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Micropropagation and Tissue Culture of Plants


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#41 euid

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Posted 30 January 2010 - 03:29 PM

Hit the wrong buttons

#42 Myc

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Posted 30 January 2010 - 04:17 PM

Thanks for the photos -
I'm back with you now - so lay a 'cucmber slice', one millimeter thick, onto the nutritive substrate and offsets occur at the nodal points?


Pardon me but I'm brand-new to this and learning at home so bear with me - no PhD here - I just tackle things (like this) and get lucky sometimes. ;)

#43 BotanyPhD

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Posted 31 January 2010 - 12:23 AM

Euid have you tried liquid culture? Do you have a stir plate or a shaker??? I got totally amazing results off of bamboo.. I bet cacti would just respond like crazy...

As soon as I find a nice contaminate syringe Ill try out that kaython mix

#44 euid

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Posted 31 January 2010 - 02:50 AM

Thanks for the photos -
I'm back with you now - so lay a 'cucmber slice', one millimeter thick, onto the nutritive substrate and offsets occur at the nodal points?


Pardon me but I'm brand-new to this and learning at home so bear with me - no PhD here - I just tackle things (like this) and get lucky sometimes. ;)



Yeah, between 1-2mm thick. It is hard to cut them when they are 90 days old, because they are so small... so mine ended up being a bit thick.

So then you lay these cross sections on your medium and they grow into a mass of cells (callus tissue growth or "callogenesis"), like this carrot callus tissue:

Posted Image


From there you move on to different medium, with different levels of auxins and cytokinins to induce growth of stems and/or roots (organogenesis), which will grow out of this cellular mass.

Lots of work ahead, but it'll be fun.

#45 euid

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Posted 31 January 2010 - 02:53 AM

Euid have you tried liquid culture? Do you have a stir plate or a shaker??? I got totally amazing results off of bamboo.. I bet cacti would just respond like crazy...

As soon as I find a nice contaminate syringe Ill try out that kaython mix


I have not ever tried liquid culture actually... I'd love to though, do you have a protocol for bamboo that you can share, so I could try it out?

Very cool.

#46 BotanyPhD

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Posted 31 January 2010 - 11:58 AM

What type of bamboo you want to do? I wish you much luck on getting it clean its about a 90% fail rate and higher because the plants are just naturally infected with everything! lol its fun though.. one thing you cant get discouraged when playing with this stuff.. expect failure.. the suprises are the accomplishments..

#47 euid

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Posted 01 February 2010 - 10:59 PM

Well, Maybe we'll save the Bamboo for another day. I am interested though.

I do have a protocol for LC with Loph, maybe i'll try that out once i've grown enough tissue to experiment with.


Thanks Botany.

#48 euid

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Posted 12 February 2010 - 08:45 PM

So here we are at ~30 days. I've initiated approximately three times the tissue I started with.

I've subcultured from the initiation medium to a growth medium that is as follows:


Volume: 100 ml (per vessel)

Contains:

MSk basal salts w/vitamins: 0.45g
Dextrose: 3g
Agar: 1g
2,4-D: 1 ppm
BA: 5 ppm


Attached are photos of the callus tissue from initiation; of which I took three pieces from each vessel. The callus tissue grown on the first medium containing liquid endosperm was very friable, which is desireable.


I will let all of these grow and I will continue to subculture the callus tissue onto the growth medium.

Once I am satisfied that I have enough tissue; I will start experimenting to establish a third and fourth medium for organogenesis to complete this process/protocol/tek, or whatever you want to call it.

Attached Thumbnails

  • NEW6VCT.JPG
  • V1CT.JPG
  • V2CT.JPG
  • SC2.JPG
  • SC1.JPG


#49 euid

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Posted 12 February 2010 - 11:11 PM

As a matter of interest, this is a comparison of the same explant on the initiation medium at 15 and ~30 days:

15 Days:

http://mycotopia.net...-callus-15d.jpg


30 Days:

http://mycotopia.net...plants-v1ct.jpg

#50 Myc

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Posted 13 February 2010 - 02:02 AM

Still watching with keen interest. :thumbup:

#51 euid

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Posted 13 February 2010 - 04:42 PM

I believe that the major roadblock people might have had in the past with culturing Lophophora, is the age of the explant tissue. As mentioned earlier it appears to be one of the "keystones" that no one cares to mention or isn't aware of in the first place.

Even then, 99% of what i've seen from others isn't a protocol at all, its just, "I used this combination of crap and it kinda worked", no mention of environment or variables or any real information at all.

On that note, here are the environmental variables:

Light: Low light, full spectrum (~500 lumens/sq ft., standard cool white CFL)
- Day Light Cycle: 10 hours

Temperature:
Day: ~25 degrees centigrade
Night: ~ 16-18 degrees centigrade



Also interesting in regard to this particular protocol that we're unfolding here; your callus tissue will be very simmilar to a live plant in alkaloid content... Do with that information what you will.


Cheers.

Edited by euid, 13 February 2010 - 07:23 PM.


#52 euid

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Posted 15 February 2010 - 06:13 PM

The subcultured tissue has taken on a translucent red appearance on the new medium. I'm not sure of the cause, possibly the 2,4-D or an error in my lighting...

Bacteria is not a suspect quite yet and it doesn't look like necrosis.

Hopefully this changes in time and I continue to see growth of green tissue.

Any thoughts here BotanyPhD?

#53 whatchamacallit

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Posted 15 February 2010 - 06:57 PM

Subscribing.. Be cool to take a piece of iboga plant and grow a whole new one. This could be a way to multiply the plant to a large enough amount, that anyone needing ibogaine treatment could get it..

#54 euid

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Posted 15 February 2010 - 10:36 PM

A Gabonese agricultural institute conducted research relating to micropropogation of Tabernanth iboga in 2005, it is a very important crop in Gabon.

I would imagine the protocol is fairly straight forward.

Are you planning to try this in the near future?

#55 BotanyPhD

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Posted 16 February 2010 - 08:53 AM

That could be a variety of things as a rule if it isnt spreading to the media it isnt bacteria or mold. My barrel cactus use to turn red when I went to a multiplication agar. I sometimes had the callus die after that. Transfer to new media every two weeks if you can it might prevent premature death. When was the last transfer? What media was it on and what media are you going to?

#56 euid

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Posted 16 February 2010 - 05:06 PM

I prepared a squash slide and examined it under my microscope; there are plenty of healthy cells within the mass...

I'm chalking this up to a stress reaction as a result of subculturing the tissue... for now.

I'm already seeing some new translucent-nodular growth on some of the explants.

We shall see what time brings.

#57 euid

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Posted 03 March 2010 - 06:06 PM

...Time brought bacteria and all of my subcultured tissue died. The moral of this story is:

"Don't rush."


At any rate; i've started a new round of callogenesis.

Back to medium #1.

#58 BotanyPhD

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Posted 08 April 2010 - 11:33 AM

Euid what the status of your work? Curious to see what you have

#59 euid

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Posted 09 April 2010 - 12:20 PM

Work is getting in my way a bit, I have been unable to move on to an attempt at inducing ogranogenesis... the initiation and multiplication medium are verified, however.


When I am able to start back up, i'm going to work with some older plants, as well (~1 year old vs. the ~4 month old plants i've been working with to date)

#60 BotanyPhD

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Posted 09 April 2010 - 03:28 PM

[quote name='euid']...Time brought bacteria and all of my subcultured tissue died. The moral of this story is:

"Don't rush."



Do you need PPM or some other wonder stuff to keep the bacteria out?




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