Tissue Culture Protocol for MJ

[BotanyPhD]

I have gotten several requests for doing my speciality tissue culture... requests have been for various strains of Cannabis.. I have cloned just about every other plant on the earth. I have not tried this but have the required information for you to get started.. There are several videos on other plants on Youtube you can watch to get some basics of how the whole tissue culture thing goes down. Banana is a fun plant to watch, although it is a monocot. The easiest protocol I have found is below from a scientific journal...

Induction of high-frequency shoot regeneration using nodal segments containing axillary buds from a 1-yr-old mother plants of Cannabis sativa was achieved on Murashige and Skoog (MS) medium containing 0.05–5.0 μM thidiazuron. The quality and quantity of regenerants were better with thidiazuron (0.5 μM thidiazuron) than with benzyladenine or kinetin. Adding 7.0 μM of gibberellic acid into a medium containing 0.5 μM thidiazuron slightly increased shoot growth. Elongated shoots when transferred to half-strength MS medium supplemented with 500 mg l−1 activated charcoal and 2.5 μM indole-3-butyric acid resulted in 95% rooting. The rooted plants were successfully acclimatized in soil.

MS is the most commonly used nutrient base in tissue culture. It can be found at Phytotech Labs website. They sell it in various packets from 1 liter dose packs to huge amounts. It is fairly cheap. You can mix it in a gallon jug and freeze what you dont use and use it next time... its fairly stable.. Part of this protocol asks for 1/2 MS or half strenght MS. Just double the water on a 1L packet.

Thidiazuron is a herbicide. It works like a plant growth regulator at this low dose. Again it can be found at Phytotech labs.

Glibberic acid is there too commonly used for busting dormancy on seeds it works well in the tissue culture media. It is found at Phytotechs Website.

Phytotechs website; http://www.phytotechlab.com/

Link to MS media for this protocol is; it runs about $1 a liter http://www.phytotechlab.com/detail.aspx?ID=452

Whats in Ms? its listed below! I might try a mushie grow with some of this in the water since I have inadvertently grown fungus on my plant medias from a wild spore. Neat stuff..

Ammonium Nitrate 1650 Boric Acid 6.2 Calcium Chloride, Anhydrous 332.2 Cobalt Chloride•6H2O 0.025 Cupric Sulfate•5H2O 0.025 Na2EDTA•2H2O 37.26 Ferrous Sulfate•7H2O 27.8 Magnesium Sulfate, Anhydrous 180.7 Manganese Sulfate•H2O 16.9 Molybdic Acid (Sodium Salt)• 2H2O 0.25 Potassium Iodide 0.83 Potassium Nitrate 1900 Potassium Phosphate, Monobasic 170 Zinc Sulfate•7H2O 8.6 Glycine (Free Base) 2 myo-Inositol 100 Nicotinic Acid (Free Acid) 0.5 Pyridoxine•HCI 0.5 Thiamine•HCI 0.1

Glibberic acid is found here. (Also called GA) At this strenght use 1/2 a mL its close enough for clone work;

http://www.phytotechlab.com/detail.aspx?ID=332

2.5 μM indole-3-butyric acid (or IBA) is found at:

http://www.phytotechlab.com/detail.aspx?ID=386


How do you get a clean plant to put in vitro??

This is the hard part and this is where people get pissed off because they get contaminated cultures. I can lessen the amount of contamination if you follow this easy cleaning protocol;

1) Excise you explant. Explant is the plant you are going to use in vitro. Here we are using the nodal segments containing axillary buds. Cut above and below the node. I suggest using surgical gloves when handling the plant to lessen contamination. Use a clean non serrated edge knife to make the node excision. I would say if you got the plants take 3 nodal cuttings.

2) Take a mason jar wide mouth quart, take the middle flat plate out. Replace it with window screen and screw the ring cap over the screen after placing your explants in the jar. Put the jar under your kitchen sink spigot and turn the water on. Let the water flow freely into the jar got 15 minutes. This will use gentle water pressure to remove gross contaminates from your explant. After it is done drain the water and get your explants to you hood or clean box quickly.

3) Next is the alcohol dip. Use regular strenght alcohol for this. Using a long pair of tweezers or a hemostat that is clean remove the explant from your screened over jar and place it in a jar that contains alcohol for about 10-15 seconds. This helps further remove contaminates.

4) Next you want to have on hand a 10% Ultra Bleach Solution. I make it in a measured shotglass that has mL on it. 10mL of bleach and 90mL of distilled water.. Make about 5 glasses full of this stuff and put it in a separate mason jar with a normal lid (dont autoclave this stuff make it up fresh). Place one drop of dish washing soap in the bleach solution. Place your explants from the alcohol dip directly into the bleach water. Now shake it up moderately for 10-15 minutes.

5) when done shaking you will use tweezers or hemostats remove explant from this solution and transfer the explant to a jar of plain distilled water. this will dilute/ wash off the bleach and stop killing good tissue. 6) Take explants out trim each end of the node (cut off bleached ends to get to nice green tissue and place each explant in a jar of your media). Thats how you clean the plant....


Ok how to make the media!! Easy stuff... Use Agar or other types of gelling agents.. I prefer agar... follow the instructions to make 1 liter... What I normally do it take 1 liter of water and place it in a teflon pot and bring it to a boil.

Once it boils chuck in the MS nutrients, shake in agar and I add a chemical called PPM (plant preservative solution anti fungal and anti bacterial... found here... cuts down on contamination http://www.hometissueculture.org/catalogHTCGsupplies.htm ( I use 1 mL/L)....... you will see the water go from cloudy to clear.. the agar is melted and the media is ready for jars.

Put media in jars and pressure cook at 15 lbs for 15 minutes. When you take it out of the Pressure cooker make sure it is still kinda hot and swirl the jar a bit to make sure the agar mixes.. if you dont you will not get a good gel set. Swirl is pretty critical..

You can learn how to do tissue culture via a kit and get a nice set of equipment to use and get a nice DVD here (I am friends with the president of the non-profit) : http://www.hometissueculture.org/catalogkits.htm

Alternative places to get required chemicals at: http://www.caissonlabs.com/

Compare prices.. Phytotech isnt always the cheapest.


Start asking questions.. I am here to help!

[weeeeeee]

damn nice thread im gonna have to subscrbe for sure and re-read when im less stupid

[nra9785]

Nice man!
And I will start to ask questions!
This is right where I want to go, I graduate with a BS in Biology in MAy and am deciding whether to go into Ecology of Bryophytes or Ecology of Fungi or just straight Mycology or Bryology

[BotanyPhD]

this transfers over to mycology pretty easily.... I am just getting into it.. Id go Ecology myself.. just because I am a eco- kinda guy... Its more expansive and interdiciplinary...

[troncotron]

Tissue culture in cannabis is not very hard once you have the regulators, media, equipment (glovebox or flowhood) and if you are familiarized with sterile techniques. Here is some photos of my experiments..
I used MS media supplemented with kinetin, AIA, sacarose and pH adjusted to 5.8 before sterilizing.
Good luck!

[TheFreak]

i just seen this in hightimes. it's pretty cool. very interesting stuff. if it is ok i can take a picture of the articles an post them. up to the mods though.

[Stonehenge]

I fooled around with this a while back. You absolutely need a glovebox at a minimum and a flowhood is better. What stopped me was constant contamination. Even jars i had sterilized and left in the pressure cooker ended up contamed. I found out later that it was from mites getting into the jars. Your jars have to have a tiny crack for ventilation and the dust mites crawl in and bring mold spores with them.

The cure for that is a type of clingy wrap they use in labs. Sorry, i can't think of the name of it off hand but somone on this board i'm sure can supply the name. You can pick it up on ebay or a lot of places. You wrap it around your jars or petrie dishes and it lets in a tiny bit of air but keeps bugs out. Dust mites are everywhere and you can't see them.

[ShroomGuerilla]

ParaFilm is what you need.

It is a breathable, sterile, membrane to put over petri dishes etc..

[zodd]

book marked

[copelandiaKidd]

Subscribed!!!!! awesome work, great info and

I Have a Chem D momma and it is a TMV infected plant . What is the odds of taking cultures until you get a specimen that is w/o the virus. I understand that the virus isn't throughout the whole plant as I have read,,but this goes against my intuition. any input on erradicating a virus from a donor?