mail:
[email protected]
5.2% and 1.6%, respectively. Southern blot analysis revealed the independent nature of the tested transgenic plants and a complex transgene integration pattern with multiple insertions. Keywords Tall fescue (Festuca arundinacea Schreb.) . Particle bombardment . Selectable markers . Herbicide . Antibiotic
Introduction Particle bombardment remains a uniquely advantageous transformation method and indeed the only one available for many species (Altpeter et al. 2005). Gene delivery to plant cells and tissues by particle bombardment has led to the production of transgenic plants from many recalcitrant species. Tall fescue is an out-crossing polyploid grass species widely used for forage and turf. Because of its agronomic importance, tall fescue has been one of the most important target grass species used for genetic transformation in the last decade. Initial success was achieved producing transgenic tall fescue plants by direct gene transfer to protoplasts (Ha et al. 1992; Wang et al. 1992; Dalton et al. 1995). More recent work has demonstrated the recovery of transgenic tall fescue plants through particle bombardment and Agrobacteriummediated transformation using established long-term suspension culture or embryogenic callus culture (Spangenberg et al. 1995; Cho et al. 2000; Dong and Qu 2005; Wang and Ge 2005; Gao et al. 2008). As DNA is introduced into only a very small portion of cells in most experiments, the chances of recovering transgenic lines without selection are usually low. Selectable marker genes have, therefore, been central to the development of plant transformation technologies because
COMPARISON OF THREE SELECTABLE MARKER GENES FOR TRANSFORMATION
they allow identification and selection of cells that express and have incorporated the introduced DNA (Miki and McHugh 2004). The nptII gene is the most frequently used selectable marker gene for generating transgenic plants. It is very efficient in model species such as Arabidopsis and tobacco. However, high natural levels of resistance to kanamycin have frequently been observed in members of the Gramineae family (Hauptmann et al. 1988). The most extensively used herbicide resistance selectable marker gene is the bar gene. Also used is the hpt gene, which confers resistance to the antibiotic hygromycin B. For forage and turfgrass transformation, the use of the nptII gene with kanamycin or paromomycin has been reported (Wang and Ge 2006), but the bar gene in combination with bialaphos or phosphinothricin and the hpt gene together with the antibiotic hygromycin B are the most frequently used selection systems. A comparison of the selection efficiency of different selectable markers has been reported in cereals (Witrzens et al. 1998) but not in forages and turfs. In this study, we compared three selectable marker genes (bar, hpt and nptII) in combination with four selection procedures (bialaphos or phosphinothricin, hygromycin and paromomycin, respectively), using particle bombardment of tall fescue. Moreover, we describe for the first time the successful genetic transformation of tall fescue using the nptII gene as a selectable marker.
Materials and Methods Plant material and callus tissue culture. Mature tall fescue seeds (breeding line ISI-8872, DLF-Trifolium A/S, Store Heddinge, Denmark) were sterilized for 1 min in 70% ethanol, followed by 45 min in 5% sodium hypochlorite with 0.08% Tween 20 and rinsed six times with sterile Milli-Q (Millipore, Billerica, MA) water. Mature embryos were dissected under the microscope and cultured on solid callus induction MS5 medium (MS [Murashige and Skoog 1962] supplemented with 5 mg/l 2,4-D, 30 g/l sucrose, 3.75 g/l Gelrite (Duchefa, Haarlem, the Netherlands), pH 5.8, autoclaved 20 min) and incubated at 24°C in the dark. After 4–8 wk, friable, white to yellowish embryogenic calluses developed from about 2% of the mature embryos. Embryogenic calluses derived from single embryos, Figure 1. Plant transformation vectors pAHC20, pAct1IHPT-4 and pJFnp.
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representing individual genotypes, were grown separately and subcultured every 3 wk. DNA constructs. For selection with the herbicide bialaphos or DL-phosphinothrincin (PPT), we used plasmid pAHC20 containing the bar gene fused to the ubiquitin 1 promoter and intron 1 from maize (Fig. 1a, Christensen and Quail 1996). For selection with antibiotics hygromycin B or paromomycin, the plasmids used were pAct1IHPT-4 (Fig. 1b; Cho et al. 1998) and pJFnpt (Fig. 1c; Altpeter et al. 2000a). Plasmid pAct1IHPT-4 contains the hygromycin phosphotransferase (hpt)-coding sequence under control of the rice actin1 promoter (Act1), its intron (Act11) and followed by the nos 3′ terminator. Plasmid pJFnpt contains the nptII selectable marker gene, encoding the enzyme neomycin phosphotransferase II under control of the maize ubiquitin promoter and first intron. Particle bombardment. Embryogenic calluses (approx. 100–150 small pieces of callus per shot) were distributed as a 1.5-cm diameter monolayer in a 5.0-cm Petri dish containing 133 s/m medium (MS basal medium plus 3 mg/ l 2, 4-D, 0.2 M sorbitol and 0.2 M mannitol) for a 4h osmotic treatment prior to bombardment. Gold particles (0.6 μm, Bio-Rad, Hercules, CA) were coated with DNA as described by Vain et al. (1993) with the following modifications. The mixture contained 1-mg gold particles per shot. To have the same amount of the DNA molecules, 1 μg plasmid pAHC20 or plasmid pAct1IHPT-4 or 2 μg plasmid pJFnpt DNA was loaded per shot. Bombardment was carried out using a Particle Inflow Gun (Vain et al. 1993). Bombardments were performed at 8 bar with a target distance of 17 cm. Selection and regeneration of transgenic plants. Twentyfour h after bombardment, calluses were transferred to callus induction medium MS5 supplemented with either 2 mg/l bialaphos, 5 mg/l PPT, 100 mg/l hygromycin, 50 mg/l paromomycin or100 mg/l paromomycin. The bombarded calluses were incubated at 24°C under a 16/8h photoperiod (light/dark, white fluorescent light, 100 μmol m−2 s−1) and were subcultured to fresh selection media every 3 wk. After 6–12-wk selection, bialaphos-, PPT-, hygromycin- and paromomycin-resistant calluses
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were transferred to regeneration medium M1G (MS basal medium, 0.2 mg/l kinetin) supplemented with the appropriate selective agent. Plants with roots developed within 9– 12 wk on regeneration media. Fully recovered plants were transferred to containers (one half-strength MS medium supplemented with 2% sucrose and 3.75 g/l Gelrite, without selective agent) for an additional 30 d growth, and green plants were subsequently transferred to soil and grown in a greenhouse. PCR analysis. For the isolation of DNA, the FastDNA kit DNA isolation system (MP Biomedicals, Solon, OH) was used. Polymerase chain reaction (PCR) was carried out with DNA, isolated from the resistant calluses and leaves to detect one of the following three selectable marker genes: (1) bar gene using bar forward (5′-GGATCTACCAT GAGCCCAGA-3′) and bar reverse (5′-TGCCTCCAGG GACTTCAG-3′); (2) hpt gene using hpt forward (5′GCTTCTGCGGGCGATTTGTGTA-3′) and hpt reverse (5′-GCTGATGCTTTGGGCCGAGGACTG-3′); (3) nptII gene using nptII forward (5′-ACAAGATGGATTGCACG CAGG-3′) and nptII reverse (5′-AACTCGTCAAGAAGGC GATAG-3′). Southern hybridization analysis. Genomic southern hybridization analyses were performed according to standard procedures (Sambrook et al. 1989). DNA digested with the restriction endonucleases BamHI or KpnI was separated by agarose gel electrophoresis and blotted onto Hybond-N membrane (GE Healthcare, Pittsburgh, PA). The bar and nptII probes were prepared by PCR using primers designed to amplify 357 and 367 bp internal fragments within the bar and nptII coding regions, respectively. The bar and nptII fragments were purified using the QIAquick® PCR purification kit (Qiagen, Hilden, Germany). The hpt probe was prepared by digesting plasmid pAct1IHPT-4 with BamHI and KpnI (GibcoBRL, San Francisco, CA) and separated on a 1.0% agarose gel. Probes were radiolabelled with α-32P-labelled dCTP (3,000 Ci/mmol) using the random primer method (Megaprime, GE Healthcare). Prehybridization, hybridization and subsequent washing steps were performed according to standard protocols (Sambrook et al. 1989). Signals were detected by exposing the blots to autoradiography films (Kodak Biomax, SigmaAldrich, St. Louis, MO) for 1 to 5 d at −70°C, depending on the signal intensity of the blots. Herbicide application. Plants regenerated from bialaphosor PPT-resistant calluses were acclimatized and tested for their response to herbicide by a leaf spray assay. All transgenic and non-transformed control plants were sprayed twice at 2-d intervals with an aqueous solution containing 0.5% (v/v) of the commercial herbicide Basta® (Hoechst
AG, Frankfurt, Germany), containing 200 g/l glufosinate ammonium on both sides of the plant leaves 1 mo after plants had been transferred to the greenhouse. The plants were scored as “dead” or “alive” according to their visible symptoms after 7 d. Statistical analysis. All Pairwise Multiple Comparison Procedures (Holm-Sidak method) were conducted to compare the following parameters: (1) selection regime difference on transformation efficiency; (2) genotype difference on transformation efficiency; (3) genotype difference on recovery frequency. A p value of
