Hi I thought I would post a few photos of my attempts at culturing Nepenthes. It has taken a lot of attempts but finally I have some limited success with N. maxima, N. mirabilis x x tiveyi and N. ampullaria. The last four photos are of N. mirabilis, N. gracilis and N. ampullaria seed in Tissue Culture If anyone is interested in Tissue Culture of Nepenthes start with some seed first and then work your way up it will save a lot of headaches.
I have never even had luck with getting them to germinate normally. Maybe seeds were damaged in transit, maybe they are just taking their time to germinate. Looks like a nice way to make tons of N. ventricosa. i would be very interested in the techniques you used to disinfect the seeds.
First thing u need fresh seed I have had very little success with seed that is older than a few months but in saying that some seed will still germinate after several months but at a very low %. seed can take from a few weeks to months I have some seed I was about to throw out but as I went to clean out the jar I noticed it had just started to germinate after six months so don’t give up on it to soon.
As for disinfecting the seeds all I can say PPM (plant preservative mixture) Google it! Forget all the other treatments like bleach or peroxide too much room for error just use PPM it can be a bit expensive but well worth the cost make up your media as listed below
: Murashige and Sskoog basal medium with Vitamins (use at half strength) : 1 lt of distilled water
after you make this use 25 ml of above mix and add 2ml of PPM this will give you a good sterilizing treatment it works every time well almost. Soak seed for 2 to 4 hours in this don’t rinse just place seed strait on to media
For the rest of the above media the 1 lt of MS you have made I add: : 2 ml of PPM : 25g sucrose (sugar) : About 5 grams of AGAR (test this out first with some plain water as some brands of agar require different ratios) : adjust PH to 5.5 - 5.68 range (use white vinegar to lower PH and bi-carb soda (baking soda) to raise PH) Add media to your containers or jars and sterilize as normal.
Hope that this helps you a bit I am no expert it has taken a lot of trial an error to get it right I still have some failures every now and then.
Just a word of warning don’t bother to try and tissue culture Nepenthes from plant material its dam hard or near impossible to do I have had a bit of luck try with seed first then once you have that worked out maybe try it but be prepared for a lot of green blue fuzzy stuff growing in your jars.
makes sense. I do know PPM. I got my self a kitchen culture kit and was playing around with it. I will need to get more PPM. I have a few pods on a female ventricosa starting to swell. Don't know who the pollen parent is.
Has anyone tried green pod culture? I remember doing this with orchids in college (a long time ago). Basically was told it was embryo culture. However, disinfecting was less difficult. Just bleach soak the mature green pod to clean outside. then cut open with sterile instruments and remove seeds. Seems like would be easier. But if it were that easy I suppose someone would have done it. I was sort of thinking about trying with some of the other CPs that I have that produce abundant seeds.
I've searched the net an uncountable amount of times for Nepenthes tissue culture, but so far: zip. All I come across is people germinating seeds in vitro. Sorry to sound disrespectful, but what I'm reading here isn't tissue culture but aseptic germination. What are the sterilisation procedures and multiplication recipes for tissue culture of Nepenthes nodes or leaf tissue ?
I thought the idea was to germinate the seeds in vitro. The seedlings can then be sub-cultured - divided and put in separate flasks to grow as clones. Since the tissue is sterile already things proceed rapidly at this point.
just to clarify: I've never seen people actually using leaf or node tissue (think samples of cubic millimetres) of Nepenthes to multiply. All references I come across germinate seed and root shoots with leaves. What I'm looking for is tissue culture from leaf sections or node samples, that's what tissue culture actually is - in this example (and all results on the net) you're just growing and cutting plants in an aseptic environment. This does speed things up, but it's not exactly tissue culture.
Just to set the record straight: I think it's wonderful to see these experiments documented and shared on public fora.
just to clarify: I've never seen people actually using leaf or node tissue (think samples of cubic millimetres) of Nepenthes to multiply. All references I come across germinate seed and root shoots with leaves. What I'm looking for is tissue culture (TC) from leaf sections or node samples
I've been in contact with many TCers and all of them say it's impossible to get Nepenthes tissue sterile.
Surely it's not impossible, but no one I know of has done it successfully and I too haven't been able to find any articles on the subject either.
The sterility issue you're referring to is in the context of sterilising tissue from ex vitro and introducing it in vitro. Have you tried taking sterile tissue from germinated seeds in IV and working with that ?
I use diluted activated bleach to sterylise Nepenthes seeds with very goood results. For about ~10 different species i had 0% contamination and germination similar as in in-vivo conditions. I read that PPM can inhibit germination in higher concentrations, but i see that it is working too
I tried to introduce Nepenthes from exlants- top parts witch top meristem continued growth after very hard sterylisation, but they was still contaminated.... After some re-passages and resterylisations i tried 10 min 50% PPM but it definitively killed plants.... Now i'm collecting material and i will try with long time sterylisation with first bleach, next low-concentration HOCl from NaDCC and next long soaking in PPM.
What medium you use to growth? 50% MS is to concentrated for Nepenthes, some species stay alive but some just die....[highlanders?] I use 0,2MS but i want to switch to knudsen medium....
Hi all just a few updated photos of my plants and a few questions answered. First thing yes growing seed in sterile conditions is not technically tissue culture but the resulting division of this tissue grown from seed is tissue culture we just cheated a bit to get the sterile tissue but after this first stage we are tissue culturing.
it's not impossible to get Nepenthes tissue sterile but very hard that’s why even the biggest of the Nepenthes labs start with seed in vitro its faster, cheaper and easier. Leaves don’t work in fact I have tried every part of the plant except root tips and green seed pods, the only part of the plant that I could get to multiply was the meristem and that is not easy the first photo is of a meristem taken from a N. mirabilis x "Viking" plant. after several months of growing it was one of five placed into tissue culture all others failed but its growing well.
this is N. maxima, N. mirabilis x x tiveyi the third photo in my original post has started to grow very fast he (or she) will be removed from the flask soon to be hardened off and grown on in my shade house to give the smaller plants in the flask room to grow.
the photo below is of N. mirabilis seed grown in vitro after about four months plants are about 30 to 40 mm across these will be allowed to grow to about 50 to 60 mm before i plant them out.
This is N. x hookeriana that will be divided and sub cultured in a month or two
meristem taken from N. izumiae x truncata
this is N. maxima just removed from TC starting to be hardened off bottle cap is for size reference
the following photos are of more seed just starting to germinate
Leaves don’t work in fact I have tried every part of the plant except root tips and green seed pods the only part of the plant that I could get to multiply was the meristem
Just out of curiosity ... 1. Do you remember whether you used leaf tissue from a leaf with a developed or undeveloped pitcher? Protein expression differs like day and night before and after pitcher development and this might also include inhibitors for tissue culture. 2. have you tried the swollen tip of a tendril? The way I see it, the leaf in Nepenthes is actually a phyllode and is not the favorite subject for TC in many families.