Efficient Agrobacterium-mediated transformation of Arabidopsis thaliana using the bar gene as selectable marker

Summary We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin acetyltransferase (PAT). Hypocotyl explants from young seedlings cocultivated with agrobacteria carrying a bar gene were selected on shoot-inducing media containing different concentrations of phosphinothricin (PPT) which is an active component of bialaphos. We found that 20 mg/l of PPT completely inhibited the control explants from growing whereas the explants transformed with the bar gene gave rise to multiple shoots resistant to PPT after 3 weeks under the same selection conditions. The transformation system could also be applied to root explants. Resulting plantlets could produce viable seeds in vitro within 3 months after preparation of the explants. The stable inheritance of the resistance trait, the integration and expression of the bar gene in the progeny were confirmed by genetic tests, Southern analysis and PAT enzyme assay, respectively. In addition, the mature plants in soil showed tolerance to the herbicide Basta.Abbreviations bar bialaphos resistance gene - CIM callus-inducing medium - DTNB 5,5-dithiobis(2-nitrobenzoic acid) - GM germination medium - HPT hygromycin phosphotransferase - MS Murashige and Skoog salts - NPTII neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin - SIM shoot-inducing medium     

Bialaphos selection of stable transformants from maize cell culture

Summary Stable transformed Black Mexican Sweet (BMS) maize callus was recovered from suspension culture cells bombarded with plasmid DNA that conferred resistance to the herbicide bialaphos. Suspension culture cells were bombarded with a mixture of two plasmids. One plasmid contained a selectable marker gene, bar, which encoded phosphinothricin acetyl transferase (PAT), and the other plasmid encoded a screenable marker for -glucuronidase (GUS). Bombarded cells were selected on medium containing the herbicide bialaphos, which is cleaved in plant cells to yield phosphinothricin (PPT), an inhibitor of glutamine synthetase. The bialaphos-resistant callus contained the bar gene and expressed PAT as assayed by PPT inactivation. Transformants that expressed high levels of PAT grew more rapidly on increasing concentrations of bialaphos than transformants expressing low levels of PAT. Fifty percent of the bialaphos-resistant transformants tested (8 of 16) expressed the nonselected gene encoding GUS.     

Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus

A gene which confers resistance to the herbicide bialaphos (bar) has been characterized. The bar gene was originally cloned from Streptomyces hygroscopicus, an organism which produces the tripeptide bialaphos as a secondary metabolite. Bialaphos contains phosphinothricin, an analogue of glutamate which is an inhibitor of glutamine synthetase. The bar gene product was purified and shown to be a modifying enzyme which acetylates phosphinothricin or demethylphosphinothricin but not bialaphos or glutamate. The bar gene was subcloned and its nucleotide sequence was determined. Interspecific transfer of this Streptomyces gene into Escherichia coli showed that it could be used as a selectable marker in other bacteria. In the accompanying paper, bar has been used to engineer herbicide-resistant plants.     

Expression of a cholera toxin B subunit in transgenic lettuce (Lactuca sativa L.) using Agrobacterium-mediated transformation system

To increase expression level of cholera toxin B subunit (CTB) in lettuce plants, synthetic CTB (sCTB) gene based on the optimized codon usage was fused with an endoplasmic reticulum retention signal, KDEL. The sCTB gene was introduced into a plant expression vector and transformed to lettuce plants using Agrobacterium-mediated transformation system. As a selection marker, a bialaphos resistance (bar) gene that encodes phosphinothricin acetyltransferase (PAT), conferring tolerance to the herbicide phosphinothricin (PPT), was used. PCR amplification of genomic DNA confirmed the presence of the sCTB gene in the transgenic lettuce plants. Expressions of mRNA and protein of sCTB were observed by Northern and Western blot analyses, respectively. The sCTB synthesized in the transgenic lettuce showed strong affinity for GM₁-ganglioside suggesting that the sCTB conserved the antigenic sites for binding and proper folding of pentameric CTB structure. The expression level of CTB was relatively high, reaching total soluble protein (TSP) levels of 0.24% in transgenic lettuce.     

Regeneration of herbicide resistant transgenic rice plants following microprojectile-mediated transformation of suspension culture cells

Summary Suspension cells of Oryza sativa L. (rice) were transformed, by microprojectile bombardment, with plasmids carrying the coding region of the Streptomyces hygroscopicus phosphinothricin acetyl transferase (PAT) gene (bar) under the control of either the 5 region of the rice actin 1 gene (Act1) or the cauliflower mosaic virus (CaMV) 35S promoter. Subsequently regenerated plants display detectable PAT activity and are resistant to BASTA^TM, a phosphinothricin (PPT)-based herbicide. DNA gel blot analyses showed that PPT resistant rice plants contain a bar-hybridizing restriction fragment of the expected size. This report shows that expression of the bar gene in transgenic rice plants confers resistance to PPT-based herbicide by suppressing an increase of ammonia in plants after spraying with the herbicide.     

High-efficiency phosphinothricin-based selection for alfalfa transformation

Despite the significant advantages of using herbicide resistance for selection of genetically engineered plants, alfalfa transformation has relied primarily on selection for antibiotic resistance. In the few studies reporting the use of resistance to the herbicide phosphinothricin (PPT), transformation efficiencies were low. The present investigation describes a PPT-based selection system for alfalfa transformation that uses the phosphinothricin acetyl-transferase (pat) gene as a selectable marker and 5.0 mg l⁻¹ of bialaphos as the selective agent. The method achieves transformation efficiencies, measured as the percentage of explants giving rise to one or more transformed plantlets, greater than 50%. These plantlets accumulated detectable amounts of PAT at levels varying from 2 to 1367 pg μg⁻¹ total protein. Transformed plants transferred to soil in the greenhouse were phenotypically normal and exhibited resistance to bialaphos leaf painting at 5 g l⁻¹ and applications of PPT equivalent to field-level use (0.5 kg ha⁻¹).     

Fertile transgenic Lotus corniculatus resistant to the non‐selective herbicide phosphinothricin

Resistance to the non‐selective herbicide dl ‐phosphinothricin (PPT) was introduced into commercial Lotus corniculatus cv. Bokor by co‐cultivation of cotyledons with Agrobacterium tumefaciens AGL1 harbouring the binary vector pDM805 which contains the bialaphos resistance gene (bar) from Streptomyces hygroscopicus encoding phosphinothricin acetyltransferase (PAT) and the uidA gene encoding β‐glucuronidase. The half‐cotyledon explants were precultured on regeneration Murashige and Skoog's (MS) medium supplemented with 6‐benzyladenine (BA) and 1‐naphthaleneacetic acid (NAA) at 0.5 mg L^?1 each, 3 days prior to infection. Upon co‐cultivation, the explants were cultured on PPT‐free regeneration medium for 10 days, and then subcultured on regeneration/selection media with increasing PPT concentrations (5–7 mg L^?1) for about 18 weeks. Out of 480 initially co‐cultivated explants, 272 regenerated shoots survived the entire PPT selection procedure. Resistant shoots were grown further, multiplied by tillering that was additionally promoted by PPT and rooted on hormone‐free MS medium containing 5 mg L^?1 PPT. Established shoot cultures, continuously maintained on the same medium, have preserved PPT resistance up to now (more than 2 years). Transformed plants assessed in vitro and in a greenhouse were tolerant to the herbicide PPT at 300 mg L^?1 equivalent to more than twofold the recommended field dosage for weed eradication. Applied PPT treatment did not affect the activities of glutamine synthetase (GS; EC 6.3.1.2) and NADH‐dependent glutamate dehydrogenase (NADH‐GDH; EC 1.4.1.2) in transformed plants. However, PPT did increase the mobility of glutamine synthetase isoforms GS1 and GS2 as well as the inhibition of an additional high mobility GS (hmGS) activity. In untransformed plants, PPT treatment reduced total GS activity by 4.4‐fold while contrary the activity of NADH‐GDH was increased by ninefold. All transformed herbicide‐resistant plants were phenotypically normal and exhibited genomic stability, as were the untransformed plants analysed by flow cytometry. Under greenhouse conditions, they grew to maturity, flowered and set seeds. Stable integration and expression of the bar gene in T0 and T1 plants were confirmed by Southern and Western blot analysis, while integration of the reporter uidA gene did not occur. The bar gene was inherited in a Mendelian fashion by the progeny, as detected by PPT resistance. The production of PPT‐resistant plants may have significant practical applications in weed control in fields of L. corniculatus.     

Production of fertile transgenic maize plants by silicon carbide whisker-mediated transformation

A simple and inexpensive system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed using silicon carbide whiskers to deliver plasmid DNA carrying the bacterial bar and uidA (gus) genes. Transformed cells were selected on medium containing the herbicide bialaphos. Integration of the bar gene and activity of the enzyme phosphinothricin acetyl transferase (PAT) were confirmed in all bialaphos-resistant callus lines analysed. Fertile transgenic maize plants were regenerated. Herbicide spraying of progeny plants revealed that the bar gene was transmitted in a Mendelian fashion.     

Transgenic fertile Scoparia dulcis L., a folk medicinal plant, conferred with a herbicide-resistant trait using an Ri binary vector

Summary Transgenic herbicide-resistant Scoparia dulcis plants were obtained by using an Ri binary vector system. The chimeric bar gene encoding phosphinothricin acetyltransferase flanked by the promoter for cauliflower mosaic virus 35S RNA and the terminal sequence for nopaline synthase was introduced in the plant genome by Agrobacterium-mediated transformation by means of scratching young plants. Hairy roots resistant to bialaphos were selected and plantlets (R0) were regenerated. Progenies (S1) were obtained by self-fertilization. The transgenic state was confirmed by DNA-blot hybridization and assaying of neomycin phosphotransferase II. Expression of the bar gene in the transgenic R0 and S1 progenies was indicated by the activity of phosphinothricin acetyltransferase. Transgenic plants accumulated scopadulcic acid B, a specific secondary metabolite of S. dulcis, in amounts of 15–60% compared with that in normal plants. The transgenic plants and progenies showed resistant trait towards bialaphos and phosphinothricin. These results suggest that an Ri binary system is one of the useful tools for the transformation of medicinal plants for which a regeneration protocol has not been established.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Transgenic herbicide-resistant Atropa belladonna using an Ri binary vector and inheritance of the transgenic trait

Summary Transgenic Atropa belladonna conferred with a herbicide-resistant trait was obtained by transformation with an Ri plasmid binary vector and plant regeneration from hairy roots. We made a chimeric construct, pARK5, containing the bar gene encoding phosphinothricin acetyltransferase flanked with the promoter for cauliflower mosaic virus 35S RNA and the 3 end of the nos gene. Leaf discs of A. belladonna were infected with Agrobacterium rhizogenes harboring an Ri plasmid, pRi15834, and pARK5. Transformed hairy roots resistant to bialaphos (5 mg/l) were selected and plantlets were regenerated. The integration of T-DNAs from pRi15834 and pARK5 were confirmed by DNA-blot hybridization. Expression of the bar gene in transformed R₀ tissues and in backcrossed F1 progeny with a nontransformant and self-fertilized progeny was indicated by enzymatic activity of the acetyltransferase. The transgenic plants showed resistance towards bialaphos and phosphinothricin. Tropane alkaloids of normal amounts were produced in the transformed regenerants. These results present a successful application of transformation with an Ri plasmid binary vector for conferring an agronomically useful trait to medicinal plants.Abbreviations CaMV cauliflower mosaic virus - NPT-II neomycin phosphotransferase II - PAT phosphinothricin acetyltransferase - PPT phosphinothricin     

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Developing phosphinothricin-resistant transgenic sunflower (Helianthus?annuus L.) plants

Most investigations on genetic transformations of sunflower have used the neomycin transferase (nptII) gene as the selectable marker. We previously reported a PPT-based selection system for sunflower transformation that uses the bialaphos resistance (bar) gene as the selectable marker and 20 mg/l of phosphinothricin (PPT) as the selective agent. Sunflower (Helianthus annuus L.) variety Skorospeliy 87 was genetically transformed via Agrobacterium tumefaciens strain EHA 105 harbouring the binary plasmid vector pBAR. Two-day-old explants from mature embryos competent for direct shooting were used. Southern blot and ELISA experiments confirmed the stability of expression in two generations of transgenic plants. Transformed plants transferred to soil in the greenhouse exhibited resistance to the herbicide Basta^? at 3 l/ha.     

Production of herbicide-resistant sweet potato plants transformed with the <Emphasis Type="Italic">bar</Emphasis> gene

Herbicide-resistant sweet potato plants were produced through biolistics of embryogenic calli derived from shoot apical meristems. Plant materials were bombarded with the vectors containing the β-glucuronidase gene (gusA) and the herbicide-resistant gene (bar). Selection was carried out using phosphinothricin (PPT). Transformants were screened by the histochemical GUS and Chlorophenol Red assays. PCR and Southern-blot analyses indicated the presence of introduced bar gene in the genomic DNA of the transgenic plants. When sprayed with Basta, the transgenic sweet potato plants was tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to sweet potato.     

Agrobacterium tumefaciens mediated transfer of Phaseolus vulgaris alpha-amylase inhibitor-1 gene into mungbean Vigna radiata (L.) Wilczek using bar as selectable marker

Morphologically normal and fertile transgenic plants of mungbean with two transgenes, bar and α-amylase inhibitor, have been developed for the first time. Cotyledonary node explants were transformed by cocultivation with Agrobacterium tumefaciens strain EHA105 harboring a binary vector pKSB that carried bialaphos resistance (bar) gene and Phaseolus vulgaris α-amylase inhibitor-1 (αAI-1) gene. Green transformed shoots were regenerated and rooted on medium containing phosphinothricin (PPT). Preculture and wounding of the explants, presence of acetosyringone and PPT-based selection of transformants played significant role in enhancing transformation frequency. Presence and expression of the bar gene in primary transformants was evidenced by PCR-Southern analysis and PPT leaf paint assay, respectively. Integration of the Phaseolus vulgaris α-amylase inhibitor gene was confirmed by Southern blot analysis. PCR analysis revealed inheritance of both the transgenes in most of the T₁ lines. Tolerance to herbicide was evidenced from seed germination test and chlorophenol red assay in T₁ plants. Transgenic plants could be recovered after 8–10 weeks of cocultivation with Agrobacterium. An overall transformation frequency of 1.51% was achieved.     

Bar-expressing peppermint (Mentha × Piperita L. var. Black Mitcham) plants are highly resistant to the glufosinate herbicide Liberty

Weed control is a substantial economic input for production of mint oils, the most commercially important of which are obtained from peppermint. The objective of this research is to obtain peppermint plants resistant to the broad-spectrum herbicide glufosinate, which can be used for development of economically efficacious weed control strategies and, perhaps, serve as a paradigm in perennial crops. The bar gene, which encodes phosphinothricin acetyltransferase (PAT) which inactivates glufosinate-ammonium or phosphinothricin (PPT), was constructed into Agrobacterium tumefaciens binary vectors under the nopaline synthase (NOS) or a chimeric promoter containing a trimer of the OCS-upstream-activating sequence (UAS) to a MAS promoter/activator region[(OCS) ₃ MAS]. A total of 142 independent transgenic peppermint (cv. Black Mitcham) plants were obtained (107 and 35 were obtained with pGPTV (and pCAS1) and pATC940 vectors, respectively) and evaluated for herbicide resistance in the greenhouse after foliar application of glufosinate herbicide Liberty as the commercial product. All transgenic plants exhibited substantially less herbicide symptom development than non-transgenic Black Mitcham or untransformed tissue cultured-derived plants, albeit variation for herbicide resistance occurred amongst the transformed lines. Plants from 35 of the 142 lines were selected at random and all were PCR-positive for the presence of bar. Five lines, that were least affected, exhibited no injury symptoms to Liberty concentrations that are 4 times the standard level for control of weeds in peppermint fields. The most resistant transgenic plants had the greatest steady-state PAT mRNA levels and PAT activities. No experimental difference in herbicide resistance was evident between plant populations obtained with pGPTV (pCAS1)-bar or pATC940-bar vector. However, 4 of 35 lines transformed with (ocs) ₃ MAS-bar exhibited maximal resistance while only 1 of 107 NOS-bar lines has comparable resistance. These herbicide resistant peppermint plants will facilitate development of post-emergent herbicide control strategies that use newer generation herbicides, like glufosinate, which have reduced environmental and product residual because of metabolism by microbes and the transgenic plants.     

Herbicide resistance of tobacco chloroplasts expressing the bar gene

The chloroplast transformation system has the potential advantages of maternal inheritance and high-level expression of heterologous genes. We studied the expression of the bar gene in tobacco chloroplasts to test these ideas. The bar gene conferring tolerance to the herbicide phosphinothricin (PPT) encodes phosphinothricin acetyltransferase (PAT). It was introduced into the chloroplast genome at a targeted site by homologous recombination. Transplastomic plantlets were selected in medium supplemented with PPT (up to 50 mg l(-1)). The polymerase chain reaction (PCR) and Southern blot analysis confirmed that bar had been inserted at the specified site in the chloroplast genome. The transplastomic plants transferred to a greenhouse proved to be resistant to 2% PPT. Reciprocal crosses between wild type and transplastomic plants confirmed maternal inheritance of the PPT resistance and high levels of PAT activity in the transplastomic plants were confirmed by assays of PAT and of ammonium evolution. The technology demonstrated here could perhaps be usefully transferred to other crop species.     

Cross-protection and selectable marker genes in plant transformation

Summary Selectable marker genes play an important role in plant transformation. The level of selection pressure is generally established by generating a kill curve for the selectable marker. In most cases, the lowest concentration which kills all explants is used. This study examined two selectable marker genes, phosphinothricin acetyl transferase (PAT) and hygromycin phosphotransferase (HPT), in transformation of tobacco leaf disks. Experiments to determine the lethal level of the herbicide, glufosinate-ammonium (phosphinothricin) (PPT) using a leaf-disk regeneration assay established that no shoots regenerated at 2 to 4 mg PPT per 1. Likewise with the antibiotic, hygromycin (HYG), no plants regenerated at 50 mg hygromycin per 1. In contrast, after cocultivation of the leaf disks withAgrobacterium tumefaciens containing either the PAT or HPT gene in combination with a Bt gene for insect resistance, plants were successfully regenerated from leaf disks at 2 to 4 mg PPT per 1 and 50 mg hygromycin per 1. However, most plants regenerated at 2 and 3 mg PPT per 1 were found to be nontransformed (95–100% escapes) by i) Southern-blot analysis, ii) herbicide application test, and iii) insect feeding bioassay. On the other hand, plants that regenerated on 50 mg hygromycin per 1 and 4 mg PPT per 1 were transgenic as determined by Southern analysis, leaf assay for PPT or HYG resistance, and death of tobacco budworms feeding on these leaves. This study showed a significant level of cross-protection and/or transient expression of the PAT selectable marker gene allowing escapes (95–100%) at selection levels of 2 and 3 mg PPT per 1 which completely kill controls. On the other hand, the HPT gene at 50 mg is efficient in selecting for T-DNA integration.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 g/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 g/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.Abbreviations gus -glucuronidase - PAT phosphinothricin N-acetyltransferase - PPT phosphinothricin - MS Murashige and Skoog medium     

Stable transformation of Phaseolus vulgaris via electric-discharge mediated particle acceleration

Summary Transgenic Phaseolus vulgaris or common bean has been produced using electric-discharge particle acceleration. The method uses particle acceleration to introduce DNA into bean seed meristems. Multiple shoots are then generated and screened to recover transgenic plants at a rate of 0.03% germline transformed plants/shoot. We have been able to recover transgenic plants using both GUS and herbicide screening to introduce the gus, bar, and bean golden mosaic virus coat protein genes into the navy bean cultivar, Seafarer. The transgenic plants have been characterized over 5 generations of self-fertilization with no loss of introduced genes or expression. In addition, several families have been crossed with non-transgenic parents and these plants also show expected inheritance patterns. The introduced bar gene has been shown to confer strong resistance in transgenic beans to basta herbicide application in the greenhouse.Abbreviations BGMV bean golden mosaic virus - PAT phosphinothricin acetyltransferase