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Article

Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies

by
Jiaqi Tang
,
Ruyan Huang
,
Dan Yuan
,
Han Sun
,
Yuping Li
,
Zekang Pei
,
Congqiao Wang
,
Chengyue Li
,
Dongliang Qiu
* and
Qingqing Chen
*
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Agronomy 2024, 14(6), 1326; https://doi.org/10.3390/agronomy14061326
Submission received: 23 April 2024 / Revised: 9 June 2024 / Accepted: 10 June 2024 / Published: 19 June 2024
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
Dendrobium officinale Kimura & Migo is a perennial herbaceous plant of the genus Dendrobium in the family of Orchidaceae with high medicinal value. Melatonin (MT) is an indole-like tryptamine with functions such as regulating plant growth and development. This experiment investigated the effects of different concentrations of MT on the growth and development of protocorms of D. officinale protocorm-like bodies (PLBs). The results showed that the changes in morphological indicators such as color, cluster size, and surface changes were more significant under 75 µM MT than those of 0 µM (CK), and the appearance of white on the PLB surface was expedited, which was more conducive to the proliferation of PLBs. MT treatment of 100 µM inhibited the differentiation of adventitious buds, and the contents of photosynthetic pigments, polysaccharides, and flavonoids were significantly increased. Moreover, as compared with CK, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PLBs increased significantly, while the content of malondialdehyde (MDA) decreased gradually with 75 µM or less. In conclusion, a concentration of 75 µM melatonin can enhance the rapid propagation rate of D. officinale in vitro, providing insights into the effects of melatonin on the growth of tissue-cultured D. officinale seedlings.

1. Introduction

Dendrobium officinale Kimura & Migo is a perennial herb in the genus Dendrobium of the family Orchidaceae. It has high medicinal value for hypoglycemia, gastric ulcer protection, anti-tumor activity [1], liver protection [2], antioxidant activity [3], anti-cataract activity [4], and enhancement of human immunity. However, due to Dendrobium’s limited reproductive capacity in natural habitats, slow growth and development under wild conditions, and high market demand, rampant harvesting has led to a sharp decline in wild Dendrobium species, pushing them to the brink of extinction [5]. Protocorms have emerged as an effective propagation system in orchid tissue culture, with standardized culture conditions leading to a significant increase in propagation rates [6]. In recent years, many experts and scholars have reported more studies on the addition of plant growth regulators in the fast propagation of protocorm tissues [7,8,9]. Studies have demonstrated the induction of flowering in Dendrobium nobile test-tube cultures through the addition of benzylamino purine (BA), thidiazuron (TDZ), and abscisic acid (ABA) to the media [10], and α-naphthalene acetic acid (NAA) has been shown to stimulate the proliferation of Chinese orchid protocorms [11]. Thus, the application of tissue culture technology in the in vitro culture of Dendrobium can protect the germplasm resources of Dendrobium and meet certain market demands [12].
Melatonin (N-acetyl-5-methoxytryptamine; MT), an indole tryptamine, was first thought to be animal-owned and was later discovered in plants [13]. As one of the endogenous plant hormones, melatonin is an endogenous plant hormone that regulates several aspects of physiological processes, including growth and development, flowering, stress tolerance, and responses to external stimuli [14,15,16,17,18]. The rooting rate of tea plant tissue culture seedlings can be improved with the addition of MT in the culture medium, and MT can promote the growth of chrysanthemum seedlings [19]. MT not only induces test-tube ball formation in histocultured lily seedlings [20], but also promotes the expansion of their bulbs and seedpods [21]. In addition, MT regulates photosynthesis and enzyme promotion in tomato under acid rain deposition by affecting the content of chlorophyll and carotenoids [22], and even improves the quality as well as antioxidant capacity of tomato under acid rain stress [23]. While previous studies have demonstrated the induction of anthocyanin production in Dendrobium ‘Sabin Blue’ protocorm-like bodies (PLBs) with MT [24], limited research exists on the morphological impact of MT supplementation in D. officinale protocorm culture. Therefore, this study aims to evaluate the effects of MT on the proliferation and growth of D. officinale PLBs in tissue culture, with the objective of accelerating in vitro plant propagation rates. The findings will contribute to a deeper understanding of MT’s role in the growth of D. officinale tissue culture seedlings.

2. Materials and Method

2.1. Experimental Materials

Undifferentiated D. officinale protocorm-like bodies (PLBs) were utilized in this experiment. PLBs with a consistent growth state were selected and transferred to a liquid medium consisting of 1/2 MS + 50 g/L potato juice + 25 g/L sucrose [25]. The cultures were maintained under shaking conditions for a duration of 4 weeks to promote mass proliferation. All media were adjusted to a pH of 5.6 ± 0.2 and autoclaved at 121 °C (105 kPa pressure) for 20 min, and the growth conditions were standardized with a light intensity of 50 µmol m−2 s−1, a temperature of 25 ± 1 °C, and a shaking speed of 1100 r/min.

2.2. Experimental Method

Five small clusters of PLBs exhibiting consistent growth were selected and transferred into culture bottles containing a base medium composed of 1/2 MS supplemented with 50 g/L potato juice, 25 g/L sucrose, and 6 g/L agar [25]. MT was not added as a control (CK). The specific parameters for each treatment are detailed in Table 1, and the pH of the medium was adjusted to 5.6 ± 0.2. Cultures were maintained under white fluorescent lamps with a photon flux of 50 µmol m−2 s−1, at a temperature of 25 ± 1 °C, and under a 12/12 h cycle of light and darkness to facilitate plantlet production. A total of 50 bottles were cultured for each treatment, and the materials were cultured for a duration of 45 days. Sampling commenced on the 25th day, with intervals of 5 days, and three bottles were randomly selected each time, with repetitions performed three times.

2.3. Measurement Methods

2.3.1. Morphological Developmental Measurements

The changes in the growth of protocorms and the structure of PLBs were observed using an electron microscope (Leica Microscope M205FA 6120772, Leica, Wetzlar, Germany).

2.3.2. Determination of Proliferation and Differentiation

The weight of each bottle with the solidified medium (G1), and the total weight of the inoculated PLBs bottle seedlings (G2) were initially measured using an electronic balance, from which the weight of the explant in the bottle (G0) was calculated. Refer to Fan [26] for the determination method.
For the four different gradient ratios of MT concentration treatments and the control group without added MT (CK) treatment of PLB group seedlings, three bottles of group seedlings were randomly selected every 5 days, and all the grown PLBs were taken out, and their fresh weights were determined to obtain the weights (G3), which were measured up to the 45th day, for a total of five measurements, and the fresh weight gain of each bottle of PLB group seedlings was calculated accordingly (G4).
In each treatment, 20 clusters of uniformly growing protocorms were randomly selected, and the number of differentiated clusters was counted to calculate the differentiation rate of the corresponding treatment.
Fresh samples were put into paper bags and dried in an electric blast drying oven (Shanghai Heng Scientific Instrument Co., Ltd., Shanghai, China) at 105 °C for 15 min, followed by further drying at 50 °C for 48 h. Subsequently, they were weighed to obtain the dry weight, and the fold drying rate was calculated. This testing was performed with three bottles for each treatment and a total of three replications. The calculations were performed as follows:
Weight of explant in bottles:
G0 = G2 − G1
Fresh weight gain of plants per bottle:
G4 = G3 − G0
Proliferation rate:
Proliferation rate = G4/G0 × 100%
Differentiation rate:
Differentiation rate = differentiation number/20 × 100%
Drying rate:
Drying rate = Dry weight/fresh weight × 100%

2.3.3. Determination of Chlorophyll

The mixture extraction method was performed according to Ji et al. [27]. A 0.1 g frozen sample of a Dendrobium bulb was weighed with an accurate analytical balance, the sample was then ground under liquid nitrogen in a light-shielded environment and transferred into a 10 mL stoppered test tube. A mixture extraction solution consisting of acetone, anhydrous ethanol, and distilled water in a ratio of 4.5:4.5:1 was added to the test tube. The mixture was thoroughly shaken and left to extract in darkness for 24 h, with shaking every 12 h until the original color of the Dendrobium bulb turned white. The resulting mixed extract served as a blank control, and the OD (optical density) values at 470 nm, 663 nm, and 646 nm were recorded to calculate the content of chlorophyll a (Chla), chlorophyll b (Chlb), total chlorophyll Chl (a+b), and carotenoids (Caro), respectively, and each treatment was repeated three times.

2.3.4. Determination of Antioxidant Enzymes

The antioxidant enzymes were determined according to the instructions of a superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and malondialdehyde (MDA) kit (micro method) produced by Fujian Herui Biotechnology Co. Approximately 0.1 g of Dendrobium PLB tissue was weighed, and 1 mL of extraction solution was added, followed by homogenization on ice. The homogenate was then centrifuged at 8000× g 4 °C for 10 min, and the supernatant was collected and kept on ice to be tested. Reagents and working solutions were prepared according to the concentrations specified in the kit. The reagents were added sequentially according to the instructions, ensuring thorough mixing after each addition. Superoxide dismutase activity was measured by absorbance at 560 nm; peroxidase, at 470 nm; and catalase, at 240 nm.

2.3.5. Determination of Polysaccharides

With reference to the adjustment and improvement of Wei Li et al. [28,29], the phenol–sulfuric acid method was used for polysaccharide determination. Initially, 0.1 g of powder was mixed with 20 mL of water in a test tube and subjected to heating reflux for 2 h. After cooling, the liquid was filtered and transferred to a 25 mL volumetric bottle. An appropriate volume of anhydrous ethanol was added to the filtrate and thoroughly shaken. Subsequently, the mixed filtrate was evaporated to obtain precipitation. The precipitate was dissolved in 2 mL of distilled water to obtain the liquid to be measured. Then, 1 mL of the liquid to be measured was taken in a test tube, to which 1 mL of 5% phenol solution and 5 mL of sulfuric acid solution were added. After thorough shaking, the tube was heated in boiling water for 20 min and removed to cool for 5 min. Using the corresponding reagent as a blank, the absorbance was measured at a wavelength of 488 nm using a UV spectrophotometer. A standard curve was constructed based on glucose concentrations.

2.3.6. Determination of Total Flavonoids and Total Phenols

Sample extraction buffer was prepared following the procedures of Karabegović et al. [30], with slight modification. Freeze-dried powder samples were accurately weighed with 0.1 g and mixed with an appropriate 2% hydrochloric acid methanol solution. Ultrasound extraction was carried out for 60 min, followed by centrifugation at 12,000 rpm for 10 min at 4 °C, and the supernatant was taken as the extraction solution.
The content of total flavonoids was determined according to Zhu et al. [31]. The NaNO2-Al(NO3)3-NaOH colorimetric method was used to determine the total flavonoid content. One milliliter of the extract was placed in a 10 mL test tube, to which 0.3 mL of 5% NaNO2 was added, shaken well, and allowed to stand for 6 min. Then, 0.3 mL of 10% AI(NO3)3 was added, shaken well, and left for another 6 min. Finally, 3 mL of 1 mol/L NaOH was added, shaken well, and left for 15 min before the determination of the absorbance value at 510 nm with a spectrophotometer. A standard curve was constructed using rutin as the standard.
The determination of total phenol content followed the procedure of Karabegović et al. [30], with slight modifications. Using the Folin–Ciocalteu method, 1 mL of the extract was placed in a 10 mL test tube, and 4 mL of distilled water and 0.5 mL of Folinol were added and mixed homogeneously. The mixture was allowed to stand for 5 min before the addition of 1 mL of 7% NaCO3 solution and thorough mixing. The solution was then stored at 25 °C in darkness for 2 h, after which the absorbance value was determined at 765 nm. A standard curve was generated using gallic acid as the standard.

2.3.7. Determination of Total Alkaloids

A reagent kit (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) was used for the determination. Approximately 0.1 g of a dried PLB sample was weighed and mixed with 0.1 mL of reagent I and 0.9 mL of 80% ethanol. After thorough mixing, the mixture was transferred to an EP tube and subjected to ultrasound extraction for 60 min. Following extraction, the sample was centrifuged at 8000× g 25 °C for 10 min, and the supernatant was taken to be measured. In addition, the EP tube was emptied, and 0.1 mL of reagent I and 0.9 mL of 80% ethanol were added, mixed evenly, and used as a blank control. The reagents were added sequentially according to the instructions, and the absorbance value A was read at 416 nm and used to calculate ΔA = A assay − A blank.
Alkaloid content (mg/g, dry weight) = 0.261 × (ΔA + 0.0005) ÷ W
Here, W is the dry weight of the sample, g.

2.4. Statistical Analysis

The analysis of the obtained experimental data was carried out using SPSS Statistics 19.0 software for one-way and multi-factor ANOVA. Later, Excel 2010 and GraphPad Prism (v8.0.2.263) were used for plotting.

3. Results

3.1. Characteristics of PLBs

A preliminary observation on the formation of protocorm-like bodies of Dendrobium officinale was made; as shown in Figure 1, it included protocorms, protocorm + healing tissue complexation, protocorm budding, protocorms of differentiated bud clusters, and other protocorm development morphology. It was found that the growth points of the somatic embryoid body of the occurrence of stem and bud existed at the same time.
It was observed that the growth stage of D. officinale PLBs not only had the morphological characteristics of embryoid bodies (Figure 2A), but also had white reticulated villi on the PLB surface (Figure 2B). Therefore, the protocorm-like bodies were in a somatogenic pathway at this time.
When the protocorms were cultured for about 35 d, most of the protocorms began to germinate and differentiate; the generation of small protrusions was observed, and some of them developed indeterminate bud clusters (Figure 3). In general, these produced leaf and stem buds from the globules of the protocorm-like bodies, so the PLBs were in the organogenesis pathway in the later stage.
As mentioned above, a protocorm-like body of D. officinale is an incomplete complex of somatic embryoid with spore organogenesis. The protocorm-like bodies are in the somatic pathway in the early stage of development and move to the organogenesis pathway in the later stage (as shown in Figure 1, Figure 2 and Figure 3).

3.2. The Morphology of PLB Tissue Culture Seedlings

At the early stage of culture, the color of Dendrobium bulbs in the combination of four different concentrations of MT treatment was light green with a yellowish color. On the 25th day, the color was mostly yellowish green. As the cultivation progressed, both the CK and the MT3 treatments gradually shifted from yellowish green to a lighter shade of green. Under the MT1 treatment, a small amount of sprouting and healing tissue appeared in the protocorm-like body mass, while fine white flocculent began to appear on the surface of the protocorms under the MT1, MT2, and MT4 treatments. On the 30th day, an increase in callus germination and the emergence of small bulges were observed across all treatments, with more pronounced sprouting under the CK and MT3 treatments, accompanied by varying degrees of bulge formation. On the 35th day, the CK began to appear sprouting buds. As MT concentration increased, the tops of the protocorm-like bodies gradually turned green, with the color changing from yellowish green to light green. The bulges enlarged, and the number of white flocculent structures began to increase. On the 40th day of cultivation, compared to the control, protocorms treated with MT3 and MT4 exhibited more white flocculent on the surface, with fewer differentiated clumps treated with MT1 and MT4. On the 45th day, the MT1, MT2, and MT3 treatments showed more differentiated clumps and white flocculent than CK. However, protocorms treated with MT4 appeared yellow in color, the degree of mass differentiation was less, and there were more white flocculent structures. The specific morphology parameters are shown in Figure 4 and Table 2. In summary, the addition of MT may promote the proliferation of protocorms.

3.3. Proliferation and Differentiation of PLBs

As depicted in Figure 5, the fresh weight gain and proliferation rate of the PLBs obviously increased with the duration of culture, while the trends of differentiation rate and fold drying rate of the protocorm group-cultured seedlings under different treatments varied. Among all treatments, the proliferation rate of MT3 surpassed that of CK, showing a significantly higher proliferation rate compared to other treatments from the 25th to the 45th day, peaking at 65.68% on the 45th day. The differentiation rate of all treatments was significantly higher than that of CK on the 25th day but significantly lower than that of CK on the 40th day. The proliferation rate of protocorms under the MT1, MT2, and MT3 treatments on the 45th day showed different trends. On the 40th day, the differentiation rate of MT1 and MT4 was significantly lower than that of CK, while the differentiation rate of the MT1, MT2, and MT3 treatments resembled that of the CK on the 45th day, with the highest differentiation rate displayed in the MT3 treatment. However, the differentiation rate of MT4 was extremely lower than that of CK and the other treatments. This indicates that lower concentrations (50 µM and below) of MT favored cluster bud differentiation of Dendrobium bulbs, while higher concentrations (50–100 µM) of MT inhibited cluster bud differentiation to some extent. Furthermore, higher concentrations (50–100 µM) of MT increased the fold drying of Dendrobium bulbs over time.
Analysis of variance and range (Table 3) revealed significant effects of MT concentration and days of incubation on fresh weight gain, proliferation rate, differentiation rate, and fold drying rate. The greatest effect on the proliferation rate of Dendrobium bulbs was found for both MT concentration and days of incubation. Moreover, a greater effect on differentiation rate was obtained for MT treatments, while the number of days of incubation primarily influenced fresh weight gain and differentiation rate. Overall, the effects of treatment concentrations on Dendrobium PLBs were not as significant as the effects of incubation time on protocorms.

3.4. Photosynthesis-Related Pigments in Dendrobium PLBs

Excluding the effect of incubation days on the growth of Dendrobium, the content of photosynthesis-related pigments in PLBs treated with MT for 30 d is shown in Table 4. The results showed that with the increase in MT concentration, the contents of chlorophyll a, chlorophyll b, chlorophyll a+b, chlorophyll a/b, and carotenoids in Dendrobium PLBs were significantly higher than those in the CK treatment.
Under the MT4 treatment, chlorophyll a, chlorophyll b, and chlorophyll a+b were increased by 136.51%, 62.31%, and 110.75%, respectively, compared with the CK treatment; under the MT3 treatment, chlorophyll a/b and carotenoid contents were increased by 76.48% and 200.00%, respectively, compared with the control.

3.5. Antioxidant Enzyme Activities of PLB Tissue Culture Seedlings

The effects of MT treatment on the 30th day on the antioxidant enzyme activities of Dendrobium PLB group-cultivated seedlings are shown in Figure 6. The results showed that the SOD, POD, and CAT enzyme activities in PLBs of the MT1, MT2, and MT3 treatments were significantly higher than those of CK, while those of the MT4 treatment were lower than those of the CK treatment, and the MDA content was significantly lower than that of the CK treatment; the SOD, POD and CAT enzyme activities in PLBs under the MT1 treatment were 40.9%, 6.15%, and 33.62% higher than those of the CK treatment, respectively; the three enzyme activities in the treatment of MT2 were 95.53%, 15.03%, and 21.83% higher than those for CK, respectively; the SOD and CAT enzyme activities of Dendrobium PLBs under the MT3 treatment were 15.82% and 33.62% higher than those of the CK treatment, respectively. Conversely, these enzyme activities of MT4 were 36.86%, 33.86%, and 33.62% lower than those of CK, respectively. Moreover, the MDA content of MT1, MT2, and MT3 treatments was significantly lower than that of the CK treatment, reduced by 36.86%, 40.30%, and 8.73%, respectively. Overall, the MDA content of the four treatments was reduced by 13.51%, 21.38%, 30.96%, and 36.60% compared with CK.

3.6. Medicinal Ingredients of PLB Tissue Culture Seedlings

The effect of MT treatment on the 30th day on the functional products of Dendrobium PLBs in histocultured seedlings is shown in Figure 7. The results showed that the polysaccharides, total flavonoids, and total phenols in PLBs of MT1, MT2, MT3, and MT4 treatments were significantly higher than those in CK treatment, by 54.09%, 97.07% and 62.65%. Moreover, the alkaloid content under the MT1 treatment was lower than that of the CK treatment by 17.50%. Conversely, under the MT2, MT3, and MT4 treatments, the total alkaloid content was notably higher than that of the CK treatment, with increases of 14.51%, 43.53%, and 26.03%, respectively.

4. Discussion

Dendrobium officinale is usually propagated by tissue culture using seeds and stems as explants [32]. The contents of medicinal components in protocorm tissues were similar to those of biennial wild Dendrobium [33]. The addition of MT can promote the development of the plant [14,17]. Under certain MT treatment concentrations, an appropriate increase in MT concentration is favorable to both the growth of fresh weight and the accumulation of dry matter in plants. This trial investigated the effects of melatonin on the tissue growth of D. officinale PLB tissue culture seedlings within the period of the 25th to 45th day, using a concentration gradient ranging from 0 to 100 μM. The results indicated that by day 45, 75 µM of MT was optimal for the proliferation and differentiation of PLBs, which easily resulted in a green color, with more white fluffy stout plants. However, the 100 µM MT treatment inhibited differentiation to a certain extent, and the change became more pronounced than that caused by the day of incubation. This is confirmed by Khan et al. [34], who showed that 600 µM of MT had the highest rate of induction for adventitious roots of Withania somnifera L. It was observed that lower concentrations of MT promoted callus proliferation, while higher concentrations inhibited plant tissue differentiation [35,36]. Similar results were reported in other plants like Zoysia matrella [37], Prunella valgaris [38], Scutellaria Dianthus [39], and Festuca ovina L [40]. Moreover, the increase in the production of fine white flocculent around the embryoid body healing tissues of these protocorms was synchronized with the proliferation of each biomass, which suggests that white flocculent is an important sign of the growth and development process of protocorms.
Between the 25th and 35th day, there is a notable increase in the abundance of white hairs on the surface of D. officinale. The increase in white hairs is indicative of growth progression. Excluding the influence of days on the tissue-cultured seedlings of D. officinale PLBs, seedlings from the 30th day, representing a positive growth phase, were selected for the determination of physiological indicators and medicinal component content. The photosynthetic pigments in plants are mainly chlorophyll and carotenoids [41]. OU et al. [42] reported that exogenous spraying of MT effectively increased the chlorophyll content in Sapotaceae seedlings. In our present study, the treatment with 100 µM MT exhibited the best effect on chlorophyll content, while the treatment with 75 µM MT had the greatest effect on photosynthetic efficiency and carotenoids. Compared with the control, the content of photosynthetic pigment in MT treatment was generally increased. Similar studies have shown that 0.125 µM MT soaking treatment delayed the color degradation of Rambutan [43], indirectly indicating that MT could increase the photosynthetic pigment content in fruit peel and delay fruit senescence. Wang et al. [44] reported similar results in grape leaves, and Ahmad et al. [45] in wheat. It can be concluded that the addition of MT promotes the synthesis of photosynthetic pigments.
Under stressful environments, cells in plants produce large amounts of reactive oxygen radicals, disrupting the balance of reactive oxygen species metabolism and causing metabolic disorders, which in turn leads to damage to tissues within the cellular membrane system and affects the normal growth and development of plants [46]. Melatonin, as an influential antioxidant, improves the plant antioxidant defense system by directly and/or indirectly scavenging reactive oxygen species, thereby slowing down the abiotic stress suffered by plants [47,48,49]. Cui et al. [50] reported that MT increased antioxidant capacity and enhanced drought tolerance in wheat seedlings. Additionally, 600 µM MT can improve the activity of antioxidant enzymes (SOD, CAT, etc.) in postharvest guava and delay fruit senescence [51]. In our study, the SOD, POD, and CAT enzyme activities in Dendrobium PLB group-cultivated seedlings under MT treatment showed an increasing trend compared with CK treatment and showed a trend of increasing and then decreasing with the increase in concentration. This suggests that MT improved the antioxidant properties of D. officinale PLBs and reduced the degree of cell damage in the plant. Concurrently, the MDA content decreased compared to the CK treatment, aligning with findings for Gerbera jamesonii [52], oilseed rape [53], and buckwheat seeds [54]. Polysaccharides, alkaloids, flavonoids, and total phenols are recognized as vital components contributing to the medicinal effects of D. officinale [55,56,57,58]. Melatonin, acting as a plant growth regulator, enhances the growth and developmental capacity of explants while promoting an increase in sugar content [59]. The enrichment of flavonoids augments plant metabolism, thereby elevating their content [60]. Phenolic substances are secondary metabolites with significant antioxidant activity, which can protect plants from oxidation. Sandoval et al. [61] reported that melatonin treatment of high-bush blueberries increased total phenol content by 27%. In this study, polysaccharides, total flavonoids, and total phenols were significantly increased in Dendrobium PLBs under MT treatment on the 30th day. This is consistent with the results of Gao Fan’s research [62] on kiwi fruit (Actinidia deliciosa), Sanie Khatam’s research [63] on citrus (Citrus reticulate Blanco), and He Xiuli’s research [64] on golden line orchid (Anoectochilus roxburghii). In addition, the alkaloid content under the MT1 treatment was lower than that of the CK treatment, while alkaloid content under the MT2, MT3, and MT4 treatments was higher than that of the CK treatment. This suggests that a lower concentration of MT may inhibit alkaloid synthesis, whereas a higher concentration of MT may promote alkaloid expression; this is consistent with the result of Agata et al. [65].

5. Conclusions

The formation of D. officinale protocorms primarily involves protocorms, protocorm + healing tissue complexation, protocorm budding, and protocorm differentiation. The fresh weight gain, proliferation rate, and differentiation rate of D. officinale PLB tissue culture seedlings peaked on the 45th day in the treatment of 75 μM melatonin, demonstrating the most effective promotion effect. Conversely, the differentiation rate was lowest in the treatment of 100 μM. After 30 days of melatonin treatment, PLBs treated with 100 μM melatonin exhibited the highest chlorophyll, polysaccharide, and total flavonoid contents, all significantly different from the control. Significant differences in SOD activity and the contents of MDA, total flavonoids, and total phenols were observed between melatonin treatments and the control. In conclusion, the addition of melatonin in a culture medium could be a new method for improving the micropropagation of D. officinale PLBs.

Author Contributions

J.T. designed and executed the experiment, wrote the manuscript, and analyzed the data. C.W., C.L., R.H., D.Y., Z.P., H.S. and Y.L. helped in experimenting. D.Q. and Q.C. conceived and supervised the work and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by a project on the molecular regulation of polysaccharide metabolism of Dendrobium officinale under the influence of temperature difference (JT180141) of Fujian Provincial Education Department of young and middle-aged teachers education research.

Data Availability Statement

The data collected in this experiment can be accessed from the corresponding author on request.

Acknowledgments

The authors acknowledge the support received from the Fujian Agriculture and Forestry University, China.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. The developmental morphology of D. officinale at various developmental stages in in vitro culture conditions of 12 h/12 h L/D, 45 days.
Figure 1. The developmental morphology of D. officinale at various developmental stages in in vitro culture conditions of 12 h/12 h L/D, 45 days.
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Figure 2. PLB development on the embryogenic callus of D. officinale in culture conditions of 12 h/12/h L/D, 45 days; (A,B) Leica microscope observations.
Figure 2. PLB development on the embryogenic callus of D. officinale in culture conditions of 12 h/12/h L/D, 45 days; (A,B) Leica microscope observations.
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Figure 3. PLBs developing adventitious buds and the newly developed PLBs of the original PLB of Dendrobium candidum in culture conditions of 12 h/12 h L/D, 35 days, were observed with a Leica microscope.
Figure 3. PLBs developing adventitious buds and the newly developed PLBs of the original PLB of Dendrobium candidum in culture conditions of 12 h/12 h L/D, 35 days, were observed with a Leica microscope.
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Figure 4. Growth of D. officinale from 25 to 45d under melatonin gradient treatment. (a–e) Growth condition under MT1 treatment. (f–j) Growth condition under MT2 treatment. (k–o) Growth condition under MT3 treatment. (p–t) Growth condition under MT4 treatment. (u–y) Growth condition under CK treatment. Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
Figure 4. Growth of D. officinale from 25 to 45d under melatonin gradient treatment. (a–e) Growth condition under MT1 treatment. (f–j) Growth condition under MT2 treatment. (k–o) Growth condition under MT3 treatment. (p–t) Growth condition under MT4 treatment. (u–y) Growth condition under CK treatment. Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
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Figure 5. Effects of different concentrations of MT on proliferation and differentiation of PLB tissue culture seedlings of D. officinale. Different lowercase letters in the bar chart during the same period indicate significant differences between different treatments (p < 0.05). Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
Figure 5. Effects of different concentrations of MT on proliferation and differentiation of PLB tissue culture seedlings of D. officinale. Different lowercase letters in the bar chart during the same period indicate significant differences between different treatments (p < 0.05). Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
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Figure 6. Effects of different concentrations of MT treatment on the 30th day on antioxidant enzyme activities of Dendrobium PLB group-cultured seedlings. Note: Different lowercase letters represent significant differences (p < 0.05), the same below; 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
Figure 6. Effects of different concentrations of MT treatment on the 30th day on antioxidant enzyme activities of Dendrobium PLB group-cultured seedlings. Note: Different lowercase letters represent significant differences (p < 0.05), the same below; 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
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Figure 7. Effect of different concentrations of MT treated for 30d on functional products of D. officinale PLB tissue culture seedlings. Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
Figure 7. Effect of different concentrations of MT treated for 30d on functional products of D. officinale PLB tissue culture seedlings. Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
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Table 1. Melatonin gradient concentration parameters.
Table 1. Melatonin gradient concentration parameters.
TreatmentThe Concentration of
MT (µM)
MT125
MT250
MT375
MT4100
CK0
Note: MT represents melatonin treatment, CK represents control.
Table 2. Effect of MT treatment on plant morphology of D. officinale PLB tissue culture seedlings.
Table 2. Effect of MT treatment on plant morphology of D. officinale PLB tissue culture seedlings.
TimeTreatmentColorMorphologyCompaction DegreeClump SizeWhite FuzzGermination/Differentiation
25 dMT1yellowish greendelicatetightbiglessnone
MT2yellowish greenfulltightbiglessnone
MT3yellowish greenfulltightsmallnonenone
MT4yellowish greenfullloosebiglessnone
CKyellowish greenfullloosesmallnonenone
30 dMT1yellowish greenfulltightmiddle, biglesslittle
MT2yellowish greendelicateloosesmall, middlelesslittle
MT3yellowish greendelicateloosemiddle, biglesslittle
MT4yellowish greenfulltightsmall, biglesslittle
CKyellowish greendelicateloosebiglesslittle
35 dMT1emerald greenfullloosesmall, middlelesslittle
MT2fresh greenfullloosemiddle, biglessmore
MT3yellowish greenfullloosebigmoremore
MT4yellowish greenfulltightbigmorelittle
CKemerald greenfulltightsmall, middlelesslittle
40 dMT1emerald greenfullloosesmall, middlelesslittle
MT2yellowish greendelicateloosesmall, middlelessmore
MT3light greendelicateloosemiddle, bigmoremore
MT4light greenfullloosesmall, bigmorelittle
CKlight greenfullloosebigmoremore
45 dMT1light greendelicateloosesmall, biglessmore
MT2light greenfullloosemiddle, biglessmore
MT3light greendelicateloosebigmoremore
MT4yellowfullloosesmall, bigmorelittle
CKdark greenfulltightsmalllessmore
Note: CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM. Big: the diameter of the clump is greater than 2 cm; middle: the diameter of the clump is 0.5 to 2 cm; small: the diameter of the clump is less than 0.5 cm. More: more than half of the total has white fuzz; less: less than half of the total has white fuzz.
Table 3. Variance and range analysis table.
Table 3. Variance and range analysis table.
TreatmentFresh Weight Gain (g)Proliferation Rate (%)Differentiation Rate (%)Drying Rate (%)
F/R
F/R		Concentration
Day		11.177 */1.248
117.824 */1.319		138.965 */14.594
819.014 */10.345		42.346 */25.67
102.514 */5.667		19.094 */0.644
69.288 */0.244
Note: F stands for variance, * p < 0.05; R stands for range.
Table 4. Photosynthetic pigment content of Dendrobium PLB group-cultured seedlings incubated with different concentrations of MT on the 30th day.
Table 4. Photosynthetic pigment content of Dendrobium PLB group-cultured seedlings incubated with different concentrations of MT on the 30th day.
Treatment Chlorophyll a
mg/mL		Chlorophyll b
mg/mL		Chlorophyll a+b mg/mLChlorophyll a/b mg/mLCarotenoids
mg/mL
CK0.504 ± 0.01 c0.268 ± 0.01 b0.772 ± 0.02 a1.888 ± 0.05 b0.015 ± 0.01 a
MT10.692 ± 0.03 b0.284 ± 0.04 b0.976 ± 0.08 c2.514 ± 0.25 ab0.017 ± 0.01 d
MT20.768 ± 0.06 b0.414 ± 0.04 a1.182 ± 0.02 b1.920 ± 0.34 b0.029 ± 0.01 c
MT31.084 ± 0.05 a0.330 ± 0.03 ab1.414 ± 0.04 a3.332 ± 0.34 a0.045 ± 0.02 b
MT41.192 ± 0.03 a0.435 ± 0.05 a1.627 ± 0.04 c2.785 ± 0.21 a0.042 ± 0.05 b
Note: Different lowercase letters represent significant differences (p < 0.05). CK: 0 µM; MT1: 25 µM; MT2: 50 µM; MT3: 75 µM; MT4: 100 µM.
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Tang, J.; Huang, R.; Yuan, D.; Sun, H.; Li, Y.; Pei, Z.; Wang, C.; Li, C.; Qiu, D.; Chen, Q. Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies. Agronomy 2024, 14, 1326. https://doi.org/10.3390/agronomy14061326

AMA Style

Tang J, Huang R, Yuan D, Sun H, Li Y, Pei Z, Wang C, Li C, Qiu D, Chen Q. Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies. Agronomy. 2024; 14(6):1326. https://doi.org/10.3390/agronomy14061326

Chicago/Turabian Style

Tang, Jiaqi, Ruyan Huang, Dan Yuan, Han Sun, Yuping Li, Zekang Pei, Congqiao Wang, Chengyue Li, Dongliang Qiu, and Qingqing Chen. 2024. "Effect of Melatonin on the Growth of Dendrobium officinale Protocorm-Like Bodies" Agronomy 14, no. 6: 1326. https://doi.org/10.3390/agronomy14061326

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