Cannabis sativa (hemp, marijuana) is a dioecious species with homogametic (XX) pistillate female plants and heterogametic (XY) staminate male plants (Moliterni et al., 2004). The species is cultivated for cannabinoids, most notably CBD and tetrahydrocannabinol (THC), fiber, and grain, from which a wide range of consumer products are derived (Small, 2015). Cannabinoids have reported medicinal value and are produced in the glandular trichomes of the plant, which are found in the greatest density on the inflorescences of female plants (Small and Cronquist, 1976). Hemp is distinguished from marijuana by the content of THC produced by the plant, which is less than 0.3% dry weight THC for hemp (Agriculture Marketing Service, 2019). Hemp fiber is produced from the stalks and the seed is harvested for grain and hempseed oil (Small, 2015). Monecious cultivars have been developed for dual-purpose fiber and grain production.
Hemp seed from open-pollinated dioecious plants can be expected to produce a 50:50 ratio of male-to-female plants (Small, 2015). During CBD hemp production, it is important for growers to remove male plants before anthesis, because pollination of female plants reduces cannabinoid yield (Meier and Mediavilla, 1998). Hemp growers prefer to use feminized seed or vegetatively propagated female clones for CBD production to eliminate the labor of removing male plants and the lost acreage from removed male plants. Hemp is wind-pollinated, and pollen can drift long distances (Small, 2015). It has been reported that hemp pollen can drift more than 300 km (Clarke, 1977). Therefore, even when hemp farmers take strict measures to grow only female plants, they can experience seed production as a result of drifting pollen from neighboring fiber and grain farms or from CBD farms that did not remove males. A distance of at least 5 km is recommended to prevent pollen drift from neighboring hemp fields (Neiden, 2020; Small, 2015). Disputes between farmers over unintended seed production from drifting pollen has led to several lawsuits (Perkowski, 2019). Pollen can also drift from wild or escaped hemp, known as ditch weed (Neiden, 2020).
Induction of polyploidy has been used by plant breeders to develop improved horticultural crops with enhanced traits such as size, vigor, and metabolite content (Alexander, 2017; Lehrer et al., 2008; Sattler et al., 2016; Wang et al., 2016; Xu et al., 2014). Tetraploids are polyploids that contain four sets of chromosomes. Compared with diploids, tetraploid purple cone flower produces more secondary metabolites and biomass (Xu et al., 2014), and tetraploid ryegrass is more drought tolerant and disease resistant (Sattler et al., 2016). Tetraploidy can be induced artificially using mitotic spindle inhibitors such as colchicine or oryzalin (Sattler et al., 2016; Wang et al., 2016). C. sativa is almost exclusively diploid (2n = 20) in the wild (Small and Cronquist, 1976). There is only one report of a natural tetraploid of C. sativa, from India (Sharma et al., 2015). Tetraploid C. sativa has been produced using colchicine on seedling shoot tips (Bagheri and Mansouri, 2015; Mansouri and Bagheri, 2017), and by using oryzalin on in vitro nodal explants (Parsons et al., 2019). Tetraploid plants produced in these studies exhibited traits such as larger leaves and greater shoot fresh weight and flavonoid content.
Tetraploid plants crossed with diploid plants can generate triploid plants, which have three sets of chromosomes (Wang et al., 2016). Triploid plants are frequently seedless, because unequal segregation of chromosome pairs during meiosis results in inviable gametes (Wang et al., 2016). Seedless triploid cultivars have been bred for hops, watermelon, banana, and citrus (Trojak-Goluch and Skomra, 2018; Wang et al., 2016). Warmke and Davidson (1944) reported crossing tetraploid and diploid marijuana and producing triploid plants; however, no cytogenetic evidence of triploidy was provided. The objective of this work was to investigate a more efficient and easy method for inducing tetraploidy in hemp, and to cross tetraploid plants with diploid plants to produce triploid hemp. Triploid hemp that does not produce seed when exposed to pollen could be a solution for the problem of pollen drift.
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