Genetic transformation

Methods of genetic transformation

1. Methods of Gene Transfer Prepared By, Prof. T. A. Pagar Dept of PHFBT, K. K Wagh College of Agricultural Biotechnology, Nashik . 1

2. Transformation Gene transfer is the uptake of foreign DNA or transgene by plant cells. It is the subsequent stable integration & expression of a foreign DNA into the genome. The method used for gene transfer is known as transformation. 2

3. Methods of Transformation There are mainly 2 methods of gene transfer: 1. Indirect or Agrobacterium -mediated gene transfer Gene transfer is done by using the bacteria Agrobacterium tumificiens . 2. Direct gene transfer By using various techniques (listed on next page) the gene is directly transferred into the host. 3

4. 4 Methods of Genetic Transformation Indirect Method of Gene Transfer Direct Method of Gene Transfer Agrobacterium mediated gene transfer Physical Electroporation Biolistic Macroinjection Microinjection Liposome mediated Chemical PEG mediated DMSO polycation DEAE Dextran Calcium phosphate co-precipitation

5. Agrobacterium mediated gene transfer Agrobacterium is soil borne, gram negative, rod shaped, motile found in rhizosphere . Causative agents of “Crown gall” in dicotyledons . Two strains of Ti-plasmid: - Octopine strains- contains two T-DNA region: T L (14 kb) and T R ( 7 kb) - Nopaline strains- contain one T-DNA region (20 kb) Size is about 200 kb Contain a vir region ~ 40 kb at least 8-11 vi r genes 5

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7. T-DNA Size 12 – 24 kb Left and right border sequence (24-bp) which will be transferred into genome of host plant Oncogenes e.g. Auxin , cytokinin , opines The T-DNA contains eight potential genes - these are eukaryotic in nature (eukaryotic promoters, monocistronic , eukaryotic polyadenylation signals, eukaryotic translation mechanisms) 7

8. Process of T-DNA transfer and integration Identify a suitable explants : Suitable plant tissue is removed and sterilized. Co-cultivate with the Agrobacterium : Small pieces of leaf tissue placed into a culture of Agrobacterium for about 30 mins . The explants then placed on MS medium without selective agent. Incubate explants with Agrobacterium for 2 days to allow transfer of the T-DNA. 8

9. Kill the Agrobacterium with a suitable antibiotic : The explants are removed from the medium and washed in cefotaxime . Select for transformed plant cells : The explant are transferred to a selective ( kanamycin ) medium with cefotaxime . Auxin , Cytokinin are used to encourage the regeneration of by organogenesis. Regeneration of whole plant : The shoot can be rooted by placing them on solid medium containing a high auxin to cytokinin ratio. 9

10. Electroporation Plant materials is incubated in a buffer solution containing DNA and subjected to high-voltage electric pulse. The DNA then migrates through high-voltage-induced pores in the plasma membrane and integrates into the genome. It can be used to transform all the major cereals particularly rice, wheat, maize. It can be used to deliver DNA into plant cells and protoplasts. 10

11. There are two systems of electroporation Low voltage – Long pulses 300-400 V cm -1 for 10-50 ms Produce high rates of transient transformation High voltage – Short pulses 1000-1500 V cm -1 for 10 μ s Produce high rates of stable transformation Transformation frequency can be improve A prior heat shock treatment to protoplast Presence of low conc. PEG (8%) 11

12. Advantages: Both intact cells and tissue can be transformed. The efficiency of transformation depends upon the plant materials Disadvantages ~40 to 50% incubated cells receive DNA ~50% of the transformed cells can survive 12

13. Microinjection A holding pipette holds the protoplast while an injection pipette injects the macromolecule. For manipulation of protoplasts without damage, protoplasts are cultured about 1 to 5 days before the injection. The injection through the partially regenerated cell wall facilitates to target particular compartments of the cell. Particularly useful for transformation of plant protoplasts with exogenous genes. 13

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15. Macroinjection Macroinjection is the method tried for artificial DNA transfer to cereals plants that show inability to regenerate and develop into whole plants from cultured cells. Needles used for injecting DNA are with the diameter greater than cell diameter. DNA injected with conventional syringe into region of plant which will develop into floral tillers 15

16. Advantages This technique does not require protoplast. Instrument is simple and cheap. Methods may prove useful for gene transfer into cereals which do not regenerate from cultured cell easily. Technically simple. Limitations 1. Less specific. 2. Less efficient. 3. Frequency of transformation is very low. 16

17. Biolistic Method Firstly used by Klein et al (1987) & Sanford et al (1987). Also called as, Ballistic method / Gene gun method / Particle bombardment / Particle gun method / Microprojectile Gene gun is developed to enable penetration of the genetic material containing a gene of interest in the cell wall . 1-2µm tungsten or gold particles are used, coated with the DNA. Acceleration is given to enter the micro-projectiles into the plant cells. Acceleration achieved by using a device varies in design & function. 17

18. Devices Pressurized helium gas or 2. The electrostatic energy released by a droplet of water exposed to high voltage. 18

19. Advantages This method can be use to transform all plant species. No binary vector is required. Transformation protocol is relatively simple. Disadvantages Difficulty in obtaining single copy transgenic events. High cost of the equipment and microcarriers . Intracellular target is random (cytoplasm, nucleus, vacuole, plastid, etc.). Transfer DNA is not protected. 19

20. Liposome mediated gene transfer Liposomes are spheres of lipids used to transport molecules into the cells. These are artificial vesicles that can act as delivery agents for exogenous materials including transgenes. They are considered as sphere of lipid bilayers surrounding the molecule to be transported and promote transport after fusing with the cell membrane. 20

21. Cationic lipids are those having a positive charge are used for the transfer of nucleic acid. Liposomes are able to interact with the negatively charged cell membrane more readily than uncharged liposomes Due to fusion between cationic liposome and cell surface results in the delivery of DNA directly across the plasma membrane. Cationic liposomes can be produced from a number of cationic lipids, e.g. DOTAP and DOTMA. These are commercially available lipids that are sold as an in vitr o transfecting agent, as lipofectin . 21

22. Advantages High degree of reproducibility. Long term stability. Low toxicity. Protection of nucleic acid from degradation. 22

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24. PEG mediated gene transfer Polyethylene glycol (PEG), in the presence of divalent cations (using Ca 2+ ), destabilizes the plasma membrane of protoplasts and renders it permeable to naked DNA. In this way, the DNA enters nucleus of the protoplasts and gets integrated with the genome. Culture of protoplasts is taken into a tube and to this tube 40% PEG 4000 (w/v) dissolved in mannitol and calcium nitrate is added slowly. Then incubated for few min. 24

25. Advantages A large number of protoplasts can be simultaneously transformed. Can successfully use for a wide range of plant species. Limitations The DNA is susceptible for degradation and rearrangement. Random integration of foreign DNA into genome may result in undesirable traits. Regeneration of plants from transformed protoplasts is a difficult task. 25

26. Calcium Phosphate co-precipitation The DNA is allowed to mix with calcium chloride solution and isotonic phosphate buffer to form DNA-calcium phosphate precipitate. When the actively dividing cells in culture are exposed to this precipitate for several hours, the cells get transformed. The success of this method is dependent on the high concentration of DNA and the protection of the complex precipitate. Addition of dimethyl sulfoxide (DMSO) increases the efficiency of transformation. 26

27. DNA Imbibition By Cells/Tissues: Some workers have seriously tried to transform cells by incubating cell suspensions, tissues, embryos and even seeds with DNA. The belief is that the DNA gets imbibed, and the cells get transformed. DNA imbibition approach has met with little or no success. 27

28. DMSO polycation It involves use of a polycation , polybrene , to increase the absorption the absorption of DNA to the surface followed by a brief treatment by 25-30% DMSO to increase the membrane permeability and enhance the uptake. The major advantage of polybrene is that it is less toxic than other polycations and a high transformation efficiency requires very small quantities of  plasmid DNA to be used. 28

29. DEAE Dextran The desirable DNA can be complexed with a high molecular weight polymer diethyl amino ethyl (DEAE) dextran and transferred. The efficiency increased to 80% when DMSO shock is given. The major limitation of this approach is that it does not yield stable transformants . 29

30. Thank you... 30