Tissue Culture success! (with seeds at least)
Well, I have successfully started seedlings in vitro now. For those of you who don't know what tissue culture is or what in vitro means, tissue culture is growing plants in containers where disease and pests can't get to them. In vitro literally means "in glass." On November the 18th, I made my first attempt at sterilizing Dionaea plant tissue and seeds along with s 3 species of Sarracenia seeds. I am using the kit ordered from Dr. Carol Stiff's website: Kitchen Culture Kits. Basically I followed the instructions for preparation of the media except that for carnivorous plants, the media needs to be at half strength. The media formula is: 1) 1 liter of distilled water2) 1 packet of standard Murashige and Skoog (MS) Once I have all of this mixed together thoroughlly, I pour 500ml into another container and add the following to each container:3) 500ml of distilled water4) 2 tablespoons of table sugar5) 1.5ml of PPM6) 3ml of BAP (leave out for seeds)7) enough baking soda or vinegar to get the PH to between 5.6 and 5.7This creates a 50% MS mixture. Sarracenia seed needs to be stratified. In order to avoid a 4 week stratification period, I used Gibberellic Acid or GA3. I soaked the seed in GA3 for 24 hours prior to sterilizing. I also did this with the Dionaea seed just to test it out. I was very successful at sterlizing the seeds with only 3 jars contaminated out of 30 or so, or about 10% contamination. I was a little less successful at sterilizing the Dionaea tissue and had about 20% contamination there. However, after a month, it appears that I might have over-sterilized the Dionaea tissue that wasn't contaminated. Nearly all of it looks black now with little to no sign of life. I'll probably leave it in the containers for another month to see what happens, but I'm not hopeful. Below are my sterilization techniques for this first attempt. For seeds:1) 10% bleach for 15 minutes2) 2% peroxide for 2 to 3 minutes3) Rinse in sterile water4) 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 60 minutes6) Transfer to media I think my downfall in the sterilization of the tissue that lead to tissue death was that I let it soak too long in the PPM (plant preservative mixture). I know that some people that do Dionaea tissue culture don't use PPM at all. Next time I think I'll cut the PPM soak time in at least half. I'll probably shoot for a PPM soak between 20 and 30 minutes. I got the majority of my seed from the International Carnivorous Plant Society seed bank. I got 1 packet of S. leucophylla "Hurricane Creek White", one packet of S. oreophila and one packet of Dionaea muscipula. I also had a Sarracenia "Dana's Delight" that flowered this year and produced a ton of seed I placed all of the tissue culture vessels 4 inches under grow lights in my terrarium. Within two weeks of placing the seeds in vitro, I noticed that some of the "Dana's Delight" seeds were starting to germinate. It's been over a month now and quite a few of the "Dana's Delight" seeds have germinated, but not much else has happened. I've noticed one oreophila seed germinated and nothing else. No leucophylla or Dionaea seed has germinated yet. It might be a little early still. I'm now wondering if the seed from the ICPS seed bank was old. I harvested the "Dana's Delight" seed myself, so I know that it is fresh. It could be that the ICPS seed was over a year old. I hope not. In any case, below are a few photos of the "Dana's Delight" seed and one of the oreophila. Not that you could actually tell at this point, but the first 3 photos are of "Dana's Delight" and the last one is of the lone oreophila seed that germinated: A "Dana's Delight" seed that I accidentally sowed too deep in the media: Another "Dana's Delight" seed that germinated. Below is an oreophila seed germinating. The only one so far.