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Methods of Gene Transfermation

The u201csharingu201d of DNA among living forms is well<br>documented as a natural phenomenon.<br>uf0ae For thousands of years, genes have moved<br>from one organism to another.<br>uf0ae For example, Agrobacterium tumefaciens, a soil<br>a bacterium is known as 'nature's own genetic<br>engineer', has the natural ability to genetically<br>engineer plants.<br>Insertion of copies of a gene into living cells in order to<br>induce synthes is of the gene's product.<br>Why Gene transfer?<br>u2756 To add a desired trait to a crop, a foreign gene<br>(transgene) encoding the trait must be inserted into<br>plant cells, along with a u201ccassetteu201d of additional<br>genetic material. The cassette includes a DNA<br>sequence called a u201cpromoter,u201d which determines<br>where and when the foreign.<br>u2756 Gene is expressed in the host, and a u201cmarker geneu201d<br>that allows breeders to determine which plants<br>contain the inserted gene by screening or selection.

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Methods of Gene Transfermation

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  1. METHODS OF GENE TRANSFERIN PLANTS Dr. Thirunahari Ugandhar Assistant Professor in Botany Govt Degree College Mahabubabad

  2. Sufficient, Nutritionally Adequate & Culturally Acceptable Food for an Active, Healthy Life. 5/20/2020 3

  3. Introduction  The “sharing” of DNA among living forms is well documented as a natural phenomenon.  For thousands of years, genes have moved from one organism to another.  For example, Agrobacterium tumefaciens, a soil bacterium known as 'nature's own genetic engineer', has the natural ability to genetically engineer plants.

  4. 5/20/2020 5

  5. Conventional breeding Plants Tissue culture Genetic engineering 5/20/2020 6

  6. What What is is Gene Genetransfer transfer Insertion of copies of a gene into living cells in order to induce synthes is of the gene's product. Why Genetransfer? ❖ To add a desired trait to a crop, a foreign gene (transgene) encoding the trait must be inserted into plant cells, along with a “cassette” of genetic material. The cassette includes a DNA sequence called a “promoter,” which determines where and when the foreign. ❖ Gene is expressed in the host, and a “marker gene” that allows breeders to determine which plants contain the inserted gene by screening or selection. additional

  7. Why genetically Engineer Plants? ➢To improve the agricultural, horticultural or ornamental value of a crop plant. ➢To serve as a living bioreactor for the production of economically proteins or metabolites ➢To provide a renewable source of energy To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes important

  8. Desired Traits Pest resistance Herbicide tolerance Viral resistance Drought resistance Increased nutritional value Improved Fruit Altered ripening

  9. Genetic Engineering Genetic engineering, also called genetic modification, is the direct manipulation of an organism's Genome using Biotechnology New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using Molecular Cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism.

  10. Engineer  An Engineer is a Professional Practitioner of Engineering, concerned Scientific Knowledge and ingenuity to develop solutions for technical problems.  Engineers design materials, structures, and systems while considering imposed by practicality, regulation, safety, and cost.  The word Engineer is derived from the Latin roots ingeniare ("to contrive, devise") and ingenium ("cleverness") with applying the limitations

  11. TRANSGENIC PLANTS ✓The process of introducing new genes into plants to produce new traits is called Plant Genetic Engineering. ✓The product of this process is called Transgenic Plants. Transgenic plants contain a gene or genes which have been artificially inserted. The inserted genes are known as transgenic. 5/20/2020 12

  12. TRANSGENIC PLANTS – AN INTRODUCTION ❖ Transformation – The process of obtaining transgenic plants. ❖ Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome ❖ Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants. ❖ Most common genes (and traits) in transgenic or biotech crops herbicide resistance Insecticide resistance, Bt genes in field corn (maize) virus-resistance (coat-protein) genes Agrobacterium and plants,

  13. WHY HAVE GM CROPS? Growing Human Population Loss of Farmable Land Remediation of Soil Enrich Nutrient Content Transgenic Wild-type

  14. Plant Transformation Methods In planta Chemical Biological Physical A. Tumefaciens A. Rhizogenes Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Microinjection Silica/carbon fibers Lazer mediated SAT PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers Virus-mediated 5/20/2020 15

  15. Genetic Transformation • PLANTS - PHYSICAL METHODS • Microinjection • Electroporation • Biolistics - gene gun • Silica/carbon fibers • Lazer mediated • SAT 5/20/2020 18

  16. 1. MICROINJECTION 5/20/2020 19

  17. 2. Electroporation This electroporator is for low-current applications such as those using small electrodes 5/20/2020 20

  18. 3. Biolistic / Gene Gun 5/20/2020 21

  19. 3. Biolistic / Gene Gun

  20. 4. SILICA/CARBON FIBERS

  21. Streptothricin Acetyl Transferase (SAT) T R A N S F O R M A T I O N 5/20/2020 25

  22. Chemical Gene Transfer 1. PEG 2. DEAE-dextran 3. Calcium phosphate 4. Artificial lipids 5. Proteins 6. Dendrimers

  23. 1. PEG Gene Transfer

  24. 2. Dead Dextran Mediated Gene Transfer

  25. 3. Calcium phosphate Transfer

  26. Artificial lipoplex Mediated Gene Transfer

  27. Biological Transformation ❑ Agrobacterium tumefaciens & ❑ Agrobacterium rhizogenes 5/20/2020 32

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  29. BIOLOGICAL TRANSFORMATION Possible plant compounds, that initiate Agrobacterium to infect plant cells. Acetosyringone, ferulic acid, gallic acid, Hydroxybenzoic acid, pyrogallic acid, vanillin etc. In monocot – not efficient Transformation frequencey – very less 5/20/2020 34

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  31. Agrobacterium Agrobacterium (disease symptomology and host range) A. radiobacter - “avirulent” species A. tumefaciens - crown gall disease A. rhizogenes - hairy root disease A. rubi - cane gall disease A. vitis - galls on grape and a few other plant species Otten et al., 1984 5/20/2020 36

  32. Cellular process of Agrobacterium–host interaction Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121 5/20/2020 37

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  36. Plant Transformation Methods Virus-mediated gene transfer (Plant viruses as vectors) Caulimo viruses – ds DNA – CaMV Gemini viruses - 2ss DNA – maize streak virus RNA plant viruses - TMV 5/20/2020 41

  37. POLYMERASE CHAIN REACTION PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA. Kary Banks Mullis (Born December 28, 1944) is a Nobel Prize -winning American Biochemist, author, and lecturer. In recognition of his improvement of the Polymerase Chain Reaction (PCR) technique

  38. PCR Animation Denaturation: DNA melts Annealing: Primers bind Extension: DNA is replicated

  39. Recombinant DNA & Plasmids Combining genes from different sources and/or species  Circular DNA from bacteria = plasmids  Target DNA recombined in plasmid  Bacteria rapidly reproduces many clones

  40. Gene transfer techniques… 46

  41. Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants 5/20/2020 47

  42. PLANT TRANSFORMATION • Plants are the easiest of higher organisms to transform • Both physical and biological methods exist for transformation • Until recently, only transgenic organisms in wide public release were plants 5/20/2020 48

  43. Which foods contain GM Product? India Approval for GM food crops •Corn •Soy •Papaya •Canola •Potato •Chicory •Rice •Squash •Sugarbeet •Tomatoes Approval does not necessarily mean these crops are distributed Database of GM crops:

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