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Micropropagation and Tissue Culture of Plants


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#21 BotanyPhD

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Posted 11 January 2010 - 01:33 PM

Well think of it this way... the plant has to reproduce in some harsh conditions. The seeds had to survive drought, being eaten whole by predators, being munched on my mice and other critters. You have to be armored. This seed is well armored. To get water in you need to break that armor down a little bit. Soaking might work, but if the nick it up with sandpaper the water can penetrate the seed a little more and start the germination process.

I have used this also with Passiflora species and it works well. They are armored pretty. Also datura can benefit from this too if you have a hard time with the seeds.


Doc

#22 Psiloman

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Posted 11 January 2010 - 02:09 PM

Hello, this is my first post here!

All in all very interesting thread! I see tissue culture as an excellent start not only for propagating plants but also for running experiments on them. Some very interesting procedures either require or can be simplified by callus culture and micropropagation.Two of them that come to my head are:

1) Protoplast fusion :
To put it quite straight forward,the cell walls of plant cells are digested so cells can fuse together. This can create polyploidy if its from the same plant species ,hybrids if it is from different plant species originating from the germ line of cells (haploid cells), or heteropolyploidy if the cells come from two different species but are somatic (not germ) cells. The above might be difficult without micromanipulation ,so for the "kitchen biochemist" it might be a bit out of reach.

2)Inducing polyploidy:
Inducing polyploidy can have many benefits, from giving more virile plants to upping production of secondary metabolites in the plant cell. While many people's notion of inducing polyploidy has stuck to the "ol' colchicine" (which is dangerous) other polyploidy inducers can be used which are safer and more effective ,for example trifluraline (search the web for trifluraline inducing polyploidy). In a cell culture cells can be exposed to the antimitotic agent and then grown as plantets. Given that the callus has quick cellular division this ups the rate of success.Such a project is within the reach of the "kitchen biochemist".

Hope you find the above interesting, i can provide further info if one is interested.

#23 Myc

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Posted 11 January 2010 - 07:14 PM

I've asked many of these questions myself over in the MJ forum.
Own a copy of Lydiane Kyte and John Kleyn's book - Plants from Test Tubes

euid,
This formula you posted is proven?? I'm curious 'cause most guys are waaay guarded with that formula. You just spoiled my plan if it works. I was gonna post a formula for the masses and piss 'em off!

I'm buckled in to watch this one.

#24 euid

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Posted 11 January 2010 - 07:40 PM

Hey Myc, I plan to verify some results with this.

I should have something in the next 30 days, i'll be starting this week and will continue to update... But yeah, if I can verify the protocol that I have, i'm all about ruining this for people who don't like to share.

Information should be free, it's the only way we can evolve and move forward.


Cheers

#25 euid

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Posted 15 January 2010 - 07:39 PM

I have now started the initiation of callus tissue growth:


Species: L. Williamsii

Medium: MSk (100ml per vessel)

Components:

- 2,4-D (1 ppm)
- Dextrose (3g)
- Agar (1g)
- Liquid Endosperm (10ml - 15ml)
- MSk Vitamins (0.5g)


Two vessels are 10% liquid endosperm, one vessel contains 15%.

Once this phase is complete and callus tissue growth has been initiated, i'll be subculturing it onto a different medium and will post the information.

We'll see how this phase progresses over the next 30 days; i'll update as appropriate.


Cheers

Attached Thumbnails

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  • Culture1.Jpg
  • Culture2.jpg
  • Culture3.jpg
  • Culture1.Jpg


#26 Psiloman

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Posted 15 January 2010 - 09:40 PM

Euid ,do you plant on experimenting with the cultures or are you going to use them utlimately for propagation of adult plants?

#27 euid

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Posted 16 January 2010 - 01:46 AM

Euid ,do you plant on experimenting with the cultures or are you going to use them utlimately for propagation of adult plants?



Propogation, ultimately... Experiments mixed in.


Cheers.

#28 Myc

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Posted 16 January 2010 - 11:13 AM

FUCK AN A BABY!!!

You had better believe that I will be watching closely.
I'm totally capable of this science.

To answer a question,
The goal here is cloning
As in MJ cloning - you can have a "mother" plant from which you can propagate....perpetually

Lopophora is very slow from seed - and from offsets - thus, its endangered status in the wild.
Sustainable laboratory/nursery propagation could relieve stress on natural populations and prevent them from becoming all-together extinct.

Read "Fingerprints of the Gods"
We aren't the only advanced civilization to populate tha planet.
I hope to distribute and leave as many wide-spread species behind as possible - for future evolutions/civilizations
Our attitude of "take what is mine..bury what's not..." is going to kill the purpose of our existance

#29 Deus Irae

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Posted 16 January 2010 - 12:09 PM

This guys youtube channel has some good vids on micropropagation. Fascinating stuff.
Thanks to everyone who's posted info so far.

http://www.youtube.com/user/fbt2007

#30 euid

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Posted 16 January 2010 - 12:51 PM

Lopophora is very slow from seed - and from offsets - thus, its endangered status in the wild.
Sustainable laboratory/nursery propagation could relieve stress on natural populations and prevent them from becoming all-together extinct.


I'm with you there man.

#31 euid

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Posted 22 January 2010 - 11:23 PM

Just for the hell of it... I saw this formulation posted by someone else (somewhere else):

Loph Media/Pedro pupping [my original formula, causes vitrification]:
1/2 MS w/ vit./L
0.7mg/L PPM [trying 1.0 mg/L]
5.0mg/L BAP
0.5mg/L NAA
25g/L sucrose [trying 20g sucrose + 5g dextrose]
~5.80 pH adjusted w/ H2SO4

Loph Media [my newest recipe, no vitrification and stronger growth]:
1/2 MS w/ vit./L
1.0mg/L PPM
5.0mg/L BAP
0.5mg/L NAA
20g/L sucrose
5g/L dextrose
~5.80 pH adjusted w/ H2SO4


I will say... I question these ones.

I also saw pictures of what was supposedly growing on this medium and it looked abhorrent... Nightmarish.

I'd be too afraid to ingest lophophora ever again, after subjecting them to that; lest the peyote spirit subject me to the same punishment.

Yikes.

#32 euid

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Posted 29 January 2010 - 09:21 PM

Day 15 and I have successfully initiated callus tissue growth.

I have lost one vessel to bacteria (which was my fault -- not dirty explants); down the road I may have to try your PPM additive, BotanyPhD.

The vessel lost was the one with the medium using 15% liquid endosperm, so i'm not very concerned as it was just an aside to see which would initiate growth faster (with my own eyes; I already have the data)

Any concentration between 10-15% will work, and the reason to hit somewhere in that range is because of the varying levels of trans-zeatin riboside in the LE, as it comes from a natural source (green coconuts).

The 10-15% range is also the range which showed the fastest growth and most friable callus tissue. This was tested in varying concentrations between 5-20%


At any rate, pic attached and i'll continue to update as it becomes relevant.

Attached Thumbnails

  • Callus-15d.JPG

Edited by euid, 29 January 2010 - 11:06 PM.


#33 euid

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Posted 29 January 2010 - 09:52 PM

An important piece of information that I have left out (unintentionally):

If you are going to try this with Lophophora Williamsii, you need to use young plants. The tissue I am using was taken from a plant that was ~90 days old.

You won't want to use a plant that is much larger than 1-1.5 cm (~1 year old)

Also, you need to use explants from the whole stem section. Slice them around 1mm thick.

End.

#34 euid

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Posted 30 January 2010 - 01:42 AM

I've washed the explant and medium that was contaminated... We'll see if it continues to develop cleanly; without having to pour a new vessel...

...I was bored. ;)

#35 Myc

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Posted 30 January 2010 - 01:57 AM


Also, you need to use explants from the whole stem section. Slice them around 1mm thick.

End.


Was following you until now...........
Pics?

#36 euid

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Posted 30 January 2010 - 02:06 AM

Was following you until now...........
Pics?


You would take 1mm thick cross sections of the whole stem, simmilar to the pic attached.

No root sections, they don't work well.

Whole stem sections, not parts or pieces.

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#37 BotanyPhD

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Posted 30 January 2010 - 03:43 AM

are you using PPM or Kathon in the media to cut down on contamination?

#38 euid

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Posted 30 January 2010 - 01:09 PM

are you using PPM or Kathon in the media to cut down on contamination?


No, though I might experiment with something like PPM down the road, when I have more tissue to work with.

#39 BotanyPhD

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Posted 30 January 2010 - 01:20 PM

Although it violates patent law.... kathon can be used it is the same as PPM just diluted and is a hell of a lot cheaper. I am going to see how it works in mushy agar and if it cuts the contams back or just halts all growth. If it just cuts contamination back that might be a good addittion to put in water for BRF initial hydration. Imagine a world without green in the jars.

#40 euid

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Posted 30 January 2010 - 01:31 PM

Although it violates patent law.... kathon can be used it is the same as PPM just diluted and is a hell of a lot cheaper. I am going to see how it works in mushy agar and if it cuts the contams back or just halts all growth. If it just cuts contamination back that might be a good addittion to put in water for BRF initial hydration. Imagine a world without green in the jars.


That would be spectacular... Please let me know how it works.

Great idea man.

Edited by euid, 30 January 2010 - 03:30 PM.





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