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.
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
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.
Last edited by canndo; 02-19-2011 at 01:03 AM.
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.
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.
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).
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.
I've always wanted to see this done. Thank you for your demonstration.
Learning to free the spirit from the ape matrix... We do not fear the flame, though it burns us; We do not fear the fire, though it consumes us; And we do not fear its light, though it reveals the darkness of our souls, for therein lies our power.
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.