Now, were are we today?
I was more than a little overzealous with the large stem transfer and I killed most of them. That is really too bad because the ones that aren't dead are growing very quickly - I don't think I included a pic of the good ones. I probably shouldn't have done that last alcohol wash, and maybe I shouldn't have done all of the bleach washes. I'll get more for my next run and we will try that one again.
The seeds. I tried this experiment once before and it was a failure. the seeds grew to just around the same situation they are at now. That time I used NAA and 2IP, 30 grms of sucrose and 2 grms of MS. They just quit. The biggest thing about seeds is that they harbor all sorts of spores. I was lucky to get even a few of the seeds clean enough last time. This time I did a fairly good clean and my medium is devoid of anything but sucrose (16 grms) and MS (1 grm). No hormones, and even no biocide (cause I forgot to put it in). Because I didn't put the biocide in, I figured I'd be even more thorough with the wash. I really don't know how much a seed will take before I destroy it but that is an experiment I will do when I have more seeds.
I am including pictures of the failures and the cuttings I am most happy with.
If things continue to go well I project that we will be looking to rooting in two weeks. So what does that mean exactly? Well we have been at this for about a month. Within that month I have taken 12 cuttings from a branch that would have yielded perhaps 2 traditional clones. Half of them died. From the 6 remaining I got 13 I believe (I don't have my notes handy). So in the one sense I got 13 clones from two (if you consider traditional methods only) - not bad for a month. In another I got 2 to 1.
If we consider that I got about 2 cuttings per plant in 3 weeks this pass then I would have 26 in three weeks. I would have 52 in another 3 weeks and 104 in another 3 weeks. That is 104 clones in 3 months without really trying. Of course that number doesn't count rooting time and hardening off but you can see how quickly this sort of cloning could pay off for a larger grower.
Included in the pictures is one of a tub. This is only for use when you are quite sure that you won't be contaminating anything. While I was doing some transfer work I discovered a bacterial contamination in one of the jars - everything I transfered from that jar failed. The biocide seems to work very well but loses its effectiveness in a matter of weeks and it doesn't seem to work against large amounts of infection, only the stray spore or two. I have tried using it as a wash on heavily contaminated plants and that does work - but this stuff is very very expensive, costing over a dollar a milliliter. If someone had a major reason to preserve his plantlet I think he might be able to use this stuff to save even the nastiest infection.
Now, the seed experiments again. My hope and plan is to get to a place where I can take the resulting plant, having been completely in a sterile environment from the beginning and use that as a "cutting". This will give me a huge advantage over traditional methods. Consider that I would only have to wait about 15 to 20 days in order to take the first cutting for a clone. Three weeks later I have two, then 4, then 8 then 16. 16 clones in 3 months (well a bit more if you count the seedling phase). From seed. I don't think that can be done using traditional methods. Now this is where the sexing in vitro comes into play. I can't think of a reason that I can't flower a rootless in vitro plant. If I can, then I could have legions of new, sexed plantlets ready for planting. If I need to root the plantlet before sexing, so be it, it will take a few extra weeks but I can still grow in tandem or parallel to the sexing without much waste of space, resources or light.
Notice that in this transplant I spared the two top shoots, they just weren't big enough. Were I to do this one again in a few weeks I would slice on the bias so that one shoot would still have a portion of the stem. From this I would have the top, one small shoot and another larger shoot. I did trim all of the bottom leaves so they won't touch the medium.
Whenever possible I like to place all the shoots I cut from the base plantlet in the same jar just so I can see how much biomass I am actually generating. This was all from a single plantlet. I could have taken more from any of these plantlets but more mass is better so I opted to try for the nicest fatest plants for rooting rather than trying to get as many cuttings as I could have.
This is a tub picture. The tubs have good points, they are easy to work with, they offer large amounts of space for your cuttings to grow, they capture a lot of air so if what I suspect is true, they should go longer without vitrification. The down side is that if there is any contamination, I lose everything in the tub.
This is just a nice, single shoot. I am anxious to see how quickly this one exhibits auxiliary shoots.
Look at the black top of this plantlet, this doesn't look like bleach damage. It is too bad I killed it, it is an apical "bud" and I think it would have not only worked great to give me a number of new shoots but it would have looked nice. Note the size of the stem is much larger than anything I have used or shown pictures of before. I do get this size stem after a month or so of in vitro growth.
And you can see at the base the fluffy stuff. I have a suspicion that the fluffy stuff could be a particular sort of mushroom - can't be sure though, I'll let it grow a few days and see if it sporulates.
So I've rambled on. The price you will pay for letting me do this while buzzed.
I've cleared the space for the flowering phase. I will put the vessels in a dark blue plastic bin with a growlux lamp in the top. I will not set it up for any sort of exotic air exchange and I will not do anything about temperature. I will set it up for 11.5 hrs light and give it three weeks, although I don't know what I will be able to see on such a miniature scale. As always, input is welcome.
So these are three pictures of a plantlet that is about 2 months old. Notice the shoot growth. What I am particularly pleased with is the girth of the stem itself. Except for the last batch, I never use stems that are even close to the size this one is. What it seems to indicate is that my ratios work for longer term preservation. I wish there were more of them though, I would like to see an average of 5 shoots.
