In the link, you used PPM in the flask media in addition to the sterilization. Do you do the same for Nepenthes?
Yes, PPM is used, both for sterilization and as a bio-static preservative of sorts, for the media . . .
What concentration of sucrose are you using to 1/4 MS? I looked at this paper:
Mekong et al 2000. Vegetative Propagation of selected Nepenthes species Borneo Science 7:1-9 They found 1/2 MS +6% sucrose had the highest germination rate (66%) with N. mirabilis, N. ampullaria. With 3% sucrose, the germination rate became less than 1/2. So I'm curious what concentration you are using (I went through the thread, but I might have missed it).
I typically use 2.5- 3% of sucrose; and have never had successful experiences at those high concentrations that you had mentioned. Also, explants grown with such high carbohydrate loads, take longer to eventually get established, ex vitro. Further, Nepenthes ampullaria and N. mirabilis are real outliers in terms of that high sucrose tolerance; and few highlanders would ever respond well to that concentration. It is also a direct invitation for algal or bacterial contamination; and it comes as little surprise to me that Mekong et al faced such issues with their cultures. They may as well have been making bread . . .
With sterilization, what is the rationale to use MS + PPM instead of water + PPM?
Mekong paper used 30% Clorox (2.5% NaClO) for 3 min. They had 10-20% contamination rate. You mentioned that Nepenthes seems to be sensitive to bleach. So I haven't decided the method. What kind of contamination rate and germination rate (ball-park) are you getting with PPM?
The Plant Preservative Media (PPM) producers have established a protocol, for their product, which involved the use of full MS salts, without pH adjustment, along with the PPM. Presumably, that combination -- along with the high solute (hypertonic) concentrations -- lowers the pH of the already acidic solution that much more; and, thereby furthers its effectiveness, as an antibacterial agent. I do recall the paper that you had mentioned; and all that I can say, is that Mekong, et al must have been using their unwashed feet, along with serious sinus infections to achieve that stellar level of contamination. Even, while at university, our contamination rates, in our first rookie experiences with micropropagation, were well below five percent. With the use of PPM and improved sterile technique, my contamination rate hovers around .87 percent; and the overall germination rate -- highly dependent upon the condition and freshness of the seed (lowland Nepenthes have seed viability which may be measured in weeks) -- is over sixty-seven percent. Forget the bleach with Nepenthes seed; they are far too sensitive, rare and fragile . . .
Final question, are you using vented flasks?
I no longer use vented flasks, since cultures tend to quickly become desiccated; the lids don't tolerate much autoclaving; and the venting is yet another invitation for contamination. Simply, swap out the cultures more often; and the gas exchange issues become moot . . .
I have flasked orchids, but I have never done it with Nepenthes, so this thread was very helpful. Thanks!