Identification of geneticaly modified soybean seeds resistant to glyphosate

Abstract

Advances in genetic engineering permit the modification of plants to be tolerant to certain herbicides that are usually not selective. For practical and commercial purposes, it is important to be able to detect the presence or absence of these traits in genotypes. The objective of this research was to develop a procedure for identifying genetically modified soybean (Glycine max L. Merr.) with resistance to the herbicide glyphosate. Two studies were conducted based on germination test. In the first study, soybean seeds were pre-imbibed in paper towel with the herbicide solutions, then transferred to moist paper towel for the germination test. In the second study, seeds were placed directly in herbicide solutions in plastic cups and tested for germination using the paper towel method. Eight soybean genotypes were compared: four Roundup Ready, that contained the gene resistant to the herbicide (G99-G725, Prichard RR, G99-G6682, and H7242 RR) and four non-transgenic parental cultivars (Boggs, Haskell, Benning, and Prichard). In the first study, the seeds were imbibed for 16 hours at 25°C in herbicide concentrations between 0.0 and 1.5% of the glyphosate active ingredient. In the second, seeds were subjected to concentrations between 0.0 and 0.48%, for one hour, at 30°C. The evaluation parameters were: germination, hypocotyl length, root length and total length of the seedlings. Both methods are efficient in identifying glyphosate-resistant soybean genotypes. It is possible to identify the genetically modified soybean genotypes after three days, by imbibing the seed in 0.12% herbicide solution, and after six days if the substrate is pre-imbibed in a 0.6% herbicide solution. The resistance trait was identified in all cultivars, independent of the initial physiological quality of the seed. Key words: transgenic, germination, detection, glyphosate IDENTIFICAÇÃO DE SEMENTES DE SOJA GENETICAMENTE MODIFICADA RESISTENTE AO GLIFOSATORESUMO: Avanços na engenharia genética têm resultado na obtenção de plantas tolerantes a certos herbicidas que usualmente não são seletivos. Para fins práticos e comerciais é importante ser capaz de detectar a presença ou ausência dessas características nos genótipos. O objetivo desta pesquisa foi desenvolver um procedimento para identificação de soja (Glycine max L. Merr.) geneticamente modificada com resistência ao herbicida glifosato. Dois estudos foram conduzidos baseados no teste de germinação. No primeiro estudo, sementes de soja foram pré-embebidas em papel toalha com solução do herbicida e após transferidas para papel toalha umedecido para o teste de germinação. No segundo estudo, as sementes foram colocadas diretamente nas soluções do herbicida em copos plásticos e avaliada a germinação pelo método do papel toalha. Oito genótipos de soja foram comparados: quatro Roundup Ready, que contém o gen de resistência ao herbicida (G99-G725, Prichard RR, G99-G6682 e H7242 RR) e quatro cultivares parentais não transgênicas (Boggs, Haskell, Benning e Prichard). No primeiro estudo, as semen...

Ajuste de metodologias para a identificação de cultivares de soja quanto à tolerância ao glifosato

“…Como em vários outros estudos sobre detecção de sementes de soja tolerante ao herbicida (Goggi andStahr, 1997, Bevilaqua et al, 2000;Torres et al, 2003;Funguetto et al, 2004;West, 2004, Cunha et al, 2005;Miranda et al, 2005), a diferença marcante entre as cultivares foi a presença e ausência de plântulas normais quando em tratamento contendo o herbicida, tanto para o bioensaio em papel umedecido (Tabelas 2 e 3), quanto para o bioensaio de pré-embebição (Tabelas 6 e 7). O princípio de ambos bioensaios consiste no fato de que o contato entre as sementes de soja convencional com a solução do herbicida (Funguetto et al, 2004;Tillmann and West, 2004;Miranda et al, 2005) resulta em plântulas anormais. Nesta situação de contato com o herbicida, sementes de uma cultivar GM resultam plântulas normais.…”

“…No que se refere à soja GM tolerante ao glifosato, bioensaios com diferentes princípios vêm sendo publicados no intuito de fornecer uma metodologia confiável para detecção de sementes de soja GM em lotes de sementes de soja não-GM. Entre os bioensaios, todos com bons resultados, podem ser citados: germinação seguida de aplicação do herbicida sobre as plantas (Goggi and Stahr, 1997); pulverização foliar com glifosato pós-germinação (Bevilaqua et al, 2000;Torres et al, 2003); umedecimento do substrato de germinação com solução do herbicida (Bevilaqua et al, 2000;Funguetto et al, 2004;Miranda et al, 2005); identificação com base na atividade de enzimas (Menezes et al, 2004); imersão de sementes em solução do herbicida (Funguetto et al, 2004;Tillmann and West, 2004); pré-embebição de sementes em substrato umedecido com glifosato (Goggi and Stahr, 1997;Funguetto et al, 2004;Tillmann and West, 2004); emergência da plântula em areia, seguida de aplicação do herbicida (Miranda et al, 2006); e, detecção em sistema hidropônico (Bertagnolli et al, 2006).…”

Detection of adventitious presence of genetically modified seeds in lots of non transgenic soybean

“…Immediately after, these rolls were then placed into a seed germinator, at 25 ºC, during 16 h, in the dark (Tillmann and West, 2004). To obtain the standard solution of the AE of the herbicide glyphosate, a formulation of the product Roundup ® WG, containing 792.5 g.kg -1 glyphosate ammonium salt, 720.0 g.kg -1 of AE of N -Phosphonomethyl (glyphosate), and 207.5 g kg -1 of other ingredients) were used.…”

“…After that period, the seeds were set to germinate; what was performed by placing the seeds, evenly distributed, on top of paper towels moistened with distilled water; with eight subsamples of 25 seeds each or 16 subsamples of 25 seeds each (depending on sample size), and then placed into a seed germinator, at 25 ºC, according to Rules for Seed Testing (Brasil, 2009). For the assessment of seedlings, the methodology described by Tillmann and West (2004) was used. Seedlings were sorted as normal (tolerant to glyphosate) and abnormal (non-tolerant to glyphosate, i.e., seedlings of non-GM soybean).…”

“…Estimates of the Associação Brasileira de Sementes e Mudas (ABRASEM) (Brazilian Association of Seeds and Seedlings) for the 2011/2012 crop season indicate that the demand for transgenic soybean seeds is still more significant, and in mean figures, countrywide, the area sowed with GM soybean has already surpassed 80%. Zito et al (2011) have reported that the decrease in the area of non-GM soybean seed production is due to the difficulty, each time greater, in producing seed exempt of transgenic seeds. Although soybean is a cleistogamic autogamous plant, the exchange of genetic material among plants in the field occurs in variable rates, according to the distance of the transgenic pollen source and of the population of insects.…”