Shaping Fire Ep. 74 - Tissue Culture and Micropropagation with guest Ryan Lee

In the days of complete cannabis prohibition, cannabis folks didn't have as much access to advanced science, to complex analytical technology, or most of the great scientists who are now feeling more comfortable to sign on to cannabis related research. Because of this, our entire scene, including heritage cultivators, newcomers to cannabis and big agriculture are all rejiggering their relationship to both cannabis and technology. Some of the science, like terpene and potency tests are clearly a win and few question their usefulness, even though we are still questioning their accuracy. The other end of the spectrum is full on genetically modified cannabis DNA, and while that is already happening in labs, the vast number of cannabis consumers are not very supportive of it. Each cannabis consumer needs to decide for themselves how much science they want in their cannabis no part of today's discussion is gmo. No part of this discussion involves genetic insertion into the cannabis genome. Today's discussion is heavy on science technique though, and much of it describes lab techniques that are new to cannabis. So even though some of the vocabulary may be new and some of the techniques may sound like science fiction, they in no way degrade the cannabis genome or the pool of available genetics. Most of the techniques actually preserve cannabis genetics for the future. Today's episode is all about tissue culture. I wanted to do this topic because I have a poor understanding of it and I have a general aversion to laboratories as well. And yet there's so much science in cannabis discussions today. And from my early research into tissue culture, I realized that most cannabis folks were using the term tissue culture really loosely, without a firm understanding of the concepts we were talking about, and including myself. The goal of today's episode is to translate tissue culture for the layperson with nothing more than a high school understanding of science. Most of the episode will focus on micro propagation, since that is mostly the sort of tissue culture we use in cannabis. But we'll round out the show in the third set by discussing all the tricks that fall under the umbrella of the general term tissue culture. So wash your hands and get ready for some time in the lab. If you want to learn about cannabis health, business and technique efficiently and with good cheer, I encourage you to subscribe to our newsletter. We'll send you new podcast episodes as they come out, delivered right to your inbox, along with commentary on a couple of the most important news items from the week, and videos too. Don't rely on social media to let you know when a new episode is published. Sign up for the updates to make sure you don't miss an episode. Also, we're giving away very cool prizes to folks who are signed up to receive the newsletter. There's nothing else you need to do to win except receive that newsletter. This month we are giving away two prize packs, each with a bag of Happy Endings Root Stimulation Tea Mix and Ocean Bounty Flowering Tea Mix from our friends at Green Bicycles. Kevin Jodry turned me onto this mix about five years ago and I've been using it in my gardens ever since. So go to shapingfire.com to sign up for the newsletter and be entered into this month's and all future newsletter prize drawings. You are listening to Shaping Fire and I am your host, Shango Loess. Today my guest is Ryan Lee. Ryan is a professional cannabis researcher and plant breeder specializing in cultivar design and the inheritance of the therapeutic compounds in cannabis. His background training is in neuroscience and studying the endocannabinoid system with a strong focus on genetics. His post grad work at the University of British Columbia was in plant breeding and biotechnology. Ryan Lee is founder of Chimaera Genetic Resource Management and the Chemovar Corporation. Chimera is essentially a seed bank and cannabis germplasm firm operating in Europe for 20 years. ChemoVAR Corporation is based in Canada and focuses on breeding specialty cannabis varieties, laboratory analytics, cultivation, consulting, cultivar selection and germplasm sourcing, licensing and importation for licensed producers. Chemovar has guided clients worldwide to navigate international regulations surrounding the movement of cannabis genetic material. Ryan recently appeared on shaping Fire, episode 64 about feminized seeds and female only breeding. Today we're going to talk about the application of tissue culture techniques in cannabis cultivation. Welcome to the show. Ryan. Welcome back brother.

Thanks so much for having me again. Shangha.

So man, let's start by clarifying what tissue culture really is, because I think most folks throw around that term pretty darn loosely. Would you give us a quick overview of like the total breadth of the term, but then hone in a bit more on what people in the cannabis industry usually intend when they use the term tissue culture.

Yeah, absolutely. I think tissue culture is really like an overarching term. It's kind of like saying cultivation. There's so many different ways that you can grow a plant and tissue culture is really the same thing. When we say tissue culture, really what we're doing is we're culturing plants or parts of plants in a glass test tube or some type of plastic or glass sterilized vessel and we call those pieces of plants explants. I guess a little bit of history might help around the turn of the 20th century, like the 1900s, scientists were starting to learn about bacteria and fungus and how they could grow these things on petri dishes. And I guess a German biologist wondered if he could do the same thing with plants or plant parts. And so he started harvesting leaves and cross sections of stems and little bits of cells. And they would take the cells and mash them all up and create something called a suspension culture. And they would see like, we can grow these very small bacteria and fungi. Can we also do the same with plant cells? And that brings up a term called totipotency, which really means the ability of any cell to regenerate a whole plant from that one cell.

Is that like stem cells?

Yeah, we have cannabis. All plants have. They're the equivalent of stem CE them. Callus, which is really stem cells, are what we call undifferentiated tissue. And usually when we make callus, it's de differentiated tissue. And so that means you might have a cell type that was like, for example. Here's a perfect example. When you make cuttings, the cells along the stem, they're at that point in time, they're stem cells. I don't mean stem cells. They're cells from the stem of a plant. And when we cut those little branches off and expose it to a high oxygen, high moisture environment, like you'd.

Puck.

Or a Rockwell cube or something, that encourages the cell to change its cell type and become a root cell. And so when we actually see roots growing out of the side of a cutting, that's de differentiation. That cell has differentiated itself from a stem type cell to a root type cell. And so we can manipulate that process, or we've learned that we can manipulate that process. And we can take very tiny pieces of plants. Like we can section out just the meristem, which is a very small structure in the tip of a plant shoot, that produces the entire plantlet. But that's where all the cell division takes place. And it's about the size of a pin. And you can cut those out and grow them on these agar mediums like we're talking about, and eventually culture that little piece into a full plant.

Is it the DNA of the plant that's driving all this? Because the instructions are there in every single cell?

Yeah, I would consider the meristem actually an organ. So it's not just the DNA, it's an organized cell type that are dividing in a specific genetic program. So it's probably a little more advanced than just the DNA. Thinking of it as just the DNA or a couple of cells. It's actually an actively dividing organ.

All right, so if we've got this big umbrella, which is tissue culture, and over the course of the show, we're going to talk about a bunch of things that are under this umbrella of tissue culture. But my, I guess my best understanding of it is that, you know, 80 or 90% of what we're doing in cannabis, as far as tissue culture goes, all falls into the subsection of micropropagation. And then the other 10 or 20% are like all this random things that we're gonna talk about mostly in the third set. But would you, you know, am I getting it right that micropropagation is really what just about everybody in cannabis is talking about when we talk about tissue off the cuff?

Yeah, that's really the focus right now. I think that serves an industry need to be able to produce large amounts of virus free, mold free, pest free plants. And we can do that using micropropagation. But there's other other types of things that we can do. And there's something called a metabolite reactor or a bioreactor. And you know, at one point in time, scientists were trying to figure out if we could get cannabis cells to produce cannabinoids without having to actually grow the plant during this crazy prohibition area that we've kind of just graduated from. But, you know, you could do something like you take some leaves off of the plant and chop them up very, very finely using a, you know, a delicate blender or some kind of sectioning device. And then you suspend those cells into a big, like a big Erlenmeyer flask, one of those big kind of triangle shaped flasks that sits on a orbital shaker or a spinning shaker, and that just kind of shakes the soup broth gently and provides lots of oxygen. And these cells just float around in the medium. And the thought was that perhaps we could get these plants to produce cannabinoids, because it's actually been, you're able to do that with other plant species. But it turned out that of course the production of cannabinoids needs the resin glands, the trichomes. So we actually can't do that. But you can use that same technique to grow embryos or produce something called artificial seeds, which are essentially little shoot tips encased in an alginate or an agar jelly. It looks like a little jelly bean. And I'll try to put some of those up on my Instagram page so people can see those while we're, we're Talking about it. Yeah. But they're just essentially little ways that we can produce different plant parts in. In these glass beakers. But like you said, the most common thing in the cannabis industry is to use those to grow plantlets, sterile plantlets, which we can either section by subculturing and multiply them into a whole army of plants.

Army of plants. I like that. You know, so many of us, we just want, you know, overgrow. Right. Army of plants. Bring it on. Yeah.

Well, I mean, the truth is, you know, these large production systems that we're seeing start to be grown all over California, Washington, they require armies of plants. I mean, you plant. You know, the grape growers are pretty lucky. They, or even hop growers are pretty lucky. You plant the crop once and then it just keeps growing year after year. Whereas cannabis, I mean, if you're. If you're planting or if you're harvesting maybe two times in a summer or three, if you're really organized, you know, you need a full complement of plants to be planted every year or every planting. So it really, the production of armies of plants will be part of this industry.

All right, so if we are talking about creating armies of plants as being like, the primary usage of tissue culture in cannabis, let's focus on this micropropagation because it's, it's, you know, it's what most everybody's talking about anyway. So this is a strategy to multiply a plant very quickly under very clean controls in a lab. And I mean, my understanding of it is essentially cloning, but with exceptionally immature plant material, like at the cellular level. Cellular level. Would you walk us through what that, you know, the science of that. Because the idea of even handling and manipulating the cells is, you know, one step removed from my understanding. So walk us through that process.

Yeah, so these, these agar containers, we'll call them, or tissue culture vessels, you really have to think of it like a plant in a pot. So just like you're making your. Transplanting any plant into a container, the. The tissue culture vessel holds. It does all the same thing. It's got a place for the plant to grow. It has the medium or the, sorry, the hormones and the food source, the nutrients that are needed by the plant. We also put some sugar in there and some other hormones that'll help the plant grow as it goes along. But it's such a perfect space and a perfect environment. You got to remember, when we put a little piece of an X plant or a little piece of a plant into that tissue culture medium, there's no roots, right. The plant has just, it's just like a little cutting. So the plant has to be able to be able to absorb everything that it needs from that, that jar. And one of the problems is that that environment is so suited to growing anything, even, even other molds and bacteria, that if the plant part is not completely sterilized before we put it in the jar, it may bring in a virus or a bacteria or a fungus with it. And that fungus or bacteria will then be able to propagate itself much, much more quickly than the, the plant part that we've put in there. And so that's what we call contamination of the vessel. So we have to autoclave these jars at very high temperature and pressure before we put the plant in there. It's all sealed up. You make your medium, you add your hormones and your food and your sugar, and that all goes in the jar. And then we put it in a pressure cooker or an autoclave. And that sterilizes the plant medium. And so the next step is cooling that material down and letting the agar harden. And then we use something called a laminar flow hood, which is essentially a very high quality HEPA filter that creates an airflow that passes through this filter process. So any air that comes in contact with the plant has actually had all the fungus and viruses and bacteria been removed from the plant.

Is that the same kind of hood that I'm talking about with people who are growing psilocybin mushrooms?

Very similar, yeah, very similar. Same kind of process. Again, the same with. The idea is that you want to maintain sterility. You really want. The only organism that you're putting into.

The pot is your target.

Yeah, your target. So either cannabis or like you said, mushrooms.

Before we go on, let's break down a little bit about this, this petri dish. So first of all, what's agar?

Agar is like, it's an extract from seaweed. If you're walking by the seaside, you can sometimes you pick up seaside and they've got these little kind of jelly balls on them, them or whatever, it's that kind of thing. So they take it and they, you know, they'll, they'll extract it from these, these plants, boil it all down and purify it. And really all of it, all that it does is it gives a matrix, it adds some solidify, it's a solidifying agent for your liquid. Like we were talking about the bioreactors before or the embryo culture reactors. If you have these little plant parts in Sugar water, with all the right nutrients and hormones and the, the, your solution is actively moving around so that it's being oxygenated. You can actually grow these plant parts just in the liquid. But the agar adds like a solid aspect to that. It just solidifies the medium and it kind of makes it more stable. You know, we can, the plant can remain upright in that agar.

So is it kind of like a neutral and inert thing?

Absolutely.

Okay, I get it. So it's just, they're kind of, kind of like to hold shit there.

Yep, it's just, it's just a medium.

Alright. So then you also said that in this agar, you're putting these, you know, tiny plant cuttings. Well, like we've, we're already talking about at a cellular level. Right. And I, you know, when I make a clone, it's a pretty big cutting. So how small of the cuttings are placed in these petri dishes? Like, are we talking about like, you know, an inch that we've cut with a scalpel or are we talking about like a sliver? Like what, how small is small?

Yeah, well, they, ideally you want them to be small because the bigger, the bigger the plant, the more opportunity that we're going to have missed some of the bacteria or the viruses or the molds in our sterilization process. So we want to get down to pretty small. Just by getting down to a pretty.

Small part, like a centimeter.

You can go that small. You can. It depends. Like again, we're talking, you know, we can do all these different types of medium. You can actually use pieces of leaf, you can use cross sections of stems, you can try to do roots, or you can grow the meristem like I was talking about, or, or even the auxiliary bud. Like, you know, if you imagine a plant that's growing, we've got the shoots coming off the top. And then you might have say, three leaves coming off, you know, two on one side of the stem and one on the other side. And everywhere that a leaf stem or the petiole meets the main stem junction, there's a space where a new branch will grow. And any grower knows this if you watch plants that the new shoots always come out between a leaf and the stem. And so that little spot on the, on the crotch of the, you can call it the crotch of the plant in between the stem and the shoot that actually has what it was called an auxiliary bud. And that is just the place on the plant where that shoot will grow. And so we can actually, we can Actually cut all the leaves off and just plant the node where that piece of the shoot will grow. And so there's no. You got to consider there's no leaves, right? We've cut them all off. And so because there's no leaves, that plant can't harvest CO2 from the air. Right? And so we've put sugar into the medium directly, because sugars are the byproduct of photosynthesis. So by putting sugar in the medium, we kind of bypass that photosynthesis step and allow the plant to have access to these. These molecules so they can just incorporate them rather than having to synthesize them themselves.

So is that little growth that occurs in the crotch, is that the meristem?

There is a meristem in the auxiliary bud. Yeah.

So define that, would you? Meristem.

So the meristem is. It's kind of like the center of generation for all the plants. Like, if you look at the top of a growing plant where the leaves are growing, and you can, you know, you can get in there and fold the leaves down and try to get to that most central point, that most central point where everything is growing out of is the meristem. So, you know, think of it like a worm. Like, I don't know if people have ever heard of this experiment where you can take a worm and cut it in half and both halves will live and they can regenerate their own parts. It's kind of the same with cannabis. This plant, anywhere that there's an auxiliary or an apical meristem, and the apical meristem is just the one on the very top of the plant. That thing can generate an entire new plant from it. So kind of think of it like the. The source code. You know what I mean? That's like the.

Is. Is the central meristem ness of the cannas plant only present in certain parts of the plant, or is it everywhere? Because, you know, like. Like, you know, I'm not super good at making clones, right? And, like, you know, I have put a lot of effort into making my cone or my clone cuts, you know, right there at the point where the, you know, the new growth of the leaves are on the side. So I'm like, oh, this is supposed to be where all the new horizontal hormones are. And I'm like, really careful. But are you telling me that, like, most of the plant can be used as a clone in kind of like a random way, or are these meristems only a few places on the plant?

No, anywhere that the stem meets a leaf Junction, there's going to be a meristem.

Oh, I see.

Okay. So. But using tissue culture, we can actually also get the plant to produce new meristems from other parts of tissues where they don't exist. So, you know. Yeah, it's kind of a weird thing, but we can take a leaf and grow what we call. People might have noticed when you. When you're rooting cuttings, for example, they go. The stem goes through a stage before the roots start growing out where they kind of swell a little bit and they become like a little bit of a rough, bumpy texture that's called callus. And callus is kind of the intermediate or what we think of as the stem cell. It's de. Differentiated tissue. It's tissue that's kind of. It hasn't been genetically destined to do anything. So it can kind of become anything at that point in time. Callus can become a root. It can be encouraged to become a shoot. But you really have to encourage the plant using hormones to do, you know, to guide it down the path that you want it to go through.

All right. All right, cool. Well, thank you for going down that with me. There's one more ingredient in those petri dishes I want to talk about. You said that we're going to add a little sugar. So I'm curious to know what kind of sugar and why sugar. I mean, we use it in compost tea, and we talk about that all the time on the show. But in this particular application, why are we using it?

So like I said, sugar. Sugar is the end result of photosynthesis. Light comes in, it hits the cells in the leaf, and there's a whole reaction that happens. But what essentially comes out the other end is sugar or is a form of sugar. And so we actually use sucrose. Just go to the grocery store and buy a couple of pounds of sugar. And that gets, you know, it gets mixed in at like. I can't even remember. It's like a few grams per liter. And so essentially, we bypass the photosynthesis and we just put the sugar directly into the food source. And that way the plant has a carbon source. Because, like I said, if you cut off all the leaves and we're just trying to grow a little auxiliary bud from a small piece of stem, and there's no leaf. There's no way for the plant to harvest CO2 from the air or the X plant to harvest CO2 from the air.

That sense makes. Makes sense.

And so we kind of bypass that process and put the sugar right into the, into the medium so that the plant can actually suck that up through, you know, the, the piece of the plant that's in contact with the medium.

So you were talking earlier about how like what an opportunity this environment is, right? You're like, okay, we're gonna, we're gonna, we're gonna try to grow cannabis, you know, plantlets in this thing. But, but it would love to grow fungus and, you know, probably bacteria and anything else that happens to be in the room. What are the, you know, what are the sterile conditions that have to be set and generally how are those made in this tissue culture environment? I'm assuming this is, you know, a tissue culture lab.

Yeah, it's all done in a lab, but I think I like to use the cut finger analogy. So I mean, if you're out working away in the garden or in your shop or whatever and you cut your finger, you get a big old deep gash on your finger. The first thing you do is you run it underwater and take the blood away. But you want to sterilize it, right? You want to clean it up so that there's no bacteria or things that are going to start growing in the cut. Because our bodies, the inside of your body, we've got all these nutrients. It's like a perfect place for a bacteria to come and set up home. And so too is the tissue culture vessel. It's kind of the same thing. So we want it to be completely sterile. And sterile just means that we've killed 100% of the bacteria, the fungal spores, and any viruses or any other organisms that might be living in the medium. And we do that by, we make a big batch of this, of our agar, and then we pour say 25ml into a small baby food sized jar. And then those get special lids on them. And then we can stack a whole bunch of those in an autoclave or a pressure cooker. For anybody that's ever done canning canned fruits or jams or even different vegetables or whatever that you can can meats, we seal up the lids in these jars and we put them in a large container like a pressure cooker. That's essentially like a big, it's more or less a bomb. It's like a big metal container that we heat from the bottom and we put water in the container. Obviously we seal it all up and then we heat it from the bottom and the heating it from the bottom creates a steam inside this big vessel. And so you have all these big really locks and tighteners and fasteners that hold the lid down. I mean, it's a slight, serious piece of equipment. And we actually, by letting the steam build up, it gets to a really high temperature, but the pressure also increases immensely inside this vessel. And that will actually sterilize anything that's in the jar. It kills all the bacteria and it kills all the funguses, fungal spores, or anything that are in the medium. So that when we take it off the stove and we let it cool back down and releasing the steam from the container, those jars can then be taken over to the laminar flow hood. You actually take the whole autoclave, or the whole. Not the whole autoclave, but the whole container over to your laminar flow hood, and we take the jars out and let them cool in the laminar flow. So once they've come out of this pressurization process, the sterilization process, they only exist in an environment where there's no bacteria.

So, like, I don't have a lot of lab experience, and this question very well may illustrate that, but it kind of sounds to me like this pressure cooker that you're talking about is the exact opposite of using a vac oven that we use in a lab as well. Like where the vac oven is, you know, trying to create different atmosphere by creating a vacuum, Whereas this is doing the opposite. It's trying to open, ultra pressurize it, also to create an unlivable environment. Am I anywhere accurate on that?

That's exactly what it does. It creates an unlivable environment. Right on. And so that anything that's gone through the process dies. And then from that point on, it's what we call sterile. And so think of, you know, when we're talking about the laboratory or the laminar flow hood, this sterile air environment, think of that literally like a surgical suite or a surgical room. You know, when the doctors and the nurses go in to do surgery, they're all gowned up, they've got special gloves on and masks on that are stopping them from breathing. You know, just like everybody knows about masks during COVID Right. I mean, it's the same process. You want your doctors to have on a mask so that they're not blowing out bacteria into the open patient that's lying in front of them. Right. And that's kind of like this. Think of. You can kind of think of the sterile jar as like, the patient. Right. Anytime you open it, there's a risk that we're going to introduce again, either a virus, a bacteria, a pest, or some fungus into it. And we really, this whole process, we want to do in the absence of all those organisms.

All right, now that we've established really well, really, really well, what are in these petri dishes, let's kind of reorient ourselves. So explain how the cannabis industry right now is using this for cultivation. Is one of these petri dishes going to make a gazillion plantlets and all these like, like little tiny ass clones, or is it. Is it going to be one petri dish per clone?

No, no, it really won't be one petri dish per clone. At the end of the stage, it gets to be that. But so when we think about it, there's really a few parts to tissue culture. The first part is the sterilization. And then what we go into something called initiation, where we actually put the sterile piece of plant material into the jar. If you put that plant material into the jar and put the lid on, take it to your sterile culture room, that plant will actually grow inside the jar for, say, two weeks. Okay. And then after two weeks, you come back along, you can take that plant part or that grown plant part out of the jar under laminar flow again in this sterile environment, and you can cut it up into probably two or three or maybe five new shoots and plant each of them again in their own jar. And that's called subculturing, where we actually take one culture vessel, we take it out, divide it into multiple plant parts, and then create five new vessels. Okay. In doing that, we've skipped the step of having to sterilize five times those individual plants that are going in. So the beauty of subculturing is that you can. You can essentially multiply your plant material without having to go through the initiation process again. So if you think of doing that, one plant goes into the jar, two weeks of vegetative growth, we take it out. You've now got a plant that we can cut out, five plant parts. Each of those goes into their own individual jar. They can go back into the culture room for another two weeks, and then what comes out is 25 plants. Right. So over.

It reminds me of mothers. Right? Just like regular old mothers.

Yeah, it is. It's very. In the same way, it is very much like the mother process, but the, the mothering process is all done in a greenhouse environment or a grow room environment, and you're subject to bacteria and molds and fungus and anything else is floating around in the air or what's brought in with, you know, the cultivators as they come in and out, maybe they're bringing bugs in on their clothes. So it's, it's just a different way of doing the same thing in a sterile environment. And you can imagine the logarithmic curve that you get, you know, after, like I said, if you go 1 to 5, 5 to 25, the next multiplication is 125. The next multiplication is, you know, we're well over 600. So it really starts to grow exponentially. And so that's, that's, it takes a period of time. I mean, you're not going to, you know, if you want to plant a million cuttings in a year, you have to start today, right? But it does allow you to hit this critical mass where the, like I said, the multiplication becomes exponential.

So. All right, so if we've got this one original plant, for lack of vocabulary, I'm going to call this the mother, right? This is the plant that we want to make a bunch of because we want to micro propagate it, because we're going to fill our field, our cultivation facility with it or whatever. You know, I would think that there are some attributes we would want this mother to have. I mean, like, sure, sure, we want it to have a good terpene profile and good potency and all the regular stuff we're used to. Right. But because we are doing it in the lab and we are doing this sorcery with it, I would think that there are other attributes that we would be looking for in our plant that makes it appropriate for micro propagation. So what do we look for in a plant that makes it especially a good candidate for micro propagation?

That's actually a really good question. And the answer is that not nobody is currently doing that.

Wait, like we don't even know, we don't even know what plants make good micropropagation plants?

No, I think so. So cannabis is a really interesting species in many, many ways. But in one of the ways it's really interesting is that like every single trait that we can consider in the plant, there's usually, there's usually almost always a great amount of variation. Like, for example, talk about height. You can have tall plants, medium plants, short plants, right? Yielding plants, low yielding, medium yielding, high yielding terpene plants, you can have low levels of terpenes, high levels of terpenes, cannabinoids, same thing. So the point that I'm trying to make is that any trait that we can look at in the plant, there's a continuum of performance for that trait. And currently, the way that we're doing Tissue culture is people are growing cannabis the way they normally do. They're selecting the plants that they want to be ideal for their production environment. And then once we've selected the plant, we get our blue dream or our cookies or OG comes out. @ the end of whatever process we're doing to select plants, we then take those plants back into the tissue culture system and see how well they perform in tissue culture. We've come to learn over the past 15 years or 20 years that there is actually different plants perform either well or poorly in micro propagation. Right. Some plants just don't like the process. They want more vitamin B or they want a specific hormone. They just don't grow quickly when you put them in the tissue culture. So it might be in the future that the question of does this plant propagate well under tissue culture is actually a criteria that we use for selecting individual plants. But currently we're not doing that.

Man, I can't think that we're too far off from that. You know, with the, with the increased interest in corporate ag and everything and these like crazy huge fields they've got in California now, I've got to think that's the future as much as it may not mean my flavor.

Yeah, well, the more, listen, the more we move towards scientists being involved in the selection of plants, these, these added characteristics are going to start being looked at before. The problem with handing it all over to sciences, they just don't get the culture right. I mean you have to, it's kind of like, you know, if you're making wine, the people that make the best wine in the world, like they consume wine, right? Because they understand the things, they understand the processes that go into production that yield quality at the end. And just the scientists aren't at that point with cannabis where they really, the vast majority of the pure scientists that are being brought in to cannabis, I mean the most successful companies that I see operating in cannabis space, they have a science team or a group of people that are bringing science to the venture. But there are also people from the cannabis community that understand all the important things that go along with, you know, cannabis production. And it's really the marriage of those two things that that makes the win.

Right? Yeah, I don't, I don't really want a scientist who has been over in ornamentals, you know, making plants for somebody's frickin landscaping to be making these choices. I want mean Gene to be making those choices, you know, somebody who I respect because they have done the propagation and the sifting in the old ways. Right. I want somebody like that but with this ridiculous science education to be making those choices. And you know, I don't know, maybe there's a handful of those people nowadays, but I would guess that those are probably the, these freaking lucky kids who are in their early 20s now who are coming up in this post prohibition environment who are going to learn that stuff and they're going to come up and, and they're, you know, they're pot smokers who also are getting science degrees.

Yeah, well, you know, I, I see plant breeding or plant improvements as really, it's not a one man sport, it's a team sport sport. Right. And it's kind of one of those situations where it's like it takes teamwork to make the dream work.

Right.

Like, you know, it's a cheesy thing, but it's like if you could pair, you know, Mean Gene with a really high advanced plant scientist that understands things about plants that maybe Mean Gene doesn't understand or someone like myself doesn't understand that, you can actually, you know, get a lot farther of the synergy between the experience, the two, those two different worlds of experience meeting together or being married together.

That's great. I can already start to imagine the teams that I want to put together now. All right, so we should probably take our first commercial break. Thank you very much, Ryan. You are listening to Shaping Fire. And my guest today is cannabis geneticist Ryan Lee with the National Hemp program influx. Due to stringent THC testing requirements, brothers Seth and Eric Crawford continue to release seeds to hemp farmers that will be legal no matter how you grow them or when you test them. These new varieties from Oregon CBD seeds have substantial amounts of CBDV, CBGV, CBCV and THCV while always staying below the 0.3% THC limit and guaranteeing compliant crops for farmers every time. Also, these new varieties cannot be pollinated by your neighbor's uncontrolled pollen or a rogue male in your own crop either. Oregon CBD seeds are non GMO certified too. Oregon CBD seeds was founded and funded in 2015 by Seth and Eric. Maxing out their personal credit cards without outside investment. They continue to refuse outside investment that would change their company culture. Oregon CBD grows tons of fresh food on their research farms for local food banks. Literally tons of food. They also give away tens of thousands of pounds of R and D flour to patients. As their company began to succeed, Seth and Eric started donating money to the cannabis medicine and hem fiber cause two by giving millions of dollars to Oregon State University in order to establish the world's leading cannabis genomics research program, and they treat their employees right. Oregon CBD pays for full health and dental coverage for their employees, a 401k program and their minimum starting wage is 20 bucks an hour. Plus everyone shares food from the farms. Seth has been on Shaping Fire a few times to talk about novel cannabinoids. You can check out episodes 25 and 37 on CBD Cultivars in the Hemp Market, episode 66 on Triploid Cannabis Genetics, and the very first Shaping fire Live episode 47 with Seth and soil expert Jeff Lowenfels talking about autoflowers. If you are a hemp farmer and you want to grow reliable seeds that are sure to thrive in pasture testing, check out oregoncbdseeds.com to learn more about buying seeds for the 2021 season. That's oregoncbd seeds.com sufficient dissolved oxygen in the root zone is a challenge Gaia's brand of ultrafine nanobubble systems will help your garden thrive in ways you may not have considered. No matter if you grow in soil hydroponics or aquaponics, you Gaia's ultra fine nanobubble systems will increase your dissolved oxygen and increase your yield. Often the first sign of inadequate oxygen supply to the roots is wilting of the plant. 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Go to Gaia's website@h2o5.com to learn more about using dissolved oxygen and how to purchase this simple yet amazing technology. That's h2o5.com as cannabis regulations become more demanding and consumers become more educated, it is increasingly important to avoid the use of chemical pesticides when cultivating cannabis beneficial Insects have been used for decades by the greenhouse, vegetable and ornamental plant industry and today many cannabis cultivators are moving from spring sprays and chemicals to beneficial insects. Kopert Biological Systems has the beneficial insects, mites and nematodes, microbials, sticky cards and air distribution units. You need to partner with nature to defend your garden. Whether you manage acres of canopy or have a simple grow tent in your home, Kopert is ready to help answer your questions and help you transition away from chemical sprays towards clean and natural solutions. Since 1967, Copert has assisted growers in identifying pests and devising reliable solutions while providing healthy insects and mites that will protect your Yield. Since the 1990s, Copert has been a leader in cannabis pest and disease control worldwide and have highly trained consultants to assist you in Canada and the US from coast to coast. No matter where you live. Copert Biological Systems can help. Visit kopert.com, choose your country and get detailed information. That's Kopert K O P p e r t.com for the most up to date cannabis related biological control information. You can also check out their Instagram pertcanada. You know, getting away from pesticides is good for health and good for business and Copert is ready to help. Visit kopert.com today. Welcome back. You are listening to Shaping Fire. I am your host Shango Los, and our guest this week is cannabis geneticist Ryan Lee. So now we have selected our mother plant, we have harvested plantlets, we have incubated and multiplied them in the agar growing medium. Let's talk about transitioning them to the garden. So how do we move the plants from the growing solution in these petri dishes to rooting in the soil and becoming plants?

Okay, it's a, it's a really good question. I think we should probably walk through the whole process. We've already started the process, but there's different phases in tissue culture. And so we start with our explant, which is the piece of the plant that we've cut off, which is then sterilized and initiated into the tissue process or into the jar that plant then grows. We talked about already the process of multiplying it and subculturing those plant parts again into their own individual jars. And that process kind of goes on over and over and over again until like I said, we have our army. But at that point in time, we still have a tiny little plant shoot in an agar medium in the jar with no roots. If we were to take that plant outside and either try to clone it or try to try to stick it into soil, it would just die because again, it needs to have that set of roots on it. And so depending on the hormone mixture that we're using in the agar, we might have to create a new specific mixture or a new specific rooting medium which we plug those just like we do with clones. We plug those little pieces of those little plant shoots into the rooting medium. And over the process of say seven to 14 days, just like a clone, these things will grow roots. But again, that little plant part is used to being in its like happy little jar sterile greenhouse. It hasn't built up the type of protections that it would need to just be taken and put it under like a thousand watt light or in a greenhouse or something. The plant would just wilt and die. And so we have this process where we take the rooted plants out, we wash away the agar medium and we put them into soil and they kind of go through this real babying phase which we call hardening off. You might think of it like the nursery in the hospital where the babies go right when they're born. It's just like a happy warm life environment with just the right amount.

That's a funny idea that the nursery in the hospital is hardening off the humans.

One of the things that happens when we grow these plants in tissue culture, they grow very small and they're just because they don't need a lot of inputs, they're not really harvest, they're pulling sugar from the agar. They're not harvesting it through sunlight and they're not exposed to wind and all these other things. Things, right. And so we have plants have, when you grow plants in a greenhouse or in a grow room environment, you can actually look tell on the plant. They've got something called the waxy cuticle. And that waxy cuticle is kind of like a, like think of it like an anti transpiration barrier. It stops the plant from just losing all of its water. And so we have to baby these plants in a way and kind of encourage them to get to toughen up enough to the point where we can actually take them and put in them into a tissue culture or sorry into a standard production environment. And so that it might be like a warm environment with lots of humidity. Right. It's just they don't go. I think the important thing to realize is that they don't go from the jar to the feed field. Right. You have to have this intermediate step.

Where this intermediate step Is this still happening in the lab?

It's kind of, it's like, it's kind of in between the lab and the field. Right. So it's less.

So it's not, it's not sterile, but it's also not exposed to the elements.

Exactly. All right. Yeah, yeah. It's kind of like a really. It's like I said, it's like a nursery. It's like. It is. It's exactly what it is, a nursery.

Right.

It's like we want to take these little babies and encourage them to grow up just enough so that when we then take those plants and move them into the production environment, either in a grow room or in a greenhouse, that they can just take off.

How big are they physically at this point?

It's not really to do with size. I mean, you can do it on very small plants, but you kind of want to do it on what we, you know, staged like three and a half, four plants. They're like, you know, maybe three, four inches tall.

So in that petri dish, a plant has grown that's about three or four inches long, and that is the size of the plant you're going to take out of the petri dish and put into your soil in this nursery.

Yeah. In the later phases of the tissue culture process, I mean, we might start with baby jars or actually even test tubes, but you can actually upgrade scale the size of the container. And so they use like those, I don't know, they're like, you know, 500 gram yogurt containers. You've probably seen them at like fast or not fast food takeouts, but like a, maybe like a slightly higher end, you know, vegetarian takeout. It's like a clear, clear polypropylene.

So, so they're actually like, like transplanting. They're potting them up even in micro propagation in the lab.

Yeah. As they get too small, you know, you're growing these little trees. It literally looks like a two foot tall bush, but it all happens in the size of a baby jar. A baby food jar. Right. And so if you want to get, if you want the plant to grow past that size, you make a bigger jar. Right. So that's why we call, that's why these tissue culture vessels come in various sizes.

All right, so. So let's talk one more moment about this waxy cuticle. So the waxy cuticle, if I understand it correctly, it is preserving like the moisture in the plant and is kind of just kind of protecting it during the hardening off process. Does the waxy Cuticle go away during the hardening off process. And is it like reabsorbed into the plant or does it like fall off and is in the bottom of the soil?

Sorry, I maybe wasn't clear when I said that the waxy cuticle is what develops in this hardening off process. So it's actually growing more thick during the. Or thicker during the hardening off.

Oh, I see. So. So it's like the outer layer. It's the defense. It's the, the outside skin. It's. The skin is toughening up during the hardening off process.

Exactly. And there's, there's actually a product that you used to be able to buy that people would take. I think it was called no Wilt, but it was a, it was a product that you could actually put on cuttings and it was kind of like a waxy spray that you'd spray on the plant and it, it kind of seals the plant up and it stops. That's one of the things when we, when we take a. Cutting off of a branch, cutting off of a plant, you've cut off the way that the plant is going to get access to water. Right. And so that's why we use a dome on the clone trays. Right. Is maintains that humidity. If we were to take the dome off and put even a fan blowing lightly across clones that were freshly planted, they'd all wilt. Because what you're doing is you're encouraging them to transpire all that water out of the plant and they can't reabsorb enough water to compensate for what they're losing. So they wilt. Right. And the idea is that we kind of create this little mini greenhouse with these domes and that prevents them, it holds the water in, it prevents them from transpiring.

So the waxy cuticle that's going to grow on the outside. This actually reminds me a lot of wax grafting tape that, you know, after you make a graft, you wrap it with this waxy tape that is semi permeable. So it closes off off the cutting spot to disease and a bunch of stuff. It kind of reminds me of the same thing how wax is generally protective.

Yeah, absolutely. And we know that these waxes are all on the plant. I mean, I don't know if you've ever seen these big CO2 extractors in the process, but they actually pull out a large amount of wax in a CO2, which is usually why they do that winterization step. Right. In the extraction process where they take the cannabis oil and they Put it in a big vat of, or a big container of ethanol, and then they drop the temperature really low, like well below freezing. And what happens is all the waxes in the extract, they precipitate out of the solution and you can actually filter the waxes away.

So those waxes, though, are coming from the trichomes and not the plant material, right?

Probably both.

Okay. Yeah. All right. So it's, it's, you know, it's. It's common in the literature to read that micropropagation produces plants that can be more robust than their traditionally cloned counterparts. Has this been your experience?

I think probably what we're seeing. I don't know if they would be any more robust than a healthy plant. I think that probably what we're seeing is that some of the plants that are coming out of tissue culture, compared to the plants that they came from, they might be a little more vigorous. But it's probably because the plants that we're comparing them to have had more. You know, they've. We're talking about like dotted plants. You've heard of these dotted plants, Right. They've got a viral or bacterial load. Like the plant has essentially become infected over years. You gotta, again, remember, cannabis plants are annuals. So their natural life cycle is to grow and live for a year, and then they set seed and the whole life cycle starts again, which is kind of a way for the plant to shed any bacteria that's growing, that's growing in the plant.

Right. But if you're. If you're growing a 19, 1996 Jack Harrer plant, chances are it's picked up some garbage along the way.

Yeah. And that's actually one of the things that we're seeing in tissue culture is that, you know, there's. We've seen some. Some groups identify, like, bacteria that are actually living within the cells of the plant or in between the cells of the plant. And, and so part of one of the things that you can do in tissue culture is play around and try to actually find a way to kill those bacteria and viruses in the plant.

So instead of it being, oh, tissue culture, plants are by definition, you know, better and more vigorous. It's really like. Yeah, it's really about the fact that the preparation that goes into creating a tissue culture. Mother. You're to going cleaning up the bacteria and the viruses, so you're just making a better plant. It's not necessarily that plants that are grown in petri dishes are somehow more vigorous.

Yeah, I suspect that's what it Is, again, I can't say that with 100% conclusivity. I think the truth is also is that while, yeah, some of these companies are starting to do tissue culture in cannabis, I would say that we're probably three to five years away, or maybe 10 years away from having a completely bulletproof system that is producing perfect plants every time.

Well, in the category of bulletproof, you know, one of the things that people who want to complain about tissue culture will. They'll point out that some cultivars do not grow true to type after they're grown out from tissue culture. Is this a failed extension expression common in cannabis, like the plant, or is this more or less common in other species? And, like, what might cause this?

Yeah, so that's something called somaclonal variation. Somaclonal variation just means the variation that we see due to possible genetic changes in the plant through the tissue culture process. And we actually know that some of the hormones that are used in the tissue culture process actually do increase the rate of genetic mutation in these plants. So we might be seeing genetic changes. It could also be that we're seeing epigenetic changes. Epigenetics is essentially, it's a way that genes are regulated by the environment. And so a plant or an organism might go through some environmental insult that changes the way that the DNA that their genes are expressed.

Is this like nature versus nurture from high school biology?

Yeah, a little bit. It has to do with that for sure.

So let's hit on that a little bit more then, because I find this interesting. So. So did you just say that, like, you know, I mean, I know people use the term epigenetics a lot, and, you know, one of my favorite breeders, modern epigenetics, is making cannabis seeds. But, you know, the word epigenetics I'm not, like, super intimate with. Are you suggesting that environmental stresses can change the DNA?

They change the way the DNA is expressed. And so yet, like, there's something called DNA methylation. And so there's these little tags that can be put on a DNA which say, transcribe this gene or don't. So the DNA itself doesn't really change, but it's what's being altered is the regulation of those genes. And so epigenetics literally means above genetics, or standing above upon genetics is really what epigenetics means. And so this is a type of regulation that occurs on the DNA without changing the DNA.

Interesting. So it's kind of like, all right, the DNA says do this, but then there's like, then there's like an epigenetic post IT note next to it that says yeah, do that but like don't, don't do that a lot or yeah, do that but do that a whole bunch.

Yeah. I mean without getting into a whole show on epigenetics, I think that that's pretty like, I think the post IT note is actually like a really good way to think about it. You've got your little piece of machinery that comes along and it reads the DNA and it encounters the post it note. And that post it note is a little instruction that says, hey, read this DNA a little bit differently.

And so the message that's on the post it notes, would that be considered somaclonal variation?

No, Somaclonal variation is like the change in the observable phenotype is how I would describe it.

So the somaclonal variation is the variation from the nurture, not the nature.

Exactly.

Alright, I got that.

It's the off type. It's like I said, it's the way we put known genetics in and 2% of those plants that come back out look different. Those are somaclonal variants.

Yeah, like, like, you know, getting, getting. If I, if I get genetics up here in the Pacific Northwest that grow totally kick ass in Humboldt and I bring it here and people are like, oh, that's not that plant. You can tell by the photograph. And I'm like, yeah, well you know, I got it from, from Jodhry and it's definitely from Humboldt. But our weather here sucks. And so I'm experiencing somoclonal variation.

Well that's, yeah, that's really. Environmental differences.

All right, I went a little too far.

Yeah, it's really the variation that we see coming out of the tissue culture process and I wouldn't try to extrapolate it past that, but we do know, for example, there's one chemical that was really used a lot in cannabis tissue culture called Thi, diazeron or tdz. And TDZ is known to cause genetic mutations or at least to increase the frequency of genetic mutations. And so by using some of these hormones in the process. Yeah, we can lead to changes in the plant. There can be changes in the process. And that's really part of what we want to do is monitor these, these types so that we can make sure that as we're cloning, you know, you don't want to take a variant or an off type and then use that to start your whole new subculturing process. Because everything that every Plant that comes from that cell line is going to be changed.

Right.

And the idea is that we want to be have them being true to type, AKA blue dream or whatever the variety is. Right.

You want, you want to use the plant that is true. The truest expression of the genetics that you want.

Exactly.

Right on. So this would be a great place for me to ask you about genetic drift because, oh my God, all the crazy stuff that is said about genetic drift and people pointing fingers and like, you know, all the bro science, God help us, you know, people argue a lot about whether or not genetic drift is even a thing. So generally people are, when they talk about genetic drift, they're like, okay, you had a mother some time ago, let's say, for the sake of this conversation, let's say that it was five years ago, there was this great plant, and then you made a clone off that mother and you grew that up and then you made a clone off that plant, and then you made a clone off that plant. So now maybe we're like seven or eight or nine clips, clones off of the original plant that was grown from seed. And what people normally tell me is like, oh, there's genetic drift because the cutting that you are growing now, nine cuttings later is so far from the mother. And the clone that you have now is the same age as that mother was way back nine cuttings ago. And so the genetics have drifted from the original expression of the seed. Is that a thing? And if so, what are the mechanics there?

Yeah, those are. Again, so those are what I would call off types. Genetic drift is actually something else. Genetic drift really means the change in the first frequency of the genes in a population from one generation to the next. And those typically the frequencies of genes. If your population is large enough, your breeding population is large enough, the frequency of those genes don't really change in the population unless we're exerting some type of selection pressure, like, you know, either through artificial selection by humans or pests or molds, or if you use a small breeding population, those are the things that can really cause a change in the genetic frequency. But that's probably different than what we're talking about. It's just the community has started using the wrong term. That term genetic drift already has a specific meaning. And I think that what the community is really talking about is more results of somaclonal variation or some type of epigenetic regulation that's either happening on the plant or the change in phenotype is being induced by something like the plant has Acquired a virus, right?

So it's not really age related, it's not time related. It's because over time, your cuttings of cuttings of cuttings are actually picking up, you know, diseases and viruses and stuff and getting weaker.

I think that that's more likely. We have shown through the sequencing that has been done in the last 10 years that clones, what we call clones, I don't know if you remember the Filos galaxy, but they used to have these clone groups. And that's really what we're talking about, is if two people, if you got one cutting from Jodri and somebody else got the same cutting from him 10 years ago and took it to the east coast as those two plants are, are now, they're divergent branches on an evolutionary tree, right? And so they actually may go through little bits of DNA mutation. You know, that's how Pinot Gris and Pinot Blanc came about. Right. Like those grape varieties actually started as Pinot Noir. And then, you know, somebody in a, in a vineyard in France 300 years after they started growing Pinot Noir found one group of branch or one group of grapes that was like slightly gray colored. And it was a, it was a white grape, not a red grape anymore. And that was just because that one branch produced a mutation. And the mutation was in the, in the gene that was responsible for the color of the pigment in the grape. Right. And so when you actually take that, if you cut that branch off and root it genetically, except for that one mutation, it's the same plant as the Pinot Noir. Right. But it has just acquired a mutation that allows it or that has caused it to have a slightly different phenotypic expression.

All right, so before we go to the commercial break, let's talk about one of the risks that people talk about with this entire process. Because, you know, you take your original mother plant, shall we say, and she is the best expression of the genetics that you want to micro propagate to make your cannabis clone army. And so then you go through the process of tissue culture to micro propagate it. And now you've got, you've got, you know, a gazillion of these plants and you're going to put 5,000 of them out in a field or, you know, in a series of warehouses or whatever. You know, one of the things that I like about growing outdoors from seed is that all of my plants are slightly different. So if I get pest, if I get crappy weather, if I get a fungus, if I get like whatever, my threat is not all of my plants are going to be equally susceptible to it. So, like, okay, I might lose some of my plants, but I'm not going to lose all of my plants because they're like slightly different people. You know, when you do this micro propagation, though, since you're targeting on this very specific expression of the genetics, you know, these 5,000 plantlets that become plants are all going to be exactly the same plant. And you know, I agree with a lot of folks that like, you're like, okay, well, that might give you a whole bunch of plants that will produce flowers that will look appropriate in the same bag because they look the same, but it also makes them susceptible as hell to all of these pests. And, you know, I think that that would decrease the, the defensive posture of all these plants, making your plant, your, your field or warehouse more at risk to everything. How realistic is that concern and how do we weigh that?

Well, that's. You're talking about monoculture. And this is, this is always the risk of monoculture. Right. When you grow one genotype in an environment, the possibility always exists that a pest is going to come in and really love the plant that you're growing on. And if that's the case, they'll run through it like locusts. Right. I mean, they literally will spread the whole way. And it's kind of one of the negative aspects of that we have in the way that we grow cannabis currently as a monoculture is that we're very susceptible to invasion of pests or even specific molds. I mean, if something shows up, it's going to run through the entire population. And really the only way to combat that is to plant some level of heterogeneity or some level of genetic diversity that it kind of acts as like a shield or a barrier to the mass expansion of the pest. Right. If you have a quarter of your production space is a plant that spider mites, for example, don't find very tasty, they're going to stay away from that one plant or that one genetic type. And so you can actually kind of create barriers throughout your, your room. You create a mosaic of different patches of genetics and you kind of set it up in a randomized way that they, you know, you don't have all your blue dream in one area, for example, so that if it gets contaminated, it's going to run through them. Right?

All right. Yeah, this is my follow up, is really freaking specific, so I hope I can get us all the way there. So here. So, so you said that we want to to decrease the risk of the monoculture by creating this mosaic with different genetics. And so my question is, is this mosaic of different genetics all cannabis plants? So, like, you know, to use your example, plant one is a blue dream, but then plant two would be skittles and then plant three would be wedding cake or whatever. And then like, you know, plant five would be blue dream again, so that plants two, three and four have got different genetics and may not be as attractive to whatever the pest or fungus is that's attacking plant one. Now, do you mean that or. We talk a ton about polyculture on this show because I'm a big fan of, you know, planting cannabis plants alongside other plants like comfrey or whatever, and because those plants might, might attract the pests or repel the pests because of their terpene profile depending on the nature of the plant. So are you saying that, that you know, the better solution for monoculture as well, been talking about it now, is to produce is to plant, you know, five or more different types of cannabis plants but interweave them, or are you saying no, no, no, you can actually do a whole field of one variety of cannabis plant, but then we want to solve it with all these different species of plants.

You can do it both ways. I think for the context of this discussion, I mean, it's more about planting different types of cannabis. I wouldn't intersperse them plant by plant. That's just not an efficient way to either plant or grow or track, you know, through production. You might do blocks of say, you know, whatever, 20 by 20 or 100 by 100 of one genetic type. And then you, you, you rotate your blocks. Right. In a completely integrated system. I think that we're going to start to see more farms, more small farms that aren't just growing cannabis, that they're adding cannabis as a value added product to their production of what they can grow on their farm. I mean, they'll grow their blueberries and their garlic or whatever flowers they're going to grow and then like a little bit of cannabis that helps with the bills. And I see that the future of cannabis being really integrated into more of a holistic system, of a holistic production system. One step less advanced, though, is even in a cannabis grow is if you're, say you're growing five varieties, that you don't just have one big block of each of these, of those five varieties, that you have many blocks of those different varieties interspersed.

Yeah. You know, that idea of growing your blueberries or whatever and then cannabis around it was what I really thought we were signing up for when we started going down this legalization path. In my head, cannabis was going to save the family farm, you know, because you could take the sales pressure off of the vegetables or the fruit that has to be harvested at a very specific time and brought to market. There's so much pressure on the family farm for these smaller margin plants for food. I love the idea of like, oh, you do a row of your fruit tree and then a row of your cannabis plant. Right. And then the cannabis plant can make sure that the more mortgage is paid and then you can make a, you know, a good profit, but at a lower margin off of whatever your food plant is. And we save the family farm. But you know, unfortunately, like, no states are, are encouraging that with their regulations. And I think that it's, I think it's a disappointment.

Yeah, we're allowing it a little bit up here in Canada. And I've seen a few smaller farms try to integrate cannabis. Unfortunately, they don't do it in a professional enough way. Like you really, you know, growers know what they're doing at this point in time. But I think that like, as an industry, we're still in the very nascent, you know, we're in the nascent stage where, you know, we're trying out big, large public companies and they're not really working out because they just produce a bunch of garbage. It's hard to quote, to maintain that level of quality when you scale it. And I think that there is going to be a sweet spot. But the smaller family farms also need to learn how to integrate that information in. And it's actually something that Oregon and Southern Washington, even Northern California, there's some people that are really doing it right. But I think that it's going to be a learned experience that people need to learn how to integrate.

Great.

A good amount of cannabis, not a small amount, I'm not talking like a small amount, but a reasonable amount of cannabis that they can grow. They can maintain the level of care and attention that is required to produce a crop of that level of quality. Because again, you can't, in today's market, you can't just produce a bunch of average bud and expect to get a decent paycheck for it on the market. I mean, there's just way too many people growing cannabis and the stuff that gets turned into THC distillate. It doesn't really matter what it looks like because again, you're just going in for that one compound. It's kind of like farming for high fructose corn sugar. But I think that we all recognize that the people that really consume cannabis and are passionate about cannabis, those aren't the products that they want to consume.

Right.

They want to consume either really high quality flowers or extracts made from really high quality flowers.

Yeah. The only time I've ever seen the interplanning of real food crops and real high quality cannabis in the way, the kind of ideal way that you're talking about is when I visited Happy Day Farms, Casey and Amber down in Mendocino, they actually do that. But, but it takes so much work and they've only got a few acres of it. But, you know, I know that they're a small craft farm and they're probably not interested in scaling up to, you know, you know, mega acreage levels, but that's what I would like to see, you know, like where you have acres of beautiful cannabis plants alongside beautiful food and we can feed everybody and get stoned at the same time while resisting pests and viruses. But I don't think we're there yet.

Well, and I don't think the small farmers are there yet. I mean, running a farm, even a couple acres of any crop, there's a lot of work there and some crops are more forgiving than others. But I don't think that cannabis is very forgiving in terms of. It's like you said earlier, when it needs to be harvested, it needs to be harvested. You can't just put it off a week or cut it a week earlier and expect that it's going to maintain that same level of quality. Right?

Yeah.

So, yeah, it's hard to juggle those acre sized balls in the air and keep everything humming, you know?

Yeah, for sure. All right, so we're going to go to commercial, but stay with us. When we come back, we're going to start talking about the other aspects of tissue culture. We've already covered micro propagation very seriously, but there are a good five or six other things that are included with tissue culture and we're gonna do that after the break. You are listening to Shaping Fire and my guest today is cannabis geneticist Ryan Lee. Sometimes the topics I want to share with you are far too brief for an entire Shaping Fire episode. In those instances, I post them to Instagram. I invite you to follow my Instagram profiles and participate online. The Shaping Fire Instagram has follow up posts to Shaping Fire episodes, growing and processing best practices, product trials, and of course, gorgeous flower photos. 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As of Today, there's over 200 videos that you can check out for free. So go to YouTube.comshangolos or click on the link in the newsletter. Welcome back. You are listening to Shaping Fire. I am your host Shango Los, and our guest this week is cannabis geneticist Ryan Lee. So we have reviewed how tissue culture can be put to work multiplying plants, but sometimes before a plant can be multiplied it needs to be cleaned up. And we've talked earlier about removing these viruses and bacteria and different things that might be in it that kind of weighs down the plant over the years, but we haven't really talked about a lot about how that's done. So Ryan, at what point in the process would we put the target plant through a PCR test to see how contaminated it might be?

Well, really, it's something that we probably want to do repeatedly and over at different points throughout the tissue culture process and that's really how we go about certifying something that's pest, fungus, or virus free. Right? We test them, we prove that these organisms aren't in the plant, and that's how we certify them. Just to clarify how PCR does that, PCR is a technique for multiplying specific genetic markers. And so, for example, you know, powdery mildew has a genetic sequence that's unique to powdery mildew, and so do spider mites and every virus, you know, Covid. We all know that the COVID test, these Covid plants can be identified through specific genetic sequencing. So we can, we can kind of do the same thing with like, the Hopps latent viroid or any of these other viruses that are affecting cannabis. We can look for, we can look and try to amplify, amplify these specific genes from those species. And if we don't show, if those, if those pieces of DNA aren't amplified, we can't multiply them, then we can say that those organisms are absent from the, you know, from the population.

So it's like turning up the volume of a song so that you can hear the instrumentation, but you turn it up and you're like, yep, I really don't hear a triangle in that. And then. And so then you can certify it's triangle free.

Yeah, it's a great analogy, in fact, because, you know, those things are just a genetic single. And that's what PCR does, is it amplifies that genetic signal. And so if the signal is free, if we're free from the signal, then we're also free from the organism that creates the signal.

All right, so after we've put these plants into the PCR and we've turned up the volume so that we can hear all the of these different signals very clearly. And we go like, oh, we hear the signal for, I don't know, powdery mildew. How do you go into the plant and separate these, These, these pollutants out?

Well, let's. I'm not exactly sure how you would do it with powdery mildew. You'd probably have to use a chemical that suppresses powdery mildew. But let's consider. What's a better example then let's use a virus. And so like the Hops Layton virus, for example, when humans get infected by a virus, one of the things that happens is we get a fever, right? And the reason that your body has learned to give it to raise your core temperature is that viruses, or at least a subset of Viruses don't actually do very well. They don't replicate well. They're not very happy when the temperature is higher than, you know, 37 degrees Celsius or whatever the ideal human temperature is, right? So your body ramps up the temperature, turns up the thermostat, you get really hot and sweaty and, you know, you maybe develop a fever, but it's actually giving your body's, your body's creating a system where your immediate immune system is going to be tougher than the virus that it's fighting at that point in time. And it gives it a chance to kind of take over. So if we apply that same logic to plants, imagine as a plant cell is growing, you've got the meristem, and so the meristem is producing new cells. That new cell is initially born without any viruses in it. And then the virus is, moves in afterwards, okay? And so if you can actually grow a plant in a raised temperature condition, as new cells are created, if the temperature is high, the virus can't actually infect those cells because it's at a disadvantage because the environment, the environmental temperature is high. And so you might have a little section of the plant where the whole tip of the plant becomes virus free because it's growing in a high temperature environment and the viruses aren't able to take over. And so if you were able to go in at that point and cut out the new healthy, the cells that hadn't been infected by a virus, and you subculture them and plant them on their own, you know, their own agar plate, grow them for two to three weeks again, monitor them with a PCR test to see if the virus is there. And if it is, you just go through the process again. But after a series of subcultures, theoretically you're able to get a group of cells that are true to the plant type but free from virus infection. And if that plant, the plant that grows from those cells then becomes your new starting line, you've now actually removed the viruses from your plant line.

I had it all wrong, man. I like, I was even close, I was like, I can see how novice or amateur my picture was, but I was literally picturing the lab scientists there with a scalpel cutting around the virus, you know, so that it's in the cutting.

But you know, in a way they're doing that, but it's just that you're using the temperature to create the barrier for the virus, right? Another way to do it, you might, I mean, if it was a bacteria, you might put like antibiotics on the growth medium, for example. Right. So you're creating a chemical situation in the, in the medium and thus in the plant where the bacteria can't grow. Right. And then you go and you do the same thing. Right.

So, so, so after you run it through the PCR and you, you've turned up the volume and you can see all the crap that your plant's got, then you create a strategy for like, okay, what are the different steps we're gonna do to this plant to allow new growth to be contaminant free? So maybe, you know, you have to turn up the heat for a while to get rid of a virus, and then maybe you need to douse it with some kind of, you know, organic chemical to get rid of something else. And like, maybe, maybe it's a three or four step process, but in the end you are getting, getting new growth that is free of these contaminants.

That's exactly it. Yeah. And then, and that's, you know, what.

A pain in the ass.

It really is. But I mean, hop latent virus is also a pain in the ass, right? I mean, it completely devastates your production space. So, or broad mites or anything, any of these types of pests that you can go through. So you can think about it. We can get down to this very small little world inside a jar where we add maybe just a very little bit of pesticide or fungicide and those cells then grow out of that. But it's a lot different than having taken a whole big tall plant and soaked it down with, you know, five liters of pesticide. Right. I mean.

Yeah. And have to get that whole plant clean when you're only trying to get a little cutting to put in your petri dish.

Exactly right. And there's less environmental damage because again, you, you've released like maybe two, three microliters worth of pesticide as opposed to, you know, 500 milliliters.

All right, so earlier I thought you misspoke when you said that we, you were talking about the things that we were going to, you know, remove from the plant. Like we talked about the virus earlier said spider mites and now you just said, I think you said russets. But like I thought that these pests are coming from the outside of the plant. The plant attracts them. Are you telling me that there are things within the plant that is making them either attract or more susceptible to these pests?

Yeah. So typically insects should be removed during surface sterilization. And so that's really like when we go through that sterilization part where before we put the plant into the jar for the first time, you know, we might cut it. Cut out a little stem section. And, you know, we use like, bleach. It's like you'll create like a 10, 15 bleach solution and just shake your little plant parts in the bleach for 15 minutes. Well, it's like really hard on the plant material. Right. I mean, when you. When you take it out, there's all this dead plant material that you actually have to cut away before you. You section out the part of the plant that you want to try to live. But that's what we're doing is we're really. And they use all sorts of nasty stuff. I mean, mean, they use like, you know, mercury compounds and all sorts of little different poisons to do that initial sterilization process. And that's kind of one of the reasons you don't want to keep reinitializing plants. Right. It's because you're always introducing them into. Into this chemical. Ideally, they go through a short chemical insult, and then what comes out from that is your starter culture material. Right.

So you could find yourself in a situation that a plant's got so much pollutant in it where you're like, man, I don't know if it's gonna be able to live through like, four or five of these treatments to get rid of all the crap. And like, I guess that's where the artisan and expert level comes on, where you can. Where you can expose a plant to these various things and actually have it, like, living on the. The far side.

Yeah, you have to have a strategy to it. Like, again, you know, if a plant's really weak and, like, really hurting, it's almost dead. That's probably not a great candidate plant for tissue culture. But, you know, there are certain forms of tissue culture where you can do essentially what's like, you know, what's called rescue. And you can, you know, by shedding the plant, the. The tips. Shoots of those plant from the rest of the. Call it carcass. Right. The infected parts of the plant, you're actually freeing it from that disease. But, yeah, it's a targeted approach, and it has to be done with care and attention.

So I know that with all the variabilities, this next question isn't really fair, but I want to talk about cost. So this all sounds incredibly expensive. Let's say that you. Let's say I don't know what's typical, but I'm just gonna assume for a Second, that a plant you need to clean up before you can micro propagate it is gonna have, like, let's say that there's a virus and a fungus in there somewhere. And you're like, okay, we need to clean. You know, we found these in the pcr. We need to clean these up so that we get a nice clean cut so that we can micropropagate it. So to take us from the PCR analysis through the clean, cleaning it up so that we have our one set of mother platelets that we're then going to micro propagate. Like, can you just give me a ballpark figure about what it's going to cost to get us through the cleanup stage?

Yeah. I don't know what it would actually cost in terms of reagent materials to do it yourself, but I know that there's companies both in Canada, the United States, that will. That will do this for you now. And I've heard quotes from anywhere of 5,000 to $25,000 per genotype. So, you know, if you have a cutting of Blue Dream and a cutting of cookies that you want to have this process done to, it's, you know, minimum 10 grand kind of thing for both.

Right, cool. That gives me a general idea.

Yeah. And it can go up from there. You know what I mean? Like, again, if you're. If these people are offering services to these public trading companies, it's going to be closer to 25.

Right? Yeah, I get you. All right, cool. Well, then let's. Let's bring this episode home. You know, we've spent the most of the show talking about micro propagation, because that is what 90% of the cannabis industry is doing under this, this umbrella of tissue culture. But we know that there's other cool stuff that is under this tissue culture umbrella that's only being dabbled in in cannabis right now, even though it is incredibly common with, like, food and ornamental crops. So, you know, you had mentioned in our, in a previous answer the issue of totipotency. The. The idea that plants have stem cells and these stem cells can become any other part of the plant using this knowledge. There are a few other more rare tissue culture applications that we're not gonna go into deeply in this show. But, you know, any. Anybody who's still listening, Right. It probably actually cares about this. Right. So would you go into a few of these different applications that fall under the tissue culture umbrella so that, like, when we end the show, people are like, okay, I understand the micropropagation that makes up 90% of tissue culture. But I also understand kind of topically or tangentially these other areas that are also under the tissue culture umbrella.

Yeah, well, there's some really neat stuff. I mean, kitchen culture is just a great way to kind of ship plants around the world and store them, you know, for long term. We have what we call ex situ storage of germplasm. So, you know, they might go into a forest and find a very rare plant, and then you can take a cutting of that plant and bring it back and keep it alive under tissue culture.

I bet you that, I bet you people are smuggling plants that way too.

Absolutely. Well, we'll get to that one. But I think the first really cool one that I think that we're going to start seeing more of is something called a process called cryophone preservation. And so cryopreservation is exactly what it sounds like. It's like, you know, on the. What's it, Futurama, they had the frozen heads, right? Yeah, yeah, it's that same kind of thing. Well, you can take like a very small shoot tip and you can subject it to a freezing process. Again, you load the plant up with sugar and these anti osmotic. If you've, if you've ever seen ice form under, under a microscope, for example, you see it start what starts off as a clear liquid and then you get these crystals growing in the, in the liquid as it turns to ice. And if you imagine that process happening in a cell, those ice crystals will actually grow and they'll pierce right through the cell wall, okay? So as soon as you stab the cell wall with like an ice crystal or a needle or whatever, the cell's done, right? Like it's going to leak all of its content. So it just, it's, it just can't take that kind of insult. But if you do it properly, you can actually get, you can, you can do something called vitrification. Whereas think of it like the life in that still, that cell going from an animated alive state to completely frozen into a glass, like, state immediately. And when.

So it just happens faster.

It happens incredibly fast and it happens through a very specific process of dropping the temperature in a, in a very specific and quick way. And if you can actually get that, that vitrification to happen, think of it like trying to walk through the mud, okay. Or walk through cement. You got like rubber boots on up to your knees and you're trying to walk through like some freshly laid cement. Like it gets stuck, right? And if you got stuck in there and you really Tried to, like, really force moving your leg, you might actually break your leg. Whereas if you were frozen solid, you're not going to break anything because you're not moving. Right. So if you can actually get a cell or a group of cells to undergo this vitrification process from essentially live to completely frozen, like in a split second that you. That's called vitrification. And it turns out that if you can actually freeze life till at that speed and size, we can then store whatever biological materials you have at like, you know, minus 120 degrees. They do it in, like, a liquid nitrogen. They have these special containers that hold liquid nitrogen and say the bottom third is filled up with liquid nitrogen, and the top third is the gas that's coming off that liquid nitrogen, which is still very, very cold. It's not as cold as the liquid, but that gaseous phase that fills up the top part of the container is cold enough where you can actually store biological samples for, you know, an extended period of time, say, indefinitely. Right. And as long as the temperature doesn't change, those. Those biological samples are safe. You can then take those things and reanimate them and grow plantlets from those shoot tips that you've. You've frozen under cryopreservation. And so it's a difficult process. It's. We've only just figured out how to do it with cannabis, but it's. It's been typically done in other species, you know, pine trees and all sorts of trees that they use for forestry. And hops. There's a place in Oregon, Hops Research center is doing all this kind of stuff with hops as well. But you can imagine, I mean, anybody that's a grower and collects, you know, special plants from all over, you know, you. You learn pretty quickly that it's like, you know, keeping 50 different genetic types and having to, like, grow them and take cuttings and cycle them. Like, that's a real process. And it takes a lot of time and energy and resources. If you could just submit those things to a frozen gene bag, knowing that you could then go and pull those plants out of cryopreservation and. Yeah, that process, to pull them out of cryopreservation, it takes like six months, right, until you're. You've got a cutting back in your hand. But if you think of, like, the space saving and the time saving and the resource saving that happens, if you can cry and preserve these plants, I mean, it's pretty. It's something pretty special.

Yeah, just. Just keeping a regular mother Library is a big difficulty. I mean, my God, I can't imagine if you were trying to, you know, take a snapshot of all the hype strains right now and like we're gonna preserve them all for the future. It's like, oh my God.

Yeah, but you could preserve them all in a, in a, you know, in like a, a small 50 pound Dewar, right? Like a small, like a, it's about the size of a propane barbecue tank, right? That you could probably store two or 300 cell lines in there, like no problem. So that's, that's pretty attractive. And as a breeder, you know, if you're doing selections, it's also a very attractive thing to be able to do that. The next step, I think the next one I think I'll mention is something a concept called artificial seeds. And that's really like a very small shoot tip. Call it like you know, three or four, eight of an inch, maybe half. Half an inch. And that gets embedded into a little jelly matrix and hardened in a, in a solution of calcium carbonate. And it's like a little mini tissue culture shoot in a seed. Like, you know, it's not, doesn't look like a seed, it looks more like a jelly bean. But those artificial seeds again, can generate a full plant. And you can think about how easy it would be to ship a small piece of plant material the size of a jelly bean around the world. I mean, you know, you get on a plane, a handful of them in your pocket and you could pretty much go anywhere, right? So those are, those are another interesting little thing. There's something called embryo rescue. People might have read in the newspaper. You know, these Russian scientists found a, a 30 year old seed in a. I'm not sure if it was an ice core or it was thawing out of a, out of a melting glacier, but they collected these 30.

You probably don't mean 30 year old. You probably mean more like 30.

Sorry, 30,000.

30,000. All right. Because as soon as you said glacier, I'm like, yeah, I think he means more than 30.

Yeah, yeah, misspoke. But yeah, 30, 30,000 year old seeds. And they actually were able to grow these things back in, to, into plants. And it took some doing. But yeah, this, this idea of being able to take seeds and, and baby them. You know, a seed, planting a seed is, you know, it's, they're tough. But again, you know, when you have a 30,000 year old seed, you really have to baby it and make sure all the conditions are perfect. And you know, Tissue culture is kind of one of those, one of those ways that you could do that.

I can't, I can't even get seeds to pop from 1978, you know, let alone 30,000 years.

Yeah, those are all possible candidates. You know, I mean once a seed is dead, it's dead. But if it's stored properly, you know, sometimes you have like a lot of what the seed is, is, it's part of the test is actually just food source for the seed. That's why you can plant the seed in the ground and they'll grow to like two sets of leaves all really eating the food that's in, in the seed already.

I've never actually heard the term dead seed before. So what is actually, do you mean from that? I mean just based on what you were just saying about food, does that mean that the food that was contained within the seed, that keeps it virile has all been used and therefore you have got genetic material in with no food in the seed and that is therefore called a dead seed?

It's not really that it's used, it's probably that we're talking a 30,000 year old seed. It might be that the starches or whatever, the compounds or sugars that are in the plant have broken down due to whatever. It could be a fungus or whatever. But yeah, within a seed there's a very tiny little embryo. Right. And so that embryo could theoretically be cut out and provided with all the nutrients and vitamins that it's need and hormones.

A typical, a typical cannabis seed. That's, let's say that somebody's got their typical cannabis seed. Let's go old school. And saved in a 35 millimeter film container. Right. Because that's how they used to be. How many years do you think that seed is going to go before its air quotes dead?

Well, it's, that's really a tough thing to say. If you, if you store a cannabis seed, it's properly dried, it's packed with a desiccant, something that's going to absorb the moisture. You can put those seeds into a fridge and we're pulling seeds out that are like 25 years old and still germinating at like 85, 90%.

Oh wow.

Right. And listen, like I said earlier, that also varies by type. Like some genetic lines are, they don't last that long and some will probably last longer. And my personal hypothesis is that it has something to do with the oil content that's in the seed before it goes through that process. But whatever it is it's just another trait that varies, right. That shows variation. So yeah, the question about it being in a container is really what, what was the temperature and what was the humidity that those that it was subject to? Right. Like, like I said, if you, if you put it in a 4 degrees Celsius fridge for you know, 20 years, they're going to be, be fine. If it's in like a 35 millimeter film camera in your attic, your attic or your trunk of your car or whatever, like your odds are going to go way down pretty quick.

Which is probably why these seeds that we're getting to pop after these thousands of years are coming out of frickin tombs.

Yeah, exactly. Yeah, exactly. They were in like a one environment that was stable for that long. Yeah. So that's, so that's really cool. There's also something called anthraculture where we, we grow what you think about when a male plant, or even a feminized reverse plant plants a female plant sprayed with sts so that it's making male flowers. Those male flowers are, you know, they go through a process until they drop the pollen and say that takes 30 days, right. So you can actually cut those branches off at like 15 days for example, and go into the forming male flowers. And within those flowers there's the cells that are growing to become pollen grains. You can actually culture those cells at that stage and, and try to culture them. And in rare circumstances, if you've done everything right, you can actually grow the pollen or the pre pollen grain cells into a plantlet and we can subject those things to a chemical called colchicine. And colchicine effectively doubles the chromosome number. And so you can imagine that a pollen grain like sperm or an egg in a female, in the female reproductive system context of an egg, when those things come together, they actually in, you know, in cannabis, when they come together, we get two sets of 10 chromosomes. So each the sperm and the egg only have one copy of the 10 chromosomes. And so that's called haploid. That's like a, essentially you need to have two halves to make the whole that when they come come together in the new organism that, that new organism has a complete genetic set. And so we can grow these plants that have half the set, double the chromosome number. And now we have a plant that is 100% true breeding for all traits. Right. And so that's, those are called dihaps.

That's some mad scientist shit right there.

It's, it's serious mad science and stuff. But this is, these are the Techniques that they use in, like, corn and wheat breeding to create seed lines where all of the seeds, you know, you can plant a hundred, like a thousand acres, and every single plant looks the same.

Do you know anybody who's doing this in cannabis now?

Yeah, there's people, there's a couple of groups that are working on, so.

So this is happening. Wow, that's some hard hardcore science.

Yeah. And you can, again, like, you know, you can. If you can figure out the process to grow these pre pollen grains into plantlets and you can, say, get a thousand of them, you can then go in and subject those thousand plants to genetic screening and find the ones that are really ideal. Right. So that's all really interesting future stuff that's coming towards cannabis that'll be. That really exceed our breeding. And the last real thing I think that's, that's probably interesting to talk about is the concept of somatic embryogenesis. And so embryogenesis is like, you can think of the word, the genesis of an embryo. It's the process of creating embryos. And so the somatic part of that word means it comes from the body of the plant. And so you can actually take, imagine you could take, for example, your favorite plant, doesn't matter what it is called skittles. You take your skittles plant, you encourage that plant to grow callus, and that callus can then be coaxed into a pre embryonic cell state. Once we have those cells, those cells can be grown and multiplied on mass quantities and then divided into little groups or clumps. And those individual clumps can then be coaxed into embryos. And so essentially what you have at the end of that process is say 10,000 embryos that all came from your skittles plant, Right. But they're what we call bipolar. They have a top chute that goes like a top meristem that grows skyward, and they have the root meristem that grows towards the soil, Right. Whereas in the context of the tissue cultures that we were talking before, you know, we had to actually put them through a separate process to get that root meristem to grow. Right. So they were really unipolar at that stage. They were only growing skyward. Right. And so having a plant part that will grow, a shoot and a root, it really makes things easy for automation because the plant's ready to just do what it does normally as it. As if it were a normal seed, right? So think of it like having a hundred thousand seeds that are all twins.

And, and do they have the virility that, you know, like when we, when we normally say we're going to grow from seeds, most people say, like, yeah, the, the a plant from a seed is going to be more energetic and more virile than a cutting. Are these going to have that. That oomph that you get from a seed?

Yeah, because again, part of that oomph comes from the having the tap root that grows straight down and it taps down into the soil and goes searching for the water table. Whereas clones, you know, when we have clones, they're coming up outside, Lace. So the roots kind of. They don't tap. They don't. They don't seem to tap down as much. They kind of. They kind of grow more along the surface of the soil. And so there really is something about having that tap root that drives straight down into the soil that has to do with vigor. Right.

Wow. You know, I'm kind of glad that we focused on micro propagation as the major umbrella, because I don't think I would have been able to follow you in a conversation with these five things that you just laid out. This is like some serious science. And I don't, you know, like, you know, there are some good people that have got capital that are probably doing these things, but this really sounds like, like mega, you know, mega ag companies that'll be doing this stuff.

Yeah, I think probably, I mean, mean, you know, someone like Mr. Mean Green, I mean, he could go and do this with a university lab and partner with someone like them. So there's. There are definitely ways to do it.

I like you interjecting hope in there. I like that. Thank you.

Yeah, well, we, you know, none of this stuff is beyond the community. I think that we've kind of felt, you know, especially in growing out of Prohibition, that it's always been like us versus these big corporations. But there are. There are opportunities for people to go. Rather than being acquired by those guys, why not go and find ways that we can partner with them? It's more of an equal relationship, not one of being told what we're going to do. And when it's like, hey, look, we have all these unique genetic resources. Can we partner with people that have knowledge of how to do these techniques so that we can advance the species?

That's a beautiful summary. I appreciate you ending this on a good note. This idea of partnering with scientists and laboratories where we're on equal footing pleases me extensively more than the idea of this idea of us having to defend ourselves because Big Ag is just going to come and steal all of our good stuff. I like this idea of we who have been in the scene for 10 or 20 years or whatever, we just go directly to scientists who smoke weed and say, all right, let's get together and let's use your. Let's use your academic knowledge. And then our ability to smell flowers and determine terpene profiles that cause joy. And then let's work together and do it ourselves before the genetics get taken and patented by somebody who doesn't have our best interest at heart.

Yeah, it's all in our hands to do. It's just, you know, being able to envision a way to make it happen. And I think that as we move into this more legal world, there is a lot of opportunity for collaboration. And the truth is, these scientists, they're not. I think the most of the people that are really, truly interested in the science, they're not out to get rich. They're out to change the world through breeding plants and, you know, making these species into things that are really valuable tools for humans. Right. And, you know, the somatic embryogenesis is maybe. Is a little more of a dream, but the di. Haploid breeding and using anthraculture, these things are. They're standard methods that are used in other crop species, other major crop species, and there's. There's no reason we can't use those same technologies. And cannabis.

Well, that's a really positive note for us to end on. Ryan, thank you so much for sharing your. Your vast experience and your time and your friendship with us. You know, your. Your. The reaction to your. Your last visit with us was just, like, so positive. I got all these POs or these private messages. They're like, you had Chimera on the show. Hell, yeah. You know, like, you are. You are a un. Unique person in our scene, and I appreciate your willingness and your ability to explain some of these really hard ideas in layman's terms so that the rest of us can follow along with you. So I appreciate your interest and your willingness to join me.

I appreciate you giving me the venue to share the knowledge, so it works for everyone. And thanks to all your listeners, and I hope someone puts this info to good use.

Awesome, brother. All right, so if you want to either, like, you know, reach out to Ryan Lee himself, or if you just want to keep up on what he's doing, the best place to do that is on Instagram. He is eating cannabis, and that's a good place to follow along. If you want to hear more from Ryan Lee, I recommend that you check out episode 64 of Shaping Fire. We did this beautiful show on feminized seeds and female only breeding, which was not only very informative, but caused a whole bunch of mind blowing and argumentation in the threads. So it's very interesting stuff. You should definitely check out episode 64. It is enlightening. And and then also the other place I'm going to mention is the Emerald cup in 2018 I was there and recorded a great panel that was titled Analytical Cannabis Breeding Programs and oh my God what a panel this was. It was Ryan Lee, who's our guest today, Seth Crawford from Oregon, cbd, plant patent guru Reggie Gaudi, Josh Wertzer from SC Labs, and then it was moderated by Jeremy Plum from Proof Cultivar. That was just awesome. A bunch of folks who really know their stuff and came together to talk serious science and trash at Emerald Crump 2018. And you can you can check that out on the Shango Los YouTube. You can find more episodes of the Shaping Fire podcast and subscribe to the show@shapingfire.com and wherever you get your podcasts. If you enjoyed the show, we'd really appreciate it if you would leave a positive review of the podcast wherever you download. Your review will help others find the show so they can enjoy it too on the Shaping Fire website. You can also subscribe to the newsletter for insights into the latest cannabis news, exclusive videos and giveaways on the Shaping Fire website. You will also find transcripts of today's podcast as well. Be sure to follow on Instagram for all original content not found on the podcast. That's shapingfire and Angolos on Instagram. Be sure to check out the Shaping Fire YouTube channel for exclusive interviews, farm tours and cannabis lectures. Does your company want to reach our national audience of cannabis enthusiasts? Email hotspotshippingfire.com to find out how. Thanks for listening to Shaping Fire. I've been your host Shango Los.

Angeles.