Tissue Culture/Micro Propagation Grow from Scratch

Thanks Wolverine, I don't know if you know this or not but I am a big cigar collector. I got thousands of cigars and each "coolerdore" has one of those in there. It is too big to fit in any of my jars. I was thinking of a probe or something like that. Again though, thanks for thinking of this project. I did three batches of establishment gel, that fills my tubs, spare jars, large jars and everything I could find to pour it into. In the morning I am going to do the largest establishment I have ever done. I have 118 jars. I got new shelves and lights for them.

Two pictures of two different plantlets, the one on the left has 8 different shoots, each one ready to cut and transfer. The one on the right is still very young but it is a vigorous one. This is just to demonstrate how well this method can work. Getting better and better.

canndo said:

Thanks Wolverine, I don't know if you know this or not but I am a big cigar collector. I got thousands of cigars and each "coolerdore" has one of those in there. It is too big to fit in any of my jars. I was thinking of a probe or something like that. Again though, thanks for thinking of this project. I did three batches of establishment gel, that fills my tubs, spare jars, large jars and everything I could find to pour it into. In the morning I am going to do the largest establishment I have ever done. I have 118 jars. I got new shelves and lights for them.

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*whistles* good luck!

There are several things I am working on here. First is a definitive side by side comparison of gelzan vs agar. Note the yellow containers vs the more clear ones. Secondly, I want to determine the best source of explants once and for all. This is my last handy plant for cuttings and so I am trying to get as many first generation explants as I can garner. I am taking apical buds and auxiliary buds. Now I am also doing this because I have to move all of my established plants to larger containers (except for the rooting experiment). This should leave me more containers for new plantlets. I also broke down today and got some magenta caps. Furthermore, I will be putting tape over the vented containers to see if Wolverine's and my idea about air exchange is still valid. Anyway, I cut up all of the explants already and am getting ready to drop these in front of the hood and get started. No point in taking pictures of most of it, you've seen the process before.

The seedlings? they are getting very leggy and I fear they won't work well in such short tubes. I know they need more intense light than they are getting now. Don't know how to give them that given the nature of the tubes themselves.

Back to work.

What kind of light are the seedlings getting? You need at least 30wpf of fluorescent light for good seedling development, and that's assuming they're kept within 6" at most from the plants.

I've had them under my flouros, I don't think I'm giving them that sort and I don't know what is getting through the plastic. I'm really don't want to set up another light system but I suppose I'm going to have to. I'm on my third set of transfers now, this is a lot of work doing this many at once.

INCREADABLY STUPID.

I always use a check list, I advise that everyone who does this, ensure that they check off every item. The only problem is that the 4 batches I did worked off of the same checklist. And I omitted the sucrose. I have a couple of hundred jars, a couple of hundred cuttings and of course all of my prospective rooting jars and none of them have sucrose in them. At all.

I have to figure out how I am going to store each plantlet long enough to take the jars they are in and refill them with the proper medium. I have to do it all in the morning. I am beside myself, I have jeapordized my entire project by being stupid and using the same checklist without actually looking at the items.

The only lucky thing is that before I began, I realized I was almost out of sugar, I made a note to myself that I would have to get more sugar for my Wed Mojitos. When I got the sugar down for the mojitos I realized I had not seen that container throughout the entire project.

Shit.

Shit.

Shit.

I'm suddenly very tired.

Well boys and girls, I took a huge hit. It seems as though I have fewer than 20 intact plantlets after my mindless stunt.

I will include pictures when I can bear to exaine everything close up.

When I discovered that I had not used the sucrose, I realized that I did not have enough jars to do a jar to jar transfer (which I now know is the only way to do this). I took the plantlets I intended to root and placed them all in a jar with a solution of biocide, then I emptied the rooting jars, made more media, plucked the plantlets out of the solution and placed them in the new - now sugared rooting mix. They are barely hanging on, none of them have that robust aura about them they had the first time.

All of the plantlets were at least two days without sugar. The looked fairly viable when I did the initital transfer and about the same when I placed them back into their final destination.


I was short of biocide and so I simply took all of the other plantlets out of the individual jars and put them in a tub - which was loaded with the sugarless media. Then I emptied the jars, cleaned them and replaced the media with new media - again, with sugar. Then I took the individual plantlets out of the tub (there were 100 or so of them in the tub) and embeded them back into the newly created jars. The vast majority of them did not survive.

I am out of biocide. More will arrive this week. Ironicaly, the 50 Magenta caps I bit the bullet and bought at a buck each arrived yesterday. I could have done a jar to jar transfer had I had the caps several days earlier.

I know all of this works, I have no doubts that a lay person can do it start to finish but the process requires a certain finesse and timeliness.

I am dissapointed but not diminished. I am going to make this into an oportunity to rebuild my shelving, reasess my environmental controls, implement an idea to make the environment more modular and do some things about light testing. Still, it would have been nice to simply be sucessful in this portion of the experiment so we could go on.

I did get the sodium alginate so as soon as things are all back on line, I will start experimenting with the encapsulation necessary for the "artificial seeds". The only problem I have now though is a shortage of raw material. All of my friend's plants are in flower and I have pretty much shown that taking cuttings from flowering plants fails.

I feel as though I've let everyone down. Well, we go again.

Sorry to hear about your setback canndo. I've been sub'd to your thread for a while now and find your work fascinating. Don't get discouraged. I'm sure with all the hard work that you're putting into this project you'll get everything ironed out soon.

Something I don't know yet is where thermal death is for these plants. We had a small heat wave and my lab gets the afternoon sun - I didn't have any thermometers in the room but I need to know how hot things can get. I have decided that this work is too important for me to leave it to the randomness of the sun and clouds. I have a couple of portable air conditioners but I just can't see running a 1000 watt machine to keep an entire room cool in the interest of just one rack of jars. I just want to keep everything in the 85 degree range tops. So I went out and got a portable evaporative cooler today. I will be building a new lab, expressly for this work. As those who have been following know, I have shared this with other projects but will no longer. Thanks Plowboy, I intend to reward those who have been following this project so stay tuned.

I went to my local Hydro shop (in order to get the cooler) and there were a number of people there, all of whom have been following my work. I am touched with the way they are all willing to contribute. I was given some seeds and a host of promises for more raw material. I had no idea there were so many people willing to support what I am doing.

So..


I will get the new lighting and shelving up in a different room. I wish there weren't carpet in there but a few days under HEPA air exchanges will help some.

First is putting all the new seeds into production - I have about 30 now.
Then a brand new set of 100 baby food containers with establishment media
Also 24 vented (and partialy taped) larger jars.
And I will take a chance and work in 10 tubs as well.


I am promised all the branches I need so I will apportion a some under a series of new sterilization protocols which I will develop and post here. This should give me a chance to refine sterilization protocols using new solutions. I have an idea for some new touches and also I will encorporate vinegar, hydrogen peroxide, denatured alcohol and bleach.

Finally, I intend to cut to the chase in this go round. I will throw new cuttings directly into root rather than wait for shoots. Of course the experiment will eventually have to involve a start to finish proceedure but I don't want to wait to work on the right rooting formula.

The people who were so willing to help me today all expressed the same hope, that I could perfect a series of protocols for them to use. They want to be able to do this without all of the lab equipment and proceedures that I have to go through now and I promised I would do that. And so we begin again from scratch.

Great work!
Since there is no way I can see to send a message to you except through a posting, can you (canndo) contact me offline?
Email: zuperclone <at> gmail <dot> com
I have some information you might find useful.
Thanks

Zuper said:

Great work!
Since there is no way I can see to send a message to you except through a posting, can you (canndo) contact me offline?
Email: zuperclone <at> gmail <dot> com
I have some information you might find useful.
Thanks

Click to expand...

You can use the message capabilities here. I would like to know what you have for me. Thanks.

To all those watching. I have rearranged my lab, I have gotten more shelving and more of petty much everything. I will be starting the second goround in a few days just as soon as I have located some new raw material. I am worried about room temperature and am working on a way to keep that down without having to spend too much money on air conditioning. This project is not abandoned, just delayed a bit, stay tuned, I think you will see some interesting and informative things.

(the seeds worked well, I am going all out on that end of the project next week)

So now, I fear heat. My room temp is fluctuating between 78 and 92. Working on that this Monday. I was put together with a man my friends thought I should meet. They said he was doing micropropagation. We spoke for quite a while and it seems that he is mixing molasses with agar and putting leaves on the surface - without treating the leaves. Furthermore, he says he is putting two leaves from different plants and getting a combination (plant) as a result. Sure doesn't seem right to me.

&#8220;You can use the message capabilities here. I would like to know what you have for me. Thanks.&#8221;
I don&#8217;t see a way to send a message to only you, Canndo. Is that an available here?
When I try to &#8220;Send Private Message&#8221; it says &#8220;you do not have permission to access this page.&#8221;

you have to have 50 posts to send pm's

Namaste'

Thanks Illum.


I am going today to pick up a tent and an air conditioning system for this project, even though it is only about 60 degrees out today.

So, I have come up with a plan for keeping the plants cool, I have purchased what I need and have managed to coherce a number of people to donate cuttings. I start on a new batch Saturday and will work through the week to get this back on track.

I have seen many journals - well a few actually - where someone was ambitious enough to start but quikly drop the project and are never heard from again. This won't happen here. I believe I will simply start from scratch yet again. I hope no one will be bored with what is essentialy a repeat of the last few months. The time should be shorter as I have dialed in some of the paramters from previous experiments.


So, we will establish as many as we are able, still varying the sterile techniques.
We will take some after they have been established and place them directly in root medium
We will take some of the rest, and take shoots and propagate from the shoot stage.
We will take some and attempt to induce flowering
We will continue the seed to propagation experiments


Of course, the hard points are:
Getting something to root
Getting a second transfer to root
Then getting a rooted plantlet to grow out into a legitimate plant.

And hopefully, I can start on the artificial "seeds" somewhere in the process.

I hope people aren't going to be bored with this the second time around.

Two more days of transfers and the like. I went ahead and remixed all of my stock solutions, I am doing about 100 more jars of course I will take pictures of my new facility. I have converted a closet into my new temperature controled room.

I have very good friends (and it seems I am making new ones on RIU).

The word went out that Canndo was seriously short on raw material and that he had killed everything with his ham handed approach to micropropagation. Lo it came to pass that he was blessed with buckets of branches of every sort and kind of flowering herb from points and places far away and close. And Canndo was rewarded with seeds and more seeds promising and fertile.

And so, we begin again, with sugar and with hopefully a cooler environment. Pictures will be forthcoming, I got what I figure is about 2000 cuttings from which I will glean only the best each of these buckets is packed with different kinds.

I should be able to parallel every experiment I want with this much material and 200 jars.

Oh, one more little thing. Some of the sad sickly things I had left I could not bear to throw away so I saved them, they weren't dead, just very sick. Well, a few of them have roots. I can't figure out how to take pictures of them because they are within the charcoal so you have to sort of twist the jar around just right in order to see them but they are definitely tiny roots, on at least 3. So my rooting formula works - but probably needs refining.

Stick with this, I said I wouldn't quit until I got it all to work.