- Mon Jul 19, 2021 9:37 pm
#385256
Hello all, been browsing this forum for years, but I don't normally post. I am no stranger to aseptic technique and have a familiarity with tissue culture in a lab environment. I recently decided to start playing around with home tissue culture, but have only had a few successes. My knowledge seems to be breaking down when it comes to Drosera spp. I apologize for this ridiculously long post. I was hoping more data would help with diagnosing where I am failing. Any assistance would be greatly appreciated.
I am assuming my issue lies in explant sterilization. In my most recent attempt I ran a sterilization trial on explants from D.adelae, D.aliciae, and D.capensis (wide leaf var). Ending in abject failure. All explants browned out 48 hours after plating. I used the following protocol:
Media:
1/3 MS (Phytotech M519)
25g Sucrose
1ml/l PPM
3g/l Gellan (Phytotech G434)
pH raised to 5.62 with NaOH
40ml Dispensed into GA-7 vessels
Pressure cooker for 25mins
After 24 hours swabbed out residual condensation on walls with sterile paper towels.
After plating, the vessels are all sealed with micropore tape.
I then used three different sterilization techniques I compiled from previous posts here on flytrapcare. 10 explants were taken for each technique; 30 total. Each explant is a full leaf, cut off the plant using a scalpel immediately before sterilization. All were reasonably new leaves with no obvious signs of damage. Approximately 1.5cm - 4cm long. All of the Drosera used were grown from cuttings indoors under humidity domes for their whole lives.
1st Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 15 minutes.
Rinsed each explant in SDW.
Placed in 20ml tubes with 3% H2O2 (created from 34% foodgrade and SDW)
Agitated for 5 minutes.
Then transferred each explant to 0.5% NaOCl (6.7% bleach) solution with a few drops of clear dish soap.
Agitated for 6 mins.
Triple rinsed with SDW and placed onto a sterile paper towel
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
2nd Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 3 minutes.
Rinsed explants and placed into 20ml vials of SDW.
Each explant was then placed into a 50ml vial of Peracetic Acid.
Agitated for 2.5 mins.
Pulled out of PAA and placed onto a sterile paper towel
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
3rd Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 15 minutes.
Rinsed each explant in SDW.
Dropped each explant into 10ml vials containing 3% PPM and SDW.
Agitated occasionally for 4-4.5 hours.
Explants were each placed onto a sterile paper towel.
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
I am assuming my issue lies in explant sterilization. In my most recent attempt I ran a sterilization trial on explants from D.adelae, D.aliciae, and D.capensis (wide leaf var). Ending in abject failure. All explants browned out 48 hours after plating. I used the following protocol:
Media:
1/3 MS (Phytotech M519)
25g Sucrose
1ml/l PPM
3g/l Gellan (Phytotech G434)
pH raised to 5.62 with NaOH
40ml Dispensed into GA-7 vessels
Pressure cooker for 25mins
After 24 hours swabbed out residual condensation on walls with sterile paper towels.
After plating, the vessels are all sealed with micropore tape.
I then used three different sterilization techniques I compiled from previous posts here on flytrapcare. 10 explants were taken for each technique; 30 total. Each explant is a full leaf, cut off the plant using a scalpel immediately before sterilization. All were reasonably new leaves with no obvious signs of damage. Approximately 1.5cm - 4cm long. All of the Drosera used were grown from cuttings indoors under humidity domes for their whole lives.
1st Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 15 minutes.
Rinsed each explant in SDW.
Placed in 20ml tubes with 3% H2O2 (created from 34% foodgrade and SDW)
Agitated for 5 minutes.
Then transferred each explant to 0.5% NaOCl (6.7% bleach) solution with a few drops of clear dish soap.
Agitated for 6 mins.
Triple rinsed with SDW and placed onto a sterile paper towel
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
2nd Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 3 minutes.
Rinsed explants and placed into 20ml vials of SDW.
Each explant was then placed into a 50ml vial of Peracetic Acid.
Agitated for 2.5 mins.
Pulled out of PAA and placed onto a sterile paper towel
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
3rd Technique:
Washed in 1000ml Erlenmeyer flasks containing sterile distilled water with 500ppm citric acid, and a few drops of clear dish soap for 15 minutes.
Rinsed each explant in SDW.
Dropped each explant into 10ml vials containing 3% PPM and SDW.
Agitated occasionally for 4-4.5 hours.
Explants were each placed onto a sterile paper towel.
Used a scalpel to cut 1-5mm off the petiole end of the leaf that was pinched by the forceps, and plated immediately.
- By wcrosman
- By optique
- By ChefDean
- By jetfire245