Vector mediated gene transfer methods for transgenesis in Plants.

Presentation include Vector mediated gene transfer methods for trans-genesis in Plants. Only Vector-based methods are covered. Vectors includes Bacteria, Viruses, transposable genetic elements. Other possible vectors for transgenesis are also covered.

1. Vector Mediated Gene Transfer Methods for Transgenesis in Plants - Akshay More, M.Sc . Biotechnology-1., Modern College, Ganeshkhind, Pune .

2. OUTLINE Vector mediated gene transfers: A. Bacteria. B. Viruses. C . Transposable Genetic Elements. D. Other Possible Vectors .

3. A) Bacteria : Agrobacterium (Nature’s little genetic engineer) - Genus include : A. tumefaciens, A. rhizogenes, A. radiobacter, A. rubi . Gram Negative, Soil Bacteria. Infect plant and causes cancerous growth in tissue. A. rubi has narrow host range as compared to others. cancerous growth is because of plasmi d. Agrobacterium tumefaciens: - Almost all strains have Ti Plasmid Ti Plasmid- → Large ds circular DNA, size- 150-230 kbp., M.W.-(120-160 MD). → Denaturation temp. is above 37 ° C , loses its tumorigenic property. → Has region encoding unusual amino acids like opine. → Has region for synthesis of opines i n its catabolism . → Has sequence for conjunctive transfer of plasmid. → Has sequence for virulence (T-DNA) and replication .

4. General Structure of Ti Plasmid

5. Types: a) Octopine: - Synthesized from Arginine. -Molecular formula is C 9 H 18 N 4 O 4 -e.g. pTi-B6, pTi-ACH b) Nopaline: -Molecular formula is C 9 H 16 N 4 O 6 e.g. pTi- C58

6. Overview of infection Process Source: http ://www.nature.com/nature/journal/v433/n7026/images/433585a-f2.2.jpg

7. Gene transfer through Ti plasmid: Desired foreign gene is isolated An intermediate vector plasmid (pBR322) is constructed using Col E1 plasmid Ti plasmid is isolated from Agrobacterium & T-DNA is separated. T-DNA +pBR 322 = Shuttle Vector (has strong promoter, replicates both in E. coli & Agrobacterium T-DNA is then cut with restriction enzymes and then prepared foreign DNA is inserted into T-DNA. Chimeric DNA is then inserted into E. coli (Competent cell preparation). Transformed E.coli + Agrobacterium , incubation, bacterial conjugation, transfer of plasmid to Agrobacterium.

8. In Agrobacterium , chimeric DNA undergoes homologous recombination with Ti Plasmid, Agrobacterium transformed. Selective screening, and selected transformed bacteria used for transgenesis. Methods for obtaining Agrobacterium transformed plant tissue : 1) Co-cultivation - protoplasts +transformed Agrobacterium , plants cells grown as callus. 2) Leaf-disk method - Leaf disk +transformed Agrobacterium , plants cells grown as callus. 3) Agroinfiltration —forcing Agrobacterium with transgenes into leaves

9. Results of gene transfer plants- Strain of Salmonella grows vigorously in media having glyophosphate. So gene is isolated from Salmonella and insert into plant Arabidopsis through Agrobacterium. Resulting plant show resistance to glyophosphate . Nif- genes are transferred to plants for nitrogen fixation, but has not given beneficial results. Kemp (1981) transferred gene for phaseolin synthesis from beans to the cells of sunflower . The transformed cells produced mRNA of phaseolin in them. The United States Department of Agriculture transferred a gene for a storage protein of maize into the cells of Sunflower . Transformed cells produced desired storage protein of maize in them.

10. B) Virus Mediated Gene Transfer: - Viral genome doesn’t integrate into plant genome . -Vectors are episomal vectors, therefore they have high copy number per cell. -The gene product is very rapidly accumulated. -Viral genome sequences are excellent source of promoters, enhancers and other components useful for designing gene vectors. -Virus is systemically spread in the plant body. -Generally have wide host range . Most Notables: 1) Caulimovirus based vectors. 2) Gemini virus based vectors. 1)CaMV based vectors.

11. Characteristics: -dS DNA virus. Packaged as nucleosome. -Mechanical and aphid mediated transmission -Virion DNA alone or cloned CaMV DNA is infectious when simply rubbed on leaves -Up to 10 6 copies per cell. 3-4 weeks for systemic infection through plant.

12. Two regions of the CaMV genome— open reading frames (ORFs) II and VII—do not seem to be essential for infection, as both can be either deleted or expanded by small inserts of foreign DNA . Results of gene transfer plants- Brisson et al. (1984) – transferred CaMV vector containing the bacterial dihydrofolate reductase gene, into turnip plant cells gives resistance to methotrexate. 2) Gemini virus based vectors

13. Characteristics: - Genome size is 2.6- 3.0 kb. - ssDNA, can cause diseases to cereals, cassava, tomato, Digitaria. Has geminated morphology, hence name. Capsids are icosahedral, dimensions= 10-20*20 nm. Most have insect mediated transmission . No mechanical transmission . Replicated by rolling circle mechanism .

14. *Gene transfer strategy: pWDV2 V2 deletion vector Results of gene transfer plants- Use of vectors based on the gemini tomato golden mosaic virus (TGMV) to introduce the neomycin phosphotransferase ( neo gene) into cereals, cassava plants . {*Source: Nature 334 , 179 - 182 (14 July 1988 )}

15. C. Transposable Genetic Elements. Structurally and genetically discrete segments of DNA capable of moving from one position in genome to other. First discovered by Barbara McClintok (1940) in Zea mays , jumping genes. Insertion of transposon interfere with expression of that gene(translation). E.g. Ac-Ds elements in maize, Insertion Elements, Tn Family, Maize Elements, Ty elements in Yeast, Copia Elements.

16. “Cis” and “Trans” Transposon Mediated Gene Delivery “ Cis” and “Trans” transposon mediated gene delivery. GOI, gene of interest; 5’TR, 5’ terminal repeat; 3’TR, 3’terminal repeat; the yellow and beige arrows indicate promoters to drive gene expression.

17. * Copia Elements- -Found in Drosophila . -e.g. FB ( F old Back), P elements. P elements- Variable size, largest element is 1907 bp long having 31 long terminal inverted repeats. Foreign DNA can be inserted into transposase element to develop recombinant P element. Recombinant P element then microinjected into fertilized egg along with normal P element. Result of gene transfer- - Gierl et al (1989) prepared recombinant P element by inserting DNA fragment of rosy gene. R ecombinant P element then inserted into plasmid along with normal P element. Both plasmids were co-injected into Drosophila embryos. 50 % progeny were rosy progeny.

18. D) Other Possible Vectors 1) Maize Mitochondrial Elements: Studies on Cytoplasmic Male Sterility have reveal several of these elements. Unlike Transposons, they replicate autonomously in mitochondria and integrate into mitochondrial genome. Size around 1-4 kb. Can be act as vector for gene transfer. 2) Nuclear Genomic Components: Constructed from yeast chromosomal DNA, integrate into host by homologous recombination. E.g. YAC, YIC. High success in mammals. Plants lack homologous recombination system, so limited success.

19. 3) Viroids : Smallest and simplest pathogenic agents. Small, 300-400 bases long, circular, single stranded having naked RNA. These non-protein coding viroids replicate in host using host’s replicative enzyme probably RNA Pol II. They are mechanically transmissible, able to move throughout the sap and infect other parts of plant. They are certainly associated with nucleus and may replicate in it. Can be act as vector for gene transfer.

20. References: R. C. Dubey, A textbook of Biotechnology,2006, first edition, S. Chand and Company Ltd. Chapter 5. Sunandan Saha and Matthew H. Wilson, chapter 11, Gene Therapy - Tools and Potential Applications, Intech , http:// dx.doi.org/10.5772/52527 . Kjemtrup S, Sampson KS, Peele C, Nguyen LV, Conkling MA, Thompson WF and Robertson D (1998). Gene silencing from plant DNA carried by a geminivirus. The Plant Journal, 14, 91-100 . Darbani B., Eimanifar A., Stewart C. N., and Camargo W., Methods to produce marker‐free transgenic plants, Biotechnology Journal. Vol. 2,2007, 83‐90. S. Ignacimuthu,sii, Plant Biotechnology, 2001, Oxford and IBH Publishing Co. Pvt. Ltd.

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