High-THC Cannabis, commonly referred to as marijuana, denotes any Cannabis sativa L. cultivar containing more than 0.3% delta-9 tetrahydrocannabinol (Δ9-THC), the compound responsible for its psychoactive effects as defined by the 2014 and 2018 US Farm Bills (House of Representatives, C, 2018). In contrast, cultivars with up to 0.3% Δ9-THC are classified as industrial hemp, distinguishing drug-type (marijuana) from fiber-type (hemp) in accordance with the same regulations. Despite this distinction, both hemp and marijuana belong to the Cannabaceae family and share characteristics such as being annual, short-day, and dioecious plants (Junior et al., 2023). Dioecious refers to plants in which male and female gametes are produced by separate individual plants (Chandra et al., 2017; Monthony et al., 2021). While hemp enjoys complete legalization in the United States and serves various purposes, including food, feed, fiber, and non-psychoactive cannabinoid production, marijuana is legalized for medicinal and recreational use in select states (Chandra et al., 2017; Monthony et al., 2021; NCSL, 2023; Junior et al., 2023). Overall, Cannabis sales are projected to reach volume of $50.7 billion by 2028, this growth is driven by the trend of lifting Cannabis prohibition (Dorbian, 2023).

The therapeutic benefits of Cannabis are attributed to its diverse array of phytochemicals, including cannabinoids, terpenoids, non-terpenoids and others with various chemical functionalities (Oswald et al., 2021; Oswald et al., 2023). These compounds have shown promise in treating conditions such as epilepsy, breast cancer, asthma, and Alzheimer’s disease, as well as possessing antifungal, analgesic, antibacterial, and anti-inflammatory properties (Gertsch et al., 2008; McAllister et al., 2012; Vuolo et al., 2015; Gulluni et al., 2018; Cox-Georgian et al., 2019; Koo et al., 2020; Robaina-Cabrera et al., 2021).

Phytochemicals are primarily concentrated in trichomes, extracellular structures found on female flowers, making female plants more suitable for phytochemical production (Lubell and Brand, 2018; Sommano, 2020). Female Cannabis plants can be propagated sexually via seeds or asexually through methods like soft-wood cuttings and tissue culture (Chandra et al., 2017; Monthony et al., 2021). While sexual propagation is more economical for large-scale commercial production, regular seeds pose limitations as they produce both male and female plants (Lubell and Brand, 2018; DiMatteo et al., 2020; Yoshimatsu et al., 2010). Failure to remove male plants before pollen release can lead to cross-pollination and diminished phytochemical yields (Small, 2015).

To maximize THC content and exclude male plants, commercial Cannabis facilities often utilize feminized seeds (Small, 2015; Lubell and Brand, 2018; Kurtz et al., 2020, 2020b). However, challenges persist, as commercially certified feminized seeds may not always yield satisfactory results. For instance, a grower in Montana state encountered substantial economic losses when approximately 30% of the plants obtained from certified feminized seeds turned out to be male (Olejar and Park, 2022). This unintended occurrence led to cross-pollination and subsequent seed production, resulting in decreased THC levels causing economic losses (Olejar and Park, 2022).

This review aims to address these challenges and presents crucial seed feminization guidelines based on seven principles such as (I) the right stock (mother) plant material, (II) the right ethylene inhibitor to enhance sex reversal, (III) the right dosage of ethylene inhibitor, (IV) the right placement of the compound within the plant, (V) the right application frequency of the compound, (VI) the right photoperiod to induce flowering, and (VII) the right management of progeny plants, aiming to enhance feminized seed production in high-THC cultivars to meet commercial standards for both medical and recreational purposes.

Feminized seeds produce exclusively female plants with all-female flowers, achieved by applying an ethylene inhibitor to female plants (Lubell and Brand, 2018). This induces sex reversal, prompting the development of male flowers on the female plants (Kurtz et al., 2020b; Flajšman et al., 2021). Ethylene, a naturally occurring ripening phytohormone, typically promotes the formation of female flowers (Lubell and Brand, 2018). However, when female Cannabis plants are treated with ethylene inhibitors such as aminoethoxyvinylglycine (AVG), cobalt nitrate (CBN), colloidal silver, silver thiosulfate, 1-Methylcyclopropene (1-MCP), they undergo sex reversal, becoming monoecious or bisexual (Lubell and Brand, 2018; Flajšman et al., 2021; Fitzgerald, 2021). In this state, they bear both male and female flowers on the same plant, facilitating self-fertilization and seed production (Lubell and Brand, 2018; Flajšman et al., 2021). The resulting seeds exclusively carry the XX female chromosome inherited from the original self-crossed female parent, ensuring that all plants derived from these seeds are exclusively female, capable of producing all-female flowers plant (Lubell and Brand, 2018; DiMatteo et al., 2020; Kurtz et al., 2020b; Flajšman et al., 2021).

Plant growth hormones such as ethylene, auxins, and cytokinin promote the development of female flowers in Cannabis, while gibberellins stimulate the formation of male flowers; controlling these phytohormones can partially reverse plant sex (Galoch, 1978; Yamasaki et al., 2005; Lubell and Brand, 2018). For instance, Flajšman et al. (2021) investigated the impact of foliar application of silver thiosulfate and colloidal silver on sex reversal and masculinization of female Cannabis plants, resulting in the induction of male flowers, self-pollination of the remaining female flowers, and production of feminized seeds. Similarly, Lubell and Brand (2018) observed that silver thiosulfate foliar application induced male flowers on treated female plants under short photoperiod conditions. Additionally, apical application of aminoethoxyvinylglycine (AVG) promoted the development of male flowers in lateral branches of dioecious female plants (Mohan-Ram and Sett, 1982).

Sex reversal in Cannabis plants is not exclusive to female plants; male plants can also undergo sex reversal by inducing female flowers for seed production (Moon et al., 2020). However, the offspring will exhibit male plants due to the inheritance of the parental heterogametic Y chromosome (Moon et al., 2020). For instance, male plants treated with ethephon foliar spray developed female flowers (Moon et al., 2020).

Manipulating phytohormones to induce plant sex reversal does not alter the underlying genetic framework governing sex determination in Cannabis plants. In Cannabis, sex determination is primarily regulated by genetic factors rather than phytohormonal influence (Yamasaki et al., 2005; Lubell and Brand, 2018). Two main mechanisms dictate sex determination in Cannabis: the active Y-chromosome pathway and the X-to-autosome (X: A) balance system (Vyskot and Hobza, 2004; Yamasaki et al., 2005). In the active Y-chromosome pathway, specific genes carried by the Y chromosome suppress female sex expression, leading to stamen development and anther maturation (Yamasaki et al., 2005). Conversely, the X: A balance system regulates sex determination based on the ratio of X chromosomes to autosomes (Vyskot and Hobza, 2004; Yamasaki et al., 2005). A ratio equal to or higher than one promotes female sex expression, while a lower ratio (up to 0.5) favors male sex determination (Yamasaki et al., 2005). Additionally, intersex and hermaphroditism occur when the X:A ratio falls between 0.5 and 1.0 (Negrutiu et al., 2001; Vyskot and Hobza, 2004; Yamasaki et al., 2005). Furthermore, advancements in genomic approaches have facilitated the identification or prediction of individuals’ sex expression in Cannabis plants. Notably, the Cannabis Sex Purity-1 (CSP-1) assay has emerged as a promising tool for accurately predicting plant sex, particularly in its dioecious populations (Galoch, 1978; Laverty et al., 2019; Toth et al., 2020);. Its efficacy extends across various Cannabis varieties, including CBD (Cannabidiol) types, grain types, and grain/fiber types (Toth et al., 2020; Laverty et al., 2019). However, uncertainties persist regarding its accuracy, as evidenced by reported recombinants in previous assays (Divashuk et al., 2014; Törjék et al., 2002).

Recent insights from Cannabis whole-genome sequencing have highlighted potential genetic markers, such as MADC6, which may contribute to the success of the CSP-1 assay (Laverty et al., 2019). The discovery of unassembled scaffolds in the male genome bearing high similarity to the MADC6 sequence underscores the potential of genomic approaches to enhance sex determination accuracy in Cannabis plants (Sakamoto et al., 2000; Vyskot and Hobza, 2004). Moreover, this approach emphasizes the intricate nature of sex determination in Cannabis, involving genetic factors like the active Y-chromosome pathway and the X-to-autosome balance system (Sakamoto et al., 2000; Vyskot and Hobza, 2004). While molecular assays like the CSP-1 sex assay provide valuable insights, a comprehensive understanding of the genetic mechanisms underlying sex determination is essential.

Literature research indicates that Silver Thiosulfate (STS) is the most effective compound for inducing sex reversal and feminizing seeds in high-THC Cannabis cultivars. Specifically, applying a single dose of 3 mM STS during the vegetative stage by foliar spraying until runoff, followed by exposure to a short photoperiod of up to 12 hours, effectively induces flowering and seed production.

Progeny plants should be evaluated for germination rates and compared to the original parent plant for growth traits, such as plant height, biomass, and Delta-9-tetrahydrocannabinol (THC) yield, to assess the negative effects of inbreeding. Following these guidelines will help growers and breeders enhance the quality and viability of feminized seeds, thereby meeting the high standards and demands of the commercial market for high-THC Cannabis cultivars.