Simulação da soja geneticamente modificada tolerante ao glyphosate por meio do cultivo de explantes

Siqueira, Sérgio C.; Moreira, Maurílio A.; Mosquim, Paulo R.; José, Inês C.; Ferreira, Francisco A.; Sediyama, Carlos S.

doi:10.1590/S0100-83581999000100009

Resumos

O objetivo do experimento consistiu na simulação in vitro da soja transgênica tolerante ao glyphosate, através do cultivo de explantes em meios de cultura contendo aminoácidos aromáticos. As avaliações basearam-se nos efeitos do glyphosate sobre sementes oriundas de explantes de soja (Glycine max (L.) Merr.) cv. UFV-16. Para tanto, explantes de soja foram cultivados em meios de cultura líquidos com pH em torno de 5,0. Cada explante constou de um legume completamente expandido contendo duas sementes de aproximadamente 100 mg, conectada a um segmento de caule de 45 mm de comprimento. Os tratamentos testados foram: A = glutamina (Gln); B = Gln + fenilalanina (Phe) + tirosina (Tyr) + triptofano (Trp); C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. O experimento foi conduzido sob irradiância de 80 mmol-2s-1 a 25oC por 204 horas. Nos tratamentos que receberam aminoácidos aromáticos e glutamina, o herbicida não afetou as massas fresca e seca das sementes, como também, não afetou seus constituintes bioquímicos (proteínas, óleo, ácidos graxos, carboidratos e clorofilas). Portanto, a suplementação exógena de aminoácidos aromáticos suprime os efeitos fitotóxicos do glyphosate sobre explantes de soja, permitindo estudos sobre o seu modo de ação e metabolismo nas sementes, uma vez que os explantes se comportaram analogamente à soja transgênica não suscetível ao herbicida.

Glycine max; cultivo in vitro; do shiquimato; aminoácidos aromáticos

The objective of this experiment consisted in simulation in vitro of the transgenic soybean tolerant to glyphosate through explant cultivation in culture medium containing aromatic amino acids. The effects of glyphosate on soybean (Glycine max (L.) Merrill) were evaluated in seeds harvested from explants of cv UFV-16. The soybean explants were cultivated in liquid medium culture with pH about 5.0. Each explant consisted of one fruit completely expanded, containing two seeds of 100 mg approximately, and connected to a stem segment of 45 mm length. The explants were submitted to the following treatments: A = glutamine (Gln); B = Gln + phenylalanine (Phe) + tyrosine (Try) + tryptophan (Trp); C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. The experiment was conducted under irradiance of 80 mmol-2s-1 at 25o for 204 hours. The treatments with aromatic amino acids and glutamine avoided the herbicide effect on seed fresh and dried weights and had no effect on their biochemical constituents (proteins, oil, fatty acids, carbohydrates and chlorophylls). Thus, an exogenous supplementation of aromatic amino acids eliminates phytotoxic effects of glyphosate on soybean explants. Therefore, it was developed an in vitro system that allows to study the mode of action and the metabolism of glyphosate in soybean seeds, since the explants behaved in a similar manner as the transgenic soybean unsusceptible to the herbicide.

Glycine max; in vitro culture; shikimate pathway; aromatic amino acids; seed composition

Simulação da soja geneticamente modificada tolerante ao glyphosate por meio do cultivo de explantes

Simulation of the transgenic soybean tolerant to glyphosate through explant cultivation

Sérgio C. Siqueira^I, Maurílio A. Moreira^II, Paulo R. Mosquim^III, Inês C. José^IV, Francisco A. Ferreira^V, Carlos S. Sediyama^V

^IEstudante de Doutorado do Depto de Fitotecnia da UFV, BIOAGRO. CEP 36571-000, Viçosa/MG

^IIProfessor Titular, Ph.D., Depto de Bioquímica e Biologia Molecular da UFV, BIOAGRO. CEP 36571-000, Viçosa/MG

^IIIProfessor Titular, D.S., Depto de Biologia Vegetal da UFV. CEP 36571-000, Viçosa/MG

^IVPesquisadora, M.S., Núcleo de Biotecnologia Aplicada à Agropecuária da UFV. CEP 36571-000, Viçosa/MG

^VProfessores Titulares, respectivamente, D.S. e Ph.D., Depto de Fitotecnia da UFV. CEP 36571-000, Viçosa/MG

RESUMO

O objetivo do experimento consistiu na simulação in vitro da soja transgênica tolerante ao glyphosate, através do cultivo de explantes em meios de cultura contendo aminoácidos aromáticos. As avaliações basearam-se nos efeitos do glyphosate sobre sementes oriundas de explantes de soja (Glycine max (L.) Merr.) cv. UFV-16. Para tanto, explantes de soja foram cultivados em meios de cultura líquidos com pH em torno de 5,0. Cada explante constou de um legume completamente expandido contendo duas sementes de aproximadamente 100 mg, conectada a um segmento de caule de 45 mm de comprimento. Os tratamentos testados foram: A = glutamina (Gln); B = Gln + fenilalanina (Phe) + tirosina (Tyr) + triptofano (Trp); C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. O experimento foi conduzido sob irradiância de 80 mmol-2s-1 a 25oC por 204 horas. Nos tratamentos que receberam aminoácidos aromáticos e glutamina, o herbicida não afetou as massas fresca e seca das sementes, como também, não afetou seus constituintes bioquímicos (proteínas, óleo, ácidos graxos, carboidratos e clorofilas). Portanto, a suplementação exógena de aminoácidos aromáticos suprime os efeitos fitotóxicos do glyphosate sobre explantes de soja, permitindo estudos sobre o seu modo de ação e metabolismo nas sementes, uma vez que os explantes se comportaram analogamente à soja transgênica não suscetível ao herbicida.

Palavras chave: Glycine max, cultivo in vitro, do shiquimato, aminoácidos aromáticos.

ABSTRACT

The objective of this experiment consisted in simulation in vitro of the transgenic soybean tolerant to glyphosate through explant cultivation in culture medium containing aromatic amino acids. The effects of glyphosate on soybean (Glycine max (L.) Merrill) were evaluated in seeds harvested from explants of cv UFV-16. The soybean explants were cultivated in liquid medium culture with pH about 5.0. Each explant consisted of one fruit completely expanded, containing two seeds of 100 mg approximately, and connected to a stem segment of 45 mm length. The explants were submitted to the following treatments: A = glutamine (Gln); B = Gln + phenylalanine (Phe) + tyrosine (Try) + tryptophan (Trp); C = Gln + glyphosate; D = Gln + Phe + Tyr + Trp + glyphosate. The experiment was conducted under irradiance of 80 mmol-2s-1 at 25o for 204 hours. The treatments with aromatic amino acids and glutamine avoided the herbicide effect on seed fresh and dried weights and had no effect on their biochemical constituents (proteins, oil, fatty acids, carbohydrates and chlorophylls). Thus, an exogenous supplementation of aromatic amino acids eliminates phytotoxic effects of glyphosate on soybean explants. Therefore, it was developed an in vitro system that allows to study the mode of action and the metabolism of glyphosate in soybean seeds, since the explants behaved in a similar manner as the transgenic soybean unsusceptible to the herbicide.

Key words:Glycine max, in vitro culture, shikimate pathway, aromatic amino acids, seed composition.

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LITERATURA CITADA

Recebido para publicação em 25/11/97 e na forma revisada em 07/08/98.

ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS - A.O.A.C. Official methods of analysis. Washington, D.C., 1975. 1094p.
BUBECK, DM; FEHR, WR; HAMMOND, EG. Inheritance of palmitic and stearic acid mutants of soybeans. Crop Sci., v.29, p.652-656, 1989.
CHANDLER, PM; HIGGINS, TJV; RANDALL, PJ; SPENCER, D. Regulation of legumin levels in developing pea seeds under conditions of sulfur deficiency. Plant Physiol., v.71, p.47-54, 1983.
COLE, DJ. Mode of action of glyphosate-a literature analysis. In: GROSSBARD, E; ATKINSON, E (eds). The herbicide glyphosate. London: Butterworths Publishers,1985, p. 48-74.
DEVINE, M; DUKE, SO; FEDTKE, C. Physiology of herbicide action. Englewood Cliffs: PTR Prentice Hall, 1993, p.251-294.
DUKE, SO. Glyphosate. In: KEARNEY, PC; KAUFMAN, DD (eds.). Herbicideschemistry, degradation and mode of action. New York: Dekker Publishers, 1988, p. 1-70.
DUKE, S.O.; HOAGLAND, R.E. Effects of glyphosate on metabolism of phenolic compounds. Phenylalanine ammonia-lyase activity, free amino acids, root-fed amino acids and glyphosate toxicity in soybean (Glycine max) seedlings. Weed Sci., v.29, p.297, 1981.
EUCLIDES, R.F. Manual para análise estatística e genética SAEG. Viçosa: UFV, CPD, 1982, 59 p.
FONTES, E.P.B; MOREIRA, A.M.; DAVIES C.S.; NIELSEN N. C. Urea-elicited changes in relative electrophoretic mobility of certain glycinin and ?-conglycinin subunits. Plant Physiol., v.76, p.840-842, 1984.
HARRISON, L.A., BAILEY, M.R., NAYLOR, M., REAM, J., HAMMOND, D.L. The expressed protein in synthase in glyphosatetolerant soybeans, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested and is not toxic to mice upon acute gavage administration. J. Nutr.,v.128, p.756-761, 1996.
HOAGLAND, D.R.; ARNON, D.I. The water culture method for growing plants without soil. Calif. Exp. Sta. Circ., v.347, 1950.
HODGE, J.E.; HODFREITER, B.R. Determination of reducing sugars and carbohydrates. In: WILSTER, R.C. & WOLFRON, M.L. (eds.) Methods in Carbohydrate Chemistry, v.1, New York, Academic Press, 1962, p.380-394.
INSTITUTO ADOLFO LUTZ. Lipídeos. Normas analíticas do Instituto Adolfo Lutz: métodos químicos e físicos para análise de alimentos. 3 ed. São Paulo: IAL..., 1985. v.1, 533 p.
KITCHEN, L.M.; WITT, W.W.; RIECK, C.E. Inhibition of aminolevulinic acid synthesis by glyphosate. Weed Sci., v.29, p.571-577, 1981.
KITCHEN, L.M.; WITT, W.W.; RIECK, C.E. Inhibition of chlorophyll accumulation by glyphosate. Weed Sci., v.29, p.513-516, 1981.
LEA, P.J.; HUGHES, J.S.; MIFLIN, B.J. Glutamine and asparagine dependent protein synthesis in maturing legume cotyledons cultured in vitro. J. Exp. Bot., v.30, p.529-537, 1979.
LICHTENTHALER, H.K. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol., v.148, p.92-102, 1987.
MARGNA, J.; VAINJÄRV, T.; LAANEST, L. Different L-phenylalanine pools available for the biosynthesis of phenolics in buckwheat seedling tissues. Phytochemistry, v.28, p.469-475, 1989.
MOSQUIM, P.R.; SODEK, L. Culture of soybean fruit explants: growth conditions and efficiency of nitrogen sources for reserve protein synthesis. Plant Cell, Tissue and Organ Cult., v.27, p.71-76, 1991.
MUGNIER, J. Behaviour of herbicides in dicotyledonous roots transformed by Agrobacterium rhizogenes. J. Exp. Bot., v.39, p.1045-1056, 1988.
NELSON, N. A photometric adaptation of the Somogy method for the determination of glucose. J. Biol. Chem., v.153, p.375-380, 1944.
PINTO, J.E.B.P., DYER, W.E., WELLER, S.C.; HERRMAN, K.M. Glyphosate induces 3-dexoxy - D - arabino - heptulosonate - 7 - phosphate synthase in potato (Solanum tuberosum L.) cells grown in suspension culture. Plant Physiol., v.87, p.891-893, 1988.
SCHULZ, A.; MUNDER, T; HOLLÄNDERCZYTKO, H.; AMRHEIN, N. Glyphosate transport and early effects on shikimate metabolism and its compartmentation in sink leaves of tomato and spinach plants. Z. Naturforsch., v.45, p.529-534, 1990.
SERVAITES, J.C.; TUCCI, M.A.; GEIGER, D.R. Glyphosate effects on carbon assimilation, ribulose biphosphate carboxylase activity, and metabolite levels in sugarbeet leaves. Plant Physiol., v.85, p.370-74, 1987.
SOMOGY, M. Notes on sugar determination. J. Biol. Chem., v.95, p.19-23, 1952.
THOMPSON, J.F.; MADISON, J.T.; MUENSTER, A.M.E. In vitro culture of immature cotyledons of soybean (Glycine max (L.) Merril). Ann. Bot., v.41, p.29-39, 1977.
VAUGHN, K.C. & DUKE, S.O. Ultrastrutural effects of glyphosate on Glycine max seedlings. Pestic. Biochem. Physiol., v.26, p.56-65, 1986.

Datas de Publicação

Publicação nesta coleção
16 Jun 2010
Data do Fascículo
Abr 1999

Histórico

Aceito
07 Ago 1998
Recebido
25 Nov 1997

This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

[1] ASSOCIATION OF OFFICIAL ANALYTICAL CHEMISTS - A.O.A.C. Official methods of analysis. Washington, D.C., 1975. 1094p.

[2] BUBECK, DM; FEHR, WR; HAMMOND, EG. Inheritance of palmitic and stearic acid mutants of soybeans. Crop Sci., v.29, p.652-656, 1989.

[3] CHANDLER, PM; HIGGINS, TJV; RANDALL, PJ; SPENCER, D. Regulation of legumin levels in developing pea seeds under conditions of sulfur deficiency. Plant Physiol., v.71, p.47-54, 1983.

[4] COLE, DJ. Mode of action of glyphosate-a literature analysis. In: GROSSBARD, E; ATKINSON, E (eds). The herbicide glyphosate. London: Butterworths Publishers,1985, p. 48-74.

[5] DEVINE, M; DUKE, SO; FEDTKE, C. Physiology of herbicide action. Englewood Cliffs: PTR Prentice Hall, 1993, p.251-294.

[6] DUKE, SO. Glyphosate. In: KEARNEY, PC; KAUFMAN, DD (eds.). Herbicideschemistry, degradation and mode of action. New York: Dekker Publishers, 1988, p. 1-70.

[7] DUKE, S.O.; HOAGLAND, R.E. Effects of glyphosate on metabolism of phenolic compounds. Phenylalanine ammonia-lyase activity, free amino acids, root-fed amino acids and glyphosate toxicity in soybean (Glycine max) seedlings. Weed Sci., v.29, p.297, 1981.

[8] EUCLIDES, R.F. Manual para análise estatística e genética SAEG. Viçosa: UFV, CPD, 1982, 59 p.

[9] FONTES, E.P.B; MOREIRA, A.M.; DAVIES C.S.; NIELSEN N. C. Urea-elicited changes in relative electrophoretic mobility of certain glycinin and ?-conglycinin subunits. Plant Physiol., v.76, p.840-842, 1984.

[10] HARRISON, L.A., BAILEY, M.R., NAYLOR, M., REAM, J., HAMMOND, D.L. The expressed protein in synthase in glyphosatetolerant soybeans, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested and is not toxic to mice upon acute gavage administration. J. Nutr.,v.128, p.756-761, 1996.

[11] HOAGLAND, D.R.; ARNON, D.I. The water culture method for growing plants without soil. Calif. Exp. Sta. Circ., v.347, 1950.

[12] HODGE, J.E.; HODFREITER, B.R. Determination of reducing sugars and carbohydrates. In: WILSTER, R.C. & WOLFRON, M.L. (eds.) Methods in Carbohydrate Chemistry, v.1, New York, Academic Press, 1962, p.380-394.

[13] INSTITUTO ADOLFO LUTZ. Lipídeos. Normas analíticas do Instituto Adolfo Lutz: métodos químicos e físicos para análise de alimentos. 3 ed. São Paulo: IAL..., 1985. v.1, 533 p.

[14] KITCHEN, L.M.; WITT, W.W.; RIECK, C.E. Inhibition of aminolevulinic acid synthesis by glyphosate. Weed Sci., v.29, p.571-577, 1981.

[15] KITCHEN, L.M.; WITT, W.W.; RIECK, C.E. Inhibition of chlorophyll accumulation by glyphosate. Weed Sci., v.29, p.513-516, 1981.

[16] LEA, P.J.; HUGHES, J.S.; MIFLIN, B.J. Glutamine and asparagine dependent protein synthesis in maturing legume cotyledons cultured in vitro. J. Exp. Bot., v.30, p.529-537, 1979.

[17] LICHTENTHALER, H.K. Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol., v.148, p.92-102, 1987.

[18] MARGNA, J.; VAINJÄRV, T.; LAANEST, L. Different L-phenylalanine pools available for the biosynthesis of phenolics in buckwheat seedling tissues. Phytochemistry, v.28, p.469-475, 1989.

[19] MOSQUIM, P.R.; SODEK, L. Culture of soybean fruit explants: growth conditions and efficiency of nitrogen sources for reserve protein synthesis. Plant Cell, Tissue and Organ Cult., v.27, p.71-76, 1991.

[20] MUGNIER, J. Behaviour of herbicides in dicotyledonous roots transformed by Agrobacterium rhizogenes. J. Exp. Bot., v.39, p.1045-1056, 1988.

[21] NELSON, N. A photometric adaptation of the Somogy method for the determination of glucose. J. Biol. Chem., v.153, p.375-380, 1944.

[22] PINTO, J.E.B.P., DYER, W.E., WELLER, S.C.; HERRMAN, K.M. Glyphosate induces 3-dexoxy - D - arabino - heptulosonate - 7 - phosphate synthase in potato (Solanum tuberosum L.) cells grown in suspension culture. Plant Physiol., v.87, p.891-893, 1988.

[23] SCHULZ, A.; MUNDER, T; HOLLÄNDERCZYTKO, H.; AMRHEIN, N. Glyphosate transport and early effects on shikimate metabolism and its compartmentation in sink leaves of tomato and spinach plants. Z. Naturforsch., v.45, p.529-534, 1990.

[24] SERVAITES, J.C.; TUCCI, M.A.; GEIGER, D.R. Glyphosate effects on carbon assimilation, ribulose biphosphate carboxylase activity, and metabolite levels in sugarbeet leaves. Plant Physiol., v.85, p.370-74, 1987.

[25] SOMOGY, M. Notes on sugar determination. J. Biol. Chem., v.95, p.19-23, 1952.

[26] THOMPSON, J.F.; MADISON, J.T.; MUENSTER, A.M.E. In vitro culture of immature cotyledons of soybean (Glycine max (L.) Merril). Ann. Bot., v.41, p.29-39, 1977.

[27] VAUGHN, K.C. & DUKE, S.O. Ultrastrutural effects of glyphosate on Glycine max seedlings. Pestic. Biochem. Physiol., v.26, p.56-65, 1986.

Brasil

Brasil

Simulação da soja geneticamente modificada tolerante ao glyphosate por meio do cultivo de explantes

Simulation of the transgenic soybean tolerant to glyphosate through explant cultivation

Resumos

Datas de Publicação

Histórico