Plant Tissue Culture

bombbudpuffa · Jul 19, 2011

Thought i'd share a few pics of one of my hobbies. Its not really that hard to do but theres a lot of info to remember. I took a couple of months to read and learn about it before attempting it. Cleanliness really is next to godliness when it comes to growing plants in a tube though.

I've tried it with cannabis with marginal success. I could get growth but my sterilization technique for canna isnt what it should be. Right now i'm practicing with carnivorous plants. They aren't only some of the easiest species to culture but also some of the funnest. I mean, they eat things

.

1. Venus Flytrap started from seed in vitro
2. Venus flytrap seedling in vitro
3. Flytrap flower stalk with lots of growth
4. Flytrap leaf with several divisions
5. Drosera or sundews germinated in vitro
6. Sundews germed in vitro

I'll probably give canna another shot once I perfect my technique a lil.

Locked · Jul 19, 2011

Very cool and very interesting.....keep us posted puff.

4u2sm0ke · Jul 19, 2011

pulling up my milk crate and have pen and papper at the read...

:ciao: give me a learning Bombbudpuffa!!!

:48:

bombbudpuffa · Jul 19, 2011

HL-Will do

4U-How about I just point you in the right direction?

This guys videos are very detailed and he tells you everything from where to get supplies down to how to do it. I will warn you that every plant needs different hormones to be tissue cultured successfully. There are usually "how tos" called protocols for all of the different plants you can do it with but I can find zero for canna so you'll have to find the right combination and strengths for canna through experimentation.

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

ozzydiodude · Jul 19, 2011

bombbudpuffa · Jul 19, 2011

If you want to learn I can throw up some links and recommend some books for you guys

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ozzydiodude · Jul 19, 2011

I don't watch vids but throw up them up for others, I'll read everything I can and put in alot of dumb looks(natural for me)

bombbudpuffa · Jul 19, 2011

Seriously Ozzy...if you want to learn watching videos would be quicker because theyre going to use terms in the books that you wont understand. The vids will let you actually see what theyre talking about and kind of be like a refresher course. Not to mention, the vids are super boring so you can imagine what the books are like

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ozzydiodude · Jul 19, 2011

But I got to buy a new comp to run videos. Right now looking thing I don't understand up is the only thing other the PT that is keeping me sanely insane

pawpaw · Jul 19, 2011

Very cool bombbudpuffa, are you using a laminar flow hood?

bombbudpuffa · Jul 19, 2011

Supplies-

hxxp://hometissueculture.org/cataloghtcgsupplies.htm#Media%20Zero

Random links for basic understanding-

hxxp://www.bio.davidson.edu/people/kabernd/seminar/2002/method/amy/aj.htm

hxxp://www.scribd.com/doc/3544465/Plant-Tissue-Culture

hxxp://tech.groups.yahoo.com/group/hometissueculture/

hxxp://www.omnisterra.com/botany/cp/slides/tc/tc.htm

hxxp://www.ccebook.org/preview/0881923613/Plants-from-Test-Tubes-An-Introduction-to-Micropropagation

hxxp://csm.jmu.edu/biology/renfromh/pop/pctc/tcstart.htm

hxxp://www.flytrapcare.com/tissue-culture-basics.html

hxxp://www.archive.org/stream/handbookofplantt00whit/handbookofplantt00whit_djvu.txt

That should be enough to keep you busy for a while. Tried to just post the easy to understand links

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bombbudpuffa · Jul 19, 2011

pp-nope

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pawpaw · Jul 19, 2011

bombbudpuffa said:
pp-nope.

Wow:holysheep:

I've got two books:
Plants from Test Tubes: An Introduction to Micropropagation [Lydiane Kyte, John Kleyn]
and
Introduction to Plant Tissue Culture [M.K.Razdan]

I have only skimmed these but the first looks quite good as a howto. The second looks even more technical than I want to get. I wouldvery much like to know which books you recommend.

Thanks.

bombbudpuffa · Jul 20, 2011

The first one you listed is the best one imo. Easy to understand with lots of info and even a section on protocols

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Locked · Jul 20, 2011

Looks like school is in session....putting on my thinking cap....

Thanks for the links puff. Good to see ya posting again.

pawpaw · Jul 20, 2011

I have two pdfs that may be of interest to you. one is titled,

INFLUENCE OF CULTIVAR, EXPLANT SOURCE AND PLANT GROWTH
REGULATOR ON CALLUS INDUCTION AND PLANT REGENERATION
OF CANNABIS SATIVA L.

hemp. Hemphil et al. (1978), Fisse et al. (1981), Mac-
Kinnon et al. (2000) and Feeney and Punja (2003)
reported that callus readily produced roots but was
unreceptive to shoot formation. Mandolino and Ranalli
(1999) reported occasional shoot regeneration from callus.
Successful regeneration and propagation has been
achieved in other fibrous plants such as sisal (Das,
1992; Hazra et al., 2002) and flax (Rakousky´ et al.,
1999; Yildiz and Ozgen, 2004). Conventional breeding
and biotechnological approaches, including tissue culture
and transformation procedures, could be extended
to hemp breeding.
This study was intended to determine the optimal
combinations of plant growth regulators for callus induction
and plant regeneration of five hemp cultivars,
using different types of explants.

The other is

Assessment of the Genetic Stability of
Micropropagated Plants of Cannabis
sativa by ISSR Markers

Inter-simple sequence repeat (ISSR) markers were used to evaluate
the genetic stability of the micropropagated plants of Cannabis
sativa over 30 passages in culture and hardening in soil for 8
months. A total of 15 ISSR primers resulted in 115 distinct and
reproducible bands. All the ISSR profiles from micropropagated
plants were monomorphic and comparable to mother plants,
confirming the genetic stability among clones and mother plants.
Chemical analysis of cannabinoids, using gas chromatography/
flame ionization detection (GC/FID), was done to further confirm
whether the qualitative and quantitative differences in the major
secondary metabolites exist between the mother plant and micropropagated
plants. Six major cannabinoids Δ9-THC, THCV,
CBD, CBC, CBG, and CBN were identified and compared with
the mother plant. Our results clearly showed a similar cannabinoid
profile and insignificant differences in THC content between
the two types of plants. These results suggest that the micropropagation
protocol developed by us for rapid in vitro multiplication
is appropriate and applicable for clonal mass propagation of
C. sativa.

I've tried to upload a zip of the first. It's 8.3 MB zipped. But after clicking on upload the Manage Attachments dialog displays the please wait msg then evetually closes with no joy. Can I have a clue?

View attachment TissueCulture Geneticstability.pdf

View attachment TissueCulture_1.zip

Mutt · Jul 20, 2011

Howdy BBP nice to see ya about.

sterilization technique

What is your lab like that your are doing cultures in?
Sterilization is limited to availability of contaminates.
Can you describe your environment in your process?
Got a lot of experience with "contamination" in "another" hobby. might be able to help ya out.

Are you PCn all of your equipment to at least 15psi for at least 10-15 minutes?

Locked · Jul 20, 2011

Mutt said:
Got a lot of experience with "contamination" in "another" hobby. might be able to help ya out.

Sounds quite Magical Mutt.........

Mutt · Jul 20, 2011

Hamster Lewis said:
Sounds quite Magical Mutt.........

it's not much difference then doing any other kind of culture.

ozzydiodude · Jul 20, 2011

That's some of the best "go to sleep " reading I had done in awhile. Very interesting was to keep strains alive in a small space. This might be the way for a person to keep a living sample of the strains that truely need to be saved for future generations

Plant Tissue Culture

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