Unrooted cuttings of Chrysanthemum cv. Vivid Scarlet provided by the National Institute of Horticultural and Herbal Science (South Korea) were transplanted into a greenhouse at 25 °C under ambient long-day conditions. After 4 weeks, the shoot tips were taken and then thoroughly washed under running tap water with 5% of liquid detergent. They were subsequently surface-sterilized with a 70% (v/v) ethanol solution for 30 s and 1% NaOCl plus Tween 20 for 5 min, followed by five rinses with sterilized distilled water. The sterilized shoot tips were then cultured on plant growth regulator (PGR)-free Murashige and Skoog (MS) medium containing 1 g L⁻¹ of activated charcoal to induce plant growth. After 6 weeks of culture, the youngest fully expanded leaves from the in vitro-grown healthy and uniform plants were used as a source of explants for further experiments.

To determine the best combination of plant growth regulators (PGRs) for optimal shoot regeneration, leaves were cut into 0.5–1-cm-long segments and cultured on MS medium supplemented with various concentrations of 6-benzyladenine (BA) and α-naphthaleneacetic acid (NAA), as shown in Table 1, along with 3 g L⁻¹ of Gelrite. The explants were cultured under condition with 16 h photoperiod of 37 μmol m⁻² s⁻¹ for 45 days.

To investigate the effects of the dark incubation period on shoot regeneration, explants were prepared as described above (0.5–1-cm-long segments), and the resulting explants were incubated for 10, 20, or 30 days in the dark, after which they were subjected to culture conditions with 16 h photoperiod of 37 μmol m⁻² s⁻¹ and kept for 45 days, including the initial dark incubation period.

Leaf segments were cultured on a MS medium supplemented with 1 mg L⁻¹ of BA and 2 mg L⁻¹ of NAA, which resulted in the highest number of shoots per explant. To investigate the effects of the gelling agents on the capacity of shoot regeneration, the induction medium was solidified with 7 g L⁻¹ of Agar (Duchefa Biochemie, The Netherlands), 4 g L⁻¹ of Agarose (Cambrex, USA), 3 g L⁻¹ of Gelrite (Duchefa Biochemie, The Netherlands), or 3 g L⁻¹ of Phytagel (Duchefa Biochemie, the Netherlands). The explants were cultured under condition with 16 h photoperiod of 37 μmol m⁻² s⁻¹ for 45 days.

A medium supplemented with 1 mg L⁻¹ of BA, 2 mg L⁻¹ of NAA, and 3 g L⁻¹ of Gelrite showed the best results for shoot induction. Therefore, different concentrations (0–5 μM) of silver nitrate were added to this shoot induction medium to test its effects on the capacity of shoot induction from leaf segments. The explants were cultured under condition with 16 h photoperiod of 37 μmol m⁻² s⁻¹ for 45 days.

To investigate the effects of gelling agents on rooting and plant growth of regenerated shoots in vitro, regenerated shoots were transferred to a plant-growth regulator (PGR)-free MS medium solidified with the following gelling agents: 7 g L⁻¹ of Agar (Duchefa Biochemie, the Netherlands), 4 g L⁻¹ of Agarose (Cambrex, USA), 3 g L⁻¹ of Gelrite (Duchefa Biochemie, the Netherlands), or 3 g L⁻¹ of Phytagel (Duchefa Biochemie, the Netherlands). The explants were cultured under conditions with a 16-h photoperiod of 37 μmol m⁻² s⁻¹ for 45 days.

All cultures contained 3% of sucrose, and each treatment consisted of 30 explants and three replicates. For shoot regeneration, the number of usable shoots (about 3 cm in height) formed per explant was recorded after 45 days of culture. Similarly, plant growth parameters were considered after 45 days of culture. All data were analyzed using ANOVA, and means were compared by DMRT (P > 0.05).

Plantlets obtained from hormone-free MS medium solidified with Gelrite were selected at random and subjected to flow cytometry, which was performed as described by Naing et al. [8]. Briefly, approximately 20 mg of tissue per sample were chopped using a sharp razor blade in a plastic Petri dish containing 500 mL of a nuclei extraction buffer (Partec, GmbH, Munster, Germany) to obtain a fine suspension. The samples were then filtered through a nylon mesh (50 μm), after which 2 mL of staining buffer (Partec, GmbH, Munster, Germany) were added and the suspension supplemented with propidium iodide. The ploidy level of each sample was then measured using a flow cytometer (CyFlow@ Ploidy Analyser, Partec, GmbH, Munster, Germany). A minimum of 2000 particles (total count) was analyzed in each sample, and the measurements were carried out using at least three samples. The leaf tissue from mother plants in the greenhouse was used as a hexaploid reference.

To determine the effect of the cultivar on shoot induction, 6-week-old in vitro grown expanding leaves of several cultivars (Borami, Peach Andy, Pink Pride, Star, Baeksun, Baekma, and Shinma) were cut into 0.5–1.0-cm-long segments, which were then cultured on a shoot induction medium supplemented with the combination of 1 mg L⁻¹ BA and 2 mg L⁻¹ NAA along with 3 g L⁻¹ of Gelrite. The explants were maintained under culture conditions, as explained above.

Leaf explants were cultured on MS medium containing different concentrations of BA and NAA under continuous light conditions without any dark initial period, after which morphogenetic changes in the explants were observed after 2 weeks of culture. Adventitious shoot primordia were visible on the surface of leaf explants (Fig. 1A). Development of shoots occurred over the next 3 weeks after initiation. Shoot regenerability of leaf explants was highly variable in response to PGR concentrations. Of the various PGR combinations tested, only a few combinations showed shoot regenerability, especially media containing higher concentrations of NAA than BA (Table 1).

Shoot regeneration occurred for all combinations of PGRs when explants were subjected to an initial dark period of 10 days (Table 2). However, PGR combinations that showed shoot induction under continuous light condition showed negative effects on shoot regenerability when the explants were cultured with the initial dark period of 10 days. Reduction of shoot regeneration was also detected for all PGR combinations when explants were incubated for 20 days in the dark (Table 3). Total inhibition of shoot regeneration was observed for some PGR concentrations upon dark incubation for 30 days (Table 4). Therefore, PGR concentration and dark incubation period had interactive effects on shoot regeneration. Our results show that a dark incubation period of 10 days resulted in a more reasonable number of shoots per explant in most PGRs combinations compared to the other periods (0, 20, or 30 days). However, the highest frequency of shoot regeneration as well as the mean number of shoots per explant were obtained using a medium containing a combination of 1 mg L⁻¹ of BA and 2 mg L⁻¹ of NAA under continuous light conditions without any initial dark period. Thus, this medium composition and culture conditions were used in further experiments.

The effects of the gelling agents on shoot formation from leaf explants of Chrysanthemum are presented in Fig. 2A and B. Shoot formation was observed in all media after 3 weeks, and the percentages of shoot formation were not significantly different among the gelling agents. However, the mean number of shoots formed per explant was dependent on the gelling agent according to the following order: Gelrite > Phytagel > Agar > Agarose. Shoots induced by Gelrite were likely to be more vigorous and healthy than those induced by other gelling agents. Explants cultured on Agarose medium curled upward and resulted in shoots with abnormal patterns, which was in contrast to explants cultured on other gelling agents. This result clearly indicates that Agarose had stronger negative effects on shoot regeneration compared to Phytagel or Agar, both of which induced lower shoot regenerability than Gelrite. Thus, Gelrite was the best gelling agent for increasing both the percentage of shoot formation as well as the number of shoots per explant.

To examine the stimulatory effects of silver nitrate on shoot regeneration from leaf explants of Chrysanthemum, explants were cultured on a shoot induction medium solidified with Gelrite. As shown in Fig. 3A and B, the presence of silver nitrate markedly reduced shoot regeneration compared to control. In addition, leaf burning was observed on shoots treated with 1 or 5 μM of silver nitrate. Elevation of silver nitrate concentrations up to 10 or 25 μM completely inhibited shoot regeneration. Based on these findings, silver nitrate had highly negative effects on shoot regeneration from leaf explants of Chrysanthemum.

The data presented in Table 5 show that plant growth parameters were influenced by the type of gelling agent. Shoots grown on Gelrite showed better parameters, especially for number of roots, plant height, and fresh weight, compared to the other gelling agents (Fig. 1C). However, Agar produced the highest average number of leaves per plantlet as well as it showed superior effects on most parameters compared to Phytagel and Agarose. Similar to its effect on shoot regeneration, Agarose had negative effects on rooting and plant growth. Further, Gelrite resulted in the greatest number of shoots per explant along with improved plant growth compared to the other gelling agents. Plantlets that reached approximately 4–5 cm in height were transferred to an acclimatization pot (Fig. 1D), and more than 90% of plants survived in the greenhouse.

Histograms based on the mother plant (control) and plantlets derived from hormone-free medium are shown in Fig. 4A and B. The flow cytometry results show that peaks of the analyzed samples occurred at nearly the same positions as those of standard hexaploid leaf cells (control), which indicates a high level of genetic stability. In addition, the means of the peaks were almost the same as that of the control. These findings demonstrate that the regeneration method had no effect on the induction of polyploids.

To examine the applicability of the established protocol to the other Chrysanthemum cultivars, shoot organogenesis was investigated in six commercially important cultivars. Shoot formation was detected in four of the cultivars, and the mean number of shoots per explant for each cultivar is presented in Table 6. The highest mean number of shoots per explant was observed in cv. Borami, followed by cv. Star, Pink Pride, and Peach Andy. No shoot formation was detected in cv. Baeksun or Baekma. Based on the results, the selected combination of PGRs could stimulate shoot formation in four out of six cultivars.