DR. PRERNA SONI

1. Production Of Transgenic Tomato Plant Through Agrobacterium Mediated Transformation Using Green Fluorescent Protein DR. PRERNA SONI ASSISTANT PROFESSOR BIOTECHNOLOGY SPCAS COLLEGE NAWAPARA RAIPUR CHHATTISGARH

2. INTRODUCTION Tomato History How to genetically modify a plant Agrobacterium mediated gene transfer methodology The process of gene transfer from Agrobacterium to plant cells implies several essential steps :- Bacterial colonization Induction of bacterial virulence system Generation of T-DNA transfer complex T-DNA transfer Integration of T-DNA into plant genome.

3. Callus Growing Plant in Culture Media Modified Plant

4. The Fluorescent ProteinGreen Fluorescent Protein (GFP) OOH Aequorin It is discovered by Shimomura in 1962. It is derived from Jellyfish Aequoreavictoria It is a bioluminescent protein Coelenterazine 3Ca2+ Ca3 – Aequorin - Coelenteramide GFP Ca3 – Aequorin – Coelenteramide + CO2 Invitro Green lightλ max = 508 nm Invitro Blue lightλ max = 470 nm

5. EXPLORING THE STRUCTURE 3D GFP FLUORESCENCE IN JELLYFISH 3D IMAGE OF A.victoria’s GFP

6. The structure of GFP was solved in 1996. • It has a novel three dimensional structure called  can • It contains a chromophore which is biosynthetically created between the amino acid residues of 65-67 modified Ser-Tyr-Gly sequence of the GFP protein. • This chromophore absorbs blue light & allows the emission of the green light

7. Expression of GFP in Tomato and Wheat root Tomato plant of the variety "Minitom" (left) and colonisation of the root by the gfp-labelled bacterium Serratialiquefaciens MG1 (middle). The labelled bacterium shows intensive green fluorescence. Right: Azospirillumbrasilense Sp7-cells with activated ipdC-gfp-reporter construct (arrows) on the surface of wheat roots.

8. Timeline of Plant Biotechnology 1700s-- Naturalists identify hybrid plants. 1860s -- Austrian botanist and monk Gregor Mendel studies pea plants and recognizes that specific traits are passed from parents to offspring - these traits are eventually discovered to be genes. 1900 -- European botanists begin to improve plant productivity using genetic theories based on Mendel's work. 1922 -- Farmers purchase hybrid seed corn created by crossbreeding two corn varieties. 1953 -- Structure of DNA is discovered - marking the beginning of modern genetic research. 1970s -- Hybrid seeds are introduced to developing countries to increase food supplies. 1973 -- Genetic engineering is used to precisely manipulate bacterial DNA. 1983 -- First GM plant is created; a tobacco plant resistant to an antibiotic. 1985 -- GM plants resistant to viruses, bacteria, and insects are field tested. 1986 -- EPA approves the release of the first GM crop (herbicide resistant tobacco). 1990 -- First successful field trial of GM cotton (herbicide resistant). 1992 -- FDA decides GM foods will be regulated as conventional foods. 1994 – FlavrSavr Tomato becomes the first GM food to be approved for sale. 1995 -- Herbicide resistant canola, corn, 2000 -- Cotton, soybeans, sugar beet as well as insect or virus resistant corn, cotton, papaya, potato, squash, tomato approved in the U.S. 2001 -- "Golden rice" which may help prevent millions of cases of blindness and death caused by Vitamin A and iron deficiencies undergoes continued testing.

9. MATERIALS AND METHODS Plasmid Isolation (pGPTV, pUC-GFP) Plasmid isolation involves four steps – I. Harvesting II. Alkali lysis III. Plasmid precipitation IV. Purification Agarose gel electrophoreusis I. Preparation of gel II. Loading of samples III. Passing of electric current.

10. Interesting, right? This is just a sneak preview of the full presentation. We hope you like it! To see the rest of it, just click here to view it in full on PowerShow.com. Then, if you’d like, you can also log in to PowerShow.com to download the entire presentation for free.