L10- How transgenic plants are made Flashcards
What are the methods of gene delivery in plants?
- Microinjection
- Electroporation
- Biolistic/ microbombatdment
- Agrobacterium
Microinjection
- Use a very fine pipette to inject DNA molecule directly into nucleus of a single living cell
Organism:
- animal cells (embryo)
- plant cells (difficult to target)
Electroporation
- An electrical current is applied to cells causing tiny holes to form in the cell membrane
- DNA molecules diffuse through into the cell
Organism:
- E.Coli
Biolistic/ microbombardment/ gene gun
Biolistic particle delivery system:
- device for injecting cells with genetic information
- DNA vector is coated onto gold or tungsten particles
- Particles are accelerated at high speeds by the gun
- DNA enters the nucleus and incorporates into chromosomes
- Integration process unknown
Organism:
- Animals and plant cells
Agrobacterium
- Ability to transfer DNA between itself and plants
Organism:
- Plant cells
What is a Ti plasmid?
Ti plasmid or tumour inducing plasmid:
- A circular plasmid that can be part of the genetic equipment that Agrobacterium tumefaciens use to transduce its genetic material to plants
What are opines?
Opines:
- low molecular weight compounds found in plant crown gall tumors or hairy root tumors produced by parasitic bacteria of the genus Agrobacterium
- The Ti plasmids are classified into different types based on the type of opine produced by their genes
What is Virulence Region?
- Genes in the virulence region are grouped into operons (functioning unit of genomics DNA containing a cluster of genes under the control of a single promoter) virABCDEFG, which code for the enzymes responsible for mediating conjugative transfer of T-DNA to plant cells
What are the three main steps of Agrobacterium mediated transformation
- Insertion of the cloned gene into Ti plasmid (or binary vetor) of Agrobacterium tumefaciens
- Infection of cells from plant with the engineered Agrobacterium tumefaciens
- Growth of transformed plant cells in culture and regeneration of plants from these cells
Random site of insertion of the T-DNA construct and multiple insertion may occur in the same nucleus
- T-DNA is randomly inserted into any position(s) in plant genome
- Single copy or multiple copies of T-DNA can be inserted into a single plant cell
What is an ideal model plant organism?
• A model organism is a species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the model organism will provide insight into the workings of other organisms
Desirable traits/ characteristics:
- Size (Small, easy to handle)
- Generation time (Fast growing)
- Accessibility
- Manipulation (eg. can it be transformed easily)
- Genetics
- Conservation of mechanisms
- Potential economic benefit
What makes Arabidopsis thaliana an ideal model plant organism?
- Small: 8 to 12 inches tall
- Fast life cycle: 6 weeks from seed to seed
- Self-fertilizing
- Makes thousands of seeds per plant
- Not particular about growth conditions
- Small, relatively succinct genome
- It can be transformed easily
- Its entire genome has been sequenced, and the sequence is freely available
What are selectable marker genes?
- A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection
- Selectable markers assist in following inheritance of transgenes
- Transformed cells are selected by killing non-transformed cells with a selective agent
What is forward and reverse genetics?
• FORWARD GENETICS
- approach of determining the genetic basis responsible for a phenotype
• REVERSE GENETICS
- a method to understand the function of a gene by analysing the phenotypic effects of specific gene sequences
- switch off gene target
