In running a molecular biology laboratory providing (among other things) cannabis genetic fingerprinting services, there’s a situation and term which consistently reappears in variations on a theme like something out of Groundhog Day. In its basic form, the situation is that a cultivator has been maintaining a plant line for an extended time through the use of a mother plant and cuttings. After some time, the mother plant seems to be less and less vigorous, and so it’s retired and one of its cuttings is retained as the new mother plant. However, it often doesn’t seem to start off quite as healthy as the original young mother plant did, and its decline occurs faster than the last generation.
Particularly over successive rounds of replacing mothers with their own cuttings, the loss in overall health and vitality is quite noticeable. Frequently in this situation, cultivators say things like, “I must be getting genetic drift.” In fact, the term genetic drift often gets used in the cannabis space to describe any change in appearance or behavior of a clone variety over time. The reality, however, is that this is most emphatically not genetic drift – firstly because the term doesn’t mean what many people think it means, and secondly because what they mean to say isn’t what’s actually happening. What really is genetic drift, why doesn’t it apply in these situations, and what actually are the – dare we say – root causes of the problems observed?
To address this, we’ll need to start off with a very brief refresher on some basic genetics, particularly as it applies to cannabis. We’re in luck here, because now you can’t make it out of high school science without having been exposed to these concepts:
• Genes are coded for in the DNA;
• DNA is organized into contiguous linear pieces called chromosomes;
• People are diploid – that is, they have two copies of every chromosome, one from their mother and one from their father;
• Therefore, people carry two copies of each gene; one each on the maternally derived and paternally derived chromosome;
• These copies can be subtly different, sometimes with one form being dominant over another (recessive). The resulting physical characteristics (phenotype) are a result of these interactions; and
• While it’s not critical for our topic today, gender is determined by the one partial exception to this – one pair of chromosomes aren’t the same, but consist of an X and Y form. If you get an XX pair you’re female, and if you get an XY pair you’re male.
Why we’re reminding ourselves of our own genetics is because it turns out cannabis plants follow exactly all those same rules – and the reason we’re lucky is because very many plants are not nearly as simple.
We also need to build on a few more concepts here. One, the spot on a chromosome where a particular gene exists is called the “locus” for that gene. Two, we noted above that in an organism, the maternal and paternal derived gene forms (properly called alleles) at a single locus may be different – but a key concept here is that there may exist in a population (an interbreeding group of one species) many more possible alleles than just two.
In fact, it’s not uncommon for there to be tens of different known allelic variants for a single locus. If we imagine a locus and it has alleleic forms we’ll call A, B, C, D, … N, every individual in the population (be it person or cannabis plant) carries only two of these possible alleles as its two copies of the loci. Three, different alleles of a single locus occur at different frequencies in a population; that is, maybe A is 17 percent of them, B is 63 percent, C is 2 percent, and so on. Across the population as a whole, it’s simple math to then say what fraction of individuals have a particular allele combination at a locus (the mathematically inclined among you will grasp, for example, that 39.7 percent of our population will be BB at this locus, and 1.26 percent would be BC – you just multiply the individual likelihoods).
Now we’re ready to define what genetic drift actually means. It is a change in the allele frequencies at a locus in a population. If you have a single plant – or even if you have a group of genetically clonal plants such as from cuttings – that’s not a population in the genetic sense of the word, there’s only (at most) two alleles for each locus, and there’s no change over time in the relative frequency of each allele – it’s either 100 percent or 50 percent.
The words “genetic drift” can only be applied to heterogenous populations of a species over normal reproductive cycles – for any of you looking for more reading, it also only occurs when said population is not in Hardy-Weinberg equilibrium, which is a fancy way of saying some form of non-random mating or selective pressure is occurring to alter allelic frequencies over reproductive generations. If all of that lost you, the bottom line is genetic drift, by definition, doesn’t occur in a single individual organism.
So, what is it people actually mean when they use the term genetic drift? At least where this author has encountered the term, the users were trying to say, “I believe I am getting an accumulation of heritable genetic changes – mutations – in my plant, and most of them are bad, because my plant isn’t as healthy as it used to be and the cuttings are worse each time.” (To be fair, if you haven’t studied population genetics and hear the term genetic drift without appreciating it has a strict definition, it’s easy to understand how one might think it’s a succinct way to describe this idea).
As warned in our opening above though, even this correctly expounded basic idea is probably wrong. No, it’s not wrong about what’s being observed; the perceived progressive loss of vigour is probably very real. What’s wrong is the assumption that these changes are arising from accumulated changes to the genomic sequence of the plant.
Recall again from some long-ago biology course that cells – the individual building blocks of any living organism – are small. Really small. (We’re going to ignore inconvenient outliers, like turkey eggs. Cells making up a cannabis plant are uniformly each very tiny).
A cannabis plant – or even a small cutting – is therefore thousands to millions of cells. Each cell carries a full organism copy of its genomic DNA, the two sets of chromosomes with all their loci discussed above. Now, it’s absolutely true that the DNA within a single cell can get mutated over time. Background radiation is one cause of this; when an energetic subatomic particle goes blasting through a cell and just happens to hit a DNA nucleotide square on, it can cause breakage and rearrangement of chemical bonds. UV light can do this too, albeit with a different specific pattern of what DNA bonds get changed. Just plain “chemistry” can occur too, in particular interaction between water molecules and amino (-NH2) groups on DNA bases leading to “deamination” – the -NH2 changes to an oxygen. In fact, every cell, whether of a cannabis plant or the person reading this, has on average many such DNA damage events every day.
Let’s do an audience poll at this point. How many of you reading this, and I guarantee you are undergoing all of these cellular level mutational events all day every day, grew a third hand in the last 24 hours? Show of (third) hands, please!
No hands shown? Why’s that? Well, in the first place, cells have really robust DNA repair mechanisms which can actually repair the vast majority of mutations rapidly and accurately. First part of our answer: most spontaneous mutations get repaired, they don’t get turned into permanent changes in the genome.
“Most,” however, lets slip that at least some of these mutations don’t get repaired and yes, they are now permanently present in the DNA. So why then are these not probably the cause of our plants losing vigour? Well, for the same reason these aren’t making you spontaneously grow a third hand. The point is, each of these mutations occurred in a single cell, in a single locus (actually, statistically speaking, most mutations occur in non-coding DNA areas and do absolutely nothing – but let’s think just about the subset in actual genes or their regulatory elements).
Recall you have two copies of each locus; part of the biological reason for that is to have ‘backup copies’ of genes so if one gets mutated to non-functional, the other allele still functions. For most genes that’s good enough, although exceptions occur – these are referred to as “haploinsufficient genes.” So what if we have a huge number of coincidences here, and we get a cell which gets mutated in a haploinsufficient gene but doesn’t repair? Well, that cell dies. One of its adjacent healthy cells then divides to fill in the space. End of story and from the whole organism level, you never knew it happened.
Well, what if you had a similar chain of coincidences, but rather than shutting down a haploinsufficent gene, you made a new allele with some sort of not-very-helpful behavior? Not so bad that the damaged cell dies, but it’s got one pretty messed up copy of some gene, and that cell is just not as vigorous as it was. Yes, that can absolutely happen, and it’s called a somatic mutation. If we now stop and think about just how much damage that one cell can do, we find the answer is, “not much!”
Think about what happens when you take a cutting from a plant; it’s made of a very large number of cells, and it will grow into a new, fully mature plant by each one of those cells dividing into a larger group of organized cells all at or near its starting location. These progeny cells divide in turn as the new clonal cutting grows, but the underlying reality is that only a very small number of cells in the adult mature plant can trace their lineage back to the mutated somatic cell. It was one of only maybe hundreds of thousands of cells in the cutting, making up a fraction of the percentage of the total; and at maturity of the grown plant derived from that cutting, it’s still more or less the same tiny fractional percentage of cells in the whole which carry that mutation. In other words, the vast majority of the final derived plant is from non-mutated cells. If you’re seeing overall loss of vigour of the whole plant, it’s not due to 0.00001 percent of the cells.
(As an aside – note this is a somatic mutation. If an equivalent mutation was what’s called a gametic mutation, that is in sperm/pollen or egg, then that single zygote cell origin of a new organism will have this mutation pass on to 100 percent of its cells, and this is a real problem. The crux here is that cloning techniques like cuttings or tissue culture don’t go down to single cells to generate new multicellular clones, while sexual reproduction does.)
So now we’re back to where we started – “genetic drift” isn’t the right term for progressive loss of vigour in a clonally propagated plant, and it’s likely not due to actual mutations in a fraction of the cells starting a clone anyway, so what is it from?
The answer is that a number of factors can contribute to progressive decline in clonal lineage health. An important one is likely to be the slow, subvisible accumulation of bacterial, fungal, viral, or viroid diseases. The mother plant and cuttings process is particularly prone to this, where over time pathogens can infect the mothers and rather than showing gross signs of disease, just gradually parasitize the plant health. Cuttings are infected too and each round generally carries a higher pathogen load, meaning less and less healthy plants. Tissue culture processes tend to be much safer than mothers and cuttings in this regard, as use of apical meristem tissue, hyperthermic growth conditions, and aseptic techniques can work together to isolate and remove pathogens, leaving just healthy plant cells to initiate each new clone.
A second cause for progressive loss of vigour may be “senescence,” which is a term for innate biological aging; that is, plants, like animals, normally have finite life spans and various cellular processes act to limit total number of cell divisions to allow for the natural dying off of older plants to make way for the next generation. While this can be observed both with mother-and-cutting and tissue culture propagation, the capacity for tissue culture systems to take and cryopreserve thousands or tens of thousands of “youthful” cells from a desirable clone means that in this context the culture can be refreshed with isogenic young starting material on a regular basis – in effect “resetting the clock” each time.
So, the next time you think your clones are getting a bit long in the tooth and not as healthy as they used to be, remember not to blame genetic drift, or in fact probably not genetic mutations at all. Your favorite clone still has its same genome and alleles, it’s just fighting some combination of pathogens and old age.
Image by Arek Socha from Pixabay
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