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Micropropagation and Tissue Culture of Plants


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#1 MurCurY

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Posted 30 November 2009 - 02:03 PM

People in chat have expressed alot of interest in this. So...I said I would do a write-up. This is going to take several days...maybe longer. I will post some sources for nutrients, hormones, and solvents for hormones. As with mushrooms, there is a learning curve in this. All I know is theory. I worked in a place that did tissue culture of plants. And I spent alot of time in the lab and asked alot of questions. Picked up the propper supplies....now for a propper writeup.
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#2 MurCurY

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Posted 30 November 2009 - 02:19 PM

Most people are interested in only TC of a few plants....

Here's the deal. All plants are able to be tissue cultured. All plants of Agro-economic value are TCed someplace in the world.

One of the questions I get alot is, "Is there a tek for XXX plant?"

The answer is....maybe.

Again, someplace in the world...i'm sure there IS someone culturing that plant. Is his process, formulas published? maybe? If that person, or group, or lab...spent alot of $ and time developing that process....probably not.

But...this is where the mind of a "Topiate" comes in. By using widely published papers on Agro-economic valued crops by widely accepted sources...like universities and extension services....you get a starting point. Again...may not be exactly your plant....but...if you find a plant that IS cultured...say...within the same family or even genus....then you have a starting point.

Say the paper calls for MS media and BAP. First you have to know what these are.
MS media
BAP
Ok...so where do you get these crazy chemicals?
First...plant hormones are really hard to dissolve. I have access to a chemical called DMSO. You...probably will not.
Most hobby culturists use KOH to dissolve the hormones.

WARNING: KOH is highly corrosive base and extreme caution should be
taken....it will turn your skin to soap...NO JOKE.

I get mine from AAA Chemical
HERE is a good video on how to weigh out and dissolve the hormones and mix the media.
And I get my hormones and media from HERE

And actually...his videos are great for starting out. The african violet is one of the easiest plants to TC...as well as begonias and probably anything else that spawns new plants easy from leaf...::cough::viridis::cough::

So....there's my start.

All chemicals supplies and sources are totally legal and legit.


More to come....
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#3 MurCurY

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Posted 30 November 2009 - 02:20 PM

Another member sent me this...can't remember who. Thanks.

This process can be applied to bunches of cactii.

Attached Files



#4 rocketman

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Posted 30 November 2009 - 07:17 PM

Awesome post! archive material when finished
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#5 roscoe

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Posted 01 December 2009 - 01:31 AM

alright im pulling up a chair. cant wait to get started on some kitchen culture.
:headbang:

#6 MurCurY

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Posted 02 December 2009 - 05:55 PM

Little update on this....

There's a book. Plants From Test Tubes
Highly recommended.

#7 thatgreenthumb

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Posted 02 December 2009 - 10:02 PM

My FOAF works in a micropropagation lab. He works mainly with embryo rescue from seed and he really enjoys it. He would like to work on some lesser studied entheogenic plants so a seat has been pulled up for this.
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#8 euid

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Posted 03 December 2009 - 01:19 AM

For Lophophora Williamsii medium:



Medium
---------

Base:

Standard Murashig and Skoog basal salt

Vitamines:

Niacin 0.5 mg/l
Thiamine Hcl 0.5 mg/l
Pyrodoxine Hcl 0.5 mg/l
I-Inostitol 100 mg/l

Dextrose:

30 g/l

Agar:

10 g/l


Combine 100ml of above with:
-----------------------------

1 ppm (1ug/ml) 2,4-D (2,4-Dichlorophenoxyacetic acid)
5 ppm (5ug/ml) BA (6-Benzylaminopurine aka. benzyl adenine)



Cheers.
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#9 euid

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Posted 03 December 2009 - 03:31 PM

To start your Lophophora tissue before you subculture onto the previously mentioned medium, use the same mix, but change the following:

include: 2,4-D (same concentration, 1ppm)

add: 10-15% liquid endosperm

exlude: 5 ppm 6-Benzylaminopurine

Lighting should be a 16/8 day/night cycle as well...

The cultures also like blue light, not so much the red.

From this initiation medium, to the subcluture medium you could experiment and find something better maybe?

#10 buteo

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Posted 03 December 2009 - 04:17 PM

ah murc thanks so much. im subscribing. i guess that cactus TC pdf was useful afterall. i didnt know if it would be bc it was a different species of cactus than we like...but i figured it was close enough.

thanks euid for the lophophora write up as well.
-b

#11 euid

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Posted 03 December 2009 - 04:42 PM

I'm pretty excited about this project, i'm looking forward to your final writeup Murc.

I hope to be able to contribute more as this moves along.

Great Idea!

#12 BotanyPhD

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Posted 03 December 2009 - 06:16 PM

When looking for a protocol type into a search engine the name of the plant common or scientific and then tissue culture or microporpagation it should list some protocols. I am vice preseident of an international micropropation organization, if you cannot find the protocol I may know someone that has done the plant and can get it for you. Just PM me.

I did a post a while back with sources for TC materials. If you are starting off dont mix your own hormones, it is simpler to get them from cassion labs or phytotech labs they are fresh and are quality controlled (not to mention not very expensive) small amount of cheap hormones can be purchased from Dr. Carol Stiff at her kitchen culture kit website she also hosts a webserve that has a wealth of information and a network of international people that work in micropropagation. She has kits,DVDs and is a phone call away for help.

I also would recommend using a chamical called PPM for use in your media if you are working at home to cut down on your contamination rates. It is fairly safe and I use it on my cloned food crops with no traceable residual properties in adult plants (confirmed from petiole grind tests).
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#13 buteo

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Posted 03 December 2009 - 09:49 PM

thank you botanyphd...you and murc always hand out such great info.:)
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#14 euid

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Posted 04 December 2009 - 05:34 PM

I've got some young lophophora williamsii that will suit this perfectly, i'll start a TC shortly and share the tek\logs\pics\info for this species as it progresses.

#15 MurCurY

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Posted 04 December 2009 - 05:43 PM

for anyone attempting this...2,4-d...is a SERIOUSLY strong plant hormone.
If you use it on broadleaf...it causes uncontrolled growth and potentially death in larger amounts. The amount your aiming for...causes the uncontrolled growth :up:

If you use this same chemical on a grass....nothing happens.
2,4-D is a common ingredient in herbicides and i believe in "weed and feed" stuff.

Is this for a slurry? I wonder if this wouldn't work for other actives as well.
Could you site this info?
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#16 euid

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Posted 04 December 2009 - 05:57 PM

I love science. :bow:

#17 euid

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Posted 11 December 2009 - 04:18 PM

Well, I just got resupplied (finally). I'll be starting a TC with my Lophophora Williamsii candidates as soon as possible.

#18 euid

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Posted 10 January 2010 - 02:40 PM

I have spent a while verifying some other individual results, and as expected i'm unable to initiate callus tissue growth from seed.

I've experimented with 10 Lophophora Williamsii seeds on varying media composition and none were successful.

I'm moving on to actual tissue now, i'll keep logs and post pics when there is something to show.

I might re-visit a seed initiation experiment in the future, by attempting to break dormancy by soaking the seeds in gibberellic acid for a duration first... Which i've not seen done before; but for now i'll move on.


Cheers.

#19 BotanyPhD

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Posted 11 January 2010 - 12:29 AM

For seeds what I normlly do (with cactus) is hit them with a little sandpaper to rough them up a little then do a 10% bleach solution soak for 10 minutes, triple wash and place them in 1/2 MS media they germinate into a nice small plant, take that plant then go for your callus proliferation. It will be sterile and ready to go.

#20 euid

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Posted 11 January 2010 - 01:18 AM

For seeds what I normlly do (with cactus) is hit them with a little sandpaper to rough them up a little then do a 10% bleach solution soak for 10 minutes, triple wash and place them in 1/2 MS media they germinate into a nice small plant, take that plant then go for your callus proliferation. It will be sterile and ready to go.


I never considered scoring the seeds to help break dormancy, not sure why. Thank you for the input on that front, I may have to try again sooner than later.

Cheers Botany, I hope you continue to track this one and provide input.
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