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To maximize yield, cannabidiol (CBD) hemp producers prefer female plants, and this is accomplished by using expensive feminized seed, vegetatively propagated female clones, or by removing male plants from dioecious seed lots. Hemp pollen drifts long distances on wind, and pollination of females reduces CBD content. Induction of triploidy is a common strategy used by plant breeders to produce sterile cultivars of agricultural crops. Triploid (3n) hemp, with three sets of chromosomes, was developed by crossing naturally diploid (2n) hemp with tetraploid (4n) hemp. Tetraploid plants used to create triploids were produced using pregerminated seeds and the mitotic spindle inhibitor colchicine. Seedlings from seeds of ‘Abacas’ × [(‘Otto2’ × ‘BaOx’) × (‘BaOx’ × ‘Colorado Cherry’)] treated with 0.05% colchicine or 0.02% colchicine for 12 hours and longer were significantly shorter than controls and ≤1 cm tall at 10 days after sowing. Surviving seedlings exhibited thickened cotyledons and hypocotyls, which indicated a potential change in ploidy. Tetraploid induction ranged from 26% to 64% for pregerminated seeds of five different hemp cultivars (Abacus × Wife, Cherry Wine, Mountain Mango, Wife, and Youngsim10) treated with 0.05% colchicine for 12 hours. Tetraploids had nearly twice the DNA content as diploids according to flow cytometric analysis. Tetraploid ‘Wife’ had larger stomates and reduced stomatal density compared with diploid ‘Wife’. Four triploid ‘Wife’ genotypes produced from crossing tetraploid ‘Wife’ with diploid ‘Wife’ were acclimated to greenhouse conditions after embryo rescue. DNA content and stomate size of triploid ‘Wife’ was intermediate between the parents. This is the first report of triploid plants of hemp. Future research will evaluate the sterility of triploid hemp.

There is demand for early-flowering cannabis (Cannabis sativa) cultivars to hasten harvest and avoid late-season detrimental weather conditions. A field study and greenhouse studies were conducted to evaluate the effect of gene dosage at the autoflowering locus on flowering timing for diploid and triploid hybrids between autoflowering and photoperiod-sensitive parents. Autoflowering × photoperiod-sensitive hybrids were all photoperiod sensitive, but their critical photoperiods were longer than for homozygous photoperiod-sensitive plants, which resulted in earlier flowering. For triploid genotypes, decreasing dosage of the photoperiod-sensitive allele (A), from AAA to AAa to Aaa, reduced the time to flowering. Flowering timing for the diploid genotype Aa was intermediate between Aaa and AAa. These results provide evidence of incomplete dominance of the A allele at the autoflowering locus. Plants of genotype Aaa flowered 32 to 40 days earlier in the field than genotypes of AA, 15 days earlier than genotype Aa, and were ready for harvest by the second week of August in Connecticut. Plants of Aaa were as tall as other diploid and triploid photoperiod-sensitive genotypes studied, which suggests that they have similar yield potential. The use of tetraploid autoflowering (aaaa) maternal plants in combination with diploid photoperiod-sensitive (AA) pollen parents to produce Aaa genotype seed is a reliable approach for developing early-flowering cultivars of cannabis for flower production purposes.

There is demand for micropropagated Cannabis sativa liner plants, because they are uniform, vigorous, and pathogen free; however, availability is limited because of challenges with in vitro culture decline and ex vitro rooting. Ex vitro rooting success of microcuttings was evaluated for ‘Abacus’ and ‘Wife’ when cultures were 6, 9, 12, 15, and 18 weeks old from initiation. Microcuttings of ‘Wife’ harvested from 6, 9, and 12-week-old cultures rooted at or above 80%, but rooting declined to 50% and 30% for 15- and 18-week-old cultures, respectively. Rooting for ‘Abacus’ remained relatively constant between 47% and 70% for microcuttings harvested from 6- to 18-week-old cultures. ‘Wife’ plants grown from microcuttings, stem cuttings, and retip cuttings (cuttings taken from new shoots on recently micropropagated plants) had equivalent total shoot length, number of shoots, and flower dry weight, whereas micropropagated ‘Abacus’ plants had less shoot length and flower dry weight than plants from stem cuttings. However, when micropropagated ‘Abacus’ plants were provided an extra week of vegetative growth to reach an initial size equivalent to stem and retip plants, all plants performed the same. Propagation method did not change cannabinoid content for both ‘Abacus’ and ‘Wife’. Retip cuttings of ‘Abacus’ and ‘Wife’ rooted at 76% to 81% without rooting hormone, which is comparable to rates reported for stem cuttings of C. sativa treated with rooting hormone. Propagators should consider retipping to expand their liner production, because retips root well and possess the same desirable attributes as micropropagated plants.

Micropropagation of hemp (Cannabis sativa) is constrained by problems with hyperhydricity and culture decline of microshoots. These problems can be reduced by increasing agar and nutrients in the media during micropropagation stages 1 and 2, respectfully. Performance of microshoots of ‘Abacus’ and ‘Wife’ hemp cultured in Driver and Kuniyuki Walnut medium (DKW) for 15 weeks (6 weeks of stage 1 + 9 weeks of stage 2), with subculturing every 3 weeks during both stages 1 and 2, or in Murashige and Skoog with vitamins medium (MS) for 6 weeks (stage 1) followed by Lubell-Brand Cannabis medium (LBC) for 9 weeks (stage 2), with subculturing every 3 weeks during both stages 1 and 2, was evaluated. In a separate study, microshoot performance of ‘Abacus’ and ‘Wife’ in MS for 3 weeks (stage 1) followed by LBC for 6 weeks (stage 2), with subculturing every 3 weeks, using boxes (Magenta GA-7) with lids featuring a vent with a diameter of 10 mm and a pore size of 0.2 µM or using microboxes (Sac O₂ O95/114 + OD95) with lids featuring a filter (Sac O₂ #10) were evaluated. Shoot multiplication rate (SMR) and explant height were greater for ‘Abacus’ in LBC than DKW. For ‘Wife’, SMR at 9 weeks was greater in LBC, as LBC provided more nutrients and water than cultures had received in MS initially during stage 1. Culture medium did not influence ex vitro rooting success, which was 75% for ‘Abacus’ and ≥ 90% for ‘Wife’. Microboxes resulted in greater hyperhydricity of shoots and a lower ex vitro rooting percentage than boxes. For cultivars that are highly prone to developing hyperhydricity, like ‘Abacus’, the microboxes were not adequate to control this condition.

Hyperhydricity of shoots initiated in vitro, poor shoot extension, inability of shoot cultures to maintain good growth over an extended time, and unsuccessful ex vitro rooting have limited the development of a commercial scale micropropagation system for hemp (Cannabis sativa). We present a culture initiation method that prevents shoot hyperhydricity using vented-lid vessels with 0.2-µm pores and medium containing agar at 1% (w/v). To optimize shoot multiplication in vitro, a control medium (medium A) and four treatment media (medium B, C, D, and E), with varying inorganic nutrients and vitamins were tested. Control medium A consisted of 1× Murashige and Skoog (MS) with vitamins plus 3% (w/v) sucrose, 0.5 mg·L⁻¹ metatopolin, 0.1 mg·L⁻¹ gibberellic acid, and 0.8% agar (w/v) at pH 5.7. The four treatment media differed from the control medium as follows: medium B, 2.5× MS with vitamins; medium C, 1× MS with vitamins plus added mesos [calcium chloride (anhydrous), magnesium sulfate (anhydrous), and potassium phosphate (monobasic) nutrients]; medium D, 1× MS with vitamins plus added vitamins; and medium E, 1× MS with vitamins plus added mesos and vitamins. Medium C and medium E produced more microcuttings than the control at 6 weeks after the initial subculture with shoot multiplication media and all other treatments at 9 and 12 weeks. Shoots grown on these two media displayed optimal extension and leaf lamina development; however, they exhibited slight chlorosis by 12 weeks after subculture with shoot multiplication media. In a separate experiment, medium E was supplemented with ammonium nitrate at 0, 500, 1000, or 1500 mg·L⁻¹, and cultures grown with 500 mg·L⁻¹ produced the most microcuttings and exhibited the best combination of shoot extension and leaf lamina development. We provide a method of prerooting microshoots in vitro that has resulted in 75% to 100% rooting ex vitro in rockwool. Using 10 recently micropropagated plants, ≈300 retip cuttings (cuttings taken from new shoots from recently micropropagated plants) were harvested over 10 weeks. The average weekly rooting was more than 90%. Retipping can produce nine-times as many plants in a similar amount of floor space as stem cuttings derived from traditional stock mother plants. The micropropagation/retipping method proposed can be a more efficient way to generate clonal liner plants for commercial-scale production.

Feminized hemp seed producers often use selfing to maintain a strain name; however, selfing may lead to inferior plants for cannabidiol (CBD) production. Using three different hemp strains as parents [Candida (CD-1), Dinamed CBD, and Abacus], two outcrosses [Candida (CD-1) × Abacus and Dinamed CBD × Candida (CD-1)] and one self-cross [Candida (CD-1) × Candida (CD-1)] were conducted to produce feminized seed. Progeny from the self-cross were significantly smaller and had less yield than outcrossed progeny. Selfed progeny were variegated and highly variable for total dry weight and floral dry weight. Discriminant analysis of principal components (DAPC) using amplified fragment length polymorphism (AFLP) separated the three progeny populations and showed that outcrossed populations clustered closer to the maternal parent, possibly the result of a maternal effect. Analysis of molecular variance (AMOVA) indicated that most variation (74.5%) was within populations, because the progeny from all three populations are half-siblings of each other. The selfed progeny population had lower expected heterozygosity (H_e = 0.085) than each of the outcrossed progeny populations (H_e ≈ 0.10). These results suggest that selfed progeny may demonstrate inbreeding depression resulting from enhanced expression of homozygous recessive traits. It may be beneficial for feminized seed producers to use outcrossing instead of selfing to generate feminized seed for CBD production.