Nepenthes Tissue Culture
 

I wonder if we can make a subforum for "nepenthes propagation" though it is not such too much a hot topic recently, maybe it might help sort out some of the topics.

Going to my main question... I am wondering, I read somewhere that IBA is a rooting hormone or chemical. IBA is used in Tissue culture.

I think I may have it from my TC kit that I was able to purhase from Kitchen culture kit.

My question is....

I wonder if I can use IBA on Stem Cuttings to help promote rooting of cuttings of Nepenthes?

Or will it be detrimental in terms of maybe promoting fungal or microbial growth on the stem cuttings, maybe even killing the cutting instead?

I really want to try out water rooting some of my Neps, which reminds me I will post a request for update on Shawn :)

TTFN
Arvin

Re: Nepenthes Tissue Culture
 

Ok Arvin. Here's your Nepenthes Propagation board. :)

Re: Nepenthes Tissue Culture
 

Thanks! David! :)

Now I hope someone will share their experties about TC and maybe reply to my questions about IBA :)

Hehehe, I just thought of something, to the Admin, maybe we can actually transfer some of the propagation posts here as well already, for instance Shawn's water rooting. :) That is if there is extra time only :)

TTFN
Arvin

Re: Nepenthes Tissue Culture
 

I had a nice email chat with an experienced TC guy, he said that he uses seeds from newly opened seed pods to make sure there is minimum contamination. He mentioned that using cuttings from adult plants usualy end up with contamination. One possible idea is to surgically cut a growth tip (forgot the scientific term for it) with a microscope in sterile environment, but no one has reported any sucess in these.

I am wondering about the system in which one seed can turn up hundreds of clones... does that mean that once the seed germinates the plant is then chopped up and then TCed while still in sterile environment? I wonder if at that stage (plantlet stage) all the parts cut from it will grow a new plantlets?

TTFN
Arvin

Re: Nepenthes Tissue Culture
 

First, to clarify something. Do you wish to discuss the process of germinating seeds on a sterile medium like agar, or tissue culture, where a small piece of tissue from the apical meristem is repeatedly grown and chopped up to obtain hundreds of plants that are genetically identical..? These 2 processes are similar, but not the same.

In the first process (germinating seed), all that we are doing is using a sterile medium instead of a natural one like moss, to ensure higher rate of germination. This is a common practice for some plants with very very tiny seeds that are usually symbiotic with a fungus (like orchids), as the agar medium can be tailored to the plant's needs.

Tissue culture means replicating the mother plant and producing hundreds of copies that are essentially the same (genetically). The apical meristem (the tissue which gives rise to new leaves) or even the root meristem tissue can be used. It is cut and placed into a flask of agar containing hormones and placed on a shaker. The blob of tissue then forms a callus, which looks like a small piece of moss. This anti-gravity environment promotes growth of the callus, but shoots and roots do not develop. This is chopped up again and reflasked. Then when there is enough tissue to produce the number of plants needed, the shaker is stopped. The effect of gravity causes leaves and roots to emerge and the clones can be grown on to adult size.

Re: Nepenthes Tissue Culture
 

IBA (indole butyric acid) is an auxin (hoemone) that promotes cell elongation. Its close cousin, indole acetic acid (IAA) is found naturally in plants and controls development of the new shoot in response to light. Auxins also promote adventitious root growth and are used as rooting hormones. There should be no problem using these to encourage new roots on cuttings. But you must use the correct strength. Too little is not effective and too much is detrimental.

BTW, IBA is not the same hormone used in tissue culture. Tisssue culture mainly uses cytokinins to pomote cell division, so that a large quantity of callus can be obtained. :)

Re: Nepenthes Tissue Culture
 

Thanks Paphioboy for replying. first of all regarding IBA, I received IBA with my TC kit, that is why I asked about the IBA. So it is okay to use IBA even if I was going to stick the cutting in potting mix? I'm just afraid that it might promote fungal or bacterial growth. I need to read up why IBA was included in my TC kit though.

As for my other question, yes sorry I didn't realize it's two questions in one... but you are correct however that I was asking about both. However....

1. I understand that for Nepenthes, what they do is to germinate seeds in a sterile environment (Agar) so that the plantlets will be sterile, then they use those to start Tissue culture. Which now I imagine is the same as what you described with the callus. I just don't know how one can get Apical Meristem from those small plantlets, unless they just chop the whole planlet up and do as what you described?

2. I was also asking about the practicality or sucess rate with using Apical Meristem from an adult nepenthes cutting, because so far I have read that usually cuttings are already contaminated by a lot of organisms that it is almost impossible to get a sterile sample to be able to do TC.

TTFN
Arvin

Re: Nepenthes Tissue Culture
 

sorry guys need some help

i want to try this tissue culture thing where do i get this "agar" thing from?

can i do it by myself ?

should i try this in some humidity environment

please explain more about seed t.c and what are the success rates ?

Re: Nepenthes Tissue Culture
 

Hi amitzauber,
agar is the thing to make gracilaria you can find them in market.
You can do it by your self but you need a perpect mix of nurtrien it need a lot of studies.
Lighting condition, Temperature is 26 - 21.
This will help you.

Re: Nepenthes Tissue Culture
 

really confusing

i would like to try myself (seed t.c)

can you tell me which things i will need for the mix ?

Re: Nepenthes Tissue Culture
 

Hi amitzauber,
I buy a mix from a garden shop they sold MS mix for growing vegatable. Here is some information for TC
http://www.omnisterra.com/botany/cp/slides/tc/tc.htm
http://en.wikipedia.org/wiki/Murashige_and_Skoog_medium
The T.C can make the plant 10x to 15x grow.
You should find some information in the internet.

Re: Nepenthes Tissue Culture
 

Tissue culture is not easy to DIY for the average CP grower. To be successful, you must have a completely sterile environment. The reason is that agar is nutrient rich, so even a tiny speck of fungus/bacteria will contaminate the entire container/flask and kill all the seedlings inside. Most commercial labs have at least an autoclave, laminar flow hood with air filter and a shaker. It is much easier to send a piece of tissue to the lab and ask if they will culture it for you for a small fee.

Arvin, the apical meristem does not come from the callus. The apical meristem is taken from the adult plant. When chopped up and under the influence of hormones, it then produces the callus, which grow into many individual plantlets.

Re: Nepenthes Tissue Culture
 

The apical meristem used should be relatively clean compared to the rest of the plant because it is hidden beneath all the other young leaves. If unsure of contamination, can soak in 5% bleach solution to surface-sterilise it before TC.

Re: Nepenthes Tissue Culture
 

Amitz you should try to order a kit from here:
http://www.kitchenculturekit.com/index.htm

That is where I got my kit, but I haven't had time to try it out yet, and so I am also still researching. Best bet to try first are VFTs and Droseras, Nepenthes apparently is hard to TC from meristem so I am asking help here in the forum about it.

It is a bit complicated as Paphioboy mentioned, but if you are the type that likes a challenge or to experiment that TC is for you. By the way I feel that the generalization that TC can make one seed into a thousand plants is a bit of an exaggeration. Well it is possible, but sure heck will need a long long time to get to a million. :) However it is true that if you do keep at it, you can grow a lot of clones from one meristem.

Paphio, the current suggestion is 10% bleach, but people still say they still get contamination, oh well, we'll see.

TTFN
Arvin

Re: Nepenthes Tissue Culture
 

Bleach will only sterilize the surface of the meristem tissue, but if your air is still not clean and contaminated with fungal spores, it is no use..

Re: Nepenthes Tissue Culture
 

for a fresh starter understanding TC can be confusing. I found a link from another forum which was very useful.
http://www.world-of-carnivores.com/tcathome.html

Re: Nepenthes Tissue Culture
 

10% was the acceptable level. when carrying out TC contamination at every level must be eliminated even after the work surface was sterilised. For example accidental drop of tweezer, forcep or cutter on the work surface they must be dip in ethynol (spirit), then place over the burner thus killing the foreign bodies.

Re: Nepenthes Tissue Culture
 

If I use IPA as a disinfectant I understand it's dangerous to have a burner in the box. Also I understand the PPM really helps a lot in controlling contamination?

Still thinking about where I will be able to do TC and if I will want to make a laminar flow hood or not bother anymore (DIY hood I mean).

I'd like to ask another thing though.. maybe this deserves another thread, but may I ask what is the recommended way of keeping the Tissue cultures? I mean.. a rack, with how much watts florecent lamp? how long photo period? Is it better to cover with glass or plastic to prevent possible contamination? etc etc.

TTFN
Arvin

Re: Nepenthes Tissue Culture