Gene Transfer methods.pptx

Gene transfer methods

1. Genetic T ransformation M ethods Dr. Saraswathi , M.S.c , Ph.D Assistant Professor , Dept. of Life Sciences, Kristu Jayanti College, Autonomous, Bengaluru

2. Genetic transformation Genetic transformation is a powerful tool and a significant strategy for studying plant functional genomics. Gene exploration, new insights into gene regulation and the analysis of genetically regulated characteristics . G enetic engineering allows the insertion of alien genes into crop plants and the accelerated creation of new genetically modified organisms.   Gene transformation and genetic engineering lead to the overall production of crops.

3. Importance of gene transfer technologies to plants i Provide resistance against viruses ii. Acquire insecticidal resistance iii. To strengthen the plant to grow against bacterial diseases iv. Develop the plants to grow in draught v. Engineering plants for nutritional quality vi. Make the plants to grow in various seasons vii. Herbicide resistant plant can be made viii. Resistance against fungal pathogens ix. Engineering of plants for abiotic stress tolerance x. Delayed ripening can be done

4. The process of transfer, integration and expression of transgene in the host cells is known as genetic transformation . A foreign gene ( transgene) encoding the trait must be incorporated into plant cells, along with a "cassette " of extra genetic material to add a desirable trait to a crop. Various genetic transfer techniques are grouped into two main categories . 1) Vector mediated gene transfer (Indirect method) 2) Vector less gene transfer (Direct method) Gene transfer technologies in plants

5. Plant transformation technique using Agrobacterium

6. In general, most of the Agrobacterium-mediated plant transformations have the following basic protocol: 1. Development of Agrobacterium carrying the co-integrate or binary vector with the desired gene 2. Identification of a suitable explant e.g. cells, protoplasts, tissues, calluses , organs 3. Co-culture of explants with Agrobacterium 4. Killing of Agrobacterium with a suitable antibiotic without harming the plant tissue 5. Selection of transformed plant cells 6. Regeneration of whole plants

7. i . It is a natural means of gene transfer ii. Agrobacterium is capable of infecting plant cells and tissue and organs iii. Agrobacterium is capable of transfer of large fragments of DNA very efficiently iv. Integration of T DNA is a relative precise process v. The stability of gene transferred in excellent vi. Transformed plants can be regenerated effectively Advantages

8. Limitations i . Host specificity: There is a limitation of host plants for Agrobacterium, since many crop plants (monocotyledons e.g. cereals ) are not infected by it. In recent years, virulent strains of Agrobacterium that can infect a wide range of plants have been developed ii. Inability to transfer multiple genes: The cells that regenerate more efficiently are often difficult to transform, e.g. embryonic cells lie in deep layers which are not easy targets for Agrobacterium iii. Somaclonal variation iv. Slow regeneration

9. Particle Bombardment/ M icroprojectile /Biolistic/Gene G un Particle bombardment is a technique used to introduce foreign DNA into plant cells Particle (or micro projectile) Bombardment is the most effective method for gene transfer, and creation of transgenic plants. This method is versatile due to the fact that it can be successfully used for the DNA transfer in mammalian cells and microorganisms .

10. The process of transformation employs foreign DNA coated with minute 0.2-0.7 μm gold (or) are tungsten particles to deliver into target plant cells. The coated particles are loaded into a particle gun and accelerated to high speed-pressurized helium gas. By electro static energy released by a droplet of water exposed to a high voltage. In order to protect plant tissues from being damaged by bombardment, cultures are maintained on high osmoticum media or subjected to limited plasmolysis .

11. Factors affecting bombardment i Nature of micro particles: Inert metals such as tungsten, gold and platinum are used as micro particles to carry DNA. These particles with relatively higher mass will have a better chance to move fast when bombarded and penetrate the tissues. ii) Nature of tissues/cells: The target cells that are capable of undergoing division are suitable for transformation . iii) Amount of DNA: The transformation may be low when too little DNA is used. On the other hand, too much DNA may result is high copy number and rearrangement of transgenes.

12. Plant material used in bombardment: 1. Primary explants which can be subjected to bombardment that are subsequently induced to become embryo genic and regenerate 2. Proliferating embryonic tissues that can be bombarded in cultures and then allowed to proliferate and regenerate

13. Plant transformation process using particle bombardment

14. Advantages of particle bombardment Gene transfer can be efficiently done in organized tissues Different species of plants can be used to develop transgenic plants. Limitations of particle bombardment I nstability of transgene expression due to gene silencing The target tissue may often get damaged due to lack of control of bombardment velocity Sometimes , undesirable chimeric plants may be regenerated

15. Electroporation Electroporation is the incorporation of DNA into the cell by exposing them to high voltage electrical pulses for a very short period of time to cause temporary pores in the plasma lemma . Plant cell electroporation generally uses protoplast, while thick plant cell walls restrict the movement of macromolecule. The plant material is incubated in a buffer solution containing the desired foreign/target DNA, and subjected to high voltage electrical impulses.

16. Electroporation

17. After entry into the cell, the Foreign DNA gets incorporated with the host genome. Resulting the genetic transformation the protoplasts are then cultured to regenerate in to whole plants. This method can be used in those crop species in which regeneration from protoplast is possible . In the early years, only protoplasts were used for gene transfer by electroporation . Now a day, intact cells, callus cultures and immature embryos can be used with suitable pre-and post-electroporation treatments.

18. Electroporation has been successfully used for the production of transgenic plants of many cereals e.g. rice, wheat, maize . (1) Isolate protoplasts from leaf tissues . (2) Inject DNA-coated particles into the protoplasts using particle gun . (3) Regenerate into whole plants. (4) Acclimate the transgenic plants in a greenhouse

19. Advantages of electroporation This technique is simple, convenient and rapid, besides being cost effective The transformed cells are at the same physiological state after electroporation. Efficiency of transformation can be improved by optimising the electrical field strength .

20. Limitations of electroporation Under normal conditions, the amount of DNA delivered into plant cells is very low Efficiency of electroporation is highly variable depending on the plant material and the treatment conditions Regeneration of plants is not very easy, particularly when protoplasts are used

21. Chemical gene transfer methods Polyethylene glycol (PEG), in the presence of divalent cations ( using Ca2 +), destabilizes the plasma membrane of protoplasts and renders it permeable to naked DNA. The DNA enters nucleus of the protoplasts and gets integrated with the genome. The procedure involves the isolation of protoplasts and their suspension, addition of plasmid DNA, followed by a slow addition of 40% PEG-4000 (w/v) dissolved in mannitol and calcium nitrate solution . As this mixture is incubated, protoplasts get transformed .

22. The naked plant protoplasts are mixed with molecules of linearized plasmid DNA containing the foreign gene. The two are mixed in a transformation medium rich in Mg2+ ions in place of Ca2+ ions, following which 20% polyethylene glycol (PEG) solution is added . After the treatment, the PEG concentration is reduced and Ca2+ concentration is enhanced. It promotes the frequency of transformation .

23. Advantages of PEG-mediated transformation i ) A large number of protoplasts can be simultaneously transformed ii) This technique can be successfully used for a wide range of plant species Limitations of PEG-mediated transformation i ) The DNA is susceptible for degradation and rearrangement ii) Random integration of foreign DNA into genome may result in undesirable traits iii) Regeneration of plants from transformed protoplasts is a difficult task. . Sometimes the foreign DNA is degraded in the cytoplasm before reaching the nucleus

24. DEAE dextran-mediated transfer 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 trans-formants.

25. Calcium phosphate co-precipitation-mediated transfer   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. Applications of Transgenic Plants The six applications are: ( 1) Resistance to Biotic Stresses ( 2) Resistance to Abiotic Stresses ( 3) Improvement of Crop Yield and Quality ( 4) Transgenic Plants with Improved Nutrition ( 5) Commercial Transgenic Crop Plants and ( 6) Transgenic Plants as Bioreactors.

27. i . Resistance to biotic stresses i.e. resistance to diseases caused by insects, viruses, fungi and bacteria . ii. Resistance to abiotic stresses-herbicides, temperature (heat, chilling, freezing), drought, salinity, ozone, intense light. iii. Improvement of crop yield, and quality e.g. storage, longer shelf life of fruits and flowers. iv. Transgenic plants with improved nutrition. v. Transgenic plants as bioreactors for the manufacture of commercial products e.g. proteins, vaccines, and biodegradable plastics.

28. Almost all the stresses, either directly or indirectly, lead to the production of reactive oxygen species (ROS) that create oxidative stress to plants. This damages the cellular constituents of plants which is associated with a reduction in plant yield . Bacillus thuringiensis ( Bt ) toxin:

29. Resistance to diseases (insect, microorganisms). ii. Improved nitrogen fixing ability. iii. Higher yielding capacity. iv. Resistance to drought and soil salinity. v. Better nutritional properties. vi. Improved storage qualities. vii. Production of pharmaceutically important compounds.

30. Delay in fruit ripening has many advantages: i . It extends the shelf-life, keeping the quality of the fruit intact. ii. Long distance transport becomes easy without damage to fruit. iii. Slow ripening improves the flavour.

31. Transgenic Plants with Improved Nutrition: Amino Acids of Seed Storage Proteins: Golden Rice —The Provitamin A Enriched Rice : Commercial Transgenic Crop Plants: