Daniell, H

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 9, pp. 855-856, Sep 1999

Environmental concerns have led to a growing public wariness of genetically modified crops around the world. A common concern is the possibility of gene escape through pollen or seed dispersal from crop plants engineered for herbicide resistance to their weedy relatives, possibly creating "superweeds" or causing gene pollution among other crops. The introduction of genes via chloroplast genetic engineering was recently advanced as a potential solution to this problem. In this issue, two new important tools are described that should facilitate the development of the field of chloroplast genetic engineering: a novel DNA delivery system for eukaryotic organelles and a fluorescent antibiotic marker to track plastid transformation.

Ohkawa, H; Tsujii, H; Ohkawa, Y

Pesticide Science [Pestic. Sci.], vol. 55, no. 9, pp. 867-874, Sep 1999

Mechanisms of herbicide resistance include (1) modified target site, (2) enhanced detoxification or delayed activation, and (3) alterations in the uptake, translocation, or compartmentalization of a herbicide. The first two mechanisms have mainly been identified in plants. Herbicide resistance genes were isolated for several herbicides of different modes of action. Genes that coded for herbicide target or detoxification enzymes were transferred into crop plants. The transgenic plants expressing these genes were tolerant of the active ingredients of herbicides. Before commercialization, the transgenic plants were tested in the field for risk assessment. In the case of crops with herbicide detoxification enzymes, including cytochrome-P450-species-metabolizing xenobiotics, the substrate specificity of the enzymes as well as the toxicological properties of the herbicide metabolites and the pattern of secondary metabolites in plants must be evaluated.

McCabe, MS; Schepers, F; van der Arend, A; Mohapatra, U; de Laat, AM; Power, JB; Davey, MR*

Theoretical and Applied Genetics [Theor. Appl. Genet.], vol. 99, no. 3/4, pp. 587-592, 24 Aug 1999

Inheritance of resistance to herbicide (300 mg/l glufosinate ammonium) up to the third (T3) seed generation was compared in two populations of transgenic lettuce (Lactuca sativa L. cv 'Evola') harbouring a T-DNA containing the bar gene, linked to either the Cauliflower Mosaic Virus (CaMV) 35S promoter, or a -784-bp plastocyanin promoter from pea (petE). Only 2.5% (4 /163) of CaMV 35S-bar plants, selected by their kanamycin resistance (T0 generation), transmitted herbicide resistance at high frequency to their T3 seed generation compared with 97% (29 /30) for kanamycin resistant petE-bar plants. In the case of 35S-bar transformants, only 16% (341/2,150) of the first seed generation (T1) plants, 22% (426/1,935) T2 plants and 11% (1,235 /10,949) T3 plants were herbicide-resistant. In contrast, 63% (190 /300) T1 plants, 83% (2,370/2,845) T2 plants and 99% (122/123) T3 petE-bar transformed plants were resistant to glufosinate ammonium. The T-DNAs carrying the petE-bar and CaMV 35S-bar genes also contained a CaMV 35S-neomycin phosphotransferase (nptII) gene. ELISA showed that NPTII protein was absent in 29% (45/156) of the herbicide-resistant T2 plants from 8/19 herbicide-resistant petE-bar lines. This indicated specific inactivation of the CaMV 35S promoter on the same T-DNA locus as an active petE promoter. The choice of promoter and T-DNA construct are crucial for long-term expression of transgenes in lettuce.

Ryals, J; Harms, C; Friedrich, L; Beck, J; Uknes, S; Ward, E

US Patent: 5942662, 24 Aug 1999

The present invention provides chemically regulatable DNA sequences capable of regulating transcription of an associated DNA sequence in plants or plant tissues, chimeric constructions containing such sequences, vectors containing such sequences and chimeric constructions, and transgenic plants and plant tissues containing these chimeric constructions. In one aspect, the chemically regulatable DNA sequences of the invention are derived from the 5' region of genes encoding pathogenesis-related (PR) proteins. The present invention also provides anti-pathogenic sequences derived from novel cDNAs coding for PR proteins which can be genetically engineered and transformed into plants to confer enhanced resistance to disease. Also provided is a method for the exogenous regulation of gene expression in plants, which comprises obtaining a plant incapable of regulating at least one gene or gene family, or at least one heterologous gene, due to the deactivation of at least one endogenous signal transduction cascade which regulates the gene in the plant, and applying a chemical regulator to the plant at a time when expression of the gene is desired A novel signal peptide sequence and corresponding DNA coding sequence is also provided. Further provided are assays for the identification and isolation of additional chemically regulatable DNA sequences and cDNAs encoding PR proteins and assays for identifying chemicals capable of exogenously regulating the chemically regulatable DNA sequences of the invention.

Anthony, RG; Reichelt, S; Hussey, PJ*

Nature Biotechnology [Nat. Biotechnol.], vol. 17, no. 7, pp. 712-714, Jul 1999

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an alpha -tubulin gene. We transformed and regenerated tobacco plants with an alpha / beta -tubulin double gene construct containing the mutant alpha -tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene alpha - and beta -tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous alpha - and beta -tubulin synthesis by up to 45% in some tissues. Transgene alpha - and beta -tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene alpha - and beta -tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the alpha / beta -tubulin pools are encoded by the transgenes, which implies that the mutant alpha -tubulin and the beta -tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.

Anthony, RG; Hussey, PJ*

Plant Journal [Plant J.], vol. 18, no. 6, pp. 669-674, Jun 1999

The repeated use of dinitroaniline herbicides on the cotton and soybean fields of the southern United States has resulted in the appearance of resistant biotypes of one of the world's worst weeds, Eleusine indica. Two biotypes have been characterized, a highly resistant (R) biotype and an intermediate resistant (I) biotype. In both cases the resistance has been attributed to a mutation in alpha -tubulin, a component of the alpha / beta tubulin dimer that is the major constituent of microtubules. We show here that the I-biotype mutation, like the R-biotype mutation shown in earlier work, can confer dinitroaniline resistance on transgenic maize calli. The level of resistance obtained is the same as that for E. indica I- or R-biotype seedlings. The combined I- and R-biotype mutations increase the herbicide tolerance of transgenic maize calli by a value close to the summation of the maximum herbicide tolerances of calli harbouring the single mutations. These data, taken together with the position of the two different mutations within the atomic structure of the alpha / beta tubulin dimer, imply that each mutation is likely to exert its effect by a different mechanism. These mechanisms may involve increasing the stability of microtubules against the depolymerizing effects of the herbicide or changing the conformation of the alpha / beta dimer so that herbicide binding is less effective, or a combination of both possibilities.

Siminszky, B; Ward, FTCER; Fleischmann, TJ; Dewey, RE

Proceedings of the National Academy of Sciences, USA [Proc. Natl. Acad. Sci. USA], vol. 96, no. 04, pp. 1750-1755, 16 Feb 1999

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR- based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast and microsomal fractions generated from these strains were tested for their potential to catalyze the metabolism of 10 herbicides and 1 insecticide. In vitro enzyme assays showed that the gene product of one heterologously expressed P450 cDNA (CYP71A10) specifically catalyzed the metabolism of phenylurea herbicides converting four herbicides of this class (fluometuron, linuron chlortoluron, and diuron) into more polar compounds. Analyses of the metabolites suggest that the CYP71A10 encoded enzyme functions primarily as an N-demethylase with regard to fluometuron, linuron, and diuron, and as a ring- methyl hydroxylase when chlortoluron is the substrate. In vivo assays using excised leaves demonstrated that all four herbicides were more readily metabolized in CYP71A10-transformed tobacco compared with control plants. For linuron and chlortoluron, CYP71A10- mediated herbicide metabolism resulted in significantly enhanced tolerance to these compounds in the transgenic plants.

Eichholtz, DA; Gasser, CS; Kishore, GM

US Patent 5866775, , 2 Feb 1999

Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate (EPSP) synthases, DNA encoding glyphosate-tolerant EPSP synthases, plant genes encoding the glyphosate-tolerant enzymes, plant transformation vectors containing the genes, transformed plant cells and differentiated transformed plants containing the plant genes are disclosed. The glyphosate-tolerant EPSP synthases are prepared by substituting an alanine residue for a glycine residue in a first conserved sequence found between positions 80 and 120, and a threonine residue for an alanine residue in a second conserved sequence found between positions 170 and 210 in the mature wild type EPSP synthase.

Thomas, T; Freyssinet, G; Lebrun, M; Bogue, M

US Patent 5824865, , 20 Oct 1998

Helianthinin is an 11S seed storage protein of sunflower embryos. The present invention is directed to the 5' regulatory regions of helianthinin genes. More particularly, the present invention is directed to specific cis-regulatory elements of this regulatory region which direct abscisic acid-responsive gene expression. The present invention provides chimeric genes comprising the cis-regulatory elements linked to a coding sequence from a heterologous gene to control expression of these genes. The chimeric genes provided by the instant invention are useful in conferring herbicide resistance and improved seed lipid quality to transgenic plants.

Schneider, R; Donn, G; Mullner, H

US Patent 5792926, , 11 Aug 1998

Virus genes, such as coat protein genes, which confer viral resistance or bring about a reduction in the signs of infection by the corresponding virus can be combined with herbicide-resistance genes, such as the phosphinothricin resistance gene, for the transformation of plants. A combination of a viral gene and a herbicide-resistance gene facilitates the selection of transgenic plants. In addition, in practical field cultivation, the vitality of the plants is increased by the virus tolerance, and an improved plant protection is possible due to the herbicide resistance gene.

Gray, AJ; Raybound, AF

Nature, vol. 392, no. 6677, pp. 653-654, 16 Apr 1998

Genetically modified crops and the food derived from them have had a bad press, but a rare piece of good news is provided by Daniell et al. in this month's Nature Biotechnology. They report a development that has profound implications for the risk assessment of genetically modified crops. Most crops are modified by inserting genes into the nucleus, and the genes can therefore spread to other crops or wild relatives by movement of pollen. By engineering tolerance to the herbicide glyphosate into the tobacco chloroplast genome, however, the authors have not only obtained high levels of transgene expression, but, because chloroplasts are inherited maternally in many species, they have also prevented transmission of the gene by pollen--closing a potential escape route for transgenes into the environment.

Metz, PLJ; Stiekema, WJ; Nap, J-P*

Molecular Breeding [Mol. Breed.], vol. 4, no. 4, pp. 335-341, 1998

The microbial bar and pat genes confer tolerance to the non-selective herbicide phosphinothricin (PPT; sold as Basta or Finale). This tolerance in plants could provide an environmental gain compared to current-day herbicide cocktails, but the safety of such a transgene approach is questioned by many. The biosafety of the presence of these herbicide tolerance genes in plants is evaluated in a 'transgene-centered approach'. Potentially, the introduction of transgenic PPT-tolerant crops could result in acquired PPT tolerance in weedy relatives of these crops. Assuming responsible use of this trait in agronomy, the ecological consequences with respect to weediness or spread of the transgenic PPT tolerance are concluded to be negligible. The key issue for the toxicological evaluation is whether or not the plant has actually been sprayed with PPT. Consumption of the gene and/or gene product from unsprayed transgenic plant material will not have adverse effects. In case of PPT-sprayed material, PPT or its derivatives could be present in food and feed and crop-specific metabolites might be formed. To date, the toxicological impact of such a putative exposure is not sufficiently clear, and further premarket testing is recommended.

Keller, G; Spatola, L; McCabe, D; Martinell, B; Swain, W*; John, ME

Transgenic Research [Transgen. Res.], vol. 6, no. 6, pp. 385-392, Nov 1997

Resistance to bialaphos, a non-selective herbicide, was introduced into cotton through genetic engineering. A gene encoding phosphinothricin acetyltransferase (bar) from Streptomyces hygroscopicus was inserted into elite varieties of cotton through particle bombardment. Based on the marker gene, beta -glucuronidase (gus) expression, a total of 18 Pima (Gossypium barbadense), 45 DP50 (G. hirsutum L.), 20 Coker 312 (G. hirsutum) and 2 El Dorado (G. hirsutum) transgenic plants were recovered. Integration of the bar gene into cotton genomic DNA was confirmed by Southern blot analysis and gene expression was confirmed by northern blot and enzyme assays. Herbicide (Basta registered ) tolerance up to 15 000 ppm was demonstrated in greenhouse trials. The newly introduced herbicide tolerance trait is inherited in a Mendelian fashion in the progenies of germline transformants. This study demonstrates the potential for particle bombardment to introduce commercially important genes directly into elite varieties of cotton. This mode of gene transfer can expedite the introduction of transgenic cotton products into world markets.

Donn, G

US Patent 5623782, , 29 Apr 1997

By selection for resistance to aryloxyphenoxyalkanecarboxylic acid herbicides, herbicide-tolerant maize cell lines, calli and plants regenerated therefrom can be obtained which pass this herbicide tolerance on to their progeny in a stable manner.

Mannerloef, M; Tuvesson, S; Steen, P; Tenning, P

Euphytica, vol. 94, no. 1, pp. 83-91, 1997

Sugar beet (Beta vulgaris L.) lines transformed with the 5-enolpyruvylshikimate-3-phosphate synthase gene (CP4 EPSPS) from Agrobacterium sp. CP4 and a glyphosate oxidase reductase gene (GOX) also isolated from bacteria resulted in the development of lines highly tolerant to glyphosate. Glyphosate (N-phosphonomethyl-glycine) is the active ingredient in Roundup registered , herbicide. The EPSPS enzyme is involved in the biosynthesis of aromatic amino acids. Glyphosate binds irreversible to the EPSPS and inhibits the pathway. GOX degrades glyphosate into non-toxic compounds. 260 independent transformants have been evaluated in greenhouse and field trials for tolerance to Roundup registered in 1993 and 1994. Variation of tolerance was recorded between different transformants, ranging from complete susceptibility to full tolerance. The Agrobacterium tumefaciens mediated transformation resulted in a negative correlation between copy number of the T-DNA insert and the level of tolerance to the herbicide. Transformants which contain a single copy insert showed tolerance to higher doses of glyphosate than transformants with multiple copies. Two transgenic lines were identified that showed agronomically useful tolerance to glyphosate.

US Patent 5498544, , 12 Mar 1996

The present invention provides an expression cassette encoding a plant acetyl CoA carboxylase gene and methods for conferring herbicide tolerance and/or altering the oil content of plants by introducing and expressing a plant acetyl CoA carboxylase gene in plant cells. An expression cassette of the invention can comprise a gene coding a plant acetyl CoA carboxylase or a functional mutant thereof or an antisense DNA sequence complementary to the sequence for a plant acetyl CoA carboxylase gene combined with a promoter functional in a plant cell. The method of imparting herbicide tolerance to a plant includes the steps of introducing the expression cassette into the cells of a plant tissue and expressing the plant acetyl CoA carboxylase gene in an amount effective to render the acetyl CoA carboxylase and/or plant cell substantially tolerant to the herbicides. The method of altering the oil content in a plant includes the steps of introducing an expression cassette into plant cells and expressing the acetyl CoA carboxylase gene in an amount effective to alter the oil content of the cells. The expression cassette can also be introduced into a prokaryotic cell to increase yield of a plant acetyl CoA carboxylase so that crystallized enzyme can be used to screen and identify other herbicides that bind to and inhibit the enzyme.

McHughen, A; Holm, FA

Canadian Journal of Plant Science/Revue Canadienne de Phytotechnie [CAN. J. PLANT SCI./REV. CAN. PHYTOTECH.], vol. 75, no. 1, pp. 117-120, 1995

Flax (Linum usitatissimum L.) is a poor biological competitor and so requires chemical weed control for optimal productivity. An alternative to current, relatively expensive combinations of herbicides might be to introduce into a commercial flax genotype, using Agrobacterium mediated transformation, a gene conferring tolerance to a non-selective herbicide. Here, we describe the transformation of Norlin flax with a PAT (phosphinothricin acetyl transferase) gene conferring tolerance to glufosinate, a non-selective herbicide (Harvest super(TM), Ignite super(TM), Agrevo). We also present results from a preliminary field trial, showing the efficacy of the PAT gene in conferring tolerance while not dramatically altering the normal agronomic traits of the parent cultivar.