Tissue Culture/Micro Propagation Grow from Scratch

[canndo]

First of all Please forgive me if I make any errors in custom or etiquette, I am quite new here.

A gentleman in another thread rightfully claimed that no one has been able to demonstrate a tissue culture grow from start to finish. I am going to try.

I will take explant cuttings from a Lemon Skunk branch I was given yesterday. I hope to establish the cuttings in vitro, multiply them once, root the resulting shoots in vitro and then transplant into soil. From there I hope to grow the plant to one foot. If after that time I gain the facilities to flower the Lemon Skunk I will do so as well. The initial rules for these grow journals say that I must invite others to post on this thread and so I do. Post any questions, jeers or taunts you wish. Let's see how this micropropagation theory works in the real world.

 

[canndo]

This is the start. I have to make the media (is it medium?), get some plant material from the branch and clean it.

 

[shmarlo]

awesome dude
I have always wanted to see this in action
did you buy a kit for this or pick everything up separately?
i was interested in this process to have a mass collection of hundreds of strains at my disposal in a relatively small area
i hope it works out

 

[canndo]

I started with the kit but found that the media was not as good as some of the later mixes I have experimented with. Don't get me wrong, the kit is fine but you need to put some other cytokinins together and as I said, I like agar and the kit doesn't have agar.

 

[canndo]

Set up the works.



Make the media



Fill the containers and pressure cook them.



Cut the explants


1/10 Bleach - and 70% Alcohol wash


Ultrasonic cleaning for 6 minutes


Stir for 20 minutes



Put the explants in the jars



And put them under the light with the others




We got 12 jars. I will try to get better pictures than these in the future. So, off we go.

 

[canndo]

Several things.

I changed the formula for the last batch - this one and added about 15 percent more gel. I did it because some of the larger plants either fell over or sank into the media and I didn't want it to happen to this batch. I believe however that the stiffer gel is slowing growth down. I'll change it to about 5 percent once they are established.

I will not include pictures of the plantlets that show no or little growth - save bandwidth and storage. So far I've not gotten a single contamination loss and if nothing of that nature happens in the next few days then I will can claim my first 100 percent sucess rate operation.




This is the first leaf, - I don't think the top will grow.



This is a set of three that I did taking a chance - if any of these are contaminated then I will lose all three. You can see new growth on every cutting.


Another set of three new distinct shoots on two of them. Well they really aren't NEW as I preserved just the base of them when I took the cuttings but as you can see they have leaves already. Note the burnt ends on all of the stems from the bleach and alcohol. This, or a bit more than this seems to indicate that you did a decent job of sterilizing - much more and the plant will die, any less and you might surely get contamination. I should have cut those ends off though.


This is shaping up to be the best one of the lot. I expect to have lots of shoots here. Lots of new growth already and it hasn't been a week yet.



Another very good one, I'll get a number of shoots off of this one in two weeks or maybe three.

 

[canndo]

I posted too soon - looks like I have two incidences of contamination, both on the plant itself.

This one has a tiny spot of mold on the right hand side of the stem just a bit down from the top.

You can see that little tuft of fuzz at the top of the foremost plantlet.

I am going to attempt to salvage these as neither has yet contaminated the agar (gelzan).

 

[canndo]

The salvages didn't work so I am now two jars dowm to 10 jars. These currently show the most growth.


Note the new shoots at the base of the foremost shoot and another set behind and to the right.

This is the best shot I could get of this plantlet. It is one of the best, showing lots of new growth.


Just can't a good picture of this one, it is one of the best ones.




There are three plantlets in here, you can only see two. There is some very decent growth here and you can see the beginnings of genuine shoots.


Another series of shoots but the two primary ones were left from the original cutting.

Some of the plantlets need a bit of work, I should top them to further encourage adventitous shooting and some of the stem material just doesn't need to be there. I see that growth is indeed slowed by the different strength of gell so rather than simply operating on them and puting them back in the same jars I may make a new batch of media. Or maybe I will give them another week.

 

[JealousGreen]

I've always wanted to see this done. Thank you for your demonstration.

 

[Stud Boy]

Hi, can you give a breakdown of all of the auxins, cytokinnins, and any other chemical that you're using in you media? Also, are you starting with the MS mix, or are you using a different base? I've had a lot of success sanitizing my explants with a mix of 50% vinegar heated to 150 F and 50 % of the 3% strength hydrogen peroxide. I get very little contamination and a lot less plant damage than with bleach.

 

[canndo]

Stud Boy - I am using TDZ, NAA and GA3 for my basic mix. My establishment is MS and I use a rather high concentration of PPM in establishment, prop is MS + I think I said that Gelzan is not my favorite but I am warming up to it cause I can spot contam more quickly, but I have been using agar for one thing or another for so long I just kinda like it.


Rooting? I havn't even started working on a decent rooting protocol - what are you using?

I like your vinegar idea but where do you get 50 percent Acetic acid? Are you diluting glacial? I can't tell you the number of plantlets I've burned to death working for a good chemical decontam. I will say that for me, ultrasonics work quite well on the mechanical aspect and I found another sort of ultrasonics device that works even better - it's an electric denture cleaner!

Oh, one more thing - I am finding that these things are highly temperature sensative and I am working on some sort of a temperature curve to find the optimum. I got lots of questions - how far along are you?

have you flowered in vitro? with or without roots? what is your seed starting protocol? What are your hardening protocols? How do you vent your caps or do you?

 

[canndo]

The best ones so far, I'll do some triming and change media in two weeks.

 

[Stud Boy]

I like your vinegar idea but where do you get 50 percent Acetic acid? Are you diluting glacial? I can't tell you the number of plantlets I've burned to death working for a good chemical decontam. I will say that for me, ultrasonics work quite well on the mechanical aspect and I found another sort of ultrasonics device that works even better - it's an electric denture cleaner!

I meant 50% by volume, not concentration. That's the beautiful part, it's just white vinegar and hydrogen peroxide from the grocery store. You just put 500 ml of vinegar into your beaker, zap it in microwave, add 500 ml H2O2 and let cool. I use it to sterilize my glove box, my equipment, and my explants!

I got lots of questions - how far along are you?

I did 3 batches, got some small plantlets to grow and a few wads of callus. I never did get the plantlets to grow roots, and I don't think anyone has figured out how to get the callus cells to switch over to organized plant cells. Each plant has its own magic formula, and I don't think that anyone has cracked cannabis. You've re-inspired me and I'm dusting off the old equipment. More importantly, I'm going to find my notebook today.

have you flowered in vitro? with or without roots? what is your seed starting protocol? What are your hardening protocols? How do you vent your caps or do you?

I never flowered in vitro, but I did want to see 1) how long I could keep a plantlet alive without adding anything and 2) how long could it remain uninfected. I'm using the vented plastic lids for baby food jars from Kitchen Culture. I then put the jar in an opened, inverted ziplock bag. That lets oxygen into the jars, but minimizes any particulate that could cause contamination. Then I kept them in a cabinet to reduce airflow with a cfl. I was able to keep the plants alive for about 6 months without contamination!

I've got some good references that I'll share once I locate my notes!

[/QUOTE]

 

[canndo]

Studboy, callus is the wrong direction, it isn't needed and it is not a geneticly stable way to do this - I have a callus protocol but it is a wasted step. What, if you don't mind me asking, was your establishment and multiplication hormones? How much sucrose did you use for a 6 month run in the same medium? I'd love to see anything you have. Thanks.

 

[canndo]

We are about at establishment with a number of jars. It looks like I may have lost another to contamination on the tip of the stem - no pictures of that till I am certain. The others are at varying degrees of completion of this phase.

 

[canndo]

Two shoots worthy of watching


Two from this one probably

Three or four, two that I could take today.



My favorite of the bunch - I believe this will be my mother plant for future Lemon skunk propagation.

 

[DarthD3vl]

Cool stuff man, never seen this before.

 

[canndo]

Today's updates:

I have decided that this one isn't going anywhere and I need the jar.

See how it kind of droops and if you compare it with others it has no new growth. I have to strike a balance in size. The larger the explant, the more likely it is to become established and the faster it grows. However, larger explants are harder to sanitize and more suseptable to contamination. This one was too small, or I took it from too old a place on the branch. I don't think the stiffness of the gelzan helped it either.

 

[canndo]

This one is going to be dumped, it didn't take for any number of reasons - too small, taken from the wrong place on the branch (not juvinile enough), not enough contact with the medium - I don't know, it happens.

View attachment 1484192

 

[canndo]

This is my latest favorite - nice shoots.