3.3 Plant Biotechnology
3.3 Plant Germplasm
3_Influence-of-Genetic-Engineering-on-Agriculture-and-Germplasm-Conservation
Exercise 3a Herbaceous Cuttings
Exercise 3b Flower Reproductive Parts Dissection
Influence of Genetic Engineering on Agriculture and Germplasm Conservation
Overview
Plant tissue cultures being grown at a USDA facility. USDA, Lance Cheung, Public domain, via Wikimedia Commons
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Introduction
Learning Objectives
- Compare conventional breeding and genetic engineering.
- List the advantages and disadvantages of plant breeding.
- Explain the steps in molecular cloning.
- List examples of genetically engireered, transgenic crops.
- Define germplasm.
- Explain the significance of germplasm conservation.
- Describe USDA-ARS National Plant Germplasm System.
Key Terms
biotechnology - use of biological agents for technological advancement
clone - exact replica of an organism, a cell, DNA molecule
contig - larger sequence of DNA assembled from overlapping shorter sequences
conventional breeding - crossing or mating the organisms with preferred traits and selecting the progeny that produces those traits or a combination of traits.
cytogenetic mapping - a technique that uses a microscope to create a map from stained chromosomes
ex-situ conservation - conserving an organism outside of its natural habitat, such as a zoo
foreign DNA - DNA that belongs to a different species or DNA that is artificially synthesized
gene targeting - method for altering the sequence of a specific gene by introducing the modified version on a vector
genetic engineering - alteration of the genetic makeup of an organism
genetic recombination - DNA exchange between homologous chromosome pairs
genetically modified organism (GMO) - an organism whose genome has been artificially changed
germplasm - a collection of all genetic material stored as seeds, tissues, and live samples.
in-situ conservation - conserving an organism in its natural habitat
recombinant DNA - combining DNA fragments from two different sources or organisms
recombinant protein - a gene's protein product derived by molecular cloning
transgenic - organism that receives DNA from a different species
Introduction
Plants are the source of food for humans as well as livestock. Farmers have historically developed ways to select plant varieties with desirable traits, long before modern-day biotechnology practices were established. conventional breeding relies on crossing or mating the organisms with preferred traits and selecting the progeny that produces those traits or a combination of traits. conventional breeding has generated many present-day crops from wild relatives over thousands of years. However, modern scientific techniques have led to faster and more efficient practices. Staples like corn, potatoes, and tomatoes were the first crop plants that scientists genetically engineered. Biotechnology creates organisms by using a targeted approach to modify specific traits, changing an organism's genomic composition or DNA. Since the discovery of the structure of DNA in 1953, the biotechnology field has proliferated through both academic research and private companies. The primary applications of this technology are in medicine (vaccine and antibiotic production) and agriculture (crop genetic modification to increase yields). Biotechnology has many industrial applications, such as increasing fermentation, treating oil spills, and producing biofuels. Similarly, the collection and maintenance of germplasm, is critical for advancements in technology. Germplasm is a collection of all genetic material stored as seeds, tissues, and live samples. The conservation and documentation of all the samples and related documentation provide vital information useful in biotechnology.
DNA and Recombinant DNA
To understand the basic techniques used to work with nucleic acids, it is important to remember a few basic facts:
- Nucleic acids are macromolecules made of nucleotides—a sugar, a phosphate, and a nitrogenous base—linked by phosphodiester bonds. The phosphate groups on these molecules each have a net negative charge.
- An entire set of DNA molecules in the nucleus is called the genome. DNA has two complementary strands linked by hydrogen bonds between the paired bases. Exposure to high temperatures (DNA denaturation) can separate the two strands and cooling can reanneal them.
- The DNA polymerase enzyme can replicate the DNA.
- Unlike DNA, located in the eukaryotic cells' nucleus, RNA molecules leave the nucleus.
- The most common type of RNA that researchers analyze is messenger RNA (mRNA) because it represents the protein-coding genes that are actively expressed. However, RNA molecules present some other challenges to analysis, as they are often less stable than DNA.
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Molecular Cloning
In general, the word “cloning” means the creation of a perfect replica; however, in biology, the re-creation of a whole organism is referred to as “reproductive cloning.” Long before attempts were made to clone an entire organism, researchers learned how to reproduce desired regions or fragments of the genome, a process that is referred to as molecular cloning. The technique offered methods to create new medicines and overcome difficulties with existing ones. Scientists have repurposed and engineered plasmids as vectors for molecular cloning and the large-scale production of important reagents, such as insulin and human growth hormone. Cloning small genome fragments allows researchers to manipulate and study-specific genes (and their protein products) or noncoding regions in isolation. A plasmid, or vector, is a small circular DNA molecule that replicates independently of the chromosomal DNA.
In cloning, scientists can use the plasmid molecules to provide a "folder" in which to insert the desired DNA fragment. Plasmids are usually introduced into a bacterial host for proliferation. In the bacterial context, scientists call the DNA fragment from the genome of the studied organism, foreign DNA —or a transgene; to differentiate it from the bacterium's DNA—or the host DNA.
Plasmids occur naturally in bacterial populations (such as Escherichia coli) and have genes that can contribute favorable traits to the organism, such as antibiotic resistance (the ability to be unaffected by antibiotics). An important feature of plasmid vectors is the ease with which scientists can introduce a foreign DNA fragment via the multiple cloning site (MCS). The MCS is a short DNA sequence containing multiple sites that different commonly available restriction endonucleases can cut. Restriction endonucleases recognize specific DNA sequences and cut them in a predictable manner. They are naturally produced by bacteria as a defense mechanism against foreign DNA. Many restriction endonucleases make staggered cuts in the two DNA strands, such that the cut ends have a 2- or 4-base single-stranded overhang. Because these overhangs are capable of annealing with complementary overhangs, we call them “sticky ends.” Adding the enzyme DNA ligase permanently joins the DNA fragments via phosphodiester bonds. In this way, scientists can splice any DNA fragment generated by restriction endonuclease cleavage between the plasmid DNA's two ends that have been cut with the same restriction endonuclease (Figure 3.3.1).
Plasmids with foreign DNA inserted into them are called recombinant DNA molecules (Figure 3.3.1) because they are created artificially and do not occur in nature. They are also called chimeric molecules because the origin of different molecule parts of molecules can be traced back to different species of biological organisms or even to chemical synthesis. We call proteins that are expressed from recombinant DNA molecules recombinant protein.
Not all recombinant plasmids can express genes. The recombinant DNA may need to move into a different vector (or host) that is better designed for gene expression. Scientists may also engineer plasmids to express proteins only when certain environmental factors stimulate them, so they can control the recombinant proteins' expression.
Genetic Engineering
Scientists have genetically modified bacteria, plants, and animals since the early 1970s for academic, medical, agricultural, and industrial purposes. Genetic engineering is the alteration of an organism’s genotype using recombinant DNA technology to modify an organism’s DNA for the purpose of achieving desirable traits. The addition of foreign DNA in the form of recombinant DNA vectors generated by molecular cloning is the most common method of genetic engineering. The organism that receives the recombinant DNA is a genetically modified organism (GMO). In the US, GMOs such as Roundup-ready soybeans and borer-resistant corn are part of many common processed foods. If the foreign DNA comes from a different species, the host organism is transgenic, Bt corn and Bt cotton are two such examples of transgenic plants.
Gene Targeting
Although classical methods of studying gene function began with a given phenotype and determined the genetic basis of that phenotype, modern techniques allow researchers to start at the DNA sequence level and ask: "What does this gene or DNA element do?" This technique is called reverse genetics, and it has resulted in reversing the classic genetic methodology. This method would be like damaging a body part to determine its function. For instance, an insect that loses a wing cannot fly. The classical genetic method would compare insects that cannot fly with insects that can fly and observe that the non-flying insects have lost wings; this would result in understanding that the function of the wing is flight. Similarly, mutating or deleting genes provides researchers with clues about gene function. We collectively call these methods they use to disable gene function – gene targeting. Gene targeting is the use of recombinant DNA vectors to alter a particular gene's expression, either by introducing mutations in a gene or by eliminating a certain gene's expression by deleting a part or all the gene sequences from the organism's genome.
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Plant Biotechnology
Plant biotechnology includes techniques used to adapt plants for specific needs or a possibility. Situations that combine multiple needs and opportunities are common. For example, a single crop may be required to provide sustainable food and healthful nutrition, protection of the environment, and opportunities for jobs and income. Finding or developing suitable plants is typically a highly complex challenge. Plant biotechnologies utilize tools and resources from genetics, genomics, marker-assisted selection (MAS), and transgenic (genetically engineered) crops to assist in developing new varieties and/or new traits in plants. This allows researchers to detect and map genes, discover their functions, select specific genes in genetic resources and breeding, and transfer genes for specific traits into plants where they are needed, for example, research and development of disease-resistant crops.
Most public research on transgenic crops focuses on one or two general objectives:
- a better understanding of all aspects of the transgenic/genetic engineering process, for enhancing efficiency, precision, and proper expression of the added genes or nucleic acid molecules
- and a wider range of useful and valuable traits, including complex traits.
National Institute of Food and Agriculture (NIFA) a U.S federal government body, funds research, training, and extension for developing and using biotechnologies for food and agriculture. Areas of work include, but are not limited to:
- genetic structures and mechanisms,
- methods for transgenic biotechnology (also known as genetic engineering),
- identification of traits and genes that can contribute to national and global goals for agriculture,
- plant genome sequences—molecular markers and bioinformatics,
- gene editing/genome editing,
- and synthetic biology.
Transgenic and genetically modified Plants
Manipulating the DNA of plants—creating GMOs—has helped to create desirable traits, such as disease resistance, herbicide and pesticide resistance, better nutritional value, and better shelf-life (Figure 3.3.2). As mentioned in the previous section, GMOs are plants that receive recombinant DNA) and transgenic plants receive DNA from other species. Because they are not natural, government agencies closely monitor transgenic plants and other GMOs to ensure that they are fit for human consumption and do not endanger other plant and animal life. To prevent foreign genes from spreading to other species in the environment, extensive testing is required to ensure ecological stability. Let us discuss some common methods used in developing tansgenic and genetically modified plants.
Explore the Nature Education article on GMOs by using this link.
Explore US Food & Drug Administration page on GMOs
Transformation of Plants Using Agrobacterium tumefaciens
Gene transfer occurs naturally between species in microbial populations. Many viruses that cause human diseases, such as cancer, act by incorporating their DNA into the human genome. In plants, tumors caused by the bacterium Agrobacterium tumefaciens occur by DNA transfer from the bacterium to the plant. Although the tumors do not kill the plants, they stunt the plants and they become more susceptible to harsh environmental conditions. A. tumefaciens affects many plants, such as walnuts, grapes, nut trees, and beets.
The artificial introduction of DNA into plant cells is more challenging because of the thick cell wall compared to animal cells. Researchers use the natural transfer of DNA from Agrobacterium to introduce DNA fragments of their choice into plant hosts. In nature, the disease-causing A. tumefaciens have a set of plasmids—Ti plasmids (tumor-inducing plasmids) —that contain genes to produce tumors in plants. DNA from the Ti plasmid integrates into the infected plant cell’s genome. Researchers manipulate the Ti plasmids to remove the tumor-causing genes and insert the desired DNA fragment for transfer into the plant genome. This newly engineered plasmid also carries antibiotic resistance genes to aid selection and researchers can propagate them in E. coli cells as well. Agrobacterium has been used as a vector to transform many GMOs such as canola, sugar beet, cotton, and soybean.
The Organic Insecticide Bacillus thuringiensis
Bt maize and Bt cotton are two examples of genetically modified crops with B. thuringiensis toxin. Bacillus thuringiensis (Bt) is a bacterium (Figure 3.3.3) that produces protein crystals (figure 3.3.4) during sporulation that is toxic to many insect species that affect plants. Insects need to ingest Bt toxin to activate the toxin. Insects that have eaten Bt toxin stop feeding on the plants within a few hours. After the toxin activates in the insects' intestines, they die within a couple of days (Figure 3.3.6). Modern biotechnology has allowed plants to encode their own crystal Bt toxin that acts against insects. Scientists have cloned the crystal toxin genes from Bt and introduced them into plants. Bt toxin is safe for the environment and non-toxic to humans and other mammals, and organic farmers have approved it as a natural insecticide. This reduces the use of synthetic spray pesticides.
Let us look at the basics of one of the techniques used in creating genetically modified plants with Bt toxin.
Step 1. Scientists identify the trait that is desired (for example, insect resistance).
Step 2. Find an organism that already has that trait - Bacillus thuringiensis (Bt) produces toxins against insects.
Step 3. Gene governing the production of toxins is excised using enzymes called restriction enzymes.
Step 4. Excised gene is utilized to create a DNA construct that includes the gene of interest or reporter gene as well as promoter and terminator sequences for proper transformation.
Step 5. DNA constructs are coated on gold particles and delivered to the undifferentiated plant cells or directly into a plant using a gene gun (Figure 3.3.5).
Step 6. The cells that absorb the DNA construct are stable and are selected and grown under with nutritive medium and treated with plant hormones to cause differentiation to form new plants.
Step 7. Newly formed young plants are grown and monitored in greenhouses and tested in fields. After a comprehensive evaluation, introduced for commercial purposes.
Study the use and impact of Bt Corn in this Nature article.
Here are some examples of successfully developed transgenic or genetically modified plants.
Flavr Savr Tomato
The first genetically modified crop on the market was the Flavr Savr Tomato, created in 1994. Scientists used antisense RNA technology to slow the softening and rotting process caused by fungal infections, which led to the increased shelf life of this tomato. Additional genetic modification improved the tomato's flavor. However, the Flavr Savr tomato did not successfully stay in the market because of problems maintaining and shipping the crop.
Golden Rice
Golden rice (Figure 3.3.8) was created to combat widespread vitamin A deficiency in children that live in developing nations, especially Africa, South Asia, and Southeast Asia (Figure 3.3.7). Golden rice is genetically modified to produce beta-carotene in the endosperm. Beta carotene is converted to vitamin A by the human body. Vitamin A is critical for normal vision, growth, and immune reaction. Night blindness is an early sign of vitamin A deficiency. Prolonged deficiency can cause complete blindness, as well as premature death. According to WHO, as many as 250,000 to 500,000 children are affected by this deficiency and about half of these children die within 12 months of losing their sight. The first country to adopt Golden Rice for production and consumption was the Philippines. However, due to misinformation and misunderstandings about genetically modified organisms, fewer countries have adopted the commercial use of golden rice.
Visit the USDA National Institute of Food & Agriculture to know more about plant biotechnology.
Kew's Millenium seed bank.
Access for free at https://openstax.org/books/biology-2e/pages/17-1-biotechnology
Plant Germplasm
Since the domestication of plants—over many thousands of years, humans have collected the seeds and other plant material for the purpose of propagation over growing seasons or years. germplasm is a collection of any plant material or data that can be utilized to conserve and investigate the genetic composition of a species. Germplasm includes seeds, vegetative parts of a plant, plant tissue culture collection samples, DNA samples, cultivars, landraces, crop wild relatives (CWR), and accessions with the relevant documentation and data on these collections (Veerala et al, 2021) (Figure 3.3.9). Genetic diversity of plants is critical, and acquisition, maintenance, research & analysis, documentation, conservation, and distribution are vital to the conservation of plant diversity.
Food security, dietary expectations, availability of feed for animals, medicine, fibers, and oils, as well as demands for fuel, continue to grow alongside the expanding human population. According to Byrne et al., 2018, a 25 to 70% increase in global agricultural production is required to meet food demand by 2050. With increased agricultural demands comes the increased risk of environmental deterioration due to soil erosion, greenhouse gas emissions, and nutrient runoff to waterways; additionally, global climate changes are presenting new challenges, such as increasing temperatures, water scarcity, and new emerging pests. Genetic engineering can aid in the needed response to these growing concerns, along with plant breeding, improved horticulture practices, integrated pest management, sustainable farming practices, and research in the various fields that inform better plant science.
Effective conservation and efficient use and access to the diversity of germplasm dictate the production of cultivars/accessions that are more suited to the various environmental conditions such as drought, flooding, soil salinity, nutrient-deficient soils as well as pathogen/pest infestation, and increased nutritional quality, and increased crop yield.
National Plant Germplasm System (NPGS)
USDA-ARS National Plant Germplasm System (NPGS) is the primary body involved in the preservation of germplasm resources in the United States. NPGS is made up of many laboratories and research stations (table 1). Multiple USDA offices and USDA Animal and Plant Health Inspection Service participate in acquiring, quarantining, and distributing of NPGS collections with collaboration. The complete and comprehensive database of NPGS collections is administered via the National Germplasm resource Laboratory, Beltsville, Maryland. NPGS is part of an international collaboration called the GRIN-Global project. (National Research Council 1991. The U.S. National Plant Germplasm System).
Visit the website of the USDA plant germplasm collection.
Collection/Facility | Location | Number of collections |
National seed storage laboratory | Fort Collins, Colorado | 230,000 accessions |
4 Regional stations | Pullman, Washington. Ames, Iowa Geneva, New York Griffin, GA
| 135,000 accessions of 4000 species |
10 National clonal germplasm repositories |
| 27,000 accessions of 3000 species |
National small grain collection | Aberdeen, Idaho | 110,000 accessions |
Interregional Research Project-1 | Sturgeon Bay, Wisconsin | 3500 potato accessions |
Multiple collections in universities/USDA laboratories |
|
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Unit 3 Lab Exercises
Exercise 3a: Herbaceous Cuttings
Students learn the techniques and procedures for propagating plants through herbaceous cuttings, including steps for selecting, preparing, and planting cuttings to ensure successful growth and development.
Exercise 3b: Flower Reproductive Parts Dissection
Students dissect a flower to identify and study its reproductive parts, including the stamen, pistil, and ovary. This exercise aims to help students understand the structure and function of these components in plant reproduction.
Attributions
Biology 2e By Mary Ann Clark, Matthew Douglas, Jung Choi. OpenStax is licensed under Creative Commons Attribution License v4.0
Priyanka, V.; Kumar, R.; Dhaliwal, I.; Kaushik, P. Germplasm Conservation: Instrumental in Agricultural Biodiversity—A Review. Sustainability 2021, 13, 6743. https://doi.org/10.3390/su13126743
Sustaining the Future of plant breeding: The critical role of the USDA-ARS National Plant Germplasm System by Patrick F Byrne, Gayle M Volk, Candice Gardner, Michael A Gore, Philipp W. Simon and Stephen Smith. Crop Science, 58:451-468 (2018). doi: 10.2135/crp[sco2017.05.0303
Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA. This is an open-access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).Published January 12, 2018
https://acsess.onlinelibrary.wiley.com/doi/10.2135/cropsci2017.05.0303
National Research Council 1991. The U.S. National Plant Germplasm System. Washington, DC: The National Academies Press. Https://doi.org/10.17226/1583