Selection of transgenic plants with antibiotics
Petri dish selection
Kanamycin (pBI101)
Media
    1/2x MS, no sucrose, 0.8 % Sigma Agar (A1296)
    40µg/ml Kanaqmycin, 100µg/ml cefotaxim.
  1. Sterilize 200µl (do  not exceed) seeds in 1.5 ml tube. 
  2. Resuspend in 0.1-0.2 % agar.  Make total volume of 1 ml.
  3. Spread 250µl seed suspension/90 mm plate.
  4. Cold-treat for 2 days.
  5. Move to room tempearature.  It will take 4-5 days to distinguish green transgenic plants in yellow untransformed plants.
Hygromycin (pFGC1008)
1/4x MS, no sucrose, 0.8 % Sigma Agar (A1296)
    20-30µg/ml Hygromycin B, 100µg/ml cefotaxim.
  1. Sterilize 200µl (do  not exceed) seeds in 1.5 ml tube. 
  2. Resuspend in 0.1-0.2 % agar.  Make total volume of 1 ml.
  3. Spread 250µl-1 ml seed suspension/90 mm plate.
  4. Cold-treat for 2 days.
  5. Move to room tempearature.  It will take 4-5 days to distinguish tall transgenic plants in short untransformed plants.
Hygromycin selection can be done in very high density (but drain the excess liquid from the media surface).  Resistance based on the root hair development can be observed under microscope after few days.
Hygromycin can loose titer over the time.  I used 20µg/ml for fresh purchase but increased to 35µg with 3 year-old stock.

Glyphosate (pMDC100-GAT)
1/4x MS, no sucrose, 0.8 % Sigma Agar (A1296)
    0.4 mM glyphosate (MW 169.07), 100µg/ml cefotaxim.
(I dissolved glyphosate in ethanol as 500x stock but will try to use methanol or DMSO to prepare higher concentration stock)
  1. Sterilize 200µl (do  not exceed) seeds in 1.5 ml tube. 
  2. Resuspend in 0.1-0.2 % agar.  Make total volume of 1 ml.
  3. Spread 250µl seed suspension/90 mm plate.
  4. Cold-treat for 2 days.
  5. Move to room tempearature.  It will take 4-5 days to distinguish green transgenic plants with their root penetrating media in yellow untransformed plants.
Basta (pSKI015)
1/4x MS, no sucrose, 0.8 % Sigma Agar (A1296)
    3 µg/ml liberty herbicide, 100µg/ml cefotaxim.
Commercial Liberty herbicide is 11.33% (113.3g/L).  Filter sterilize and add 26.4µl/L to the media.
  1. Sterilize 200µl (do  not exceed) seeds in 1.5 ml tube. 
  2. Resuspend in 0.1-0.2 % agar.  Make total volume of 1 ml.
  3. Spread 250µl seed suspension/90 mm plate.
  4. Cold-treat for 2 days.
  5. Move to room tempearature.
Asulam (pCB301-Sul)
1/4x MS, no sucrose, 0.8 % Sigma Agar (A1296)
    30 µg/ml Asulox herbicide, 100µg/ml cefotaxim.
Commercial Asulox herbicide is 355.89g/L.  Filter sterilize and add 84.2µl/L to the media.
  1. Sterilize 200µl (do  not exceed) seeds in 1.5 ml tube. 
  2. Resuspend in 0.1-0.2 % agar.  Make total volume of 1 ml.
  3. Spread 250µl seed suspension/90 mm plate.
  4. Cold-treat for 2 days.
  5. Move to room tempearature.
Soil selection
Basta (pSKI015)
  1. Prepare soil.  Make sure to include Banlot to prevent Pithium infection, and malathon to prevent insects. 
  2. Sow seeds with high density.
  3. Mix seeds (1ml seeds/big tray) and sands (1:1) in salt shaker and shake onto the surface of soils.
  4. Water from the bottom with Banlot containing water.  Make sure the surface became wet.  If needed, drain excess water.
  5. Keep in cold room for 2-5 days. 
  6. Move to growth chamber set to 21-24C.
  7. After 1 week, spray with 30µg/ml Liberty solution for 3 consecutive days.  After 5days interval, treat with 3 more sprays to eliminate escapes.
  8. Untransformed plant start to die after second spray of 3 sprays.
Glyphosate (pMDC100-GAT)
(This is not a final protocol)
  1. Prepare soil.  Make sure to include Banlot to prevent Pithium infection, and malathon to prevent insects. 
  2. Sow seeds with high density.
  3. Mix seeds (1ml seeds/big tray) and sands (1:1) in salt shaker and shake onto the surface of soils.
  4. Water from the bottom with Banlot containing water.  Make sure the surface became wet.  If needed, drain excess water.
  5. Keep in cold room for 2-5 days. 
  6. Move to growth chamber set to 21-24C.
  7. After 1 week, spray with 4 mM glyphosate (0.8g/L) solution for 3 consecutive days.  After 5days interval, treat with 3 more sprays to eliminate escapes.