Tissue Culture/Micro Propagation Grow from Scratch

**canndo** · 02-18-2011, 01:41 PM

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** · 02-18-2011, 01:44 PM

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

**shmarlo** · 02-18-2011, 11:25 PM

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** · 02-18-2011, 11:57 PM

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** · 02-22-2011, 10:14 AM

Set up the works.

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Make the media

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Fill the containers and pressure cook them.

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Cut the explants
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1/10 Bleach - and 70% Alcohol wash
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Ultrasonic cleaning for 6 minutes
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Stir for 20 minutes

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Put the explants in the jars

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And put them under the light with the others

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

**canndo** · 02-23-2011, 12:45 PM

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.

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

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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.
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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.
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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.

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

**canndo** · 02-24-2011, 11:57 AM

I posted too soon - looks like I have two incidences of contamination, both on the plant itself.
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This one has a tiny spot of mold on the right hand side of the stem just a bit down from the top.
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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** · 03-01-2011, 12:32 PM

The salvages didn't work so I am now two jars dowm to 10 jars. These currently show the most growth.
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Note the new shoots at the base of the foremost shoot and another set behind and to the right.
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This is the best shot I could get of this plantlet. It is one of the best, showing lots of new growth.

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

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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.
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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** · 03-05-2011, 01:06 AM

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

**Stud Boy** · 03-05-2011, 10:55 AM

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.