ORIGINAL ARTICLES
Effects of In Vitro Culture Conditions on Growth and Overwintering Bud Formation in a Gentian Leaf Culture
2025 Volume 94 Issue 3 Pages 393-400
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2025 Volume 94 Issue 3 Pages 393-400
In vitro culture is essential for propagating F1 cultivar parents and vegetatively propagated gentian cultivars. In vitro overwintering bud (OWB) formation is also crucial to maintain and propagate these plants. This study examined the effects of temperature, medium composition, and photoperiod on in vitro growth, including OWB formation, using Gentiana triflora and G. scabra as model species in leaf cultures, a practical propagation method. The growth parameters of the G. triflora line TY1-11-4-9 were assessed under different temperature and sugar composition conditions, revealing positive and negative correlations between OWB and rosette-shaped shoot (RSS) formation and between OWB and flower bud formation, respectively. Consequently, further investigations focused on these growth parameters. Temperature effects on four cultivars/lines revealed interspecific differences: at all temperatures, G. triflora had a higher rate of flower bud formation than G. scabra, whereas G. scabra showed increased OWB and RSS formation. Culturing under low temperature (15°C/15°C: regeneration stage/OWB induction stage, which was also applicable below) conditions, which may be impractical owing to slow growth effects, suppressed flower bud formation in G. triflora, but promoted RSS and OWB formation. Similar effects, albeit milder, were observed at 20°C/15°C, and these conditions were further explored as a practical method combined with other treatments to induce OWB formation. The medium sugar composition had negligible effects on in vitro growth, whereas gibberellin at 1.0 mg·L−1 in the medium composition suppressed OWB and RSS formation in G. scabra. Photoperiod variations affected several growth parameters, with a 12-h photoperiod promoting a high rate of OWB formation in the G. triflora line TY1-11-4-9. In conclusion, low-temperature treatments after a certain amount of growth, along with combined treatments such as photoperiod adjustments and gibberellin inhibitor administration, show promise for promoting OWB formation in gentians. Furthermore, the interspecific differences observed in flower bud formation, RSS production, and OWB formation contribute to a better understanding of gentian physiology and genetics.
Gentiana are popular herbaceous perennials, commonly used as cut flowers and pot flowers, with high demand in Japan where they are used as offerings at the Bon Festival, the autumnal equinox Ohigan in late September, and other occasions. Gentians represent a major cut flower, ranking third in planted area after chrysanthemums and lilies and eighth in shipment volume (Ministry of Agriculture, Forestry and Fisheries, 2021). Regions with cool climates are ideal for gentian cultivation, with Iwate, Akita, Yamagata, and Fukushima prefectures in northern Japan producing over 80% of the national output (Ministry of Agriculture, Forestry and Fisheries, 2021). In recent years, the introduction of new color varieties, including white and pink flowers, has increased their use in flower arrangements and casual decor. Additionally, Japanese gentian cut flowers, known for their numerous flowers on long, multitiered stems, are highly regarded globally. Research on gentians is active worldwide; over 1,600 papers (according to Web of Science) have been published on Gentiana, with >90% of these published by researchers outside Japan.
F1 breeding is now common in gentians, with early to late maturing F1 cultivars developed from Gentiana triflora and G. scabra (both native to Japan), as well as their hybrids. Maintaining and propagating parental stocks is crucial for a stable seed supply in F1 cultivars. However, preserving parental stocks through repeated self-fertilization in fields and pots is challenging owing to gentians’ strong inbreeding depression (Nishihara et al., 2018; Suzuki et al., 2004). Therefore, vegetative propagation techniques are required to obtain clones, with in vitro culture being an efficient method (Sakuma, 1997; Suzuki et al., 2004). In vitro culture is also effective for propagating vegetatively propagated cultivars, making it indispensable for gentian production.
Raising gentian seedlings in the field takes one to two years, during which time they form overwintering buds (OWBs) to facilitate dormancy, acquire low-temperature tolerance, overwinter, and sprout the following year (Samarakoon et al., 2012). OWBs can also be formed in vitro. Empirical evidence shows that in vitro plants with OWBs are easier to maintain and propagate compared with those lacking OWBs (Sakuma, 1997). Therefore, in vitro OWB formation is pivotal for gentian propagation, making it essential to study conditions that promote this process. Along with OWB formation, in vitro flowering is also vital for gentian breeding (Zhang and Leung, 2000).
Previous studies have investigated a single condition for OWB formation during gentian in vitro culture using G. triflora (Imamura et al., 2014; Takahashi et al., 2012); however, few studies have examined various culture conditions using multiple species. Therefore, the present study investigated the effects of multiple factors, including temperature, medium composition, and photoperiod, on OWB formation, flower bud formation, and rosette-shaped shoot (RSS) formation in vitro, using both G. triflora and G. scabra as model species. Flower bud and RSS formation were included because their relationship with OWB formation (e.g., OWB formation from RSS and death after flowering) is empirically known in in vitro culture models. Using this combination of culture conditions and cultivars/lines, we aimed to elucidate the physiological mechanisms underlying OWB formation and develop techniques to promote it. Notably, apical buds or leaf segments can be used for in vitro propagation of gentians. Leaf culture is advantageous because of the ease with which a large quantity of materials at uniform growth stages can be obtained as well as its ability to use individuals lacking viable apical buds due to poor growth. Therefore, leaf culture was chosen as a practical culture method in this study, and this is a distinctive feature of this research.
Gentiana triflora TY1-11-4-9 (GT-TY), G. triflora ‘Fukushimahonoka’ (GT-FH), G. scabra ‘Amanogawa’ (GS-AM), and G. scabra G103 (GS-G1) were used in this study. These cultivars/lines were bred in Fukushima Prefecture, Japan, in a field with GT-TY flowering early, GT-FH mid-late, and GS-AM and GS-G1 late.
2. In vitro culture for shoot regeneration and rooting (regeneration stage)Unexpanded leaves were collected from the upper nodes of gentian plants grown in the field and washed with mild detergent, rinsed with water, immersed in 70% ethanol for 30 s, sterilized in a sodium hypochlorite solution (approximately 1% effective chlorine) for 7 min, and washed four times with sterile water. Leaves were then cut into 5–6-mm-wide segments and cultured on MS medium (Murashige and Skoog, 1962) supplemented with 1-(2-chloro-4-pyridyl)-3-phenylurea (1.0 mg·L−1), 1-naphthylacetic acid (0.5 mg·L−1), 3% sucrose, and 0.2% gellan gum. After approximately two months, formed calli were transferred to the same medium without plant growth regulators to promote shoot formation. After around one month, shoots were transplanted into the same medium containing half-strength inorganic salts and no plant growth regulators to promote rooting. Cultures were maintained in test tubes (ø25 × 100 mm) with 10 mL of medium at 15°C or 20°C under a 18/6-h (light/dark) photoperiod with a photosynthetic photon flux density (PPFD) of 50 μmol·m−2·s−1.
3. Experiment 1: Effects of temperature and sugars on in vitro growth in GT-TY (OWB induction stage)GT-TY plantlets from the regeneration stage were transferred to MS medium containing 0.2% gellan gum with half-strength inorganic salts (MS-1/2I medium). The temperature was set at 20°C/10°C (regeneration stage/OWB induction stage, also applicable below), 20°C/15°C, 15°C/15°C, 20°C/20°C. Sugar concentrations were set at 3% sucrose (standard), 6% sucrose, and 3% fructose. PPFD and photoperiod were consistent with the regeneration stage.
4. Experiment 2: Effect of temperature on in vitro growth in different cultivars/lines (OWB induction stage)Plantlets of the four cultivars/lines from the regeneration stage (15°C or 20°C) were transferred to MS-1/2I medium. The temperature was set at 20°C/10°C, 20°C/15°C, 20°C/20°C, 15°C/10°C, 15°C/15°C. In the 15°C treatment during the regeneration stage, plantlets were temporarily (1.5 to 2.0 months) incubated at 20°C to promote callus formation during the regeneration stage. The sugar concentration was set at 3% sucrose. PPFD and photoperiod were consistent with the regeneration stage.
5. Experiment 3: Effects of sugars and plant growth regulators in the medium on in vitro growth (OWB induction stage) 1) Experiment 3-1: Effects of sugars in the culture mediumPlantlets from the regeneration stage (20°C) were transferred to MS-1/2I medium. Sugar concentrations were set at 3% sucrose, 6% sucrose, 3% fructose, and 3% trehalose. The effects of fructose and trehalose were compared with those of sucrose. Fructose was chosen for its growth-promoting effects that were observed in preliminary experiments, whereas trehalose was included for its reported ability to extend incubation periods in Torenia (Yamaguchi et al., 2011). The temperature was set at 15°C. PPFD and photoperiod were consistent with the regeneration stage.
2) Experiment 3-2: Effects of plant growth regulators in the culture mediumPlantlets from the regeneration stage (20°C) were transferred to MS-1/2I medium. The plant growth regulators added included 2.0 mg·L−1 ancymidol, 0.1 mg·L−1 gibberellic acid 3 (GA3), 1.0 mg·L−1 GA3, or 0.5 mg·L−1 benzyladenine. A control with no plant growth regulators was also included. The sugar concentration was set at 3% sucrose, and the temperature was set at 15°C. PPFD and photoperiod were consistent with the regeneration stage.
6. Experiment 4: Effect of photoperiod on in vitro growth (OWB induction stage)At the regeneration stage (20°C), cultures were subjected to 6-, 12-, and 18-h photoperiods. Plantlets were then transferred to MS-1/2I medium, and cultures were maintained under the same three photoperiods during the OWB induction stage. The sugar concentration was set at 3% sucrose, and the temperature was set at 15°C. PPFD was consistent with the regeneration stage.
7. Measurements and statisticsEach experiment was performed in triplicate, with one plant per tube and five plants per replicate. Except for Experiment 4, growth parameters were measured 6 months after the start of the OWB induction stage. In Experiment 4, measurements were taken 5 months (shoot length), 6 months (flower bud and RSS formation), and 9 months (OWB formation) after the OWB induction stage began. Shoot length was calculated as the average of the longest shoot per plant. The rates of flower bud, RSS, and OWB formation were calculated as the percentage of plants forming each structure out of the total number of plants. Statistical analysis was performed using Statcel-the Useful Add-in Software Forms on Excel (4th ed.; OMS Publishing), with percentage data subjected to arcsine transformation.
The effects of temperature and sugars on the in vitro growth of G. triflora GT-TY are shown in Table 1. Analysis of variance (ANOVA) revealed differences among temperatures for all growth parameters, including shoot length and the formation of flower buds, RSSs, and OWBs. ANOVA also showed differences among sugars in their effects on shoot length. For all sugars, shoot length was longest at 20°C/20°C, whereas the lowest rate of flower bud formation occurred at 15°C/15°C. Conversely, the rates of RSS and OWB formation tended to be lowest at 20°C/20°C and highest at 15°C/15°C. Pearson’s correlation coefficients using all data in Table 1 showed significant relationships at the 1% level: a positive correlation of 0.80 between the rates of OWB and RSS formation, a negative correlation of −0.70 between the rates of OWB and flower bud formation, and a negative correlation of −0.94 between the rates of RSS and flower bud formation.
Effects of temperature and sugars in the culture media on the growth of GT-TY in vitro (Experiment 1).
The effects of temperature on the in vitro growth of the four cultivars/lines are shown in Table 2. ANOVA indicated differences among cultivars/lines and temperatures for shoot length as well as the rates of flower bud and OWB formation, along with significant interactions between these factors. Differences in the rates of RSS formation were observed among cultivars/lines. Growth tended to differ between G. triflora and G. scabra. Specifically, G. triflora had longer shoot lengths and higher rates of flower bud formation compared with G. scabra, whereas G. scabra showed higher rates of RSS and OWB formation. Across all cultivars/lines, shoot length was longest at 20°C/20°C. The rates of flower bud formation tended to decrease at lower temperatures during the regeneration and/or the OWB induction stages in G. triflora, whereas they were consistently low in G. scabra, except at 20°C/20°C. The rates of RSS formation did not differ significantly among temperatures, but tended to be higher at lower temperatures during the regeneration and OWB induction stages in G. triflora, while being generally higher in G. scabra. The rates of OWB formation were significantly higher at 15°C/15°C in GT-TY, whereas no OWBs were formed in GT-FH. In GS-AM, these rates were higher at 20°C/10°C, whereas GS-G1 showed increased rates at 20°C/15°C, 20°C/10°C, and 15°C/15°C. Table 2 shows the effects of temperature combinations for both stages as a set, where specific comparisons were made within each cultivar/line. Meanwhile, multiple temperature treatments for each stage enabled comparisons of the effects of these temperature treatments for each stage (Supplementary Table S1). This comparison revealed significant differences in the rates of flower bud and OWB formation at the regeneration and/or OWB induction stages in some cultivars/lines, as well as in the rates of flower bud formation during the OWB induction stage in all cultivars/lines.
Effects of temperature on the growth of four gentian cultivars/lines in vitro (Experiment 2).
ANOVA revealed differences in the effects of sugars among cultivars/lines for all growth parameters, whereas no differences were observed among sugar treatments (Table 3). Consistent with Experiment 2’s findings, G. triflora exhibited higher rates of flower bud formation than G. scabra, whereas G. scabra showed higher rates of RSS and OWB formation than G. triflora. There were no discernible differences among sugar treatments within each cultivar/line. The differences in growth parameters observed between the 3% sucrose treatment of GT-FH (Table 3) and the 20°C/15°C treatment of GT-FH (Table 2) remain unexplained and warrant further investigation.
Effects of sugars in the culture media on the growth of four gentian cultivars/lines (Experiment 3-1).
In Experiments 1, 2, and 3-1, findings were presented in tables with ANOVA results to highlight overall trends and relationships among cultivars/lines and growth parameters. This approach clarified species-specific differences and parameter relationships. For Experiments 3-2 and 4, results are illustrated with graphs to focus on specific growth parameters in particular cultivars/lines (e.g., the effect of 1.0 mg·L−1 GA3 on OWB formation in GS-AM and GS-G1) rather than general trends. Figure 1 shows the effects of plant growth regulators on growth, comparing each treatment to the untreated control. Across all cultivars/lines, shoot length was longest with 1.0 mg·L−1 GA3. Despite plant growth regulator treatments, the rates of flower bud formation were higher in G. triflora compared with G. scabra, reaching 80% in most treatments. Notably, the rates of flower bud formation in G. triflora were lowest in ancymidol treatment, with GT-FH showing a significant difference compared with the control. Conversely, the rates of flower bud formation in G. scabra remained consistently below 10% across all treatments, with no significant differences observed among treatments.
Effects of plant growth regulators on the growth of four gentian cultivars/lines (Experiment 3-2). Shoot length, the rates of flower bud, RSS, and OWB formation were measured in plants cultured in vitro in a medium containing 2.0 mg·L−1 ancymidol (ANC), 0.1 mg·L−1 GA3 (GA0.1), 1.0 mg·L−1 GA3 (GA1.0), 0.5 mg·L−1 benzyladenine (BA), or no plant growth regulator (control). Error bars show standard errors of means. * and ** indicate significant differences at P < 0.05 and P < 0.01, respectively, between the control and each plant growth regulator treatment (Dunnett’s test).
Despite plant growth regulator treatments, the rates of OWB formation were generally higher in G. scabra compared with G. triflora (Fig. 1), although no OWBs were formed in G. scabra with 1.0 mg·L−1 GA3. The rates of OWB formation were generally low and showed no significant differences between the control and treatment groups for G. triflora, with ancymidol treatment yielding the highest rates: 27% for GT-TY and 7% for GT-FH. These OWB formation results were supported by RSS formation data.
4. Experiment 4: Effect of photoperiod on in vitro growthDue to poor growth during the regeneration stage under the 6- and 12-h photoperiods, GS-G1 was not included in Experiment 4. In GT-TY, shoot lengths and the rates of flower bud formation decreased with shorter photoperiods, whereas the highest RSS and OWB formation rates were observed under a 12-h photoperiod (Fig. 2). Conversely, no significant differences in growth parameters were observed among photoperiods in GT-FH (Fig. 2). In GS-AM, despite different photoperiods, no flower buds were formed, whereas the rates of RSS formation reached 100%. The rates of OWB formation were higher under an 18-h photoperiod than under a 6-h photoperiod.
Effects of photoperiod on the growth of three gentian cultivars/lines (Experiment 4). Shoot length and the rates of flower bud, RSS, and OWB formation were measured in plants cultured in vitro under 6-, 12-, and 18-h photoperiods. Measurements were taken 5 months (shoot length), 6 months (flower bud and RSS formation), and 9 months (OWB formation) after the OWB induction stage began. Error bars show standard errors of means. Different letters in each cultivar/line indicate significant differences between photoperiods at P < 0.05 (Tukey’s test).
The effects of four temperature combinations on GT-TY growth were initially assessed using media containing three different sugar compositions (Table 1). As the data in Table 1 includes the largest number of treatments per line in this study and shows a wide range of OWB formation rates, the data were considered appropriate for analyzing correlations between OWB formation rates and other growth parameters. Therefore, this dataset was pivotal for examining relationships between the rates of the OWB formation and other growth parameters. Pearson’s correlation coefficients based on all data in Table 1 revealed positive relationships between the rates of OWB and RSS formation, alongside negative relationships between the rates of OWB and flower bud formation and between the rates of RSS and flower bud formation. Moreover, these findings suggest that the rates of RSS and flower bud formation could serve as indicators of OWB formation. Based on these results, we focused on OWB formation, along with RSS and flower bud formation, to further investigate the effects of cultivars/lines and culture conditions on in vitro growth.
The results presented in Experiments 1 and 2 underscore the significant effects of temperature on all growth parameters, highlighting its efficacy in regulating in vitro growth. Previous studies typically used 22°C and 23°C as standard temperatures for in vitro culture (Hoshi et al., 2011; Imamura et al., 2014; Zhang and Leung, 2002). In the present study, the effects of 20°C/20°C and other temperature combinations were compared. Notably, in GT-TY, the 15°C/15°C treatment effectively reduced flower bud formation while increasing RSS and OWB formation. This result is supported by findings by Hoshi et al. (2011), who found that 15°C was effective for OWB formation, albeit with different starting plant materials and objectives compared with our study. Nevertheless, lower temperature treatments, such as 15°C/15°C, 20°C/10°C, and 15°C/10°C, moderately suppressed flower bud formation in G. triflora, although such low-temperature treatments are impractical for efficient OWB induction and subsequent cultured seedling production owing to their tendency to slow growth rates. Supporting this concept, in vitro plants with OWBs had higher biomass compared to those without OWBs (Supplementary Table S2), suggesting that OWBs form in well-developed plants, necessitating specific temperature conditions to simultaneously promote growth and OWB formation.
Although the impact of the 20°C/15°C temperature treatment on the rates of flower bud, RSS, and OWB formation was less pronounced than that of 15°C/15°C, significant differences were observed in those parameters compared with 20°C/20°C in certain cultivars/lines. Therefore, 20°C/15°C was further explored in combination with other treatments as a practical method for inducing OWB formation. In Experiment 4, conducted at 20°C/15°C, OWB formation ranged from 26.7% to 80.0% in the two G. triflora cultivars/lines (Fig. 2). The rates of OWB formation in G. triflora were notably higher in Experiment 4 compared with the other experiments, and this may be attributable to an extended nine-month OWB induction period. Despite the relatively high rate of OWB formation under the 20°C/15°C treatment compared to Experiments 1 and 2, optimizing treatments other than temperature are necessary to shorten the induction period.
In Experiment 3, regarding the medium composition, the effects of sugar on in vitro growth were initially examined. Previous studies have suggested that increasing the sucrose concentration from 3% to 5% or 6% enhances OWB formation (Imamura et al., 2014) and suppresses flowering (Zhang and Leung, 2002) in G. triflora. Based on these findings, a 6% sucrose concentration was compared with the standard 3%. However, no significant effects of sugar composition were observed, except for shoot length in GT-TY. Discrepancies relative to previous studies, i.e., differences in the effects of increased sugar concentrations in the medium on in vitro growth, OWB and flowering, may be due to variations in cultivars/lines, starting materials, and culture conditions, although definitive conclusions remain elusive. Fructose and trehalose were also tested owing to their growth-promoting effects in preliminary experiments and their extended incubation benefits, previously reported by Yamaguchi et al. (2011). However, no significant growth differences were observed with these sugars.
Zhang and Leung (2002) demonstrated that benzyladenine, but not GA3, induces flower bud formation during the in vitro culture of G. triflora. Therefore, the effects of gibberellin, its inhibitor ancymidol, and cytokinin were next investigated. In G. scabra, which exhibited higher rates of RSS and OWB formation compared with G. triflora in Experiments 2, 3-1, and Experiment 3-2 (control), GA3 at 1.0 mg·L−1 markedly reduced these formation rates, highlighting gibberellin’s negative impact on RSS and OWB formation. Conversely, G. triflora rarely exhibited RSS and OWB formation across all treatments, except with ancymidol treatment. Additionally, the rates of flower bud formation decreased in GT-FH when treated with ancymidol. Previous greenhouse experiments showed that gibberellin suppresses crown bud formation (presumed OWBs), whereas gibberellin synthesis inhibitors promote such formation (Samarakoon et al., 2015). Although the applicability of greenhouse results to in vitro culture experiments is uncertain, the previous study may support the current results suggesting a relationship between gibberellin and the rates of flower bud, RSS, and OWB formation. Further investigation into gibberellin’s role in in vitro growth, including improved gibberellin inhibitors and their application, could be beneficial for promoting OWB formation.
GT-TY exhibited flower bud formation when promoted under longer photoperiods in vitro. Shoot length in GT-TY did not differ between 6- and 12-h photoperiods (Fig. 2), and no increase in leaf number, plant fresh weight, or plant dry weight was observed under extended photoperiods (data not shown), suggesting that the promotion of flower bud formation under longer photoperiods was not due to enhanced vegetative growth. In contrast, the effect of photoperiods was less clear in the other two cultivars, indicating variability in their responses to photoperiods for flower bud formation during in vitro culture.
In GT-TY, the rates of OWB formation were higher under a 12-h photoperiod compared with an 18-h photoperiod. This enhanced OWB formation under short-day conditions relative to long-day conditions has been reported in greenhouse cultivation (Samarakoon et al., 2015), supporting our results for GT-TY. In contrast, in GS-AM, OWB formation rates were higher under an 18-h photoperiod compared with a 6-h photoperiod, and photoperiod had no effect on OWB formation in GT-FH. Despite the photoperiod appearing to influence OWB formation, further study is necessary to clarify the different responses observed between cultivars/lines. Although photoperiod effects are not usually considered in in vitro culture, our results suggest that the photoperiod warrants close attention.
This study revealed the influence of various culture conditions, including temperature, gibberellin administration, and photoperiod, on the growth of G. triflora and G. scabra. Regarding OWB formation, treatments such as low temperature (15°C) after a certain amount of growth and combination treatments involving photoperiod modification and gibberellin inhibitors show promise. Moreover, the distinct responses between species, and the variability in flowering, RSS production, and OWB formation among gentians, provide valuable insights for future physiological and genetic studies focusing on these plants.
We would like to thank Dr. Bunzaemon Kanke for his valuable advice. We would also like to thank the many researchers and technicians in the Crops and Horticulture Department of the Fukushima Agricultural Technology Center for their support and cooperation.
