Tissue Culture/Micro Propagation Grow from Scratch

Wolverine97

Well-Known Member
Same batch Wolverine, a friend handed me a big old bag - I think there is a picture of it somewhere. If this last test goes south (or doesn't go anywhere) then I will have to conclude that he must have done something horrible to those seeds. I think I am showing that I am a patient guy in this thread but this seed thing is trying my nerves something horrible. I want to try this method out very badly, propagating from seedlings is a sure way to go (aside from being unable to confirm phenotypes).

I also figure that it would help others who want to start doing this by avoiding the difficulties of serility. I have demonstrated that you can sucessfuly do propagation in a kitchen without a hood of any kind if your technique is down and you use a decent biocide and are willing to take some initial contamination hits.

We know that even if our sucess rate is as little as 25 percent in the establishment phase multiplication will make up for the loss in a matter of three weeks. But, I believe that going directly from seedling will give the newbies confidence. I don't want anyone to refuse to do this sort of work because "it's too hard" or "you have to be too clean" or "it is too expensive" and a part of that is GETTING THOSE DAMN SEEDS TO SPROUT.
I hear you. Your patience absolutely astounds me honestly, I've had some serious doubts about whether I'd be able to put in the necessary time. Hopefully this all turns out well, and some of us here can learn from your hard work. As to the seeds, they must be bad. I can think of no reason why you wouldn't get good germination in the STG once you got the temps raised up a bit, they must be bad. I hope that didn't exhaust your willingness to keep at the seed thing...
 

canndo

Well-Known Member
I hear you. Your patience absolutely astounds me honestly, I've had some serious doubts about whether I'd be able to put in the necessary time. Hopefully this all turns out well, and some of us here can learn from your hard work. As to the seeds, they must be bad. I can think of no reason why you wouldn't get good germination in the STG once you got the temps raised up a bit, they must be bad. I hope that didn't exhaust your willingness to keep at the seed thing...

Hey. I have a thing about people saying something can't be done. The challenge here is not my patience but if I can get everything to work dependably for others - one of the reasons for my still standing offer. This will all turn out, everything is sound. My dream is to be the Oss and Oric of the micropropagation field. One more day and I am declaring the seeds doa. I think I will sacrifice the hog seeds after all.
 

canndo

Well-Known Member
Had a friend tell me that my seeds were most likely originaly from a mexican brick. He says that they oven dry those bricks and the seeds smell like that oven drying process. My brick days are a long way behind me so I havn't a clue. Does this sound reasonable to anyone? Not a single sprout in what must be 40 or so seeds total.
 

Wolverine97

Well-Known Member
Had a friend tell me that my seeds were most likely originaly from a mexican brick. He says that they oven dry those bricks and the seeds smell like that oven drying process. My brick days are a long way behind me so I havn't a clue. Does this sound reasonable to anyone? Not a single sprout in what must be 40 or so seeds total.
In my experience they dry their crops outdoors in the sun, never head of anyone using ovens. It's typically the handling/storage process that destroys seed viability. I'm convinced that's your issue. You need new seeds canndo.
 

canndo

Well-Known Member
Well... at least it isn't my technique. I have put the word out for seeds. Lots of people want to help with cuttings, but not many with seeds.
 

Wolverine97

Well-Known Member
Well... at least it isn't my technique. I have put the word out for seeds. Lots of people want to help with cuttings, but not many with seeds.
Yeah, that's an unfortunate, and pretty common problem these days. If I had any I could part with I'd share, but the only beans I keep around are the top notch stuff that's in line for the show. Good luck.
 

canndo

Well-Known Member
I'm going to go with my 9, I am not going to do anything until the brown truck arrives with the goods for my next run - no seeds, I am going to up the dosage and add acid washed activated charcoal (or is it all acid washed?)
 

Wolverine97

Well-Known Member
I'm going to go with my 9, I am not going to do anything until the brown truck arrives with the goods for my next run - no seeds, I am going to up the dosage and add acid washed activated charcoal (or is it all acid washed?)
Your 9? I'm not sure what that is...
 

canndo

Well-Known Member
Wolverine, I have several "moms" that are hog. I've always liked that strain and it seems to do well in vitro. The last time I spread a bunch of seeds out on a paper towel between two plates I forgot about it. I just looked and out of maybe 30 seeds I have three with spouts. Now I know that I am no longer remotely sterile but I took and put them in three tubes that had failed seeds in them previously. Then I put the 9 more in their respective tubes.

Everything is slowing down, wilting and a bit of browning besides. We are still waiting for the brown truck for charcoal, more large vessels and some more hormones. I want to mix a fresh batch of rooting stock but it is getting dicey. I will have to juggle. Make a batch of rooting gel for the few free jars I have, transplant to those, take those jars, clean them and then make a fresh batch of multiplication formula, then move the most recent transplants into there. I hate having to do that but the Magenta "B" caps I use have been out of stock at Phytotech for over a month and I simply refuse to get them from any other place. Phyto sells them for 9 bucks for 25. Everyone else sells them for 22 or more per 30. Someone gave me an entire, 3 foot high plant of G13 (so they say) to do with as I wish. It would be great to deal in a number of different sterilization strategies with the same plant at the same time but the plant is languishing in the dark in my garage waiting for me to get something in order here. But, I don't have nearly enough jars for these procedures. I don't want to put more than one plant at a time in the rooting approach. I don't want to put more than one plant at a time in the establishment jars

Finally for the evening, I'm sure everyone saw the nice vented jars I used for the last establishment phase and my confidence that these things would grow much better in a vented, lower humidity environment. Nope. Wrong. I will try to include pictures in the morning. The tips, at all levels have turned black on the vented jars but not on the identical jars that are not vented.

Now the vents are about the size of a nickle and I suspect that they should be smaller, next time I will block some of the opening with tape. I also am beginning to believe that there need be no venting in the establishment phase, only multiplication and rooting.

I got a line on an orbital stirrer and will probably buy it in the morning in anticipation of all the other things finally working - although I am telling myself that if I am willing to spend all that money on a machine I should be willing to spend a few extra dollars to get some magenta caps NOW.

Sorry I am filling this journal with issues that have nothing to do with the endeavor at hand but.. what can I say hm?
 

canndo

Well-Known Member
I see out side that the brown truck just pulled up! Now it is a big day. (except he didn't have anything for me - DAMN)

More news, I got a sprout - a single one. And more, I am confident now that apical buds are the least desirable explants to use. Very few have advanced to multiplication stage compared to auxiliary buds.

DSCF1279.jpgThis is a recent addition of a pure indica, now three weeks old, note that we already have shoots ready for splitting up.

DSCF1280.jpgNote the dried out portions of leaves, this is one of the vented vessels.

DSCF1281.jpgMore dried leaves, another vented vessel.

DSCF1282.jpgWell look what we have here. Now I think a balance of light is needed, I must have some decent growth in order to take an explant. It can't stretch too much. More waiting I'm afraid.
 

Wolverine97

Well-Known Member
I see out side that the brown truck just pulled up! Now it is a big day. (except he didn't have anything for me - DAMN)

More news, I got a sprout - a single one. And more, I am confident now that apical buds are the least desirable explants to use. Very few have advanced to multiplication stage compared to auxiliary buds.

View attachment 1574594This is a recent addition of a pure indica, now three weeks old, note that we already have shoots ready for splitting up.

View attachment 1574597Note the dried out portions of leaves, this is one of the vented vessels.

View attachment 1574595More dried leaves, another vented vessel.

View attachment 1574596Well look what we have here. Now I think a balance of light is needed, I must have some decent growth in order to take an explant. It can't stretch too much. More waiting I'm afraid.
Strange that the vents are causing more issues, I was in agreement with you that they were probably too humid. I'll check in this weekend, I'm on a crazy deadline for a project I'm working on.
 

canndo

Well-Known Member
Wish I had a teeny tiny way to measure the interior for RH. As I said, I am just going to tape the top and try not to ruin the filter. No truck, no luck, I'm on the edge here, these shipments take a loooong time. Still no caps.

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canndo

Well-Known Member
The truck came after all. I am changing some formulas, planting more seeds, switching jars, and making lots of new explants.
 

canndo

Well-Known Member
A question comes up about the longevity and volitility of IBA. I have two stock mixtures of IBA, both have been kept in the dark in a refrigerator. one at .1 mg/L the other at 1 mb/L. One manufacturer recommends the mixture remain at 0 degrees C. Another says 45 degrees F. I am concerned that I may be using old solution now. I really don't want to go through the pain of preping more solution but...If I don't, I may doubt the validity of the next series of tests. On the other hand, we've all been patient this long, whats another 10 days or so? If it doesn't work, I can make the solution then. If it DOES work after that, then I will know the aproximate shelf life of an IBA solution. I know the G3 is good refrigerated. I know the NAA is good, I know the TDZ is good (well... I think it is, it's been working) I know the IAA is ok. I'm pretty sure the biocide is fine. Well, I've convinced myself - besides, this solution is pretty expensive.
 

canndo

Well-Known Member
My changed rooting protocol is in the pressure cooker. Jar juggling is probably the most frustrating and time wasting task of this series. I keep thinking that people don't want to read my whining but then figure it is better that you all see the more simple problems that have nothing to do with protocols, light levels, and the costs of this chemical or that.

On activated charcoal. I understand the principle. Activated charcoal has tiny tubules running through it which tend to trap contaminants, or in this case, Cytokinins. This all makes sense and I have used AC for other projects in order to clarify or decolor extracts. But this charcoal is the finest I've ever seen. It reminds me a lot of laser printer toner. Now, if it is this fine, how can there be those tubules? Ah well, the reason I got this stuff at full price from a chemical supplier is because I didn't want any doubts. Ordinarily I would get to an aquarium store or break open a water filter or something like that. This time I want exactly what the big boys use - and with that comes more doubt.

I am seriously tweaking the Chinese protocol. My interpretation of it, after almost a month doesn't work. The plants, again, after a two week show of vigor and another week of solid presence, are beginning to turn brown. This is the same cycle and when I finally go into true production or support of a DNA library I will change the medium every 3 weeks - no stretching it. I've tried varying sugar content and it seems to make little difference. You can see somewhere in this journal where I've experimented with active life of the biocide, and it is about 2 to 3 weeks. I can't know exactly how long the hormones work well but if the sugar runs out then it doesn't make any difference anyway. Three weeks is about all, I suppose it is a small price to pay for the results of getting hundreds and hundreds of "clones" and keeping many dozen "moms" and you can't have everything. The refrigeration experiments I have done seem to work as I had anticipated. We can place plants into suspended animation for at the very least, weeks - so in the event someone can't change out their medium in the requisite 3 weeks, they can place their project in the fridge.

I've been using GA3 as a quasi hormone in balance but am replacing that with NAA in this round. Of course there is the IBA as well, and activated charcoal. I am sticking with MS full strength and dropping the vitamins. It is fairly easy to detect if they are vitamin deficient but it seems to take a while for that to become apparent and I figure before this happens they should be generating their own in soil (or whatever I put them in).

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Seeds. So I have three seedlings. Seems that my test tubes may be too short (that's a pity because they are virtually useless for other sorts of micropropatation work). I am doing what I can to keep from getting too much stretch - sort of like the constant struggle of indoor cannabis cultivators everywhere - but in miniature. I want the material above the cotyledons and I have to have enough to be immersed in medium while the first set of adult leaves above can still be clear. Seems that the plastic doesn't let a whole lot of light in no matter what I do. This isn't a huge problem, when I get more seeds I will likely just use a different container and put more than one in each.

Well, I have work for the rest of the day, I will take pictures of some of it because I suspect this thread is probably pretty boring if it is just words and speculation and fret on my part.
 

canndo

Well-Known Member
I am very unhappy. The activated charcoal is so fine that it permeates the medium. Good in the sense that everything is homogeneous but bad because I won't be able to see the roots. I suppose, I suppose, it would be ok to use this protocol if and when I know about how long to wait before I have roots. It isn't imperative that I be able to see them... except for the first time.

I've pulled out my more granular charcoal and am re-making the medium with it. I will do the two batches in parallel. Of course the granules drop out of suspension quickly so there is that problem, and I don't know if there is enough coverage over all. If I see roots in the granular jars I can presume there are roots in the opaque jars. ah well. Nothing wrong with the method, I just like to see what happens.
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Wolverine97

Well-Known Member
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