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#1539588 - 05/27/09 12:22 PM Re: Mothers in micro-culture **** [Re: omentheduck]
Matador Offline
Stranger

Registered: 05/26/09
Posts: 3
Hey:

Just read thru the post and I recalled that in the cannagram site: http://www.cannagram.20m.com/magazine.html#
there is an article that mentions immersing the xplants in a 5% calcium hypochlorite solution for 15 minutes as it was done in a successful 2005 study. I emailed Cannagram for links to the study itself and will post it if they get back to me with the info.

Keep up the good work guys,

Matador

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#1539617 - 05/27/09 03:08 PM Re: Mothers in micro-culture [Re: Matador]
RRog Offline
Carpal Tunnel
***

Registered: 08/26/08
Posts: 2326
Wow. An oldie but goodie resurrected
_________________________
I am not an Expert. I have so many posts because I ask a lot of questions.




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#1626954 - 03/25/10 11:47 AM Re: Mothers in micro-culture [Re: Loki]
Summerhouses Offline
Stranger

Registered: 07/09/09
Posts: 3
Wondering if Loki or OSM are still out there working on micro-propagation? Did we get flowers from any in-vitro cuttings yet? This is fucking inspiring.

So, I'm setting up a sterile lab right now with pretty similar ambitions: small mothers that are easy to carry around, and virus-free to boot if I can pull off cell culture like you have Loki. Apparently the meristem cells aren't connected to the vascular system of the plant yet, so cuttings from the very tips haven't been infected with any viruses or diseases that the plant may have. Can clean up old mothers. And I'm really thinking about applying this to a future vineyard, and seeing if vines from explants are just as vigorous as traditional cuttings -- in a few years I'm hoping to have a library of red wine grapes on my book shelf. I've already got 30 varieties of cuttings to start taking explants from when they break bud next month. I'm fucking stoked!

So I'm setting up the sterile lab now, and want to share some of the books and online resources that are helping me along, and hope there are some seasoned micro-propagators still around to throw questions at every now and then.

This book has an amazingly clear, DIY guide to tissue culture: "Growing Gourmet and Medicinal Mushrooms" by Paul Stamets (http://www.fungi.com/books/stamets.html)

And on the same website there's "Plants from Test Tubes: An introduction to micropropagation" which has medium recipes for 24 different plants and outlines the basics. Haven't finished this book yet, but it's been a very good primer on the subject, and very approachable with just a high school chemistry background. Stamets' book has a more detailed look at setting up a sterile lab though; it's aimed at putting together a professional grade set-up, or adapting the professional set-up to the hobbyist scale. Which is what I'm working on now. Bloody expensive hobby to start up on, but if you DIY, it doesn't have to cost your first-born's college tuition. So far I've spent 70 bucks on the raw materials for a large plastic tent (1" pvc for a 4'x6'x6' frame, duct tape, 6 mil plastic,and velcro for a door) and I built a laminar flow hood (http://www.fungi.com/tools/airfilters.html) about the size of the series II for the cost of a sheet of furniture grade ply, a flange for my 6" inline fan, and the $230 bucks for a 2'x2' HEPA filter. Sealed it up tight with wood glue and caulk. I already have the fan to blow thru the laminar flow hood (a 6" can/filter combo; keeping a carbon filter downstream of the HEPA filter to prolong it's life). So those 1200 dollar units on the website can really be made at home for 300-odd dollars, plus a fan.

So at this point I have a small, airtight space I can spray down with a bleach solution before I work, and a filter that screens the air down to .3 microns with 99.99% efficiency: the air in that workspace is effectively sterilized of airborne germ and mold spores, and in a tent, it's being recirculated/resterilized a couple of times a minute. I still need to pick up a pressure cooker, agar, scalpels, alcohol lamp, petri dishes... but I should have it all together within a month. And then the mad experiments!

Hope some of the old heads are still around. Take it easy.

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#1642402 - 06/04/10 04:19 AM Re: Mothers in microculture [Re: sad old man]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
Originally Posted By: sad old man
Hello everyone. I'm new here and I'd like to pose a question to the group.
Does anyone out there know what the plant specific recipe for the agar mix used to put a cannabis cutting into in-vetro microculture is? I would like to know the amount of the hormones in each type of culture, shoot developement and rooting phase. The ammount of finished medium is to be 1 liter. Any help would be appreciated. I know I'll get some help here,so thanks in advance.


Hi.

I live in the UK and am interested in this technique.

I REALLY don't want to approach conventional suppliers and want an easy recipe for agar mixes where:

MEDIUM A) STIMULATE ROOT
MEDIUM B) STIMULATE SHOOT
MEDIUM C) STIMULATE DIVISION / MAINTENANCE

DIY RECIPES WELCOME!

Using the following webpages as guidelines:
http://www.thcfarmer.com/forums/f9/micropropagation-cannabis-19773/
http://www.shroomery.org/23/Agar
http://www.shroomery.org/9427/Grocery-Store-Agar-Tek


I see that I need to be able to obtain / manufacture two groups of hormones:
CYTOKININES
AUXINS

Also I am unable to locate a DIY M&S (MS or Murashige or Skoog) culture medium recipe.
Worse still am lost on how to locate the chemicals and micronutrients required. Currently considering liquid fertiliser.

LIST OF REQUIRED CHEMICALS FOR CULTURE MEDIUMS:

MS or Murashige or Skoog addative to Agar Agar?
HELP! Still no easily obtained substitute.
Will any of the mixes listed on the following link be a good substitute?
http://www.shroomery.org/9393/FastFreds-Media-Cookbook

NAA (Auxin)
naphthylacetic acid (NAA)
SOLVED! DIY rooting powder active ingredient NAA 0.25% w/w

IAA (Auxin)
indole-3-acetic acid (IAA)
HELP! Any ideas on products that contain quantities of this hormone?
Is it an Auxin or a Cytokinine?
If this really is an Auxin what products should I seek out for a suitable cytokinine?


Answers on a postcard please!

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#1642858 - 06/06/10 07:31 PM Re: Mothers in microculture [Re: Bloo]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
Bump!

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#1642894 - 06/07/10 04:23 AM Re: Mothers in microculture [Re: Bloo]
Earl Offline

Pot Head
****

Registered: 04/07/07
Posts: 3714
Loc: Synthetic farm
I think you should look into some biology courses at a college near you.

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#1643189 - 06/08/10 03:37 PM Re: Mothers in microculture [Re: Earl]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
Thanks E. I'll get right on that!

In the meantime - products that contain suitable citokinins?

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#1643396 - 06/09/10 03:06 PM Re: Mothers in microculture [Re: Bloo]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
E. Might be right.

A course would give me access to equipment and know-how.

Bit long-in-the-tooth for it maybe!

In terms of this experiment, I'm resolved to 'trial and error' but any input is welcome.

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#1643418 - 06/09/10 04:29 PM Re: Mothers in microculture [Re: Bloo]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
Originally Posted By: Bloo
In the meantime - products that contain suitable cytokinins?


CYTOKININS Solved?
"Nitrozyme" £8.00 100ml 400ppm
http://www.progrow.co.uk/acatalog/info_282.html



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#1643570 - 06/10/10 11:09 AM Re: Mothers in microculture [Re: Bloo]
Bloo Offline
Stranger

Registered: 05/26/10
Posts: 8
Urticaceae anyone?
http://www.thcfarmer.com/forums/f9/micropropagation-cannabis-19773/

"This regeneration technique can also be used to create new hybrids: ... the generation of protoplasts (vegetable cells without their cell wall), and the fusion of two protoplasts from two different plants. So we obtain a cell, with both the two genomes, expressing all the characteristics of the two plants.

In addition, these cells are at minimum tetraploïd (or a lot more), so the stock of growing and blooming hormones is double.

You now just have to regenerate these merged protoplasts with micropropagation, and you will obtain gruesomely hardy and productive plants. And you could clone or micropropagate them..."

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