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AGROBATERIUM MEDIATED GENE TRANSFER

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AGROBATERIUM MEDIATED GENE TRANSFER

  1. 1. Genetic Engineering Kuldeep Gauliya Research Scholar, Dept. of Biotechnology DHSGU, Sagar, M.P. Altering God’s Creation…. Lecture I – Introduction to Agrobacterium Nov. 18, 2022
  2. 2. What are TRANSGENIC PLANTS ?  “A transgenic plant is a modified organism where genes are transferred from one organism to another through genetic engineering techniques”.  This may involve changing a single base pair (A-T or C-G), deleting a region of DNA or adding a new segment of DNA 28 November 2022 2
  3. 3. What is Genetic Engineering ? Genetic engineering (also called genetic modification) is a process that uses laboratory-based technologies to alter the DNA makeup of an organism. 28 November 2022 3
  4. 4. Methods of production of Transgenic plants  Microprojectile bombardment: shooting DNA-coated tungsten or gold particles into plant cells.  Electroporation: use of a short burst of electricity to put the DNA into cells.  Agrobacterium tumefaciens-mediated transformation. 28 November 2022 4
  5. 5. What is Agrobacterium?  Agrobacterium is a soil pathogenic bacterium. This plant pathogen causes crown-gall disease or hairy root disease in infected dicotyledonous plants.  The galls or tumors are formed at the junction between the root and the stem of infected plants. 28 November 2022 5
  6. 6. How does Crown Gall Disease looks like ? 28 November 2022 6
  7. 7. What is Agrobacterium-mediated transformation ? Agrobacterium-mediated transformation is a process of using Agrobacterium to transfer a gene of interest into the plant cells, generating transgenic plants. 28 November 2022 7
  8. 8. Why is Agrobacterium used to make transgenic plants? • Agrobacterium is a useful tool bcz it can carry, transfer, and integrate a gene of interest into the plant genome. • In the development of transgenic plants, this system allows plants to stably harbor and pass a particular gene of interest to the next generations relatively quicker than by using the more traditional plant breeding method. • This method is relatively inexpensive and easy to perform. In addition, it provides convenient way to screen and select the transformed plant tissues. 28 November 2022 8
  9. 9. 1. The ability of A. tumefaciens to induce crown galls in plants is controlled by genetic information carried on Ti plasmid (tumor-inducing plasmid). 2. Ti plasmid has two components the T-DNA (Transferred DNA) and 3. the vir region, which are essential for the transformation of plant cells. 4. During the transformation process, the T-DNA is excised from the Ti plasmid, transferred to a plant cell, and integrated into the plant cell genome. 28 November 2022 9 How does Transformation occurs…?
  10. 10. 28 November 2022 10 Fig. shows the Plant -Agrobacterium Interaction. It depicts how does the T-DNA gets incorporated into the plant cell that ultimately causes Crown Gall Disease.
  11. 11. Structure of Ti Plasmid 1. T-DNA: In nopaline-type Ti plasmids the T-DNA is a 23,000- nucleotide-pair segment. 2. It carries 13 known genes including genes encoding enzymes that catalyze the synthesis of phytohormones (the auxin indoleacetic acid and the cytokinin isopentenyl adenosine). 3. These phytohormones are responsible for the tumorous growth of cells in crown galls. 4. The T-DNA region is bordered by 25-nucleotide-pair imperfect repeats. 5. One of this must be present in cis for T-DNA excision and transfer. 28 November 2022 11
  12. 12. Fig. Structure of the nopaline Ti plasmid pTi C58, showing ori, origin of replication. 1. Tum, genes responsible for tumor formation; 2. Nos, genes involved in nopaline biosynthesis; 3. Noc, genes involved in the catabolism of nopaline; 4. vir, virulence genes required for T-DNA transfer. 28 November 2022 12
  13. 13. Structure of Ti Plasmid: The vir (virulence) region • It contains the genes required for the T-DNA transfer process. • These genes encode the DNA processing enzymes required for excision, transfer, and integration of the T-DNA segment. • They are expressed at very low levels in A. tumefaciens cells growing in soil. • Exposure of the bacteria to wounded plant cells or exudates from plant cells induces enhanced levels of expression of the vir genes. • This induction process is very slow for bacteria, taking 10 to 15 hours to reach maximum levels of expression. • Phenolic compounds such as acetosyringone act as inducers of the vir genes. 28 November 2022 13
  14. 14. 28 November 2022 14 Fig. Structure of Ti Plasmid Vector.
  15. 15. 28 November 2022 15 Fig. Showing the Agrobacterium mediated gene Transformation
  16. 16. Ti plasmid vector for creating transgenic plants • Foreign genes could be inserted into the T-DNA and then transferred to the plant. • In the modified Ti plasmid the genes responsible for tumor formation are deleted. • Selectable markers added along with appropriate regulatory elements. 28 November 2022 16
  17. 17. Ti plasmid vector for creating transgenic plants • The kanr gene from the E. coli transposon Tn5 has been used extensively as a selectable marker. • It encodes an enzyme called neomycin phosphotransferase type II (NPTII), it detoxify the kanamycin. • The NPTII coding sequence are provided with a plant promoter and plant termination and polyadenylation signals. • Such constructions with prokaryotic coding sequences flanked by eukaryotic regulatory sequences are called chimeric selectable marker genes. 28 November 2022 17
  18. 18. 1. One widely used Chimeric selectable marker gene contains the cauliflower mosaic virus (CaMV) 35S promoter, the NPTII coding sequence, and the Ti nopaline synthase (nos) termination sequence; this chimeric gene is usually symbolized 35S/NPTII/nos. 2. The Ti vectors used to transfer genes into plants have the of the plasmid replaced with a chimeric selectable tumor- inducing genes marker gene such as 35S/NPTII/nos. 28 November 2022 18 Ti plasmid vector for creating transgenic plants
  19. 19. 28 November 2022 19
  20. 20. 28 November 2022 20
  21. 21. Ti plasmid-based vectors: Components 1. A selectable marker gene, such as neomycin phosphotransferase, put under the control of plant (eukaryotic) transcriptional regulation signals, including both a promoter and a termination–polyadenylation sequence. 2. An origin of DNA replication that allows the plasmid to replicate in E. coli . 3. A polylinker to facilitate insertion of the cloned gene into the region between T- DNA border sequences. 4. A “killer” gene encoding a toxin downstream from the left border to prevent unwanted vector DNA past the left border from being incorporated into transgenic plants. 5. If this incorporation occurs, and the killer gene is present, the transformed cells will not survive. 28 November 2022 21
  22. 22. Binary vector system 1. The binary cloning vector contains either the E. coli and A. tumefaciens origins of DNA replication or a single broad host range origin of DNA replication. 2. All the cloning steps are carried out in E. coli before the vector is introduced into A. tumefaciens. 3. The recipient A. tumefaciens strain carries a modified (defective or disarmed) Ti plasmid that contains a complete set of vir genes but lacks the T-DNA region, so that this T-DNA cannot be transferred. 4. With this system, the defective Ti plasmid synthesizes the vir gene products and acts as a helper plasmid. 5. This enables the T-DNA from the binary cloning vector to be inserted into the plant chromosomal DNA. 6. Since transfer of the T-DNA is initiated from the right border, the selectable marker, is usually placed next to the left border. 22
  23. 23. Binary vector system 28 November 2022 23
  24. 24. Limitations as routine Ti plasmid vectors 1. The production of phytohormones by transformed cells prevents them from being regenerated into mature plants. 2. A gene encoding opine synthesis is not useful to a transgenic plant and may lower the final plant yield. 3. Ti plasmids are large (approximately 200 to 800 kb). 4. Ti plasmid does not replicate in Escherichia coli, therefore it cannot be cloned in E. coli. 5. Transfer of the T-DNA, which begins from the right border, does not always end at the left border. Rather, vector DNA sequences past the left border are often transferred. 28 November 2022 24

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