Cannabis (Cannabis sativa L.) is a multi-purpose plant in the Cannabaceae family that has garnered significant scientific attention due to its complex chemical profile and diverse industrial [1], medicinal [2], horticultural [3], and recreational [4] applications. The plant is composed of various chemotypes with distinct phytochemical compositions, primarily driven by the presence and ratios of cannabinoids, terpenoids, and flavonoids [5]. Despite the growing interest in cannabis cultivation and its expanding legal status in various regions, the scientific understanding of cannabis genetics and breeding techniques lags behind that of many other crops [6].

The recent wave of cannabis legalization in various regions has spurred considerable scientific interest in optimizing cultivation techniques, developing new cultivars, and exploring potential medical applications [7]. However, plant breeding technologies in cannabis are still in their nascent stages, with limited information and research accessible to producers in the field [8]. This knowledge gap presents a significant challenge for producers seeking to develop new cultivars with specific traits, such as a higher cannabinoid content, disease resistance, or tailored aroma profiles [6]. A promising approach for cannabis breeding involves the process of polyploidization, in which the number of copies of each chromosome is increased [9,10,11]. In some studies, artificially induced polyploids, and in particular triploids, have been reported to have an increased yield and cannabinoid content [12]. However, other reports indicate that polyploidy has variable impacts on plant growth and development, likely due to the genetic variation in the starting material, and much more work is needed [13].

While cannabis is typically a diploid species, instances of presumably natural triploid (3×) [14] and tetraploid (4×) [15] plants have been observed in some studies. Further, some elite clonal cultivars used in commercial production have been identified as triploids (i.e., Mac1), with no indication that they were artificially produced (unpublished data). Despite their rarity, naturally occurring polyploid cannabis plants offer valuable insights into the plant’s genetic diversity and adaptation to varying environmental conditions [14]. Understanding the genetic consequences of natural polyploidy in cannabis is of significant interest to both researchers and breeders [9]. These polyploid individuals may exhibit distinct traits, such as altered morphology or growth patterns, which could have implications for their ecological niche or potential utility in cultivation [16]. Furthermore, investigating the stability and reproductive viability of naturally occurring polyploid cannabis plants is essential for comprehending the long-term evolutionary dynamics of this species [14]. Research in this area can provide valuable insights into the adaptive potential of cannabis populations and inform breeding efforts aimed at improving various aspects of cannabis cultivation, including the yield, cannabinoid content, and environmental resilience [17].

Somatic doubling or the generation of unreduced gametes are the main mechanisms known to result in natural polyploidy [18] (Figure 1). Somatic doubling is linked to mitotic processes (e.g., endoreduplication or endomitosis), which can take place within apical meristematic tissues or zygote cells, ultimately leading to the development of entirely polyploid organisms or mixoploid individuals [19]. However, it is widely acknowledged that somatic doubling is of relatively minor significance in the natural formation of polyploids, despite this being the primary mechanism used for the induction of artificial polyploids [20].

The predominant mechanism leading to polyploidy in plants is understood to involve the production and subsequent fusion of unreduced gametes [20]. In this instance, the unreduced gamete is diploid, containing two copies of each gene, instead of the standard haploid state with only one copy. The ability to generate unreduced reproductive cells is a hereditary trait observed in numerous plant species [19]. In addition to genetic regulation, environmental factors (e.g., nutrient deficiencies, water scarcity, injury, herbivory, and temperature) can exert an influence on unreduced gamete formation [20]. Once created, the unreduced gamete possesses the capacity to merge with another unreduced gamete to produce a tetraploid, a process known as bilateral polyploidization, or with a gamete that has undergone reduction to produce a triploid, termed unilateral polyploidization (Figure 1A) [18].

From a cytological perspective, depending on the meiotic stage at which the restitution event occurs, two distinct processes can give rise to the production of unreduced reproductive cells (i.e., the first division restitution (FDR) and the second division restitution (SDR)) (Figure 1) [18]. The FDR encompasses errors that occur during meiosis I. These errors can stem from the failure of homologous chromosomes to segregate correctly during anaphase I and/or the lack of chromosome pairing during the zygotene/pachytene stage (Figure 1B) [21]. Within the SDR process, chromosome pairing and division proceed typically during meiosis I, but an anomaly arises in anaphase II where sister chromatids fail to segregate as expected [22] (Figure 1B). These irregularities facilitate the restoration of the somatic chromosome number within the gametes, leading to the formation of dyads or triads [18]. The genetic outcomes related to the maintenance of heterozygosity vary between each meiotic restitution mechanism [18]. Unreduced gametes produced via the FDR typically contain two non-sister chromatids and may maintain a level of heterozygosity similar to that of their parent cells [21]. In contrast, unreduced gametes resulting from SDR contain two sister chromatids, leading to a lower heterozygosity level compared to their parent cells [22]. The disparity in heterozygosity levels plays a pivotal role in influencing the potential success or failure of a recently formed polyploid, whether within wild populations or in the context of plant breeding [18].

The focus of this study was to determine the extent of natural polyploidy in cannabis. To achieve this, natural diploid crosses were made to generate a series of populations that were screened to determine if they contained any polyploid individuals and estimate the natural rate of occurrence. Naturally occurring triploids, but no tetraploids, were identified at a low rate in most populations. These results clearly demonstrate that polyploidy is a naturally occurring phenomenon in cannabis, likely as a result of unreduced gametes. These results have significant implications for future breeding efforts and help to explain the presence of polyploids within landrace populations and commercial production.