Abstract
Melatonin plays an important role in plant resistance to stress. The role of melatonin in the propagation of plant tissue, such as rhizome proliferation, differentiation, and seedling rooting in Cymbidium species, remains unknown. In this study, we selected C. goeringii and C. faberi as experimental materials and attempted to understand the effect of melatonin on this process. We found that 1.0 μM melatonin was beneficial for rhizome proliferation of C. goeringii, with a proliferation rate of 5.52. In terms of C. faberi, the highest proliferation rate of 8.29 was observed in the medium with 0.5 μM melatonin. In proliferation, the cut rhizome of C. goeringii is more likely to cause browning phenomenon than that of C. faberi. The addition of melatonin can significantly inhibit the browning phenomenon and improve the survival rate of C. goeringii rhizome. The highest number of shoot buds per explant (3.11 after 60 days) was observed in the medium with 1.0 μM melatonin. The number of shoot buds per explant (3.28 after 60 days) was significantly higher for C. faberi in the medium with 5.0 μM melatonin than that for the control. Furthermore, culture medium incorporated with 1.0 μM of melatonin had the best comprehensive effect of seedling height and root number and length of C. goeringii. By contrast, 0.5 μM melatonin significantly promoted root elongation of C. faberi, reaching 1.77 cm, whereas it was 0.28 cm in the control. We demonstrated that melatonin in specific concentrations effectively promote rhizome proliferation, differentiation, and seedlings rooting in the rapid propagation of C. goeringii and C. faberi.
Cymbidium, one of the most important orchid genera in horticulture, has ≈52 epiphytic and terrestrial species (Islam et al., 2015; Kumar et al., 2022). The terrestrial seedlings are difficult to propagate compared with epiphytic species (Liu et al., 2017). A few terrestrial Cymbidium species, such as C. goeringii, C. faberi, C. sinense, C. ensifolium, and C. kanran, are widely cultivated for their beautiful and fragrant flowers (Huang et al., 2012). Owing to human activities and global environmental changes, these species are threatened with extinction in the wild (Popova et al., 2016).
In recent years, tissue culture has been introduced in Cymbidium for the mass production of seedlings to meet market demand (Islam et al., 2015). Terrestrial Cymbidium seeds form protocorms after germination and develop into rhizomes in vitro. Rhizomes are recalcitrant to regeneration and differentiation; therefore, the shoots grow slowly and inconsistently, even after induction (Paek and Kozai, 1998). Additionally, rhizomes quickly turn brown, limiting further growth (Huang and Fang, 2021). Their rooting phase is also difficult. Species, plant growth regulators, and culture environment can affect the ability of rhizome proliferation, differentiation, and rooting. Among numerous factors, plant growth modulators such as auxins and cytokinins play key roles in vitro (Shimasaki and Uemoto, 1990). Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthaleneacetic acid (NAA) are effective in inducing rhizome proliferation in Cymbidium (Guha and Rao, 2012; Park et al., 2018; Shimasaki and Uemoto, 1990). Besides, it was reported that activated charcoal (AC) can effectively promote rhizome proliferation in C. sinense (Gao et al., 2014). For rhizome differentiation, it was revealed that medium supplementing 6-benzylaminopurine (6-BA) and thidiazuron (TDZ) was most effective for shoot buds induction of C. faberi and C. kanran, respectively (Fukai et al., 2000; Tao et al., 2011). Furthermore, a medium incorporated with 20 μM 2,4-D and 2 μM TDZ was most effective in inducing adventitious buds in C. goeringii (Park et al., 2018). Moreover, an increased level of indoleacetic acid (IAA) is required for high-frequency shoot regeneration in Cymbidium hybrids (Liu et al., 2017). In the C. goeringii rooting stage, 2 μM of NAA works best, and the mean of 5.3 ± 1.1 roots per shoot was achieved after 12 weeks of culturing (Park et al., 2018). Recently, it was demonstrated that certain combinations of amino acids can effectively promote the proliferation and differentiation of rhizomes during the rapid propagation of C. goeringii (Huang and Fang, 2021).
Melatonin (N-acetyl-5-methoxy-tryptamine) is an indoleamine that was initially isolated from the bovine pineal gland and was structurally characterized (Lerner et al., 1958, 1959). Melatonin was further detected in several higher plants such as tomato, banana, cucumber, and tobacco (Dubbels et al., 1995). Subsequently, it was confirmed that melatonin is widely present in bacteria, fungi, algae, animals, and plants (Hardeland et al., 2011). Although melatonin content in plants is low, it plays a vital role in regulating growth and development of plants. Melatonin regulates the growth of roots, shoots, and explants to activate seed germination and rhizogenesis and to delay induced leaf senescence (Arnao and Hernández-Ruiz, 2014, 2015). Furthermore, it is used commercially in the postharvest management of fruits (Jayarajan and Sharma, 2021).
Increasingly, studies have demonstrated that melatonin can act as a hormone and functionally synergize with other plant hormones (Hwang and Back, 2018; Zhang et al., 2019). In tissue culture systems, few advances have been made in understanding the role of melatonin in vitro (Hernández-Ruiz et al., 2021; Litwińczuk and Wadas-Boroń, 2009; Malik et al., 2021; Zhang and Zhang, 2021). In tissue culture systems, melatonin promotes root regeneration, but the explants used in these experiments were mostly from woody plant species, such as Prunus cerasus and Punica granatum (Sarropoulou et al., 2012; Sarrou et al., 2014). Recently, it was reported that melatonin improves in vitro production of anthocyanin from protocorm-like bodies of Dendrobium (Malik et al., 2021) and survival chance from axillary buds of cryopreserved Ludisia discolor (Burkhan et al., 2022).
The effect of melatonin on tissue culture and rapid propagation is largely unknown. Therefore, we chose C. goeringii and C. faberi as the representatives of famous Chinese traditional flowers for the effect of melatonin on the growth and development of rhizome and seedling. We determined the effects of melatonin on rhizome proliferation, differentiation, and seedling rooting during the rapid propagation of C. goeringii and C. faberi to develop an efficient method for in vitro propagation of Cymbidium.
Materials and Methods
Mature capsules (150 d after pollination) of C. goeringii and C. faberi were harvested. The capsules were surface-sterilized by dipping in 70% ethanol for 30 s, immersed in 0.1% (w/v) HgCl2 solution for 10 min, and rinsed five times with sterile distilled water. The seeds were scraped out and then placed in a culture bottle containing Murashige and Skoog (MS) medium, containing 0.5 mg·L−1 NAA, 30.0 g·L−1 sucrose, 2.0 g·L−1 activated charcoal (AC), and 100 mL·L−1 coconut water (CW). Seeds germinated and developed into protocorms in 150 d and then transformed into rhizomes after another 30 d.
The rhizomes were cut into 1.0-cm segments with a scalpel, and each tissue culture bottle was inoculated with 15 of the preceding materials. The 1.0-cm rhizome apical segments were placed on medium P1, P2, P3, P4, P5, and P6, containing 0, 0.1, 0.5, 1.0, 5.0, or 10.0 μM of melatonin, respectively. Melatonin solution was prepared by dissolving melatonin with absolute ethanol, then diluted with water to 1.0 μM, filtered, sterilized, and added to the culture medium.
The media were supplemented with 1.5 g·L−1 Hyponex NO.1, 1.5 g·L−1 Hyponex NO.2, 2.0 mg·L−1 NAA, 1.0 g·L−1 AC, 30.0 g·L−1 sucrose, 8.0 g·L−1 agar, and 50.0 mL·L−1 CW. There were three replicates of each treatment, with 150 rhizomes per replicate. The rhizome proliferation rate by weight was calculated as the rhizome fresh weight after 60 d of incubation compared with that before. The proliferation rate was calculated by the new growth point on each rhizome.
After determining the effects of melatonin on rhizome proliferation, the effect of different concentrations of melatonin and 6-benzylaminopurine (6-BA), NAA, and sodium citrate on rhizome differentiation were examined using the same methods. All the media were supplemented with 1.5 g·L−1 Hyponex NO.1, 1.5 g·L−1 Hyponex NO.2, 20.0 g·L−1 sucrose, and 8.0 g·L−1 agar. The specific culture medium for rhizome differentiation named D1-D10 is shown in Supplemental Table 1. The number and length of newly grown shoot buds on each rhizome were recorded after 60 d. The number of bit shoot buds/bottle refers to the number of buds longer than 1.0 cm in each tissue culture bottle.
The in vitro–produced shoot buds were cultured on a 1/2 MS medium incorporated with 0.1 mg·L−1 6-BA, 2.0 mg·L−1 NAA, 1.0 g·L−1 AC, 30.0 g·L−1 sucrose, 8.0 g·L−1 agar, 50.0 g·L−1 banana homogenate (BH), and 0, 0.1, 0.5, 1.0, 5.0, or 10.0 μM of melatonin for seedlings rooting. These culture media were named R1, R1, R3, R4, R5, and R6, respectively. The experiment was conducted in triplicates with 50 shoot buds for each treatment. The rooting percentage, the number and length of roots, shoot height, and growth status were observed and calculated after 60 d of culture.
The pH of all media were adjusted to 5.4 before autoclaving at 121 °C for 20 min at 1.06 kg·cm−2. All cultures were incubated in a controlled environment growth room at 25 ± 2 °C with a 16-h photoperiod under cool white light (45 μmol·m−2·s−1). All experiments were set according to a completely random design. The data were analyzed using one-way analyses of variance and Duncan’s multiple range tests at the 5% level. The degree of browning phenomenon of C. goeringii and C. faberi in the process of rhizome proliferation, differentiation, and seedling rooting are described in Supplemental Table 2.
Results
Effects of melatonin on rhizome proliferation of C. goeringii and C. faberi.
To investigate the effects of melatonin on rhizome proliferation of C. goeringii and C. faberi, rhizomes were cultured in media supplemented with different concentrations of melatonin for 60 d. The survival rate, rhizome proliferation rate by weight, and number of added growth points per explant were measured. The rhizome of C. goeringii exhibited a lower survival rate, whereas that of C. faberi exhibited a higher survival rate (Fig. 1A and D). Various concentrations of melatonin significantly improved the survival rate of C. goeringii rhizome, but melatonin had no significant effect on the survival rate of C. faberi rhizome (Fig. 1A and D). The degree of browning phenomenon of C. goeringii in the process of rhizome proliferation showed that medium devoid of melatonin was moderately browning, whereas medium supplemented with melatonin was slightly browning (Supplemental Table 2). However, no browning phenomenon of C. faberi was observed (Supplemental Table 2). The highest survival rate (>90%) of C. goeringii rhizome, ≈50% higher than that of the control, was achieved in the 0.5 μM melatonin medium (Fig. 1A). Furthermore, the highest rhizome proliferation rate by weight and number of added growth points/explant of C. goeringii rhizome were documented in the 1.0 μM melatonin medium with 3.94 and 5.52, respectively, which were significantly different from those in the control (Figs. 1B, 1C, 4A, and 4B).
Effect of melatonin on rhizome proliferation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), rhizome proliferation rate by weight (B), and number of added growth points/explant (C) of C. goeringii. Survival rate (D), weight proliferation rate (E), and number of added growth points/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on rhizome proliferation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), rhizome proliferation rate by weight (B), and number of added growth points/explant (C) of C. goeringii. Survival rate (D), weight proliferation rate (E), and number of added growth points/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on rhizome proliferation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), rhizome proliferation rate by weight (B), and number of added growth points/explant (C) of C. goeringii. Survival rate (D), weight proliferation rate (E), and number of added growth points/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
The highest proliferation rate of the weight of 2.97 of C. faberi rhizome was achieved in 5.0-μM melatonin medium (Fig. 1E), whereas the number of added growth points per explant reached 8.29 at 0.5 μM melatonin, which was significantly higher than that in other treatments (Figs. 1F, 4C, and 4D). Therefore, C. goeringii rhizomes had a better survival rate than C. faberi when subjected to melatonin. A low melatonin concentration (0.5–1.0 μM) had a better effect on the rhizome proliferation rate by weight and number of added growth points per explant of C. goeringii rhizomes, whereas higher melatonin (5.0 μM) concentration was required to increase the proliferation rate of C. faberi. Therefore, melatonin can promote the proliferation of Cymbidium rhizomes, but the desirable concentration varies with species.
Effects of melatonin on rhizome differentiation of C. goeringii and C. faberi.
To investigate the effects of melatonin on differentiation of C. goeringii and C. faberi rhizomes, rhizomes were cultured in a medium supplemented with different concentrations of melatonin for 60 d. The survival rate, number, length of shoot buds/explant, and number of big shoot buds per bottle were recorded. The results showed that the survival rate of C. goeringii rhizomes significantly improved at 0.5 and 1.0 μM melatonin compared with other treatments at the stage of rhizome differentiation (Fig. 2A). Different melatonin concentrations did not significantly promote the survival rate of differentiated rhizomes in C. faberi (Fig. 2E).
Effect of melatonin on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), length of shoot buds/explant (C), and number of big shoot buds/bottle (D) of C. goeringii. Survival rate (E), number of shoot buds/explant (F), length of shoot buds/explant (G), and number of big shoot buds/bottle (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), length of shoot buds/explant (C), and number of big shoot buds/bottle (D) of C. goeringii. Survival rate (E), number of shoot buds/explant (F), length of shoot buds/explant (G), and number of big shoot buds/bottle (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), length of shoot buds/explant (C), and number of big shoot buds/bottle (D) of C. goeringii. Survival rate (E), number of shoot buds/explant (F), length of shoot buds/explant (G), and number of big shoot buds/bottle (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
The number of shoot buds per explant of C. goeringii rhizome was highest at 1.0 μM melatonin, reaching 3.11 (Figs. 2B, 4E, and 4F), and the browning phenomenon degree of rhizome was the lowest (Supplemental Table 2). The length of shoot buds/explant (Fig. 2C) and the number of big shoot buds/bottle (Fig. 2D) were 0.44 cm and 1.6, respectively, at 0.5 μM melatonin, which was significantly different from other treatments. This result indicates that a higher concentration (1.0 μM) of melatonin promoted the number of shoot buds per explant differentiated from the rhizome of C. goeringii, but a lower concentration of melatonin (0.5 μM) boosted the length and size of buds of C. goeringii. The number of shoot buds per explant of C. faberi rhizome in 5.0 μM melatonin was 3.28, which was considerably higher than that in other treatments (Figs. 2B, 4E, and 4F). There was no significant difference in the length of shoot buds/explant (Fig. 2G) and number of big shoot buds per bottle under all melatonin treatments of C. faberi (Fig. 2H). Melatonin had little effect on the rhizome differentiation of C. faberi, and a high concentration (5.0 μM) was required to stimulate the rhizome to differentiate into buds.
Combined effects of melatonin, plant growth regulator, and sodium citrate on rhizome differentiation of C. goeringii and C. faberi.
To investigate the effects of melatonin combined with plant growth regulators and sodium citrate on rhizome differentiation of C. goeringii and C. faberi, the rhizomes were cultured in a medium supplemented with different melatonin concentrations for 60 d. The survival rate, number, and length of shoot buds/explant were measured. The survival rate (80%) of C. goeringii was the highest under 1.0 μM melatonin + 5.0 mg·L−1 6-BA + 0.2 mg·L−1 NAA + 1.0 g·L−1 sodium citrate treatment (Fig. 3A; Supplemental Fig. 1B). Similarly, the number and the length of shoot buds per explant were the highest under the same treatment (Fig. 3B and C; Supplemental Fig. 1B). The survival rate of C. faberi reached to 90.37% under 0 μM melatonin + 5.0 mg·L−1 6-BA + 0.2 mg·L–1 NAA + 0 g·L−1 sodium citrate treatment (Fig. 3D; Supplemental Fig. 1K). Similarly, the number of shoot buds per explant was the highest under the same treatment (Fig. 3E; Supplemental Fig. 1K). Both the number of shoot buds per explant of D7 and D8 treatments were 0, whereas others were not (Fig. 3B and E; Supplemental Fig. 1), which means that the rhizomes of C. goeringii and C. faberi did not differentiate into buds without 6-BA and NAA. D7 and D8 treatments differ only in the addition of melatonin, indicating that melatonin could not replace the combination of 6-BA and NAA to induce rhizome differentiation into buds. Additionally, for both C. goeringii and C. faberi, the length of shoot buds per explant of D1 and D4 treatments were longer than D9 and D10 treatments, implying that the addition of 1.0 g·L−1 sodium citrate promote bud elongation (Fig. 3C and F; Supplemental Fig. 1).
Effect of melatonin, 6-benzylaminopurine, naphthaleneacetic acid, sodium citrate on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), and length of shoot buds/explant (C) of C. goeringii. Survival rate (D), number of shoot buds/explant (E), and length of shoot buds/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin, 6-benzylaminopurine, naphthaleneacetic acid, sodium citrate on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), and length of shoot buds/explant (C) of C. goeringii. Survival rate (D), number of shoot buds/explant (E), and length of shoot buds/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin, 6-benzylaminopurine, naphthaleneacetic acid, sodium citrate on rhizome differentiation of Cymbidium goeringii and Cymbidium faberi. Survival rate (A), number of shoot buds/explant (B), and length of shoot buds/explant (C) of C. goeringii. Survival rate (D), number of shoot buds/explant (E), and length of shoot buds/explant (F) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Rhizome proliferation and differentiation of Cymbidium goeringii and Cymbidium faberi after 60 d. Rhizome proliferation of C. goeringii on (A) Hyponex medium and (B) Hyponex medium containing 1.0 μM melatonin. Rhizome proliferation of C. faberi on (C) Hyponex medium and (D) Hyponex medium containing 0.5 μM melatonin. Rhizome differentiation of C. goeringii on (E) Hyponex medium and (F) Hyponex medium containing 1.0 μM melatonin. Rhizome differentiation of C. faberi on (G) Hyponex medium and (H) Hyponex medium containing 5.0 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Rhizome proliferation and differentiation of Cymbidium goeringii and Cymbidium faberi after 60 d. Rhizome proliferation of C. goeringii on (A) Hyponex medium and (B) Hyponex medium containing 1.0 μM melatonin. Rhizome proliferation of C. faberi on (C) Hyponex medium and (D) Hyponex medium containing 0.5 μM melatonin. Rhizome differentiation of C. goeringii on (E) Hyponex medium and (F) Hyponex medium containing 1.0 μM melatonin. Rhizome differentiation of C. faberi on (G) Hyponex medium and (H) Hyponex medium containing 5.0 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Rhizome proliferation and differentiation of Cymbidium goeringii and Cymbidium faberi after 60 d. Rhizome proliferation of C. goeringii on (A) Hyponex medium and (B) Hyponex medium containing 1.0 μM melatonin. Rhizome proliferation of C. faberi on (C) Hyponex medium and (D) Hyponex medium containing 0.5 μM melatonin. Rhizome differentiation of C. goeringii on (E) Hyponex medium and (F) Hyponex medium containing 1.0 μM melatonin. Rhizome differentiation of C. faberi on (G) Hyponex medium and (H) Hyponex medium containing 5.0 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effects of melatonin on seedling strengthening and rooting in C. goeringii and C. faberi.
To observe the effects of melatonin on seedling strengthening and rooting in C. goeringii and C. faberi, we cultured shoots in a medium supplemented with different concentrations of melatonin for 60 d. The explant height, rooting rate, number, and length of roots were recorded. The explant height of C. goeringii reached the maximum under 1.0 μM melatonin compared with other treatments, with an average value of 5.58 cm (Fig. 5A). The rooting of C. goeringii seedlings was easy, and all treatments were 100% effective (Figs. 5B, 6A, and 6B). The number of roots reached the highest (2.8) at 5.0 μM melatonin (Fig. 5C), and the length of roots was more than 2 cm under 0.1 to 1.0 μM melatonin (Figs. 5D and 6C). The result showed that 1.0 μM melatonin has the best comprehensive effect on seedling height and root number and length of C. goeringii.
Effect of melatonin (MT) on seedling rooting of Cymbidium goeringii and Cymbidium faberi. Explant height (A), rooting rate (B), number of roots (C), and length of roots (D) of C. goeringii. Explant height (E), rooting rate (F), the number of roots (G), and length of roots (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin (MT) on seedling rooting of Cymbidium goeringii and Cymbidium faberi. Explant height (A), rooting rate (B), number of roots (C), and length of roots (D) of C. goeringii. Explant height (E), rooting rate (F), the number of roots (G), and length of roots (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin (MT) on seedling rooting of Cymbidium goeringii and Cymbidium faberi. Explant height (A), rooting rate (B), number of roots (C), and length of roots (D) of C. goeringii. Explant height (E), rooting rate (F), the number of roots (G), and length of roots (H) of C. faberi. The different lowercase letters indicate significant differences at P < 0.05 level.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on seedling rooting of Cymbidium goeringii and Cymbidium faberi after 60 d. Seedling rooting of C. goeringii on (A) 1/2 Murashige and Skoog (MS) medium, (B) 1/2 MS medium containing 10.0 μM melatonin, and (C) 1/2 MS medium containing 1.0 μM melatonin. Seedling rooting of C. faberi on (D) 1/2 MS medium, (E) 1/2 MS medium containing 10.0 μM melatonin; and (F) 1/2 MS medium containing 0.5 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on seedling rooting of Cymbidium goeringii and Cymbidium faberi after 60 d. Seedling rooting of C. goeringii on (A) 1/2 Murashige and Skoog (MS) medium, (B) 1/2 MS medium containing 10.0 μM melatonin, and (C) 1/2 MS medium containing 1.0 μM melatonin. Seedling rooting of C. faberi on (D) 1/2 MS medium, (E) 1/2 MS medium containing 10.0 μM melatonin; and (F) 1/2 MS medium containing 0.5 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Effect of melatonin on seedling rooting of Cymbidium goeringii and Cymbidium faberi after 60 d. Seedling rooting of C. goeringii on (A) 1/2 Murashige and Skoog (MS) medium, (B) 1/2 MS medium containing 10.0 μM melatonin, and (C) 1/2 MS medium containing 1.0 μM melatonin. Seedling rooting of C. faberi on (D) 1/2 MS medium, (E) 1/2 MS medium containing 10.0 μM melatonin; and (F) 1/2 MS medium containing 0.5 μM melatonin (bar = 1.0 cm).
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Except a high concentration of 10.0 μM, different concentrations of melatonin had no significant effect on the rooting rate of C. faberi (Figs. 5F, 6D, and 6E). A concentration of 0.5 μM melatonin was beneficial to the rooting number and length of C. faberi seedlings (Figs. 5G, 5H, and 6F). Therefore, melatonin has a specific effect on the rooting of C. faberi, with a concentration of 0.5 μM having the best effect on root number and root length.
Discussion
The results of this study have shown that certain concentrations of melatonin added to the culture medium significantly improve the proliferation and differentiation of C. goeringii and C. faberi (Figs. 1–3). The optimal melatonin concentrations suitable for rhizome proliferation and differentiation of C. goeringii were 1.0, and 0.5 and 5.0 μM for C. faberi, respectively. The optimal concentration of melatonin required for the growth and development differed between C. goeringii and C. faberi (Figs. 1–3), indicating that different Cymbidium species have different demands for melatonin at various growth and development stages. Our results are similar to those in an earlier study showing that different rhizomes lines obtained from the hybridization of C. sinense required different concentrations of NAA and 6-BA (Lee et al., 2011). Melatonin effectively mimics plant hormones to regulate the proliferation and differentiation of Cymbidium rhizomes because of its similarity to auxins (Murch et al., 2001; Murch and Saxena, 2002). Furthermore, melatonin and IAA have more similar effects and promote the formation of axillary buds and adventitious buds of blueberries (Litwińczuk and Wadas-Boroń, 2009).
Melatonin can help plants resist abiotic stress (Lei et al., 2004). As a broad-spectrum and effective antioxidant, melatonin protects plants from various environmental stresses by enhancing the activity of plant antioxidant enzymes, reducing the content of reactive oxygen species and peroxides, and scavenging reactive oxygen free radicals (García et al., 2014; Zhang et al., 2014). Previous studies have shown that melatonin treatment is beneficial in reducing the surface browning phenomenon of litchi, banana, lotus seeds, and fresh-cut tuberous root of sweetpotatoes (Li et al., 2022; Luo et al., 2020; Wang et al., 2020, 2021). In our study, we found that the browning phenomenon of rhizomes is generally severe due to the strict cutting of rhizomes and the removal of branches The addition of melatonin to the culture medium significantly inhibit the browning phenomenon during the proliferation and differentiation of Cymbidium rhizomes and improve the survival rate (Figs. 1A, 2A, 3A, 3D, and 4; Supplemental Table 2). Melatonin inhibits browning phenomenon during the cultivation of plant tissue (Dan et al., 2015), suggesting that it is an effective antioxidant that can inhibit the oxidation of phenolic compounds.
In addition, melatonin can promote the formation of lateral roots and adventitious roots in Arabidopsis thaliana, but it has little effect on the growth of taproot and the development of root hair (Pelagio-Flores et al., 2012). Melatonin does not seem to affect rooting formation in C. goeringii and C. faberi (Fig. 5), possibly because of the difference in the roots of Cymbidium and A. thaliana. Our results showed that melatonin has a certain effect on the rooting of C. faberi, and its concentration of 0.5 μM has a better effect on the root number and length (Fig. 5). The primary mechanism of how melatonin aids in rooting is that it promotes the elongation and expansion of cells by enhancing the biosynthesis of IAA, which results in enhanced growth of roots and development of lateral and adventitious roots. Because melatonin and auxin both are synthesized from tryptophan, they may have similar functions (Murch and Saxena, 2002; Park et al., 2012; Shi et al., 2015). Furthermore, most of the IAA-regulated genes were coregulated by melatonin (Yang et al., 2021), indicating that melatonin and IAA regulated a similar subset of genes. In addition, a moderate concentration of melatonin (0.5 μM) can promote the rooting of C. goeringii and C. faberi, but a high concentration of melatonin can inhibit the rooting (Figs. 5 and 6). Previous studies have also shown that the growth in monocotyledons was blocked under a higher concentration of melatonin (Hernández-Ruiz et al., 2004, 2005). The formation and growth of lateral and adventitious roots of Lupinus micranthus Guss. were induced at a low concentration (≤10 μM) of melatonin, whereas the growth was inhibited at high concentrations of melatonin (≥100 μM) (Arnao and Hernández-Ruiz, 2007). Concentration-dependent effects of melatonin have also been described in Hypericum perforatum and Brassica juncea (Chen et al., 2009; Murch et al., 2001), indicating that low-concentration melatonin promotes the growth and development of plant roots. We speculate that a high concentration of melatonin inhibits the accumulation and transport of auxin (Wang et al., 2016).
Conclusion
This study demonstrated that specific concentrations of melatonin effectively promote rhizome proliferation, differentiation, and seedling rooting during the rapid propagation of C. goeringii and C. faberi. The required melatonin concentration differed greatly in all propagation stages of the two species. The addition of melatonin significantly inhibits the browning phenomenon in tissue culture and improve the survival rate of C. goeringii rhizome, but it has no effect on C. faberi rhizome. Melatonin is a promising growth regulator for use in the culture medium, which improves the efficiency of tissue culture and rapid propagation of Cymbidium.
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Rhizome differentiation of Cymbidium goeringii and Cymbidium faberi dominated by melatonin (MT), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and sodium citrate after 60 d. Basic medium supplemented with (A) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (B) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (C) 1.0 g·L−1 sodium citrate, (D) 1.0 μM MT +1.0 g·L−1 sodium citrate, (E) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (F) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. goeringii. Basic medium supplemented with (G) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (H) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (I)1.0 g·L−1 sodium citrate, (J) 1.0 μM MT +1.0 g·L−1 sodium citrate, (K) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (L) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. faberi (bar = 1.0 cm). Basic medium component was shown in Supplemental Table 1.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Rhizome differentiation of Cymbidium goeringii and Cymbidium faberi dominated by melatonin (MT), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and sodium citrate after 60 d. Basic medium supplemented with (A) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (B) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (C) 1.0 g·L−1 sodium citrate, (D) 1.0 μM MT +1.0 g·L−1 sodium citrate, (E) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (F) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. goeringii. Basic medium supplemented with (G) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (H) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (I)1.0 g·L−1 sodium citrate, (J) 1.0 μM MT +1.0 g·L−1 sodium citrate, (K) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (L) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. faberi (bar = 1.0 cm). Basic medium component was shown in Supplemental Table 1.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Rhizome differentiation of Cymbidium goeringii and Cymbidium faberi dominated by melatonin (MT), 6-benzylaminopurine (6-BA), naphthaleneacetic acid (NAA), and sodium citrate after 60 d. Basic medium supplemented with (A) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (B) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (C) 1.0 g·L−1 sodium citrate, (D) 1.0 μM MT +1.0 g·L−1 sodium citrate, (E) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (F) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. goeringii. Basic medium supplemented with (G) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (H) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA+1.0 g·L−1 sodium citrate, (I)1.0 g·L−1 sodium citrate, (J) 1.0 μM MT +1.0 g·L−1 sodium citrate, (K) 5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA, (L) 1.0 μM MT+5.0 mg·L−1 6-BA+0.2 mg·L−1 NAA for rhizome differentiation of C. faberi (bar = 1.0 cm). Basic medium component was shown in Supplemental Table 1.
Citation: HortScience 57, 9; 10.21273/HORTSCI16704-22
Culture medium component of Cymbidium goeringii and Cymbidium faberi for rhizome proliferation, differentiation, and seedling rooting.
The degree of browning phenomenon of C. goeringii and C. faberi in the process of rhizome proliferation, differentiation, and seedling rooting.
