How GM Plants Conquered the World by romer-labs

Issue 6

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A Romer Labs® Publication

How GM Plants Conquered the World The 7 Sins of GMO Testing How to Change the DNA of a Plant

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Contents

4-9

How GM Plants Conquered the World

It only took GMOs 30 years to go from trial to covering more than 185 million hectares in 26 countries worldwide. Spot On takes a closer look at this revolution in food production. By Donna HOUCHINS, Research & Development Specialist, Romer Labs® USA

How to Change the DNA of a Plant Spot On is a quarterly publication of Romer Labs Division Holding GmbH, distributed free-of-charge. ISSN: 2414-2042

Editors: Joshua Davis, Cristian Ilea, Simone Schreiter

Contributors: Kurt Brunner, Donna Houchins, Pavlo Futernyk Graphic: GraphX ERBER AG Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

©Copyright 2023, Romer Labs® All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder. All photos herein are the property of Romer Labs or used with license.

A quick look at Agrobacterium tumefaciens and biolistics (the “gene gun”), two of the most common ways to transform the DNA of agricultural plants.

Cauliflower Mosaic Virus In 35S, the virus provides scientists with an effective promoter, but it also poses risks to cruciferous plants.

GMO Proteins Important to the Grain and Seed Industry What they are called and what they do.

The 7 Sins of GMO Testing

8-9 10-11

The use of GM crops is growing, as are the risks and challenges they bring to the analysis industry. One expert talks about what not to do in this fast-developing area. By Pavlo FUTERNYK, PhD, Managing Director, Romer Labs® Ukraine

Spot On Issue 6

Editorial Using Science to Help Feed the World We rely on agriculture to supply enough food to sustain life. Though the world's population is increasing, the amount of land available for growing crops and producing livestock is not getting any bigger. In some areas, it is actually getting smaller. Producing more crops with less space is a perpetual problem for the agricultural industry, especially in light of increasing pressure from the changing climate and environment. Adverse weather conditions and an everincreasing range of diseases all exert pressure on crop production. With advances in technology, crops can now be genetically modified so that they express characteristics such as herbicide tolerance, disease resistance and drought tolerance, promoting survivability and maximising yields. The possibilities for genetic modification are endless, but any changes made to crops bring a new set of challenges to how we analyze them. If regulations and labeling laws are any indication, the world is far from a consensus about genetically modified organisms (GMOs), with some countries opting for strict controls and others adopting a more laissez-faire approach. In light of such diversity in international opinion, it has never been more important for food and feed producers to test for GM ingredients in their products. In this issue of Spot On, the Romer Labs experts provide you with an overview of GMOs, highlighting their use around the world and the possibilities they offer the agricultural industry. We then provide some insight into the field of analysis by sharing what our experience has shown to be the “seven sins” of GMO testing. We hope that these suggestions can help you prevent your lab from falling into some common traps, ensuring that your results are accurate and reliable. Enjoy reading this issue of Spot On.

Kurt Brunner DI Dr., Division Research Officer, Romer Labs®

A R moam g ae zr i n L ea bosf® RPoumbel irc a L taibosn®

Spot On Issue 6

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How GM Plants Conquered the World By Donna Houchins - Research & Development Specialist, Romer Labs® USA

Genetically modified organisms (GMOs) are widely used in agriculture to give plants beneficial traits such as herbicide and pesticide resistance as well as quality traits that optimize growth and nutritional content. First trialed in the 1980s, GMO use is now widespread with over 185 million hectares of GM crops planted in 2016.

A Romer Labs® Publication

Some GM plants may express quality traits that allow them to be tolerant of environmental conditions such as drought, or to improve their nutritional content.

What are GMOs? Organisms that are genetically modified have undergone alterations to their genetic material in a process known as genetic engineering. These alterations result in the organism expressing a trait or traits that would not naturally occur in that organism. GMOs currently exist in bacteria, animals and plants, and are used in a wide range of applications including biological and medical research, the production of pharmaceuticals, and agriculture.

Why GMOs? One of the most widely adopted uses of GMOs is in agriculturally important crops. In these plants, alterations to the genetic material are often accomplished by inserting DNA material from a different organism into

the target organism. This results in the plant (and any seeds harvested from the plant) expressing novel traits, such as herbicide or insect resistance, or quality traits such as drought tolerance. For example, genetic modifications have been made so that plants are resistant to herbicides such as glyphosate or glufosinate, allowing a field to be sprayed with the herbicide to kill off weeds without harming the crop. Genetic modification can also involve the transfer of a trait or traits that allow the plant to produce endotoxins originating from the soil bacterium Bacillus thuringiensis, known as “Bt”. This confers insect resistance. These endotoxin proteins have been used as sprayon insecticides since the 1920s. They target certain insect species while having no effect on non-target

How to Change the DNA of a Plant

Using the bacterium Agrobacterium tumefa ciens provides a natural mechanism for transformation. The bacterium infects injured plant tissue and transfers its Ti plasmid to the plant’s chromosome. The Ti plasmid naturally contains genes that cause the plant tissues to overexpress plant hormones and nutrients for the bacteria, leading to plant tumors. The Ti plasmid may be modified to delete unwanted effects and add desirable traits, along with a selectable marker, which is then integrated into the plant’s chromosome during bacterial infection. However, not all species are susceptible to infection by this bacterium. Over the course of the last decade, a second method of transformation has grown more popular than Agro bacterium: biolistics, also known as the “gene gun” method. In this method, plasmid DNA is coated onto small tungsten or gold beads. These micron-sized beads are

Photo: Fotolia_Vitstudio

The transformation of plants can be accomplished in several ways. Two of the most common methods in agricultural crops are the use of Agrobacterium tumefaciens, and biolistics (the “gene gun”).

then “shot” into the plant tissue. Some of the cells in the plant tissue may successfully take up the new DNA and integrate it into a chromosome. This method has proven effective for integrating DNA into the cell nucleus as well as into organelles such as chloroplasts, and works in almost all species researched. Other methods that have been used include microinjection and electroporation. Spot On Issue 6

species such as humans, wildlife, and beneficial insects. When ingested, these proteins form pores in the mid-gut epithelium of the larvae of susceptible insect species (which feed on the crops, causing damage). This causes paralysis of the gut, and the affected insect stops feeding and succumbs to starvation. Non-target species have no receptors in the gut for the protein, and thus the protein has no effect on them. In addition, GM plants may express quality traits that allow them to be tolerant of environmental conditions such as drought, or to improve their nutritional content.

GMO naming conventions GMOs may be referred to in one of three ways. First, they may be identified by their event name, which is the name of the unique DNA recombination experiment that occurred in the laboratory in which one plant cell successfully incorporated a desired gene. That cell is subsequently used to regenerate whole plants and is the “foundation” of a GMO strain. For example, one event name for herbicide-tolerant corn is NK603. Second, GMOs may also be identified by the unique protein they express. In the case of event NK603, the protein expressed is CP4 EPSPS. Thirdly, the GMO may be identified by the trade name under which it is sold commercially.

GM crops around the world – then and now Current GMO production mainly comprises four crops: soybeans, maize, cotton, and rapeseed/canola. Global trade of these crops and their main derivatives is dominated by material of GMO origin. In addition, global planting of these four crops includes a very high percentage of biotech seed (78% of soybean, 64% of cottonseed, 33% of maize, and 24% of rapeseed/canola globally; ISAAA 2016). Within these crops, there are several GMO proteins currently important to the grain and seed trade. The cultivation of GM plants is increasing globally, as is the utilization of stacked traits - including two or more novel traits in the same plant. The first field trials of GM plants began in the United States and France in 1986, with herbicide-resistant tobacco. The first country to allow commercialized GM plants was China, which introduced a virus-resistant tobacco in 1992. The first GM crop approved for sale in the US was the FlavrSavr tomato in 1994. In that year, the European Union also approved its first GM plant for sale, which was a herbicide-tolerant tobacco. Commercialized cultivation of GM plants such as corn and cotton began in 1996. In 2016, 11 different types of GM crops were commercially grown on 457 million acres (185 million hectares) in 26 different countries around the world.

The global trade in soybeans, maize, cotton, and rapeseed/canola is dominated by material of GMO origin.

Cauliflower Mosaic Virus Genes that encode proteins (traits) need genetic elements called promotors in order to initiate the expression processes.

A very effective promotor is known as 35S. This promotor is derived from a common plant virus, the cauliflower mosaic virus. It is common practice in DNA-based analysis to detect the 35S promotor instead of the encoding gene as many different genetic modifications use the same promotor. The problem is that unmodified plants can be infected by the actual cauliflower mosaic virus. Such plants would be GMO-positive in a DNA-based analysis without ever having been transgenic. Cauliflower mosaic virus (CaMV) infections are widespread among cruciferous plants, including canola. To be sure, screen for CaMV in the DNA analysis. A Romer Labs® Publication

Cauliflower mosaic virus, Brunt et al. (1997): Plant Viruses Online: Descriptions and Lists from the VIDE Database. Retrieved from http://sdb.im.ac.cn/vide/descr184.htm

The most commonly approved GMOs are herbicide-tolerant traits. Since 2007, the number of approvals for stacked events has been more than for

The leading cultivators of GM crops Table 1 shows the global cultivation of GMOs in 2016. Adoption rates of GM crops in the countries where they are planted is often high. USDA survey data from 2016 shows that herbicide-tolerant soybeans comprised 94% of the planted acreage in the United States, herbicide-tolerant cotton comprised 93% of the planted acreage, and herbicide-tolerant corn comprised 92% of the planted acreage. The corn plantings comprised 3% insect-resistant, 13% herbicide-tolerant, and 76% stacked insect-resistant/herbicide-tolerant varieties. The cottonseed plantings comprised 4% insect-resistant, 9% herbicide-tolerant, and 80% stacked insect-

single events. Table 1. List of countries where GM crops are cultivated. Rank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Country

United States

Brazil

Argentina

72.9 49.1

23.8

Canada

11.6

Paraguay

3.6

India

Pakistan China

South Africa Uruguay Bolivia

Australia

Philippines Myanmar Spain

Sudan

Mexico

Colombia Vietnam

10.8 2.9

2.8

2.7 1.3 1.2 0.9 0.8 0.3 0.1 0.1 0.1 0.1

<0.1

Honduras

<0.1

Portugal

<0.1

Chile

Bangladesh

<0.1 <0.1

Costa Rica

<0.1

Czech Republic

<0.1

Slovakia

Source: ISAAA brief 52-2016

Area (million hectares in 2016)

<0.1

GMO Proteins Important to the Grain • CP4 EPSPS The expression of CP4 EPSPS transgenic protein in plants results in glyphosate herbicide tolerance. This protein is expressed in commercial varieties of creeping bentgrass, sugarbeet, rapeseed/canola, soybean, cotton, alfalfa, potato, wheat, and corn. • Bt-Cry1F The expression of Bt-Cry1F transgenic protein results in insect resistance. This protein is effective against the larvae of lepidopteran pests such as the tobacco budworm, beet armyworm, soybean looper, cotton bollworm/corn earworm, European corn borer, southwestern corn borer, fall armyworm, and black cutworm. This protein is expressed in commercial varieties of corn and cotton.

• Bt-Cry34Ab1 The expression of Bt-Cry34Ab1 transgenic protein in plants results in insect resistance. This protein is effective against the larvae of coleopteran pests such as the corn rootworm. This protein is expressed in commercial varieties of corn.

• Bt-Cry1Ab, 1Ac, & 1A.105 The expression of Bt-Cry1Ab, Cry1Ac, and/or 1A.105 transgenic protein results in insect resistance. The proteins are effective against the larvae of lepidopteran pests such as the European corn borer, tobacco budworm, cotton bollworm/corn earworm, pink bollworm, beet armyworm and soybean looper. These proteins are expressed in commercial varieties of corn, cotton, and tomato.

resistant/herbicide-tolerant varieties. The US also planted biotech soybean (herbicide-tolerant), canola, sugarbeet, alfalfa, and others. In Brazil, approximately 96.5% of soybean acreage was biotech. 36.7% of the acreage was herbicide-tolerant, and 59.8% was stacked insect-resistant/herbicide-tolerant. Approximately 88.4% of maize in Brazil is biotech, with the majority containing stacked traits. Approximately 79% of the cottonseed crop there was biotech. In Canada, approximately 93% of rapeseed/canola acreage is herbicide-tolerant. 94% of the soybean, 92% of the corn, and nearly 100% of the sugar beet is biotech. In India, approximately 96% of cotton planted is modified by the Bt bacterium. In China, biotech cotton comprised 95% of the acreage in 2016. In Paraguay, biotech resistant corn was first commercialized in 2013, Spot On Issue 6

GM crops have

and Seed Industry • Bt-Cry3Bb1 The expression Bt-Cry3Bb1 transgenic protein results in insect resistance. The protein is effective against the larvae of coleopteran pests such as the corn rootworm. This protein is expressed in commercial varieties of corn. • eCry3.1Ab The expression of eCry3.1Ab transgenic protein results in insect resistance. The protein is effective against coleopteran and lepidopteran insects. This protein is expressed in commercial varieties of corn.

• Bt-Cry2Ab The expression of Bt-Cry2Ab transgenic protein results in insect resistance. The protein is effective against the larvae of lepidopteran pests such as the cotton bollworm, pink bollworm and tobacco budworm. This protein is expressed in commercial varieties of cotton. • PAT The expression of PAT transgenic protein in plants results in Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium. It is also often used as a selectable marker for genetic transformation. This protein is expressed in commercial varieties of corn, rapeseed/canola, cotton, chicory, sugarbeet, and rice.

• VIP3A The expression of VIP3A transgenic protein in plants results in insect resistance. This protein is effective against the larvae of lepidopteran pests such as the cotton bollworm/corn earworm, tobacco budworm, pink bollworm, fall armyworm, beet armyworm, soybean looper,

and the adoption rate was already 44% by 2016. In Pakistan, 97% of cotton acreage was biotech.

Numerous approvals Many other countries do not cultivate GM crops, but have approved them for import as food and feed. In 2016, there were 115 food approvals, 87 feed approvals, and 49 cultivation approvals, for a total of 251 approvals. These approvals are divided among 87 events from seven crops. The most commonly approved GMOs are herbicide-tolerant traits. Since 2007, the number of approvals for stacked events has been more than for single events, and in 2016, 82.6% of the approved events were stacked. The trait distribution of approved events in 2016 was as follows: 14% insect-resistant, 15% product quality, 19% herbicide-tolerant, 6% herbicide-tolerant + pollination control, 3% herbicide-tolerant + product quality, A Romer Labs® Publication

cabbage looper, cotton leaf perforator, black cutworm, and the western bean cutworm. This protein is expressed in commercial varieties of corn and cotton.

• PMI The PMI protein (phosphomannose isomerase) is expressed by a gene derived from E. coli. This protein allows growth on mannose and is often used as a selectable marker in GM corn.

• NPTII The NPTII protein (neomycin phosphotransferase) is expressed by a gene derived from E. coli. This protein allows resistance to aminoglycoside antibiotics such as kanamycin, neomycin, paromomycin, and geneticin (G418). It is a commonly used selectable marker.

become a component of a strategy for many countries and companies looking to feed rapidly growing populations.

• cspB The cspB (cold shock protein B) protein is expressed by a gene derived from Bacillus subtilis. This protein allows for improved performance under water stress conditions. • DMO The expression of the DMO protein (dicamba monooxygenase) results in dicamba herbicide tolerance. This protein is expressed in commercial varieties of soybeans and cottonseed.

• aad-12 The expression of the aad-12 (aryloxyalkanoate dioxygenase 12) protein results in 2,4-D herbicide tolerance. This protein is expressed in commercial varieties of cotton and soybeans.

3% disease-resistant, 3% insect-resistant + disease-resistant, 31% herbicide-tolerant + insect-resistant, and 6% others. The global use of biotech crops is increasing. These technologies can help provide new crops with higher yields, higher disease resistance, resistance to adverse environmental conditions, and increased nutritional value. World opinion about the value of GMOs, however, is divided. Yet it is indisputable that GM crops have become a component of a strategy for many countries and companies looking to feed rapidly growing populations.

Reference ISAA brief. (2016). Global Status of Commercialized Biotech/GM Crops: 2016 ISAAA Brief 52-2016, February 23, 2016. Retrieved October 12, 2017.

It is imperative to thoroughly clean any grinding equipment after use, as crosscontamination is a huge problem in GMO testing.

The Sins of GMO Testing By Pavlo Futernyk, PhD – Managing Director, Romer Labs® Ukraine

The use of genetically modified (GM) crops is increasing around the world. This presents many opportunities to the agricultural industry while it brings many challenges to the analysis industry. One Romer Labs expert shares his knowledge about testing GM crops so that you can avoid making common mistakes in your laboratory. 1. Identifying the wrong target Do your homework: this is perhaps the most basic habit any scientist can learn. This is particularly necessary for genetically modified organism (GMO) testing, where doing your homework means knowing which events comprise your targets so that you can avoid false positives and erroneous results. The problem relates to traits. A trait is a protein derived from a genetic modification which gives a special feature to the plant. Genetic modifications may be present in different combinations that produce similar—or entirely different—traits. Among the most popular elements of genetic modifications are promoters (p35S, FMV), terminators (NOSt), coding genes for certain useful traits (cp4 epsps, pat, bar, Cry1A, and others), and genes coding selective markers (NPTII, PMI). Your technicians need to consider many different factors in defining the target for any GMO analysis. Make sure you consider the territory the product originates from as well as the possible events, whether these events are authorized in the region in question, and whether the plant of interest has commercially available biotech traits. It can be tricky: sometimes the modification exists but is not authorized or planted in certain regions. However, it may be widely used in another region. In addition, there have been cases of unapproved events escaping containment and making their way to the field.

2. Choosing a faulty or insufficient method It can be a risky decision: what should be the scope of any GMO screening? Laboratories can narrowly target a few genetic elements or proteins, or they can expand the scope to several events or proteins, which can be expensive. Finding the method that fits your needs and ensures accurate results is essential. DNA-based methods are time-consuming and depend on high-quality DNA extraction and proper controls.

Copy number variation and polyploidy can also cause problems in samples and should be taken into consideration when performing DNA analysis. A look at some common plants will illustrate the point. In the case of soybeans, it is not enough to test only for promoters and terminators, because only a few of the 15 potential soybean events contain these common genetic elements. Maize is a different story al together: broadly screening for these elements is almost completely sufficient, because most events in maize contain these elements. While DNA-based methods are expensive and used only in analytical service labs, protein identification is relatively cheap and is useful in screening for common traits on-site. The majority of GM events has its own level of modified protein expression. Be careful when carrying out protein analysis of soybean and canola with the same lateral flow device (LFD) strips. Table 1 highlights different expression rates of CP4 EPSPS, making it necessary to use different LFD detection methods in soybean and canola. The right approach uses different tests and adjusts the sensitivity for different commodities to reach a similar level of detection.

3. Not fully understanding different degrees of test sensitivity DNA-based methods are very sensitive and are able to detect a single copy of the target gene. The sensitivity for DNA-based methods is limited by the number of seeds tested. It is usually 0.01% or one genetically modified (GM) seed in a set of 10,000 non-GM seeds. Such sensitivity is more than enough to be 99.9% sure of any specific level of detection down to 0.1%. On the other hand, protein-based methods have a limit of detection (LOD) as low as 0.1%, or 1 GM seed in 1,000 non-GM seeds. Yet this sensitivity is not enough to guarantee a result with 99% certainty. The best you can get with a single protein test with an LOD of 0.1% is Spot On Issue 6

Table 1. Different expressions rates of CP4 EPSPS in soybean and canola. Commodity

Event

Trait

Average Level

Soybeans

GTS-40-3-2

CP4 EPSPS

280 ug/g

GT73

CP4 EPSPS

28 ug/g

Soybeans Canola

MON89788

CP4 EPSPS

140 ug/g

Source: ILSI Research Foundation, 2010.

a level of 0.46% at 99% confidence. The non-uniform distribution of the seeds in real samples leads to this discrepancy. To be 99% certain that the level of GMO contamination is lower than 0.1%, we recommend that you carry out at least four tests of 1,000 kernels at a sensitivity of 0.1% each. For 95% confidence at 0.1%, carry out three tests of 1000 kernels all with negative results.

4. Removing impurities or treating the sample Grain labs often remove impurities from samples before conducting a test. However, the purification of GMO samples is not allowed. If some residues containing traces of GM plants are left in the samples, they should be detected. For example, if a corn sample contains some residues of GM soy (kernels, shells, dust), washing and manual cleaning will remove these residues and produce an incorrect result. Do not subject the samples to any kind of heat treatment, not even in order to dry excess moisture. For protein-based methods in particular, this could result in the denaturation of the trait proteins and lead to the complete loss of test sensitivity, as the target protein would have changed. Antibodies used in these applications would then no longer be able to recognize their targets.

5. Using an improper procedure to prepare the sample Your method of DNA isolation should be carefully designed and followed to avoid DNA degradation, which could skew your results. Therefore, it is important to verify the quality and quantity of purified DNA. In addition to the DNA extraction reagents used, the grinding method is also very important. Blade mills give the best results, as they grind finely enough to enable DNA extraction. However, it is imperative to thoroughly clean any grinding equipment after use, as cross-contamination is a huge problem in GMO testing. Equipment that is not properly cleaned could produce false negative results in subsequent batches. Protein extraction requires a coarser grind. It is A Romer Labs® Publication

enough simply to crush all seeds in the sample. If you grind seeds too finely, it will take too long for the extract to settle, and the sample will contain too many solid particles, which will stick to the LFDs and inhibit the flow of liquid. Mills used for extraction before LFD analysis should be set to avoid causing fractions that are too fine. The shape of the extraction vessel should also be considered: it should not be flat, and it is better to use disposable vessels to avoid cross-contamination. Blenders are very commonly used for grinding for protein testing because they are very easy to clean, therefore avoiding problems with cross-contamination.

Make sure you thoroughly read all packaging and documentation.

6. Customizing testing procedures or failing to read the package insert This could well be the most serious sin that operators commit. While reference testing labs employ trained staff throughout the whole year, grain labs often have a seasonal employment policy and therefore employ staff with less expertise in testing. This could lead to the testing procedure being misread, incorrectly followed or altered in the lab. This can affect all parts of the testing process, beginning with extraction and affecting testing elements such as the volumes of liquid required for the test, development times, and analysis procedures. Make sure you thoroughly read all packaging and documentation before carrying out any analysis to ensure reliable results.

7. Insufficiently cleaning your equipment (causing cross-contamination) Make sure that the mill you use for GMO analysis is easy to clean. The best way is to use removable jars and blades so that you can clean them separately from the mill motor. Every component of your equipment should be washable. Blenders are commonly used for grinding GMO samples as they are easy to clean. Mills have to be dismantled and all the parts washed separately. Wash all equipment with liquid soap and rinse with water afterwards. Do not use ethanol as a cleaning agent; its only use is to accelerate the drying process. Each lab should validate its cleaning procedure to ensure efficiency. We recommend the use of gloves when handling all samples. Changing your gloves and washing your hands between samples will also help to avoid cross-contamination. Make sure you clean any tools that may have come into contact with the extract of ground material. Another solution is to use disposable equipment as much as possible.

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