Glyphosate Selection Research Articles

Selectivity of glyphosate in tank mixtures for RR soybean in sequential applications with mixtures only in the first or second application

Tank mixtures among herbicides of different action mechanisms might increase weed control spectrum and may be an important strategy for preventing the development of resistance in RR soybean. However, little is known about the effects of these herbicide combinations on soybean plants. Hence, two experiments were carried out aiming at evaluating the selectivity of glyphosate mixtures with other active ingredients applied in postemergence to RR soybean. The first application was carried out at V1 to V2 soybean stage and the second at V3 to V4 (15 days after the first one). For experiment I, treatments (rates in g ha-1) evaluated were composed by two sequential applications: the first one with glyphosate (720) in tank mixtures with cloransulam (30.24), fomesafen (125), lactofen (72), chlorimuron (12.5), flumiclorac (30), bentazon (480) and imazethapyr (80); the second application consisted of isolated glyphosate (480). In experiment II, treatments also consisted of two sequential applications, but tank mixtures as described above were applied as the second application. The first one in this experiment consisted of isolated glyphosate (720). For both experiments, sequential applications of glyphosate alone at 720/480, 960/480, 1200/480 and 960/720 (Expt. I) or 720/480, 720/720, 720/960 and 720/1200 (Expt. II) were used as control treatments. Applications of glyphosate tank mixtures with other herbicides are more selective to RR soybean when applied at younger stages whereas applications at later stages might cause yield losses, especially when glyphosate is mixed with lactofen and bentazon.

Growth, Phenology, and Intraspecific Competition between Glyphosate-Resistant and Glyphosate-Susceptible Horseweeds (Conyza canadensis) in the San Joaquin Valley of California

Experiments were conducted in 2006 to 2008 to study growth, phenology, and competitive ability of glyphosate-resistant (GR) and -susceptible (GS) biotypes of horseweeds from San Joaquin Valley (SJV), CA. When grown alone, in pots, the GR horseweeds consistently developed more rapidly than the GS weeds, as evidenced by their earlier bolting, flowering, and seed set; the GR horseweeds set seeds nearly 25 d (approximately 190 fewer growing degree days) sooner than the GS horseweed. At seed set, the relatively slow-developing GS horseweeds had amassed 40% more shoot dry matter than the GR weeds at the same phenological stage, but neither biotype was consistently more fecund than the other. Although the GR biotype had lower shoot dry mass than the GS biotype when grown alone, in mixed populations under increasing levels of competition (in a replacement series design) and limited resources (mainly moisture), the GR weeds were not only taller, but also accumulated more dry matter than the GS weeds. Thus, the GR biotype was more competitive than the GS biotype, particularly when grown at high densities and under moisture-deficit stress. Therefore, under California conditions there is no apparent fitness penalty for this particular GR horseweed biotype, and it is likely to persist in the environment and outcompete the GS biotypes regardless of further glyphosate selection pressure. If so, this biotype of GR horseweed is likely to become increasingly common in the SJV until effective management strategies are developed and adopted.

Evolution of glyphosate resistance in a Lolium rigidum population by glyphosate selection at sublethal doses

The majority of the documented cases of field-evolved herbicide-resistant weed biotypes established that single major genes confer glyphosate resistance. However, the contribution of minor genes endowing substantial plant survival at sublethal herbicide doses may be a potential complementary path to herbicide resistance evolution in weed populations under selection. Here, we subjected a number of susceptible individuals of Lolium rigidum to recurrent glyphosate selection to test the potential for sublethal glyphosate doses to additively select for glyphosate resistance. After 3-4 cycles of glyphosate selection in two distinct environments, the progenies of the initially susceptible population were shifted toward glyphosate resistance. The results indicate progressive enrichment of minor gene trait(s) contributing toward plant survival in the glyphosate-selected progenies. After three generations of selection, the estimated LD(50) values were doubled compared with the original population and up to 33% plant survival was obtained in the glyphosate-selected progeny at the recommended glyphosate label rate. This level of resistance probably was the maximum shift achievable with sublethal glyphosate dose selection in this small population. Cross-pollination was a crucial factor enabling the rapid rate of accumulation of minor glyphosate resistance gene trait(s) that are likely to be present at a relatively high frequency in a small susceptible population. The mechanistic basis of the moderate glyphosate resistance level selected by sublethal glyphosate doses remains unknown and warrants future research. Studying the main factors influencing the evolution of resistant weed populations is crucial for understanding, predicting and managing herbicide resistance.

Opinion of the Scientific Panel on genetically modified organisms [GMO] related on an application (Reference EFSA GMO UK 2004 08) for the placing on the market of products produced from glyphosate tolerant genetically modified sugar beet H7‐1, for food and feed uses, under Regulation (EC) No 1829/2003 from KWS SAAT AG and Monsanto

EFSA JournalVolume 4, Issue 12 431 OpinionOpen Access Opinion of the Scientific Panel on genetically modified organisms [GMO] related on an application (Reference EFSA GMO UK 2004 08) for the placing on the market of products produced from glyphosate tolerant genetically modified sugar beet H7-1, for food and feed uses, under Regulation (EC) No 1829/2003 from KWS SAAT AG and Monsanto European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author European Food Safety Authority (EFSA), European Food Safety Authority (EFSA)Search for more papers by this author First published: 14 December 2006 https://doi.org/10.2903/j.efsa.2006.431Citations: 4 Adoption date: 5 December 2006 Published date: 14 December 2006 Question number: EFSA-Q-2004-164 AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References ACNFP, 1994. Annual Report 1994, Appendix 4, Advisory Committee on Novel Foods and Processes, London, UK. www.foodstandards.gov.uk/multimedia/webpage/acnfp_report_1994 CAC, 2003. Codex principles and guidelines on foods derived from biotechnology. Joint FAO/WHO Food Standards Programme, Food and Agriculture Organisation, Rome. ftp://ftp.fao.org/codex/standard/en/CodexTextsBiotechFoods.pdf Dewar, A., May, M. and Pidgeon, J. (2000) GM sugar beet – The present situation. Br. sugar Rev., 68: 22– 27. EC, 2003. Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ, L268, 1– 23. europa.eu.int/eur-lex/pri/en/oj/dat/2003/l_268/l_26820031018en00010023.pdf EFSA, 2003a. Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the Notification (Reference CE/ES/00/01) for the placing on the market of herbicide-tolerant genetically modified maize NK603, for import and processing, under Part C of Directive 2001/18/EC from Monsanto (Question No EFSA-Q-2003-003), the EFSA Journal (2003), 10, 1– 13. www.efsa.eu.int/science/gmo/gmo_opinions/176/opinion_gmo_03_final_en1.pdf EFSA, 2003b. Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the safety of foods and food ingredients derived from herbicide-tolerant genetically modified maize NK603, for which a request for placing on the market was submitted under Article 4 of the Novel Food Regulation (EC) No 258/97 by Monsanto (Question No EFSA-Q-2003-002), the EFSA Journal (2003) 9, 1– 14. www.efsa.eu.int/science/gmo/gmo_opinions/177/opinion_gmo_02_final_en1.pdf EFSA, 2004. Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic resistance genes as marker genes in genetically modified plants the EFSA journal (2004) 48, 1– 18. www.efsa.europa.eu/etc/medialib/efsa/science/gmo/gmo_opinions/384.Par.0001.File.dat/opinion_gmo_05_en1.pdf EFSA, 2006. Guidance document of the Scientific Panel on Genetically Modified Organisms for the risk assessment of genetically modified plants and derived food and feed, the EFSA Journal (2006) 99, 1– 100. www.efsa.europa.eu/etc/medialib/efsa/press_room/publications/scientific/1497.Par.0005.File.dat/gmo_guidance%20gm%20plants_en.pdf. Hartnell, G.F., Hvelplund, T., Weisbjerg, M.R., 2005, Nutrient digestibility in sheep fed diets containing Roundup Ready or conventional fodder beet, sugar beet, and beet pulp. J, Anim. Sci., 83, 400– 407. May, M.J., Champion, G.T. and Qi, A. (2003) Novel weed management options in GM herbicide tolerant sugar beet. In: GM Herbicide Tolerant sugar Beet, Proceedings of the 1st joint IIRB-ASSBT Congress, San Antonio (USA), p. 77– 90. Metcalfe, D.D., Astwood, J.D., Townsend, R., Sampson, H.A., Taylor, S.L., Fuchs, R.L., 1996. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Crit. Rev. Food. Sci. Nutr., 36(S), 165– 186. OECD, 2002. Consensus document on compositional considerations for new varieties of sugar beet: key food and feed nutrients and antinutrients. Series on the Safety of Novel Foods and Feeds, No. 3, Organisation for Economic Co-operation and Development, Paris. SCP, 1998a. Opinion of the Scientific Committee on Plants regarding the genetically modified cotton, tolerant to glyphosate herbicide notified by the Monsanto Company (notification C/ES/97/01). europa.eu.int/comm/food/fs/sc/scp/out17_en.html SCP, 1998b. Opinion of Scientific Committee on Plants regarding submission for placing on the market of fodder beet tolerant to glyphosate notified by DLF-Trifolium, Monsanto and Danisco Seed (notification C/DK/97/01). Wevers, J., May, M., Hermann, O. and Petersen, J. (2005) Efficacy and selectivity of glyphosate and glufosinate in genetically modified sugar beet. In: Genetic Modification In sugar Beet, J. Pidgeon, R.M. Molard, J.D.A. Wevers and R. Beckers (Eds.), Advances in sugar Beet Research, Vol. 6, IIRB, p. 45– 60. Citing Literature Volume4, Issue12December 2006431 ReferencesRelatedInformation

Controle de Brachiaria brizantha com uso do glyphosate após o estabelecimento de Tifton 85 (Cynodon spp.)

A eficiência do glyphosate no controle de Brachiaria brizantha cv. Marandu em áreas de cultivo do Tifton 85 foi avaliada utilizando o delineamento experimental em blocos casualizados, com oito doses de glyphosate (0, 90, 180, 360, 720, 1.080, 1.440 e 1.800 g ha-1), e quatro repetições. Cada parcela foi constituída de um vaso com duas plantas de B. brizantha cv. Marandu e duas plantas de Tifton-85. A aplicação do herbicida foi feita quando as plantas de B. brizantha apresentavam cerca de 40 cm de altura. O nível de intoxicação nas plantas de Tifton 85 e a eficiência do herbicida no controle de B. brizantha foram avaliados aos 15, 30 e 60 dias após aplicação (DAA). Aos 60 DAA, as plantas foram colhidas ao nível do solo e secas em estufa. A rebrota foi avaliada, do mesmo modo, aos 60 dias após o corte (DAC). Obeservou-se controle superior a 90% de B. brizantha, a partir da dose de 738,28 g ha-1 de glyphosate, enquanto a intoxicação para as plantas de Tifton 85 foi de apenas 12,05. Aos 60 DAA, houve redução na produção de massa seca de braquiária a partir da dose de 90 g ha-1. Doses superiores a 720 g ha-1 diminuíram o crescimento e desenvolvimento do Tifton 85, afetando sua produção, sem, no entanto, ocasionar a morte das plantas. Os resultados evidenciam boa tolerância do Tifton 85 até a dose de 720 g ha-1 de glyphosate.

Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy

5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C(317)-->T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops.

MicroTom—a high-throughput model transformation system for functional genomics

We have developed a high-throughput Agrobacterium-mediated transformation model system using both nptII and the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens strain CP4 (cp4) based selections in MicroTom, a miniature rapid-cycling cherry tomato variety. With the NPTII selection system, transformation frequency calculated as independent transgenic events per inoculated explant ranged from 24 to 80% with an average of 56%, in industrial production scale transformation experiments. For CP4, with glyphosate selection, the average transformation frequency was 57%. Stable transformation frequency was positively correlated with transient expression (R=0.85), and variable with the genes of interest. DNA integration and germline transformation were confirmed by biological assay, Southern Blot analysis, and R(1) phenotype segregation. Transgene expression was observed in leaf, root, stem, flower, and fruit tissues of the transgenic plants. Ninety-five percent of transgenic events coexpressed two introduced genes based on beta-glucuronidase (GUS) and neonmycin phosphotransferase II (NPTII) expression. Seventy-five percent of transgenic events contained one to two copies of the introduced uidA (GUS) gene based on Southern analysis. Transgenic plants from the cotyledon explants to the transgenic plants transferred to soil were produced within about 2-3 months depending on the genes of interest. The utility of this MicroTom model transformation system for functional genomic studies, such as identification of genes related to important agricultural traits and gene function, is discussed.

Agrobacterium-mediated transformation of seedling-derived maize callus

Efficient production of seedling-derived Type I callus was demonstrated for several corn genotypes including commercial inbred lines. Seeds were germinated on MS-based medium containing 10 mg l(-1) picloram and 3 mg l(-1) 6-benzylaminopurine, which induced the development of axillary buds in the area of coleoptilar node. Nodal sections of 7-10-day old seedlings were isolated, split longitudinally, and placed on callus induction medium supplemented with 2.2 mg l(-1) picloram and 0.5 mg l(-1) 2,4-dichlorophenoxyacetic acid. For lines L4 and L9 the frequency of embryogenic callus induction was 38-42% based on calli per split nodal section. Frequency of callus induction from split nodal sections of seeds germinated on media without growth regulators was 0-3%. Seedling-derived callus of five genotypes was used for Agrobacterium-mediated transformation. Two constructs containing the green fluorescence protein gene and genes for either neomycin phosphotransferase II or glyphosate selection were used in transformation experiments. Transformation frequency varied from 2 to 11% and about 60% of the T(0) plants had 1-2 copies of transgenes.

Weed Control in Sugar Beet using Genetically Modified Herbicide‐tolerant Varieties – A Review of the Economics for Cultivation in Europe

AbstractGenetically modified sugar beet varieties have been developed to be tolerant to glyphosate and glufosinate. To date, research regarding other active ingredients did not result in additional herbicide‐tolerant varieties and the approval of glufosinate‐tolerant varieties for market access has been withdrawn by the applicant. Therefore, only glyphosate‐tolerant varieties could be introduced for cultivation in the short run. Results concerning efficacy and cost of weed control using these varieties and the complementary herbicides were extensively reported in various contributions, mostly on a national level. Based on these results, the economics of weed control for sugar beet production in Europe were reviewed and aspects of integrated control, risk management, and issues of sustainable development of crop production are discussed. Efficient weed control is possible in almost any field situation with glyphosate at about 2 kg a.i. ha−1, compared with conventional herbicides at 6 kg ha−1 or higher, depending on weed infestation level. Cost savings for weed control with glyphosate would amount to an average of €150 ha−1, without any great deviation across different sites and states. A technology fee of about €40 ha−1 is assumed. The high selectivity of glyphosate may result in a 1–3 % higher yield performance of the crop. All assumptions being considered, total cost savings of €180 × 106 year−1 were calculated for the area of 1.7 × 106 ha in the main EU sugar beet‐growing countries. A risk management by implementing a monitoring programme is compulsory, and systems of identity preservation or quality assurance are needed in order to enable the production of conventional and genetically modified sugar beet in coexistence. To date, costs are unknown for these measures. Because of the favourable ecotoxicological behaviour of glyphosate and the possibility of threshold‐based weed control, this new technology could provide an excellent option towards sustainable development of the crop. However, political reasons and the lack of acceptance of genetically modified varieties by the consumer have prevented the market entry of GMHT sugar beet to date, so that conventional herbicides will continuously be used in the future.

Inheritance of evolved glyphosate resistance in Conyza canadensis (L.) Cronq.

N-(phosphonomethyl)glycine (glyphosate) resistance was previously reported in a horseweed [Conyza (=Erigeron) canadensis (L.) Cronq.] population from Houston, DE (P (0) (R) ). Recurrent selection was performed on P (0) (R) , since the population was composed of susceptible (5%) and resistant (95%) phenotypes. After two cycles of selection at 2.0 kg ae glyphosate ha(-1), similar glyphosate rates that reduced plant growth by 50%, glyphosate rates that inflicted 50% mortality in the population, and accumulations of half of the maximum detectable shikimic acid concentration were observed between the parental P (0) (R) and the first (RS(1)) and second (RS(2)) recurrent generations. In addition, RS(1) and RS(2) did not segregate for resistance to glyphosate. This suggested that the RS(2) population comprised a near-homozygous, glyphosate-resistant line. Whole-plant rate responses estimated a fourfold resistance increase to glyphosate between RS(2) and either a pristine Ames, IA (P (0) (P) ) or a susceptible C. canadensis population from Georgetown, DE (P (0) (S) ). The genetics of glyphosate resistance in C. canadensis was investigated by performing reciprocal crosses between RS(2) and either the P (0) (P) or P (0) (S) populations. Evaluations of the first (F(1)) and second (F(2)) filial generations suggested that glyphosate resistance was governed by an incompletely dominant, single-locus gene (R allele) located in the nuclear genome. The proposed genetic model was confirmed by back-crosses of the F(1) to plants that arose from achenes of the original RS(2), P (0) (P) , or P (0) (S) parents. The autogamous nature of C. canadensis, the simple inheritance model of glyphosate resistance, and the fact that heterozygous genotypes (F(1)) survived glyphosate rates well above those recommended by the manufacturer, predicted a rapid increase in frequency of the R allele under continuous glyphosate selection. The impact of genetics on C. canadensis resistance management is discussed.

Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat

Factors influencing the Agrobacterium-mediated transformation of both monocotyledonous and dicotyledonous plant species have been widely investigated. These factors include manipulating Agrobacterium strains and plasmids, growth conditions for vir gene induction, plant genotype, inoculation and co-culture conditions, and the selection agents and their application regime. We report here a novel physical parameter during co-culture, desiccation of plant cells or tissues post-Agrobacterium infection, which greatly enhances transfer DNA (T-DNA) delivery and increases stable transformation efficiency in wheat. Desiccation during co-culture dramatically suppressed Agrobacterium growth, which is one of the factors known to favor plant cell recovery. Osmotic and abscisic acid treatments and desiccation prior to inoculation did not have the same enhancement effect as desiccation during co-culture on T-DNA delivery in wheat. An efficient transformation protocol has been developed based on desiccation and is suitable for both paromomycin and glyphosate selection. Southern analysis showed approximately 67% of transgenic wheat plants received a single copy of the transgene.

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.

Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.

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.

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.

Progeny Analysis of Glyphosate Selected Transgenic Soybeans Derived from Agrobacterium -Mediated Transformation

Glyphosate selection has been implemented into an Agrobacterium-mediated transformation system for soybean [Glycine max (L.) Merr.]. Cotyledonary explants were excised from 5-d-old seedlings and inoculated with an Agrobacterium strain carrying the CP4 gene, which confers tolerance to the herbicide Roundup, and the β-glucuronidase (GUS) gene. Explants were selected on sublethal levels of glyphosate during shoot initiation and elongation. Glyphosate tolerant shoots were identifiable after 8 to 12 wk of selection. Coexpression of GUS occurred in approximately 80% of the primary transformants (R θ ). The R θ plants were fully fertile, with no apparent phenotypic abnormalities. Progeny analysis from 81 independent transgenic soybean events derived from glyphosate selection demonstrated that the majority of the transformants transmitted the transgenes as a single functional locus.

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

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.

Characterization of the glyphosate selection of carrot suspension cultures resulting in gene amplification

When Daucus carota L. (carrot) suspension cultures, which had been under gradual selection for increased glyphosate resistance, whre plated in glyphosate-containing medium, the populations became gradually more resistant with I 50 values about equal to the final selection concentration used in the liquid medium. Wild type cells did not grow on plates with 2 mM glyphosate, but some colonies formed after 90 days. Cells in these colonies, after growth away for the inhibitor to obtain sufficient cells, had increased glyphosate resistance and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) activity and usually had amplified EPSPS genes. One step selection in liquid or solidified medium with 10 mM glyphosate was not successful. However, in liquid medium with 2 mM glyphosate, the cell viability decreased to about 30% by 30 days and then increased as growth became evident. Kinetic analysis indicates that at least 0.8% of the cells grow. The growth rate seen when the cells are subjected to a gradual selection scheme, where glyphosate was about doubled at each step, also indicates that a high proportion of the original population survives to form the new population. These results indicate that the gene amplification which causes the glyphosate tolerance in carrot cell cultures is a gradual and relatively stable process that occurs in a relatively high proportion of the cell population.

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

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.

Selectivity of glyphosate in creeping red fescue and reed canarygrass*

Summary:The differential tolerance of resislant creeping red fescue (Fes‐ tuca rubra L, var, rutra) and susceptibie reed canarygrass (Pha‐ laris arundinacea L.) seedlings to glyphosate [N‐(phosphono‐ meihyDglycine) was confirmed under growth chamber condilions. The absorption, transiocation and metabolism of 14C‐glyphosate was examined in both species to determine if differences in these processes could account for the observed selectivity, Creepmg red fescue actually absorbed more glyphosate than did t‐eed canarygrass, and both species rapidly translocated the herbicide throughout their respective tissues. No metabolism of glyphosate was detected in either species. Differential interception and retention of the glyphosate spray can probably be eliminated as possible selectivity mechanisms. The relative tolerance of creeping red fescue to glyphosale appears to be related to its ability lo regenerate roots and shoots from the crown of the plant, but the mechanism of resistance remains obscure.