1. B. Tech. (Biotechnology) III Year V th Semester
Unit II Lecture 4
B. Tech. (Biotechnology) III Year V th Semester
EBT-501, Genetic Engineering

2. Unit II Restriction modification
Enzymes used in recombinant DNA technology endonucleases, ligases and other enzymes useful in gene cloning
PCR technology for gene/DNA detection, cDNA,
Use of Agrobacterium for genetic engineering in plants
Gene libraries; Use of marker genes.
Cloning of foreign genes: DNA delivery methods - physical methods and biological methods,
Genetic transformation of prokaryotes: Transferring DNA into E. coli –Chemical induction and Electroporation,

3. Gene Transfer in Plants
The Ti plasmid of Agrobacterium tumefaciens is an important tool for transferring genes into plants.

4. Agro-bacterium tumefacians
Agro bacterium tumefacians is a bacterium that causes a disease known as crown gall in plants.
Infects plants by transferring its genetic material into plant cell.
Agrobacterium transformation is the most common technique for genetically engineered plants

5. Production of Transgenic Plants
Microprojectile bombardment
Electroporation
Agrobacterium tumefaciens-mediated transformation
Plant cells are totipotent, so an entire plant can be regenerated from a genetically engineered plant somatic cell.

6. Agrobacterium tumifaciens Infection

7. The Ti Plasmid

8. Transformation of Plant Cells by the Ti Plasmid

9. Key steps from natural Agrobacterium to “useful” Agrobacterium
Some vir genes deleted--disarmed
Opines not going to be produced
Deleting tumorogenesis function
Choosing strains that transfer DNA in lab
Clone in genes of interest, antibiotic resistance genes, etc.
Binary system-- two plasmids are better than one Ti plasmid

10. Plant transformation with the Ti plasmid of Agrobacterium tumefaciens
Tumor formation is the result of the transfer, integration and expression of genes on a specific segment of A. tumefaciens plasmid DNA called the T- DNA (transferred DNA)
The T-DNA resides on a large plasmid called the Ti (tumor inducing) plasmid found in A. tumefaciens

11. The Ti plasmid of Agrobacterium tumafaciens and the transfer of its T-DNA to the plant nuclear genome

13. Ti plasmid: structure & function
The infection process:
Wounded plant cell releases phenolics and nutrients.
Phenolics and nutrients cause chemotaxic response of A. tumefaciens
Attachment of the bacteria to the plant cell.
Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir gene transcription and allow for T-DNA transfer and integration into plant chromosomal DNA.
Transcription and translation of the T-DNA in the plant cell to produce opines (food) and tumors (housing) for the bacteria.
The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens to use opines as a C, H, O, and N source.

14. The binary Ti plasmid system involves using a small T-DNA plasmid (shown below) and a disarmed (i.e., no T-DNA) Ti plasmid in A. tumefaciens

15. DNA-delivery methods Method Comment
Ti plasmid-mediated gene transfer *
Excellent and highly effective, but limited to dicots
Microprojectile bombardment *
Easy and effective; used with a wide range of plants
Viral vectors
Not very effective
Direct gene transfer into plant protoplasts
Only certain protoplasts can be regenerated into whole plants
Microinjetion
Tedious and slow
Electroporation *
Limited to protoplasts that can be regenerated into whole plants
Liposome fusion

16. Microprojectile bombardment or biolistic-mediated DNA transfection equipment
equipment (a) lab version (b) portable version
When the helium pressure builds to a certain point, the plastic rupture disk bursts, and the released gas accelerates the flying disk with the DNA-coated gold particles on its
lower side. The gold particles pass the stopping screen, which holds back the flying disk, and penetrate the cells of the plant.

17. Some plant cell reporter and selectable marker gene systems
Enzyme activity
Selectable marker
Reporter gene
Neomycin phosphotransferase (kanr)
Yes
Hygromycin phosphotransferase (hygr)
Nopaline synthase No
Octopine synthase
-glucuronidase (GUS)
Firefly luciferase
-galactosidase
Bromoxynil nitrilase
Green fluorescent protein (GFP)

18. Genetically Modified Organisms