Development of antibiotic marker-free creeping bentgrass resistance against herbicides

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1 Acta Biochim Biophys Sin 2011, 43: ª The Author Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: /abbs/gmq106. Advance Access Publication 9 December 2010 Original Article Development of antibiotic marker-free creeping bentgrass resistance against herbicides Ki-Won Lee 1,2, Ki-Yong Kim 1, Kyung-Hee Kim 2, Byung-Hyun Lee 2, Jin-Seog Kim 3, and Sang-Hoon Lee 1 * 1 Grassland and Forages Division, National Institute of Animal Science, Rural Development Administration, Cheonan , South Korea 2 Division of Applied Life Science (BK21 Program), Gyeongsang National University, Jinju , South Korea 3 Korea Research Institute of Chemical Technology, Daejeon , South Korea *Correspondence address. Tel: þ ; Fax: þ ; sanghoon@korea.kr Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacteriummediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species. Keywords creeping bentgrass; CP4-EPSPS; Agrobacterium tumefaciens; herbicide; transformation Received: June 28, 2010 Accepted: September 30, 2010 Introduction Creeping bentgrass (Agrostis stolonifera L.) is used as forage [1] and is one of the turf species most widely used in golf greens around the world. When the golf green is established, weeds hinder the early growth and settlement of the grass, decrease the productivity of the green and interfer with the later growth of the grass. When creeping bentgrass is used, co-mixture with weeds is a direct cause of degradation and irregularity of the grass surface. Perennial grasses such as cape weed, chickweed, clover and creeping oxalis that become weeds in golf greens are among the most difficult grasses to control. In most cases, there are no herbicides for the selective eradication of perennial grassy weeds from lawns. Applying more than one herbicide to lawns would be better for an effective weed control. Therefore, engineering dual-herbicide-resistant genes may help reduce the appearance of resistant weeds by enabling the use of two herbicides at low concentrations, rather than the commonly used single highconcentration herbicide. The development of new herbicide-resistant species is impossible by traditional breeding methods. The only alternative to these breeding methods is molecular genetic engineering, a field which has recently undergone rapid development. In 1996, Monsanto Company launched the Roundup Ready bean in the USA, prompting the development of herbicide-resistant crops via genetic transformation. Herbicide-resistant plants have thus far been developed and commercialized to specifically target the non-selective herbicides (glyphosate and gluphosinate ammonium) on corns, cottons, canola, rice and other crops [2]. Nearly every herbicide for weed control may be resisted by a single herbicide-resistant gene, which is typically delivered on engineered vectors that also encode antibiotic-resistant selection markers. The technology of herbicide resistance has become so generalized that 77% of the transformation crops in the world are based on the technology. The principles of breeding herbicide-resistant crops can largely be divided into two types of methods. One method utilizes transformation with microbial or mutated plant genes encoding resistant variations of the herbicide targets [3 5]. The other utilizes transformation with herbicideinactivating genes [6 10]. To prevent the emergence of herbicide-resistant weeds when only one herbicide is in use, we sought to develop a new dual-resistant creeping bentgrass to facilitate a mixture processing with much lower density or systematic ( progressive) processing through a combination of non-selective herbicides. Acta Biochim Biophys Sin (2011) Volume 43 Issue 1 Page 13

2 Materials and Methods Preparation of plant materials and explants Mature seeds of creeping bentgrass (A. stolonifera L., Penncross) were dehusked, and sterilized with 70% ethanol for 1 min and 5% (w/v) sodium hypochlorite (NaOCl) for 30 min. Seeds were thoroughly rinsed with sterile water and placed on callus induction medium, containing MS basal salts and vitamins [11], 2.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/l 6-benzylaminopurine (BA), 0.5 mg/l proline, 30 g/l sucrose and 3 g/l Gelrite, and incubated at 258C under dim-light conditions (5 10 mol/m 2 /s, 16 h light). After 4 weeks of incubation, callus tissues with shiny and compact structures were selected and maintained by subculturing on the same medium at 3-week intervals. Actively growing embryogenic callus tissues were visually selected, separated into small pieces and used for transformation experiments. Vector construction and genetic transformation The expression vector was generated by introducing the glyphosate-resistant gene, CP4-EPSPS, into the pcambia3300 binary vector. The transfer (T)-DNA fragment of this binary vector contained the phosphinotricin acetyltransferase (bar) and Enol-pyruvylshikimate-3-phosphate synthase (CP4-EPSPS) genes, which were originally cloned from Streptomyces hygroscopicus and Agrobacterium strain CP4, respectively. Both genes were controlled by the CaMV 35S promoter and nos terminator (Fig. 1). Agrobacterium tumefaciens strain EHA105 was used for genetic transformation in all of the experiments. The recombinant vector, pcambia3300 CP4-EPSPS, was introduced into Agrobacterium strain EHA105 by the freeze thaw transformation method [12]. Agrobacterium transformants were inoculated into the YEP liquid medium, containing 50 mg/l kanamycin and grown overnight at 288C. Inoculation and co-cultivation A. tumefaciens cells were harvested by centrifugation at 2500 g for 10 min and resuspended in a liquid MS Figure 1 Schematic diagram of the plasmid construct used for transformation RB, right border; LB, left border; 35S, CaMV 35S promoter; bar, phosphinotricin acetyltransferase gene coding region; CP4-EPSPS, Enol-pyruvylshikimate-3-phosphate synthase; T 35S, CaMV 35S terminator; T NOS, nopaline synthase terminator; HindIII and EcoRI, restriction sites. Bold bar used in Southern analysis. medium, containing 30 g/l sucrose and 100 mm acetosyringone until the OD 600 attained 0.6. Batches of 200 callus explants were immersed in 30 ml of A. tumefaciens suspension for 30 min, blotted with filter paper to remove excess bacteria and transferred to a co-cultivation medium, which consisted of an MS medium, containing 2.5 mg/l 2,4-D, 0.1 mg/l BA, 1 g/l casein hydrolysate, 30 g/l sucrose and 3 g/l Gelrite, with 100 mm acetosyringone. Co-cultivation was performed at 258C under dark conditions for 5 days. Selection and regeneration of transgenic plants Following co-cultivation, calli were washed with sterile distilled water, containing 250 mg/l cefotaxime, transferred onto the MS basal medium, containing 1 g/l casein hydrolysate, 3 mg/l 2,4-D, 0.1 mg/l BA, 30 g/l sucrose, 3 g/l Gelrite and 250 mg/l cefotaxime and then cultured at 258C. After 5 days, the calli were moved to the selection medium, which consisted of the N6 medium [13], containing 1 mg/l 2,4-D, 3 mg/l BA, 1 g/l casein hydrolysate, 30 g/l sucrose, 3 g/l Gelrite, 300 mg/l cefotaxime and 10 mg/l phosphinothricin (PPT). Calli were subcultured every 3 weeks onto the fresh medium of same composition and incubated at 258C under 16 h light photoperiod (70 mol/m/s). After 6 8 weeks, regenerated putative transgenic shoots were separated and transferred onto a rooting medium which consisted of half-strength MS medium containing 30 g/l maltose, 3 g/l Gelrite and 10 mg/l PPT. After 3 4 weeks, PPT-resistant plants were transferred to soil. Molecular analyses of transgenic plants PPT-resistant plants were selected for molecular analyses by PCR and Southern hybridization. Genomic DNA was isolated from the leaf tissues of putative transgenic (T0) and wild-type plants, as described previously [14]. Internal fragments of CP4-EPSPS and bar were amplified by two individual PCRs. The EPSPS-specific forward (5 0 -ACGA TTTCGACAGCACCTTC-3 0 ) and reverse (5 0 -GTGACA GGGTTTTCCGACAC-3 0 ) primers yielded a 928-bp fragment inside the synthetic EPSPS gene. A separate reaction was carried out using the bar-specific forward (5 0 -CGGTCTGCACCATCGTCAACC-3 0 ) and reverse (5 0 -GTCCAGCTGCCAGAAACCCAC-3 0 ) primers, yielding a 443-bp fragment inside the bar gene. The transgenes CP4-EPSPS and bar were also amplified in the same reactions. Integration of CP4-EPSPS into the genome of transgenic plants was confirmed by Southern blot analysis. Genomic DNA (20 mg) was digested with HindIII and separated by electrophoresis in a 1.2% agarose gel, then transferredtoahybondnþ (Amersham, Buckinghamshire, UK) membrane. The earlier CP4-EPSPS-specific PCR product was used as a probe and Southern blot hybridization was carried out as described previously [14]. Acta Biochim Biophys Sin (2011) Volume 43 Issue 1 Page 14

3 RNA isolation and Northern blot analysis Northern blot analysis was carried out to investigate the expression levels of the CP4-EPSPS and bar genes in transgenic bentgrass plants. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer s instructions. RNA samples (10 mg) were separated on a 1.2% agarose gel containing formaldehyde. The 928-bp CP4-EPSPS and 443-bp bar PCR products were labeled with [a- 32 P]-dCTP and used as probes. Northern hybridization was performed as described previously [15]. ELISA assays ELISA assays were used to quantify CP4-EPSPS protein, essentially as described previously [16]. Leaf samples from non-transgenic plants and independent transgenic lines were collected. Then, extracts from 0.2 g of the crushed plant material were added to the MEB sample buffer (1:20) and poured into plates pre-coated with anti-cp4 EPSPS antibody (Agdia, Inc., Elkhart, IN, USA), 100 ml per plate, and kept for 1 h at room temperature. The plates were washed six to seven times with PBST washing buffer and left to soak in PBST buffer for 3 min at room temperature. The peroxidase enzyme conjugate (100 ml) was added at 1:100, and the plates were kept for 1 h at room temperature. They were washed six to seven times again with the PBST washing buffer. A TMB substrate solution (100 ml) was added for the color reaction, and incubated for 20 min before the color was measured at 650 nm. Analyses of herbicide tolerance For herbicide tolerance analyses, non-transgenic plants and three independent T0 transgenic tall fescue lines, E1, E2 and E3, were used. The CP4-EPSPS and bar transgenic lines and non-transformed control plants were analyzed for glufosinate and basta resistance. Plants were grown in a growth chamber and sprayed once daily for 7 days with a 0.5% (v/v) solution (900 mg/l glufosinate ammonium) of the commercial herbicide Basta (Bayer Crop Science Ltd., Monheim, Germany), a commercial formulation of glufosinate ammonium salt (18%). Milli-Q water was used as a control. The treatment procedure was performed according to that described by Lee et al. [16]. The plants were observed over 1 week to determine resistance or susceptibility to the herbicide. In all cases, biosafety regulations were followed strictly according to Korean Government s Biosafety Policy. Results and Discussion Production of transgenic creeping bentgrass Transformation efficiency is largely influenced by the quality of the callus. Embryogenic callus tissue derived from mature seeds has previously been used and is considered to be the best target tissue for Agrobacterium-mediated transformation of creeping bentgrass [17,18]. In this study, we performed Agrobacterium-mediated transformation on embryogenic calli derived from the scutellum of mature seeds. Over the past several years, consumers and environment-protective organizations have expressed concerns about the use of antibiotic-resistant genes from an ecological and food safety perspective. Antibiotics are known to inhibit the growth and regeneration of transformed cells, and thereby decrease the transformation frequency [19]. Avoiding antibiotic selectable markers would be, therefore, a better strategy to develop herbicide-resistant plants. The constructed gene containing a bar and a CP4-EPSPS gene driven by CaMV 35S promoter was introduced into creeping bentgrass genome. Four-week-old calli of creeping bentgrass were co-cultivated with Agrobacterium EHA105 carrying the bar and CP4-EPSPS recombinant plasmid, pcambia3300. More than 16 PPT-resistant, putative transformants were regenerated, transferred to pots and successfully acclimated to glasshouse conditions (Fig. 2). Results from independent experiments are summarized in Table 1. Stable transformation efficiency in these experiments averaged 9.4% and ranged from 8.8% to 9.8%. A 5% 60% transformation efficiency has been reported in creeping Figure 2 Production of transgenic creeping bentgrass plants (A, B) Calli initiated from seeds after 4 weeks. (C) Embryogenic callus after 8 weeks, used for Agrobacterium-mediated transformation. (D, E) Regenerated transgenic resistant green shoots compared with dead calli after being transfered to selective regeneration medium for 4 weeks. (F, G) Root growth of transgenic shoots. (H) Well-rooted in vitro transgenic plants after transferring the regenerated shoots to rooting medium. (I) Greenhouse-grown transgenic creeping bentgrass plants after Agrobacterium-mediating transformation. Acta Biochim Biophys Sin (2011) Volume 43 Issue 1 Page 15

4 Table 1 Efficiency of Agrobacterium tumefaciens-mediated transformation of creeping bentgrass in three independent experiments Experiment No. of calli infected No. of transgenic plants a Transformation frequency (%) Total a Total number of independent transgenic creeping bentgrass events was confirmed by PCR analysis and 0.5% (v/v) Basta application. bentgrass [17,20]. These differences could be attributed to the use of different vectors and gene constructs. There were no apparent phenotypic or developmental differences between the non-transgenic and transgenic plants. Figure 4 Southern blot analysis of the transgenic creeping bentgrass plants Genomic DNA was digested with HindIII and hybridized with [a- 32 P] dctp-labeled CP4-EPSPS probe. WT, untransformed control plant; 1 10, transgenic creeping bentgrass plants. Molecular analysis of the transgenic plants The presence of the transgenes was confirmed by PCR analysis. PCR analysis confirmed the integration of the foreign gene into the genome of the putative transgenic creeping bentgrass plants (Fig. 3). PCR amplifications produced the expected 928-bp fragment of CP4-EPSPS and 443-bp fragment of the bar gene from both the putative transgenic lines and the plasmid DNA used as a positive control. Transgene DNA was not detected in nontransgenic wild-type plants. Integration of CP4-EPSPS was further confirmed by Southern blot analysis of independent transgenic lines. Each line exhibited a specific hybridization pattern, and hybridization with the CP4-EPSPS probe for copy number reconstruction showed the presence of only one copy of the gene. This result reinforces the hypothesis that the Agrobacterium-mediated method of transformation is better than the biolistic method, which frequently produces transformants with multiple copies of the transgene [21]. No cross-hybridizing band was seen in the wild type (Fig. 4). Figure 3 PCR analysis of T-DNA insertion into the genome of creeping bentgrass Two primer sets (EPSPS and bar) were used to amplify from 500 ng plant genomic DNA and/or 10 pg EPSPS vector. Mw, 1-kb DNA ladder; P, plasmid DNA; W, genomic DNA derived from the untransformed control plants; 1 10, transgenic creeping bentgrass plants. Arrows indicate the expected EPSPS and bar fragments. Figure 5 Northern blot analysis of transgenic creeping bentgrass plants Hybridized with [a- 32 P]dCTP-labeled CP4-EPSPS and bar probes. WT, untransformed control plant; 1 10, transgenic creeping bentgrass plants. Northern blotting was used to verify the expression of CP4-EPSPS in transgenic plants. CP4-EPSPS transcripts were expressed at very high levels in transgenic creeping bentgrass plants (Fig. 5). ELISA test of the transgenic plants ELISA is a quantitative, diagnostic technique that uses antibodies to detect proteins. To observe CP4-EPSPS expression in 10 independent lines, ELISA was conducted using CP4-EPSPS antibody. Each test consisted of three replications. The results demonstrated that the transformed plants expressed the protein at high levels. Each transformed line showed a different level of protein expression (Fig. 6), indicating that the amount of expressed protein was dependent upon the location of the gene insertion. The absorbance levels derived from the transgenic plants were almost 13 times greater than the negative control and greater than the positive control. Herbicide tolerance of the transgenic plants All transgenic lines exhibited similar transgene expression at the protein level; therefore, we tested three independent uniform-sized transgenic lines (E1, E2 and E3) and nontransgenic control for herbicide tolerance. Glyphosate and Acta Biochim Biophys Sin (2011) Volume 43 Issue 1 Page 16

5 Figure 6 ELISA protein detection in greenhouse-grown transgenic plants Qualitative (top panel) and quantitative (bottom panel) determination of proteins detected by ELISA. Quantitative data are means (SE) of three replicates as described in Materials and Methods section. N, negative control; P, positive control; 1 10, transgenic creeping bentgrass plants. glufosinate are non-selective, show no special contamination risk to the environment and show lower toxicity than postemergent herbicides. The use of dual-herbicide resistance might help reduce the appearance of resistant weeds by enabling the use of two herbicides in lower concentrations, rather than the commonly used single high-concentration herbicides. There was no significant difference in the growth of wild-type and transgenic plants in the absence of herbicides. However, significant differences were observed between the wild-type and the transgenic plants in the presence of glyphosate and/or glufosinate. Non-transgenic plants treated with glyphosate exhibited necrosis within 2 days, leaf drying appeared on Day 3 and most of the plants had withered by Day 5, while the transgenic plants remained largely unaffected. Non-transgenic plants treated with glufosinate exhibited leaf drying, etiolation and chlorosis on Day 2, and withering death of all units by Day 4. The transgenic lines did not show any significant susceptibility to the herbicide treatments (Fig. 7). The transgenic lines exposed to a combination of glyphosate and glufosinate had characteristics similar to those observed with the single-agent glufosinate test. In conclusion, the transgenic creeping bentgrass exhibited equal resistance to both types of herbicide, in both single and combined treatments. In conclusion, the developed transgenic creeping bentgrass plants had significant resistance against two herbicides glyphosate and glufosinate. Dual-herbicideresistant gene allows an effective weed control using two herbicides at low concentrations. This strategy would be useful in reducing the cost of weed management as well as soil toxicity. References Figure 7 Herbicide resistance in transgenic lines E1, E2 and E3 Plants were sprayed with glyphosate and glufosinate once daily for 7 days. Photograph was taken at the 8th day. 1 Wang Z, Hopkins A and Mian R. Forage and turf grass biotechnology. Crit Rev Plant Sci 2001, 20: Padgette SR, Kolacz KH, Delannay X, Re DB, LaVallee BJ, Tinius CN and Rhodes WK, et al. Development, identification, and characterization of a glyphosate-tolerant soybean line. Crop Sci 1995, 35: Comai L, Facciotti D, Hiatt WR, Thompson G, Rose RE and Stalker DM. Expression in plants of a mutant aroa gene from Salmonella typhimurium confers tolerance to glyphosate. Nature 1985, 317: Haughn GW, Smith J, Mazur B and Somerville C. Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides. Mol Gen Genet 1988, 211: Mousdale DM and Coggins JR. Subcellular localization of the common shikimate-pathway enzymes in Pisum sativum L. Planta 1985, 161: Barry G, Kishore G, Padgette S, Taylor M, Kolacz K, Weldon M and Re D, et al. Inhibitors of amino acid biosynthesis: strategies for imparting glyphosate tolerance to crop plants. In: Singh BJ, Flores HE and Shannon JC, eds. Current Topics in Plant Physiology. Vol 7: Biosynthesis and Molecular Regulation of Amino Acids in Plants. Rockville, MD: American Society of Plant Physiology 1992, Acta Biochim Biophys Sin (2011) Volume 43 Issue 1 Page 17

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