Meristem cloning

[tulasi132]

HI! Well, I've been trying to research this online but cant seem to find more than a cursory explanation. Does anyone have any info on meristem cloning. I know this is a commercial method and I would like to know how it is done. I know the meristem tip is dissected form the growing tip of a spike, cane, or leaf. I know it is cultured in medium that is kept in motion to prevent differentiation of the primordial cells, but I don't know much more. What kind of medium under what conditions? Are there chemicals used to induce different reactions? I am new to this idea and am a student of bio-chem. I would like a garage project that would give me experience in a field with a hobby I love. Anything would be great.

[dabblin-n-orchids]

from what i understand it is as you said above. the cells are kept in a liquid nutrient media and I would assume there would be some form of an auxin in the media to stimulate cell division, or these cells would be placed on a solid agar based nutrient media to divide and form what is known as a callus, basically a mass of undifferentiated cells which are separated off the callus with a mixture of proteolytic enzymes called EDTA. these are then placed on a growth media and treated with different plant hormones to induce the differentiation of root, stem and leaf cells. . . . i'm sure that was clear as mud . . .I used to be much more knowledgeable on the subject but have since forgotten . . . .and someone else has probably beaten me to all of this while I racked my brain . . ..

[dabblin-n-orchids]

mmmmk
auxins induce root growth
cytokins induce bud and shoot growth
those are the important two of the 5 plant hormones and the liquid media would NOT contain either merely micro- and macronutrients and sucrose as an energy source if the intent were to create a mass of undifferentiated cells. A chemical inhibitor might also need to be used to prevent cell differentaition
I think I jut made EVERYTHING worse . .. SORRY FOR THE CONFUSION!

[dabblin-n-orchids]

gibberellins are the chemical inhibitor (one of the 5 plant hormones)

[dabblin-n-orchids]

almost forgot to cite my source SHAME ON ME!
http://aggie-horticulture.tamu.edu/s...ation-2002.pdf

[tulasi132]

Thats a great start, thank you. One of the things I found on the net said that the undifferentiated cells were kept in motion to inhibit differentiation into roots and stem because they require a "sense" of gravity and light source to do so. But the info on chemical inhibition is exactly the kind of info I'm looking for, please give me any thoughts on this idea of motion. I appreciate the info on hormones also. I cant appreciate any info at all enough thanks again dabblin, you've already increased my power,...er... understanding alot......mua mua muahahahaha...oh. Seriously though. Thanks, I'm facinated.

[dabblin-n-orchids]

I can see how constant motion could inhibit gravotropism . . .fascinating . .this might end up on my ever expanding project list. I wonder too if the motion would prevent them from clumping and forming a callus. so mabey chemical inhibition is only needed in a gelled media . ... .this bears further research . .. .

[Undergrounder]

Hi DnO, get to the library and find a book called 'The Micropropagation of Orchids' by Joseph Arditti, it has detailed lists of chemicals used and basic procedures for most cloning practices.

I think you're right on the basic procedure, with a sterile area for work, good sterile practices and materials, a shaker, correct liquid medium and properly disected meristem tissue, you could probably try cloning and see if it works.

For the media i suggest you get a hold of J. Arditti's book, or look online for a pre-mixed media, you can get orchid multiplication media without agar from different vendors.

[newflasker]

Google for "meristem tip culture" you will find ton of information. In simple words about meristem clone, you cut a shoot tip and grown it. Shoot tip contains young cells that give you more chance to grow cells and they become plants. You may have few shoots but if you want more than that then you can use hormones to induce shoots then cut new shoots to induce more. Most people use combination of auxin and cytokinin in medium which contain rich macro and micro-nutrient plus sugar (in vitro) to induce root and shoot depend on the ratio between them.

EDTA is not an enzyme. It's a chelating chemical which is used to dissolve some heavy metals such as iron and copper in solution so plants can absorb them easily.

[dabblin-n-orchids]

oops my bad on the EDTA . . . . isn't there a mixture of proteolytic enzymes used to separate cells or am i making this up (which is completely probable)