Lecture 4 - Genetic Engineering in Agriculture PDF

Introduction to Genetic Engineering in Agriculture: Exploring the Foundations and Techniques Lecture 4 Dr. Hadil Alahdal KEY QUESTIONS How is a gene isolated and amplified by cloning? How are specific DNAs or RNAs identified in mixtures? How is DNA amplified without cloning? How is amplif...

Introduction to Genetic Engineering in Agriculture: Exploring the Foundations and Techniques Lecture 4 Dr. Hadil Alahdal KEY QUESTIONS How is a gene isolated and amplified by cloning? How are specific DNAs or RNAs identified in mixtures? How is DNA amplified without cloning? How is amplified DNA used in genetics? How are DNA technologies applied to agriculture Lecture outline Introduction Foundations of Genetic Engineering Techniques in Genetic Engineering Applications of Genetic Engineering in Agriculture Examples of Genetic Engineering in Agriculture Ethical, Legal, and Social Considerations Genetic engineering in agriculture It is the process of introduction of a gen with a particular character inside the chromosome of a cell Usually, it helps to improve crop variety Ensures food security Introduce beneficial traits Raise the quality and yield of crops Introduction To date, scientists have engineered bacteria that produce medication-grade drugs, crops with built-in pesticides, and beagles that glow in the dark. While these are all relatively recent advances in scientific technology, humans have been altering the genetics of organisms for over 30,000 years. How did the original practice of selective breeding evolve into the concept of genetically modified organisms, as we know it today? Innovators, motivated by some of the world’s most critical problems, have paved the way for GMOs — a path that leads to an unimaginable array of benefits, but also raises extremely important questions.  Genetic engineering Genetic engineering, also called genetic modification or genetic manipulation, is the modification and manipulation of an organism's genes using technology. It is a set of technologies used to change the genetic makeup of cells, including: The transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesizing the DNA. A construct is usually created and used to insert this DNA into the host organism. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly or targeted to a specific part of the genome. History The first GMO was a bacterium generated by Herbert Boyer and Stanley Cohen in 1973. Rudolf Jaenisch created the first GM animal when he inserted foreign DNA into a mouse in 1974. The first company to focus on genetic engineering, Genentech, was founded in 1976 and started the production of human proteins. Genetically engineered human insulin was produced in 1978 and insulin-producing bacteria were commercialized in 1982. Genetically modified food has been sold since 1994, with the release of the Flavr Savr tomato. The Flavr Savr was engineered to have a longer shelf life, but most current GM crops are modified to increase resistance to insects and herbicides. GloFish, the first GMO designed as a pet, was sold in the United States in December 2003. In 2016 salmon modified with a growth hormone were sold.  Ancient Genetic Modification The dog is thought to be the first organism our ancestors artificially selected. Around 32,000 years ago, wild wolves in East Asia joined groups of humans as scavengers. They were domesticated and then artificially selected to increase docility. Artificial selection has also been utilized with a variety of plants dates back to 7800 BCE in archaeological sites found in southwest Asia, where scientists have found domestic varieties of wheat. one of the most dramatic alterations in plant genetics has occurred in corn. Corn, or maize, began as a wild grass called teosinte that had tiny ears with very few kernels. A similar process has given us large heads of broccoli, bananas with nearly unnoticeable seeds, and apples that are sweet and juicy. The Birth of Modern Genetic Modification Examples of Genetic Engineering in Agriculture Golden Rice: A solution to vitamin A deficiency Bt crops: Effective pest management Herbicide-tolerant crops: Weed control in agriculture Disease-resistant crops: Protecting plants from pathogens  Other examples: UNDERSTANDING GMOs Source: GMOAnswers.com :https://gmoanswers.com/sites/default/files/GMOA-GeneticTraits10crops-4x6_Postcard-Jan2018.pdf Examples of genetic modification tools: Techniques in Genetic Engineering Gene cloning and recombinant DNA technology Genome editing using CRISPR-Cas9 RNA interference and gene silencing Plant tissue culture and micropropagation DNA technology DNA technology depends on two basic foundations of molecular biology research: The ability of specific proteins to recognize and bind to specific base sequences, within the DNA double helix (Figure 11-1). The ability of complementary single-stranded DNA or RNA sequences to spontaneously unite to form double- stranded molecules. Examples are the binding of the sticky ends and the binding of the primers.  Generating recombinant DNA molecules Type of donor DNA Genomic DNA. This DNA is obtained directly from the chromosomes of the organism under study. It is the most straightforward source of DNA. It needs to be cut up before cloning is possible. cDNA. Complementary DNA (cDNA) is a double stranded DNA version of an mRNA molecule. In higher eukaryotes, an mRNA is a more useful predictor of a polypeptide sequence than is a genomic sequence, because the introns have been spliced out. Chemically synthesized DNA. Sometimes, a researcher needs to include in a recombinant DNA molecule a specific sequence that for some reason cannot be isolated from available natural genomic DNA or cDNAs. 1-Cutting genomic DNA Restriction fragments  Restriction enzymes? Restriction enzymes cut DNA into fragments of manageable size, and many of them generate single stranded sticky ends suitable for making recombinant DNA. 2-Attaching donor and vector DNA 3-Amplification inside a bacterial cell Vectors for DNA cloning How can scientists recognize if the insertion is successful? Take away message! Gene cloning is carried out through the introduction of single recombinant vectors into recipient bacterial cells, followed by the amplification of these molecules as a result of the natural tendency of these vectors to replicate. What is the process of GM?  Ways to GMOs are the product of a specific type of plant breeding where precise changes are made to a plant’s Have Better DNA to give it characteristics that cannot be achieved Harvests through traditional plant breeding methods. Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/WhatIsGMO_Infographic_1224x792px-Jan2018_0.jpg  The GMO Process Step 1: Trait Identification Fun fact: For every one trait that is brought to market, more than 6,000 others are screened and tested. Scientists conduct research to identify the specific genes responsible for beneficial traits that make crops resistant to disease, pests or drought. Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/GMOA%2011x17%20Handout_0.pdf  The GMO Process Step 2: Transformation Fun fact: There are many ways to transform a cell. One common method uses agrobacterium - a natural bacterium that passes genes to plants. Once the desired gene has been identified, scientists transfer the gene into a plant seed. The result is a genetically modified organism or GMO. Researchers can also turn off or move a gene within a plant to create a GMO. Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/GMOA%2011x17%20Handout_0.pdf  The GMO Process Step 3: Regulatory Science Although the regulatory review process begins here, it will continue throughout the GMO process and carry on through the life cycle of the product. Safe to grow Crop exhibits expected Fun fact: characteristics (e.g. insect resistance) A new biotech seed product takes an average Safe for the environment and of 13 years and beneficial insects $136 million in R&D before coming to market.2 Safe to eat Same nutrients as non-GM crops & No new dietary allergens More than 75 different studies are performed on each new biotech product before commercialization to ensure that they are safe for people, animals and the environment.1 Sources: Phillips McDougall 2011; GMOAnswers.com https://gmoanswers.com/sites/default/files/2018- 11/How%20Does%20a%20GMo%20Get%20to%20Market_0.JPG  The GMO Process Step 4: Greenhouse Testing Fun fact: Only after several years of rigorous testing are the top performing plants and traits selected to advance to field testing and further regulatory review. After a GMO is developed in the lab, the seedlings are moved to greenhouses where further tests are performed. Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/GMOA%2011x17%20Handout_0.pdf The GMO Process Step 5: Field Testing Fun fact: More than 90 government bodies in more than 60 countries globally review and approve GMOs. In many countries, multiple agencies are involved in the regulation of GMOs. Field trials are an important part of developing new products. They provide critical scientific and performance data. Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/GMOA%2011x17%20Handout_0.pdf  The GMO Process Step 6: Getting Seeds to Farmers Fun fact: In 2018, 17 million farmers globally chose to plant GMO seeds for better harvests, improved crop quality and the ability to use sustainable farming practices, such as no-till. Farmers choose seeds that are best for their farms and businesses. Both GM and non-GM seeds are available options for farmers. Source: ISAAA.org https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp  How a GM Seed Gets to Market GMOs are required to be reviewed. Even before the new AG INNOVATION seed goes through the review process, years of testing and research take place. Source: Phillips McDougall 2011  Genetically Modified Crops (GMOs) UNDERSTANDING GMOs 6.7 billion acres of farmland used for GMO crops since 1996 35+ years that GMO crops have been researched and developed 71 countries where GM crops have been found safe for growing or import Sources: ISAAA.org https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp; Phillips McDougall 2011; biofortified.org  Better Harvests Between 1996 and 2018, Crop Biotechnology was Responsible for an Additional: UNDERSTANDING GMOs 32.6 278 498 Million Tonnes Million Tonnes Million Tonnes of Cotton Lint of Soybeans of Corn Source: pgeconomics.co.uk https://www.tandfonline.com/doi/full/10.1080/21645698.2020.1779574  Economic Benefits Economic gains of ~U.S. $225B were generated UNDERSTANDING GMOs globally by biotech crops between 1996 to 2018. 30% Due to reduced production costs 70% Due to substantial yield gains In 2018, farmers in developing countries received $4.41 as extra income for each extra dollar invested in GM crop seeds Sources: pgeconomics.co.uk https://www.tandfonline.com/doi/full/10.1080/21645698.2020.1779574; ISAAA.org https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp  Environmental Benefits The reduction in pesticides from 1996 to UNDERSTANDING GMOs 2018 was estimated at 776 million kilograms or 8.6% reduction In 2018 alone, biotech helped prevent an estimated 23 billion kg of CO2 emissions, equivalent to removing 15.3 million cars from the road for a year. Without biotech, it would take an additional 60 million acres to produce the same amount of food produced in 2018. Sources: pgeconomics.co.uk https://www.tandfonline.com/doi/full/10.1080/21645698.2020.1779574; ISAAA.org https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp  Is it safe? UNDERSTANDING GMOs GM crops are reviewed by hundreds of independent risk assessors and scientists. Every credible U.S. and international food safety authority that has studied GM crops has found that they are safe & no health effects attributable to their use. Since 1992, 71 countries have granted more than 4,485 commercial use approvals for 403 different biotech events in 29 biotech crops. In many countries, multiple regulatory authorities (up to seven in one country) are responsible for assessing a particular aspect of safety. Source: ISAAA.org https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp In the U.S., Three Regulatory Agencies have Oversight for GM Crops UNDERSTANDING GMOs Is it safe for the Is it safe for humans Is it safe for environment? and animals to eat? humans and the Nutritional changes environment? Compositional changes Globally, >90 additional government bodies also review each product before it can be commercialized.  GMO safety in KSA UNDERSTANDING GMOs The Kingdom of Saudi Arabia’s regulations allow the importation of biotech plant products, but they are required to be labeled if they contain more than one percent genetically engineered (GE) plant ingredients. This process is mostly covered by the Saudi Food and Drugs Association (SFDA) However, Saudi Arabia imports large quantities of biotech U.S. corn, soybeans, and their products. KSA as well as Gulf Standardization Organization (GSO) regulations prohibit the import of genetically modified animals, birds, fish, and their products. Meanwhile, biotech planting seeds are permitted for import. Currently, there are no ongoing commercial development activities for GE plants in Saudi Arabia.  GMO Research, Review and Regulation UNDERSTANDING GMOs Source: GMOAnswers.com https://gmoanswers.com/sites/default/files/2018-11/How%20Does%20a%20GMo%20Get%20to%20Market_0.JPG  It is not easy! The goal of predicting how genetic engineering will affect UNDERSTANDING GMOs organisms that live and disperse outdoors under variable biotic and abiotic conditions is a major challenge. Phenotypic characteristics, such as an organism's size, health, and reproductive capacity, are determined by complex interactions among its genes and its surroundings. It is important to ask how the phenotypes of transgenic organisms differ from those of their non-transgenic counterparts, and whether these phenotypes can be characterized adequately in small scale experiments. Also, how will receiving populations, ecological communities, and ecological processes be affected when vast numbers of genetically engineered organisms (GEOs) enter managed and unmanaged habitats? This type of ecological knowledge is crucial for reaching defensible decisions about how to regulate and monitor transgenic organisms. ECOLOGICAL EFFECTS OF GMOs? Unintended phenotypes Past incidents! UNDERSTANDING GMOs Off-target Bt-maize on monarch butterflies UNDERSTANDING GMOs N4640-Bt reared larvae eat less, grow slower and have higher mortality and predicted N4640-Bt maize to have significant off target effects and significantly impact Monarch populations raising both the public's and biotech companies' consciousness about possible off-target Bt- maize on monarch butterflies. However further attempts to extrapolate their results to other Bt and GM crops have been with moderate success, with current evidence suggesting effectiveness in insect control without off-target effects.25 The effect of NK-603 Roundup Ready Maize (NK-603 RR Maize) on rats UNDERSTANDING GMOs Significant chronic kidney deficiencies representing 76% of altered parameters. 3–5x higher incidence of necrosis and liver congestions in treated males. 2–3-fold increase in female treatment group mortality. High tumour incidences in both treated sexes, starting 600 days earlier than control (only one tumour noted in control). However, This paper drew heavy criticism for its flawed experimental design, animal type used for study, statistical analysis and data presentation deficiencies and overall misrepresentations of science and was retracted  pest resistance due to gene overexpression leading to pest evolution via natural selection. An analysis of 77 studies depicted reduced Bt- UNDERSTANDING GMOs crop efficacy caused by field evolved pest resistance for 5 out of 13 (38.4%) major pest species examined in 2013, compared to just one in 2005,32. Such resistance can be evolved over several generations in a relatively short time as most insects have shorter life spans. In maize, S.frugiperda and B.fusca resistance was reported after just 3 and 8 years respectively, consistent with the worst case scenarios. In the former, it led to crop withdrawal in Puerto Rico and was reported to still affect maize growers in 2011, 4 years after crop withdrawal. In India, Pink bollworm resistance currently affects ∼90% Bollgard-II Bt-hybrid cotton growers and ∼35% of cultivable cotton area in key regions. Environmental impact assessment UNDERSTANDING GMOs Is the assessment of the environmental consequences of a plan, policy, program, or actual projects prior to the decision to move forward with the proposed action. In this context, the term "environmental impact assessment" is usually used when applied to actual projects by individuals or companies and the term "strategic environmental assessment" (SEA) applies to policies, plans and programmes most often proposed by organs of state. The purpose of the assessment is to ensure that decision-makers consider the environmental impacts when deciding whether or not to proceed with a project. must provide certain information in seven key areas UNDERSTANDING GMOs Description of the project Alternatives that have been made Description of the environment Description of the significant effect on the environment Mitigation Nontechnical summary Lack of know how Environmental threats do not respect national borders Health aspects of the final genetically modified organism UNDERSTANDING GMOs The risk assessment must include the donor and recipient organism, insert and vector, as well as the properties of the final GMO. Significant features of the GMO, including substances that are formed or may be formed as a result of the genetic modification, have already been described. An assessment of the final GMO must include both human health and environmental considerations as well as a description of methods for monitoring. In addition, assessment of health aspects of the final GMO must include the following aspects: 1. Expected toxic or allergenic effects of the GMO and its metabolic products. 2. Product risks, including the formation of toxins or highly biologically active substances. 3. A comparison between the GMO and the recipient organism, or parental organisms, in terms of pathogenicity. 4. Known and expected habitats. More environmental considerations UNDERSTANDING GMOs Ecosystems, into which the organism might escape from the laboratory or facility – the contained use. The GMO's expected survival, reproduction and spreading in the identified ecosystems. Expected outcome of the interaction between the GMO and the organisms that may be exposed in case of unintended release into the environment. Known or anticipated effects on plants and animals, such as pathogenicity, toxicity, allergenicity, healthy pathogen carrier, altered antibiotic resistance patterns, altered tropism or host specificity and colonisation ability. Known or anticipated participation in biogeochemical processes. UNDERSTANDING GMOs Beyond traditional transgenic technology: UNDERSTANDING GMOs Alternative technologies for generating improved crop plants References Chapter 11 https://sitn.hms.harvard.edu/flash/2015/from-corgis-to-corn-a-brief-look-at-the-long- history-of-gmo-technology/ https://www.nature.com/scitable/topicpage/genetically-modified-organisms-gmos- transgenic-crops-and-732/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5790416/ [email protected] https://www.douglascollege.ca/course/biol-2301 Saudi Arabia: Agricultural Biotechnology Annual https://gmoanswers.com/ask/what-countries-have-banned-gmos https://at.dk/en/regulations/guidelines/risk-assessment-genetic-engineering-c-0- 5/#:~:text=The%20risk%20assessment%20must%20include,modification%2C%20have%2 0already%20been%20described. Quiz Which of the following is NOT a common technique used in genetic engineering in agriculture? A Polymerase Chain Reaction (PCR) B Gene editing using CRISPR-Cas9 C Selective breeding D RNA interference (RN Which of the following is an example of a genetically engineered agricultural product? A Organic fruits and vegetables B Herbicide-resistant crops C Conventional livestock breeds D Non-GMO grains  Which of the following techniques is commonly used in genetic engineering in agriculture? A PCR (Polymerase Chain Reaction) B ELISA (Enzyme-Linked Immunosorbent Assay) C Western Blotting D CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated 9)