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PARTICLE BOMBARDMENT IN RICE PLANT .pptx
- 1. PARTICLE BOMBARDMENT IN RICE ANANTHU S B 2020-44-010
- 2. Particle Bombardment Particle bombardment is the most important and most effective direct gene transfer method in regular use. In this technique, tungsten or gold particles are coated with the DNA that is to be used to transform the plant tissue.
- 3. Biolistic transformation of rice • PDS-1000/He system • 2 plasmids • pOZ (5000bp) – carries transgene of interest • pHAG (9040bp) – carries the selectable marker and reporter gene • Microcarriers • Plant material for transformation
- 5. Macrocarrier Rupture disc Macrocarrier Holder Stopping screen MATERIALS AND INSTRUMENTATION
- 6. Preparation of Microcarriers • Pre-treatment of beads with ethanol and distilled water • Attachment of Plasmid DNA to microcarriers • 2.5mol/l calcium chloride • 0.1mol/l spermidine • DNA • Microcarriers • Application of prepared micro carries to macro carrier membrane
- 7. Preparation of plant material • Sterilization using ethanol and distilled water • Cultured in MS media of 2.5g/l phytagel, supplemented with 2.5mg/l 2,4-D and 30g/l maltose • Cultures are incubated in dark for 2 weeks at 25C • These are then transferred to MS media with 1mg/l 2,4-D, 14% w/v maltose, 2.5g/l phytagel • Further incubated for 4hrs
Editor's Notes
- PDS-1000/He™ System The PDS-1000/He system accelerates nucleic acid–coated gold or tungsten microparticles (0.6–1.6 µm) to velocities necessary to transfect cells, tissues, or organelles. The system uses a burst of high-pressure helium gas to accelerate a plastic macro carrier disk carrying microparticles toward target cells. The helium pressure used to propel the macro carrier is determined by the choice of rupture disk, a plastic seal designed to burst at a specific pressure. A stopping screen retains the macro carrier while allowing the microparticles to pass through and penetrate the target cells. To increase the efficiency of the process, the chamber may be evacuated to sub-atmospheric pressures, reducing the frictional drag on the microparticles as they travel toward the target cells The plasmids used in this experiment are of e-coli origin. pOZ gene carries the gene of interest, but lacks a selectable marker pHAG carries the selectable marker, hyg and reporter gene, gusA hyg – confers resistance to the antibiotic hygromycin gusA – can be assayed histochemically
- pOZ (5000bp) – carries transgene of interest pHAG (9040bp) – carries the selectable marker and reporter gene Plant material for transformation Microcarriers Gold or Tungsten particle Suspensions of microcarriers can be stored as aliquots at 40c for gold and at -200c for tungsten
- pOZ (5000bp) – carries transgene of interest pHAG (9040bp) – carries the selectable marker and reporter gene Plant material for transformation
- Macro carries are then applied to the macro carrier membrane in an ethanol suspension and are allowed to dry on the membrane
- After washing with ethanol, distilled/ sterile water is used to remove any trace of ethanol form the seeds. MS media stands for Murashige and Skoog medium, the major Plant growth media used in tissue culturings Phytagel is a solidifying agent used while preparing the gel for culturing