In Vitro Propagation of Humulus lupulus through the Induction of Axillary Bud Development

Humulus lupulus (hop) is a necessary material for beer brewing. Improved breeding cultivars of hops with enhanced tolerance to environmental stresses, such as drought and heat stress, accompanying climate change have been developed. However, a propagation system, which is needed for the proliferation of new cultivars, is not currently available for hops. In this study, we found that treatment of stem explants with 0.01–0.05 ppm gibberellic acid (GA3) induced the development of axillary buds in the hop cultivar Kirin-2, resulting in the proliferation of shoot branching. Additionally, 0.01 ppm benzyl adenine (BA) enhanced the development of axillary buds formed in response to 0.05 ppm GA3 in various hop cultivars, particularly Nugget. The development of axillary buds was strongly repressed by the application of 0.05 ppm BA at a concentration equal to the 0.05 ppm GA3 concentration, which showed the possibility that a high concentration of cytokinin preferentially prevents the effect of GA3 on the development of axillary buds in hops. These results indicated that combined treatment of stem explants with GA3 and cytokinin at appropriate concentrations is effective for the propagation of proliferated hop cultivars through shoot branching.


Introduction
Humulus lupulus var. lupulus (hop) is a perennial, dioecious, and climbing plant belonging to the Cannabaceae family. Hop is widely cultivated in areas under a microthermal climate worldwide and is used as a key ingredient in beer brewing because it brings a floral aroma, bitterness, and foam stability [1].
The female flower of the hop called the "hop cone" contains a unique structure and function. In hop cones, the base of bracts surrounded with strig and bracteoles possess glandular trichomes known as lupulin grands, which secrete cells and biosynthesize various kinds of bioactive secondary metabolites, such as prenylated flavonoids (xanthohumol and desmethylxanthohumol), bitter acids (humulone (α-acid) and lupulone (β-acid)) and essential oils [2]. The secondary metabolites derived from the hop cone benefit not only beer brewing but also animal and human diseases. For example, xanthohumol shows multifunctional anticancer activity on various organs of the body [3]. Additionally, isoα-acids, which are hop-derived bitter acids in beer, suppress amyloid β deposition in a mouse model of Alzheimer's disease, suggesting that iso-α-acids prevent dementia [4,5].
The demand for high-quality hops has increased with the diversification of the brewing industry and preferences for beer. In addition, it is expected that the quality and yield of hops will decrease in the production area due to global warming [6][7][8]. To solve this problem, improved cultivars of hops have been developed using classical intercrosses to increase their quality and yield in warm areas. Because hops are propagated vegetatively, the rapid production or proliferation of new cultivars requires many plantlets but not seeds. The basic methods for the tissue culture of hops are applicable in various cultivars, but there are few reports on the propagation system of hops [9,10]. In propagation systems of some species of plants, the induction of shoot branching based on the development of axillary buds is effective in increasing the numbers of shoot apexes and stem nodes, which are used as materials for plantlets [11][12][13]. In this study, we present a method for inducing the development of axillary buds in hop cultivars Kirin-2, Cascade, and Nugget. We found that the formation of axillary buds in stem explants was promoted by gibberellic acid (GA 3 ). Additionally, the application of cytokinin enhanced the elongation of axillary buds formed with GA 3 , which indicated that the combination treatment of GA 3 and cytokinin is useful for the proliferation of hops through the induction of shoot branching.

Glucose Is Suitable as a Sugar Source in the Axillary Bud Development of Hop
Sugar plays an important role in regulating growth and development in plant tissue culture. In the tissue culture of hops, different types of sugar are used as a sugar source by each hop cultivar [9,10,14]. Thus, we checked the response of hops to glucose, sucrose, and fructose with respect to the axillary bud development of stem explants using the hop cultivar Kirin-2, which is an improved cultivar of hop cv. Shinshu Wase in Japan. The hop cultivar Kirin-2 shows a higher yield and larger amounts of αand β-acids than the hop cultivar Shinshu Wase. At 3 weeks after incubation, the length of axillary buds of stem explants treated with glucose was 3 times longer than that of stem explants treated with glucose and fructose (glucose: 1.4 cm, sucrose: 0.48 cm, fructose: 0.47 cm) (Figure 1a,b). This result suggested that glucose was most suitable as a sugar for the induction of axillary bud development in the hop cultivar Kirin-2.
Plants 2022, 11, x FOR PEER REVIEW 2 of 10 of some species of plants, the induction of shoot branching based on the development of axillary buds is effective in increasing the numbers of shoot apexes and stem nodes, which are used as materials for plantlets [11][12][13]. In this study, we present a method for inducing the development of axillary buds in hop cultivars Kirin-2, Cascade, and Nugget. We found that the formation of axillary buds in stem explants was promoted by gibberellic acid (GA3). Additionally, the application of cytokinin enhanced the elongation of axillary buds formed with GA3, which indicated that the combination treatment of GA3 and cytokinin is useful for the proliferation of hops through the induction of shoot branching.

Glucose Is Suitable as a Sugar Source in the Axillary Bud Development of Hop
Sugar plays an important role in regulating growth and development in plant tissue culture. In the tissue culture of hops, different types of sugar are used as a sugar source by each hop cultivar [9,10,14]. Thus, we checked the response of hops to glucose, sucrose, and fructose with respect to the axillary bud development of stem explants using the hop cultivar Kirin-2, which is an improved cultivar of hop cv. Shinshu Wase in Japan. The hop cultivar Kirin-2 shows a higher yield and larger amounts of α-and β-acids than the hop cultivar Shinshu Wase. At 3 weeks after incubation, the length of axillary buds of stem explants treated with glucose was 3 times longer than that of stem explants treated with glucose and fructose (glucose: 1.4 cm, sucrose: 0.48 cm, fructose: 0.47 cm) (Figure 1a,b). This result suggested that glucose was most suitable as a sugar for the induction of axillary bud development in the hop cultivar Kirin-2.

Cytokinin Inhibits the Development of Axillary Buds in Hop
Cytokinin induces axillary bud development in a wide range of plant species [15]. To investigate the effect of cytokinin on the development of axillary buds in hop, we observed sensitivity to benzyl adenine (BA) or trans-zeatin (tZ) in stem explants of the hop cultivar Kirin-2. Under normal conditions, axillary buds developed from stem explants in liquid culture (Figure 2a,b,d,e). In contrast, the application of BA or tZ inhibited the formation of axillary buds in stem explants (Figure 2b,e). The length of shoots in stem explants treated with BA and tZ was 2.5 times shorter than that of stem explants without BA and tZ (Figure 2c,d,f). Additionally, the discolouration of explants was induced by tZ treatment ( Figure 2d). These results suggested that cytokinin inhibited the formation of axillary buds and shoot development in hop.

Cytokinin Inhibits the Development of Axillary Buds in Hop
Cytokinin induces axillary bud development in a wide range of plant species [15]. To investigate the effect of cytokinin on the development of axillary buds in hop, we observed sensitivity to benzyl adenine (BA) or trans-zeatin (tZ) in stem explants of the hop cultivar Kirin-2. Under normal conditions, axillary buds developed from stem explants in liquid culture (Figure 2a,b,d,e). In contrast, the application of BA or tZ inhibited the formation of axillary buds in stem explants (Figure 2b,e). The length of shoots in stem explants treated with BA and tZ was 2.5 times shorter than that of stem explants without BA and tZ (Figure 2c,d,f). Additionally, the discolouration of explants was induced by tZ treatment (Figure 2d). These results suggested that cytokinin inhibited the formation of axillary buds and shoot development in hop.

Induction of Axillary Bud Formation with Gibberellic Acid
In several species of plants, GA 3 treatment induces the development of axillary buds [16,17]. Thus, we examined whether the development of axillary buds was induced with GA 3 in hop cv. Kirin-2. Under normal conditions, axillary buds were not confirmed at stem nodes near the shoot apex ( Figure 3a). However, the formation of axillary buds was observed at stem nodes near the shoot apex in explants treated with GA 3 . The number of axillary buds in stem explants treated with GA 3 was twice as large as that in stem explants without GA 3 (Figure 3b). GA 3 treatment did not induce internode elongation in the main stem explants, leading to the GA 3 -induced promotion of axillary bud formation in hops (Figure 3c).

Induction of Axillary Bud Formation with Gibberellic Acid
In several species of plants, GA3 treatment induces the development of axillary buds [16,17]. Thus, we examined whether the development of axillary buds was induced with GA3 in hop cv. Kirin-2. Under normal conditions, axillary buds were not confirmed at stem nodes near the shoot apex ( Figure 3a). However, the formation of axillary buds was observed at stem nodes near the shoot apex in explants treated with GA3. The number of axillary buds in stem explants treated with GA3 was twice as large as that in stem explants without GA3 (Figure 3b). GA3 treatment did not induce internode elongation in the main stem explants, leading to the GA3-induced promotion of axillary bud formation in hops (Figure 3c). Different letters indicate significant differences. (c) Relative shoot length of stem explants treated without and with GA3. The shoot length of explants treated with GA3 was normalized against the shoot length of explants without GA3 for 3 weeks (n = 10, individual experiment, no significant difference; Tukey-Kramer test). Different letters indicate significant differences.

Combined Treatment with Gibberellic Acid and Cytokinin Strongly Promotes the Development of Axillary Buds
To verify that BA enhances the effect of GA3 on axillary bud development in hop cv. Kirin-2, we observed the development of axillary buds in stem explants treated with both GA3 and BA. The detection frequency of axillary buds in stem explants treated with both GA3 and BA was comparable with that in stem explants treated with only GA3 (Figure 4a,b). In addition, combined treatment with GA3 and BA markedly increased the length of axillary buds by 1.7 times compared with that obtained with the GA3 treatment (Figure 4c). These results suggested that cytokinin enhanced the elongation of axillary buds formed by GA3 treatment in hop.

Combined Treatment with Gibberellic Acid and Cytokinin Strongly Promotes the Development of Axillary Buds
To verify that BA enhances the effect of GA 3 on axillary bud development in hop cv. Kirin-2, we observed the development of axillary buds in stem explants treated with both GA 3 and BA. The detection frequency of axillary buds in stem explants treated with both GA 3 and BA was comparable with that in stem explants treated with only GA 3 (Figure 4a,b). In addition, combined treatment with GA 3 and BA markedly increased the length of axillary buds by 1.7 times compared with that obtained with the GA 3 treatment (Figure 4c). These results suggested that cytokinin enhanced the elongation of axillary buds formed by GA 3 treatment in hop.
We then investigated the effect of GA 3 and cytokinin on axillary bud development in hop cultivars other than Kirin-2 using Cascade and Nugget, which are mainly cultivated in the US and Germany and show resistance to downy resistance [18,19]. Cascade shows a low amount of α-acid and an aroma with medium strength. Additionally, Nugget is the variety which has high α-acid and mild aroma. The combined treatment with BA and GA 3 increased the number of axillary buds in stem explants of Cascade and Nugget by 2 and 2.5 times, respectively, compared with that without BA and GA 3 (Figure 5a,b). The length of axillary buds in stem explants of Cascade and Nugget treated with BA and GA 3 was 2.7 and 4.3 times, respectively, higher than that of stem explants without BA and GA 3 (Figure 5c). In addition, the shoot length in stem explants in Nugget but not in Cascade increased 1.6 times after the GA 3 and BA treatments, which indicated that GA 3 and cytokinin might have the ability to enhance the development of axillary buds in various hop cultivars, unlike the results obtained for shoot development (Figure 5d). We then investigated the effect of GA3 and cytokinin on axillary bud development in hop cultivars other than Kirin-2 using Cascade and Nugget, which are mainly cultivated in the US and Germany and show resistance to downy resistance [18,19]. Cascade shows a low amount of α-acid and an aroma with medium strength. Additionally, Nugget is the variety which has high α-acid and mild aroma. The combined treatment with BA and GA3 increased the number of axillary buds in stem explants of Cascade and Nugget by 2 and 2.5 times, respectively, compared with that without BA and GA3 (Figure 5a,b). The length of axillary buds in stem explants of Cascade and Nugget treated with BA and GA3 was 2.7 and 4.3 times, respectively, higher than that of stem explants without BA and GA3 (Figure

Rooting and Transplanting in Hop
Finally, we checked whether the induction of axillary buds was useful in the plantlet propagation of hops. The rooting of stem explants derived from propagated plants through the induction of axillary buds development was observed with 1-Naphthaleneacetic acid (NAA) treatment ( Figure 6a). Additionally, transplanting of stem explants with roots was confirmed in a greenhouse under natural photoperiod conditions, suggesting that our findings might help increase the efficiency of plantlet propagation of hops (Figure 6b,c).

Rooting and Transplanting in Hop
Finally, we checked whether the induction of axillary buds was useful in the plantlet propagation of hops. The rooting of stem explants derived from propagated plants through the induction of axillary buds development was observed with 1-Naphthaleneacetic acid (NAA) treatment ( Figure 6a). Additionally, transplanting of stem explants with roots was confirmed in a greenhouse under natural photoperiod conditions, suggesting that our findings might help increase the efficiency of plantlet propagation of hops (Figure 6b,c).

Rooting and Transplanting in Hop
Finally, we checked whether the induction of axillary buds was useful in the plantlet propagation of hops. The rooting of stem explants derived from propagated plants through the induction of axillary buds development was observed with 1-Naphthaleneacetic acid (NAA) treatment ( Figure 6a). Additionally, transplanting of stem explants with roots was confirmed in a greenhouse under natural photoperiod conditions, suggesting that our findings might help increase the efficiency of plantlet propagation of hops (Figure 6b,c).

Discussion
In this study, our sugar application test first revealed that glucose is most suitable as a sugar source for the induction of axillary bud development in stem explants of the hop cultivar Kirin-2 (Figure 1a,b). Different types of sugar were used as a sugar source in tissue culture of each hop cultivar in previous studies. For example, sucrose is used as a sugar source in the hop cultivars Hallertauer Mitterlfrüeh, Columbus, and Chinook [14]. In contrast, glucose is supplemented in the growth medium of the hop cultivars Cascade and H138 [9,10]. Thus, it might be important to select a suitable sugar source for the induction of axillary bud development in each hop cultivar.
We revealed that combined treatment with GA 3 and BA induces the development of axillary buds in stem explants of hop. The molecular mechanism underlying the development of axillary buds has been studied for several decades using model plants [20]. In Arabidopsis thaliana (A. thaliana), BRANCHED1/2 (BRC1/2), which belongs to the TCP transcription factor family and is related to TEOSINTE BRACNCHED1 (TB1) in Zea mays and FINE CULM1 (FC1) in Oryza sativa, is a negative regulator of axillary bud development [21][22][23]. The expression of BRC1/2 is activated by increasing the concentration of auxin transported from the shoot apex in the main stem through PIN-FORMED (PIN) auxin-efflux facilitators [21]. In addition, high levels of cytokinin reduced the expression of BRC1/2, resulting in induction of axillary bud development, which suggested that auxin derived from the shoot apex inhibits axillary bud development through the repression of cytokinin biosynthesis at stem nodes. Recent studies showed that the homologue of BRC1/2 also plays an important role in the development of axillary buds in the climbing plant Cucumber sativus [24]. Cucumber BRANCHED1 (CsBRC1) reduces the expression levels of PIN1/3 through direct interaction with their promoter regions, and knockdown mutants of CsBRC1 exhibit overshoot branching [25].
Although cytokinin induces the development of axillary buds in Cannabis sativa, which belongs to the Cannabaceae family, as well as in most species of plants, our phytohormone application test showed that axillary buds developed with GA 3 but not BA in hops [26]. GA 3 inhibits the outgrowth of axillary buds through regulation of the auxin levels and transport in model plants such as A. thaliana and Populus [27,28]. In contrast, the development of axillary buds is promoted by GA 3 in Phaseolus vulgaris, citrus, and snapdragon, as well as in hops, which suggests that the application of GA 3 might be useful for the induction of axillary buds in plant species that are insensitive to cytokinin application [12,13]. The combined treatment with BA and GA 3 induces the development of axillary buds in stem explants to a greater degree than in plants treated with only GA 3 in woody plants, such as Jatropha curcas (J. curcas) [29]. Although BA enhanced the elongation of axillary buds induced with GA 3 in stem explants of hops as well as J. curcas, the formation of axillary buds was strongly inhibited by the application of BA at a concentration equal to the GA 3 concentration (Figure 4a). These results show the possibility that a high concentration of cytokinin preferentially prevents the effect of GA 3 on the induction of axillary buds in hops.
Previous studies have shown that GA 3 is involved in the development of hop cones in various cultivars. GA 3 application increases the number of hop cones with changes in the α-acid contents in the hop cultivars Saazer, Hüller Anfang, and Hallertauer in the field compared with the variation in the number of hop cones observed with GA 3 in the cultivar Fuggle [30,31]. Our phytohormone application tests also showed that the sensitivity to GA 3 of shoot development differs between the hop cultivars Cascade and Nugget (Figure 5d). These findings suggest that the molecular mechanisms controlling growth and development involving GA 3 are complicated in hop cultivars. Recently, highquality draft genomes have been constructed through sequencing and de novo assembly in some hop cultivars [32,33]. This information might contribute to revealing the molecular mechanism underlying growth and development in the presence of GA 3 .

Plant Materials and Growth Conditions
Humulus lupulus cv. Kirin-2, Cascade, and Nugget were used as materials in this study. To obtain the in vitro plants, stem explants were isolated from seedlings and sterilized with 70% (w/v) ethanol and 1% (w/v) sodium hypochlorite for 1 min and 5 min, respectively. After being washed 3 times with sterilized water, the stem explants were transferred to 1/2 Murashige and Skoog (MS) medium supplemented with 2% (w/v) glucose, 0.01 ppm benzyl adenine (BA), and 0.8% (w/v) agar at pH 5.8. The samples were moved into an incubator set at 20 • C with a 16 h light/8 h dark photoperiod. After 3 weeks, developed axillary buds with stem explants were transferred to 1/2 MS medium supplemented with 2% glucose and 0.8% agar to establish the in vitro plants in hormone-free conditions. The in vitro plants were subcultured every 1.5 months.

Sugar Application Test
Stem explants isolated from 1.5-month-old in vitro plants were incubated in 1/2 MS medium supplemented with 0.8% agar and 2% glucose, sucrose, or fructose. At each week for 3 weeks, the length of axillary buds was measured. At least two independent experiments were performed, and similar results were obtained.

Phytohormone Application Test
Stem explants isolated from 1.5-month-old in vitro plants were incubated in 1/2 MS liquid medium supplemented with 2% glucose, BA, trans-zeatin (Tokyo Chemical Industry, Tokyo, Japan), and GA 3 (FUJIFILM Wako, Japan). After 3 weeks, the shoot length, axillary bud length and number of axillary buds of the explants were measured using ImageJ software. To calculate the relative shoot and axillary bud lengths, the shoot and axillary bud lengths of explants treated with each phytohormone were normalized against the shoot and axillary bud lengths of explants without phytohormones. At least two independent experiments were performed, and similar results were obtained.

Rooting and Transplanting
Stem explants isolated from 1-month-old propagated plants through the induction of axillary bud development were incubated on papers containing 1/2 MS medium supplemented with 2% glucose and 0.05 ppm NAA (FUJIFILM Wako, Japan) in an incubator at 20 • C. After 2 weeks, stem explants with roots were transferred to the cell trays with soil consisting of Metro-Mix 350 (Sun Gro Horticulture, Agawam, MA, USA), vermiculite, and red ball earth (=3:1:1). Plantlets were grown in a greenhouse (25 • C) under natural photoperiod conditions.

Statistical Analysis
Statistical analysis was performed using Student's t test and the Tukey-Kramer test. The sample size and p value are presented in the Figure Legends Funding: This study was supported by the Kirin Holdings Company, Ltd.

Data Availability Statement:
The data presented in this study are available on reasonable request from the corresponding author.