1. Plant Biotechnology
Chapter 6

2. Motivation for genetically engineered crops
Agriculture is the biggest industrial sector in the world
$1.3 trillion of products/year
Over past 40 years, world population has doubled while agricultural land area has increased by only 10%

3. Plant transgenics
Transfer of genes to plants directly accelerates selective breeding practices used in the past.
Cotton fiber strength
increased 1.5% per year through conventional breeding
Increased 60% by inserting a single gene into the plant
Corn and soybean have been targets of much genetic engineering

4. Genetic engineering techniques applied to plants
Cloning
Many types of plants can regenerate from a single cell, similar to a bacterium.
The resulting plant is a clone or replica of plant from which original cell was taken.
Protoplast fusion
Introducing a gene into a “denuded” plant cell and generating a new plant

5. Protoplast fusion Leaf fragment technique Plant A Plant B Hybrid plant
Callus cells
Protoplast fusion

6. Agrobacterium tumefaciens as a vector for transferring foreign genes into plant chromosome
This bacterium naturally infects plant cells causing cancerous growths - crown gall disease
Infection (vir) genes carried on Ti plasmid
Type of bacteria that infects plant cells, integrating into its DNA to ensure its survival causing crown gall disease in many ornamentals
Two forms of DNA:
1)bacterial chromosome
2)Ti plasmid
--transferred stretch of DNA “T-DNA” (20 kb long)
--vir (virulence) genes that direct infection

7. Infection Process Vir genes copy T-DNA
Open channel in bacterial cell membrane for T-DNA to pass through
T-DNA enters plant through wound, integrates itself into plant chromosome
When infecting, vir genes
Copy T-DNA
Attach product to copied T-DNA
Add proteins along length of T-DNA
Open channel in bacterial cell membrane for T-DNA to pass through
Then enters plant through wound, integrates itself into plant chromosome
**Transformation: inserting foreign DNA into bacteria -- Removed tumor-inducing section of T-DNA, replace with transgene (between T-DNA border regions)

8. Leaf fragment technique used to introduce foreign genes into plant
Small discs are cut from plant leaf
Discs are cultured to start a new plant
Early in the regeneration process, the bacterium Agrobacterium tumefaciens carrying a Ti plasmid is introduced into the culture
The plasmid DNA combines with the plant chromosome
Discs are treated with hormones to encourage shoot and root development and then the new plant is planted in the soil

9. Leaf fragment technique
Make leaf discs
Transfer to shoot stimulating medium
Agrobacterium
with Ti plasmid
with foreign gene
Briefly culture
discs with
genetically
modified
Agrobacterium
Transfer to filter
paper over nurse
cells
Culture 2-3 days

10. stopped
Gene
gun
Gene guns

11. Antisense technology Used to produce the Flavr-Savr tomato in 1994.
Enzyme polygalacturonase breaks down structural polysaccharide pectin in wall of a plant.
This is part of the natural decay process in a plant
Monsanto identified the gene than encodes the enzyme and made another gene that blocked the production of the enzyme.

12. Antisense molecules
-- Enzyme polyglacturonase (PG) digests pectin in the wall of the plant
-- Identified gene that encodes PG, removed gene from plant cells, produced complementary copy
-- Agrobacter as vector, transfer new gene into tomato cells
-- Gene encodes an mRNA molecule (antisense) that unites with/inactivates normal mRNA (sense molecule) for PG production.

13. Natural insecticide produced by soil bacterium
The bacterium Bacillus thuringiensis produces a crystalline protein that if ingested by insects acts as a toxin and kills the insect
Farmers used to spray their fields with spores of the bacteria to inoculate the plant leaf surface before it is attacked by the insect

14. Insecticide biotechnology
The gene coding for the crystalline protein (Cry) was transferred from the bacterium to the plant.
How can they do this?
Now the plant produces the toxin protein, so don’t need to inoculate with bacterium
This strategy kills the pest before it kills the plant

15. Plant vaccines
Plants are susceptible to diseases caused by viruses (tobacco mosaic virus)
Virus surface protein induces an immune response in the plant against the virus protein
Researchers inserted the virus protein into the plant genome using the Ti plasmid/ Agrobacter vector
Now the plant produces a small quantity of the protein which elicits an immune response by the plant the way a vaccine does

16. Concerns about genetically modified foods
Human health
Unsuspected allergens
What other issues are there?
Environment
Messing up the gene pool of non-target species in the environment
Lateral gene transfer in nature
Still poorly understood in nature

17. National biofuels initiative
In 2007, the federal government increased funding for research in biofuels (ethanol and biodiesel)
Idea was to
produce ethanol from corn
convert agricultural waste products (lignocellulose) to ethanol
This requires re-engineering natural biochemical pathways in plants or microbes to produce more fuel as an end-product

18. How is ethanol produced?
Most ethanol is produced using a four-step process:
The ethanol feedstock (crops or plants) are ground up for easier processing
Sugar is dissolved from the ground material, or the starch or cellulose is converted into sugar
Microbes feed on the sugar, producing ethanol and carbon dioxide as byproducts
The ethanol is purified to achieve the correct concentration.

19. Metabolic engineering to improve efficiency of biological production of biofuels
Natural metabolic pathway
1 ton of Feedstock X product A product B product C gal Ethanol
product G gal Butanol
New enzyme
Genetically engineered metabolic pathway
1 ton of Feedstock X product A product D product E gal Ethanol

20. Benefits of ethanol
Overall, ethanol is considered to be better for the environment than gasoline.
Ethanol-fueled vehicles produce lower carbon monoxide and carbon dioxide emissions, and the same or lower levels of hydrocarbon and oxides of nitrogen emissions.
Ethanol is widely available and easy to use
Ethanol is good for the economy

21. Drawbacks of ethanol as a biofuel
Creating plant-based biofuels requires too much farmland to be practical or sustainable—land that would be better used to grow food.
Producing ethanol and other biofuels takes more energy than the fuel can generate.

22. Summary
Variety of techniques are available to introduce genes into plants and have the plants express the gene
Such genetic engineering is used to
Improve disease resistance
Flavor of product
Nutrition of product
Shelf life of product
Any other property of plant that improves its value

23. Advances in biotechnology of biofuel production will be the next greatest application of our knowledge of biology to address challenges to our society and high-maintenance lifestyles.