2. Introduction
• 25,000 field safety and production trials
• 60 genetically engineered plants and animals
• 45 countries
• 6500 in the USA alone (18,000 sites)
• 74% of the soybeans
• 71% of the cotton
• 32% of the corn
3. Introduction
• Has been practiced for hundreds of years
• Slow and uncertain
• Sexual cross between two lines, back-cross
with offspring and one of the parents until a
plant with desired characteristics is obtained.
• Limited to only plants that can sexually
hybridized
4. Introduction
• Recombinant DNA technology
• Offers unlimited possibility
• Greater specificity
• Genes from incompatible plants, animals, bacteria or
insects
• Sexual compatibility becomes irrelevant
• Process is faster since the gene can be directly selected
5. Introduction
• We will together discuss how foreign genes can be
introduced into plant
• Using Ti-plasmid system (Agrobacterium tumefaciens)
• "Gene gun"
• Also some major products of genetic engineering in
plants
• Production of insect-tolerant and herbicide-tolerant
crops
6. Ti-plasmid System
• Ti-plasmid as vector
• Agrobacterium tumefaciens is a plant pathogenic
soil bacterium
• Causes crown gall disease
• The key is 200kb circular DNA plasmid - Ti plasmid
• During infection, T-DNA is transferred and inserted
randomly into the genome of the host plant
7. Ti-plasmid System
• The T-DNA contains genes for the synthesis of:
1. Food for the bacterium (amino acids - opines)
2. Plant hormones (auxin and cytokinin)
3. Vir genes
• Both are expressed at high level
• Overexpression of hormones leads to?
8. Ti-plasmid System
• Virulence genes:
Located at 35kb region on the Ti-plasmid
Needed for the production of trans-acting proteins
that essential for plant cell transformation
25 genes in 7 operons
Switched on by chemicals (acetosyringone)
produced by wounded plant cells
9. Ti-plasmid System
• The signals perceived by virA protein,
transduced to virG protein
• Activating upstream region of other vir genes
and activates the expression of the other vir
genes
• The activation of vir genes will excised T-DNA
from the plasmid
11. Ti-plasmid System
• Flanked by two borders
• Sequence called RB and LB
• Involve in excision of the T-DNA
sequence
• Starts at RB follows by a nick at LB
as a single strand
• Coated by virE proteins (coded by
virE gene in vir-regions
• Transported into plant cells via
membrane channel formed by virB
protein
17. The binary vector system
• Developed from the concept of Ti-plasmid system
• Essential functions for transfer are supplied
separately.
• Namely?
A. ?
B. ?
18. The binary vector system
• Neomycin phototransferase II
resistance to kanamycin
• Contains polylinker
Facilitate insertion of
the DNA
Contains series of unique
restriction sites
20. The Gene Gun
• Using standard bacterial vectors
• 1 micrometer in diameter
• Tungsten or gold
• Coated with DNA (gene of interest + selectable marker)
• Fired with a gun with velocities over 400 mps directly
into plant tissue of interest
• Then transferred to a nurse cell culture plate
21. The Gene Gun
• Major advantages:
Possibility of adding a transgene to
organelles that have their own DNA genome
Need special markers:
1. Spectinomycin
2. Streptomycin
23. Insect resistance: Bt-crops
• Bacillus thuringiensis - apore forming soil bacterium
• Crystals consists of one or more delta-endotoxins or cry proteins of
130kDal
• Upon digestion by larvae, proteases in the insect's gut cleave the
protein into an active N-terminal 65-79 kDal
• The toxin acts by binding to receptors on the cell membrane of the
midgut epithelial cells
• Inserts itself into the membrane, forms pores that kill the epithelial cells
• Eventually kills the insect by colloid osmotic lysis
