what base recipe do you reccomend? I have nearly completed collection of all the equipment that i need but just cant work out how much of each ingrediant. I joined this website after i came across this thread via google. Good work and i have just spent today reading through the 20 pages that this thread has come to so far.
Tissue Culture/Micro Propagation Grow from Scratch
- Thread starter canndo
- Start date
canndo
Well-Known Member
Foolery, there are a number of different protocols depending on what you want to do. I don't think you want to do calus, but there is an establishment protocol and a mulitplication protocol and of course rooting and hardening - finally I am working on the artificial seed formula as well. I just finished 200 some odd jars using 4 different variations on my primary protocol, this one uses TDZ and GA3. Another one I am experimenting with uses IBA and TDZ and a third uses NAA and TDZ.
The room is still in disaray from all of the proceedings and I have to move everything into my new air conditioned closet space. As you have read, I am starting over. If you look closely you will see 4 different colors of media, one for each of the formula tweaks. Beyond that, since I had so many cuttings I figured I'd try to refine my sterilization system. Each of the little colored dots represent a different approach. A reader and fan suggested I use hydrogen peroxide and so I have, along with a new one I found at a beer brewing store, I don't remember the name. I am trying, as you know, to make this easy for a lay person to perform, hence the alterations in methods. If you contact me (and anyone else for that matter) I will send you an establishment kit. I need to triangulate my methods with others.
The room is still in disaray from all of the proceedings and I have to move everything into my new air conditioned closet space. As you have read, I am starting over. If you look closely you will see 4 different colors of media, one for each of the formula tweaks. Beyond that, since I had so many cuttings I figured I'd try to refine my sterilization system. Each of the little colored dots represent a different approach. A reader and fan suggested I use hydrogen peroxide and so I have, along with a new one I found at a beer brewing store, I don't remember the name. I am trying, as you know, to make this easy for a lay person to perform, hence the alterations in methods. If you contact me (and anyone else for that matter) I will send you an establishment kit. I need to triangulate my methods with others.
I had read that there are three protocols for this, I think for now I will try and get the shoot method going so that i have enough to then go onto mulitplication. Im currently trying to fill a notebook with all these written protocols so i have something to read from so that each time I make a jar up I do it exactly the same as the previous one
I also want to create a strain library preferably rarer strains to keep those strains alive, currently i only have a white widow plant that I can get plant material from.
How do I get hold of you? being a new member I can't PM you.
I also want to create a strain library preferably rarer strains to keep those strains alive, currently i only have a white widow plant that I can get plant material from.
How do I get hold of you? being a new member I can't PM you.
That's true, i can get that 50 posts up pretty easily. cheers
canndo, hey first thanks for this thread. This is good stuff. I am rooting for you.
Second, looks you are fighting some environmental and contamination difficulties and I had a suggestion or three for you.
Have you ever thought about making yourself a DIY incubator? Kludge a $50 craigslist freezer [I think freezers are preferred more than refrigerators for this type of use (cycle duration/frequency maybe?)] with a digital thermostat ($60) and then add in some lights...
**poof**mad scientististy incubator**poof**
I know you're trying to stay away from buying all sort of expensive lab kit, but this sounds like a simple cheap and effective solution. This type of incubator is used by some homebrew, cheese and mushroom folks. I like this option if you have a room that isn't air conditioned and it's too much to add AC just for your PTC.
also, I know you said that you aren't going to buy a glove box or a flow hood, but what about a simple cheap DIY glove box? You could fairly easy build yourself one from a 20gal aquarium($5 yardsale), some lexan ($20) and some glove box sleeves...
...I know glove box sleeves are expensive, but I've seen some for $100...
"Still Too Expensive!!!" you declare!?!?!?... You could make yourself pair of DIY neoprene iris(es) arm seals for next to nothing.
If you're totally against a DIY solution you could go the super simple rubbermaid box route. I have noted several sources recommending a simple plastic bin as a makeshift hood in lieu a glove box or flow hood.
Keep up the good work.
Second, looks you are fighting some environmental and contamination difficulties and I had a suggestion or three for you.
Have you ever thought about making yourself a DIY incubator? Kludge a $50 craigslist freezer [I think freezers are preferred more than refrigerators for this type of use (cycle duration/frequency maybe?)] with a digital thermostat ($60) and then add in some lights...
**poof**mad scientististy incubator**poof**
I know you're trying to stay away from buying all sort of expensive lab kit, but this sounds like a simple cheap and effective solution. This type of incubator is used by some homebrew, cheese and mushroom folks. I like this option if you have a room that isn't air conditioned and it's too much to add AC just for your PTC.
also, I know you said that you aren't going to buy a glove box or a flow hood, but what about a simple cheap DIY glove box? You could fairly easy build yourself one from a 20gal aquarium($5 yardsale), some lexan ($20) and some glove box sleeves...
...I know glove box sleeves are expensive, but I've seen some for $100...
"Still Too Expensive!!!" you declare!?!?!?... You could make yourself pair of DIY neoprene iris(es) arm seals for next to nothing.
If you're totally against a DIY solution you could go the super simple rubbermaid box route. I have noted several sources recommending a simple plastic bin as a makeshift hood in lieu a glove box or flow hood.
Keep up the good work.
canndo
Well-Known Member
Alexi, I didn't say I wouldn't buy a glovebox or hood, I have both, but I want to keep others from having to do so. I used the hood with my latest batch just because I am not sure when I will get more raw material so I wanted to get every advantage. I am afraid that all the cuttings I was given are pretty sketchy, I don't know how many will make it. I had thought of the incuabation thing but I don't know how I will have it cool at temperatures about 83.
canndo
Well-Known Member
So, we are back in full force, this is one of the shelves, there is another that I don't want to photo yet. As I said, there are different color media for different variations. I will probably settle on a favorite after this. I moved the lights a bit closer. Seems my worms don't particularly like all that baby food dumped on them at once and the population has taken a dramatic drop. I used to have an estimated 3 lbs and now I doubt I have half a lb left. Oh well, So I put the rest of the babyfood into my other compost - stinks though.
how much power does your set up use? if I am going to persuade my parents to let me build one of these shelving units in the shed then I am going to need build up an argument for it.
I found out the other day that my Girlfriend had a fish tank hanging around in her back garden so im going to make a glove box out of that.
Could you not make up some kind of composter for the baby food? mix it with some soil so its more diluted? im glad I have friends who are more than happy to give me empty baby food jars, i have loads of them.
I need to source some cuttings from somewhere for different strains. Canndo where do you get all of yours from? do you rely on donations or your own plants
I found out the other day that my Girlfriend had a fish tank hanging around in her back garden so im going to make a glove box out of that.
Could you not make up some kind of composter for the baby food? mix it with some soil so its more diluted? im glad I have friends who are more than happy to give me empty baby food jars, i have loads of them.
I need to source some cuttings from somewhere for different strains. Canndo where do you get all of yours from? do you rely on donations or your own plants
canndo
Well-Known Member
Each 24 inch grow light uses 17 watts, these are on 16 hours a day. I have a hepa air filtration system that I run continuously in the room, that is 40 watts. I am using a evaporative cooler between the hours of 3 and 6 in the afternoon and it uses 60 watts. So each rack of about 80 jars uses 17 watts. The whole thing doesn't use much electricity at all as you can see.
Foolery, I have several compost systems, a static one, a "rolling bin" and of course my favorite worms. I got just short of 5 gallons of pear puree, peas and rice and other assorted and typical baby food things. I just poured them all in the static bin. I don't quite know how one would go about making a glove box out of an aquarium but there must be many ways. I operate on the assumption that none will be necessary. You must have seen some of the earlier tests where I could open the jar up in my kitchen for a few minutes with no contamination. A decent biocide is key.
Where do I get my material? I have a number of good friends who are constantly growing something. This time however, I got in just before everyone started either flowering or moving to a new place. I got a number of flowering cuttings and used them anyway but I have never had any luck at all in growing plantlets from flowering plants. I figured I'd try again just to verify but even the act of sterilization is made much more difficult with flowers on the cutting.
Foolery, I have several compost systems, a static one, a "rolling bin" and of course my favorite worms. I got just short of 5 gallons of pear puree, peas and rice and other assorted and typical baby food things. I just poured them all in the static bin. I don't quite know how one would go about making a glove box out of an aquarium but there must be many ways. I operate on the assumption that none will be necessary. You must have seen some of the earlier tests where I could open the jar up in my kitchen for a few minutes with no contamination. A decent biocide is key.
Where do I get my material? I have a number of good friends who are constantly growing something. This time however, I got in just before everyone started either flowering or moving to a new place. I got a number of flowering cuttings and used them anyway but I have never had any luck at all in growing plantlets from flowering plants. I figured I'd try again just to verify but even the act of sterilization is made much more difficult with flowers on the cutting.
unfortunately for me I dont have any friends that are into growing. need to make some more friends I think.
I think im going to build up something then for the shed to put these jars on.
I know that my white widow plant material will be female as the seeds were femised. I cant wait to start this project, What does your establishment kit consist of?
I think im going to build up something then for the shed to put these jars on.
I know that my white widow plant material will be female as the seeds were femised. I cant wait to start this project, What does your establishment kit consist of?
canndo
Well-Known Member
Foolery, I am not selling anything although I might consider it in the future. Presently I want to find out if others are sucessful with the formulas I have created and I would like to get a take on other's experiences. The "kit" would be the cytokinins and balancing auxin plus a biocide in a bottle of distilled water, there would be a packet of MS + and another packet of gelling agent. All you have to do is mix it in the right orderm add sucrose and adjust the PH to 5.7 (for one formula) and 5.8 (for another) - seems that agar alters the PH more than the other stuff I am also using. It would be enough to prepare one liter. Depending on the jars you are using a liter can make as many as 50 starts, or more if you use test tubes which I don't recommend.
As far as your not having much material, I am working on going direct from seed as you probably know from reading the thread.
There is a sterilized seed in sterilized STG in each of those test tubes.
Although I have not done it yet, I am quite certain that you can take the top most portion of a seedling and get a damn good start. Beyond that, it is easier to sterilize a seed than it is an explant so you get sterile conditions from the very beginning. The problem comes with lighting. So far the few(cound them, 3) I have managed to get to germinate have grown too tall. This setup doesn't give nearly enough light to keep the seedlings from streching. Anyway, I believe this to be a damn good way to get your initial material.
As far as your not having much material, I am working on going direct from seed as you probably know from reading the thread.
There is a sterilized seed in sterilized STG in each of those test tubes.Although I have not done it yet, I am quite certain that you can take the top most portion of a seedling and get a damn good start. Beyond that, it is easier to sterilize a seed than it is an explant so you get sterile conditions from the very beginning. The problem comes with lighting. So far the few(cound them, 3) I have managed to get to germinate have grown too tall. This setup doesn't give nearly enough light to keep the seedlings from streching. Anyway, I believe this to be a damn good way to get your initial material.
I am interested in this kit, to be able to help prove that it works, but being in the UK could be an issue so Im happy to get my own ingredients if you tell me what I need and in what amounts,
i had noticed that you were struggling with getting the seeds to germinate, have you had anymore luck with that?
Im going to send a few texts around to see if people are growing it and then see if they will get me plant material for my science project
i had noticed that you were struggling with getting the seeds to germinate, have you had anymore luck with that?
Im going to send a few texts around to see if people are growing it and then see if they will get me plant material for my science project
canndo
Well-Known Member
Wow, I wrote a HUGE post and included all sorts of new pictures but got called away before I posted and I am afraid I lost it. DAMN.
Damn. Ok, I'll have to do it again.
Damn.
Foolery, I found that the seeds were crap. I finally got some more and I suspect they will be ok. There is a problem with the ingredients that you should be aware of. You have to buy lots of stuff. If you want to work with TDZ (and you will) then the smallest amount you can buy is 100 mg. You then have to make a stock solution down to .1 mg per milliliter. Now you have this huge amount of TDZ solution that begins to degrade the moment you mix it up. I happen to have a very precise scale so I can make rather small amounts at a time but for most this part is difficult.
I am really depressed I lost all that posting work.
Maybe I'll just send the pictures now
Ok.
You will need:
Napthaleneacetic acid
Thidiazuron (the 95% pure will do)
Indole-3 Butyric Acid
Gibberellic Acid
Adenine
Activated Charcoal
Murashige & Skoog Basal Medium (I have not yet determined if the MS + vitamins are necessary)
You can try gelzan, gelrite or Agar. I started with agar and swore by it but it isn't clear, it is expensive and I suspect it changes my ph. I have come to the point of using 1.7 grams of gelzan and .5 grams of Agar. The point is to find something that allows the chemicals to be mobile and still be stiff enough to support the plant.
Damn. Ok, I'll have to do it again.
Damn.
Foolery, I found that the seeds were crap. I finally got some more and I suspect they will be ok. There is a problem with the ingredients that you should be aware of. You have to buy lots of stuff. If you want to work with TDZ (and you will) then the smallest amount you can buy is 100 mg. You then have to make a stock solution down to .1 mg per milliliter. Now you have this huge amount of TDZ solution that begins to degrade the moment you mix it up. I happen to have a very precise scale so I can make rather small amounts at a time but for most this part is difficult.
I am really depressed I lost all that posting work.
Maybe I'll just send the pictures now
Ok.
You will need:
Napthaleneacetic acid
Thidiazuron (the 95% pure will do)
Indole-3 Butyric Acid
Gibberellic Acid
Adenine
Activated Charcoal
Murashige & Skoog Basal Medium (I have not yet determined if the MS + vitamins are necessary)
You can try gelzan, gelrite or Agar. I started with agar and swore by it but it isn't clear, it is expensive and I suspect it changes my ph. I have come to the point of using 1.7 grams of gelzan and .5 grams of Agar. The point is to find something that allows the chemicals to be mobile and still be stiff enough to support the plant.
canndo
Well-Known Member
I am unsure of what you are asking. I doubt you could use such a cloner because the shoots are so tiny they wouldn't fit into the thing. If they did I don't think they would survive long enough to establish roots. I don't understand your M+S,
I know the feeling when you lose a whole post that you've wrriten, done it plenty of times usually because i press the wrong button.
I looked at that list of chemicals that you posted and for about 5 mins was confused but ive got used to talking in their abreviations. The issue I have found so far in sourcing all the correct chemicals is that there is not much of a network of suppliers for tissue culturing here in the UK unlike the US which is lucky enough to have the kitchen tissue culture company there, the only way that I can justify buying from here is if I purchase massive amounts but I think I am going to have to do that.
Ive not heard of this gelrite, is it abit like gelatine?
Wolverine97
Well-Known Member
Glad to see you're still at it canndo. I've had some issues lately, so I haven't been able to stop in and check on things. Hope you get the bugs worked out!