Gene Transfer
Methods in plants
Gene transfer methods
•Transfer of a gene from one
DNA molecule to another DNA
•Genetic transformation
•Transient and stable
transformation
•Transgene and transgenic
Indirect gene transfer method
Agrobacterium tumeficians:
•Soil born gram negative bacteria
•Rod shaped and motile
•Belongs to bacteria family of
Rhizobiaceae
•Causes crown gall disease
Agrobacterium mediated gene transfer
Ti plasmid and Vir gene:
•T DNA region
•Virulence region
•Opine catabolism region
Transformation of cell by A.tumefaciens
1. Signal induction to
Agrobacterium
2. Attachment of
Agrobacterium to plant
cell
3. Production of virulence
protein
4. Production of single
stranded T DNA
5. Transfer of T DNA out of
Agrobacterium
6. Integration
Applications
•Generate a large number of
transgenic plants
•Introduce many new traits
•Herbicides resistance
•Disease resistance
•High growth rate
Direct gene transfer method
Biolistic method:
•Also known as particle bombardment
•Use accelerated microprojectiles
•DNA coated with gold or tungsten
filament
•Coated beads are attached to the end of
plastic bullet
•Beads pass through the cell wall
Application
•Used successfully to transform
soya bean, cotton, spruce,
sugarcane, papaya
• Transformation of Mitochondria
of plants and chloroplast of
chlamydomonas
•Manipulation of subcellular
organelles
Microinjection method
•Artificial DNA transfer to cereals plants
•Diameter of needle greater than cell
diameter
•Around 0.3 ml of DNA solution is
injected
•Timing of injection is important
•Fourteen days before meiosis.
Microinjection method
Application
•Method is effective in transforming primary cells as
well as cells in established culture
•Technique is ideally useful for producing transgenic
animal quickly
•Procedure is important for gene transfer to
embryonic cells
•Method has been successfully used with cells and
protoplast of tobacco, alfalfa etc.
Liposome mediated method
•Liposomes are spheres of lipids used to
transport molecules into the cells
•Artificial vesicles
•Cationic lipids
•Fusion
•aqueous solution of DNA molecules.
•self-organizing process
Liposome mediated method
Application
•Germline trangenises
•Transfection of ES cells
•vaccination against virus
infection
•In tumor therapy
•In multi drug resistance
PEG mediated method
•Utilized for protoplast
•Stimulates endocytosis
•Protoplast are kept in the solution containing
PEG
•Molecular weight of PEG is 8000D with final
conc. Of 15%
•Addition of cacl2
•Glucose and sucrose act as buffering agent
•Culturing of protoplast to form cells with
walls and colonies
Application
•Vaccine development
•Enhancing resistance in
plant
•Treatment of cancer
•Gene discovery
•Gene therapy
Electroporation
•It uses electrical pulse to
produce transient pores in the
plasma membrane
•Microscopic pores are induced
in biological membrane
•These pores are known as
electro pores which allow the
molecules, ions and water to
pass from one side of the
membrane to another
•Electropores reseal
spontaneously and the cell
can recover
Application
•Effective method of introducing exogenous DNA into
embryonic stem cells
•It transfer genes into cultured mammalian embryos at
defined stages of development
•Electroporation has been reported to enhance the level of
gene expression
•It significantly improve immune responses elicited to DNA
vaccines
Natural gene transfer method
Conjugation
•Require the presence of special plasmid called F plasmid
• F+ and F- plasmids
•F plasmid consist of 25 genes code for production of sex
pilli
•Conjugation event occur when the male cell extends his
sex pilli and attaches to female cell
•Attached pilus acts as a cytoplasmic bridge
•High frequency cell
Bacterial transformation
•Transformation is direct uptake of exogenous DNA from its
surrounding
•Development of competence,
•Binding of DNA to the cell surface
•Processing and uptake of free DNA
•integration of the DNA into the chromosome by
recombination.
Transduction
•Genetic transfer from one bacteria to another
through a virus
•virus uses the host machinery to make multiple
copies either directly by the lytic cycle
•Assembly of bacteriophages
• lysogenic cycle
•Specialized transduction
•General transduction
Application
•Transduction is used to insert the genes of choices in
animals and plant cells to modify the genetic constituents
and get the desired characteristics
•It can be used for gene therapy
•It has huge potential to cure genetic diseases
