| Summary: | Hemp (<i>Cannabis sativa</i> L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of <i>C. sativa</i> crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of <i>C. sativa</i> polyploids. In this study, colchicine was applied to two proprietary, inbred diploid <i>C. sativa</i> inbred lines, ‘TS1-3’ and ‘P163’, to produce the tetraploids ‘TS1-3 (4<i>x</i>)’ and ‘P163 (4<i>x</i>)’. The diploid, triploid, and tetraploid F<sub>1</sub> hybrids from ‘TS1-3’ × ‘P163’, ‘TS1-3 (4<i>x</i>)’ × ‘P163’, and ‘TS1-3 (4<i>x</i>)’ × ‘P163 (4<i>x</i>)’ were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid <i>C. sativa</i> cultivars in commercial agriculture.
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