Plant Report BIOL - The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification PDF

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The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants. Third lab report for BIOL 1050....

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The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants Erin Whyte BIOL 1050, Section 108 1056313 November 12, 2018

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

ABSTRACT The Glycine max (L.) Merr plant, also known as the soybean, has widely been genetically modified with the CP4 EPSP synthase gene. Because of this, it is immune to the glyphosate herbicide RoundupTM, and will not die when sprayed. Genetically modified plants generally have no physical differences visible to the naked eye. The GM soybean plant and organic soybean plant look identical. Since the GM soybean plant contains a transgenic trait, the CP4 ESPS synthase, an ELISA test can be used to determine if a plant has been genetically modified. In this experiment, the ELISA test used was the Agdia extraction kit. From a random set of soybean seedlings, leaves from each plant were tested using the Agdia kit to determine if the individual plants were genetically modified or organic. After determining and recording the presence of GM, the set of soybeans was sprayed with the RoundupTM herbicide. After a week, the plants were observed again, and it was recorded which were alive and which were dead. From this, false negatives and positives were able to be found, and the efficacy of the Agdia test determined. It was discovered that the Agdia test kit is around 90% effective in determining the presence of genetic modification. Understanding these findings is central to understanding how the effectiveness of genetic modification testing. INTRODUCTION Genetic modification is a type of genetic engineering which involves the direct modification of the genetic material in plants or animals (Uzogara 2000). Many plants and animals today contain some form of genetic modification. The use of genetic modification in plants can increase yield and improve resistance to pests, toxins and weather. Glycine max (L.) Merr, also know as the soybean, is just one of many plants that have been widely genetically modified. Reasons for genetic modification in soybeans include increased crop yield and allergies. Soybeans are used for a variety of things, from medical and food to industrial purpose. Because of the many uses for the plant, it is in high demand and a large crop yield is needed. Many other types of vegetation compete with soybean crops, so an herbicide must be used to obtain the largest yield possible. A popular herbicide is glyphosate, or RoundupTM. Glyphosate is toxic to all plants, including soybeans. For the soybeans to survive the spraying of glyphosate, they had to be genetically modified. pg. 2

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

Glyphosate works by inhibiting the EPSPS enzyme, which produces synthesizes essential aromatic amino acids (Taylor et al. 1999). Without the production of these amino acids, plants die. To counteract this effect, a glyphosate tolerant enzyme, CP4 EPSPS, was introduced into the genome of some soybeans. This enzyme is not killed by the herbicide and is sufficient enough in producing the required EPSPS products that the plant will continue to grow and develop, even after EPSPS is inhibited. The CP4 EPSPS enzyme is classified as a transgenic trait, because it was derived from an organism other than the soybean. CP4 EPSPS was derived from Agrobacterium sp. Strain CP4 (Padgette et al. 1995). This trait is also called the RR gene, as it is resistant to the glyphosate herbicide RoundupTM, and the soybean plants resistant to it are coined RoundupTM Ready. With the invention of genetic modification, GM testing follow. Being able to test for the presence of genetic modification is extremely important, as many products must be tested to determine if they are GM or non-GM. Chromatographic and electrophoresis techniques have proved successful in determining GM but can be expensive and time consuming (Asensio et al. 2008). ELISA, or Enzyme-Linked ImmunoSorbent Assay, is another reasonable and widely used type of GM testing. It works by using an enzyme (protein) to detect certain antibodies and antigens specific to the genetically modified species. When preforming a test, non-GM plant would test negative as it does not produce the new protein, where as a GM plant would test positive. In this experiment, the ELISA technique was used by testing the soybeans with and Agdia test kit, a type of lateral flow test which uses and ImmunoStrip. This kit works by using colloidal gold nanoparticles and specific enzymes with antibodies. When the strip is placed in the homogeneous solution of plant and buffer, it will cause the gold nanoparticles to attach to the enzyme-antibody combination. This will cause a line of colour to appear, GM or non-GM. Then, pg. 3

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

if the plant is GM, the antigens in it will attach to the enzyme-antibody-gold combination and a second line of colour, above the first will appear. Two lines means the test is positive and the organism has GM presence, while one means negative and the plant is non-GMO Hypothesis It was hypothesized that the Agdia strip test can be used to detect the level of GM presence in a population of soybean plants. Objective The objective of this study was to determine the level of the Roundup Ready gene present in a sample of soybean plants and determine the efficacy of an Agdia strip test as measured by the frequency of false positives and false negatives of the test in comparison to the reaction of the plants to the herbicide glyphosate.

METHOD AND MATERIALS The following description of the materials and methods are for The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification experiment for determining the effectiveness the Agdia test kit has when testing for genetic modification in soybeans. This experiment can be found in the Biology 1050 Laboratory Manual Module 3, pages 6-8 and 15.

Glycine max (L.) Merr Step 1: preparing to test for genetic modification in Glycine max (L.) Merr using the Agdia kit. Following the manufacture’s instructions, the test kit was prepared. This included cutting open the top of the extraction bag, which contains the buffer and the two mesh sheets, right above the mesh sheets. Then a mature Glycine max (L.) Merr (soybean) leaf from a random population of pg. 4

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

soybeans, grown at 22oC with 16 hours of light and 8 hours of darkness cycle, was obtained and placed between the two mesh sheets in the kit. The blunt end of a pen was then used to homogenize the leaf between the sheets and mix with the solution. The mixture was left to sit for 30 seconds.

Step 2: testing the solution with the ImmunoStrip. After the mixture sat for 30 seconds, an ImmunoStrip was removed from the container. The strip was held at the end with the test name. It was then carefully placed in the extraction bag, with the end of the strip submerged less than 5 millimetres in the solution. The strip was held in the extraction bag for a total of 3 minutes. After three minutes the strip was removed. If two lines of colours appeared on the test strip, the test was recorded as positive for that individual Glycine max (L.) Merr (soybean) plant. If one line of colour appeared on the test strip, the test was recorded as negative for that plant. The test was then repeated on all the soybean plants in the random population and results were recorded.

Step 3: spraying the Glycine max (L.) Merr plants with RoundupTM. After all the soybean plants from the population were tested, they were put back in their original growing environment. The plants were then sprayed with a glyphosate herbicide called RoundupTM a concentration of 540 g ai L-1. The plants were sprayed at a rate of 2.6 L ha-1. After the population was sprayed, it was left for one week.

Step 4: determining false negatives and false positives. After a week had passed, the population of Glycine max (L.) Merr (soybean) plants was removed from the growing environment to be observed. Plants that had died from the glyphosate were pg. 5

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

recorded with a negative, and plants that survived were recorded with a positive. From these results and the results from the Agdia testing, false negatives and false positives were able to be determined. If a plant tested positive for the presence of genetic modification but died, it was recorded as a false positive. If a plant tested negative for the presence of genetic modification but survived, it was recorded as a false negative. From the number of false positives and negatives and the total amount of soybean plants tested, the efficacy of the Agdia test kit was determined.

RESULTS The table shows the results from the experiment of The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants. From the Table 1, it can be determined that there were two false positives and four false negatives. The plants that were false positives were F5 and D8. Both plants tested positive for the presence of genetic modification but died when sprayed with the RoundupTM. The plants that were false negatives were E1, F2, C4 and E8. These plants all tested negative for the presence of genetic modification but lived after being sprayed with RoundupTM. It can also be seen from Table 1 that all the soybean plants which tested positive for the presence of genetic modification lived, and all the other plants which tested negative died.

Row

Column

A B C D E F

1 1 1 1 1 1

Detection of RR Gene in Soybeans Test Strip RoundupTM Spray Result (+ if plant alive, - if plant (+ if gene detected, - if gene not dead) detected) + + + + + + + pg. 6

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

A B C D E F A B C D E F A B C D E F A B C D E F A B C D E F A B C D E F A B C D E F A B C

2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 4 4 5 5 5 5 5 5 6 6 6 6 6 6 7 7 7 7 7 7 8 8 8 8 8 8 9 9 9

+ + + + + + + + + + + + + + + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + + + + pg. 7

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

D E F A B C D E F

9 9 9 10 10 10 10 10 10

+ + + +

+ + + + Table 1

DISCUSSION The ability to test organisms for the presence of genetic modification can be crucial. The utilization of genetic modification in food is a controversial topic, as many believe it is harmful (Grunert et al. 2001). Because of this controversy, food must not only be tested for the presence of GM, but also the percentage of genetic modification (Uzogara 2000). Food is then able to be labelled non-GM if the amount of GM present is under a certain percentage. Food not labelled non-GM is assumed to contain genetic modification. Since the regulations for food are so tight, a reliable form of testing must be used. One form of testing, as seen in this experiment, is ELISA. The purpose of the experiment was to determine the efficacy of ELISA testing using the Agdia test kit. From the results, it was determined that the Agdia test was only 90% effective, as there were 60 plants tested and 6 false negatives and false positives. Though this seems quite effective, when it comes to testing food it would not be acceptable. The ELISA tests also do not give the percent of genetic modification, which is needed to determine if a food is GM or nonGM. For the Agdia test strips to test positive, they require the Glycine max (L.) Merr plant to contain at least 0.25% of the genetically modified trait. Because of this requirement, it is possible that the soybean plants which did not test positive actually contained the CP4 EPSPS trait, just not enough to be detected. This would explain the presence of the multiple false negatives. The plants E1, F2, C4 and E8 all tested negative because they may not have had enough of the trait to pg. 8

The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

be detected, but enough for the plants to survive the spraying of the RoundupTM. The false negatives may also have been cause by human error in the testing of the soybeans. The plants F5 and D8 were false positives. This could have been because the samples from these plants were contaminated from the Agrobacterium itself, as it is found in soil. Though it may be unlikely, the Agrobacterium sp. CP4 may have been in the soil, and because this is the organism that the CP4 EPSPS trait is derived from and would be detected by the Agdia test. This would explain why the plants tested positive but died after being sprayed as they did not actually contain the trait. Conclusion In conclusion, the Agdia test strip kit would be suitable for field testing, but not much more. It is not suitable for determining the presence of genetic modification of the Glycine max (L.) Merr plant in the food sector, as it requires much more precise measurements. It was hypothesized that the Agdia test strip could be used to determine the presence of genetic modification. This was proved to be somewhat correct, as it determined the presence of the CP4 EPSPS gene in most of the soybean plants, but there was 10% error. Overall, the Agdia test strips would work well for quick testing, but not when determining any precise measurements to do with genetic modification.

REFERENCES Asensio, L, González, I, García, T, Martín, R. 2008. Determination of food authenticity by enzyme-linked immunosorbent assay (ELISA). Food Control. 19:1–8. Taylor, N.B, Fuchs, R.L, Macdonald, J, Shariff, A.R, Padgette, S.R. 1999. Compositional Analysis of Glyphosate-Tolerant Soybeans Treated with Glyphosate. Journal of Agricultural and Food Chemistry. 47:4469–4473. Uzogara, S.G. 2000. The impact of genetic modification of human foods in the 21st century. Biotechnology Advances. 18:179–206.

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The Efficacy of Agdia Test Strips in Determining the Presence of Genetic Modification in Glycine max (L.) Merr Plants

Grunert, K.G, Lähteenmäki, L, Nielsen, N.A, Poulsen, J.B, Ueland, O, Åström, A. 2001. Consumer perceptions of food products involving genetic modification—results from a qualitative study in four Nordic countries. Food Quality and Preference. 12:527–542. Padgette, S.R, Kolacz, K.H, Delannay, X, Re, D.B, Lavallee, B.J, Tinius, C.N, Rhodes, W.K, Otero, Y.I, Barry, G.F, Eichholtz, D.A. 1995. Development, Identification, and Characterization of a Glyphosate-Tolerant Soybean Line. Crop Science. 35:1451. APPENDIX Plant ID A1

Positive or Negative (-/+) for RR gene +

pg. 10...