Callus with Plant Tissue Culture

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MC

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Feb 18, 2017, 12:40:14 AM2/18/17
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I'm a TC hobbyist and I have a fairly basic question about calluses. In doing some research, it's my impression that callus are important for research and transformation purposes. My understanding also is that callus can be induced to differentiate into shoots or roots.

So from a hobbyist perspective, what's the best course of action with seedlings or whole plant shoots that are beginning to callus? Should I plate them to plain media to let the shoot develop and grow regardless of the callus? Or if I plate it to a cytokinin, will the callus produce additional shoots despite already being on a plant?

Cory Tobin

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Feb 18, 2017, 2:59:17 AM2/18/17
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Should I plate them to plain media to let the shoot develop and grow regardless of the callus? Or if I plate it to a cytokinin, will the callus produce additional shoots despite already being on a plant?

If I understand correctly you have a seedling with callus growing from the hypocotyl.  I would cut the callus from the hypocotyl and plate it by itself.  Then you can keep growing the seedling in soil.  I would grow the callus on plain media.  MS basal salts, phytoagar, 3% sucrose, no hormones.

It may be tempting to grow callus on "callus media" but that is for inducing new callus from differentiated tissue.  Typically contains 2,4-D and some other growth regulators like 6-benzylaminopurine, indole-3-acetic-acid, etc depending on the species.  This will often inhibit growth of callus even though it induces the formation of callus in the first place.

Once you have as much callus as your heart desires, transfer to plates containing shoot inducing hormones, then to plates with root inducing hormones.  The exact recipe will vary by species.  Some guess work may be required for less well studied species.



> Or if I plate it to a cytokinin, will the callus produce additional
> shoots despite already being on a plant?

That *might* work but it will also mess with the seedling.  So I would grow the seedling and callus separately if you want the seedling to continue growling normally.


-Cory

MC

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Feb 18, 2017, 3:20:52 AM2/18/17
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Thanks for the input!

 I would grow the callus on plain media.  MS basal salts, phytoagar, 3% sucrose, no hormones. 

So a callus will be able to self-differentiate into normal shoots without hormonal induction?

That *might* work but it will also mess with the seedling.  So I would grow the seedling and callus separately if you want the seedling to continue growling normally.

Out of curiosity, how will it mess with the seedling? I have some Drosera aliciae for example, seedlings that are much too small to separate from what appears to be developing callus cells.  

Cory Tobin

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Feb 18, 2017, 5:16:10 AM2/18/17
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 I would grow the callus on plain media.  MS basal salts, phytoagar, 3% sucrose, no hormones. 

So a callus will be able to self-differentiate into normal shoots without hormonal induction?

Not very efficiently.  On plain, hormone-free media you just grow the callus to a much larger size.  That way you can split it up, expand it to cover more plates, etc.  If you want shoots and roots you'll need hormones.  You might get some shoots without hormones but it will work much better with the right hormone mix.

 
That *might* work but it will also mess with the seedling.  So I would grow the seedling and callus separately if you want the seedling to continue growling normally.

Out of curiosity, how will it mess with the seedling? I have some Drosera aliciae for example, seedlings that are much too small to separate from what appears to be developing callus cells.  

Depends on which hormones you're using and how Drosera responds to it.  Cytokinins, auxins, GAs, etc are all critical for proper developmental patterning and environmental responses in plants.  By introducing hormones to the media you short circuit signaling pathways that are critical to the plant.

In my experience with creating callus from Arabidopsis seedlings, the seedling never lives.  But if the health of the seedling is not an issue and you can't separate the callus, just put it on a callus inducing media, get some callus, (optional) transfer to plain media to bulk it up, then go to shoot and then root inducing media.

To answer your original question which is "will the callus growing on a seedling create more shoots with the addition of hormones?"  I would imagine if the callus is big enough you could get some shoots to initiate with the right combo of hormones.  But different species of plants have all sorts of weird reproduction and growth habits. So, maybe, maybe not?  I have some plants on my balcony, Kalanchoe daigremontiana, that grow somatic embryos on their leaf margins.  Imagine a human growing clone babies all over his or her arms and legs :)  Some plants behave weirdly. I don't have any practical experience with carnivorous plants to have much intuition about this, though.

-Cory

MC

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Feb 18, 2017, 1:12:18 PM2/18/17
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That way you can split it up, expand it to cover more plates, etc.

One more practical question, must a callus be cut up? Or can the whole thing by dropped on induction media? It's one of those little things of basic technique I miss out on for not taking horticulture labs in school...

This is all great information, thanks for your help. I have a ton of the D. aliciae seedlings so I think I'm going to put them all in different media and see what happens. I have a kalanchoe as well, at first I thought it was cool, but now it's growing like weeds and I'm finding plantlets all over the patio! BTW, I'm the one who brought the ants for K.G. at The Lab a few months ago if you remember. Hopefully I can become a member once I start getting into more advanced stuff!

Cory Tobin

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Feb 20, 2017, 1:26:42 PM2/20/17
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One more practical question, must a callus be cut up? Or can the whole thing by dropped on induction media? It's one of those little things of basic technique I miss out on for not taking horticulture labs in school...

Its not necessary.  You'll end up with more material if you do, though.

 
This is all great information, thanks for your help. I have a ton of the D. aliciae seedlings so I think I'm going to put them all in different media and see what happens. I have a kalanchoe as well, at first I thought it was cool, but now it's growing like weeds and I'm finding plantlets all over the patio!

Haha, yeah, kalanchoe is pretty prolific.

 
I'm the one who brought the ants for K.G. at The Lab a few months ago if you remember. Hopefully I can become a member once I start getting into more advanced stuff!

Oh right, I remember you.  I look forward to seeing you around again :)

-Cory

 

MC

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Feb 23, 2017, 2:23:37 PM2/23/17
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Just to confirm I'm identifying things correctly, this is a Drosera aliciae seedling with what I believe is a callus. I had sprouted these as well as a few other common species on a variety of media with different combinations of BAP and NAA for experimentation. If this isn't a callus, what could it be?

My plan is to let it grow out a little more before chopping it up to experiment with induction media.


Cory Tobin

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Feb 25, 2017, 4:55:14 AM2/25/17
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If this isn't a callus, what could it be?

Hmm, not sure.  All the callus I have experience with has been white or light green.  I'm not sure why it looks so red.  Honestly, if I didn't know I was looking for callus I would have guessed that was seed coat.
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