5.3. Hormonal and environmental factors affection sex determination

Beyond genetic consideration, the sex expression in Cannabis can be shaped by environmental factors. It is well established that silver can be used to induce the formation of male flowers on female plants (Mohan Ram and Sett, 1982). This treatment is used by breeders to self female dioecious plants, which results in offspring with exclusively XX sex chromosomes (Clarke and Merlin, 2016). Because all of the progeny from such a selfed plant will be female, the ‘feminized’ seeds produced that way are usually much more valuable than conventionally produced seeds (Small, 2015). Silver is a known ethylene inhibitor, and there is also evidence that ethylene induces the development of female flowers on male plants (McDaniel and Binder, 2012; Ram and Jaiswal, 1970). Together, this therefore suggests that ethylene is involved in controlling the sex expression inCannabis .
In addition to ethylene, also other phytohormones have been shown to be capable of altering the sex expression in Cannabis(Ainsworth, 2000) . Auxin, for example, can lead to the development of female flowers on male plants. Indeed, male flower development can be entirely repressed by auxin treatment (Heslop-Harrison, 1956). Cytokinin also has a feminizing effect (Chailakhyan and Timiriazev, 1979), whereas gibberellic acid triggers the formation of male flowers on female plants (Ram and Jaiswal, 1972). Together, the hormonal effects are very similar to what is observed for sex expression in the well analysed Cucurbitaceae family (Li et al., 2019; Pawełkowicz et al., 2019; Schilling et al., 2020a). Cucurbitaceae are only distantly related to Cannabaceae, the two lineages separated more than 100 million years ago (Figure 3) (Magallón et al., 2015). Also, many of the studied Cucurbitaceae are monoecious and thus do not possess sex chromosomes (Boualem et al., 2015). Nevertheless, we hypothesize that similar developmental genetic pathways may have been co-opted to control sex expression in Cannabis and the Cucurbits. Melon, cucumber, pumpkin, and their relatives might thus serve as an excellent model to unravel the molecular intricacies of sex expression and sex determination in Cannabis .
Cannabis is a short-day plant, and flowering is initiated if day length is below 14 h (see below), but it is well established that sex expression is also affected by day length. It was reported some 100 years ago already that dioecious plants grown under short day conditions only (i.e. without an initial period of long day growth) can develop male as well as female flowers ((Tournois 1911, 1912, as cited by (Heslop-Harrison, 1957), (Schaffner, 1923)). A lot of other environmental factors like nitrogen availability or carbon monoxide also seem to have an influence on sex expression (Freeman et al., 1980; Heslop-Harrison, 1957; Small, 2015). Together, this yields a very complex picture of different environmental factors influencing sex expression in different directions that is far from being completely understood.
In summary, the picture emerges that, although sex determination is genetic, hormonal and environmental influences have a significant effect on sex expression. Because the flowers of female plants are the main source of phytocannabinoids, a more detailed study of the sex determination and sex expression mechanism of Cannabis is one of the main areas of future research. For example, creating male sterility would be very beneficial, as phytocannabinoid production is highest in unpollinated female plants. Studying both sex chromosomes, their gene content as well as the molecular intricacies of the sex determination mechanisms will certainly provide valuable insights for breeders as well as researchers.

6. Timing is everything - the complex network of floral initiation and hints for Cannabis