November Tissue Culture update
Today is February 14th and I made my first tissue culture attempt on November 18th, so it's been a few months now since I made my first attempt at tissue culture. I learned quite a bit and had moderate success germinating seeds in vitro, but explants are a different story. Micropropagation is quite hard! All of my explants were unsuccessful because the plant material died. Here are the sterilization techniques that I used:For seeds:1) 10% bleach for 15 minutes2) Rinse in sterile water3) Transfer to media For tissue:1) Dip the cutting in the 90% alcohol quickly. Hold it above the alcohol to let any excess drain.2) Drop the cutting in 3% peroxide for 2 minutes3) Drop the cutting in 10% bleach mix for 15 minutes4) Drop the cutting in sterile water for 10 minutes5) Drop the cutting in 2% PPM (plant preservative mixture) solution for 30 to 60 minutes6) Transfer to media Things I learned from my first attempt at tissue culture: PPM (plant preservative mixture) is toxic to small Dionaea explants at 2% concentration. I'm fairly certain that a 30 to 60 minute soak in 2% PPM is what killed my explants and caused extreme phenolic oxidation. However, it could also have been that I over sterilized the tissue. Seeds are quite easy to sterilize. I had no contamination of Dionaea seed and only minimal contamination of Sarracenia seed. Something that I did prevented nearly all of the Dionaea seed from germinating. My guess is one of four things: 1) The overnight soak in Gibberellic Acid (GA3) prior to doing the sterilization wasn't good. 2) The fact that the media I sowed the seed on was a little too hard which prevented the seeds from soaking up the nutrients in the media. 3) The seed wasn't viable to begin with. 4) I over sterilized them (not likely). Well, after waiting for months, I've only had 3 out of 30 Dionaea seeds germinate and they just germinated within the last couple of weeks (sometime near the end of January). After seeing how well hackerberry's (Jun) in vitro seedlings are doing after replating, I'm planning on replating mine in the next couple of days. I just wanted to take some photos now for a bookmark. Here they are: A germinated Dionaea Seed:Another Dionaea seedling:One More: And onto the Sarrs. Here is some Dana's Delight seedlings: and two shots of my best two jars of Sarracenia oreophila: Hope you enjoyed the photos! Next time you see these little guys, they'll be in new cultures on new media and hopefully growing like crazy!