Episode 150: Plants Under Pressure: How Stress Shapes Secondary Metabolites (w/ Dr. José Leme)

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 and drawn top experts from around the industry to share their wisdom and knowledge. My guest this week is Dr. Jose Lemay. Dr. Lema graduated with his bachelor's degree in agronomic engineering from Universidad Estadual de Londrina. For his master's degree, he studied precision agriculture at Auburn University, followed by his PhD at Virginia Tech with a focus on plant sciences. He started at Southern Illinois University in fall 2021, bringing his expertise in cannabis science and indoor cultivation systems. He has a joint appointment in the School of Forestry and Horticulture and the School of Biological Sciences. He is the principal investigator in the cannabis Biology and Cultivation systems labs focusing on areas of controlled environment, agriculture, plant secondary metabolites, nutraceutical pharmaceutical biology, phenomics, plant physiology, biotic abiotic stress, crop steering and growing practices. His team aims to determine how to leverage environmental controls, plant nutrition, biostimulants and hormones to improve plant production. Dr. LeMay will be speaking at the Cannabis Cultivation and Science Conference this September 5th through 7th and tickets are still available. You can learn more about this event on the KISS Organics website. This is going to be the educational and networking event of the year and it includes a facility tour of collective elevation, two days of amazing speakers, and a third day with an included tasting event sponsored by the Montana Living Soil Alliance. I'm looking forward to seeing people there. It's coming up fast. Now on to the show. All right, Dr. Lemay, welcome to the show.

Thank you. I'm happy to be here finally.

Yeah, it's great. I'm glad we got to connect. Can you tell me a little bit more about yourself and about your programs?

So, yeah, I'm. I'm assistant professor of Cannabis Biology and Controlling Environmental Agriculture here at Southern Illinois University. And I have a. I split appointment in a school of Forestry and Horticulture and a school of Biological science. So I have 51 in horticulture, 49 in plant biology. So our lab, you know, is. It's very diverse and I would say in terms of not only nationalities and genders, but also scientific mindset. So. But basically we're working with, you know, indoor cultivation of plants and testing different, you know, abiotic biotic stressors, environmental controls to, to improve cannabis quality and, and production because I think, you know, differently than other plants cultivated indoors. You know, for example, like food crops like Tomatoes, cucumbers and basil that, you know, the end goal is pretty much biomass or fresh fruit. When you're producing cannabis, you know, it's a fine line, you know, or, or a balance between production of biomass and secondary metabolites. So when you're producing biomass, you're prioritizing optimal conditions. But when you're producing plant secondary metabolite stress or a stimulus, that is kind of controversial, some people don't really like to talk about, to use the term stress, especially in crop steering, because they see more as a, as a stimulus, you know, it's not actually detrimental for the plant. So then when you're producing cannabis, you know, it's unique because you're producing biomass, but it's also secondary metabolites. So. And that's pretty much your, one of your main goals in our, in our lab here at siu.

Yes, you're at Southern Illinois University. You, you brought up a really good point. Point I think in perspective, I haven't really thought about. But when we're growing a traditional crop, we're growing it for the, for the fruit, we're looking to eat it. But when we're growing cannabis, yes, we want biomass, but the only reason that we want that biomass is to increase the concentration of secondary metabolites. So essentially to get, you know, higher concentrations of THC and cannabinoids and terpenes associated with cannabis flower or, or inflorescence, as it's called in the research community. So. And you talk about stress. I did a really interesting podcast on plant stress, and I think it's a really fascinating topic because as you know, and as you stated, secondary metabolite production. So the production of, you know, THC and various cannabinoids is highly linked to plant defense as a plant defense mechanism, but also plant stress. And there's a variety of different things that are. Can fall under plant stress. Do you want to kind of talk a little bit about that?

Yes, yes. And the thing is, even the term stress, not more recently, I'm, I'm realized that it can be controversial for me, was not really controversial. But yeah, I'm seeing, especially in the cannabis scene, I'm seeing a lot of people that don't like to use the term stress, especially because when you are trying to use the, you know, a crop steering approach, you're seeing it as a, it's just a stimulus because it's not, you're not actually stressing. It's just, it's just a trigger to increase the production of plant secondary metabolites. And then, and then it's not really being detrimental for the plant. For the studies, for the publications that we might talk later in this podcast, for most of them I decided to use the term stress because we actually used extreme water death stress. So it was not just a stimulus. And then again it's hard to know when it's just a stimulus and not a stress, you know, so. But yes, you know, as, as you mentioned, like when you're producing cannabis, you know, it's a balance between the biomass production and, and the production of plant secondary metabolites. So that's, and again that's why it's important to have that trigger and not just promote the optimal conditions for the plant because of course, you know, naturally the plant will produce plants a condabolite. But if the plant is not under stress and it can be, you know, water deficit stress, temperature stress, humidity stress, nutrient deficiency, light intensity, different light spectrum, light recipes, so and even mechanical stress that we might talk as well, it can, you know, trigger the production of crystal high, you know, higher production of secondary metabolite. Like a good example that I like to use is like, is like adrenaline. Like secondary metabolite is know as the term is saying is not primary. So the plant can survive without the supplant secondary metabolite. And, and it's like adrenaline. If you're not, if your body is not under stress or like a, some like a weird condition, you're not producing adrenaline. And it's the same thing for plant. If the plant is, if you're not creating a stimulus or a trigger, the plant is not going to increase the production of secondary metabolites. But again that's, but it's hard to find the right spot of you know, stimulus trigger or you know, or even stress.

Yeah. So essentially stress comes in a lot of different ways or stimulus either either term here and you mentioned a bunch of different, you know, abiotic stress stress or triggers like you know, environment humidity, temperature, light, you know, spectrum watering, you know, moisture, water access is a huge one. I'd add herbivory, you know, pest, pest pressure and pest stress. You know, all of those things. And I guess for me, one thing that I've kind of said for years on this podcast, and maybe that will change as research improves, is that just through the production of a plant in a, in a facility or in your home grow, you probably have multiple stress events occurring or multiple stimulus happening that are going to trigger second secondary metabolite production. And a lot of the intentional stress stuff that we See, or even unintentional, let's say we forget to water or our lights are too high, a lot of those things will have a dramatic impact on biomass production or our overall yield. So if we, if we have too much spider mite pressure or our light intensity is too high relative to our environment or I don't know, fertility, if we, if we don't give the plant the right amount of fertility, we're going to see, we're going to see increased secondary metabolite production but we're going to see a dramatic decrease in yield potentially too. So like you said, it's really about getting that optimal balance there. And I don't know that we've. One thing I've said on here, and maybe this is no longer true, is that I don't know that we figured out especially with the genetic diversity in cannabis, where those optimal trigger points or stress points are. We could say hey, week three, we want to do a water restriction or we want to you know, raise CO2 or, or all of these different things to really optimize it from a stress response, secondary metabolite production perspective. I don't know, what do you think about that?

100%. 100%. Because, because also it, it, it's so many moving parts because it's not only about one stress. Because like as we mentioned like light intensity, okay, you can know if you increase increasing light intensity but you're not increasing CO2, you create an imbalance. So it, so you're creating a stress. So and if you increase CO2 and light intensity also another factor that can be affecting is the spectrum. So talking about water deficit, stress or, or stimulus or you know, so the dryback that you know we talk in, in, in crop steering, like what is the magnitude of that stress is like you're pretty much drying that entire rock roll cube or that the entire pot or you, you know, it's, you're letting you know the pot to you know, with a little bit of water or for, for how many days is gonna, you know, last that stress. What is the best, you know, grow stage is early flower, late flower, mid flower. And, and again as you also mentioned, the genetics is that genetic, very responsive to the, that specific stress. So it's so many moving parts and then it's, it's hard. That's why I can see different results out there in terms of what is good, what is bad is beneficial, if it's not beneficial, if it's changing anything or if it's just losing biomass and it's not gaining any, you know, increasing, you know, cannabinoid concentration. So Yeah, I agree 100%. It's, it's so many moving parts. And to find, you know, a specific answer, it's, it's not easy.

Yeah. So what I'm getting is stress or these stimulus triggers will cause an increase in secondary metabolite production, but they will potentially reduce yields. We don't have it all figured out and it's very complicated and probably tied more specifically right now to the genetics that you're, you're running. It's not necessarily going to be consistent across the board. So we can't make broad sweeping claims about all cannabis plants and hopefully we'll just continue to see more research on it. Now, you recently published a paper in June of this year on the effect, I'll just read the title. The Effect of Water Deficit Stress on Cannabis Sativa Development and Production of Secondary Metabolites Review. And I think this paper is really interesting. I don't want to give away all of it because you're going to be presenting at our upcoming cannabis Cultivation and science conference specifically about this paper. So I want to keep some of it, I want to keep some of it, some of the surprises for that. But can you give us a little overview of kind of what you were looking to achieve and a little bit about what your findings were maybe?

Yes. Yeah. Actually this paper is really cool. This project was conducted by one of my master's degree students, that Shiksha Sharma. She just graduated now she's at the University of Tennessee. And that review is actually her literature review that she's pretty much summarizing is the introduction of the two other chapters. It's the first chapter of a three chapter master thesis. So she's just, you know, introducing, you know, the concept and it's pretty cool because the first time she did the experiment, we stressed the plants for only five, five to seven days. And then when we ran the analysis, it came back all not significant, not statistically significant at all.

So you didn't want, you didn't allow give the plants access to water for up to seven days and you didn't see any difference?

No, the plants were coming back like as nothing was happening. And no statistically significant result in terms of biomass and cannabinoid or in terpene secondary metabolites in general. And, and then I talked to her and, and she was like, well, I mean, I want to publish something, you know, like with these results, it's not much I can publish because it's nothing significant. So then we decide to create or promote a very extreme water death stress. So I was like okay, let's. Because she was also worried about killing the plants, you know. So and then, and this is actually a two part study. So the first study we are trying to find the best frequency of water depth stress during the flowering stage. So the first study we test control no water stress like regular irrigation, 1 period of water stress, 2 period of water deficit stress and 3 period of water deficit stress. So then after this study finding the best frequency, we want to find the best timing in the growing stage because again I'm not going to give much away but during. And again that's going to be my presentation at the conference throughout this study. But before getting the cannabinoid analysis we have the biomass, we had the trichome density counting main inflorescence weight throughout that. When we got all that results we kind of realized that one period of water depth stress was the optimal because we are not losing total and bucket biomass. And the trichom density was increasing. I was like okay, this is promising biomass not a statistically significant different when compared to the control. And we are increasing statistically significant higher trichome density. At that point I was like okay, this is promise we can start the timing study. But then you see that later everything changed. And we'll talk about that in the, in the conference. But yeah, so then the second study we had the studies pretty much different timings, you know, like no water deficit stress, early early flowering, middle flowering and then late flowering. Also actually is, we have actually five treatment control, early flower, mid flower, late flower without rewatering and late flower without rewatting. So we are kind of giving some time to the plant to recover from that stress. So and that's the, so that's the second study that we conducted. We are also working on this data.

Now just for listeners so they can better understand the framework of your methodology. What media were you growing in and what were you using for nutrition?

So yeah, across the board we use a regular standard fertilizer, liquid fertilizer, we use peat and perlite as a media. We try to be, you know, to use an inert. We are not using living soil because we know when you're dealing with living soil it's really complicated.

You.

Yes. And also we, we are not work using rock roll because it's so quick, quick. Any, you know, it change so quickly. You know, it's, it's all, of course it's awesome for crop steering but when you're conducting a Study with many students that are, you know, helping. You know, sometimes it's too quick, you know. So peat perlite is a good media for us. It's still inert. And so, yeah, we use a standard fertilizer regime for veg and flowering across the board for all the pots. And so then pretty much the only difference where the water depth is stressed, which is also consequently nutrient deficit stress. Because when you're not adding, when you're not fertigating the plant, we are also not adding, you know, nutrients. You're not feeding the plant.

Yeah, they don't have access to nutrients if there's no water. And as that water is drying out, you're getting an increase in electrical conductivity in EC in that media as well, which also can be a stressor. Good point.

So the other thing, ph didn't change that much throughout the study.

And you're using a mineral salt or chemical fertilizer as well for this?

Yes, yes. Imbalance like ph balance fertilizer. Yes.

Yeah. No, I would add that most, most research that we're going to see on any crop, but with cannabis as well is going to be a similar either using rockwool or an inert media like Pete Perlite and mineral salts. Because of all the complications and variability in living soils, it's just much harder for a scientist to draw any sort of statistically significant conclusion because you can't re. You can't remove all that variability. So as someone who grows in living soil, I have to take this research and then start figuring out how to apply it to, you know, what we're doing and what I'm doing in the organic living soil world. So. Yeah, I just, I just wanted to share that. But let's also, let's also touch on the fact that. Because the thing I was thinking about with your media being peat perlite, that's going to hold on to the physical properties of it, it's going to hold on to water better, I would assume than say rockwool, but maybe not as good as something that, like living soil, where you have more biological activity. That's also. These microbes are going to hold water in the. Within their cellular bodies. So how often were you watering? I guess in your Pete Perlite mix. Approximately. And I realize that's going to vary during the flower cycle. Before you did these drawback, these drybacks.

So. Well, for the, for the control, we are keeping the water, we had like a volumetric sensor.

Yeah.

So we are keeping the soil, the volumetric level in the pot between 30 to 40%. So when we're reaching that range, 30 to 40%, we're adding water again for the controls as a regular fertigation regime. And then for the treatments for the plants going under the water, that's stress. Then, you know, as soon as we reach that, you know, flowering stage that we want or the frequency that you, you have in the protocol, we are just stop irrigating, you know, for. So then after the first study that we test for five to seven days, the second study, we actually stress the plant for 10 to 12 days. There's no water. Wow. Zero. Yeah. And the plant survived. The plan survived. And again, something that I told my, my student was like, okay, if all the results are not good or if they don't know what we are doing is not that valuable. At least we are learning that if you're a home grower and you want to travel for like two weeks, let's say, and you have nobody to take care of your plants, you can just go, come back after, you know, 12 days, rewater the plant and the plants will survive, at least survive. You know.

Yeah, I mean, I think that's going to be somewhat dependent on, you know, evapotranspiration rates like your, your vpd, you know, if you're running really high temperature, high, higher humidity, I could, I would assume that you're going to dry out even or see plant stress sooner than a plant in a cooler, less humid environment. But yeah, I think that's really interesting and I think we do need failed experiments because that teaches us sometimes as much or more as successful ones.

Yes. Yeah, I mean, VPD was not a parameter. So we kept the VPD and the other growing parameters equal for all and optimal for all, including control. But yeah, if you're not really regulating your VPD and transpiration is pretty high. Yes. Maybe seven days will be detrimental and maybe if you come back after a week trip, your, your plant will be die, you know, but if you're controlling the vpd, which is possible to do it, you know, online or you don't need to be there to control the vpd, it's, you know, again, it's, you know, we might be able to come back and your plant be, you know, alive.

Yeah. When I think about when I screw up in, have VPD swings or forget to water and the, and the soil gets too dry, I just assume I'm increasing secondary metabolite production. Um, so I try and, try and spin it positively, but yeah, it is potentially hurting the yield. Um, yes. Cool. Well, I'm excited to hear about those results. I don't want to spend too much more time on it because you have a bunch of other research coming out of your lab that I think is really interesting. But I'm looking forward to seeing you at the conference and hearing about that.

Yes, yes, I'm excited too.

All right, so this next paper that you sent me contained a word I'd never heard of before. But once I read the paper, I could understand what you were saying. The word is thigmomorphogenesis. Do you want to talk about what thigmomorphogenesis is? What it means?

Yeah. This is also a pretty cool project that is a collaboration with a company from North Carolina, Glenn Holland. He's a great supporter. And so basically, thigmomorphogenesis, the first time that this term appeared in the literature were in 1973, something like that. And the description of tigomorphogenesis is the effect of mechanical stress in plants. You know, it can be like a wind, it can be a bug that is flying around, you know, the, the plants, any type of mechanical stress or even like a damage. Mechanical damage. So that under this description of sigma morphogenesis, we decided to create a study where we are testing vibration, like mechanical vibration and super cropping or, or as, you know, as a mechanical stress.

High stress training, I think.

Yeah, yeah. Some people know as a high stress training, some people know as a super cropping. So again, both are mechanical stressors. And in high stress training is actually a, it's a, it's a common practice in commercial facilities.

Yes.

But there's not much studies proving that if it's beneficial, if it's not beneficial. So then we decide to, you know, to, to test this. Both mechanical stress. Another interesting thing about mechanical stress is. And I, I mean, people talk a lot about sound. You know, people plant how sound can affect, you know, plants and. But again, sound, you know, the sound waves is still a mechanical stress or mechanical stimulus influence. Yeah, but it's minor. It's really minor. So it's really hard when you have students that want to, you know, publish something and, you know, and, and then start testing, you know, sounds. So then when you're vibrating the plant, actually vibrating the plant, this, this is something more effective, I would say. And so, and in this study, again, it's, it's not published yet. We're still working on it. We just got the, the, the cannabinoid analysis and we are working in the, in the, the statistics. But yeah, Four treatments. We had four treatments control only vibration. And the vibration was promoted by a grid that, you know, with a transducer that promoted like 200 hertz and 15 decibels, 15 minutes per hour during the entire cycle of the plant. So the other treatment was super cropping or high stress training, and the fourth treatment with the combination of both vibration and super cropping. So so far the results we have, we clearly could see some morphological differences. And of course, when you're super cropping or high stress training, you know, you're breaking that apical dominance. So the, you know, it's really changing the morphology of the plant. The plant is growing more like a, like a reverse umbrella instead of like Christmas tree. But you know, in terms of secondary metabolites, we don't have the results yet, but we are, you know, looking for, to see that, you know, to see if this stressors, mechanical stressors are changing, you know, the production of secondary metabolites.

Yeah. So just to back up when we talk about mechanical stimulation, just for folks that are listening, there's a variety of different things that would qualify for that. Like you mentioned wind. So in an indoor facility, having a fan that's lightly moving the branches back and forth, we know that that will increase the, it will stimulate some root production to help hold the plant in place as well as increase the thickness of the stalk, I believe, just so the plant will not fall over. Essentially, if you have a plant that gets no stimulation whatsoever in that form, a lot of times you have weaker stalks and it can sometimes fall over. This is just something I know from coming out of the nursery in landscape industry growing up, when we plant trees, other forms of mechanical stimulation where would also include pruning. When we prune a plant, we're wounding it and that stimulates it. If an insect, like a spider mite, punctures that leaf, that's called herbivory. That's also a mechanical stimulation.

Let's see, low stress training.

Low stress training. Yes.

Another one.

LST is another one. So all of these things fall under this. They go morphogenesis. Such a mouthful just for folks who are thinking about it. And we know that like these things will increase secondary metabolite production for the most part. Like there's research to show that with, I believe, other crops and other plants, and there might even be some with cannabis. I'm not sure what surprised me though is that no one has looked at super cropping or high stress training on the university side because it's such an accepted practice that knowing that we want an even canopy in indoor facilities so that we can get better yields is fairly well accepted at this point. That uniformity of canopy leads to much higher yield production. I guess I would say anecdotally, if there's no research around it, but it's fairly accepted practice. What did you find from your study? I would think that at the very least, high high stress training by itself would beat the control. Um, did the vibration by itself or in conjunction with high stress training really make a difference?

So in terms of biomass, we did not find any statistically significant results. You know, all the really biomass, again, we found numerically differences, differences, but the statistics didn't catch it. So. But again, numerically is numerically. You know, sometimes it can affect, you know, the financial impact, you know.

Yeah.

Like if, you know, the stats might say, no, all these treatments are equal. But you know, if you have a numerical difference, you know, you can, you know, the finances can be impacted. What we found, I mean, I have to, I don't remember exactly, but we also found a difference in a person, again, numerically person difference in terms of biomass stem ratio among the treatments. Okay. And. And if I'm not mistaken, I think.

I have it in front of me actually. Yeah.

I think the combination of all. The combination of high stress training and vibration had the highest biomass stem ratio. Right. I think 3 or 4%.

Yeah. So you list the control at 71.89% biomass, whereas the high stress training and vibration came in at 74.54% biomass. So what, what you're saying is essentially. Yeah, that's about a. Let's just call it a 3% increase, which in a commercial facility, 3% could be the difference between profitability and failure. I mean, 3% of a crop can be a dramatic amount of income. But for your experiment, you run a series of mathematical statistical equations and those may show you that that difference is not considered statistically significant by the scientific community based on the standards that everyone uses.

Yes. Yeah. 95% confidence.

Yeah.

Yes. Sometimes you use, you know, 90%, but yeah, the scientific community accept 95% confidence.

Yeah. I would want to, I would want to dive, like really dig into this experiment and find out, like how full that canopy was across the board. Because I would assume that the high stress training in conjunction with an optimal veg cycle to fill that canopy and give you a more even canopy would show an increase over a control where the plant's growing with, you know, strong apical dominance.

No. Yes. And also, you know, as we conduct that in, in a Controlling environment, growth units, you know, that can affect as well, you know, if you're doing that like experiment in a, you know, indoor facility with more plants because then we have more space to open the canopy and actually have instead having light competition, we will have more light exposure, you know, and so, but that's are some of the limitations that we have, you know, in a, you know, in our research or in our research settings. But, but another thing that was actually very interesting is that it's well known in the literature that mechanical vibration can decrease inflorescence elongation. There are many studies showing that and we found that because the treatment under vibration, only vibration presented the heaviest cola, the heaviest main inflorescence. So in our assumption is that as vibration, mechanical vibration is decreasing the main inflorescence, you're creating a more compact, you know, more compact cola or heavier cola because you're not actually elongating the cola. So in that same size, you know, cola, it's, it's a heavier one, you know, you know, it's increasing the weight. So that was actually an interesting result too.

So, so denser buds with vibration essentially is what you're saying. Yes, yes, that's interesting. And I think, I think you bring up a good point that we need to take these studies and really rather than just saying, you know, drawing any strong conclusions, like I can't look at this and now say, okay, high stress training doesn't work because you know, Dr. Lemme did this trial and the biomass results, the yields were the same. I think we have to dive into them as someone who's reading a study and say, okay, well maybe we need to look at like how many A quality buds versus B quality buds did we get between these two and experiment with different genetics and veg times. And so what I think these studies show us though is like, okay, this is, this is really interesting to me. Now I want to do a trial incorporating some type of vibration to see if I see in my environment an increase in yields or an increase in COLA or bud density. That would be really interesting. So I think studies like this really spark innovation and give us things to trial as growers in our environment or in our facility.

And. But also that's the price we pay when you're conducting research in cannabis. Like we are being a pioneer, like we are just, you know, it's the first studies, you know, and it's funny that that specific study that you mentioned about the, the water deficit stress, when I post about it, something about it, somebody comment, oh, Kaplan. And I know we cited that paper. Kaplan from Guelph University of Guelph already did this study and was like, so what? Like, for other plants, people are redoing so many studies. Just change tweaking the protocol a little bit. So why we cannot do that in cannabis? It doesn't mean that if you conduct one study and it's done deal, you know.

Yeah, that's a good point. We need multiple studies and we need multiple replications of studies to really form any sort of, like, scientific consensus about this plant. Because especially in this crop, compared to other traditional crops, we have way more genetic diversity. Like, it's not even close. You know, in corn we have just a few varieties that most people, A few cultivars that most people are growing. We'll talk terminology here in a second. Whereas in cannabis we have, I don't even know, thousands of cultivars probably. I mean, the genetic diversity is massive. So I would expect different results with different cultivars just based on that genetic diversity. But maybe over time we'll learn, yeah, maybe over time we'll learn that, you know, OG Kush grows best using stimulation and high stress training, whereas, I don't know, cookies grows best using very little mechanical, you know, mechanical stimulation whatsoever. It really, it really, I think, could be cultivar specific down the road.

Not only cultivar specific, but also the, the rest of the growing environment, light spectrum, fertilization, recipe, water, you know, that schedule, you know, how much water you're adding. So there are so many other moving parts that also can affect, you know, the fire, you know, the final result. So, and for these other plants, we have, you know, hundred years, you know, people are studying and changing and learning more and, you know, and changing their mind and redirecting the research. But for cannabis, it's just the beginning. So we don't have much background information. So again, that's why, you know, that's why my students are struggling to, for, you know, to write their, their literature review. Because it's like, hey, we don't have much papers, you know, the paper. And now we have recently getting better. But it's still, it's not like if you're writing a literature review for, for soybean, you know.

Yeah, so there's too many papers. Yeah.

Yes, exactly, exactly.

So real quick, before we move on, it sounds like your potential conclusions or findings with this study is that while the yields did not change across all the different treatments or variables on this, you did see an increase potentially in secondary metabolite and terpene production. So essentially higher Levels of cannabinoids and terpenes from this thing, amorphogenesis, this mechanical stimulus.

Yeah, I guess our assumption or hypothesis like as far as we know as biomass was not affected statistically not significant. If we have a higher cannabinoid concentration or higher terpene concentration, plant secondary metabolites in general, it will be equal to higher productivity because you know, same biomass, you're not actually losing biomass due to the stress. So because you know, the other result could be, okay, these stressors are actually decreasing biomass. So if I, if my cannabinoid increase a little bit is going to be equal to the plant under optimal condition or non stress condition because you know, the biomass was higher. And then when you're getting, you know, the total biomass, total cannabinoid per plant, it end up being equal, you know, so that's why it's the balance biomass and 10 secondary metabolites.

So our goal with any of these stress or stimuli is to increase trichome density without dramatically lowering biomass production or yield. Because if we have more trichomes being produced, then in, in theory we'll have higher levels of cannabinoids in the same amount of biomass. That in the sense what we really are going for here are cannabinoids. When we grow cannabis, that'd really be considered a win. I suppose so.

Well, okay, so what is your opinion about trichome density? Do you think higher trichome density is directly correlated to higher cannabinoid concentration?

Well, and correct me if I'm wrong, my understanding is if I have higher trichome density, I may not have higher levels of THC necessarily, but I'd be harvesting something that would have more overall THC and cannabinoids in something with lower trichome density. Is that, is that a, is that a wrong assumption?

Well, I mean the, the thing is that's the general assumption. And the thing is people actually even get mad when mainly legacy growers, they get mad when you do not agree with that. But unfortunately, or trichome density counting is not being correlated, directly correlated to cannabinoid concentration. And this is something that already I've been in a panel with Dr. Allison justice, with Dr. Nireet, and we are kind of finding the same results. And this is something that I addressed in my very last publication or I think we need to improve our trichome density counting methods. Because the thing is, let's say when you're doing the trichome density count, you cannot count all the trichomes. You know, in the flower. So you take a representative part of the plant and extrapolate that to the rest of the plant. So then with that, the. The first issue is where. What inflorescence structure you're gonna be using as a representative part. Sure.

What part of the flower?

Yeah. What part of the flower? The bract. The entire flower. Just a random pot in the inflorescence. The sugar leaf, the fan leaf. So we have mip. People are using different plant structure. And then. So that's the first issue. And then again, the fan leaf can have higher trichome density than the bract, or the bract can have a higher trichome density than the fan leaf. And then when you're correlated, that density with the cannabinoid concentration is not being directly correlated. Another issue is when you're counting trichomes, we're counting basically numbers. You're not counting the size of the trichome head, how tall is the trichome. So when you're counting numbers, let's say five big trichome heads could be equal to ten small trichome heads, you know, but what the trichome density is showing that 10 trichomes is higher than 5 trichomes, you know?

Yeah.

That's why that correlation. We are not really finding that correlation. And so that's why we need to improve our methodology to be more representative or somehow, you know, correlate to the cannabinoid levels.

Would you agree, though, that the same genetics, same cultivar with higher trials, trichome density, should, in theory, have more cannabinoids and more THC than what? Than the exact same plant with lower trichome density? Or does that not even hold true?

I would not. I would not make that statement based on what I'm seeing.

We're still. We're still learning that. Wow, that's really interesting. So trichome density may not really matter is essentially what you're saying. We don't know.

No, basic. I think it's a very important metric. I think it's a very important parameter, but basically for aesthetics. Because the thing is, one thing, you cannot lie. If you have a higher trichome density, you have a frostier butt. That's for sure.

Yeah, but that's what we look for as growers and as consumers. People see that and they, you know, people want those genetics just because of the trichome density. You know, how frosty it looks, as you said.

Yeah, that. That's why. That's why it's still a very important parameter, because it's A quality parameter. But to correlate it with that, with cannabinoid, again, you also need to consider the size of the trichome heads. You know, it's. It's also what part of the plant you're using as a representative sample. We use here in our lab, we use the bract because so far we're seeing that the bract is the most homogeneous structure. So, and again, we cannot be all day counting trichomes, you know, you know, counting the entire, you know, all the trichomes. So another thing that I'm suggesting that paper is to use more than one part of the plant, the bract, the fan leaf, the sugar leaf, and then you can kind of create an average of all.

We do the same thing in the microscope world, you know, it's called mpn, and essentially it's an equation. So we'll have a slide that's broken up into this looks like graph paper, let's say. And then you count essentially, you know, a certain number of the little squares. And then you take that and extrapolate how much bacteria you have based on a math equation, and it gives you a reasonably good idea of where you're at, you know, in terms of microbial biomass, because you, like, you can't count all the bacteria. And the same thing here, like you're describing with trichomes. So that's. That's really interesting. Yeah, so I guess that's a good assumption.

Yeah. And if you're using a 1 millimeter square, and then in 1 millimeter square, we can count from like 5 to 30 trichomes, so it really rang. It's a big range, you know, so, you know, and again, totally different size in terms of globular trichomes head. So, yeah, again, you know, and again, going back to the genetics, we always go back to the genetic. Some genetics might produce more trichomes in the fan leaves or in the sugar leaves than in the brackets. Some genetics will be create more trichomes in the brackets. So you cannot just generalize for all the genetics, you know.

Yeah. And I think just to further complicate things, I don't know that trichome density is a good tool for evaluating the cannabis experience or the user experience in terms from either a medical or a recreational perspective. I think we have to look much closer at cannabinoids and terpenes and what people call the entourage effect in really evaluating cannabis quality. But unfortunately, right now, at least in the commercial market, it's really tied to trichome density, visual aesthetic appeal. Like you said, and it's tied to THC levels in most what I would call immature markets, recreational or medical markets. They just want the highest level of THC because they think that's best. And if you talk to anyone who's been in the industry or is a much heavier consumer, they will tell you that that has very little impact. Like, it's not always about chasing the highest THC level. It's about finding that Entourage effect that really gives you the best consumer experience.

Yes, yes. You know, you definitely see more and more the, you know, the, the threshold is the THC level. But, but, you know, people are seeing more and more that that's not what matters, you know. Yeah, the entire, you know, all the, the three pins and all the, you know, the Entourage effect, that what matters in terms of experience. So. And another thing that is well known talking about trichomes is how tall is the trichome? Because as the trichome grows, it creates more cells to produce energy for this little biofactory that is in the trichome head to produce secondary metabolites. So again, and when you're counting trichomes, how tall is the trichome is also not being taken into consideration. So that's another factor. That's why you're seeing this kind of. The correlation is not being directly correlated to cannabinoid concentration.

Yeah. Now, you didn't send me any research on this, but I feel like users or listeners will be upset with me if I don't ask you, in terms of tricho maturity, do you have any information or research? Because, you know, people say, you know, you want it to be, you know, cloudy versus amber, you know, and you know, versus clear. Like, there's a lot of different opinions on that. Most people I know want the majority of the, of the trichome heads to be cloudy with, with some amber in there. Have you seen any research on trichomaturity in reference to secondary metabolite production or optimization?

I mean, surprisingly, there is only one study that I found that is. Exactly. They're actually studying cloudy amber and crystal trichome. I just, so far, I mean, so far I just found one study and it's actually kind of old study. I think it was done in the 90s. And that study was literally taking a needle and measuring the cannabinoid concentration in a very cloudy amber and crystal trichomes. And in that study it was found that the cloudy trichomes, it was higher cannabinoid concentration. So. And there are other studies not being that specific, but they're showing that when the, you know, the trichome is becoming amber, you know, you're degrading, so you're losing cannabinoid. So that's why I would say in my opinion so far, you know, it's the, the cloudy trichome is the best harvested harvest time, you know, because that's another issue, you know. Okay, when should we harvest? Should you just do like a, like an automatic or regular schedule? Okay, after eight weeks, nine weeks, ten weeks, we are harvest. Doesn't matter. Or should we check the trichome collar? Should we check other parameters? So that's another thing that we need research, you know.

Yeah, I do know that. I've heard. I guess I shouldn't say I know anything here because this is not an area that I have as much interest as I do in soil science. But I have heard that the, the, the secondary metabolites, the compounds inside the trichome head, while you said, you mentioned degrade, they also change chemical, the chemical constituents of them, so you will get a different effect. So one could say that while the, the trichome is technically degrading as it turns amber, I'm going to get different chemical compounds in there that are being produced that will give me an effect that I may prefer over purely milky or clear trichome heads.

Yeah. Like. Okay, is it, is, is THC being degraded to cbn? But okay, that's what I want. I want something to help me to sleep. That's, you know, that's my goal. So. Okay, I will delay my harvest. Again, I'm not saying, I'm not stating that. I'm just saying that if that it could be something like that that you're talking about.

Yeah. That'd be a, an example that.

Yes.

No one, no one should take as the truth. It was just a hypothetical example. Yes.

Like, yeah, potential. Yeah. A potential, you know, circumstance.

Yeah.

And as you mentioned, like the publication that I mentioned about the trichome density counting is actually, it was just published. I forgot to send to you. It's called Bracks, Buds and Biases.

Oh, I have it. I do have it.

Oh, you got it.

And I will, I will put links to these in the, on the podcast page for folks who want to go and check them out themselves. Yeah. Because these are publicly available, which is really, really nice. I love open access stuff. Well, let's change directions. I'd be remiss not to ask you about your work now with jasmonic acid or methyl jasminate. This is something that I heard about, oh, gosh, like 15 years ago, there was a product called Jazz Spray that was a jasmonic acid product for roses that people were spraying on roses. And there was a lot of folks that wanted to experiment with it with cannabis because it is, it's a naturally occurring chemical that we find in a variety of different plants. I think jasmine being one of the higher level plants that has higher levels of it, which is why it's called jasmotic acid. Correct me on any of this if I'm wrong, but it can't. It would have. It works sort of like a, it a plant hormone essentially. And so it impacts the growth of the plant and the way it, I guess, allocates its resources. What you did multiple studies with jazz with jasmonic acid. What are sort of your findings and how does it impact cannabis?

Okay, good. I mean, that's, I mean, I'm fascinated about that too, because basically when you're applying like exogenous jasmonic acid in plants, you're pretty much fooling the plant. You're like, as we're talking about stress, you know, when the plant is naturally under stress, the plant will produce, you know, this volatile compound. No jasmonic acid or jasminate or methyl jasminate. And then. But when we exogenously apply. You're fooling the plant. You're like, you're telling the plant that the plant is under stress without being under stress.

So, so, so when you. Just to clarify for listeners, when you say like in exogenous, that means it's, it's a external application of this plant hormone, whereas endogenous is one that's produced inside the plant itself by the plant. So essentially the plant thinks it's producing it, but you're adding it from outside.

Artificially.

Yes, artificial. I just want to clarify for listeners who may not be familiar with all this terminology because it's, it's confusing.

Yeah, exactly. No, because, because I'm pretty sure, like, for example, that study that I just, we just talked about, the, the water deficit stress. If we measure jasminate, you know, release, the plants will be. The plants under stress will be releasing much more jasminate, you know, than the, the control. Yeah. So the plant naturally is producing that. So our idea or hypothesis was like, okay, why not instead promoting a controlled stress or stimuli, instead doing that, we can just pretend or fool the plant as they are under stress without being under stress. Okay, we, so that was the idea and we thought that it was very promising and really cool to test. But again, as everything Else in cannabis, I mean we have, you know, jasmonic acid and jasminates are being tested in other plants for, for a long time. But for cannabis is very new. So for other plants we have, you know, more, much more background information in terms of when to apply the rates and in everything else. And for cannabis we're just looking, okay, how much should we apply? What is the optimal rate? So then we read some, you know, we had some studies, you know, addressing other plants and they're like, okay, maybe this range sounds, sounds okay. So okay, let's try that and let's create a protocol of when we apply. So the results came back not as promising as we thought. Again, I have to read those papers again, it's just so many other papers. But it increase trichome density. But the cannabinoid was not statistically the difference. You know, it did not increase in a statistically significant level. So but again I think the answer, or you know, general answer is like we need to refine the best rate because again when you're talking about rate, we're talking about how much you stress the plant, you know, and also timing. So I would assume that for these specific studies that we address methyl jasminate, perhaps it was not the optimal application rate.

So just looking at this first one that you did with ammonia bicarbonate, the methyl jasminate treatment led to a significant, significant 32% increase in trichome density. However, it did not significantly alter CBD or THC concentrations. So this, this supports your earlier statement saying that the increasing trichome density does not increase overall THC levels or secondary metabolites. But this would say you had a frostier looking flower that may have had greater bag appeal. Correct. Is that safe to say?

Yeah, I mean it could be a stimulus, you just to force the plant to create more X structures or more protective X structures. But maybe the what is inside the globular head was not as concentrated. You know, so the numbers, the higher number was not equal to higher cannabinoid concentration.

Yeah, I had a conversation with some folks in the cannabis department over at Cornell and essentially they talked about this idea of a dilution effect. A plant can only produce so many cannabinoids genetically. It has just this genetic potential. Once we maximize that potential, now we're diluting it. If you picture, let's just say a three foot plant and then a six foot plant that are both the same genetic cultivar, they're the exact, they're clones of each other, that six foot plant and that three Foot plant may have the exact same total cannabinoid level because that six foot plant may have just spread out the cannabinoids within the trichomes over a greater surface area, just essentially diluting it versus the three foot plant that might be more concentrated but have the same levels. Does that sound like a reasonable explanation? How would you explain that?

100 and I actually, I use that a lot in my papers because I'm actually seeing many papers actually from Dr. Nirit Bernie Stein that she used this dilution effect and the dilution effect is pretty much what he said. But I also would add that even biomass, you know, if you're producing biomass or more biomass, you are, this dilution effect can, can occur because you know you're increasing biomass and maybe that the, the trichomes are being more sparse, you know, farther apart. Another thing that can also affect dilution effect is water deficit stress. Because you know, when the plant is under stress it starts shrinking. You know, all the surface area of the plant starts shrinking and then the trichomes might be closer, you know, apart and this will increase the trichome density and then maybe a plant, a healthier plant or plant that is not going under water depth stress, the surface will be like a normal surface and this dilution effect won't happen. So again I'm seeing a lot that, and I'm using a lot of, you know, of this term to explain some of our results.

Yeah, that's, that's really interesting And I want to clarify something else too. Like the example that I gave was sort of a linear example in the idea that these two plants are producing the exact same. They're both maximizing their genetic potential, potential for cannabinoid production. And I think in reality that's rarely the case. So I think in the, in the average facility or the average cans cannabis plant out in the wild, increasing yield will also increase cannabinoid production overall. Cannabinoid biomass. It's just when you get to ex, you know, further and further along there is a point where it's going to maximize. But I think we rarely get there in most facilities, just in real world practice.

And this also happens with other plants too. Like the genetic potential of the plant, like, like for corn or soybean, you can be growing the plant, you know, even outdoors and is the optimal condition. Perfect rainfall, perfect soil texture, perfect fertility and the plant is not going to go up that know, with genetic potential that's the max and that's it, you know.

Yeah, that's a good Point. So I'm looking at this other study that you have. The role of jasminates in modulating growth, trichome density and cannabinoid accumulation in cannabis sativa. It's a little different than the other study. Did you want to talk any about that or do you want me to share a little bit from the abstract?

I mean if you.

Since I know you don't have it in front of you. I'm kind of cheating here.

Yeah.

So in the abstract you say that jasminates have emerged as a prominent elicitor for enhancing trichome development. Cannaboid, Cannabinoid production in cannabis sativa evidence suggests that moderate methyl jasminate concentrations can effectively boost trichome density and enhance key biosynthetic enzyme activity. I'm paraphrasing here. Ultimately increasing THC and CBD content. However, excessively high levels produce the risk of excessive vegetative stunting. So that's really to timing. Timing and interaction with hormones like gibberellin, salicylic acid and ethylene further shapes the plant's stress yield, stress responses and secondary metabolism. Do you want to explain that?

Yeah, let's go back. You know, that goes back to the. The right. The optimal rate. So we found that and that is, you know, it can be a, you know, a wide range. But we found in. In other plants is also new other plants that 50 to 100 micromoles are somehow working for some genetics. So in some other plants. But again we goes back to the genetic, you know, specific results. So. But one thing that is very clear and very well known in the literature is when you apply too much method jasminate or jasminate, you create this stunning effect. So you will really decrease biomass. So that's why some people even use that to actually make more compact plants, produce more compact plants. So. And again, that also goes back to what we talked in the very beginning of this conversation. Biomass and plant secondary metabolite production. It's a balance, you know, not. Not lose too much biomass and not. But also create a trigger that can increase secondary metabolites.

Is jasmonic acid totally safe for Cuban consumption? And are there organic sources of this for people who are organic growers that wanted to play around with it?

Well, I don't think there is any issue with the application of jasmonic acid or method jasminate.

Okay. I haven't looked into it too much myself, so I was just curious. I know with some of these plant growth hormones and regulators there, there can be, but yeah, okay. And maybe that's not something you've received.

Hormone, you know, method jasminates is an organic compound, you know, and I guess if it's organic label, you know, if the product that you're saying is organic label, I, I, I would assume that is, they won't have any issues. And of course it changed from one state to another because it's changing, you know, across the board. But it's, it's, and again, and that's why you want, that's, that's why we decide to, you test it, because it could be alternative for producers to, to use it as a potential yield increase.

Okay, interesting. Yeah. I haven't spent a lot of time looking into it, so just curious. Okay, well, last topic. I know we've been on here for a little while, but one of the things that you brought up in email that I thought we should talk about was terminology. What is the proper terminology for the plants that we see, you know, in cannabis? Because traditionally the term strain has been really popular for describing different cannabis. What I would say are cultivars. We know that, we know that we have different definitions for all of these things. Do you want to talk a little bit about that or do you want me to give, actually, let me give a quick altar.

Yeah, yeah, I want you to say, I want to, I want to learn. What is your opinion about that?

So when we talk about, you know, proper terminology here or classification, you know, we have species, variety, cultivar, and strain as all potential names here. Like humans were species. When we talk about varieties, for example, varieties are naturally occurring within a species. So for me, variety doesn't really apply to cannabis because it's not, it's not naturally occurring. Cannabis is obviously a naturally occurring, but, but we have cultivated. So a cultivar being a cultivated variety, that's something that's created and maintained through human intervention, I think really fits for the majority of what we see in the cannabis industry nowadays. So I've been using the term cultivar for a really long time and, and think that is, and then strain, you know, like I said, we've used it for years. I don't really think it's accurate. It more applies to viruses and a genetic variant within or subtype within a cultivar or within a species. So it is complicated. But I don't know what, what's the academic world have to say about it?

Well, so I agree with you. I mean, my, from, for all the papers that we are writing using the term cultivar because, you know, cultivar, you know, it's it means that it's being bred. You know, it's a, you know, the human. It's interacting and changing the traits. And by the. And you have a. There is like an end goal. There are some specific traits that you're looking for when you buy a specific cultivar. But some people say that to be. And again, doing this meeting with some Cornell folks, again, I thought that I was saying something that was.

Already agreed, commonly accepted.

Yeah, like, yeah, calmly accept. And like, this is not going to be controversial. And they're like, well, Jose, what are you talking about? Then I was like, well, I mean, it's not going to be a variety because variety is something that is natural coin when it's not being bred, you know, and there's not really any human action to create a specific trait and strain. I was like, well, strain, you know, you can. And then some of them, like, no, you can actually use the term strain for plants. There are some strain of rice. And then I was like, whoa. And, and then during that meeting, I was actually inviting someone for our cannabis symposium here that is going to be after the. Your conference in late September. And there was like, I think this is a perfect topic to bring up because I know especially here at Southern Illinois University, there are some people very vocal about the use of cultivar. But when they started discussing and not really agree with me, I was like, well, I guess there are some different mindsets out there. And, and again, I, I was trying to defend my, my opinion about using the, the term cultivar.

Yeah.

But, you know, it's. It was interesting to discuss with them that, you know, some people don't really agree with that and they have different opinions about it. So.

Yeah, I think that's really interesting. At the, at the end of the day, I mean, does it matter? Probably not, but I think that it would be good to see some consistency across scientific literature. At the very least, like, would be good for the academic community to agree on it. Whether or not the average grower or cannabis user calls it a strain or cultivar probably does not matter. But I think ultimately, I think like a really good taxonomist outside the cannabis industry would be the best person to ask about that and get some sort of consensus around it. But yeah, so we can narrow it down to two.

They're defending their idea about not using the term cultivar because they said that in. In order to use the term cultivar, it need to be registered in the. In the International Code of Nomenclature for Cultivated Plants, the ICN cp I see.

Okay.

You Know, and then they're like, oh, if it's not in this, you know, the code is not registered in the. In this organization or institution is not a cultivar. But. But I was like, well, but the thing in my opinion is more than that is the goal, like, you're breathing something with some specific traits that, you know, you want the consumer to have. So, you know it's not. Because if you're saying that is not a cultivar is so then it's nothing. So that means that wherever you're buying, there is not really consistency at all. Of course, we know that there is not much consistency. I. We know that, you know, if you test, you know, the, the blue dream from here, Illinois and from Colorado, the, the profile, the cannabinoid profile will change. But at least you know, when you're buying blue drink, you. You. That's what you buy. At least you're trying to buy. So then I guess to minimize that conversation and to kind of find like a middle ground, I was like, how about talk. Use the term line instead? Cultivar or variety or strength. How about use, like, line, you know, line with these specific traits. And then you cite the name of that specific line because then you're not claiming anything. You're just saying that it's a line. So they like, oh, it might be a better term because then you're not claiming too much.

So, so essentially, the International Cultivation Registry Authority is responsible for the registration of all cultivars. So they're saying if something's not registered there, then it's not truly a cultivar. I. I guess so. I. I don't know. I don't know. I would, I would agree with you on this one. Strains not typically used to describe plants, as I'm reading here. Also, it says it does sometimes show up in breeding, but mostly as it relates to genetic modification. So if genes of the wheat plant are altered, the offspring of that modified plant might be deemed a strain. And that makes sense to me. But this level of just intentional breeding or selection by humans on a plant traditionally in the horticulture world is called a cultivar. So I agree. We can rule out species, we could rule out variety. Those definitely don't fit. But if you want to keep calling it a strain, there are some folks out there in the academic world that sounds like would agree with you if you want to keep calling it a cultivar, join our club. That would work too. And hopefully, hopefully someday we'll have some.

Consensus or just a line that, you know, you're gonna be avoiding, you know, any confrontation. There you go.

Yeah.

Because the thing is for the controversial, like cannabis being cannabis activa, being a species or subspecies or, you know, the Ruderalis and the indica, all that controversial thing, I would say that now we all agree that cannabis sativa is a one species and we are not even debating more. But this cultivar, variety and strain, I guess is something that is still being debated, you know.

Yeah.

So.

Yeah. Yeah. Awesome. Well, I, I enjoyed the conversation today. I can't wait to see you at the conference. I'm really excited for all the wonderful people that are going to be attending and yeah, looking forward to it. Thanks for your time today.

Yes, thank you. Thank you. I appreciate it. It was awesome to talk to you and you know, I'm looking forward to meet you in Montana.

That was Dr. Jose Lemay and you are listening to the Cannabis Cultivation and science podcast. Dr. Forget to check out the podcast page at www.kisorganics.com just click on the Learn tab, then podcast. And just a friendly reminder that we do have a Patreon channel with a free 7 day trial and tiers as low as $5 a month. We do book club, special cultivation tips and tricks and monthly hangouts to connect and share your grow and even give out seed packs and other gifts at certain tier levels. Your support at Patreon or sourcing your products through Kiss Organics is what supports the podcast and I really appreciate it. We also have the upcoming Cannabis Cultivation and Science Conference in Bozeman, Montana this year in September. More information available on our website. Thanks for listening.