Foolery, England has a huge tradition of nutty gardeners, find some of those and ask them lol. Seriously though, I would call up Kew Gardens, your local orchid club or some random university professor and ask them. Although, I think your final outcome will be ordering from Holland.
Tissue Culture/Micro Propagation Grow from Scratch
- Thread starter canndo
- Start date
a dog named chico
Well-Known Member
where did you get the initail kit from? i have been looking for one for a while
i guess we do have a huge tradition of nutty gardeners now i think about it. When i visited kew they had a tissue culture lab will get in touch with them.
canndo
Well-Known Member
I was wondering where you were Wolverine, I was afraid you had lost interest. Yes, this is no longer a "bug" thing as I am now just doing refinements - except of course the temperature issue, and my lack of constant material.
canndo
Well-Known Member
Initial kit is from PlantTC. They guy is very nice. His kit has been maligned by many. I will tell you and everyone else that I did a major research endeavor before I started, I looked at every site like this one on the net (and found this one to be the best over all btw). I found many beginning threads about micropropagation but no one, not one, ever managed more than a few pages. I never saw extended pictures or extended growing. I couldn't believe that I was actually the first but here it is. Now, my point is that in almost every thread there were people saying that it can't be done by a lay person. It is too expensive, and the PlantTC kit is a waste of time.
It isn't, The jars, the foreceps, the tray, the lids and even his gell kits, which are about as general as you can get actually work with our plant of choice - though not at all well. He offers a decent little instruction book and even a DVD. If you do get the kit however, don't do what I did. (the second time, I actually bought two kits). I left the second kit out on the floor for a week or so - well, I didn't, the vendor I got it from did. The gell kits have to be refrigerated and they don't tell you that unless you open the box. I don't want to look like a shill for PlantTC so I will give you a list of initial ingredients.
I did something on general requirements some pages back but this one will address only the starter kit - and I am thinking of putting together a book about this. I believe that most of the contents will wind up in these threads but not everyone reads RIU and it might be nice to have everything in one place. I am spending a hell of a lot of money on this and my wife is beginning to call on me to deal with it. I am not going to quit, I am far too proud.
Speaking of books, I ran into someone who says he is working on a book with Rosenthal about this very thing. He also said that I couldn't get this done for under $10,000 dollars and 3 years work. I got some information from him that indicated that he uses organic methods, coconut milk, banana extract and the like.
I appologize to the organic purists who are reading this but I simply can't go that route, it is too imprecise for me. I hope you all will forgive me and presume that by the time you actually start growing your plantlets out, your organic methods will be enough to call the entire process "organic". But... even though lots of people are making that particular claim, the truth is that we are using some very non-organic chemicals here, even if they are being used in minute amounts. In the hundreds and hundreds of plants I've grown out I doubt I have used more than 5 milligrams of hormones and most of that gets thrown out.
I am going to post this and go on, you know why.
canndo
Well-Known Member
Ok, To start:
50 babyfood jars, 100 is better. You want enough to have a cycle going. One liter makes enough gell for about 50 jars. You set a few of the jars aside for recovery and over the course of the first three weeks you will discard the contents of the jars that didn't work out, either they died or they got contaminated but you won't have enough to make another complete set of 50. You are going to want that set when you do your first divisions. It is likely that you won't use them all just yet though. Remember the point of all of this is to ESTABLISH. Once you get those plants to the point where they are stable and sterile you are past that first large hurdle and if you don't do something foolish or overheat your collection, you can have those plants for ever. Now remember that eventually you are going to want to root them and that will take a batch of 50 jars as well. I have found that you will want a high degree of overkill early on, afterward, you will get good enough to have the confidence I have in the start of this thread.
"But Canndo, where will I get so many baby food jars?" First, if you are in a hurry, check ebay. They are always selling them but they can be expensive and shipping is a bitch. Next, take a trip to your local 99 cent type store, the places that buy damaged containers or expired food items, every town has one at least. These places will often have jars. Mothers don't like to feed their children expired food (it isn't bad at all actually but you know moms) so you may be able to find lots of them. I found hundreds at one of those shops near me and was so confident that no one else would buy them that I only got a few cases (12 in a case) at a time. They cost me 20 cents each. I went back last month and found that he had a dispute with his landlord and was moving out. I could have made a deal with him and bought him out but I didn't. In order to do my last batch I had to go to Big Lots and buy THEM out, at 45 cents each. When you get your jars (pay attention, this will save you a lot of time and agrivation), empty the contents into a bucket and dump the bucket into your compost - don't give it all to your worms, your worms will die if you do. Now soak all the jars in warm water until the labels float off or are very soft. Take the labels off and put the jars in acetone. After soaking in acetone for 20 minutes or so, take an abrasive sponge to them and then rinse them with water. This way the jars won't stick to each other and your fingers and won't annoy you or drop off onto the floor and break. The jars will be clean enough so that if you want to take pictures and publish them on RIU, the plants can actually be seen.
a similar amount of Magenta B caps - You will pay as little as 8 dollars for 25 and as much as 22 dollars for 25. (I am planning on purchasing several thousand in the near future because supplies are so spotty and you will always always need more)
Forceps/tweezers - This is your primary tool. Get several and don't skimp, cheap tweezers suck. Get 8 inch or more long. You will be reaching into rather deep jars and the further away your hands are from your working objects the better (within reason). Get at least two so you can switch them out as you work, sterilizing one while you use the other. I got mine in the kits, try ebay. DON'T skimp on this, don't look at the dollar tweezers, don't do it. You will cry if you do.
Scalpel - I am not really happy with any solution I have tried with scalpels. You can try the metal ones and get disposable blades, they are the cheapest but they are a pain and I find that the blades snap when I am working with some of the larger stems. You can try the completely disposable ones but these violate my conservative sensability. They are somewhat expensive and you wind up throwing the whole thing out. I had a single disposable scalpel that I found I could sterilize. I suppose that would be a good deal but I've yet to find them again. You can even use single edged razor blades, they are the cheapest and I have yet to get a scalpel that was as sharp. You will have to discover your preference for the shape of the blade, I like a rounded, rather wide blade myself, a #10 I believe. You are going to have to go to Ebay for these. I believe you can get them at your hydro store as well but, well, your hydro store will likely charge a whole lot more than you should pay.
Scissors - try to find a cheap pair of all metal scissors, small.
Turkey baster - I've spoken about this, get yourself a good one. Mine is all glass but the point is that you don't want one that leaks. Don't get the one at the 99 cent store, it will suck (or not suck, as the case may be)
Trays - I didn't much care about trays that hold 20 or 30 jars at a time but now I do. If you go to your local gardening center and ask nicely you will likely be able to get their plastic gardening trays, many times for free - they work quite well.
Operating plate - just some ceramic or glass plate that is completely flat at the center, get several, they work well as your cutting base covered with a sterile paper towel. 99 Cent store will have what you want.
Round screen or mesh - This is probably the single most useful thing in the kit. It is inserted in small mouth canning jars and is used to quickly drain liquids from your cuttings. Yes, you can simply punch lots and lots of holes into a canning jar lid. I have three. You can get them at your local health food store, look at the sprouting kits.
That is about all that was in the kit, except for the instructions and the DVD and the gell kits. You have the instructions right here on RIU, you don't need the damn DVD, we are already too dependant on videos and the gell kit? Well, Canndo has offered to GIVE people the kit, so if all you are waiting for is that pressure cooker, well we can talk about that.
The pressure cooker is not part of any kit. You have to have a pressure cooker. You can get a 23 quart pressure cooker at Amazon for 82 bucks. But wait. You don't need 23 quarts of capacity. What will you be sterilizing? Bundles of instruments, the longest is about 8 inches - it will fit in a 6 quart. You can fit 20 baby food jars in a 6 quart stacked 2 high. I have seen new 6 quart cookers sell for as little as 15 dollars. Now don't get me wrong, I am not advocating that you settle for something that will make more work for you. (you will have to go 3 rounds in order to sterilize all 50 jars) but you can get in this for cheap if you go that route. Furthermore, if you don't chose to continue, a 6 quart will cook 2 artichokes to perfection in under 15 minutes. If you do continue, then get a larger cooker and use the smaller one for your instruments or for sterilizing water in pint jars.
I should tell you though, if you get the larger one, you can easily sterilize 7 quart jars at a time. And leave that old PF tek in the dust.
50 babyfood jars, 100 is better. You want enough to have a cycle going. One liter makes enough gell for about 50 jars. You set a few of the jars aside for recovery and over the course of the first three weeks you will discard the contents of the jars that didn't work out, either they died or they got contaminated but you won't have enough to make another complete set of 50. You are going to want that set when you do your first divisions. It is likely that you won't use them all just yet though. Remember the point of all of this is to ESTABLISH. Once you get those plants to the point where they are stable and sterile you are past that first large hurdle and if you don't do something foolish or overheat your collection, you can have those plants for ever. Now remember that eventually you are going to want to root them and that will take a batch of 50 jars as well. I have found that you will want a high degree of overkill early on, afterward, you will get good enough to have the confidence I have in the start of this thread.
"But Canndo, where will I get so many baby food jars?" First, if you are in a hurry, check ebay. They are always selling them but they can be expensive and shipping is a bitch. Next, take a trip to your local 99 cent type store, the places that buy damaged containers or expired food items, every town has one at least. These places will often have jars. Mothers don't like to feed their children expired food (it isn't bad at all actually but you know moms) so you may be able to find lots of them. I found hundreds at one of those shops near me and was so confident that no one else would buy them that I only got a few cases (12 in a case) at a time. They cost me 20 cents each. I went back last month and found that he had a dispute with his landlord and was moving out. I could have made a deal with him and bought him out but I didn't. In order to do my last batch I had to go to Big Lots and buy THEM out, at 45 cents each. When you get your jars (pay attention, this will save you a lot of time and agrivation), empty the contents into a bucket and dump the bucket into your compost - don't give it all to your worms, your worms will die if you do. Now soak all the jars in warm water until the labels float off or are very soft. Take the labels off and put the jars in acetone. After soaking in acetone for 20 minutes or so, take an abrasive sponge to them and then rinse them with water. This way the jars won't stick to each other and your fingers and won't annoy you or drop off onto the floor and break. The jars will be clean enough so that if you want to take pictures and publish them on RIU, the plants can actually be seen.
a similar amount of Magenta B caps - You will pay as little as 8 dollars for 25 and as much as 22 dollars for 25. (I am planning on purchasing several thousand in the near future because supplies are so spotty and you will always always need more)
Forceps/tweezers - This is your primary tool. Get several and don't skimp, cheap tweezers suck. Get 8 inch or more long. You will be reaching into rather deep jars and the further away your hands are from your working objects the better (within reason). Get at least two so you can switch them out as you work, sterilizing one while you use the other. I got mine in the kits, try ebay. DON'T skimp on this, don't look at the dollar tweezers, don't do it. You will cry if you do.
Scalpel - I am not really happy with any solution I have tried with scalpels. You can try the metal ones and get disposable blades, they are the cheapest but they are a pain and I find that the blades snap when I am working with some of the larger stems. You can try the completely disposable ones but these violate my conservative sensability. They are somewhat expensive and you wind up throwing the whole thing out. I had a single disposable scalpel that I found I could sterilize. I suppose that would be a good deal but I've yet to find them again. You can even use single edged razor blades, they are the cheapest and I have yet to get a scalpel that was as sharp. You will have to discover your preference for the shape of the blade, I like a rounded, rather wide blade myself, a #10 I believe. You are going to have to go to Ebay for these. I believe you can get them at your hydro store as well but, well, your hydro store will likely charge a whole lot more than you should pay.
Scissors - try to find a cheap pair of all metal scissors, small.
Turkey baster - I've spoken about this, get yourself a good one. Mine is all glass but the point is that you don't want one that leaks. Don't get the one at the 99 cent store, it will suck (or not suck, as the case may be)
Trays - I didn't much care about trays that hold 20 or 30 jars at a time but now I do. If you go to your local gardening center and ask nicely you will likely be able to get their plastic gardening trays, many times for free - they work quite well.
Operating plate - just some ceramic or glass plate that is completely flat at the center, get several, they work well as your cutting base covered with a sterile paper towel. 99 Cent store will have what you want.
Round screen or mesh - This is probably the single most useful thing in the kit. It is inserted in small mouth canning jars and is used to quickly drain liquids from your cuttings. Yes, you can simply punch lots and lots of holes into a canning jar lid. I have three. You can get them at your local health food store, look at the sprouting kits.
That is about all that was in the kit, except for the instructions and the DVD and the gell kits. You have the instructions right here on RIU, you don't need the damn DVD, we are already too dependant on videos and the gell kit? Well, Canndo has offered to GIVE people the kit, so if all you are waiting for is that pressure cooker, well we can talk about that.
The pressure cooker is not part of any kit. You have to have a pressure cooker. You can get a 23 quart pressure cooker at Amazon for 82 bucks. But wait. You don't need 23 quarts of capacity. What will you be sterilizing? Bundles of instruments, the longest is about 8 inches - it will fit in a 6 quart. You can fit 20 baby food jars in a 6 quart stacked 2 high. I have seen new 6 quart cookers sell for as little as 15 dollars. Now don't get me wrong, I am not advocating that you settle for something that will make more work for you. (you will have to go 3 rounds in order to sterilize all 50 jars) but you can get in this for cheap if you go that route. Furthermore, if you don't chose to continue, a 6 quart will cook 2 artichokes to perfection in under 15 minutes. If you do continue, then get a larger cooker and use the smaller one for your instruments or for sterilizing water in pint jars.
I should tell you though, if you get the larger one, you can easily sterilize 7 quart jars at a time. And leave that old PF tek in the dust.
just a FYI since phytotech is still out of b-caps, caisson has some pretty good prices on B-caps $30/100. They also sell what sounds like a knock-off parafilm for crazy cheap $5/1.5" x 1000' roll. I haven't tried it so I can't say for sure, but $5 for 1000' beats $28 for 250' of parafilm.
www.caissonlabs.com
www.caissonlabs.com
Wolverine97
Well-Known Member
No, I haven't lost interest, but I am seriously questioning whether this would be a viable thing for me to attempt. I know I could "do it", but I really doubt I'd have the time, I barely find the time to maintain my garden as it is now. Thanks for keeping this going though, excellent thread!
Out of all these ultrasonic cleaners in this ebay search what do you reckon will be suitable? will one of the cheap ones be pretty good? http://shop.ebay.co.uk/i.html?_kw=ultrasonic%20cleaner&_ds=1&_fcid=3&_localstpos=&_stpos=&gbr=1
I still need magenta caps, and you guys in america find them much more easiier than I can, can i go into a group buy with you or something?
I still need magenta caps, and you guys in america find them much more easiier than I can, can i go into a group buy with you or something?
foolery I just noticed there is a distributor link on the phytotech website and they have a pair of UK distributors... www.apolloscientific.co.uk looks like the winner of the two. Hope that helps a bit.
Right well I will get one of the cheapy ones then as theres a few on there that are cordless.
And thans for that link to a Uk supplier, just looking at them now, I noticed that they are based near where I go to uni so on a 2.50 return bus trip to that town
cheers guys for your continuing help
And thans for that link to a Uk supplier, just looking at them now, I noticed that they are based near where I go to uni so on a 2.50 return bus trip to that town
cheers guys for your continuing help
upthearsenal
Well-Known Member
Wow. Okay. I'm subbed but I've got a lot of reading to catch up on 
Awesome thread man.
Awesome thread man.
canndo
Well-Known Member
Ok, we now have a steady, and fairly inexpensive source for Magenta B caps. I am assured that the proprietor will ship anywhere and he is a damn good guy. Is it proper to post such sources? I don't know the rules here.
UP, welcome to the club. We are moving on to better things in the next few weeks. I'm getting a refrigerator this week and am looking into modifying it for our incubation purposes. I'll include pictures when it comes.
UP, welcome to the club. We are moving on to better things in the next few weeks. I'm getting a refrigerator this week and am looking into modifying it for our incubation purposes. I'll include pictures when it comes.
I think the b cap link should be fine, it's a community service.
I found a relatively painless way to control humidity fairly precisely and cheapish too. get yourself an electronic cigar humidifier. one rated for 60 cu.ft. is like $150 and one for a mini-frig is $80 (I didn't shop prices those are retail from a cigar website, so I'm sure those price could come down some) You could easily modify the water res to increase it's capacity, so you could pretty much set it and forget it. I'm frightened at the prospective costs of possibly having to monitor and control the co2 at some point in time. I wish there was a cheap and easy co2 controller, some arduino guru should create one.
I found a relatively painless way to control humidity fairly precisely and cheapish too. get yourself an electronic cigar humidifier. one rated for 60 cu.ft. is like $150 and one for a mini-frig is $80 (I didn't shop prices those are retail from a cigar website, so I'm sure those price could come down some) You could easily modify the water res to increase it's capacity, so you could pretty much set it and forget it. I'm frightened at the prospective costs of possibly having to monitor and control the co2 at some point in time. I wish there was a cheap and easy co2 controller, some arduino guru should create one.
canndo
Well-Known Member
Alexi, I really haven't put ambient C02 concerns into any matrix. In fact, damn you, I haven't even thought about it. Not gonna. I got too many other variables left to clean up.
As far as humidity is concerned, I have a good set up for that but it shouldn't be necessary. I have used tape on the vents and they work quite well, you need only a 16th of an inch hole. As we start refining things I am going to get lids with vents made for baby food jars and for 1/2 pint canning jars with autoclavable clear lids. I've found what I need but they won't make them for me in quantities of less than 5,000.
I have my establishment gel dialed in and I am not going to tamper with it again. Establishment can happen in as little as a week but after two weeks my cuttings are either showing definite shoots or they are dead. I am indeed dividing up establishment and multiplication. Now, some news. We are having another spell of heat and it is treacherous. I have some thriving but not nearly as many as I need. This is likely going to be a long summer with over 100 degree temps so I have gotten a traditional size fridge, it will be here next week.
I need some help. How in the hell am I going to wire the thing to come on when the temps exceed 80 degrees? What sort of thermistat should I use in here? I have looked at conversions for incubators but they are for raising the heat and not necessarily lowering it. I know that the lights give off some heat so I can't just fake the thing. Anyone have any ideas? Anyone done anything like this before? This is the only way I am going to be able to continue.
I have gotten a tremedous amount of interest in this, far more than I had ever thought and people are beginning to donate things although they are still rather tight with the cuttings. NONE of those 5 tubs of cuttings worked. The reason is that they fermented in the water, they were just too contaminated and even my most stringent methods failed. If I can get a few more things nailed down and get more cuttings, I estimate 6 months will get us a rugged, dependable series
As far as humidity is concerned, I have a good set up for that but it shouldn't be necessary. I have used tape on the vents and they work quite well, you need only a 16th of an inch hole. As we start refining things I am going to get lids with vents made for baby food jars and for 1/2 pint canning jars with autoclavable clear lids. I've found what I need but they won't make them for me in quantities of less than 5,000.
I have my establishment gel dialed in and I am not going to tamper with it again. Establishment can happen in as little as a week but after two weeks my cuttings are either showing definite shoots or they are dead. I am indeed dividing up establishment and multiplication. Now, some news. We are having another spell of heat and it is treacherous. I have some thriving but not nearly as many as I need. This is likely going to be a long summer with over 100 degree temps so I have gotten a traditional size fridge, it will be here next week.
I need some help. How in the hell am I going to wire the thing to come on when the temps exceed 80 degrees? What sort of thermistat should I use in here? I have looked at conversions for incubators but they are for raising the heat and not necessarily lowering it. I know that the lights give off some heat so I can't just fake the thing. Anyone have any ideas? Anyone done anything like this before? This is the only way I am going to be able to continue.
I have gotten a tremedous amount of interest in this, far more than I had ever thought and people are beginning to donate things although they are still rather tight with the cuttings. NONE of those 5 tubs of cuttings worked. The reason is that they fermented in the water, they were just too contaminated and even my most stringent methods failed. If I can get a few more things nailed down and get more cuttings, I estimate 6 months will get us a rugged, dependable series
The big reason I'm on the full environmental kick is all the research I've been doing, the same idea of adjusting environmental factors to help induce rooting and also comes into play during hardening off. I figure all these phds must be on to something 
plus I've always been of the mindset that environmental controls will make up your largest yield gains.
Summer temps are of a worry to me as well as my temps generally go into the lower 100s. I have been looking into various DIY "environmental boxes" constructions and designs be they cheese caves, kegerators, mushroom growth chambers or off grid homesteaders using chest freezers as refrigerators. I have set my sights on some sort of a craigslist frost-free upright freezer coupled with a digital thermostat. ie. http://www.amazon.com/gp/product/B00368D6JA You can find ones like these at a homebrewer's supply.
I did happen to find a DIY arduino co2/humidity/temp controller that looks like it should work, I have an electrical engineer buddy that I'm going to beat over the head to help me figure out. It's very spartan, but it could work eventually http://www.instructables.com/id/Environmental-Mushroom-Control-Arduino-Powered/ and for trillions less than the full blown controllers (sentinel, etc.) that they sell for full size rooms. I'm not too worried about humidity controls as that is very easily adjusted using open containers of water in the growth chamber, but co2 is the one I'm more apt to be concerned with as that seems to have the most control during rooting and hardening, but I need to get to a stage close to where you are before I have to worry about co2 controls (at least by my estimations)
plus I've always been of the mindset that environmental controls will make up your largest yield gains. Summer temps are of a worry to me as well as my temps generally go into the lower 100s. I have been looking into various DIY "environmental boxes" constructions and designs be they cheese caves, kegerators, mushroom growth chambers or off grid homesteaders using chest freezers as refrigerators. I have set my sights on some sort of a craigslist frost-free upright freezer coupled with a digital thermostat. ie. http://www.amazon.com/gp/product/B00368D6JA You can find ones like these at a homebrewer's supply.
I did happen to find a DIY arduino co2/humidity/temp controller that looks like it should work, I have an electrical engineer buddy that I'm going to beat over the head to help me figure out. It's very spartan, but it could work eventually http://www.instructables.com/id/Environmental-Mushroom-Control-Arduino-Powered/ and for trillions less than the full blown controllers (sentinel, etc.) that they sell for full size rooms. I'm not too worried about humidity controls as that is very easily adjusted using open containers of water in the growth chamber, but co2 is the one I'm more apt to be concerned with as that seems to have the most control during rooting and hardening, but I need to get to a stage close to where you are before I have to worry about co2 controls (at least by my estimations)
canndo
Well-Known Member
Just a report, no pictures today. Things are very slow, I got some more cuttings but am hesitant to do anything with them because the heat may come back (well, it will but right now it is perfectly cool). I am still waiting for my refrigerator and I still don't know what I am going to do with it but I have some ideas. I don't want anyone to think this is in any way a dead project... oh no, it is very much alive. I am working with a Hydro store vendor to have him offer supplies and I expect him to have quite a number of things that I use being offered on his page. I'll post the address when everything is up.