1. Lecture 13
Plant Biotechnology
Dr. Aparna Islam

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

3. Improvements needed in the field of agriculture
Improving crop yield including
Reduce dependence on pesticide
Reduce vulnerability
Improving nutritional quality
Improve texture, test, appearance

4. PLANT GENETIC ENGINEERING
Product Concepts and Technical Feasibility
Building the Transgenes
Plant Transformation
Event Selection
Plant Breeding
Seed Production and Marketing
Detection of GMO Crops in the Commodity Chain

5. Building the Transgenes
ON/OFF Switch
Makes Protein
stop sign
CODING SEQUENCE
INTRON
poly A signal
PROMOTER
Plant Selectable
Marker Gene
Plasmid DNA
Construct
Plant Transgene
bacterial genes
antibiotic marker
replication origin

6. DNA Delivery to Target Cells
Plant Transformation
The introduction and expression of
genes into plants is a three step process:
DNA Delivery to Target Cells
Selection and Regeneration
Event Selection

7. Plant Transformation – DNA Delivery
microprojectile bombardment
“biolistics” or “gene gun”
Agrobacterium tumefaciens
natural property of Agrobacterium to transfer DNA to host plant cells is exploited to introduce genes of interest
difficult with cereal crops
simple DNA integration patterns
tiny DNA-coated particles are shot into plant cells
versatile method
complex DNA integration patterns: tandem arrays of fragmented molecules

8. Plant Transformation – Target Cells
All Crop Transformation Protocols Deliver
DNA to Plant Cells in Tissue Culture
Tissue cultures allow regeneration of fertile plants from single cells
Large number of target cells available for DNA delivery
Establishment, maintenance and plant regeneration

9. Plant Transformation – Selection
At best only 1 in 1000 cells integrate delivered DNA
Transformed cells (events) are marked by co-introducing gene that provides resistance to selective agents
Transformed cells are selected by killing non-transformed cells with selective agent.
Two main types of selective agents:
antibiotics
herbicides
Selectable markers assist in following inheritance of transgenes.
tissue culture cells
under selection
Herbicide Leaf Paint Assay
transgenic non-transgenic
resistant susceptible

10. Goal: Identify transgenic lines that stably exhibit desired phenotype
Event Selection
Goal: Identify transgenic lines that stably exhibit
desired phenotype
Typically only 1 in 100 events are commercialized
Transgene expression varies
transgene insertions are generally unstable
Transgenic line must satisfy regulatory requirements:
USDA, EPA, and FDA each review product
no novel toxic or allergenic proteins or metabolites
genetic stability
documented expression profile

11. 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

12. Pest resistant crop (Bt Cotton)
Bt cotton was created through addition of Cry groups of endotoxin into plant cells
When insect attack and eat cotton plants the cry toxins are dissolved due to the pH level of insect stomach
Within minutes the toxin binds to the receptors in the gut wall and the insect stop feeding
Within hours the gut wall breaks down
In 1-2 days the insect dies of septicemias as spores and gut bacteria proliferate in the blood
Bt Cotton
Recently India has become the number one global exporter of cotton and the second largest cotton producer in the world

13. Bt brinjal Controversies Developed by using the same technology
Bangladesh is the first country in the world which released Bt brinjal to the farmers
On October 30, 2013 Bangladesh Agricultural Research Institute (BARI) received permission to release four varieties such as; Bt Uttara, Bt Kajla, Bt Nayantara, and Bt ISD006
Controversies
Criticized by anti-GMO activists as not safe for human consumption
India cleared Bt brinjal for commercialization on 14 October 2009. 
Stopped the release following concerns raised by some scientists, farmers and anti-GMO activists
The government of India officially announced that it needed more time before releasing Bt brinjal

14. Transgenics: Pest resistant crop (Bt Cotton)
Insertion of Cry gene from Bacillus thuringiensis into corn genome-root expression leads to root protection
Benefits Limitations
• Simplicity • Resistance development
• Consistency/efficacy • Refuge requirements
• Reduced insecticide use • GMO marketing concerns
and chemical exposure

15. Based on
cost to the producer,
yield benefits,
efficacy/consistency,
simplicity, and
environmental implications,
transgenics potentially hold the most economic value for producers

16. Herbicide resistant crop
Excessive weed growth forces crop to compete with sunlight, water and nutrients, often leading to significant loss
Occupy space and harbor insect and disease pest
Clog irrigation and drainage system
Deposit weed seeds into crop harvests
If left uncontrolled, weeds can reduce crop yields significantly.
Several crops have been genetically modified to be resistant to non-selective herbicide  
These herbicides target key enzymes in the plant metabolic pathway, which disrupt plant food production and eventually kill it

17. Glyphosate-tolerant crops
Glyphosate herbicide kills plants by blocking the EPSPS (5- enolpyruvylshikimate-3-phosphate synthase) enzyme
EPSPS is an enzyme involved in the biosynthesis of aromatic amino acids such as, phenylalanine, tyrosine and tryptophan via the shikimate pathway in bacteria, fungi and plants. 
Not found in the mammalian genome
It is the biological target for the herbicide glyphosate
Glyphosate competitively inhibit EPSPS and plants die due to a lack of aromatic amino acids required for their survival
A version of the enzyme that both was resistant to glyphosate and that was still efficient enough to drive adequate plant growth was identified in an Agrobacterium strain called CP4
This version of enzyme, CP4 EPSPS, is the one that has been engineered into several genetically modified crops.

18. Glufosinate-tolerant crops
Glufosinate herbicides contain the active ingredient phosphinothricin
phosphinothricin kills plants by blocking the enzyme responsible for nitrogen metabolism and for detoxifying ammonia, a by-product of plant metabolism.
Crops modified to tolerate glufosinate contain a bacterial gene that produces an enzyme that detoxifies phosphonothricin and prevents it from doing damage.

19. Herbicide Tolerant Crops – Benefits
Easy to use
Full spectrum weed control
Environmentally safe
Excellent crop safety
Wide application window (crop stages & weed stages)
Control of weeds

20. Herbicide Tolerant Crops - Concerns
Spray drift –non-target crops
Multiple applications may be required
Premium price for seed
Public non-acceptance of genetically altered crops

21. Salt tolerant rice varieties
Approximately 576 million tons of rice are produced globally each year, with about 90% produced and consumed in Asia
Salinity is a major constraint limiting agricultural productivity on nearly 20% of the cultivated and irrigated area worldwide
In Bangladesh, one million hectares out of a total nine million hectares of cultivable land are salinity affected
The vulnerability is more profound during the dry seasons.
High salt concentrations affect seed germination, water deficit and ion imbalance resulting in ion toxicity and osmotic stress
Salt stress has been reported to cause an inhibition of growth and development, reduction in photosynthesis, respiration and protein synthesis in sensitive species

22. Salt tolerant rice varieties
Salt tolerant rice varieties have been developed by introducing pea helicase gene in four high yielding rice varieties.
DNA helicases are motor proteins that catalyze the unwinding of duplex DNA in an ATP-dependent manner and thereby play an important role in most of the basic genetic processes including replication, repair, recombination, transcription and translation
A pea DNA helicase 47 gene called (PDH47) which is induced in cold and salinity stress has recently been introduced rice varieties to create transgenic salt tolerance rice
Developed by Prof. Zeba Islam Seraj and her team

23. Golden rice A variety of rice produced through genetic engineering
It was designed to produce beta-carotine a precursor of vitamin A
The rice plant usually produce beta-carotene in leaves but not in the endosperm (the part we eat)
Golden rice was produced by inserting two genes in rice plant:
psy (phytoene synthase) gene from daffodil
Crt1 (phytoene desaturase) gene from a soil bacterium Erwinia uredovora
These two genes were introduced to put back the beta-carotene biosynthetic pathway in endosperm
Golden rice is at the moment in open field trial in Bangladesh.

26. GMOs: Why the Controversy?
Genetic engineering is a powerful new technology that is in general poorly understood and whose long term effects are unknown.
GMOs are an innovation that have and will continue to impact all facets of the global agricultural economy.
Production
Processing
Commodity Handling
Consumer Products

27. GMO Crops: Three Major Issues
1. Food safety and environmental impacts
2. Global trade
3. Increased corporate control of agriculture

28. GMOs and Food Safety
Genetic engineering creates novel genetic combinations
Potential exists for undesirable effects of allergenicity or toxicity
All GMOs are tested extensively for food safety prior to sale
foods for human consumption and animal feed
agricultural products (meat, dairy, fresh produce)
To Label or Not to Label?
Labels must give accurate information on product composition
Identity preservation – methods, tolerances, costs

29. GMOs and Environmental Impacts
Genetic engineering creates novel genetic combinations
All GMOs are tested for potential environmental impacts prior to sale
influence on soil and water composition
insect resistance management
gene/trait transfer to weedy relatives
interactions with agricultural environment
GMO Crops Have Many Significant Environmental Benefits
Reduced chemical pesticide and herbicide use
More sustainable pest management
Better erosion control through no-till practices

30. GMOs and Global Trade
GM Commodity Crops Highlight Differences in Culture and Economic Systems
Education level and awareness of agriculture and biotechnology
Feelings toward food and agriculture as a way of life
Governmental policies on the regulation of GM crops – imports, sales
Agricultural economies

31. Increased Corporate Control of Agriculture
The Development of GM Crops is Expensive
Intellectual property and patent protection
Consolidation/vertical integration increases ability to capture profits
Ag-biotech is a recent example of a century-old trend