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Thread: In vitro shoot tip culture of N. campanulata

  1. #9

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    Quote Originally Posted by BigBella View Post
    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 . . .
    Wow, I didn't know that. Thanks so much for the pointer!

    Quote Originally Posted by sromes1 View Post
    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
    Hm, that sounds interesting! Yes, I think it'll be great if the minimum concentration required to kill the microbes could be determined, in order to minimize damage to the explants/seeds. When I used 6 mL bleach in 100 mL water to sterilize the shoot tip explants, all were free from contamination, but the explants themselves were quite damaged, so I was wondering if that was an overkill. Which is why I decided to reduce the bleach in half for my second attempt.

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    Oh, and it also depends so much on what stuff of interest is to be disinfected. A sterilization protocol that is too harsh for one type of explant, may be perfectly fine for others.... For instance, here are shoot tip cultures of chocolate cosmos sterilized with 6 mL household bleach in 100 mL water for 15 min. After just a week of culture, they are growing strongly and seem to be unharmed at all.


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

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    Quick update:

    One-week old shoot tip cultures, using 3% instead of 6% household bleach (v/v) as surface sterilant


    Three of the cultures look pretty good.


    Two have bacterial contamination, not sure if it's internal infection or just some bacteria that the surface sterilization simply missed.




    Five-week old culture of shoot tips, using 6% household bleach (v/v) as surface sterilant

    After 3 weeks in hormone-free medium, plus 2 weeks in multiplication medium, only the lower right explant shows definite growth.



    Progress of 5-week old culture using intact shoot as explant

    Before and after photos, after 2 weeks in multiplication medium.

    Cloudiness in the "after" photo is condensation on test tube surface. The black patch on the leaf is 5-week old damage incurred during surface-sterilization of the explant with bleach.

    Here's a clearer shot taken from a different angle.
    Last edited by hardy; 07-29-2017 at 08:41 AM. Reason: photos re-uploaded

  4. #12
    BigBella's Avatar
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    Very nice work -- and I would expect that the contamination was simply a surface sterilization issue. I don't think that I've ever seen internal bacterial issues from with a dissected plant or basal . . .
    “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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    (Hardy is taking down notes and really grateful for the input....)

    Thank you

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    An update:

    3-week old shoot tip cultures, using 3% instead of 6% household bleach (v/v) as surface sterilant

    Out of five explants, only two sterile cultures are obtained. The one on the left has begun to grow. The other one still looks pretty good.


    The contaminated cultures.

    The leftmost culture seemed clear of any infection in the first 2 weeks of culture, but later developed fungal growth from within the explant itself. The whole tube was taken over within a week. The other two developed bacterial growth within the first week of culture, but the infection seems benign. In fact one of the explants is positively growing despite the bacterial contamination.




    7-week old culture of shoot tips, using 6% household bleach (v/v) as surface sterilant

    After 3 weeks in hormone-free medium, plus a month in multiplication medium, some of the shoot tips are dead and the surviving ones definitely don't look happy. Perhaps I shouldn't have transferred these to the multiplication medium so soon.




    7-week old culture using intact shoot as explant

    After 3 weeks in hormone-free medium, plus a month in multiplication medium, there is one confirmed axillary shoot, which I'm very glad to see There are other protuberances on adjacent leaf bases, but it's still too early to tell if those are axillary shoot or callus.


    Last edited by hardy; 07-29-2017 at 08:46 AM. Reason: photos re-uploaded

  7. #15
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    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!

  8. #16
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    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 . . .
    “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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