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Thread: In vitro shoot tip culture of N. campanulata

  1. #17

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    Quote Originally Posted by thez_yo View Post
    Good accessible thread Thanks for also documenting those that failed.

    Also, I'm excited and hope this means there will be more campies on the market one of these days!
    Thanks for the kind words. Yup yup, I do hope to mass-propagate it and one day spread it far and wide so that everyone can grow this very nice plant




    Quote Originally Posted by BigBella View Post
    Very nice progress. I ran into that same issue, having introduced shoot tips into multiplication media, either too early; or, with an excessive level of cytokinins . . .
    Thank you, BigBella. I've been patiently waiting for the shoot tips to grow in hormone-free medium. So far they're still alive but extremely slow....

    Here's the only two shoot tip explants that are sterile and still survive after 8 weeks



    More patience needed I guess!


    On the other hand, taking intact basal shoot as explant is so much faster. I did not document this the first time round, but here's the method I use to prepare a basal shoot explant.




    I cut the basal shoot taking with it a sliver of wood from the mother stem. The sliver of wood was removed from the explant after the last rinse in sterile water, just before the explant was stuck in the agar medium.


    Here's an update of the lone basal shoot explant that survives, looking good so far. When the culture was 3 weeks old, and the same culture 9 weeks later at 12 weeks old.


    In comparison with the shoot tip cultures, this one has grown by leaps and bounds. I've found that the multiplication rate is 4X during these 3 months. Meaning, I can take 4 intact shoot explants from the culture and start the process all over again. But the biomass has definitely more than quadrupled during this period. There's some proliferation of callus on top of the 4 shoots produced. So if I can get the callus to form shoots then the multiplication rate is theoretically greater than 4X.

    Strong shoots originating from lateral buds.


    There's also some callus near the leaf bases beginning to form what looks like tiny shoots. Possible somaclonal mutants among them?


    In this pic you can see the lower leaf axils cracked from the expanding stem girth. There's also a couple roots growing from the callus.


    Root close-up


    I think this culture will soon be ready to divide.
    Last edited by hardy; 07-29-2017 at 09:05 AM. Reason: photos re-uploaded

  2. #18

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    This is beautiful work Hardy. What is the strength of your benlate solution? Also if you don't mind me asking, is your "household bleach" 5% NaClO? Lately I've noticed that household bleach in the US is now 8%, instead of the ~5% it used to be. Thanks in advance--

  3. #19

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    Thanks for your kind comment Well believe it or not, I don't measure the concentration of the benlate solution. I simply strive to prepare a milky suspension so I can't say for sure but it's definitely more than the recommended 1g/L for spray applications. In my experience benlate has very low toxicity to plants. I even generously apply the powder or as concentrated paste to open wounds of my nep, orchid, roses etc with little if any ill effects. If you want to be conservative, then you may like to start with 1 g/L but I think you can safely double or triple that. Here's some old photos, which shows how I prepare benlate solution for rose grafting.













    As you can see, I use much lower concentration in that case and strive to get just cloudy suspension for rose grafting, but for disinfecting nep explants I strive for 'milky'.

    As for household bleach I think it's between 5% - 6%? I will have to double check at the lab tomorrow. Will be glad to clarify if you have more questions
    Last edited by hardy; 07-29-2017 at 09:10 AM. Reason: photos re-uploaded

  4. #20

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    I've checked and the concentration of the household bleach is 5%-6% NaOCl. Cheers.

  5. #21

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    Very grateful Hardy, thank you for sharing your methods and results. Years ago I tried soaking Cephalotus leaves in a milky captan solution, and then bleaching them, but it never worked for me. (However my bleach solution was much stronger than yours.) I'll have to try this with Nepenthes--

  6. #22

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    Just an update, here's two of the best-looking cultures.



    These were subcultures starting with similar-sized single shoots, using multiplication medium. One has produced fewer but larger shoots, the other has proliferated more into a mass of tiny shoots.



    I haven't tried dividing and subculturing single shoots onto hormone-free medium. Based on the experience of others this would cause the divided shoot to grow normally and begin pitchering instead of proliferating. I will try it soon.




    Last update as of 2017/7/30: unfortunately all cultures are dead due to neglect.
    Last edited by hardy; 07-29-2017 at 09:18 AM. Reason: photos re-uploaded, info updated

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