Introduction
Lilium dauricum, a perennial herb in Korea, belongs to the family Liliaceae. The bulb of the species contains various bioactive compounds, and it is used as a functional food and medicinal agent in Northeast Asia (Mimaki et al. 1992). Multiple uses, high demand, and destructive harvesting have led to habitat shrinkage of the wild populations of L. dauricum. This species is currently categorized as endangered in the IUCN Red List (Korean Red List 2014). The species is regionally protected as Endangered Wildlife by the law. L. dauricum regenerates poorly through seed germination and bulb scale in the wild; therefore, it is necessary to develop a method of in vitro micropropagation to preserve and develop this rare species.
Micropropagation of Lilium species has been successfully established and developed to propagate and improve in quality of lilies (Bakhshaie et al. 2016;Dhyani et al. 2014). An efficient micropropagation protocol for L. longiflorum using pseudo-bulblets and transverse thin-cell layers of the young stem has been established in various species of lily (Bui et al. 1999;Nhut et al. 2001). Direct bulblet regeneration using various explant types such as bulb scale, leaf, and stem or callus has been performed for L. leucanthum, L. candidum, and L. polyphyllum (Dhyani et al. 2014;Saadon and Zacai 2013;Tang et al. 2010). Mori et al. (2005) reported callus formation from cultured explants and plant regeneration from induced calli in 33 Lilium genotypes. Bulblet scale has been found to be the most common explant type during the regeneration of bulblets in Lilium spp.
Recently, advances have been made in plant biotechnology via plant cell and organ culture for not only micropropagation but also production of bioactive compounds, especially in medicinal plants (Murthy et al. 2014a;Park and Paek 2014). It can be used to directly mass produce a bioactive compound from different culture systems such as cell suspension, somatic embryogenesis, adventitious roots, and hairy roots as well as mass multiply medicinal plants (Park and Paek 2014;Sridhar and Aswath 2014). Callus culture is a beneficial system for the production of bioactive compounds, and it is an attractive source of phytochemicals because of the rapid growth and biosynthetic stability of calli. Therefore, the aims of this study were to establish an in vitro protocol for bulblet culture for mass proliferation of L. dauricum and develop callus culture for the production of bioactive compounds. Additionally, skin care efficacies of the extracts of bulblet and callus were investigated in order to develop novel plant-based biomaterials that contain high-value bioactive compounds for the cosmetic and pharmaceutical purposes.
Materials and Methods
Plant materials
Lilium dauricum was grown in a greenhouse at the Floriculture and Biotechnology Laboratory, Chungbuk National University, and used for initial culture (Fig. 1a - c). The bulbs were surface-sterilized with 70% (v/v) ethanol for 30 s and 2% (v/v) sodium hypochloride for 20 min and then rinsed thoroughly six times with sterile distilled water. The bulb-scale segments (0.3 × 0.5 cm) were placed on full-strength MS medium (Murashige and Skoog 1962) supplemented with 1.0 mg·L-1 BA, 0.1 mg·L-1 NAA, 30 g·L-1 sucrose, and 2.6 g·L-1 gelrite for plantlet regeneration. The cultures were maintained at 24 ± 1°C and 16 h photoperiod. The plantlets were subcultured every 6 weeks. The bulblets were used for further experiments.
Bulblet induction
In vitro bulb-scale segments (0.3 × 0.5 cm) were cultured on MS medium supplemented with different concentrations of BA (0 - 2.0 mg·L-1) to investigate the effect of BA concentration for bullet formation. To investigate the efficient culture system, in vitro bulblet scales were cultured using 3 different culture systems; petri-dish, Erlenmeyer flask, balloon type air-lift bioreactor. The cultures were conducted in MS medium supplemented with 0.5 mg·L-1 BA, 3% (w/v) sucrose. All cultures were maintained at 24 ± 1°C in d ark for 4 week s.
Bulblet enlargement
Small bulblets (around 5 mm in diameter) were culture in the basal MS medium supplemented with difference sucrose concentration (3, 5, 7, and 9%). The experiment was carry out in 1L BTBB containing 0.5 L medium at at fixed temperature (24 ± 1°C) and dissolved oxygen at 0.1 vvm (air volume/culture volume min) under dark conditions. The biomass (fresh and dry weight), growth index and bulblets diameter were investigated after 6 weeks of the culture period.
Callus induction
Callus was induced from the bulblet scales on MS medium supplemented with various concentrations of 2,4-D or picloram (0 - 2 mg·L-1) in combination with BA or Kin (0 - 0.5 mg·L-1). The explants were cultivated on solid MS medium containing 2.6 g·L-1 gelrite in petri dishes (diameter, 9 cm) and maintained in the dark at 24 ± 1°C. Further, leaves and small scales were cultured on selection medium to compare callus induction from different explant types.
Callus proliferation
Callus were cultured on MS medium supplemented with various concentrations of 2,4-D or picloram (0 - 1.5 mg·L-1) in combination with BA, NAA, 4-CPA or Kin (0 - 2 mg·L-1) in order to select the suitable medium for callus proliferation. All the treatment was supplemented with 250 mg·L-1 casein hydrolysate to enhance the ability of callus proliferation.
Quantification of phenolic compounds from bulblet and callus
The bulblets and calli (dry weight) of L. dauricum were reduced to powder, and 50 mg samples were sonicated (Sonicator, Mujigae, Korea) for 3 h with 80% methanol to ensure complete extraction of phenolic compounds. The extracts were filtered using filter paper (110 mm; Advantic, Japan), and the solvent was evaporated. The dried residue was dissolved in 10% methanol and fractionated twice with 10 mL diethyl ether:ethyl acetate (1:1) prior to evaporation to dryness under vacuum. The residues of both fractions were combined and dissolved in methanol prior to filtration with a membrane filter (0.2 μm pore; Whatman, UK). A photodiode array equipped a high-performance liquid chromatography (HPLC) system (2690 Separation Module; Waters Chromatography, Milford, USA) was used to measure the levels of phenolic compounds. The separation was performed using a Fortis C18 column (5 μL, 150 × 4.6 mm). Acetonitrile (A) and 0.1% aqueous acetic acid (v/v) (B) were used as the mobile phase with linear gradients of 8% A at 0 min, 10% A at 2 min, 30% A at 27 min, 90% A at 50 min, 100% A at 51 - 60 min, and 8% A at 70 min. Re-equilibrium for 10 min was performed between different injections at a flow rate of 1.0 mL·min-1 flow rate. Sample aliquots (20 μL each) were injected into the HPLC system one at a time. Individual phenolic compound standards were purchased from ChromaDex (USA). The calibration plots were created by measuring the peak areas. UV absorption spectra and retention time were used as criteria to identify individual compounds. The phenolic contents were calculated as follows: Phenolic contents (mg·g−1 DW) = [Phenolic fraction concentration (mg·L−1) × Dissolved solvent volume (L)]/Sample dry weight.
Determination of antioxidant activity
The antioxidant capacity of the callus and bulblet of L. dauricum was measured using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method (Hanato et al. 1998); 0.8 mL of 200 μM DPPH radical solution was added to 0.2 mL of methanolic root extract, and 40% methanol served as the control. The solutions were incubated for 5 min at room temperature, and absorbance at 517 nm was measured using a spectrophotometer (Optizen POP; Mecasys Co., Ltd., Korea). The results were presented as follows:
Statistical analysis
All experiments were performed using a completely randomized design and at least three replicates. The mean and standard error (SE) values were calculated. Statistically significant differences were determined using Duncan’s multiple range test in SAS software (version 9.4, SAS, USA).
Results and Discussion
Direct regeneration of bulblet from the scales
The establishment of an effective in vitro plant cell and tissue culture is very important for lily propagation. Lilies can be propagated using various types of explants, such as pollen, somatic embryo, stem node, pseudo-bulblet, bulblet, and bulb scale (Bakhshaie et al. 2016;Nhut 2003). In the present study, the MS medium supplemented with 0.5 mg·L-1 BA was found to be suitable for bulblet induction from the scales of L. dauricum, with 85% induction rate and 1.6 bulblets per explant (Table 1 and Fig. 1d - g). Direct bulblet regeneration from the scales cultured in vitro was observed within 2 weeks of culture, and the number of bulblets gradually increased in 4 weeks (Fig. 1d - g). These findings are similar to those of Saadon and Zaccai (2013), who reported that bulblet regeneration of L. candidum was most efficient from the scales. Numerous studies have also reported that bulb scales are the most common explant type for the regeneration of bulblets in Lilium spp. (Bakhshaie et al. 2016;Mori et al. 2005;Nhut 2003;Tang et al. 2010). Our results suggest that successful regeneration of L. dauricum by using bulb scales is possible by mass micropropagation for preservation and shortening the breeding program of this rare species.
Bulblet induction and proliferation in difference culture system
Rapid mass propagation and regeneration of plants using biotechnological tools is often of considerable interest to lily breeders (Bakhshaie et al. 2016;Paek et al. 2005). In this experiment, in vitro bulblet induction and enlargement of L. dauricum from bulblet scale segments, were studied using various culture system. Although the percentage of bulblet formation was higher in solid cultures, an increased growth rate, and increased bulblet diameter were observed in BTBB cultures. The MS medium, supplemented with 0.5 mg·L-1 BA and 3% sucrose, had the highest bulblet production, compared to other treatments (213 bulblets per bioreactor, 18 g·L-1 dry weight, and 12.53-fold in growth rate) (Table 2 and Fig. 2). The characteristic of bullet also better in bioreactor culture, with 6.39 mm in diameter, especially bulblet weight reach to 640 mg per bulblet, approximately 7-fold heigher than in petri dish (93 mg per bulblet) (Fig. 2).
The use of bioreactor for micropropagation has proved applicable to many species and plant organs including shoots, bulbs, microtubers, corms and somatic embryos (Paek et al. 2001, 2005). Compare to solid and liquid culture, the bioreactor culture enable a constant supply of nutrients as well as aeration to explants leading to the bulblet formation. Lian et al. (2003a) reported that more than 1,000 bulblet of Lilium Oriental Hybrid ‘Casablanca’ were harrvested in each bioreactor culture, thus bioreactor was facilitates scaling up of bulblet culture. Therefore, the use of bioreactor culture was suitable for bulblet propagation of Lilium dauricum.
Effect of sucrose concentration on bulblet enlargement
The biomass and diameter of bulbet were increased and expanded with an increasing of sucrose concentration (except at 9%) in bioreactor culture (Table 3 and Fig. 3). At the medium supplemented with 7% sucrose, the bulblet reach to the highest enlargement with 8.79 mm in diameter and 925.8 and 266.1 mg/bulblet of fresh and dry weight (Fig. 3a, b). The total biomas (g·L-1 meidum) also highest at 7% sucrose (92.47 and 26.3 g·L-1 of FW and DW, respectively). In the other hand, the percentage of dry weight increase in higher sucrose concentration, up to 9% (33.79%), whereas approximately 19.54% at 3% sucrose concentration.
The effect of sucrose on the formation and expansion of Lilium spp. was obvious and dominant (Gao et al. 2018). Sucrose can be used as the necessary source of carbon and energy during the growth and development of plants, especially, the formation and development of bulblets are crucial to the Lilium genus, since the process are closely related to carbohydrate metabolism (Gao et al. 2018;Martínez et al. 2015;Zhang et al. 2017). Varshney et al. (2000) indicated that sucrose was effective at bulblet multiplication not only is the concentration of energy source, but also with which the cultivar that is being used. They found that 6% is optimal sucrose concentration for bulblet multiplication of cultivar Gran Paradiso, wheares cultivar Sanciro, 9 % sucrose was optimum. L. sargentiae bulblet was enlarged at 6% sucorse. In bioreacor culture of Lilium Oriental hybrid ‘Casablanca’, Lian et al. (2003b) reported that the size of the bulblets was improved on a medium fortified with 9 % sucrose. In addition, sucrose played a physiological role and might also play a signal role in enlargement of bulblets (Gao et al. 2018). In the present study, 9% sucrose inhibited bulblet growth and also showed the numerous abnormal bulblets. Thus 7% sucrose was optimum for bulblet enlargement of L. dauricum.
Callus induction
Plant growth regulators had a significant effect on the percentage of callus formation on the callus formation media. A combination of 2,4-D and BA or Kin showed a low percentage of callus induction (Fig. 4a, b). Moreover, bulb formation was observed in this medium. Therefore, 2,4-D in combination with BA or Kin is not suitable for callus induction. The highest percentages of callus induction from the scale (100%) were obtained on MS medium containing 1 mg·L-1 picloram and 0.5 mg·L-1 Kin without any bulblet formation (Fig. 4c, d). The increase in picloram concentration inhibited callus formation in L. dauricum. In this study, a callus of L. dauricum was induced from both scale and leaf of the in vitro plants (Fig. 4).
Leaves and scales have been successfully used as explants for callus induction in L. leucanthum (Tang et al. 2010), which is consistent with our results. Mori et al. (2005) also reported that scales, followed by leaves, displayed the greatest ability to form callus in Lilium genotypes. A combination of 2,4-D and BA is suitable for callus induction and regeneration of Lilium species. However, this combination was unsuccessful in establishing the callus culture of L. dauricum because both calli and bulblets were observed in this medium. The aim of our study was the induction and proliferation of callus for producing biomass and bioactive compounds in L. dauricum. Thus, MS medium containing 1.0 mg·L-1 picloram in combination with 0.5 mg·L-1 Kin was optimized for callus induction in L. dauricum. Fig. 5
Callus proliferation
In order to select the suitable medium for callus proliferation, callus were cultured on MS medium supplemented with various concentrations of 2,4-D or picloram (0 - 1.5 mg·L-1) in combination with BA, NAA, 4-CPA or Kin (0 - 2 mg·L-1). Fig. 6 showed difference affected of auxin and cytokonin in combination on callus proflifereation of Lilium dauricum. There are no significane difference in biomass production among all treatments (Fig. 6a). However, a combination of 2,4-D and BA, NAA, or 4-PCA, Kin was unsuccessful in propagation of the callus culture because both calli and organ formation (bulblet, root, etc.) were observed in this medium (Fig. 6b). Whereas the medium supplement of picloram and Kin, no organ was obtain in this medium. A combination of 1 mg·L-1 pic and 0.25 mg·L-1 Kin show the most suitable for callus propagation with 2.35 g/petri dish and no organ were observed. A combination of auxin and cytokinin effected on the callus induction and proliferation in Lilium species; however, the optimum concentrations may vary among species (Bakhshaie et al. 2016;Sahoo et al. 2018). In this case 2,4-D combine with BA or NAA were most effective in callus proliferation in many Lilium species (Mori et al. 2005;Sahoo et al. 2018;Tang et al. 2010). In contrast, our study show that the medium supplemented with 2,4-D combine with BA, NAA, 4-CPA or Kin were poor to proliferate of callus; the develop of organogenesis inhibited callus growth in this case (Fig. 6b). Whereas 1.0 mg·L-1 picloram combine with 0.25 mg·L-1 kinetin is most suitable for callus proliferation in L. dauricum in this study.
Comparison of bioactive compound production and antioxidant activity in bulblet and callus
The qualitative and quantitative levels of phenolic compounds from the bulblet and callus culture of L. dauricum were determined using HPLC analysis. The calli showed greater accumulation of phenolic compounds than the bulblets (not only total phenolic content but also each phenolic group and individual phenolic compound; Fig. 7a, b). A significant difference was observed in the total phenolic content of the calli (2.31 mg·g-1 DW), and it was 1.55-fold higher than that in the bulblet (1.49 mg·g-1 DW). The flavonol content was higher than that of another group in both calli and bulblets (0.67 and 0.37 mg·g-1 DW, respectively), followed by hydroxynamic acid (0.49 and 0.26 mg·g-1 DW, respectively). Physcion (0.15 and 0.15 mg·g-1 DW), quercetin (0.22 and 0.13 mg·g-1 DW), myricetin (0.42 and 0.19 mg·g-1 DW), and salicylic acid (0.24 and 0.20 mg·g-1 DW) were the dominant phenolic compounds accumulated in both calli and bulblets, and their contents were higher in the calli than in the bulblets. These results indicate that the callus culture contained larger amounts of phenolic compounds than the bulblet culture. Therefore, callus could be a potential material for large-scale production of bioactive compounds in L. dauricum.
Plant cell, tissue, and organ culture (e.g., callus, embryo, adventitious root, hairy root, and shoot) for the production of bioactive compounds in various medicinal plant species have been widely reported (Ho et al. 2018b;Murthy et al. 2014a). For example, shoot culture of Bacopa monnieri was established for the production of bacoside A (Praveen et al. 2009). Adventitious root and hairy root cultures were successfully established for the production of phenolic compounds in Polygonum multiflorum (Ho et al. 2017, 2018a, b). Cells and adventitious roots have been used for enhancing ginsenoside content in Panax ginseng (Le et al. 2018;Murthy et al. 2014b;Thanh et al. 2014). In the present study, both callus and bulblet cultures of L. dauricum showed high contents of phenolic compounds. These results have a great significance for not only for micropropagation of L. dauricum, a rare and endangered medicinal plant in Korea, for preservation and breeding but also production of bioactive compounds. When compared with the bulblet culture, callus culture was found to be a potential source for scale-up production in a bioreactor for the accumulation of biomass and bioactive compounds.
In addition, we investigated the antioxidant activity on the basis of DPPH free-radical scavenging in the callus and bulblet cultures (Fig. 7c). DPPH activity is generally associated with high levels of bioactive compounds. Similar to the yields of the phenolic compounds, DPPH radical-scavenging activity was significantly higher in the calli than in the bulblets. Bioactive compounds, particularly phenolic compounds, significantly contribute to the antioxidant activity of plant tissues (Ferrat et al. 2003). Ho et al. (2018a) reported that the accumulation of phenolic compounds in P. multiflorum adventitious root cultures may be responsible for enhancing DPPH radical-scavenging activity. Similarly, a positive correlation was reported between DPPH activity and accumulation of phenolic compounds in Vitis vinifera (Cai et al. 2012). The higher accumulation of phenolic compounds may have resulted in higher levels of DPPH activity in the callus culture than in the bulblet culture of L. dauricum.
Conclusions
In our study, a simple and efficient protocol for bulblet and callus induction from the bulb scale of L. dauricum was established. Direct bulblet regeneration was observed from the scales cultured in vitro. The scales also showed a higher frequency for callus induction in medium supplemented with picloram and Kin. In addition, the callus showed higher accumulation of bioactive compounds than the bulblet.
