Mohd bin Mahmud @ Mansor, FIAT

Transgenic plants

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

• 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

Introduction

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

• 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

Introduction

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

• 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?

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

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

Ti-plasmid System

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

Ti-plasmid System

Ti-plasmid System

Ti-plasmid System

Ti-plasmid System

Ti-plasmid System

Ti-plasmid System

The binary vector system• Developed from the concept of Ti-plasmid system

• Essential functions for transfer are supplied separately.

• Namely?

A. ?

B. ?

• Neomycin phototransferase II

resistance to kanamycin

• Contains polylinker

Facilitate insertion of

the DNA

Contains series of unique

restriction sites

The binary vector system

The binary vector system

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

• Major advantages:

Possibility of adding a transgene to organelles that have their own DNA genome

Need special markers:

1. Spectinomycin

2. Streptomycin

The Gene Gun

The Gene Gun

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

• We have the information, so now what can we do? Discuss.

Insect resistance: Bt-crops