Gene Transfer techniques in plants
Gene transfer or uptake of DNA refers to the process that moves a specific piece of
DNA into cell. The directed desirable gene transfer from one organism to another
and the subsequent stable integration & expression of foreign gene into the genome
is referred as genetic transformation. The transferred gene is known as transgene and
the organism that develop after a successful gene transfer is known as transgenic.
Transgenic plant is the plant that carry the stably integrated foreign genes. These
plants may also be called transformed plants.
The transferred DNA may be expressed for only short period of time following the
DNA transfer process and this is called transient expression. Stable transformation
occurs when DNA is integrated into the plant nuclear genome expression occurs in
regenerated plant and is inherited in subsequent generations.
STEPS IN TRANSFORMATION
a) Identification of useful genes:
Desirable genes located in wild species, unrelated plant species, unrelated organism
and animals.
b) Designing gene for insertion:
The gene of interest is isolated from the donor source and cloned in the laboratory.
The cloning is done generally using plasmid.
c) Insertion of gene into target plant: The cloned gene i.e., multiple copies of the
gene of interest are inserted into host plant or the recipient plant.
d) Identification of transgenic cells: Transformed cells are identified using selectable
marker and are regenerated into whole plant in nutrient medium.
e) Regenerate plant compared with plant variety. It should look like parent variety
except gene of interest.
GENE TRANSFER METHODS
1. Vector Mediated Gene Transfer
,2. Vectorless or Direct Gene Transfer Methods
A) Vector-mediated gene transfer: (Agrobacterium-mediated gene transfer)
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil-borne, Gram-
negative bacteria.
These are phytopathogens (that cause infection in plants) and are treated as the
nature’s most effective plant genetic engineer.
A. tumefaciens induces crown gall disease and A. rhizogenes that induces hairy root
disease in plants.
Crown Gall Disease- Ti plasmid Almost 100 years ago (1907), Smith and Townsend
postulated that a bacterium was the causative agent of crown gall tumors, although
its importance was recognized much later.
As A. tumefaciens infects wounded or damaged plant tissues, it induces the
formation of a plant tumor called crown gall.
The entry of the bacterium into the plant tissues is facilitated by the release of certain
phenolic compounds (acetosyringone, hydroxyacetosyringone) by the wounded
sites.
Formation of a Crown Gall Tumor Crown gall formation occurs when the bacterium
releases its Ti plasmid (Tumor- inducing plasmid) into the plant cell cytoplasm. A
fragment of Ti plasmid, referred to as T-DNA, is actually transferred from the
bacterium into the host where it gets integrated into the plant cell chromosome (i.e.
host genome). Thus, crown gall disease is a naturally evolved genetic engineering
process.
The T-DNA carries genes that code for proteins involved in the biosynthesis of
growth hormones (auxin and cytokinin) and novel plant metabolites namely opines-
amino acid derivatives and agropines-sugar derivatives. The growth hormones cause
plant cells to proliferate and form the gall while opines and agropines are utilized by
A. tumefaciens as sources of carbon and energy.
Thus, A. tumefaciens genetically transforms plant cells and creates a biosynthetic
machinery to produce nutrients for its own use. As the bacteria multiply and continue
infection, crown gall develops which is a visible mass of the accumulated bacteria
and plant material. Crown gall formation is the consequence of the transfer,
, integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens in
the infected plant.
Organization of Ti plasmid:
The Ti plasmids (approximate size 200 kb each) exist as independent replicating
circular DNA molecules within the Agrobacterium cells.
The T-DNA (transferred DNA) is variable in length in the range of 12 to 24 kb,
which depends on the bacterial strain from which Ti plasmids come.
Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb while octopine
strains have two T-DNA regions referred to as TL and TR that are respectively 14
kb and 7 kb in length.
The Ti plasmid has three important regions.
1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs) and is flanked by left and right borders. These
three genes-aux, cyt and ocs are referred to as oncogenes, as they are the
determinants of the tumor phenotype. T-DNA borders — A set of 24 kb
sequences present on either side (right and left) of T-DNA are also transferred
to the plant cells. It is now clearly established that the right border is more
critical for TDNA transfer and tumori-genesis.
2. Virulence region or vir region The genes responsible for the transfer of T-
DNA into the host plant are located outside T-DNA and the region is referred
to as vir or virulence region. Vir region codes for proteins involved in T-DNA
transfer. At least nine vir-gene operons have been identified. These include
vir A, vir G, vir B1, vir C1, vir D1, D2, D4, and vir E1 and E2.
3. Opine catabolism region: This region codes for proteins involved in the
uptake and metabolisms of opines. Besides the above three, there is ori region
that is responsible for the origin of DNA replication which permits the Ti
plasmid to be stably maintained in A. tumefaciens.
T-DNA transfer and integration:
The process of T-DNA transfer and its integration into the host plant genome is is
briefly described below:
Signal induction to Agrobacterium:
Gene transfer or uptake of DNA refers to the process that moves a specific piece of
DNA into cell. The directed desirable gene transfer from one organism to another
and the subsequent stable integration & expression of foreign gene into the genome
is referred as genetic transformation. The transferred gene is known as transgene and
the organism that develop after a successful gene transfer is known as transgenic.
Transgenic plant is the plant that carry the stably integrated foreign genes. These
plants may also be called transformed plants.
The transferred DNA may be expressed for only short period of time following the
DNA transfer process and this is called transient expression. Stable transformation
occurs when DNA is integrated into the plant nuclear genome expression occurs in
regenerated plant and is inherited in subsequent generations.
STEPS IN TRANSFORMATION
a) Identification of useful genes:
Desirable genes located in wild species, unrelated plant species, unrelated organism
and animals.
b) Designing gene for insertion:
The gene of interest is isolated from the donor source and cloned in the laboratory.
The cloning is done generally using plasmid.
c) Insertion of gene into target plant: The cloned gene i.e., multiple copies of the
gene of interest are inserted into host plant or the recipient plant.
d) Identification of transgenic cells: Transformed cells are identified using selectable
marker and are regenerated into whole plant in nutrient medium.
e) Regenerate plant compared with plant variety. It should look like parent variety
except gene of interest.
GENE TRANSFER METHODS
1. Vector Mediated Gene Transfer
,2. Vectorless or Direct Gene Transfer Methods
A) Vector-mediated gene transfer: (Agrobacterium-mediated gene transfer)
Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil-borne, Gram-
negative bacteria.
These are phytopathogens (that cause infection in plants) and are treated as the
nature’s most effective plant genetic engineer.
A. tumefaciens induces crown gall disease and A. rhizogenes that induces hairy root
disease in plants.
Crown Gall Disease- Ti plasmid Almost 100 years ago (1907), Smith and Townsend
postulated that a bacterium was the causative agent of crown gall tumors, although
its importance was recognized much later.
As A. tumefaciens infects wounded or damaged plant tissues, it induces the
formation of a plant tumor called crown gall.
The entry of the bacterium into the plant tissues is facilitated by the release of certain
phenolic compounds (acetosyringone, hydroxyacetosyringone) by the wounded
sites.
Formation of a Crown Gall Tumor Crown gall formation occurs when the bacterium
releases its Ti plasmid (Tumor- inducing plasmid) into the plant cell cytoplasm. A
fragment of Ti plasmid, referred to as T-DNA, is actually transferred from the
bacterium into the host where it gets integrated into the plant cell chromosome (i.e.
host genome). Thus, crown gall disease is a naturally evolved genetic engineering
process.
The T-DNA carries genes that code for proteins involved in the biosynthesis of
growth hormones (auxin and cytokinin) and novel plant metabolites namely opines-
amino acid derivatives and agropines-sugar derivatives. The growth hormones cause
plant cells to proliferate and form the gall while opines and agropines are utilized by
A. tumefaciens as sources of carbon and energy.
Thus, A. tumefaciens genetically transforms plant cells and creates a biosynthetic
machinery to produce nutrients for its own use. As the bacteria multiply and continue
infection, crown gall develops which is a visible mass of the accumulated bacteria
and plant material. Crown gall formation is the consequence of the transfer,
, integration and expression of genes of T-DNA (or Ti plasmid) of A. tumefaciens in
the infected plant.
Organization of Ti plasmid:
The Ti plasmids (approximate size 200 kb each) exist as independent replicating
circular DNA molecules within the Agrobacterium cells.
The T-DNA (transferred DNA) is variable in length in the range of 12 to 24 kb,
which depends on the bacterial strain from which Ti plasmids come.
Nopaline strains of Ti plasmid have one T-DNA with length of 20 kb while octopine
strains have two T-DNA regions referred to as TL and TR that are respectively 14
kb and 7 kb in length.
The Ti plasmid has three important regions.
1. T-DNA region: This region has the genes for the biosynthesis of auxin (aux),
cytokinin (cyt) and opine (ocs) and is flanked by left and right borders. These
three genes-aux, cyt and ocs are referred to as oncogenes, as they are the
determinants of the tumor phenotype. T-DNA borders — A set of 24 kb
sequences present on either side (right and left) of T-DNA are also transferred
to the plant cells. It is now clearly established that the right border is more
critical for TDNA transfer and tumori-genesis.
2. Virulence region or vir region The genes responsible for the transfer of T-
DNA into the host plant are located outside T-DNA and the region is referred
to as vir or virulence region. Vir region codes for proteins involved in T-DNA
transfer. At least nine vir-gene operons have been identified. These include
vir A, vir G, vir B1, vir C1, vir D1, D2, D4, and vir E1 and E2.
3. Opine catabolism region: This region codes for proteins involved in the
uptake and metabolisms of opines. Besides the above three, there is ori region
that is responsible for the origin of DNA replication which permits the Ti
plasmid to be stably maintained in A. tumefaciens.
T-DNA transfer and integration:
The process of T-DNA transfer and its integration into the host plant genome is is
briefly described below:
Signal induction to Agrobacterium:
