Episode 167: Genetic Degradation in Propagation: Why Clones Lose Vigor Over Time

Welcome to the Cannabis Cultivation and Science Podcast. I'm your host, Tad Hussey of KISS Organics. This is the podcast where we discuss the cutting edge of growing from a science-based perspective and draw on top experts from around the industry to share their wisdom and College. Now, before diving into the show, just a friendly reminder that we are hosting the Cannabis Cultivation and Science Conference in Bozeman, Montana this year on August 28th through the 30th at Collective Elevation. It's going to be an amazing event with many of my favorite podcast guests attending and speaking. We are going to tour the largest living soil facility in Montana and possibly the United States, as well as have some incredible speakers, networking, and a sensory event that Saturday where folks can sample and enjoy a guided experience of what Montana cannabis has to offer. Tickets are available on KissOrganics.com, and we create an all-new website just for the conference, which is at cultivation.science. Now on to the show today. We are diving into a topic that every serious cannabis cultivator and nursery operator has speculated about for years: clonal degradation and genetic drift. We've all seen an elite mother plant seemingly lose its punch, its vigor, or its terpene profile over time. The prevailing assumption has always been that cloning gives us an exact 100% identical copy of the mother plant, But as many of you know, it's much more complicated than that, and mutations can and do occur. My guests today are the scientists behind the paper Genome Degradation in Plant Tissue Culture, which was recently published in the journal PNAS. They tracked clonal plant lineages across 5 decades and discovered that certain lab-based tissue culture methods can cause a shocking 3,000%+ increase in mutation rates, whole chromosome duplication, and the activation of jumping genes. Joining me today is Dr. Jay Grey Monroe. Grey is an associate professor in the Department of Plant Sciences and the Genome Center at UC Davis. His lab focuses on how mutations arise across genomes, DNA repair, epigenomics, and plant genome evolution with direct applications in crop improvement and protection. You can check out his work and contact information through the Monroe Lab website. We are also joined by Matthew Davis, Matt is a 5th-year PhD candidate in the Plant Biology graduate group, co-advised by Gray and Pat J. Brown. Matt's research focuses specifically on somatic mutation in orchard crops, an understudied genetic process that directly impacts some of California's largest agricultural industries. If you want to read their peer-reviewed paper or check out some of the excellent general audience articles summarizing their work, I have put the titles to those resources in the show notes and on the podcast page on the Kiss Organics website. Including the UC Davis article, "Not All Clones Are Created Equal." This was a fascinating, highly technical conversation about what actually happens to DNA inside the lab and how those findings might fundamentally change how we look at cannabis micropropagation, tissue culture, clonal propagation, and genetic screening. Now on to the show. Hi, Gray and Matthew. Thank you so much for coming on the show today.

Thanks for having us.

Yeah, thanks so much.

Yeah, I, I was really excited. So, uh, Kevin McKiernan from Medicinal Genomics sent me a link to your paper, and right when I saw it, I wanted to reach out to you guys, uh, to get some feedback on it. But, uh, why don't we just start diving right in? So talk to me about what your paper is and why you decided to research this topic in the first place.

Um, well, like, uh Cannabis and many other crops are clonally propagated, and for a while people have speculated that that process produces new mutations that can affect traits. You can get new weird varieties through clonal mutation, and we were interested in this question. And so I'll let Matt tell you about how we came across this really unique material in walnut to do this study.

So yeah, yeah, so, um, It was really when I started grad school. I was working with the walnut and pistachio breeder as well as with Gray. And it turned out that they had these clonally propagated walnuts that had been through different kinds of clonal propagation and in tissue culture, and they'd kept them in culture, some of them since 1985. And we were really just interested in what kind of mutation might be happening. There had been reports since all the way back in the early '70s that, you know, as we move things through tissue culture, that we end up with things that should be clonal looking very different after, after like a long time in culture. And this seemed like the perfect system to look at that.

So yeah, go ahead.

I was gonna say, so, so the theory is that when we, when you do clonal propagation, the, the idea is we're getting the exact same genetic plant as our, our mother plant. However, with somewhere in that process, there's the possibility of some level of mutation, and, and it's kind of quantifying what that level is. It's, it's low, but over time, if we do successive generations of clonal propagation, we may be introducing some level of, of, of, uh, mutation.

Yeah, exactly. I mean, people call it different things, you know, somaclonal variation. Sometimes people call it genetic drift. I've heard different ways to describe this. And, I've talked to people from different crop, like people in apple, for example, will say, "Oh, yeah, that variety is just not what it used to be. It's changed over time through successive clonal propagation events." I mean, if you think about it like this, all living organisms with DNA have the capacity to accumulate mutation. And so, plants are just the same. And so, they're just accumulating mutation in sort of a time over time. And, you know, sometimes those mutations can be dramatic. And one of the things that this paper we addressed was, do different types of tissue culture affect the mutation rate? And what was so surprising is that there's a lot of variation in the type of tissue culture that's being used and its effect on mutation rate that might be probably of, like, interest to people who are, you know, working in this area.

Yeah. So your work was with walnuts. So I realize you know, this podcast is primarily going out to cannabis growers. However, I think the— this is really interesting. So let's— and I have, I have a couple questions. The first thought I had, just kind of jump around a little bit, was I saw some research recently from Dr. Zamir Punja talking about endophytic microorganisms and how they're finding more and more, you know, endophytes inside the tissue itself, which means potentially when we're cloning or propagating or even doing tissue culture, if we're removing— specifically with certain types of tissue culture, we may be losing that endophytic relationship. And maybe that's why some of that, you know, that apple doesn't turn out the way it had before, which just further complicates this. I know you guys were looking specifically at genetic mutations, but how do you think that might play a role too in all of this?

Yeah, I mean, it's, it's a super cool idea. I think we don't really have a great idea about, uh, sort of these relationships on the whole. They're, they're really challenging systems to like piece apart, pull apart and study.

Um, yeah, I mean, we haven't looked at like, you know, bacteria, bacteria changing or something like that, like endophytes. Um, but you can imagine that like there's lots of things that are reset when you, it depends, again, it depends on the tissue culture method too. Like if I don't know what in cannabis what, you know, I know there's a lot of micropropagated shoot cultures done, but I don't know the extent to which people are doing more like callus-based cultures or somatic embryo cultures, which are the types of tissue culture systems that we find to be the most mutable. And those might also have different effects on the outcome of like this maintenance of endophytes as well. Like if you're purging the entire plant structure by going through a callus phase, you might be doing a lot of changes across lots of— not just the DNA, but the epigenetics, if people are familiar with that as well.

Yeah. And, you know, the environment is so different even in shoot culture too. I guess I wouldn't be shocked if there's dramatic changes even if you're just propagating a shoot over and over again because the actual environment in these culture scenarios is so different.

So, let's talk about your paper. Starting off here, so what was the sort of the experimental design around the research that you did?

Yeah, so really we start with a plant that was released in 1979. And so, it was probably bred in the early '60s and it has only ever been clonally propagated from that point forward. Now, we have plants that have only ever been clonally propagated out in the field. And so, that's like we're going out, taking a cutting of it, growing that cutting just all out in the field. We also have plants that were introduced into shoot tissue culture. So, I think like our traditional tissue culture method that we think of where we'll take a cutting from that plant, clean it off really well, and then introduce it into media and just grow that whole plant. And I think, I think maybe the most dramatic form of clonal propagation that we, that we had available was these somatic embryo cultures. And so that's when you take some, some somatic tissue. And so from those cells, you can actually induce embryogenesis. And so these are clonal embryos that we're creating. They aren't created through sexual reproduction like normal., and those were generated in 1995, and they've been maintained ever since. And so we have this really nice time scale of different clonal propagation methods, uh, to, to look at. Um, and then from there, you know, we, we sequenced all of our material and started to, uh, identify what, what mutations were happening.

Were there any anecdotal reports that the original tree from the '70s was very different than what expression you were seeing now with the trees?

So, in this case, no. So, the kind of like punchline of the paper is that the trees actually seem like they're doing pretty well. It's the stuff that went through extreme tissue culture where we see the most mutations actually. And so, like, for example, the stuff that went through somatic embryo culture, it looks weird and looks different if you try to regenerate it into a plant. And if you were, you know, like a walnut grower, you probably wouldn't want that plant, you know, in your— it's not, it's probably not ideal. And so, so those, those material look weird. And we have, we work on other cropping systems too, where we know that mutations happen that produce weird off-types. And just through, again, it's just, it's just the process of mutation just happening. And so this, this study was the first, like, The ability to sequence DNA at scale is finally catching up with the biological questions. Like, we can do the sequencing now for these kind of systems that was just basically not attainable even just like a few years ago. And so we can finally, yeah, address these questions across like many different systems.

So like you said, the punchline is basically your, your, the, the current commercial practice of field-grown cutting propagation was actually had a very low mutation baseline mutation rate.

Yep.

Um, and the genetic integrity of those plants was very high. But when you moved on to the, what the somatic embryo propagation method through, through tissue culture, you saw, uh, completely different results. Do you wanna go ahead and just explain that?

Yeah, it's like a, we have like a 3,000% increase in mutation rate in those material. There's also a higher mutation rate in the micropropagated shoot cultures as well. They're higher than the trees in the field too, because they're just being propagated quickly. It's a lot.

So yeah. Yeah. So it's actually, that's, that's a cool result too. So the shoots that are in micropropagated shoots that are in tissue culture, um, have a pretty, uh, linear increase of mutation with time, which, you know, is to be expected, but it is more mutations than we see out in the field. Um, the somatic embryos also had a lot of other, I would say, like crazy genomic phenomena going on. So they had like whole chromosomes that were duplicated. Um, they had losses of, of those duplicated chromosomes in some case. Uh, they had a lot of, uh, transposable element activation. And so these are, these are genes that can move themselves around, but you know, you can imagine you probably don't want pieces of DNA, uh, inserting itself randomly in your own genome, right? So plants and, and humans and, uh, really living things have a way of, of, of shutting these transposable elements down. Uh, but in these, in these embryo cultures, they were, they were activated and they, they were moving, uh, all over the place again. So there's just a lot of, uh, genetics happening in these, in these cultures.

Now, 3,000-plus percent sounds like a lot. I, I realize though, if the actual like n-value or the, the amount of, of, uh, mutation, let's say in clonal propagation, which was your baseline, is super low, you could have a very high percentage with actually a very small increase in actual mutations. What, what does that look like to a layperson in terms of, is this significant?

Um, I think it can be significant. Yeah. So like, especially so That clock, that 3,000% increase, you know, that's a sort of just way to quantify the increase. But what Matt was just talking about, like, this is the more alarming or sort of the thing that I would be more concerned if, you know, if I was planning on growing this stuff is the fact that you get not only increases in these small mutations, which is what defines that clock, but the increase of these events that are like whole chromosome duplications. Like, in humans, you know, These are called trisomies. And in humans, like, they're almost all lethal except for trisomy 21, which causes Down syndrome. So like, for most organisms, trisomies are not well tolerated. Like, they're very deleterious, we would say. And so like that, it's that kind of dramatic change that we only saw in the somatic embryos that I think would be the one that like raises the most red flags.

Yeah. And, you know, I guess just from like a real practical perspective of trying to grow these embryos back out, you know, we have a really, really hard time doing that. We can eventually get them to grow and produce little plants, but they don't grow into adult plants at all. They're really, really challenging to work with. And we think that that could be due to these really large-scale mutations that we were seeing.

Has anyone tried this research you know, maybe not with DNA sequencing, but with just, you know, looking at these types of propagation within walnuts or other crops in terms of the research you did before doing your research?

I mean, people have been interested in this idea of somaclonal variation or whatever for quite a while. And in fact, like there was some interest for a long time that you could leverage this new variation to discover like beneficial mutations. Well, and I will say that mutations that originate in clonal material sometimes have produced a lot of the varieties that we love in a lot of crops. A lot of citrus is produced by somaclonal mutations, a lot of apples, grape varieties. There's a ton of grape varieties that are all just derived from somaclonal mutations. And so, there are plenty. So, that was of interest for quite a while was taking— it's like this is new variation, we can select for it. And I wouldn't be surprised if So, you know, when you talk to the actual growers, like, I'm sure there's people in the cannabis industry who are like seeing interesting new stuff pop up in clonal material. And I'm sure people have noticed this before. And, and, you know, now we can sort of look at it at the genetic level, though. We can sequence and see like, okay, what is changing? Like, how much has it changed? You can, you can essentially like use somatic mutation to say like, how old is this clone? Basically, I mean, do just do things like that. Um, and so I think it's a new frontier in, in like a lot of, uh, cropping systems.

Yeah. So to give a little context of the cannabis industry, so this is something, cuz there hasn't been as much research, uh, compared to other crops. And what, what we've seen is anecdotally, a lot of older growers who have been doing this for a long time will say, you know, if they've been growing the same cultivar for, You know, or strain as, as we like to call it for a really long time, what you'll find is it will start to just lose vigor.

Yeah.

And so the, there's two prevailing theories, um, or methods that people do. And, and one of 'em is to say, to do meristematic tissue culture will help, uh, regenerate, remove some of those potential endophytes or things that might have built up in the plant or just restore it to its original genetic condition. I haven't really seen it. Personally done effectively. The stuff that I've seen going through meristematic tissue culture still came out kind of weird, or it didn't really have that same level of vigor. Um, did actually, again, just personally. But the other one is to get that, that plant that's been grown indoors for 20 years is to take it and do it, run it outside for a while. And somehow the being in an outside environment, I don't know if it's exposed to different microbes or the sunlight, something in there they say will also help refresh those genetics. Again, I don't, I don't, I don't put a lot of stock into this yet cuz we don't have any research, but those are the sort of the two prevailing things in cannabis is how people address this sort of overtime vigor issue.

So I think what you're describing like sounds familiar to me in talking with people like in strawberry, for example, is another like major clonally propagated cropping system where I, I've heard similar anecdotes and I've realized like when you hear these anecdotes, like you, you like my ears always perk up because I feel like it's the people who are growing the things that notice stuff, that that's where they're encountering all this really cool biology. And so, I'm sure there's something going on. I mean, so what I would do in those situations, I would say, let's sequence some of those regenerated plants, let's sequence some of the other ones, let's see what's changing. It could be epigenetic as well, especially if it's shifting through meristem culture. You might be resetting the epigenome, which is a whole other That's a little bit different than what we did in this study where we're looking at like genetic mutations, DNA changes. There could be changes to like, uh, I don't know. Yeah. The epigenetics. I'll just leave it there. Yeah.

Can you, can you talk a little bit about that for people who may be less, less familiar with some of these terms that I know are so common for you guys?

Yeah. So like, you know, genetics, let's just say is, you know, it's like DNA, A, T, C, and G, right? Like it's just the basic chemical building blocks of the DNA code. But those molecules, A, T, C, and G, can be chemically modified, for example, by what we call a methylation mark, for— it was a common one. So if you add a methylation to a C, to a cytosine, this is a type of epigenetic change. And these things affect the way that— they affect the biology of the plant. They affect how the DNA is expressed, we would say. And those things can change over time. And they can affect the way genes are expressed and affect traits and can be another source of sort of instability over time. I don't know if that— does that sound—

yeah, yeah. I mean, essentially it's like, it's like you're putting something on top of the DNA so that changes the way that you can have the DNA read, right? So it's like if you put like a, I don't know, a bookmark on top of the page, now you can't read that. Or you can take that bookmark off and now you can read the words that are there.

So the way the plant expresses itself can be different even with the same genetic structure.

Definitely.

So, so it's not an actual me— it's not an actual mutation, it's just the way the plant accesses or expresses itself.

Yep.

Again, I'm not, I'm not into genes either. This is, this is tough. This is a tough interview for me too.

Well, that's exactly it. That's it. You got it. That's it. It's not the, the DNA sequence has not changed, but the way that the DNA sequence is being read by all of the proteins that are involved in turning that DNA into something that the, you know, making pro— they, they, they're doing all this biology by reading the DNA and their ability to sort of read the DNA is affected by these like additional chemical modifications we call epigenetic marks.

Yeah. Okay. And, and the plant's environment, and when I say environment, I'm, I'm talking not just about actual, but I mean like its relationship with the microbial communities, potentially, I guess maybe in the soil, but more specifically inside its tissue could affect that expression?

Yeah. I mean, for sure. Yeah. I mean, the expression of the genome is very dependent on the environment the plants are in for sure.

Okay.

Yeah. And so, whether it's the sunlight they get, whether it's the water they have or the other microbes they're interacting with, they can all affect things.

So, so within cannabis, the, the main types of tissue culture that we're seeing is, is sort of mass micropropagation, um, for, for cleaning up genetics from things like Hoplaiten viroid, for example, um, or meristem tip culture. Did you guys do anything with, with meristem tip culture looking at how that might impact mutation rates?

Um, we have not, and it's on the— be super fun to do. So, okay. Yep.

Yeah, definitely interested in like all forms of clonal propagation, uh, to look into.

So what are the implications of your research beyond this, you know, the specific walnut variety? Um, how do you think your research can be applied to other, other crops?

Um, well, so any, I mean, we, we sort of unlock the toolkit, I would say. We have like the tools that are necessary for monitoring the genetic integrity of clonal material now. We know how it works. We know how to use it. And given a species with the right, like with the need for it, like we know how to do it. So if you had like material that was weird or you wanted to do experiments to determine what causes new mutations to accumulate faster, like we have, so it's the DNA toolkit to actually like measure that process for the first time and give some diagnostic insight to whatever system is underway.

Yeah. Yeah. And, you know, I don't know if you guys do any like transformation or gene editing work, but, you know, it's, it's a good opportunity to, to like sort of assess your materials as you go. You know, I think quite often we think, oh, you know, like the plant itself is totally fine. We're not worried about— we're not worried about the integrity, the genetic integrity of the plant that we're using. We're more interested in the, in the things that we're trying to do. But I don't know if cautionary tale is the right word, but, you know, it's, it's maybe a good reminder that we also need to be checking our plant as well and making sure that our plant is, is genetically, genomically healthy before we move on to whatever, whatever we want to work with.

So basically, your research created the method, the scientific methodology or the framework that would allow us to test not just walnuts, but in this case, really any plant species to see how it would react to these different types of propagation. And you, and you could do meristematic tissue culture as well.

Sounds good. Yeah, for sure. That would be super interesting. Yep.

Yeah, absolutely. And, and you know, like also this, this phenomena of somaclonal variation where we're seeing these different things appear from genetically identical individuals introduced into tissue culture, it's been reported in a bunch of plants for decades. So, I don't think that this is a phenomenon that is exclusive to walnuts. I think that probably start to see mutation in a bunch of other—

Yeah. I mean, the walnut is just, well, we have this unique material here to do because it's just long-term collection. But, But it's more, yeah, the principles, you know, are, we think are, there's principles here that can apply like across lots of different plant systems, colonial systems. And there's different, but I think I would say the other thing, I guess I will add a little like caution against like being like, like I don't want people to like come away from this thinking like, oh, all tissue culture is bad and it makes everything mutated. Because like, you know, this isn't, we didn't study all species. We don't have every type of tissue culture in this experiment. We looked at an extreme case where this has been in tissue culture for a very long time, you know, so it serves as a useful experimental system. But I would say, I think the verdict is still out on like, you know, is this like, yeah, I mean, it's a scientific question. Like, that's why we need to, you know, sequence new material and actually ask like in a cannabis system, like what is the mutation rate in a meristem like clonal system or in micropropagated shoots in cannabis? Those are all just questions that just require, you know, new experiments with, with material that like may already exist, basically.

I, I think you brought up a really good question that exists across all scientific research, and in terms of the applicability of that research to not just the, the topic at hand, because realistically with any research, like, I would— it would be great to see someone replicate your experiment and get similar results. And the more replications we have of that experiment, the more confidence we can have that this particular walnut variety will give this sort of level of mutation. And then how well can we apply that research to other crops like you mentioned? Well, it appears based on your research and the other research that's been done and what you've been hearing from other people that there could be potentially pretty similar results across a variety of different plants. But I think that is a good caution that we can't just automatically assume because of your research that like all we're gonna see 3,000% increases in mutations on, in raspberries and cannabis and, you know, whatever other plants are, are, you know, propagated in this manner.

Totally. And it's like, it's, you know, and, uh, yeah, so it's, these are, it's a research question, you know, and it's a, it's a sci— we gotta do the, we gotta do the actual, we gotta do the science to test each of these, each of these cases. Um, and that's where like hearing from people like you, like who, you know, work in this in, cuz it's always, It's just super helpful to hear like what people are seeing because it gives you so much insight into the kind of like what may be going on. So, yeah.

So the challenge of the cannabis industry is on scale, at least, you know, propagation. So, so there's two issues with propagation. One is preservation of genetics. You know, if we have something that we really love that we want to keep going at the same, you know, with the same vigor and expression over time, what is the potential risk of losing that through clonal propagation? You know, typically how it's done versus say doing, moving to something like meristematic tissue culture, which may or may not now introduce more mutations. We don't know. The assumption was always that it was gonna produce a cleaner, healthier, better plant, but it's costly. It's not accessible to all growers. And now this just brings up some questions that I think are really fascinating around that. The other issue is micropropagation. If we want to just create a whole bunch of plants really quickly, that's why it's used in so many other, in agriculture and horticulture in general. But if we're not getting that same consistent plant over time, then maybe it's not, you know, this, what we thought it was, I guess.

I guess like, I'm curious, 'cause you know, so like, you know, in a tree crop, you know, there's like this one mother tree that's been sitting, you know, people keep these trees alive for 50 years and it's, they have the same source and they can go back and start new cultures out of that material. But I guess, so like in cannabis, I mean, what is the current state? Do people, everybody have their own mother plant? Like there's no, you know, how old do they keep the mother plants alive that are like, that people can go back to, you know?

So, and typically, and there's, there's going to be some variety here, uh, but typically what, what we see people do is a mother plant will stay for anywhere from 2 to 6 months. After that, they can get pretty woody or you just end up with a plant with, with just too many shoots off of it and it just, it becomes harder and harder to clone over time and it's harder to keep healthy. So I do think there is that sort of pathogen or endophyte buildup in, in this annual plant over time that does create issues if you were to keep it going longer. But that's just, that's a hypothesis on my part, but typically 2 to 3 months. So what you'll do is you'll, you'll clone. Off of your vegetative plants and then put those plants into flower, and then you'll keep a couple clones extra, and then that will become your mother culture for the following crop. So you're continually going through successive generations of plants fairly rapidly, 'cause we'll go through, we'll do about 5 cycles a year in an average indoor facility or grow. So there's quite a bit of turnover.

Is there like reintroduction? So you've put these plants from micropropagation back out into your indoor grow facility. Then do you just keep some in micropropagation and keep propagating those, or do you go back out to that facility and take some new material?

So not a lot of people are doing micropropagation. I see. Most is, almost everyone's doing either from seed or clonal propagation. There is a little bit of tissue culture happening. A lot of it is, you know, falling under what proprietary stuff. I mean, we're still figuring out the media for it. I had Dr. Adel Zarai on, he's living in Australia now, and he was one of the first people to really develop a tissue culture media for cannabis. But we also, so the other issue with cannabis is we have massive genetic diversity. To where we are seeing so much, it's really hard to make any sort of inferences or generalizations because the plant will respond so differently. We'll have some plants with very tight internodal spacing, we'll have very long internodal spacing, we'll have plants that take much longer to reach harvest than others. So that's a challenge. And then the other challenge is the market's constantly changing. So we have these like hype strains that are currently popular, that may not be popular 6 months, a year, 2 years from now. And so the genetics are constantly being changed as people are searching for like the next great thing.

So when someone makes a new strain, you know, and they can— it's clonal, you— is it— how does that— what happens to it next? Does it get distributed to people? Patent— is there— do people get doing like variety patents in cannabis strains? Is that a common thing?

It's not a common thing. We're not seeing it. Typically people will try to hold that, build hype around it, and then a breeder will like release it all at once as a way of trying to maximize their profit on that. Again, it's all a little bit gray too in terms of how that works federally. So it's a challenge for breeders right now. I think over time that will change. But we are seeing, you know, now people are producing, you know, at university, even some, some AAA genetics for breeding. We're seeing some companies releasing much more homogeneous seed crops for hemp or even for Type 1, which is the THC variety of cannabis. So yeah, the industry is evolving.

Definitely. Mm-hmm. So people are— people doing— is it— is like hybrid seed a popular option for varieties or strains where people are making hybrids and selling those like as seed? Or is it always mostly still clonal for those kind of things?

Yeah, everything's a hybrid now. I mean, that's the whole thing. Like, we don't really have—

but the— sorry, but this— but if you purchase seed, I guess you're purchasing hybrid seed.

Yeah. Yeah.

Thinking about like, yeah, like a lot of most seed-based, uh, you know, breeding systems involve making hybrids. Um, and I guess the same is true for cannabis.

Yeah. Yeah. And people are, we're still working, working towards better genetic stability even and consistent genetic expression.

Yeah.

Uh, so there's, there's a lot of challenges there for sure.

Yeah. Well, I mean, I guess if someone was like thinking for practical thoughts or whatever. Like, I mean, I guess if you have a system of clonal propagation and you like have periods during your system where you have a small number of plants that are— that have the potential to be founders for a large population of stuff, like that would be the time to think about any kind of genetic testing or whatever. If you know, like that, that's where you have the most bang for your buck in terms of like checking your material. Because if like, let's say you have 5 plants, one of them has one of these duplications, you know, and the others don't, well, you would, you know, being able to eliminate that plant early from your system might prevent some, you know, prevent things from spreading. But again, I think, I mean, it'd be, I think it'd be great to look at this in cannabis and see what the rate is and across these different systems. Seems interesting.

Yeah, we've recently in cannabis identified specific markers for powdery mildew resistance. And the issue, like you brought up, like, yes, DNA sequencing is great. The problem is, we had a company do this privately and they said they were going to do this. And, and, um, so people sent their genetics to them and then they were able to create a database that allowed them to better analyze and create new genetics that were in direct competition to the breeders who sent in their genetics in the first place. Oh, uh, so that created a little bit of controversy. And it's kind of like the whole 23andMe thing, you know, you give people your— you give them your genome and then they, they take that, collate the data, and are able to better understand the human genome and, and, and sell that data or make— I mean, they're not making human clones, but you get the idea of where this, where this gets complicated or, um, challenging.

Um, yeah, maybe the benefit of, of working with academics, right, is that, you know, we, we aren't in it to, to make money as a company, right? Like, the whole point of the university system is to like expand knowledge and do this as like something that expands knowledge. And especially at universities like land grants, like that's literally your job, right? Yeah.

Yeah. No, definitely. Is, is UC Davis able to work with cannabis or is that not on?

I believe so. Yeah, I think we can. I mean, I think Well, it's like, I feel like it's one of those spaces that's constantly changing. You know, we were, there was a period where we were going to have like, try to develop like a cannabis genetic repository, germplasm repository, but then, you know, things, politics shift and rules change and it's kind of complicated. But I know there are people, there are definitely people at UC Davis who I know have, have worked. So I believe it's possible. So I mean, you know, I'm not sure what the there's probably like certain specifications. Like, you can work with like a leaf tissue that, you know, that we get DNA out of, but that's different from like growing from seed, you know what I mean? And so it's like, you know, like just talking to people and figuring out what people— if people want to collaborate or something, it's like, you know, what do they— what do people have? And like, we can figure something. I'm sure we can like figure something out. Or people do like DNA extractions externally, and then, you know, it's like, so we're just like analyzing data or whatever. People like find ways to, to be helpful without, uh, you know, working around things.

So, and yeah, maybe you can clarify for me, but isn't there, isn't there some changes with the scheduling happening right now that might open up new research avenues?

I think it's a little murky, and I, being on the cultivation side, I don't claim I totally understand it other than to say I know it is going to change the confidence or ability a lot of breeders have in mailing and selling seeds online. So it's, um, it, it's, as I understand it, it's going in the, the wrong direction for the cannabis industry as a whole, based on what, based on what I'm hearing. But I haven't, um, I, I can't say that I fully understand what the implications are, and I don't know that anyone does. From what I'm seeing, it's still, we're still figuring that out, and we may not know till this fall.

Okay.

Um, but I wanted to tie this back to to cannabis specifically and some of the questions— I had a few more questions I wanted to ask you guys. So if a cannabis facility were to use, um, single cell regeneration, so micropropagation, to produce like 10,000 clean clones so they don't have any pathogens, um, but that founder cell that you started from had a mutation that say knocked out like a key enzyme in the THC or terpene pathway, every single one of those 10,000 production plants would permanently lose that trait. Is that, is that right?

Basically, yeah.

So this is why mutations are so important to, uh, to understand, or I guess screen for ahead of time. Is that— how would someone avoid something like this, I guess?

I mean, sequence— DNA sequencing Yeah, but yeah, I mean, DNA, yeah, or, um, or not doing that, like, ever, you know. I guess not, you know, I don't know. Well, you said, so you said single cell. I mean, that's it. So what is that? Uh, I don't know what kind of system that is.

Okay, sorry, let— yeah, maybe that was the wrong terminology here, and again, I'm working outside of my range. So like, let's just go— so you didn't do meristematic tissue, so I guess let's just say micropropagation in general.

Yeah, yeah. You'd say, I mean, the same principles apply. Um, yeah, the, the mutation becomes, it's like all of the descendants of that thing can carry that, will, you know, in principle carry, yeah, will carry that mutation.

So, so any sort of, I, I guess this applies to any form of, of propagation really, but, um, if we know that the risk is potentially higher with micropropagation, we could in theory screen that material, that callus, correct, uh, for specific mutations prior to doing a lot of, you know, putting a lot of effort into the, the time it would take to do the full micropropagation process and replication.

Yeah, yeah, absolutely. Um, so if you— I guess if you're going to say, say put a plant through and try to generate a lot of them, maybe a good first step is take a little plantlet, right? But right before you try to regenerate a bunch of them, if you're doing, say, this propagation from meristematic tissue, regenerate a plant, get a couple of them, and then just sequence them. You can sequence those and just do a quick screen and make sure everything looks okay. And that way you avoid the problem of having say 10,000 individuals that, you know, all are missing either these key genes that were knocked out or removed in some way, or maybe even have a whole chromosome duplicated.

Yeah. I mean, you'd want to, yeah, early in that process would be ideal while you still have the chance to like know that, you know, this is going to be the founder for everything. I mean, I will say one point I'll add though for practical value, like You know, we could, you know, one could screen, we can like, our lab does this, you know, it's like we can measure for chromosome duplications and other like dramatic mutations that, but there, I will say this, there will always be mutation. There will always be some, it's maybe small, it's like a single base change because that's just like life being life and mutating naturally. So you could screen, but you always just have to be aware that you, you know, you might prevent the catastrophic chromosome duplication, but you, you, you're gonna see some number of mutations and some of them could do something. But if you have candidates, like if you know the THC biosynthesis pathway, you'd have some like obvious ones. Like these are mutations to look for, you know, it's like you, especially like if, yeah, in a system like that where you have good idea of what you should check, then it's, you have, uh, I think a little bit better information, which is possible in cannabis cuz I think the THC pathway is like pretty well described.

And okay. So we, so we don't know, like you'll say there's some mutation, the mutation level may be low, but we won't know exactly what the implications are for those mutations, how they're going to express themselves necessarily.

Yeah. It's just a caveat. Like, isn't, you know, it's kind of like, um, it's like getting a medical checkup, you know, it's like you can do a checkup and it's like, it's better than not, you know, but it's like, it's not, you can't guarantee that everything is, you know, 100%, like you, people do genetic testing, like personalized medicine, you know, and it's like you can find those mutations that are maybe, you know, known to do something, but there may be unknown unknowns, I guess is what I'm saying. But in a system, yeah, but, but what we found so far, it seems like the weird stuff, the stuff that like I would be worried about if I was in this space, like I would, it's the chromosome duplications, it's the big dramatic things that are really, we can detect those and like that's the low-hanging fruit that can be eliminated from like a, uh, like a cult system. Yeah.

And, you know, maybe, maybe it's good to just point out too, is it's often that we use mutation as sort of like this bad word, right? Like if a mutation happens, it's probably bad. But, you know, most of the time these, these mutations are, are happening across the genome in places that aren't really going to affect, uh, the way that, that genes, uh, are expressed, right? And so, you know, a lot of the times these smaller mutations, like if one, one base pair is changed, they'll happen somewhere where it's, it's not really gonna affect anything. It's also possible that, you know, these mutations lead to a, a good beneficial outcome like we talked about with apples or oranges.

Yeah. So yeah, that's a good point. Yeah.

It's just so complicated because not only do you have the genetic material, but then you have like we said, the environment that the plant is grown in. So how it expresses those genetics can be dramatically different based on stress events and, you know, how the plant is grown. And, and so it's just, it, it's hard. It's hard as a grower to really know where to start, if I'm being honest. And then I wonder like how accessible or, or affordable is genetic testing for the average grower?

It's a good question. I mean, the cost of DNA sequencing has dropped by like 1,000-fold from what it was, you know, 15, 20 years ago or something like that. But that doesn't mean it's like the same as like buying yourself a cup of coffee either. It's like, you know, to do like a whole genome, like what we— okay, I'll put it this way. Like what we did in this paper we just published in Walnut, I mean, it's like a single plant, you know, I don't know, probably like, like $500 of sequencing costs for a plant like that, something, something, you know, $500 to $1,000, something like that. And that's, but that's like, you know, we're like seeking the academic, like the highest standard, like we want to get like every mutation possible, you know, very like high confidence accuracy. But for screen, like say you only wanted to check for trisomies. You could probably devise like a sequencing strategy that would be a lot more high throughput and less cost.

That's honestly not as terrible as I thought you were going to say in terms of cost. So, um, but then it's also finding someone who has the knowledge and equipment to actually do that testing and can do it. Like I said, you know, in terms of like regulations and legalities too.

So, well, I mean, if it's fair game, you know, if people are listening and they're interested, hit us up. Um, we know we do this. This is what our lab does, you know, we're interested in the biology and we're interested in working on new material. You know, it's like we— it's, it's cool science. And so we're happy to like collaborate. I mean, that's kind of what we've realized we can do is like we can collaborate with people who have interesting material and come up with an arrangement that suits everybody's needs. And, you know, this is what— yeah, this is like what our lab does.

So yeah, if you ever want to get into the cannabis space or do some research with cannabis, I'd be happy to connect you with some some folks on that front. So cool.

Yeah.

I'm really interested in how the, the, the role of like pathology and genetics and how that creates expression and impacts genetic expression. I think that's what I find most fascinating right now is sort of how these, how microbes interfere or affect plant the expression of plant genetics. Yeah, yeah.

I mean, I guess along the same line, I mean, I suppose like it's probably possible actually to do genomic sequencing to identify not only the mutations but also the status of like whatever microbes are present because they have DNA, you know. And so we pick up the DNA like when we do the sequencing, we actually find like the DNA of the microbes that were living on the plants that we sequence. If you like you find them in the data. And so you can actually like infer like what's there. So it's like another layer of information.

That, that's the research that Dr. Zamir Punja has been doing recently. And that, that's what I think is how it relates to your research around genetic mutation, I think is really fascinating in terms of how those two worlds connect.

Yeah. I mean, there are two, two things that can occur.

Yeah, absolutely. Yeah. And it's, it's, uh, it is interesting to think about too, like how, how do these change? How do these, uh, interactions change as we change how we're, how we're propagating these things, right?

So yeah, so if there's one fundamental, I guess, assumption around plant cloning that you guys feel like you challenged with this paper, um, what do you think that is? And how do you think this— what, what do you think the implications or impact of this paper could be in terms of agriculture or cannabis or plants that are propagated? In this method?

So I think that we sort of addressed a longstanding intuition that people had about the mutagenic nature of some forms of tissue culture, which like revealed like a sort of disturbing possibility of a high rate. But at the same time, I think I also see some optimism because now we realize like we have the toolkit necessary to like understand this process better and to like check for it, for example. And so, in a world where there's a lot of uncertainty around what's this plant doing, why is it changing, I'm looking ahead and thinking, okay, now it's like the genetic DNA testing is on board, is sort of online now to provide some answers to questions that people have had for a long time.

Yeah, I think maybe the positive message is we can potentially prevent these things through through screening, right? All of these, all of these challenges that people have been identifying for decades, you know, propagating these things out so many different times and ending up with a totally diverse genetics at the end of them when they're supposed to be identical. Well, we actually have the ability to, to now stop that early so that that doesn't happen.

That— yeah, that's really interesting. And so potentially over time, we can, we can learn how these mutations are, you know, beneficial or deleterious and kind of make decisions based off that.

That, and I think that like, I think that we're going to have like a, um, a sort of guideline, like we'll have, we'll have an understanding of like, what is the best tissue culture method to prevent mutation? You know, like some practical knowledge as we do more experiments and test more material. It's like, what is the, like, you know, which type of tissue culture method is going to produce the most genetically, you know, pure, like, lineage going forward, you know. And so, I think there's a future in which, like, there's better knowledge about making decisions in these kind of culturing processes. It's like, okay, I'm going to do this instead of that because I know that makes more mutation than this, you know, that kind of, I think there's practical just knowledge to emerge from this.

So yeah, what's next for you guys then in terms of research and career pathways?

DNA, tons of DNA sequencing. We're working on a lot of systems. We have like, you know, people doing DNA extractions right now in the lab preparing for the next sequencing round of different plant species.

And yeah, yeah. So we're, you know, crop agnostic, you know, we're excited, excited about these, these mutations that are happening in all the plants that are clonally propagated. And so we've got probably over a dozen experience with over a dozen different plants now, and happy to just continue to, to do this kind of work and look at different propagation systems.

Yep. And Matt's a PhD student, he's graduating sometime in the next—

yeah, so very exciting.

We need—

yeah, we need more smart people coming out of the coming into the plant science world. So I love that. On that note, are you seeing any surprising findings in your current research that you can talk about right now or anything interesting you want to share before we call it a day?

Well, yeah, we're finding some interesting cases where there are mutations that originate in tissue culture and other crops that produce off-types that affected like the industry in a dramatic way. I'll just leave it at that.

So, and so the results you're finding on your current research is supporting your published research just as a general, very broad statement.

Yeah. It's, if anything, it's just, yeah. So it's strengthened our like understanding of it. Yeah, for sure.

It's definitely, yeah. We're, we are, I think, convinced that this is a good a good thing for people to be studying.

Yeah.

Are you going to look at meristematic tissue culture at all in your future?

It's an idea for sure. Yeah. I mean, we're interested in every type of tissue culture. I mean, we, you know, UC Davis is a great university for this type of work because it's one of the like most, you know, there's so many plant scientists here and there's all kinds of interesting material for us to work on. And so, yes, I would say I mean, yeah, I actually— yeah, I'm quite confident we are talking with other people. Yeah.

Yeah, that would be— that would be the really the deciding factor for cannabis in terms of the main ways that we're seeing it propagated.

Yeah, that's cool. Great.

Or preserved.

Yeah.

Yeah.

But like I said, if people are out there are interested in, you know, collaborating or whatever, let us know.

Oh yeah, I can definitely put you in touch with some people if people don't reach out, but I will put a link to your paper. A link to, you know, including your emails as well, if you're comfortable with that. And yeah, I thank you so much for your time today. I learned a ton, and I hope that more people can find out about your research and continue to expand on it.

Thanks a lot. Thanks for your interest in having us.

Yeah, it was great to hear about how the industry works, and we just had a good time.

That was Dr. Graham Monroe and Matthew Davis from UC Davis, and you were listening to the Cannabis Cultivation and Science Podcast. If you like the podcast, please leave us a rating and review and give us a follow on Instagram and YouTube. And don't forget, at Kiss Organics, we have a wide array of products we offer on our website, ranging from soils, amendments, and beneficial insects to soil test analysis and commercial consulting. We carefully research all the products we offer and manufacture to make sure they are science-based and meet our highest standards of quality. Thanks for listening.