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Thread: In vitro shoot tip culture of N. campanulata

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    In vitro shoot tip culture of N. campanulata

    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/ne...on-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.


    Last edited by hardy; 07-29-2017 at 08:20 AM. Reason: photos re-uploaded

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    Just as an aside, tissue culture of nepenthes with leaf explant is possible.
    It has been done:
    http://www.tbg.org.tw/tbgweb/cgi-bin...=19&topic=9227
    Apparently it involves the use of 2,4-D, but the details of the protocol remain undisclosed.

    Yeah, so that's it for now. Will update when something interesting happens with my N. campanulata cultures.

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    i dont do pots. amphirion's Avatar
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    very nice to see. thanks for sharing!
    " You keep using that word. I do not think it means what you think it means." -Inigo Montoya
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    Congratulations on your success so far. I too have managed to introduce meristematic tissues and leaf primordia in vitro; and I do agree that those explants are particularly sensitive to sterilization.

    I did find PPM to be useful however . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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    Thank you, BigBella. When searching the net for information a while back, PPM sounds like THE thing to have for some particular sterilization work. And now hearing you say it, hm, it might be worth the effort to import the stuff in I guess.

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    This morning I harvested five more basal shoots from my plant and cultured the shoot tips in vitro. This time round I managed to take some photos of the disinfection process, so here they go.

    The tiny basals were soaked in Benlate solution immediately after they were cut.


    I removed all scales from the shoots and also any leaves that have unfurled.


    Then I put the shoots in fresh Benlate solution to soak further for a total of 1 h.


    From Benlate solution they went straight into bleach solution for 15 mins. Finally they were rinsed briefly with sterile distilled water. After this final rinsing I discarded the basal portion of the shoots.


    The excised shoot tips were then cultured in hormone-free half-strength MS medium.


    I made a slight modification to the disinfection procedure by halving the concentration of the bleach solution. This time round I used 3 mL household bleach in 100 mL water, instead of 6 mL bleach in 100 mL water. The explants were looking good and the lower concentration of the bleach solution clearly did less damage to these tender shoot tips.




    Last edited by hardy; 07-29-2017 at 08:26 AM. Reason: photos re-uploaded

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    Sounds like a very sound protocol and continued good luck to you; though I would be cautious, in the long run, of 1:2 MS, since the ammonium nitrate concentration, toxic to Nepenthes, is quite high -- some 1650 mg / L at full strength in some formulations. I generally limit it to 1:4 or 1:3; or make my own media . . .
    “Sì perché l'autorità dell'opinione di mille nelle scienze non val per una scintilla di ragione di un solo . . ."

    -- Galileo "Biff" Galilei

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    I've just been doing either seeds or small seedlings, so this is encouraging. It would be nice to develop a good disinfecting protocol. I been trying out various
    disinfectants/times with a diluted solution of live bacteria and some trichoderma fungal spores, since it hard to judge the methods even side by side, if the explants/seeds etc are relatively clean. I think using a standard solution of known contaminates would be useful in determining how effective a method is. Any opinions concerning this?
    steve

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