Optimized Chemical Masculinization and SCAR Marker‐Assisted Screening for Feminized Seed Production in Hemp ( Cannabis sativa L.)

Hemp ( Cannabis sativa L.) is a dioecious plant of increasing economic importance due to its high‐value cannabidiol (CBD) production. Commercial CBD production requires a uniform stand of female plants, with male plants being removed to prevent pollination. It is a labor‐intensive task and reduces yield potential per unit area due to decreased plant population (up to 50%). Feminized seed production and early sex determination are thus essential for establishing female‐dominant crops. This study optimized chemical induction of male flowers on genetically female plants and validated early sex identification of F₁ progeny using sequence‐characterized amplified region (SCAR) markers. Female plants were treated with silver thiosulfate (STS), silver nitrate (AgNO₃) and cobalt chloride (CoCl 2 ) in graded concentrations. STS (250–750 ppm) and AgNO₃ (250–500 ppm) treatments induced functional male flowers, whereas CoCl 2 produced malformed, sterile buds. STS masculinization yielded the earliest flowering onset (113 days), prolonged male floral duration (up to 66 days) and greater pollen germination (48%–52%) relative to AgNO₃, although induced pollen exhibited lower viability than that of genetically male plants. Pollen from chemically induced male flowers successfully fertilized female plants, producing selfed seeds. Heat map cluster analysis revealed synchronized floral performance and male flower induction efficiency to chemical treatments, showing strong positive correlations among masculinization traits. Molecular screening of F₁ seedlings with male‐specific SCAR markers (MADC2 and MADC5) confirmed 100% feminization under STS treatments (500–750 ppm) and 88% female progeny with AgNO₃ (250 ppm). Our findings demonstrate a scalable strategy integrating chemical masculinization with marker‐assisted screening to produce feminized seed, offering labour and cost efficiencies while improving CBD crop uniformity.