Plant transformation
Problems in classical plant breeding
• Slow, not precise, repeated generations of selection and crossing needed
• Only transfer between plants that can sexually hybridize
• Relatively small gene pool
• Undesirable genes transferred (linkage drag)
• Several generations to cross out of the plants
Solutions
Molecular Plant Breeding
• MAS: (Molecular) Marker Assisted Selection
• WGS: Whole Genome Sequencing
• Genetic Modification
• Plant transformation
Plant transformation
What is it?
Plant transformation is a way to insert DNA from another organism- normally
another plant, into the genome of a plant of interest.
There are different methods of plant transformation:
• Direct methods, where no mediator is used
○ Protoplast fusion
○ Protoplast transfection (chemicals, electroporation or microinjection
○ Microprojectile bombardment (particle gun)
• Indirect methods, where a bacterial mediator is used
○ Agrobacterium tumefaciens (updated to: Rhizobium radiobacter)
○ Agrobacterium rhizogenes (updated to: Rhizobium rhizogenes)
How it is done?
Agrobacterium infects plant tissue and can be used as a mediator- to put a gene of
interest via a plasmid.
• Agrobacterium tumefaciens causes crown gall disease (callus formation)
• Agrobacterium rhizogenes causes root proliferation
• Hormones cytokinins and auxins!
Callus is an undifferentiated tissue growing on media with the plant hormones auxin
and cytokinin.
, • Agrobacterium rhizogenes causes root proliferation
• Hormones cytokinins and auxins!
Callus is an undifferentiated tissue growing on media with the plant hormones auxin
and cytokinin.
Opines
Opines are produced in the galls by the plant cells and are a food source of the
Agrobacterium. They cannot be metabolized by the plant or by any other bacteria.
How does this plant suddenly makes this compound?
Agrobacterium transfers some of its own DNA into the plant cells
The transferred DNA encodes enzymes that:
• are involved in the synthesis of auxin and cytokinin, which promote cell
division
• are needed to synthesize opines (these are not encoded in plant genomes)
Tumor inducing plasmid • Ti plasmid
Root inducing plasmid • Ri plasmid
Agrobacterium Ti plasmid
• T-DNA carries the genes that promote tumour production and opines
production
• Virulence genes, located on seven operons encode proteins that are required
for mediating the T-DNA transfer
• T-DNA is bordered by 24-bp DNA sequences on the left and right borders of
the T-DNA
• Ori of replication of the plasmid
• Vir genes required for infection
• Genes enabling opine metabolism
Transformation process
• Wounded plant secretes Acetosyringone (phenolic
Problems in classical plant breeding
• Slow, not precise, repeated generations of selection and crossing needed
• Only transfer between plants that can sexually hybridize
• Relatively small gene pool
• Undesirable genes transferred (linkage drag)
• Several generations to cross out of the plants
Solutions
Molecular Plant Breeding
• MAS: (Molecular) Marker Assisted Selection
• WGS: Whole Genome Sequencing
• Genetic Modification
• Plant transformation
Plant transformation
What is it?
Plant transformation is a way to insert DNA from another organism- normally
another plant, into the genome of a plant of interest.
There are different methods of plant transformation:
• Direct methods, where no mediator is used
○ Protoplast fusion
○ Protoplast transfection (chemicals, electroporation or microinjection
○ Microprojectile bombardment (particle gun)
• Indirect methods, where a bacterial mediator is used
○ Agrobacterium tumefaciens (updated to: Rhizobium radiobacter)
○ Agrobacterium rhizogenes (updated to: Rhizobium rhizogenes)
How it is done?
Agrobacterium infects plant tissue and can be used as a mediator- to put a gene of
interest via a plasmid.
• Agrobacterium tumefaciens causes crown gall disease (callus formation)
• Agrobacterium rhizogenes causes root proliferation
• Hormones cytokinins and auxins!
Callus is an undifferentiated tissue growing on media with the plant hormones auxin
and cytokinin.
, • Agrobacterium rhizogenes causes root proliferation
• Hormones cytokinins and auxins!
Callus is an undifferentiated tissue growing on media with the plant hormones auxin
and cytokinin.
Opines
Opines are produced in the galls by the plant cells and are a food source of the
Agrobacterium. They cannot be metabolized by the plant or by any other bacteria.
How does this plant suddenly makes this compound?
Agrobacterium transfers some of its own DNA into the plant cells
The transferred DNA encodes enzymes that:
• are involved in the synthesis of auxin and cytokinin, which promote cell
division
• are needed to synthesize opines (these are not encoded in plant genomes)
Tumor inducing plasmid • Ti plasmid
Root inducing plasmid • Ri plasmid
Agrobacterium Ti plasmid
• T-DNA carries the genes that promote tumour production and opines
production
• Virulence genes, located on seven operons encode proteins that are required
for mediating the T-DNA transfer
• T-DNA is bordered by 24-bp DNA sequences on the left and right borders of
the T-DNA
• Ori of replication of the plasmid
• Vir genes required for infection
• Genes enabling opine metabolism
Transformation process
• Wounded plant secretes Acetosyringone (phenolic
