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  Section: Biotechnology Methods » Tissue Culture Techniques
 
 
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Agrobacterium Culture and Agrobacterium — Mediated transformation

 
     
 
Content
Tissue Culture Techniques
  Tissue Culture Methods
  Plant Tissue Culture
  Plant Tissue Culture (Cont.)
  Many Dimensions of Plant Tissue Culture Research
  What is Plant Tissue Culture?
  Uses of Plant Tissue Culture
  Plant Tissue Culture demonstration by Using Somaclonal Variation to Select for Disease Resistance
  Demonstration of Tissue Culture for Teaching
  Preparation of Plant Tissue Culture Media
  Plant Tissue Culture Media
  Preparation of Protoplasts
  Protoplast Isolation, Culture, and Fusion
  Agrobacterium Culture and Agrobacterium — Mediated transformation
  Isolation of Chloroplasts from Spinach Leaves
  Preparation of Plant DNA using
  Suspension Culture and Production of Secondary Metabolites
  Protocols for Plant Tissue Culture
  Sterile Methods in Plant Tissue Culture
  Media for Plant Tissue Culture
  Safety in Plant Tissue Culture
  Preparation of Media for Animal Cell Culture
  Aseptic Technique
  Culture and Maintenance of Cell Lines
  Trypsinizing and Subculturing Cells from a Monolayer
  Cellular Biology Techniques
  In Vitro Methods
  Human Cell Culture Methods


Materials
  • Infiltration media selection plates
  • 5X MS salts 1X MS salts
  • 1X B5 vitamins .3% sucrose
  • 5% sucrose 8 g TC agar
  • 044 mM benzylamino purine
  • 03% Silwet L-77
  • Autoclave and add appropriate antibiotics (for pCGN add Kan 50 µg/mL; timentin 200 mg/µL).

Procedure
Plant Growth
  1. Plant seeds of appropriate genotype on top of cheesecloth-covered soil, making sure the soil fills the pot. Use a rubber band to secure the cheesecloth.
  2. After plants have bolted, clip off the primary bolt to encourage the growth of secondary bolts. Perform infiltration 4-8 days after clipping. Vacuum infiltration 1. Grow a large liquid culture of Agrobacterium (such as ASE strain) carrying the appropriate construct. Start a 25 mL overnight (LB+ antibiotics) 2 to 3 days ahead of infiltration. One day prior to infiltration, use this culture to inoculate a 400-mL culture.
  3. After 24 hours of growth, cells are usually at a density of at least 1 OD. Harvest cells by centrifugation and resuspend to an OD of .8 in infiltration media.
  4. Use a crystallization dish for the actual infiltration—but the pots do not fit exactly, so any ideas/improvements in this area are encouraged. Invert the pot and stick it into the infiltration solution. Put into the vacuum oven at RT. Infiltrate for 10–15 mins at 10–15 in3 Hg.
  5. Release vacuum and remove pot, lay it on its side in the tray, and cover it with Saran wrap. Remove the cover the next day and stand upright.
  6. The Agro infiltration solution can be reused for multiple pots.
Selection of Transformants
  1. Pour selection plates.
  2. Sterilize seeds in 50% water/50% bleach/drop of Tween. Rinse 3–4 times with sterile water.
  3. Plate seeds by resuspending in room temperature .1% agarose and pipetting onto selection plates. Dry plates in laminar flow hood until set. Use 1 mL agarose for every 500–1000 seeds. Plate 2000–4000 seeds per 150 × 15 mm plate. Put a small number of control seeds from a known transformed plant on one of the plates for comparison.
  4. Vernalize plants for 2 nights in cold room. Move plates to growth chamber.
  5. After about 7 days, transformants should be clearly visible as dark green plants with roots that extend over and into the selection media.
  6. Transfer seedlings to soil.
 
     
 
 
     




     
 
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