Stability and expression of amplified EPSPS genes in glyphosate resistant tobacco cells and plantlets

Summary The stability and expression of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) genes was examined in glyphosate resistant tobacco cells grown in glyphosate-free medium, and in plantlets regenerated from resistant cells. Amplified DNA was maintained in resistant cells grown in the absence of glyphosate for three years. Amplified EPSPS genes were retained in regenerated plantlets at levels comparable to those observed in the resistant cells, and EPSPS mRNA was overexpressed (compared to unselected plantlets). However, glyphosate resistance in cell lines grown in glyphosate-free medium declined 7-fold, and in regenerated plantlets approximately 20-fold, compared to resistant cells maintained under glyphosate selection. In plantlets, reduced resistance correlated with lower levels of EPSPS mRNA. Plantlets regenerated from resistant cells exhibited morphological variation, and had an approximate doubling of their nuclear genome size.     

Glyphosate selected amplification of the 5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells

Summary CAR and C1, two carrot (Daucus carota L.) suspension cultures of different genotypes, were subjected to stepwise selection for tolerance to the herbicide glyphosate [(N-phosphonomethyl)glycine]. The specific activity of the target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), as well as the mRNA level and copy number of the structural gene increased with each glyphosate selection step. Therefore, the tolerance to glyphosate is due to stepwise amplification of the EPSPS genes. During the amplification process, DNA rearrangement did not occur within the EPSPS gene of the CAR cell line but did occur during the selection step from 28 to 35 mM glyphosate for the C1 cell line, as determined by Southern hybridization of selected cell DNA following EcoRI restriction endonuclease digestion. Two cell lines derived from a previously selected glyphosate-tolerant cell line (PR), which also had undergone EPSPS gene amplification but have been maintained in glyphosate-free medium for 2 and 5 years, have lost 36 and 100% of the increased EPSPS activity, respectively. Southern blot analysis of these lines confirms that the amplified DNA is relatively stable in the absence of selection. These studies demonstrate that stepwise selection for glyphosate resistance reproducibly produces stepwise amplification of the EPSPS genes. The relative stability of this amplification indicates that the amplified genes are not extrachromosomal.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiothreitol - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase - I₅₀ 50% inhibitory concentration - Kb Kilobase (pairs) - PEP phosphoenolpyruvate - PMSF phenylmethylsulfonyl fluoride - PVPP polyvinylpolypyrrolidone - S-3-P shikimate-3-phosphate     

A novel 5-enolpyruvylshikimate-3-phosphate synthase shows high glyphosate tolerance in Escherichia coli and tobacco plants

A key enzyme in the shikimate pathway, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) is the primary target of the broad-spectrum herbicide glyphosate. Identification of new aroA genes coding for EPSPS with a high level of glyphosate tolerance is essential for the development of glyphosate-tolerant crops. In the present study, the glyphosate tolerance of five bacterial aroA genes was evaluated in the E. coli aroA-defective strain ER2799 and in transgenic tobacco plants. All five aroA genes could complement the aroA-defective strain ER2799, and AM79 aroA showed the highest glyphosate tolerance. Although glyphosate treatment inhibited the growth of both WT and transgenic tobacco plants, transgenic plants expressing AM79 aroA tolerated higher concentration of glyphosate and had a higher fresh weight and survival rate than plants expressing other aroA genes. When treated with high concentration of glyphosate, lower shikimate content was detected in the leaves of transgenic plants expressing AM79 aroA than transgenic plants expressing other aroA genes. These results suggest that AM79 aroA could be a good candidate for the development of transgenic glyphosate-tolerant crops.     

Length of time in tissue culture can affect the selected glyphosate resistance mechanism

Usually, stepwise selection of plant suspension cultures with gradually increasing concentrations of the herbicide glyphosate results in the amplification of the target enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) gene that leads to resistance by increasing EPSPS mRNA and enzyme activity. We show that glyphosate selection with newly initiated suspension cultures can produce resistant lines with resistance mechanisms other than gene amplification and that usually as the cultures age gene amplification becomes the predominant mechanism. Gene amplification did not occur in 3 lines selected from 5-month-old Datura innoxia Mill. cultures but did occur in all 10 lines selected after 52 months. Selection with Nicotiana tabacum L. (tobacco) less than 5 months old produced 2 lines out of 24 with no EPSPS amplification while all 17 lines selected from older cultures contained amplified genes. Lines selected from the oldest culture (35 years) also exhibited amplification of several different genes, indicating the expression of different EPSPS genes or an enhanced gene amplification incidence. None of the 15 lines selected from 2 different 5-month-old Daucus carota L. (carrot) lines exhibited amplification while amplification led to the resistance of all 7 lines selected from one of the original carrot lines (DHL) after 3 years. However, the other line (Car4) was exceptional and produced only non-amplified lines (9 of 9) after 8 years in culture. These results show that plant tissue cultures change with time in culture and that several different new mechanisms can result in glyphosate resistance.Abbreviations AHAS acetohydroxyacid synthase - EPSPS 5-enolpyruvylshikimate-3-phosphate synthase     

Allele exchange at the EPSPS locus confers glyphosate tolerance in cassava

Effective weed control can protect yields of cassava (Manihot esculenta) storage roots. Farmers could benefit from using herbicide with a tolerant cultivar. We applied traditional transgenesis and gene editing to generate robust glyphosate tolerance in cassava. By comparing promoters regulating expression of transformed 5‐enolpyruvylshikimate‐3‐phosphate synthase (EPSPS) genes with various paired amino acid substitutions, we found that strong constitutive expression is required to achieve glyphosate tolerance during in vitro selection and in whole cassava plants. Using strategies that exploit homologous recombination (HR) and nonhomologous end‐joining (NHEJ) DNA repair pathways, we precisely introduced the best‐performing allele into the cassava genome, simultaneously creating a promoter swap and dual amino acid substitutions at the endogenous EPSPS locus. Primary EPSPS‐edited plants were phenotypically normal, tolerant to high doses of glyphosate, with some free of detectable T‐DNA integrations. Our methods demonstrate an editing strategy for creating glyphosate tolerance in crop plants and demonstrate the potential of gene editing for further improvement of cassava.     

Plastid-expressed 5-enolpyruvylshikimate-3-phosphate synthase genes provide high level glyphosate tolerance in tobacco

Plastid transformation (transplastomic) technology has several potential advantages for biotechnological applications including the use of unmodified prokaryotic genes for engineering, potential high-level gene expression and gene containment due to maternal inheritance in most crop plants. However, the efficacy of a plastid-encoded trait may change depending on plastid number and tissue type. We report a feasibility study in tobacco plastids to achieve high-level herbicide resistance in both vegetative tissues and reproductive organs. We chose to test glyphosate resistance via over-expression in plastids of tolerant forms of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Immunological, enzymatic and whole-plant assays were used to prove the efficacy of three different prokaryotic (Achromobacter, Agrobacterium and Bacillus) EPSPS genes. Using the Agrobacterium strain CP4 EPSPS as a model we identified translational control sequences that direct a 10,000-fold range of protein accumulation (to >10% total soluble protein in leaves). Plastid-expressed EPSPS could provide very high levels of glyphosate resistance, although levels of resistance in vegetative and reproductive tissues differed depending on EPSPS accumulation levels, and correlated to the plastid abundance in these tissues. Paradoxically, higher levels of plastid-expressed EPSPS protein accumulation were apparently required for efficacy than from a similar nuclear-encoded gene. Nevertheless, the demonstration of high-level glyphosate tolerance in vegetative and reproductive organs using transplastomic technology provides a necessary step for transfer of this technology to other crop species.     

Glyphosate selection of gene amplification in suspension cultures of 3 plant species

Stepwise selection was carried out with increasing glyphosate concentrations to produce suspension cultures of Medicago sativa L. (alfalfa), Glycine max L. (Merr.) (soybean) and Nicotiana tabacum L. (tobacco) (two lines) that were at least 100-fold more resistant than the original culture as measured by the I₅₀. The selection process required from 8 to 11 transfers to fresh medium over a total period from 161 to 312 days. The alfalfa and soybean lines contained 62- and 21-fold higher activity levels of the glyphosate target enzyme, 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), respectively. The tobacco lines had EPSPS enzyme activity levels more than 800-times higher than the original cultures. The EPSPS gene copy number and mRNA were increased in all of the lines as measured by southern and northern hybridization, respectively. Thus, as has been found before with most glyphosate-resistant suspension cultures, the resistance is caused by high EPSPS enzyme activity due to EPSPS gene amplification. Alfalfa and soybean EPSPS gene amplification and the very high EPSPS enzyme activity increases found in the tobacco cultures have not been reported before. These studies show that EPSPS gene amplification can occur in many plant species to confer glyphosate tolerance.     

Glyphosate-Tolerant Crops: Genes and Enzymes

This review focuses on the genes for the enzymes 5-enolpyruvyl-3-phosphoshikimlc acid synthase (EPSPS) and the glyphosate oxidoreductase (GOX). These genes have been used to genetically engineer plants that are resistant to the herbicide glyphosate. Overproduction of glyphosate-insensitive.EPSPS in transgenic crops has been used to overcome the deleterious effuts of this herbicide. The introduction into plants of GOX also confers glyphosate tolerance to plants and augments the tolerance of transgenic plants already expressing a glyphosate tolerant EPSPS. These genes also provide a method for selecting transformed plant tissue using the glyphosate tolerance as the selectable marker in the presence of inhibitory concentrations of glypllosate. Glyphosate tolerant transgenic plants of beet, corn, cotton, lettuce, poplar, potato, rapeseed. soybean, tobacco, tomato, and wheat have already been field tested and are entering agriculture.     

Expression of glyphosate resistance in carrot somatic hybrid cells through the transfer of an amplified 5-enolpyruvylshikimic acid-3-phosphate synthase gene

Summary A Daucus carota cell line selected as resistant to N-(phosphonomethyl)-glycine (glyphosate) was found to have increased levels of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) activity of 5.5 times over wild-type carrot and an EPSPS protein level increase of 8.7 times as confirmed by Western hybridization analysis. Southern blot hybridization using a petunia EPSPS probe showed increases in the number of copies of EPSPS genes in the glyphosate-resistant line which correlated with the higher levels of the EPSPS enzyme. The mechanism of resistance to glyphosate is therefore due to amplification of the EPSPS gene. To examine the stability of the amplified genes, cloned lines selected as doubly resistant to Dl-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with the amplified EPSPS glyphosate-resistant cell line. Somatic hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semi-dominant traits, in a line-specific manner. The hybrid lines possessed additive chromosome numbers of the parental lines used and no double minute chromosomes were observed. The glyphosate-resistant parental line and most somatic hybrids retained the amplified levels of EPSPS in the absence of selection pressure over a 3-year period.     

Novel AroA from Pseudomonas putida confers tobacco plant with high tolerance to glyphosate

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1(optimized)) at the 5' end. The PparoA1(optimized) gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T(1) progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1(optimized) gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future.     

Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of <Emphasis Type="Italic">E. coli</Emphasis> (k12) and transformation of rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) in order to make tolerance to glyphosate

Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). This is a key enzyme in the aromatic amino acid biosynthesis pathway of microorganisms and plants. The manipulation of bacterial EPSPS gene in order to reduce its affinity for glyphosate, followed by its transfer to plants is one of the most effective approaches for the production of glyphosate-tolerant plants. In this study, we chose to focus on amino acid residues glycine96 and alanine183 of the E. coli (k12) EPSPS enzyme. These two amino acids are important residues for glyphosate binding. We used site directed mutagenesis (SDM) to induce point mutations in the E. coli EPSPS gene, in order to convert glycine96 to alanine (Gly96Ala) and alanine183 to threonine (Ala183Thr). After confirming the mutation by sequencing, the altered EPSPS gene was transferred to rapeseed (Brassica napus L.) via Agrobacterium-mediated transformation. The transformed explants were screened in shoot induction medium containing 25 mg L⁻¹ kanamycin. Glyphosate tolerance was assayed in putative transgenic plants. Statistical analysis of data showed that there was a significant difference between the transgenic and control plants. It was observed that transgenic plants were resistant to glyphosate at a concentration of 10 mM whereas the non-transformed control plants were unable to survive 1 mM glyphosate. The presence and copy numbers of the transgene were confirmed with PCR and Southern blotting analysis, respectively.     

甘薯草甘膦抗性基因EPSPS克隆和序列分析

EPSPS基因编码5-烯醇式丙酮酰莽草酸-3-磷酸合成酶,该酶是芳香族氨基酸合成的关键酶,该基因在细菌、真菌、藻类和植物中被广泛克隆和研究。EPSPS酶是草甘膦除草剂的靶点酶,过量表达EPSPS基因可以提高作物的草甘膦抗性。该研究根据甘薯基因组数据库设计引物,以‘广薯87’为材料提取RNA,通过RT-PCR方法扩增甘薯IbEPSPS基因,测序后进行生物信息学分析和表达分析。结果表明:(1)成功克隆获得甘薯IbEPSPS基因,该基因全长CDS为1569 bp,编码522个氨基酸,其中在第98~113、173~183位氨基酸序列具有2个EPSPS的保守结构域。(2)系统进化树分析结果表明,甘薯IbEPSPS基因与三裂叶薯(Ipomoea triloba)、打碗花(Calystegia hederacea)、田旋花(Convolvulus arvensis)和牵牛(Ipomoea nil)聚在一类,其中与三裂叶薯的亲缘关系最近。(3)实时荧光定量PCR分析结果表明,甘薯IbEPSPS基因在茎、叶和茎尖表达量较高,同时受到草甘膦胁迫后IbEPSPS基因表达量提高。该研究结果为进一步探讨甘薯IbEPSPS基因的功能及甘薯对草甘膦的耐药性机制奠定了基础。     

植物莽草酸途径EPSPS蛋白的分子进化和基因结构分析

EPSPS既是植物、微生物和真菌等生物芳香族氨基酸生物合成途径——莽草酸途径中的关键酶,也是除草剂草甘膦的靶标酶。EPSPS的克隆能为草甘膦抗性转基因作物的研发提供候选基因。该研究运用比较基因组学方法,通过对41种不同植物的43条EPSPS蛋白序列进行进化分析,取得主要结果如下:(1)不同植物EPSPS蛋白的相似性很高,且具有相同的结构域、保守基序和保守位点,但是其叶绿体转运肽序列差异显著;(2)系统发育分析表明,EPSPS基因按照双子叶植物纲和单子叶植物纲分为2个大的分支,各个小的分支又按照植物的种属亲缘关系进行分支和聚类;(3)基因结构分析表明,植物EPSPS基因基本都含有8个外显子和7个内含子,且所对应外显子的长度相当,而内含子的长度差异很大,说明在植物基因组进化过程中造成EPSPS基因结构差异的主要因素是内含子的改变。研究结果将为揭示植物EPSPS蛋白的结构功能提供参考。     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.     

Structure of the amplified 5-enolpyruvylshikimate-3-phosphate synthase gene in glyphosate-resistant carrot cells

The structure of amplified 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) DNA of carrot suspension-cultured cell lines selected for glyphosate resistance was analyzed to determine the mechanism of gene amplification in this plant system. Southern hybridization of the amplified DNA digested with several restriction enzymes probed with a petunia EPSPS cDNA clone showed that there were differences in fragment sizes in the amplified DNA from one highly resistant cell line in comparison with the parental line. Cloning of the EPSPS gene and 5 flanking sequences was carried out and two different DNA structures were revealed. A 13 kb clone contained only one copy of the EPSPS gene while a 16 kb clone contained an inverted duplication of the gene. Southern blot analysis with a carrot DNA probe showed that only the uninverted repeated DNA structure was present in all of the cell lines during the selection process and the inverted repeat (IR) was present only in highly amplified DNA. The two structures were present in about equal amounts in the highly amplified line, TC 35G, where the EPSPS gene was amplified about 25-fold. The presence of the inverted repeat (IR) was further verified by resistance to S1 nuclease hydrolysis after denaturation and rapid renaturation, showing foldback DNA with the IR length being 9.5 kb. The junction was also sequenced. Mapping of the clones showed that the size of the amplified carrot EPSPS gene itself is about 3.5 kb. This is the first report of an IR in amplified DNA of a target enzyme gene in selected plant cells.     

Selection of Glyphosate-Tolerant Tobacco Calli and the Expression of this Tolerance in Regenerated Plants

From nonmutagenized haploid suspensions of Nicotiana tabacum L. cv Wisconsin 38 cells, 51 cell lines capable of growth in the presence of 1 millimolar glyphosate (N-phosphonomethyl glycine) were initially isolated at a frequency of 2.3 × 10⁻⁸. Eighteen cell lines retained tolerance when grown on selective medium for 3 years. Tolerance persisted for at least 14 months in six cell lines cultured in the absence of glyphosate. Some plants regenerated from four glyphosate-tolerant cell lines were tolerant. Glyphosate-tolerant tissue was isolated from some sensitive as well as some tolerant regenerated plants. Six of the tolerant cell lines were also tolerant to the herbicide amitrole (3-amino-1,2,4-triazole). Five cell lines selected for amitrole tolerance were glyphosate tolerant. Some plants regenerated from three of these five cell lines were glyphosate tolerant and glyphosate-tolerant tissue was obtained from several of these regenerated plants. Amitrole uptake in suspension cultures of several variants was assessed in terms of influx rate constants. This parameter was not sufficiently different indicating that altered membrane properties could not account for the herbicide tolerance.     

转基因抗草甘膦油菜的检测方法

目的:建立检测田间转基因抗草甘膦油菜的方法。方法:用CTAB法提取DNA,经PCR分别扩增内参基因BnACCg8,及抗草甘膦外源基因CP4-EPSPS、FMV 35S启动子和E9 3’终止子等4种基因的片段,以琼脂糖凝胶电泳对扩增产物进行分析比对。结果:抗草甘膦油菜中分别扩增出与外源基因FMV 35S启动子、E9 3’终止子和CP4-EPSPS大小一致的片段。结论:该方法能有效地用于转基因抗草甘膦油菜的检测。     

Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops

Summary. Acetohydroxyacid synthase (AHAS) inhibitors interfere with branched-chain amino acid biosynthesis by inhibiting AHAS. Glyphosate affects aromatic amino acid biosynthesis by inhibiting 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Glufosinate inhibits glutamine synthetase and blocks biosynthesis of glutamine. AHAS gene variants that confer tolerance to AHAS inhibitors have been discovered in plants through selection or mutagenesis. Imidazolinone-tolerant crops have been commercialized based on these AHAS gene variants. A modified maize EPSPS gene and CP4-EPSPS gene from Agrobacterium sp. have been used to transform plants for target-based tolerance to glyphosate. A gox gene isolated from Ochrobactrum anthropi has also been employed to encode glyphosate oxidoreductase to detoxify glyphosate in plants. Glyphosate-tolerant crops with EPSPS transgene alone or both EPSPS and gox transgenes have been commercialized. Similarly, bar and pat genes isolated from Streptomyces hygroscopicus and S. viridochromogenes, respectively, have been inserted into plants to encode phosphinothricin N-acetyltransferase to detoxify glufosinate. Glufosinate-tolerant crops have been commercialized using one of these two transgenes.     

A Comparative Risk Assessment of Genetically Engineered,Mutagenic, and Conventional Wheat Production Systems

Wheat (Triticum aestivum L.) varieties produced using modern biotechnologies, such as genetic engineering and mutagenic techniques, have lagged behind other crop species, but are now being developed and, in the case of mutagenic wheat, commercially grown around the world. Because these wheat varieties have emerged recently, there is a unique opportunity to assess comparatively the potential environmental risks (human health, ecological, and livestock risks) associated with genetically engineered, mutagenic, and conventional wheat production systems. Replacement of traditional herbicides with glyphosate in a glyphosate-tolerant (genetically engineered) wheat system or imazamox in an imidazolinone-tolerant (mutagenic) wheat system may alter environmental risks associated with weed management. Additionally, because both systems rely on plants that express novel proteins, the proteins and plants themselves may impose risks. The purpose of our study was to examine comparatively the multiple aspects of risk associated with different wheat production systems in the US and Canada using the risk assessment paradigm. Specifically, we used tier 1 quantitative and qualitative risk assessment methods to compare specific environmental risks associated with the different wheat production systems. Both glyphosate and imazamox present lower human health and ecological risks than many other herbicides associated with conventional wheat production systems evaluated in this study. The differences in risks were most pronounced when comparing glyphosate and imazamox to herbicides currently with substantial market share. Current weight-of-evidence suggests that the transgenic CP4 EPSPS protein present in glyphosate-tolerant wheat poses negligible risk to humans, livestock, and wildlife. Risk for mutated AHAS protein in imidazolinone-tolerant wheat most likely would be low, but there are not sufficient effect and exposure data to adequately characterize risk. Environmental risks for herbicides were more amenable to quantitative assessments than for the transgenic CP4 EPSPS protein and the mutated AHAS protein.     

Evaluation of three herbicide resistance genes for use in genetic transformations and for potential crop protection in algae production

Genes conferring resistance to the herbicides glyphosate, oxyfluorfen and norflurazon were developed and tested for use as dominant selectable markers in genetic transformation of Chlamydomonas reinhardtii and as potential tools for the protection of commercial‐scale algal production facilities against contamination by organisms sensitive to these broad‐spectrum herbicides. A synthetic glyphosate acetyltransferase (GAT) gene, when fitted with a strong Chlamydomonas promoter, conferred a 2.7×‐fold increase in tolerance to the EPSPS inhibitor, glyphosate, in transgenic cells compared with progenitor WT cells. A mutant Chlamydomonas protoporphyrinogen oxidase (protox, PPO) gene previously shown to produce an enzyme insensitive to PPO‐inhibiting herbicides, when genetically engineered, generated transgenic cells able to tolerate up to 136× higher levels of the PPO inhibitor, oxyfluorfen, than nontransformed cells. Genetic modification of the Chlamydomonas phytoene desaturase (PDS) gene‐based gene sequences found in various norflurazon‐resistant organisms allowed production of transgenic cells tolerant to 40× higher levels of norflurazon than nontransgenic cells. The high efficiency of all three herbicide resistance genes in producing transgenic cells demonstrated their suitability as dominant selectable markers for genetic transformation of Chlamydomonas and, potentially, other eukaryotic algae. However, the requirement for high concentrations of glyphosate and its associated negative effects on cell growth rates preclude its consideration for use in large‐scale production facilities. In contrast, only low doses of norflurazon and oxyfluorfen (~1.5 μm and ~0.1 μm , respectively) are required for inhibition of cell growth, suggesting that these two herbicides may prove effective in large‐scale algal production facilities in suppressing growth of organisms sensitive to these herbicides.