Cannabis sativa L. (Cannabaceae) is a dioecious plant, producing male and female flowers on separate unisexual individuals (Sinoto, 1929; Valle et al., 1968). Although both male and female plants are capable of producing cannabinoids in equal concentrations (Valle et al., 1968), female plants produce greater floral biomass than male plants (Ohlsson et al., 1971) and thus are exclusively used in commercial marijuana production facilities. Moreover, after pollination, female plants alter their relative investment in phytochemicals by reducing the production of secondary metabolites like cannabinoids, flavonoids, and terpenoids (Pijlman et al., 2005). In the absence of pollen, stigmas on female plants continue to grow and thus produce more surface area on which cannabinoids can be produced (Small and Naraine, 2016). Because of this negative impact of pollination on cannabinoid yield, industrial growers rarely maintain male plants in production facilities, and instead propagate their stock of female plants by vegetative cloning (Flores-Sanchez and Verpoorte, 2008; Decorte, 2010). However, the "mother" plants used to produce clones eventually become non-regenerative and new mother plants are grown from seed, which necessitates pollination (Valle et al., 1968). Therefore, careful consideration must be given as to the most effective and efficient ways to collect pollen for controlled crosses while preventing pollen escape into production areas. Cannabis is anemophilous (wind-pollinated) (Small and Antle, 2003), and therefore relies on air movement for pollen transfer from male to female plants, sometimes across long distances (Small and Antle, 2003). Pollen dispersal mechanisms often reflect pollen ornamentation, as seen in C. sativa's smooth exine layer, triporate (i.e., three aperture) morphology, and low mass-features intended to maximize pollen dispersal distance and chance of successful ovule fertilization (Hesse et al., 2009). The aerodynamic morphology of C. sativa's pollen highlights the difficulty associated with controlling its movement, as any airflow following anther dehiscence can result in pollen movement, a frequent issue when studying dispersal in anemophilous species (Whitehead, 1969, 1983). It is therefore important to determine the most efficient method of capturing windborne pollen upon anthesis, in terms of both the number of pollen grains collected and the time spent collecting pollen. Procedures for controlled pollen capture are typically required in crop breeding programs to ensure precise knowledge of paternity so as to breed progeny with preferred traits (Richey, 1950; Briggs and Knowles, 1967; Allard, 1999). For example, standard methods for maize breeding were established in the early 1900s, with an abundance of literature outlining the procedure for controlled crosses (

Some economically important crop species are dioecious, producing pollen and ovules on distinct, unisexual, individuals. On-the-spot diagnosis of sex is important to breeders and farmers for crop improvement and maximizing yield, yet diagnostic tools at the seedling stage are understudied and lack a scientific basis. Understanding sexual dimorphism in juvenile plants may provide key ecological, evolutionary and economic insights into dioecious plant species in addition to improving the process of crop cultivation. To address this gap in the literature, we asked: can we reliably differentiate males, females, and co-sexual individuals based on seedling morphology in Cannabis sativa, and do the traits used to distinguish sex at this stage vary between genotypes? To answer these questions, we collected data on phenotypic traits of 112 C. sativa plants (50 female, 52 male, 10 co-sexuals) from two hemp cultivars (CFX-1, CFX-2) during the second week of vegetative growth and used ANOVAs to compare morphology among sexes. We found males grew significantly longer hypocotyls than females by week 2, but this difference depended on the cultivar investigated. Preliminary evidence suggests that co-sexual plants may be distinguished from male and female plants using short hypocotyl length and seedling height, although this relationship requires more study since sample sizes of co-sexual plants were small. In one of the cultivars, two-week old male plants tend to produce longer hypocotyls than other plants, which may help to identify these plants prior to anthesis. We call for increased research effort on co-sexual plants, given their heavy economic cost in industrial contexts and rare mention in the literature. Our preliminary data suggests that short hypocotyl length may be an indicator of co-sexuality. These results are the first steps towards developing diagnostic tools for predicting sex using vegetative morphology in dioecious species and understanding how sexual dimorphism influences phenotype preceding sexual maturity.

Pollen grains are male gametophytes, an ephemeral haploid generation of plants, that commonly engage in competition for a limited supply of ovules. Since variation in reproductive capabilities among male gametophytes may influence the direction and pace of evolution in populations, we must be able to quantify the relative fitness of gametophytes from different sires. To explore this, we estimated the relative fitness of groups of male gametophytes in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing the non-abortion rate (measured via chemical staining) and viability (measured via in vitro germination) of pollen from multiple sires. Pollen viability quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen non-abortion rates declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both viability and non-abortion rate analysis displayed no significant correlation, implying that researchers cannot predict pollen viability from non-abortion rates, nor infer male gametophytic fitness from a single measure. Our work demonstrates two independent, differential approaches to measure proxies of male fitness in C. sativa.

Pollen grains are male gametophytes, an ephemeral haploid generation of plants, commonly engaging in competition for a limited supply of ovules. Since differential male fertility may influence the direction and pace of population evolution, the relative fitness of pollen is regularly estimated as either pollen viability, the proportion of pollen containing intact cytoplasm’s and regenerative nuclei, or pollen fertility, the frequency of pollen germinating under standardized conditions. Here, we estimated the relative fitness of pollen in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing pollen fertility and viability from multiple sires. Pollen fertility quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen viability declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both fertility and viability analysis displayed no significant correlation, implying researchers cannot predict pollen fertility from pollen viability, nor infer male gametophytic fitness from a single measure. Our work demonstrates two approaches to measure proxies of male fitness in C. sativa, and identifies new questions around what are valuable estimates of male fitness in plants.