Plant tissue culture as a way to clone on a mass scale??

Uncle Ben

Well-Known Member
Ben, so far as my experiences are concerned, shipping plantlets rooted in media is asking for failure - hardening off in vitro plants is not for the faint of heart and I can't see very many people popping their newly purchased purple kushie kush OG into a plug and taking it from there.

Your quote however, is a little misinformed from my perspective. Firstly, leaves make poor propagation material in that leaves require one to produce callus and then from there shoot and finally root. I have not been able to figure out the combinations of auxins and cytokinins to bring callus back to shoots. I am reminded of the land before Oss and Orick published a little book on how to grow mushrooms - before then, it was just too monumental a task. They managed to reduce the process to something everyone could do in their kitchen. Sterile conditions seemed insurmountable but they are not. I have never used an authentic autoclave and my pressure cooker works just fine.

Beyond that, there are chemicals available that limit contamination in the medium to the point where I believe no glove box or hood is even necessary. As far as the need for fresh material? Well, I havn't needed such yet but we await final results.

Note what I said though, I would think that a large facilty might use these techniques internaly. I have never believed that tissue propagation would replace seeds and I don't think that anyone would believe such a thing. I am beginning a new experiment (it is nice that this hobby takes so little time, space and money in that I can run multiple experiments simultaniously). I am going to take seeds and grow entirely in vitro from seedling to rooted plantlet. If I can do this, I suspect that I can clone dozens of plants before traditional methods can even yield a single cutting. The trick here is the ability to sex plantlets - I intend to start that this week. Thanks for the ongoing discussion Ben. I would be curious to know what you think of my personal efforts in this realm.
You're not describing the mericloning process I'm thinking about. Check out orchid mericloning and you'll see what I mean. I talking about taking the apical meristem material, putting it into agar, spinning it in a centrifuge to induce asexual cell reproduction, spinning it again so they divide again, repeating the process until you have hundreds of identical, tiny plantlets after which you carefully......
 

canndo

Well-Known Member
Ben, I don't go to the single cell, that, in my case would be redundant. I needn't isolate the meristem alone primarily because I do not fear viral infection. One needn't go down to that level in order to get mothers and clones - micropropagation. I suppose I could but then your quote would be more accurate - it would take months to get enough biological material. Again, there is no need to do this on a cellular level as far as I can see. I am now in the 5th generation from cutting to explant to plantlet with no degeneration I can see other than what I've experienced with hyperhydration. It seemed to me that the fellow you were quoting fears sterile conditions more than is warranted. Bioreactors have their place but now we are talking about either collecting secondary metabolites which won't much work with this particular plant - or generating many more hundreds of thousands of clones than anyone might need for this sort of work. There is a process whereby pollen is grown into haploid plants or treated with chemicals in order to make them diploid that seems interesting but I can't see the point in that except to preserve the exact genetics of a single remaining male plant.
 

Wolverine97

Well-Known Member
I'd really like to see this discussion furthered, I'm intensely interested.

I have a "golden child" that exists only in clone form, and I believe it may be infected with a mosaic virus. It grows fast enough that it never becomes an issue, but if I try to keep plants in a quasi suspended animation it becomes evident. I'd dearly like to clean the genetics if possible, and it'd be worth a modest investment to do so.
 

canndo

Well-Known Member
Woverine, in that case meristemic tissue is your answer. As you have probably already noticed, the vascular system of the plant grows slightly faster than the virus can infect so the very tips of your growing shoots are virus free (probably). It is a dicey move but you can afford to fail as your plant is still alive. If you wish I can take you throgh the steps and see if it might be right for you.
 

Wolverine97

Well-Known Member
Woverine, in that case meristemic tissue is your answer. As you have probably already noticed, the vascular system of the plant grows slightly faster than the virus can infect so the very tips of your growing shoots are virus free (probably). It is a dicey move but you can afford to fail as your plant is still alive. If you wish I can take you throgh the steps and see if it might be right for you.
Oh for sure, yeah, that's actually why I've been looking into tissue culture because it's always been described to me as using the apical meristem, to obtain the pure and clean genetics. Couldn't I just disinfect a very small, soft-tip cutting of the apical meristem, and root it as normal? I actually did this with my last round of cuttings, but then I didn't isolate that clone from the rest. The ones I took may have been too large though (2-3" overall), as I'm not sure what's the largest I could do and still ensure that it's clean.
 

canndo

Well-Known Member
I really don't know how small the bit of meristem you could root conventionaly. One of the problems I had to resolve is exactly what size is the right size to do this. Too small and it takes too long to grow - it also is easier to just kill the tiny explant with all the harsh treatment involved in sterilization. We have a secret advantage over most micropropagators in that the vast majority of the plants we grow are in clean conditions from the start - we don't have to go outside in all that filth. You could further protect your meristem by putting a clean baggie over a prospective tip and let it grow in even more sterile conditions. Too large and you will include the virus in the plant and gain nothing. I am not sure what size would be best in your situation - you can see what size I have found to work best in my experience - and it is far larger than all the texts I have read suggest. I get my best results with 1.5 to 2 inch, juvinile as possible, lower stem cuttings with a bit of larger stem still attached for surface area. But you need something much smaller I suspect.
 

canndo

Well-Known Member
Ben, Wolverine asked "Couldn't I just disinfect a very small, soft-tip cutting of the apical meristem, and root it as normal? I actually did this with my last round of cuttings, but then I didn't isolate that clone from the rest". And I answered in the way I did.
 

canndo

Well-Known Member
Ulis. I have not been able to do anything with leaves, they form callus but nothing else. I have found recent studies that indicate rooting from the leaf callus but shoots from that callus are if I recall, in the 1 percent range. But the other answer to your question is that if you do use a leaf to generate callus, it won't work if the leaf is frozen or dried - it has to be alive and the fresher the better.
 

ulis5714

Member
OK. I was looking at a plant tissue culture kit from Discount-Hydro.com, but it could be beyond my capabilities. I best leave such things to the experts!
 

ulis5714

Member
I'm a new member, and I thought I replied, but maybe I have not learned to use this site properly yet. So, I have been looking at a PTC kit online, and though it looks good, cloning is probably the way to go.
 

canndo

Well-Known Member
Ulis, this all depends upon what you wish to do. Take some cuttings off of a few moms, then no, this may not be "beyond" you but there is no reason to go through the trouble and expense.
 

canndo

Well-Known Member
One more thing, as I said, by all means the kit is a good thing and it will certainly help, but the media mix is not even close to being appropriate for our plant of interest.
 

ulis5714

Member
It's good to be able to ask for good advice! I'm strictly a small-scale grower, everything I grow will be only for me, and PTC seems to be for commercial growers! Once upon a time I thought I could freeze a good plants leaves for later, but thanks for setting me straight!
 
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