Introduction to Biotechnology: Plant Biotechnology
Introduction to Biotechnology Fourth Edition Chapter 6 Plant Biotechnology
Chapter Contents
6.1 Uses of Biotechnology to Enhance Selective Breeding
6.2 Genetic Engineering of Plants
6.3 Practical Applications
6.4 Health and Environmental Concerns
6.1 Uses of Biotechnology to Enhance Selective Breeding (1 of 7)
The world population has nearly doubled in the past 40 years, while the available land for agriculture has only increased by 10 percent.
Improved crop breeding through traditional methods has allowed humanity to feed a growing population.
Recently, development of new, more productive crops has been accelerated by direct transfer of genes:
Today’s crop plants are superior to their predecessors—more productive, more resistant to disease, and able to grow under a wider range of conditions.
6.1 Uses of Biotechnology to Enhance Selective Breeding (2 of 7)
Figure 6.1: Global Area of Biotech Crops in Million Hectares (1996-2015)
6.1 Uses of Biotechnology to Enhance Selective Breeding (3 of 7)
New selective breeding techniques have revolutionized the process, making it much faster and more efficient.
Marker-Assisted Selection:
Identify genetic variants associated with desired traits.
Identify genetic markers located near these alleles.
Can also be used to select for traits that are difficult to measure or exhibit low heritability.
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Mutation Breeding:
Definition: Process of exposing seeds to mutation-causing chemicals or radiation.
From 1930 to 2014, more than 3,200 plants were released with mutagenesis in their background.
Crop plants account for 75 percent of mutation-derived species, with the remaining 25 percent being ornamentals or decorative plants.
Conventional Selective Breeding and Hybridization
Cloning Techniques:
Protoplast fusion
Leaf fragment technique
Gene guns
Chloroplast engineering
Antisense technology
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Conventional Selective Breeding and Hybridization:
Definition: Sexual cross between two lines with repeated backcrossing between the hybrid offspring and parent.
This process can take years.
Polyploid Plants: Plants with multiple chromosome sets greater than normal, potentially increasing desirable traits, especially size.
This method allows for whole chromosomes to be transferred rather than single genes.
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Cloning Techniques:
Definition: Growing plants from a single cell.
Protoplast Fusion:
Definition: Fusion of two protoplast cells from different species.
A protoplast is a callus cell whose cell wall has been dissolved by the enzyme cellulase.
The fusion of the two protoplast cells creates a cell that can grow into a hybrid plant.
Example: Broccoflower, a hybrid of broccoli and cauliflower.
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Figure 6.2: Protoplast Fusion and Regeneration of a Hybrid Plant
Steps illustrated in the figure:
Treat callus cells with cellulase.
Culture protoplasts and induce fusion.
Transfer to shoot-stimulating medium.
Transfer to root-stimulating medium.
Grow engineered plant.
6.2 Genetic Engineering of Plants (1 of 14)
Plant Transformation:
Definition: Process of inserting new genes into the target plant, creating a transgenic plant.
Using Agrobacterium to Insert Genes:
Small discs are cut from leaves and cultured in a medium containing genetically modified Agrobacterium (Agrobacterium tumefaciens).
Definition of Agrobacterium: A soil bacterium that infects plants and contains a genetically modifiable plasmid known as the Ti plasmid.
The DNA from the Ti plasmid integrates with the DNA of the host cell.
Leaf discs are treated with plant hormones to stimulate shoot and root development.
6.2 Genetic Engineering of Plants (2 of 14)
Leaf Fragment Technique:
Uses T-DNA transferred into plant cells:
Small discs of a leaf are cultured briefly in a medium containing genetically modified Agrobacterium.
6.2 Genetic Engineering of Plants (3 of 14)
Table 6.1: Current Genetically Modified Crops
Crop: Alfalfa
Traits: Resistance to glyphosate or glufosinate (herbicide resistance)
Genetic Modification: Genes added; planted in the US 2005–2007; deregulated 2011.
Canola/rapeseed:
Traits: Herbicide resistance
Genetic Modification: Genes added; 2005; 21–87%.
Corn:
Traits: Herbicide resistance; Bt (insect resistance); amylase added for ethanol production (biofuel)
Genetic Modification: 2013 herbicides; 2013 stacked genes; 26–85%.
Additional Crops and Traits: Includes cotton, papaya, potatoes (food and starch), soybeans, zucchini, sugar beet, sugarcane, sweet peppers, tomatoes, wheat, Arctic apples, eggplant, and white mushroom, detailing specific traits and percentages for each crop.
6.2 Genetic Engineering of Plants (4 of 14)
Figure 6.3: Process of Crown Gall Formation
Ti plasmid (T-DNA): Integrated into the plant nucleus from the Agrobacterium during infection, forming a crown gall tumor in the plant tissue.
6.2 Genetic Engineering of Plants (5 of 14)
Figure 6.4: Transfer of Genetically Modified Ti Plasmid
Illustration on the transfer of the Ti plasmid to susceptible plants through tissue culture.
6.2 Genetic Engineering of Plants (6 of 14)
Cloning Techniques:
Gene Guns:
Used to blast tiny metal beads coated with DNA into an embryonic plant cell.
Aimed at the nucleus or the chloroplast of the cell.
Utilize marker genes for the identification of transformed cells (e.g., antibiotic resistance).
Effective in plants resistant to Agrobacterium.
6.2 Genetic Engineering of Plants (7 of 14)
Figure 6.5: Gene Guns
Display of the mechanics of a gene gun including various components and how it targets plant cells.
6.2 Genetic Engineering of Plants (8 of 14)
Chloroplast Engineering:
DNA in chloroplasts can accept several new genes simultaneously.
High percentage of genes remain active; separates from pollen-based genes, reducing environmental dispersion risks.
6.2 Genetic Engineering of Plants (9 of 14)
Figure 6.6: Chloroplast Genetic Engineering - Diagram showcasing the integration of a Ti plasmid into chloroplasts for genetic modification.
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Gene Inactivation Using CRISPR-Cas Technology:
This technology can delete a gene from a cell accurately and precisely.
In 2015, the EU determined that plants with deleted genes through genetic engineering do not fall under GMOs (genetically modified organisms).
The U.S. APHIS does not consider CRISPR-Cas9 edited crops like white button mushrooms, waxy corn, and delayed-ripening bananas as deregulated, as transformations are achieved through gene inactivation without introducing foreign DNA sources.
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Antisense Technology:
Process of inserting a complementary copy of a gene into a cell.
Creates mRNA molecules known as antisense molecules which bind to normal mRNA (sense molecule), inactivating it.
Example: Flavr Savr tomato, developed using this technology.
6.2 Genetic Engineering of Plants (12 of 14)
Gene Stacking:
Goal of gene stacking is to introduce multiple desired genes into a plant simultaneously.
Definition of gene stack: Combinations of two or more inserted genes achieved through crossing parental lines that inherit separate genes, then selecting offspring.
Molecular stacking involves moving genes via genetic engineering methods.
6.2 Genetic Engineering of Plants (13 of 14)
Figure 6.8: Creating a Gene Stack
Illustration showing hybridization between an insect-resistant line and a herbicide-tolerant line to create a plant resistant to both.
6.3 Practical Applications (1 of 13)
Protecting Plants from Viruses
Genetic Pesticides
Herbicide Resistance
Enhanced Nutrition
Future Perspectives: Encompassing applications from pharmaceuticals to fuel generation.
6.3 Practical Applications (2 of 13)
Protecting Plants from Viruses:
Crop plants are susceptible to numerous viruses.
Vaccines encoded in the plant’s DNA (e.g., gene from Tobacco Mosaic Virus (TMV) inserted into tobacco plants), stimulate the plant's immune response, rendering the plant invulnerable to the virus.
6.3 Practical Applications (3 of 13)
Figure 6.9: Virus Protection for Plants
Depiction of the process of creating a plant immune to TMV by encoding a viral coat protein into the plant’s DNA.
6.3 Practical Applications (4 of 13)
Genetic Pesticides:
Bacillus thuringiensis (Bt):
A bacterium that produces a protein lethal to specific harmful insects.
Has been utilized as a natural pesticide for over 50 years.
Bt genes can be inserted into plant DNA, granting built-in protection.
Controversy exists regarding impacts on Monarch butterflies, although studies indicate minimal risk from Bt corn exposure.
6.3 Practical Applications (5 of 13)
Figure 6.10: How Bt Toxin Works
Mechanism by which the Cry protein from Bt attaches to the gut wall of insects, ultimately causing their death after consumption of engineered corn.
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Safe Storage:
Transgenic corn expressing avidin shows resistance to insect infestations during storage, preventing major crop losses.
Avidin works by blocking biotin, essential for insect growth.
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Herbicide Resistance:
Traditional herbicides often harm both desirable crops and weeds; however, genetic engineering can produce crops resistant to these herbicides.
Enables more environmentally friendly and milder chemical weed control.
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Glyphosate Resistance:
Glyphosate works by inhibiting the EPSPS enzyme, essential in a key biochemical pathway.
Transgenically modified crops can produce an alternative enzyme not affected by glyphosate.
Most soybeans currently grown contain these herbicide resistance genes.
The emergence of glyphosate-resistant weeds has become a challenging issue.
6.3 Practical Applications (9 of 13)
Figure 6.11: Engineering Herbicide-Resistant Plants
Illustrates the process of engineering plants that remain unaffected by glyphosate while still expressing essential functions.
6.3 Practical Applications (10 of 13)
Enhanced Nutrition:
Genetic engineering has created Golden Rice, rich in beta carotene, which the body converts to vitamin A.
As of 2011, Golden Rice has yet to be planted by farmers due to environmental concerns.
6.3 Practical Applications (11 of 13)
Biopharming:
Definition: The use of plants as efficient protein factories.
Transgenic crops can yield large amounts of valuable proteins, including:
Protein drugs and vaccines
Economical edible vaccines
“Molecular farming” of phytochemicals beneficial to human health (e.g., antibodies, cytokines, growth factors).
6.3 Practical Applications (12 of 13)
Engineered Deletion of Gene Promoters:
CRISPR gene editing can modify promoter sequences, influencing the expression of existing genes.
Researchers at Cold Spring Harbor Laboratory utilized these techniques to enhance tomato yield via gene regulation.
6.3 Practical Applications (13 of 13)
Figure 6.12: CRISPR/Cas9-induced Targeted Mutagenesis
Diagram demonstrating methods for generating long-shelf-life tomato varieties through gene editing, using phenotypic comparisons of mutant versus wild-type fruits.
6.4 Health and Environmental Concerns (1 of 3)
Transgenic Plant Concerns:
Questions surrounding their potential harm to humans and safety for consumption.
Potential environmental hazards:
Allergic reactions due to foreign genes.
Risk of antibiotic-resistance marker genes spreading to pathogenic bacteria.
Unsubstantiated fears of cancer from transgenic plants.
6.4 Health and Environmental Concerns (2 of 3)
Antibiotic Resistance Concerns:
A report in the journal Science indicates a very low probability of antibiotic-resistance genes transferring from plants to bacteria.
While antibiotic resistance is a significant issue, transgenic plants are unlikely contributors to this problem.
6.4 Health and Environmental Concerns (3 of 3)
Environmental Concerns:
Potential for transgene spread to weeds due to pest or herbicide resistance; few experts anticipate this scenario, advocating further research.
Regulatory Entities:
FDA: Oversees food safety.
USDA: Manages agricultural practices.
EPA: Controls the use of Bt proteins and related pesticides.