ChemDogLover
Well-Known Member
Is this environment clean enough? Apparently it’s working for her. Makes me want to try.
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Tissue Culture : Small Indoor Setup
- Thread starter mistergrafik
- Start date
Hollatchaboy
Well-Known Member
Drop That Sound
Well-Known Member
Yes it can be. It's standard practice now days for home mycologist to utilize makeshift still air boxes to help produce contaminant free cultures, etc. TC isn't much different in regards to it's use, so yeah. They work as long as you use them correctly, with proper sterile technique. A cheap but effective alternative to laminar flow hoods. Lots of info out there on SABs and how to use them with success.
I would recommend using an instant pot or pressure cooker instead of the microwave to sterilize the media though. Maybe a few other corners I wouldn't cut, and an extra $100 I would spend.. but over all she laid it out pretty good. You really don't need a fancy lab to pull if off, especially on a small time scale. It's a time consuming process to get viable clones through all the stages though, so the last thing you want is to mess anything up. There are plenty of cannabis protocols floating around though, and it's not a big secret to figure out the steps\recipes anymore. Follow the directions and prepare to keep trying and you should be good
ChemDogLover
Well-Known Member
Very cool, thanks for the reply. This is something that interests me, so I plan on pursuing it on 2024.Yes it can be. It's standard practice now days for home mycologist to utilize makeshift still air boxes to help produce contaminant free cultures, etc. TC isn't much different in regards to it's use, so yeah. They work as long as you use them correctly, with proper sterile technique. A cheap but effective alternative to laminar flow hoods. Lots of info out there on SABs and how to use them with success.
I would recommend using an instant pot or pressure cooker instead of the microwave to sterilize the media though. Maybe a few other corners I wouldn't cut, and an extra $100 I would spend.. but over all she laid it out pretty good. You really don't need a fancy lab to pull if off, especially on a small time scale. It's a time consuming process to get viable clones through all the stages though, so the last thing you want is to mess anything up. There are plenty of cannabis protocols floating around though, and it's not a big secret to figure out the steps\recipes anymore. Follow the directions and prepare to keep trying and you should be good
SCJedi
Well-Known Member
If you insist on using deli tubs it helps to add a micropore filter (or two) on the lid to allow gas exchange. I can see the condensation in your tubs. It also helps to allow media to cool completely before you put your lids on
formularacer
Well-Known Member
Many moons ago there was a tissue culture project involving bamboo. Now most temperate bamboo is of the running type. There are mountain bamboo which are clumpers and don't run. These are very desirable in the garden. There was a project start to propagate these by tissue culture. Millions were spent land was purchased in Mexico. I got two samples of these plants it never got bigger than a foot tall after ten years, so needless to say the whole project failed. Pretty pointless for a plant that normally gets to 6 to 8 feet tall to be less than a foot tall easy to trip over.
What started the whole project was a bamboo species flower at the same time world wide. There were giant displays at public gardens that all went to seed. The previous time for flower was 135 years ago.
So just a little trivia.....
What started the whole project was a bamboo species flower at the same time world wide. There were giant displays at public gardens that all went to seed. The previous time for flower was 135 years ago.
So just a little trivia.....
Drop That Sound
Well-Known Member
How Can The Biocoupler™ Help Your Plant Business Grow? - Plant Cell Technology
Boost your plant production with Plant Cell Technology's Biocoupler™, an efficient temporary immersion bioreactor. It multiplies the number of plants you can grow per sq ft, requires no electricity, and helps produce healthy, disease-free plants.
www.plantcelltechnology.com
Check out these bio coupler fittings, that allow you to temporarily immerse the plants (cannabis approved btw) during the multiplication stage, but without a fancy contraption containing solenoids/pumps, etc.. Just simply toss it a few times (or make a spinning tumbler rack with a timer?), and still allow gas exchange through the filter, and therefor have maximum air flow to the roots. You make a liquid media without the agar, and so its more like a hydroponic method that will speed up growth it seems. It apparently solves many issues you might experience with using a gelled media during that stage... They are cheap, but also probably can easily be replicated by gluing 2 jar lids together with epoxy putty, and using your own screen.
Turpman
Well-Known Member
I’ve been experimenting.
Just used rtv to glue both caps together. The large baby bottle didn’t handle the pressure cooker well but is intact, just a bit hazy.
Stainless wires keep explant in the top container. The black tubing is stuffed with Polly wool as a filter. Must keep it dry. So you slowly rotate keeping the vent up as you move the liquid to the other end to wet the explant.
I purchased a small dissecting microscope to see if I can get down to meristem. I did about 8 today. It’s fricken hard. The meristem is reeeely small.
Phytotecknologys has all the media nutrients. Even a few with hormones for cannabis. I’ve been mixing my own hormones. Some are not heat stable so a syringe filter is nessesary to add hormone after the agar has cooled enough. No one will share the protocols. Most have $$ signs in their eyes with the hop virus cleanup in mind. Lots of info on the net just takes some searching.
Some plants can have pathogens inside and are very hard to get sterilized. The haze around the base of this explant may be from internal. Could be from me to but it’s infected.
Meristem culture is where it’s at. You start with such a smal piece of the very tip of the plant plantpathogens are left behind. So you can clean a strain that has been infected. Even with the hop virus.
Not sure on the storage time but it is one of the reasons to get your mother in TC. It is clean steril and you can slow growth by cooling and dimming light. Probly twice a year reculturing would be needed. Plus space needed is much less.
I make beer so yeast culturing is handy as well as the odd funji run. Same tools. I just recultured some yeas and funji that was on plates on the fridge for 6 years. Blew my mind they were still alive.
This yeast plate was 6 years in the fridge crisper. Took right off when I replated it.
Just used rtv to glue both caps together. The large baby bottle didn’t handle the pressure cooker well but is intact, just a bit hazy.
Stainless wires keep explant in the top container. The black tubing is stuffed with Polly wool as a filter. Must keep it dry. So you slowly rotate keeping the vent up as you move the liquid to the other end to wet the explant.
I purchased a small dissecting microscope to see if I can get down to meristem. I did about 8 today. It’s fricken hard. The meristem is reeeely small.
Phytotecknologys has all the media nutrients. Even a few with hormones for cannabis. I’ve been mixing my own hormones. Some are not heat stable so a syringe filter is nessesary to add hormone after the agar has cooled enough. No one will share the protocols. Most have $$ signs in their eyes with the hop virus cleanup in mind. Lots of info on the net just takes some searching.
Some plants can have pathogens inside and are very hard to get sterilized. The haze around the base of this explant may be from internal. Could be from me to but it’s infected.
Meristem culture is where it’s at. You start with such a smal piece of the very tip of the plant plantpathogens are left behind. So you can clean a strain that has been infected. Even with the hop virus.
Not sure on the storage time but it is one of the reasons to get your mother in TC. It is clean steril and you can slow growth by cooling and dimming light. Probly twice a year reculturing would be needed. Plus space needed is much less.
I make beer so yeast culturing is handy as well as the odd funji run. Same tools. I just recultured some yeas and funji that was on plates on the fridge for 6 years. Blew my mind they were still alive.
This yeast plate was 6 years in the fridge crisper. Took right off when I replated it.
ChemDogLover
Well-Known Member
How Can The Biocoupler™ Help Your Plant Business Grow? - Plant Cell Technology
Boost your plant production with Plant Cell Technology's Biocoupler™, an efficient temporary immersion bioreactor. It multiplies the number of plants you can grow per sq ft, requires no electricity, and helps produce healthy, disease-free plants.
Check out these bio coupler fittings, that allow you to temporarily immerse the plants (cannabis approved btw) during the multiplication stage, but without a fancy contraption containing solenoids/pumps, etc.. Just simply toss it a few times (or make a spinning tumbler rack with a timer?), and still allow gas exchange through the filter, and therefor have maximum air flow to the roots. You make a liquid media without the agar, and so its more like a hydroponic method that will speed up growth it seems. It apparently solves many issues you might experience with using a gelled media during that stage... They are cheap, but also probably can easily be replicated by gluing 2 jar lids together with epoxy putty, and using your own screen.
Interesting
Turpman
Well-Known Member
Now I read you can take an immature seed and start in tc rather than wait for it to ripen on the plant. This bean was probly on the mature side but what the hey. Sooooo
I just sterilized it for 15 rinsed in sterile water and plunked it into multiplication media. Pobluy should have removed the embryo? Probly shouldn’t have had hormones in it but will be a good experiment. Pre 98 bubba kush S1
Been having lots of problems with internal contamination, endophytes. Most likely bacillus. They can be benificial to a regular plant but once you plunk it into a sugar rich media they take over. You can’t kill them because they are inside the plant tissue. Just received some new media and transferred some merristem onto it and it’s looking promising. Usually take a week or 2 for the endophytes to show. You can bacically watch the media turn hazy right at the cut end of the explant. Wish I could get a better pick of the merristem but they are about the size of a grain of salt. Even smaller.
I just sterilized it for 15 rinsed in sterile water and plunked it into multiplication media. Pobluy should have removed the embryo? Probly shouldn’t have had hormones in it but will be a good experiment. Pre 98 bubba kush S1
Been having lots of problems with internal contamination, endophytes. Most likely bacillus. They can be benificial to a regular plant but once you plunk it into a sugar rich media they take over. You can’t kill them because they are inside the plant tissue. Just received some new media and transferred some merristem onto it and it’s looking promising. Usually take a week or 2 for the endophytes to show. You can bacically watch the media turn hazy right at the cut end of the explant. Wish I could get a better pick of the merristem but they are about the size of a grain of salt. Even smaller.
Drop That Sound
Well-Known Member
I read about it too, but they would extract just the tiny little premature embryo from the pod. If I recall, besides speeding the process up, there are other benefits too, that are complete game changers to the current cannabis breeding techniques. Kinda like artificial human womb technology, we will be able to select traits and alter the genetics of the plants soon, via artificial seed pods..
I'm very interested to see how culturing the whole pod will work out, and hope it stays free of contams for you.
Did you check out how to make synthetic seeds yet? Looks like a fun process more than anything! I hope to try making some in the near future..
www.plantcelltechnology.com
I'm very interested to see how culturing the whole pod will work out, and hope it stays free of contams for you.
Did you check out how to make synthetic seeds yet? Looks like a fun process more than anything! I hope to try making some in the near future..
Pros and Cons of Synthetic Seeds - Plant Cell Technology
Synthetic seeds are man-made or artificial seeds which include encapsulated somatic embryos, shoot buds, cell aggregates, or any other meristematic tissue. Synthetic seeds have the ability to form a whole plant in either in vitro or ex vitro conditions. Introduction To Synthetic Seeds Synthetic...
www.plantcelltechnology.com
Drop That Sound
Well-Known Member
Not all "hazing" and or browning of the plant material or the media is completely abnormal right? Not necessarily some form of contamination or bacteria that is..
" Browning is one of the major issues in plant tissue culture processes. Browning of tissue culture media is caused by phenolic compounds released from plant tissues. Enzymatic browning, facilitated by polyphenol oxidases (PPOs), further contributes to discoloration. The situation hinders the preservation and propagation of our valuable plant genetic resources.
This can be controlled by either using charcoal, antioxidants, or polyphenol inhibitors in your media. Frequency subculturing also helps with the problem by avoiding the accumulation and penetration of the phenolic compounds in the media and explants. "
" Browning is one of the major issues in plant tissue culture processes. Browning of tissue culture media is caused by phenolic compounds released from plant tissues. Enzymatic browning, facilitated by polyphenol oxidases (PPOs), further contributes to discoloration. The situation hinders the preservation and propagation of our valuable plant genetic resources.
This can be controlled by either using charcoal, antioxidants, or polyphenol inhibitors in your media. Frequency subculturing also helps with the problem by avoiding the accumulation and penetration of the phenolic compounds in the media and explants. "
nxsov180db
Well-Known Member
How small of a piece of meristem are we talking? And do you have to use a special tool to cut it so small?
Turpman
Well-Known Member
It’s real small I think like 0.5mm. I use a dissecting microscope. Picked up a set of 3 tweezers that are very small at the end and a scalpel. You can barely see it when your done and I Probly haven’t got small enough to be an actual merristem. I need a few more scalpels. I use a hot bead sterilizer. So the need to cool. You pretty much have to switch out every cut, when your getting close. As every cut you do contaminates both the tweezers and the scalpel with whatever you are trying to get rid of.
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Turpman
Well-Known Member
Ya I’ve seen the synthetic seed thing. Neet but I read they are quite delicate.I read about it too, but they would extract just the tiny little premature embryo from the pod. If I recall, besides speeding the process up, there are other benefits too, that are complete game changers to the current cannabis breeding techniques. Kinda like artificial human womb technology, we will be able to select traits and alter the genetics of the plants soon, via artificial seed pods..
I'm very interested to see how culturing the whole pod will work out, and hope it stays free of contams for you.
Did you check out how to make synthetic seeds yet? Looks like a fun process more than anything! I hope to try making some in the near future..
Pros and Cons of Synthetic Seeds - Plant Cell Technology
Synthetic seeds are man-made or artificial seeds which include encapsulated somatic embryos, shoot buds, cell aggregates, or any other meristematic tissue. Synthetic seeds have the ability to form a whole plant in either in vitro or ex vitro conditions. Introduction To Synthetic Seeds Synthetic...
I’m more interested in cleaning up old strians. Kind of give them a new life. Genetic storage, and the immature seed thing is pretty cool. You can probly knock of a couple months off if you are breeding. Phytotec has a canna media out that you can pressure cook and not have to add heat sensitive hormones with a syringe filter. Supposed to be good for multiplication and rooting. Not sure how but I’m giving it a go.
I do have streptomycin but just for testing. I’d rather not use it. Last batch I did with the new media I didn’t use it so far no contams on the merristem plates. They look nice and green. I rinsed a few in sterilized water with a bit of peroxide. The merristem went white. Dead but no contam. Haha
I’ll see if I can get a couple picks tomorrow.
I have ECSD rez cut and triangle kush merristems in media. I’m hoping it gives them some spunk if it works out. They are a bit off. Have some white strips show up if they are under fed. May be normal. But I’m suspect. They were tested twice for HLV but I don’t think they test for anything else.
Oh I brewed today with a lager yeast I plated out and stepped up from a single colony. Hope it takes off. Pics on a shaker table.
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Drop That Sound
Well-Known Member
Maybe you could use a very thin .5mm inner diameter tube or syringe like a biopsy punch tool, and poke it in to take a core meristem tissue sample in the shape of a cylinder, and then trim the ends toward the center of it. Perfect sized meristem tissue "pucks" every time with the quickness!
I'm currently working on a 600w water cooled ZVS induction sterilizer, just for all my lab tools (still collecting them up). Also going to experiment with wrapping some of my tool (scalpels, picks ,etc) handles with thin 1/16" coiled copper tube water jackets, and try liquid cooling them as well. Crazy huh.
I can instantly sterilize most any size tool with my large diameter induction coil, and hopefully they will never build up too much heat as they will have there own remote\standalone mini pump/res/radiator cooling kit constantly pulling the heat away too, but without messing with the airflow in a SAB or flow hood. Not sure why but i'm the only one that seems to be trying to engineer such devices, yet I have no real previous sterile lab experience to even know what I'm working with compared to normal, or how good I'll actually have it, lol..
I'll be sure to have dual water cooled forceps, for picking apart the plant (and fungi ) materials when TC'ing. I'll be shaving loads of time off having to wait for cool downs, or swapping extra tools, etc. Might not even have to dip the tips in media to cool them.. I'll find out soon enough.
I can't help it either, i'm dreaming of setting up a rack full of custom DIY temporary immersion reactors ( each chamber sized to fit in an instant pot for sterilization) with timers, pumps and a solenoid, and skipping having to hand shake crudely made bio couplers with RTV'd lids daily when multiplying the explants. I've seen single jar setups where they just use motorized shelves that tilt back and forth, which sloshes the media from one side of the jar to the other with aquarium gravel as a medium, which appears to work well too. Or come up with a rack that flips bio couplers every da, which could would be nice..
I have finally gotten rid of all my plants, and cleaning my grow room out. My flower room is becoming my new small indoor lab\clean room over the winter. Ahh ya
I'm currently working on a 600w water cooled ZVS induction sterilizer, just for all my lab tools (still collecting them up). Also going to experiment with wrapping some of my tool (scalpels, picks ,etc) handles with thin 1/16" coiled copper tube water jackets, and try liquid cooling them as well. Crazy huh.
I can instantly sterilize most any size tool with my large diameter induction coil, and hopefully they will never build up too much heat as they will have there own remote\standalone mini pump/res/radiator cooling kit constantly pulling the heat away too, but without messing with the airflow in a SAB or flow hood. Not sure why but i'm the only one that seems to be trying to engineer such devices, yet I have no real previous sterile lab experience to even know what I'm working with compared to normal, or how good I'll actually have it, lol..
I'll be sure to have dual water cooled forceps, for picking apart the plant (and fungi
) materials when TC'ing. I'll be shaving loads of time off having to wait for cool downs, or swapping extra tools, etc. Might not even have to dip the tips in media to cool them.. I'll find out soon enough.I can't help it either, i'm dreaming of setting up a rack full of custom DIY temporary immersion reactors ( each chamber sized to fit in an instant pot for sterilization) with timers, pumps and a solenoid, and skipping having to hand shake crudely made bio couplers with RTV'd lids daily when multiplying the explants. I've seen single jar setups where they just use motorized shelves that tilt back and forth, which sloshes the media from one side of the jar to the other with aquarium gravel as a medium, which appears to work well too. Or come up with a rack that flips bio couplers every da, which could would be nice..
I have finally gotten rid of all my plants, and cleaning my grow room out. My flower room is becoming my new small indoor lab\clean room over the winter. Ahh ya
Turpman
Well-Known Member
Maybe you could use a very thin .5mm inner diameter tube or syringe like a biopsy punch tool, and poke it in to take a core meristem tissue sample in the shape of a cylinder, and then trim the ends toward the center of it. Perfect sized meristem tissue "pucks" every time with the quickness!
I'm currently working on a 600w water cooled ZVS induction sterilizer, just for all my lab tools (still collecting them up). Also going to experiment with wrapping some of my tool (scalpels, picks ,etc) handles with thin 1/16" coiled copper tube water jackets, and try liquid cooling them as well. Crazy huh.
I can instantly sterilize most any size tool with my large diameter induction coil, and hopefully they will never build up too much heat as they will have there own remote\standalone mini pump/res/radiator cooling kit constantly pulling the heat away too, but without messing with the airflow in a SAB or flow hood. Not sure why but i'm the only one that seems to be trying to engineer such devices, yet I have no real previous sterile lab experience to even know what I'm working with compared to normal, or how good I'll actually have it, lol..
I'll be sure to have dual water cooled forceps, for picking apart the plant (and fungi
I can't help it either, i'm dreaming of setting up a rack full of custom DIY temporary immersion reactors ( each chamber sized to fit in an instant pot for sterilization) with timers, pumps and a solenoid, and skipping having to hand shake crudely made bio couplers with RTV'd lids daily when multiplying the explants. I've seen single jar setups where they just use motorized shelves that tilt back and forth, which sloshes the media from one side of the jar to the other with aquarium gravel as a medium, which appears to work well too. Or come up with a rack that flips bio couplers every da, which could would be nice..
I have finally gotten rid of all my plants, and cleaning my grow room out. My flower room is becoming my new small indoor lab\clean room over the winter. Ahh ya
If your explant was clean id say the hole punch may work. But any infection would tag along i would think. Once you have something clean it may work fine.
Induction.
Nice all of these tecs were pretty much figured out by trial and error by some crazy. Its good to experiment.
I like the idea of the induction heaters. The beads are a pain in the ass. If your tool is a little damp the moisture heads up and condences on the tool the beads stick to it. So you then to have little beads all over haha. And they are perfectly round shit will roll right off the table haha. Not sure if there are bigger beads, may help. Mine look like glass beads out of a sand blasting cabnet. They have great contact but tend to stick. Dry tool is fine.
I did orchid and flyntrap TC about 15 maybe 20years ago fuck time flys.
Fun stuff trying to sterilize seeds as fine as pepper. I came up with a tec of my own. Id dump some seed into a syringe. Stuff polly wool in the needle big end befor puting it on the syringe as a filter. Suck up sterilant shake, then rinse in sterile water. All while keeping the seed in the syringe. When done i just dripped seeds on to the plate.
Ya as far as bio reactors. Im not sure if weed likes beeing moved around a bunch. I still have the coupler going. I dont think i have the proper hormones. Or my MS is maybe too old. She is quite plale. Ill try again with this new media.
I may set up an air pump operated bio. Just with a couple vesels and tubing pump etc.
conor c
Well-Known Member
Cool so have you ever done any embryo rescue with old seeds at all?
Turpman
Well-Known Member
No I haven’t. I haven’t read anything on it. I’m not sure if there are hormones used or not.
I’m guessing the seed casing would have to be removed. Sterilized then put on medium.
I do have a protocol for seed germination. Also have a few hormones on hand.
I could give it a go with a couple seeds. I’m in Canada though.