Here's the result so far of my recent attempt at propagating N. campanulata by culturing the shoot tips in vitro. I used small basal shoots as explant source and referred to these links for disinfection methods:
http://icps.proboards.com/thread/4891
http://www.flytrapcare.com/phpBB3/nepenthes-meristem-introduction-t19147.html
Having referred to the links above, I made adjustments and came with my own disinfection method: 1-h in Benlate, followed by 15-min in bleach solution, and then briefly in sterile distilled water (i.e. no alcohol, no PPM).
I tried two types of explants, single 'cone' of unexpanded leaf containing the shoot tip meristem or intact basal shoot. I harvested eight basal shoots in total. For three of them, I cultured them intact, and for the remaining five, I excised only the shoot tips as explants. Both methods seem to work for obtaining sterile cultures, and here's some results I'd like to share.
After three weeks of culture, all the shoot tips cultured were free from contamination (first five tubes from the left). Two of the intact shoot cultures were contaminated with fungus within a week of culture and were discarded, leaving one remaining (last tube on the right).
Based on my observation so far, the tiny shoot tips can be damaged easily during disinfection. The one in the leftmost tube is completely blackened, while the rest show varying amount of blackening, but at least there's still some green on them. Intact shoots seem to withstand the disinfection process better, but are more difficult to disinfect completely (2 out of 3 were contaminated).
Here are two explants that have shown positive growth after three weeks of culture:
One of them was a shoot tip explant. It was a completely tight 'cone' when cultured. Now it has begun to unfurl, revealing a cream-colored 'cone' within.
The other one was an intact shoot explant. It had two expanded leaves with a tight 'cone'. Now after three weeks that 'cone' has unfurled to become the largest leaf on the left. In addition, another leaf and a new 'cone' was produced.
I used hormone-free half-strength MS medium for the first three weeks of culture. Now that these remaining explants seem free from microbial contamination, I've transferred them to multiplication medium containing 1 ppm BAP and 0.1 ppm NAA.
http://icps.proboards.com/thread/4891
http://www.flytrapcare.com/phpBB3/nepenthes-meristem-introduction-t19147.html
Having referred to the links above, I made adjustments and came with my own disinfection method: 1-h in Benlate, followed by 15-min in bleach solution, and then briefly in sterile distilled water (i.e. no alcohol, no PPM).
I tried two types of explants, single 'cone' of unexpanded leaf containing the shoot tip meristem or intact basal shoot. I harvested eight basal shoots in total. For three of them, I cultured them intact, and for the remaining five, I excised only the shoot tips as explants. Both methods seem to work for obtaining sterile cultures, and here's some results I'd like to share.
After three weeks of culture, all the shoot tips cultured were free from contamination (first five tubes from the left). Two of the intact shoot cultures were contaminated with fungus within a week of culture and were discarded, leaving one remaining (last tube on the right).
Based on my observation so far, the tiny shoot tips can be damaged easily during disinfection. The one in the leftmost tube is completely blackened, while the rest show varying amount of blackening, but at least there's still some green on them. Intact shoots seem to withstand the disinfection process better, but are more difficult to disinfect completely (2 out of 3 were contaminated).
Here are two explants that have shown positive growth after three weeks of culture:
One of them was a shoot tip explant. It was a completely tight 'cone' when cultured. Now it has begun to unfurl, revealing a cream-colored 'cone' within.
The other one was an intact shoot explant. It had two expanded leaves with a tight 'cone'. Now after three weeks that 'cone' has unfurled to become the largest leaf on the left. In addition, another leaf and a new 'cone' was produced.
I used hormone-free half-strength MS medium for the first three weeks of culture. Now that these remaining explants seem free from microbial contamination, I've transferred them to multiplication medium containing 1 ppm BAP and 0.1 ppm NAA.
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