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Chapter 2
Plant transformation
Plant transformation:
It is the production of a plant containing a new gene (foreign
gene) or a DNA sequence which is added to the plant genome
and this sequence must be stable and can be inherited with
the rest of the plant genome
There are many ways to perform plant transformation but all of
them must follow the same main steps:
1- Preparing the plant tissue needed to be transformed.
2- Inserting the foreign DNA to the plant tissue
3- Screening cells and selecting the transformed ones
4- The production of a whole plant using tissue culture
5- Examining the produced plant and studying the ability of the
transformed character to be transferred successfully from one
generation to another
1- Preparing the plant tissue needed to be transformed:
-Not all tissues or plant cells are equal in their ability to accept
a new DNA.
-The selected plant tissue must has the ability to regenerate
and differentiate to a full plant.
-For the transformation to occur the plant tissue must be
wounded, and the response of the different plant to wounds
differ (Dicotyledonous plants when wounded a callus tissues
are formed at the site of injury and this callus can be
transformed, while monocotyledonous plants when wounded
a site of dead cells are formed around the site of injury which
cannot be transformed)
That’s why Embryogenic tissues are used when monocot.
Plants are needed to be transformed
Examples for the tissues used in plant transformation:
- Protoplast: which is a plant cell without its cell wall,
transformation of a nude cell is much easier than cells
with cell wall.
- Leaf disks: leaf disks are taken from the mother plant and
the main idea of taking the disks is to injure the edges of
the leaf disks.
- Cotyledon sections: Used in Soybean
- Embryogenic tissues: It is used in transforming monocot.
Plants.
2- Inserting the foreign DNA to the plant tissue:
1- Direct DNA insertion: Plant protoplasts are incubated with the
foreign DNA in the presence of Polyethyleneglycol or using
Electroporation, both will cause the increase in permeability of
the plasma membrane allowing the DNA to pass through it to the
inside of the cell.
2- Microinjection: The nucleus or the cell is injected under
microscope with the foreign DNA. Performing this technique is
very difficult and takes a long time to inject a number of cells but
one of its benefits is the ability of the micro-injector to penetrate
the cell wall.
3- The Gene gun or microprojectile: The easiest way to perform
transformation to a plant tissue, its idea is built on the fast
movement of DNA coated golden particles toward the target
tissues, the gold particles will penetrate the cells causing
transformation.
4- Agrobacterium mediated transformation : Agrobacterium
tumefaciens is a bacteria abundant in the soil related to family
Rhizobium this becteria causes the formation of tumors on the
stem at the soil surface those tumors are called Crawn galls.
The main cause of the tumor is
the transfer of a DNA
sequence from the bacteria to
the plant tissues, this
sequence has the ability to
combine with the plant DNA
and express its characters.
-The Agrobacterium contains a
large plasmid called the Ti
plasmid (Tumor inducing), this
plasmid contains genes which
interfere with the plant hormones
production. The bacteria infects
the plant through wounds at the
soil surface.
-The bacterial genes cause an
abnormal growth and the
formation of tumors at the site of
injury.
-The tumor cells can grow on a
medium free of cytokinins and
auxins.
- This means that the infected
cells had been naturally
transformed.
The Ti plasmid consists of many regions:
1- T-DNA or (Transferred DNA) which is the region that is
transferred to the plant genome, this region is confined between
2 DNA sequences, one from the left and is called the left border L
and one to the right and is called the right border R. (any
sequence between the L and the R can be transferred to the
plant genome)
-In the wild type of the bacteria
the T-DNA contains Opine
gene which is responsible for
the catabolism of Octopine
and Nopaline and use them as
a source of N and
carbohydrates also this region
contains the gene responsible
for the production of auxin and
cytokinins (Oncogenes)
2- Origin of replication
3- Virulence region: which is responsible for the cause of
infection
-Scientists were able to remove
the genes responsible for the
production of auxins and
cytokinins aldo the genes
responsible for the Opine
catabolism and a Binary plasmid
was formed.
- A marker gene (mostly antibiotic
resistance gene) and the foreign
gene can be added to the T-DNA
by the use of restriction and
ligating enzymes.
Mechanism of Agrobacterium infection:
First if the plant is injured at the soil surface phenolic
exudates are excreted from the wound in order to protect the
wound from external infection. The phenolic compounds are
the main trigger that activates the virulence genes ( VirA,
VirB, VirC, VirD, VirE, VirF, VirG) at the virulence region, The
virulence genes cause the formation of a protein membrane
between the bacteria and the host plant.
-The Bacteria forms a single strand of the T-DNA and it
moves toward the plant cell through the protein membrane.
- The T-DNA (single strand) enters the plant nucleus and then
becomes a double helix and binds with the plant DNA.
- Auxin and cytokinin are produced in large quantities leading
to the production of tumors and abnormal growth.
-Screening and selecting transformed cells
- Producing a whole plant from the successfully
transformed cells
Analyze transformants:
1- Southern hybridization: by this way the existence of the gene
and its number of copies can be detected.
2- mRNA hybridization: the gene expression can be detected
because some times the gene is transferred to the plant
genome but it doesn’t express its self as protein.
3- Studying the enzymes and proteins (proteomics): some
times the mRNA is produced but not translated to protein due
to mutations.
4- Biochemical and morphological studies: must be done in
more than one generation in order to make sure of the stability
of the transferred gene.
5- Reporter gene: It is a gene added to the T-DNA with the
marker gene responsible for showing a morphological
character (radiate or glowing under UV)
Chapter 4
Plant pathogen interaction
Introduction:
- The relation ship between plant and pathogen is one of the
most important plant studies due to its effect on plant growth
and crop productivity.
- Most of the pathogens are very specific to a certain plant or
species, but after the full infection the pathogen will cause a
great damage to the cultivated crop.
-Pathogens are either Necrotrophic pathogen (Affects the dead
tissues and had a wide range of hosts) or Biotrophic pathogen
(Affects living cells and had a very narrow range of hosts)
- The outcome of the plant pathogen relationship is either plant
infection or plant resistance, plant resistance appears as
natural borders (increasing cuticle, increasing cell wall
thickness, production of antioxidants, phenolic
compounds…etc.)
The relationship between plant and pathogen
plant
pathogen
Relationship
Result
Susceptible
Virulent
Compatible
Infection
Resistant
Avirulent
Incompatible
Resistance
The resistance to a pathogen refers to the speed of the
immunity system of the plant to respond toward the
pathogen and activate the pathways leading to the
formation of thick cell wall, cell death, phenolic compounds
production…
Gene to gene hypothesis (the old theory):
“ In each plant and pathogen there are genes for the
resistance and virulence and they are either dominant or
recessive, depending on the relationship between the genes
whether it is compatible or incompatible the infection or the
plant resistance occur”
The plant is resistant if it has the dominant resistant gene R
which interact with the gene of the pathogen which is also
dominant gene Avr (Avirulence gene)
- Each of the R gene and Avr gene produces an intermediate
protein compound and they are responsible for the activation
of the resistance pathways.
The new theory for plant resistance:
“ the relationship between the R gene and the Avr gene is not a
direct relation, but it needs an intermediate compound which is
a protein produced by the host plant and this protein activates
the NB-LRR region”
Example:
The R gene in Arabidopsis (RPM1) which resists
Pseudomonas syringae the Pseudomonas produces a protein
called RIN4 (RPM1- interacting protein) which is the
intermediate protein responsible for causing the infection. The
resistant plants deactivate the RIN4 and signal the plant to
produce salicylic acid (SA) to increase cell wall thickness.
Plant defense mechanisms:
1- Phytoalexins: they are compounds with small molecular
weight. Phytoalexins are produced by large quantities during
stress conditions which cam reach 10% of the plant dry
weight.
The genes responsible for the production of phytoalexins
becomes active only during infection or stress since the
phytoalexins are poisonous substances for pathogen and
plant cells as well. One of the genes responsible for the
phytoalexin production is the PAL gene (phenylalanine
ammonia lyase)
PR protein:
Some proteins are produced by the plants and they are
specific to attack certain pathogen and they are called related
protein pathogenesis.
Example: the chitinase and glucanase are enzymes
responsible for the analysis of the chinitin and glucane forming
the fungal cell wall.
Those proteins are not poisonous to plant cells and they are
stored in the cell vacuole therefore scientists are trying to
transfer the gene responsible for the production of the RP
protein to different plat types.
3- Cell wall:
Most pathogens produce cellulase and pectinase enzymes
to analyze the plant cell wall, after infection and the
beginning of the cell wall analysis some genes become
active and start to produce Callose synthase enzyme which
increase the cell wall thickness.
4- Salicylic acid and (SAR) system acquired resistance :
The salicylic acid is a natural compound produced by the
plant, during infection the conc. of salicylic acid increases
and translocated to different plant tissues through the xylem
and the phloem causing a signal and activates the PR
protein production.
Also it was found in some plants that
after infection other parts of the plant
become resistant to the pathogen also
the neighboring plants become
resistant to that pathogen .. By studying
this they found that the plant produces
a compound called Jasmonic acid
which causes signal to the plant and
the other neighboring plants to become
resistant to that pathogen (acquired
resistance)
Example:
Resistance gene in tomato plant is called Pto this gene can
produce a protein called Serine-threonine protein kinase which
can cause phosphorylation to other amino acids. The
phosphorylation will cause the stop of the AA action.
The binding of the Pto and the AvrPto caused a signal and the
beginning of the resistance pathways
So what is the reason of the existence of the Avrgene in the
pathogen?
-It is an important gene for the life and the existence of the
pathogen.
-Selection of the weak population.
R gene:
Consists of 3 main parts:
1- Leucine rich repeats LLR: 24 AA it is important in the
binding of the protein product of the R and Avr genes also it
plays a role in the activation of the resistance pathways.
2- Nucleotide binding site NB: Resposible for the production
of ATP’s and any mutation occurs at that part will cause the
deactivation of the gene.
3- Toll interleukin receptor similarity TI: Activates the
resistance pathways leading to the production of
antioxidants, phenols, salsilic acid…