Download Genetic Modification of Plants using Agrobacterium

Genetic Modification of Plants using Agrobacterium
Agrobacterium tumefaciens is a rod-shaped aerobic soil
bacterium that can infect dicot plants especially apple,
pear, peach, cherry, almond, raspberry, rose &
grapevines. It’s method of infection is unique, for it
transfers some of its DNA to the chromosomes of the
plant cell, causing a tumour to grow & causing the plant
to make special organic food molecules for the
bacterium.
Agrobacterium tumefaciens
Crown Gall
The process begins when a plant is wounded, often
near the base of the stem. This causes the release of
compounds that attract Agrobacterium to the damaged
cells. Transcription of genes on one of the bacterium’s
plasmids begins, transferring a 20 kbase piece of singlestranded T-DNA into the plant nucleus. Here the TDNA randomly integrates into the plant chromosome,
translating 2 groups of substances:1. One group of chemicals causes an overproduction of plant hormones, leading to crown
gall, which is an uncontrolled growth of cells, a cancer. Galls can develop any-where on
stems & roots but are usually near the soil line & vary from pea-size up to tennis-ball-size.
2. The other group of substances directs the plant to make unusual compounds called opines,
which the bacterium can use as a food source.
The genes for all these substances are under the control of plant gene regulators. This infection
does not kill the plant, it merely alters the functions within the plant.
The plasmid which controls this infection process is
the Ti (tumour-inducing) plasmid. It is a very big,
double-stranded, circular piece of DNA, which was
Opine
sequenced in 2001. It contains a number of
Gene
Wild Type
important parts:s
Ti plasmid
1. Oncogene – this is the gene that causes crown
200kb
gall tumour
L
R
2. VIV – the origin of DNA replication
3. Opine gene – this is the gene that codes for
the
making of the opines, the bacterial food source.
4.
A
Left & a Right border - 25b repeats needed
VIV
for the DNA to transfer into the plant.
As these bacteria naturally carry DNA into plants, they
have been co-opted by biotechnologists as vectors of
recombinant DNA. To do this:1. The plasmid is “disarmed”, by deleting the oncogene & opine genes.
2. The Left & a Right border are used as delineating edges of the inserted genes. The next 3
insertions are placed between these borders.
Oncogene
3. A selectable marker gene is inserted. This codes for an enzyme that detoxifies the
antibiotic kanamycin, thus giving the genetically modified plant resistance to this antibiotic.
Kanamycin will normally kill plants so only those plants with the gene will survive.
4. A restriction enzyme recognition site is also inserted.
5. The gene of interest can now be inserted in with sticky ends to match the recognition site.
All of these deletions & insertions have genetically modified the Agrobacterium (it is now a
Genetically Modified organism).
6. A culture of this GM Agrobacterium is grown.
7. Transfection – the bacterium is made to infect plant tissue (this process can also be called
transformation). This can be done in 3 different ways: Soaking leaf discs in the Agrobacterium culture & then growing these discs on agar
containing kanamycin & rooting hormones. The leaf tissue which takes up the Ti
plasmid grows into a miniature GM plant.
 Some plants will take up the Ti plasmid after soaking their seeds in the culture.
 Or the Agrobacterium can be incubated with protoplasts which are allowed to form a
tissue culture.
Whichever method is used each cell of the plant grown now contains the inserted gene &
so it is a GM plant.
This vector has been used for about 20 years to genetically modify dicots including these plants
below as well as tobacco:Crop
Name of variety
Company
Novel Traits
Tomato (1994)
Flavr Savr
Calgene
Vine-ripened flavour, longer shelf life
Cotton
Potato (1996)
Soybean
Canola
Cotton (1996)
Bollgard
New Leaf
Monsanto
Bt toxin for insect resistance
Roundup Ready
Monsanto
Glyphosphate herbicide resistance
(Release Date)
The Flavr Savr tomatoes are widely recognised as one of the first GM crops grown. They did not
express the gene for polygalacturonase, an enzyme that degrades pectin, which leads to the
softening of the fruit tissue. As a result these tomatoes can be left longer on the vine to
accumulate flavour & do not rot quickly so transport better from field to consumer.
Several crops have been engineered to express a gene from the bacteria Bacillus thuringensis (Bt).
This bacteria makes a protein that dissolves the gut of insects & so the expression of this gene
confers resistance to insect pests. The organism Bt is a biopesticide that has been used as a
“natural” pesticide by organic farmers for many years.
Crops have also been engineered for resistance to herbicides such as glyphosphate, so the
herbicide can be sprayed all over the crop to kill weeds but will not damage the crop. This should
mean less application of the herbicide.
The main problem with this whole process has been the inability of Agrobacteria tumefaciens to
infect monocots (which include most of our main cereal crops which provide the staple food in our
diet e.g. wheat, rice, corn). So, to introduce new genetic information into these crops requires
other methods such as the gene gun. However, Agrobacteria has recently been used to transform
rice & maize embryos (Chan 1998), so the problem is not universal & we may see more monocot
species being genetically modified in this way.