The Influence of Varying Wavelengths of LED Light on the Development, Physiology Response, and Metabolism Activities of Micropropagated Dendrobium Hybrid ‘Shuijing’ Plantlets

: Dendrobium hybrids have a significant role in the present floral sector. The aim of this research was to evaluate how various light qualities affect the physiological and biochemical traits of Dendrobium ‘Shuijing’. In order to determine the optimal light quality for in vitro cultivation of Dendrobium plantlets, we examined the correlations between growth, antioxidant capacity, and nutrient and chlorophyll levels, as well as chlorophyll fluorescence. The growth rate was compared by using different light qualities emitted by the LED light source. These included red light (R), blue light (B), and three ratios: 8R:2B, 7R:3B, and equal proportions of both colors, known as white, fluorescent light (CK). The combination of 7R:3B resulted in noticeable enhancements in leaf count, root length, root activity, fresh and dry weight measurements, antioxidant capability, as well as chlorophyll content and fluorescence. Specifically, the mixture of red and blue LED lights at a ratio of 7R:3B led to increased leaf number, root length, root activity, fresh and dry weight measurements, antioxidant ability, and chlorophyll content with improved fluorescence. In order to explore the effect of light quality on the growth and development of Dendrobium , the chlorophyll content and chlorophyll fluorescence parameters of plants under all light quality conditions were analyzed by using a linear regression model with other physiological and biochemical indexes. A significant correlation between non-photochemical quenching (NPQ) and leaf length was also observed. The content of chlorophyll b showed significant correlations with both root number and leaf number. Furthermore, chlorophyll a , along with its ratio to chlorophyll b , significantly correlated with root length. Chlorophyll b and the relative electron transport rate of PSII (ETRII) significantly correlated with root activity and the free proline content (FPC) and catalase (CAT) activity. The photochemical quenching coefficient (qP) significantly correlated with total soluble sugars content (SSC) and peroxidase (POD) activity. The correlation between the quantum yield of PSII (Fv/Fm ratio) and superoxide dismutase (SOD) activity was found to be significant. Similarly, the effective quantum yield of PSII photochemistry ( Φ PSII) showed significant correlations with fresh weight, dry weight, soluble protein content (SPC), and ascorbate peroxidase (APX) activity. Through a principal component analysis (PCA), it was observed that plants cultivated under the 7R:3B light treatment achieved significantly better comprehensive scores compared to those grown under different light treatments. In conclusion, growth achieved under an LED emitting a ratio of 7R:3B light yielded the most robust Dendrobium hybrid plantlets within a controlled environment.


Introduction
Light quality is a crucial environmental factor that plays a significant role in regulating plant growth, development, and stress tolerance [1].LEDs are recognized as unique light sources due to their remarkable efficiency, minimal emission of radiant heat, low energy consumption, remarkable energy conversion efficiency, customizable wavelength selection, prolonged lifespan, and rapid start-up capability.LEDs demonstrate superior performance compared to traditional light sources like High-Pressure Sodium Lamps (HPSL), Metal Halide Lamps (MHL), and Incandescent Lamps (IL) [2].LEDs can be set to specific radiant wavelength(s) to optimize the biological processes of a specific species [3].The ability to adjust LEDs allows for the creation of a personalized lighting setting, which has the potential to impact plant structure and chemical processes, leading to enhanced plant development and the synthesis of beneficial substances [4].Many studies have indicated that the combination of LEDs of different colors may favor plant growth and development [2].
Ninety percent of the pigments utilized in the process of plant photosynthesis have a propensity to absorb light within the red and blue regions [2].The efficiency of triggering photosynthesis is maximized by red light due to its proximity to the peak absorption wavelength range of chlorophyll, which falls between 600 and 700 nm [5].Plant stem elongation, leaf growth, and soluble sugar content are all affected by monochromatic red light, which also promotes flowering and fruiting and prolongs the flowering period.On the other hand, blue light is essential for photosynthesis and affects the stomatal density, photosynthetic rate, and chlorophyll content [6].Plants cultivated in the presence of blue light exhibit a higher abundance of chloroplasts compared to those exposed to red light, and these chloroplasts possess enlarged basidia that enhance the efficiency of photosynthesis.Several studies have shown that mixed red and blue light can promote higher plant photosynthesis and productivity and is more favorable to the growth and development of plants than monochromatic light.According to Liu et al. [7], plants cultivated solely under red light sourced from LED exhibited leaves with distorted morphology and a diminished rate of photosynthesis.The photosynthetic rate of tomato plantlets cultivated under monochromatic red light exhibited a reduction of at least threefold in comparison to those grown under a red-to-blue ratio of 10:1 [7].Interestingly, the impaired leaf photosynthetic machinery caused by monochromatic red light can be recovered and restored with as little as 7% of blue light exposure [8].The efficacy of plant growth is enhanced more effectively by mixed light, compared to monochromatic light, as indicated by several studies [8].The utilization of a combination of red and blue light during the cultivation of Populus euramericana plantlets in vitro has been observed to enhance cell division, as opposed to using monochromatic LED light or fluorescent light [9].These recent findings highlight the adaptability of plants in harnessing a wide spectrum of light wavelengths for photogenically regulating responses through their photoreceptor [10].
Globally, orchids are a significant crop, with hybrids of the Dendrobium species, such as Dendrobium 'Burmese Ruby' × Dendrobium 'Mae-klong', among the most well-liked varieties.Dendrobium hybrids (DHs) are widely used in decorative gardens due to their lengthy flowering season, large range of beautifully shaped and colorful flowers, and year-round availability [11].These orchids are micro-propagating for the flower industry.At present, a large number of papers have shown that LED lighting can affect their overall development and physiological efficiency in vitro [12], while the light quality requirements varied among different species or varieties [13].Chlorophyll fluorescence and other photosynthetic markers can be measured in plants to assess the dynamic fluctuations in photosynthesis and identify the impact of different external stimuli on plant growth [3].This experiment was interested in how the small plants of the in vitro-cultured Dendrobium hybrid 'Shuijing' respond to different qualities of LED light.The physiological-biochemical parameters, chlorophyll fluorescence, and growth indices were measured to determine an ideal light quality for the in vitro culturing of 'Shuijing'.The results obtained from this research can be effectively employed to enhance the technical strategy for mass-scale in vitro manufacturing of this commercially cultivated blossom.

General Growth Conditions
The trials were carried out in the Tissue Culture Laboratory, Key Laboratory of Biology of Tropical Characteristic Flowers and Trees at the College of Forestry on the Danzhou Campus of Hainan University.Sixth-generation in vitro plantlets of the Dendrobium hybrid 'Shuijing' were provided by the Hainan Tropical Quality Institute (Danzhou, Hainan, China) and were grown in transparent glass culture bottles (250 mL capacity, four plants per bottle) containing MS 4 g•L −1 + 6-BA 1 mg•L −1 + agar powder 6 g•L −1 + banana powder 3 g•L −1 + sucrose 30 g•L −1 ; the seedlings were kept in a rootless state (a root length less than 0.3 cm is considered rootless).Under the fluorescent lamp, the temperature condition was 25 ± 2 • C, the tissue culture seedlings were 60 cm away from the light source, the relative humidity was 70 ± 5%, and the illumination time was 12 h/d.After 45 days, 10 bottles of test-tube plantlets were randomly selected from each treatment to study the effects of different light qualities.

Experimental Design
In order to measure and calculate the photosynthetic photon flux density (PPFD), we use the American Apogee Instruments company's MQ-500 handheld optical quantum measuring instrument.(Apogee Instruments, Logan, UT, USA) The approximate value measured at the height of 60 cm from the bottom of the LED plant growth lamp is about 50 ± 10 µmol•m −2 •s −1 .The main wavelength of the LED red light (R) was 640 nm, and the main wavelength of the blue light (B) was 464 nm.Five LEDs (120 cm × 70 cm; iGrowLite Co., Ltd., Guangzhou, China) with light quality combinations: 100% red (R), 100% blue (B), 80% red + 20% blue (8R:2B), 70% red + 30% blue (7R:3B), 50% red + 50% blue (5R:5B), and white, fluorescent lamps using TL-D 36w/54 cold fluorescent lamps (Philips, Shanghai, China) as a control group (CK) were used (Figure 1).The test-tube plantlets with the same growth (seedling height of about 3 cm, each plant containing 2-3 leaves) were randomly selected as test materials.The test-tube seedlings were placed under the culture frame of the LED plant growth lamp and the cold fluorescent lamp for 6 treatments.Each treatment was placed in 30 bottles of test-tube seedlings (3 plants per bottle).The irradiation period was 12 h•d −1 , and the irradiation time was 6:00-18:00, which was controlled by a timer.The culture frame adopts a steel structure with a width of 1.5 m, and shading materials are set between different light quality treatments to prevent the influence of the surrounding light-quality treatments on it.

Morphological Assessment
After 45 days of incubation, 10 randomly selected seedlings from each treatment group were analyzed morphologically.A vernier caliper was used to measure plant height (in centimeters) and leaf and root lengths (cm).The number of roots and leaves were counted.The plantlets were weighed to determine their fresh weight (FW) and then baked for 30 min at 105 • C before drying for 48 h at 60 • C to determine their dry weight (DW).

Assessment of Root Activity
The root activity was determined by using triphenyltetrazolium chloride.An appropriate number of fresh samples were placed in a solution containing 0.4% vaporized triphenyltetrazolium chloride and 66 mmol/L phosphate buffer (PH = 7.0).The roots were all immersed in the solution.The beaker was placed in a constant temperature of 37 • C.After 1 h, sulfuric acid with a concentration of 2 mol/L was added to terminate the reaction.The roots were ground and the filtered extract was obtained in the 485 nm band.The blank test was used as a reference [14].Three samples were collected for each treatment, and three experiments were repeated.

Morphological Assessment
After 45 days of incubation, 10 randomly selected seedlings from each treatment group were analyzed morphologically.A vernier caliper was used to measure plant height (in centimeters) and leaf and root lengths (cm).The number of roots and leaves were counted.The plantlets were weighed to determine their fresh weight (FW) and then baked for 30 min at 105 °C before drying for 48 h at 60 °C to determine their dry weight (DW).

Assessment of Root Activity
The root activity was determined by using triphenyltetrazolium chloride.An appropriate number of fresh samples were placed in a solution containing 0.4% vaporized triphenyltetrazolium chloride and 66 mmol/L phosphate buffer (PH = 7.0).The roots were all immersed in the solution.The beaker was placed in a constant temperature of 37 °C.After 1 h, sulfuric acid with a concentration of 2 mol/L was added to terminate the reaction.The roots were ground and the filtered extract was obtained in the 485 nm band.The blank test was used as a reference [14].Three samples were collected for each treatment, and three experiments were repeated.

Antioxidant System and Nutritional Indicators
The contents of soluble protein (SP) and soluble sugar (SS) in leaves were determined according to the experimental method of Li et al. [15].Determination of plant antioxidant system indexes: the SOD activity was determined by using a nitroblue tetrazolium chromogenic method [16], the POD activity was determined by using a guaiacol oxidation method, the CAT activity was determined by using a H 2 O 2 ultraviolet absorption method [16], the MDA content was determined via a thiobarbituric acid method [17], and the APX activity was evaluated according to the Nakano and Asada methods [18].Three samples were collected randomly for each index, and the average value of the three measurements was used as the content of each index.

Chlorophyll and Carotenoid Contents
After extraction with 95% propanol at room temperature and dark for 24 h, the absorbance values at 665, 649, and 470 nm were measured via an ultraviolet visible spectrophotometer.The contents of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid were calculated according to the obtained absorbance values [19].Three samples were collected randomly for each treatment and three experiments were repeated.Three representative plants per treatment were selected for the experiment.The portable modulated chlorophyll fluorescence spectrometer (MIN-PAM.II, WALZ, Effeltrich, Germany) was used for determination: Fv/Fm, ΦPSII, ETRII, qP, NPQ, and maximum photochemical quantum efficiency (Fv/Fo) of photosystem II (PSII) under dark adaptation conditions [20].

Statistical Analysis
Microsoft Excel 2022 was used to arrange all data, and SPSS 24.0 (IBM Inc., Chicago, IL, USA) was used for analysis.The analysis conducted by Duncan involved multiple comparisons and one-way ANOVA.The observed statistical significance was attributed to differences with p-values below 0.05.To explore the relationships between growth, physiological-biochemical parameters, and chlorophyll fluorescence-related factors, a Multiple Stepwise Regression Analysis was employed.Additionally, a principal component analysis (PCA) was utilized to evaluate all variables and determine the optimal light quality for the in vitro cultivation of Dendrobium hybrid plantlets.Origin 2019 software was used to create the histogram and biplot (OriginLab Inc., Hampton, MA, USA).

Impact of Diverse Light Spectrums on the Growth Characteristics of 'Shuijing' Dendrobium Hybrid Plantlets Cultured In Vitro
The in vitro growth of 'Shuijing' plantlets was found to be significantly impacted by the light quality, as depicted in Figures 2 and 3.The plantlets varied considerably in height depending on the wavelengths of light.The plants achieved their maximum height when exposed to monochromatic red light (R), followed by the treatment of eight parts red light and two parts blue light (8R:2B), while they exhibited the shortest height under monochromatic blue light (B).The highest leaf length was observed under blue light, with the combination of eight parts red light and two parts blue light ranking second.The combination of seven parts red light and three parts blue light resulted in the highest values for root length, fresh weight, and dry weight.The greatest number of roots was found under the combination of five parts red light and five parts blue light, whereas it was lowest under both the fluorescent (CK) lighting condition and monochromatic red light.The highest root activity was observed in plantlets grown under a 7R:3B light ratio, reaching 223.98 ng•g −1 •h −1 , more than twice the activity under fluorescent (CK) and monochromatic R light.The 7R:3B and 5R:5B combinations yielded the highest leaf count, exhibiting no statistically significant distinction between them.Table S1 lists the specific values of growth characteristics in each treatment group.

Effects of Different Light Qualities on the Contents of Soluble Sugars (SSC), Soluble Proteins (SPC), and Free Proline of Dendrobium Hybrid 'Shuijing' Planets In Vitro
The 'Shuijing' plantlets exhibited significant differences in the levels of soluble sugars (SSC), soluble proteins (SPC), and free proline (FPC) under different light qualities (Figure 4).The SSC reached the highest amount, 30.73 mg•g −1 , under the 7R:3B light treatment, followed by the 8R:2B treatment (30.73 mg•g −1 ), with the lowest value, 18.64 mg•g −1 , reached under blue light only (B).The SPC exhibited higher values in all distinct combinations of LED lights, as compared to the control group (CK), with the highest value observed under B alone at 5.81 mg•g −1 , followed by 7R:3B at 5.77 mg•g −1 .Significant differences were found in the FPC among different treatments, with the maximum value, 56.05 µg•g −1 , reached under the R only treatment, with the CK in second place, at 55.70 µg•g −1 .These values were all higher than the value under the red and blue combination treatments.Table S2 lists the specific values of soluble sugar (SSC), soluble protein (SPC) and free proline content in each treatment group.served under B alone at 5.81 mg•g −1 , followed by 7R:3B at 5.77 mg•g −1 .Significant differences were found in the FPC among different treatments, with the maximum value, 56.05 µg•g −1 , reached under the R only treatment, with the CK in second place, at 55.70 µg•g −1 .
These values were all higher than the value under the red and blue combination treatments.Table S2 lists the specific values of soluble sugar (SSC), soluble protein (SPC) and free proline content in each treatment group.

Effects of Various Light Spectrums on the In Vitro Antioxidant Capacity of Leaves from Dendrobium Hybrid 'Shuijing' Plantlets
The MDA content and antioxidant enzyme activities in the leaves of 'Shuijing' plantlets cultured in vitro were significantly influenced by different light qualities (Figure 5).The highest MDA content was observed under blue light, while the lowest was observed under the 5R:5B light ratio.The POD activity was highest under the 7R:3B ratio, second highest under the 8R:2B ratio, and lowest under fluorescent light (CK).The activities of SOD under all LED treatments were higher than those under the fluorescent CK, reaching the highest level under 7R:3B, which was almost 1.5 times higher than under CK.The responses of CAT varied significantly under different light qualities.The CAT values under the combined red and blue light treatments were higher than those of CK and monochromatic R or B light, and reached the highest values under the 5R:5B ratio, with 7R:3B in second place.Revealing a resemblance to CAT, the APX activities exhibited a notable increase when subjected to a blend of red and blue light, in contrast to CK and monochromatic R or B light treatments.The 7R:3B treatment yielded the most heightened activity.Table S3 lists the specific values of Antioxidant Capacity in each treatment group.

Effects of Various Light Spectrums on the In Vitro Antioxidant Capacity of Leaves from Dendrobium Hybrid 'Shuijing' Plantlets
The MDA content and antioxidant enzyme activities in the leaves of 'Shuijing' plantlets cultured in vitro were significantly influenced by different light qualities (Figure 5).The highest MDA content was observed under blue light, while the lowest was observed under the 5R:5B light ratio.The POD activity was highest under the 7R:3B ratio, second highest under the 8R:2B ratio, and lowest under fluorescent light (CK).The activities of SOD under all LED treatments were higher than those under the fluorescent CK, reaching the highest level under 7R:3B, which was almost 1.5 times higher than under CK.The responses of CAT varied significantly under different light qualities.The CAT values under the combined red and blue light treatments were higher than those of CK and monochromatic R or B light, and reached the highest values under the 5R:5B ratio, with 7R:3B in second place.Revealing a resemblance to CAT, the APX activities exhibited a notable increase when subjected to a blend of red and blue light, in contrast to CK and monochromatic R or B light treatments.The 7R:3B treatment yielded the most heightened activity.Table S3 lists the specific values of Antioxidant Capacity in each treatment group.

Impact of Diverse Light Qualities on the Concentrations of Chlorophyll and Carotenoids in Cultured Dendrobium Hybrid Plantlets
The levels of chlorophyll a, chlorophyll b, overall chlorophyll, and carotenoids, as well as the chlorophyll a/b ratio in the leaves of Dendrobium hybrid plantlets were significantly influenced by the varying light conditions (Figure 6).The plants cultivated under LED light exhibited significantly higher levels of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids compared to those grown under fluorescent light.Among the different treatments, the 7R:3B combination resulted in maximum values for carotenoids and chlorophyll a.Interestingly, there was no noticeable distinction between the 8R:2B and 5R:5B combinations in terms of producing the highest amount of chlorophyll b.The treatments with the 8R:2B and 5R:5B ratios yielded the highest levels of total chlorophyll.Notably, R monochromatic light led to the highest value for the chlorophyll a/b ratio, while both 7R:3B and B monochromatic light tied for second place.Table S4 lists the specific values of concentrations of chlorophyll and carotenoids in each treatment group.

Impact of Diverse Light Qualities on the Concentrations of Chlorophyll and Carotenoids in Cultured Dendrobium Hybrid Plantlets
The levels of chlorophyll a, chlorophyll b, overall chlorophyll, and carotenoids, as well as the chlorophyll a/b ratio in the leaves of Dendrobium hybrid plantlets were significantly influenced by the varying light conditions (Figure 6).The plants cultivated under LED light exhibited significantly higher levels of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids compared to those grown under fluorescent light.Among the different treatments, the 7R:3B combination resulted in maximum values for carotenoids and chlorophyll a.Interestingly, there was no noticeable distinction between the 8R:2B and 5R:5B combinations in terms of producing the highest amount of chlorophyll b.The treatments with the 8R:2B and 5R:5B ratios yielded the highest levels of total chlorophyll.Notably, R monochromatic light led to the highest value for the chlorophyll a/b ratio, while both 7R:3B and B monochromatic light tied for second place.Table S4 lists the specific values of concentrations of chlorophyll and carotenoids in each treatment group.The parameters of chlorophyll fluorescence, including the maximum quantum yield of photosystem II (Fv/Fm), the effective quantum yield of photosystem II (ΦPSII), photochemical quenching (qP), non-photochemical quenching (NPQ), and ETRII were measured

The Influence of Various Light Characteristics on the Fluorescence of Chlorophyll in Dendrobium Hybrid Plantlets Cultivated in a Controlled Laboratory Setting
The parameters of chlorophyll fluorescence, including the maximum quantum yield of photosystem II (Fv/Fm), the effective quantum yield of photosystem II (ΦPS II ), photochemical quenching (qP), non-photochemical quenching (NPQ), and ETR II were measured in the leaves of Dendrobium hybrid plantlets cultivated in vitro to investigate whether variation in the quality of LED light could induce alterations in photosynthetic electron transport (Figure 7).The results demonstrated the significance of utilizing various combinations of red and blue LED light signals in order to promote the growth and development of plants.Plants that were exposed to a blend of red and blue light exhibited higher values of Fv/Fo, Fv/Fm, ΦPS II , qP, and ETR II compared to those subjected solely to either blue or red light.These values peaked under the 7R:3B regime.The NPQ was significantly greater under the 8R:2B and B monochromatic treatments, with no significant difference between them, and the 7R:3B and 5R:5B treatments were in second place, also with no significant difference between them.

Relationship between Growth and Antioxidant Parameters and the Chlorophyll and Fluorescence Parameters of Dendrobium Hybrid Plantlets Grown In Vitro
For the correlation and regression analyses (Durbin-Watson 2.00 ± 0.20; VIF < 2.00), we examined various growth and antioxidant parameters, including plantlet height, leaf length, leaf number, root number, root length, root activity, fresh weight, dry weight, soluble sugar content (SSC), soluble protein content (SPC), free radical content (FRC), malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and ascorbate peroxidase (APX) activity.Additionally, chlorophyll and fluorescence parameters such as chlorophyll a, chlorophyll b, total chlorophyll content, carotenoids content, chlorophyll a/b ratio, ratio of variable to maximum fluorescence yield in the dark-adapted state (Fv/Fo), maximum quantum efficiency of photosystem ll photochemistry in the dark-adapted state (Fv/Fm), effective quantum yield of PSII photochemistry under actinic light conditions (ΦPSII), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and electron transport rate through PSII (ETRII) were considered as independent variables.These relationships Results are presented as mean values ± standard deviation (n = 3).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.

Relationship between Growth and Antioxidant Parameters and the Chlorophyll and Fluorescence Parameters of Dendrobium Hybrid Plantlets Grown In Vitro
For the correlation and regression analyses (Durbin-Watson 2.00 ± 0.20; VIF < 2.00), we examined various growth and antioxidant parameters, including plantlet height, leaf length, leaf number, root number, root length, root activity, fresh weight, dry weight, soluble sugar content (SSC), soluble protein content (SPC), free radical content (FRC), malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity, peroxidase (POD) activity, catalase (CAT) activity, and ascorbate peroxidase (APX) activity.Additionally, chlorophyll and fluorescence parameters such as chlorophyll a, chlorophyll b, total chlorophyll content, carotenoids content, chlorophyll a/b ratio, ratio of variable to maximum fluorescence yield in the dark-adapted state (Fv/Fo), maximum quantum efficiency of photosystem ll photochemistry in the dark-adapted state (Fv/Fm), effective quantum yield of PSII photochemistry under actinic light conditions (ΦPS II ), photochemical quenching coefficient (qP), non-photochemical quenching coefficient (NPQ), and electron transport rate through PSII (ETR II ) were considered as independent variables.These relationships between growth and antioxidant parameters along with chlorophyll fluorescence parameters can provide preliminary insights into the responses of plantlets to their light quality environment (Table 1).For example, there was a significant association between leaf length and the NPQ (p < 0.05).Moreover, root number and leaf number exhibited a significant correlation with chlorophyll b content (p < 0.05).The root length showed a significant relationship with both chlorophyll a content and the ratio of Chlorophyll a/b (p < 0.05).Furthermore, the root activity demonstrated a significant correlation with both chlorophyll b and ETR II parameters (p < 0.05), while ETR II also exhibited correlations with other parameters, such as FPC and CAT activities (p < 0.05).Moreover, the ΦPS II value demonstrated significant correlations with fresh weight, dry weight, and APX (p < 0.05).Furthermore, both the ΦPS II value and chlorophyll a content were significantly correlated with SPC (p < 0.05).There was a notable association (p < 0.05) observed between qP and the levels of chlorophyll a + b and SSC, while a significant relationship (p < 0.05) was found between qP and Fv/Fm, as well as POD activity.Additionally, Fv/Fm exhibited a significant correlation (p < 0.05) with SOD activity.Plant height did not closely relate to either the chlorophyll content or the parameters of chlorophyll fluorescence.

Linear Regression Models Were Constructed by Integrating Physiological and Biochemical Parameters, along with Chlorophyll Fluorescence Measurements
A PCA was conducted on the 27 observed attributes across different lighting scenarios.The first four principal components, PC1, PC2, PC3, and PC4, all exhibited eigenvalues exceeding 1 and collectively accounted for a cumulative contribution rate of 91.815%.Consequently, these primary factors elucidated approximately 92% of the variability present in dataset (Table 2, Figure 8A).The initial principal component (PC1) explained 62.128% of the overall variability and displayed a significant association with both chlorophyll concentrations and ΦPSII-related chlorophyll fluorescence parameters such as Fv/Fm, chlorophyll a + b, chlorophyll a, chlorophyll b, Fv/Fo, ETRII, qP, NPQ, and carotenoids (R = 0.781-0.975)and to the original growth indices of dry weight, APX, fresh weight, SOD, leaf number, root activity, CAT, number of roots, and SPC (R = 0.786-0.979).The correlation index (R) value of FPC was −0.803, indicating a negative relationship with the original variation.Additionally, the second principal component (PC2) explained 13.424% of the overall variability and demonstrated significant correlations with variables such as SSC and plantlet height (R = 0.885-0.934),thereby reflecting the growth parameters of the plantlets.The correlation coefficient (R) value of leaf length was −0.553, indicating a negative relationship with the original variation.PC3 contributed to 10.652% of the overall variance and displayed a significant correlation with both MDA levels and root length (R = 0.628-0.681).The root number had a negative correlation with the original variation (R = −0.541).The fourth principal component, PC4, explained 5.611% of the total variation and exhibited a strong correlation (R = 0.512) with the original variable of FPC, indicating the plantlets' antioxidative capacity under different light qualities.

Comprehensive Scores of Dendrobium Hybrid Plantlets Grown In Vitro
According to the comprehensive scores of the first four principal components (PC1-PC4), the overall quality of the Dendrobium hybrid plantlets cultured under different light treatments was numerically evaluated (Figure 8B).The comprehensive scores were significantly higher for plantlets cultured under the 7R:3B light treatments (1.25) compared to those grown under other treatments.This suggested that the 7R:3B ratio was the most suitable LED illumination regime for the in vitro culture of Dendrobium hybrid plantlets.

Discussion
Light serves as a crucial environmental cue that exerts influence on plant photosynthesis and photomorphogenesis.Light signals play a role in regulating various physiological, growth-related, and metabolic processes through factors such as spectral quality, light intensity or quantity, and photoperiod or duration [21,22].The spectral quality of light has a significant impact on the physiological and biochemical adaptability of plants.Photoreceptors, including cryptochrome, phytochrome, and phototrophins, absorb light signals that influence photomorphogenesis [23].The light spectrum that plants predominantly absorb for photosynthesis falls within the photo-synthetically active region (PAR), which ranges from 400 nm (blue spectra) to 700 nm (red spectra) [23].The blue light receptors, phototrophins, possess the capability to regulate both stomatal aperture movement and phototropism [24,25].Red light is efficiently absorbed by plant pigments such as chlorophyll and carotenoids, which can impact endogenous phytohormones and induce secondary metabolite production in plants [26].The impact of light quality on photomorphogenesis varies across different species.Numerous investigations have indicated that the combination of blue and red LED lights can provide optimal light quality for plants [27,28].Therefore, it is very important to choose the right hybrid LED lights [29].This study delved into the impact of various qualities of LED-generated light on the in vitro growth of Dendrobium hybrid 'Shuijing' plantlets, revealing noteworthy disparities in the results.The maximum numerical values for root length, root activity, leaf count, fresh weight, and dry weight were observed when exposed to a red/blue light ratio of 7:3, while other indicators responded well in comparison to other treatments.Subsequent investigation showed that the development of the plantlets was significantly impacted by the percentage of B light in the R/B combination.The addition of 30% blue light to the monochromatic red light led to a beneficial modification in the growth indices.However, further adjustments to the proportion of blue light led to a decrease in the values of other growth indicators, except for root activity and root number.This may happen because R light can control how the plant photosynthetic system functions and how assimilates are transported, whereas blue light is crucial for chlorophyll synthesis, chloroplast growth, and stomatal opening [2,3].In general, the optimal blend of R and B LED-based light was found to be 7R:3B, as it exhibited the highest positive impact on the in vitro development of Dendrobium hybrid.
A study conducted by Matsuda et al. [30] revealed that the quality of light can potentially impact the soluble sugar content (SSC), due to variations in light absorption during carbohydrate synthesis.The findings of Lin et al. [31] yielded similar outcomes, suggesting a potential correlation between phytochromes and the regulation of sucrose-metabolizing enzymes.The findings of Lin et al. [31] demonstrated that the utilization of R light emitted by LED sources promoted the accumulation of SSC, while simultaneously inhibiting the accumulation of soluble proteins (SPC).Our experimental results align with this conclusion.Soluble protein accumulation was facilitated by the presence of blue light, potentially attributing to heightened protein synthesis due to the elevated energy level associated with this specific wavelength [32].The SSC and SPC were significantly higher when utilizing the optimal combination of red and blue LED-generated light (7R:3B) compared to using 8R:2B or 5R:5B.The findings suggest that the individual wavelengths of R or B contribute to the accumulation of soluble sugars or proteins, respectively.However, a suitable combination of these wavelengths would enhance both soluble sugars and proteins.This phenomenon has also been observed in Cucumis sativus [33] and Withania somnifera [32].The FPC exhibited a decrease in response when subjected to a blend of LED light, as opposed to monochromatic red light and CK treatment.These results imply that the combined utilization of red and blue wavelengths is more advantageous for enhancing the in vitro growth of Dendrobium hybrid plantlets.
The process of lipid peroxidation results in the production of malondialdehyde (MDA), which serves as an indicator for the degradation of unsaturated fatty acids and offers valuable insights into the antioxidant capabilities exhibited by plants [32].The MDA levels were found to be lower under the 5R:5B and 7R:3B light regimes in the present study, suggesting that these specific wavelengths do not induce damage to cell membranes.This discovery aligns with the outcomes documented by Yu et al. [29].The highest level of MDA concentration was detected when exposed to B light, indicating that the membrane lipids in Dendrobium hybrids undergo higher oxidation levels in response to increased energy from short-wavelength lights.This discovery aligns with prior investigations carried out on Triticum aestivum [33].The potential decrease in plant productivity of up to 50% can be attributed to the detrimental impact of oxidizing free radicals on crop yield.Reactive oxygen species (ROS) primarily arise from the inefficient utilization of light energy during photosynthesis [34].Superoxide dismutase (SOD) acts as the main protective mechanism against oxidative stress and lipid peroxidation by promoting the transformation of O 2− into H 2 O 2 and O 2 , while peroxidase (POD) and catalase (CAT) eliminate H 2 O 2 from peroxisomes and cytoplasm [31].As a crucial component of the ascorbate-glutathione cycle, ascorbate peroxidase (APX) plays a vital role in neutralizing H 2 O 2 to protect lipids from undergoing peroxidation [35].The process of lipid peroxidation results in the production of malondialdehyde (MDA), which serves as an indicator for the degradation of unsaturated fatty acids and offers valuable insights into the antioxidant capabilities exhibited by plants [36].The MDA levels were found to be lower under the 5R:5B and 7R:3B light regimes in the present study, suggesting that these specific wavelengths do not induce damage to cell membranes.This discovery aligns with the outcomes documented by Wang et al. [33].The highest level of MDA concentration was detected when exposed to B light, indicating that the membrane lipids in Dendrobium plantlets undergo higher oxidation levels in response to increased energy from shortwavelength lights.This discovery aligns with prior investigations carried out on Triticum aestivum [37].Our results indicate that the utilization of LED-generated light with a ratio of 7 parts red to 3 parts blue significantly increased the levels of superoxide dismutase (SOD) and ascorbate peroxidase (APX), along with the content of peroxidase (POD), surpassing all other treatments.Conversely, catalase (CAT) activity reached its peak in the 5R:5B treatment.These results suggest that an appropriate ratio of red and blue LED light can effectively enhance the antioxidant capacity of Dendrobium hybrid plantlets and delay their senescence.Furthermore, our study supports previous research by Ahmadi et al. [36], which highlighted how an LED lighting system enables precise coordination between spectral composition and plant photoreceptors to optimize growth.
The chlorophyll molecule plays a crucial role as the central component of the photo synthetic machinery in almost all green plants, directly influencing both the efficiency of photosynthesis and the synthesis of primary metabolites [38].Numerous research studies have indicated that light quality affects the concentration of chlorophyll and the process of photosynthesis in plants [39,40].For example, phototrophins, which are blue light receptors, possess the ability to regulate stomatal aperture movement and phototropism [33,41].Similarly, red light is efficiently absorbed by plant pigments such as chlorophyll and carotenoids, which can influence endogenous phytohormones and induce secondary metabolite production in plants [42].The results of this study showed that monochromatic B light was better than monochromatic R light for chlorophyll synthesis, and that red and blue light was better for chlorophyll biosynthesis than monochromatic B light.Which demonstrated that, in addition to a single spectrum, the combined light quality of red and blue light could further enhance the photosensitivity of plants.This is in agreement with the findings of Gitelson et al. [38] on Brassica campestris seedlings.Moreover, altering the red to blue light ratio induces modifications in the concentrations of chlorophyll a, chlorophyll a + b, and carotenoids.All the components, except for chlorophyll b and the chlorophyll a/b ratio, achieved their peak values when subjected to the 7R:3B treatment.Furthermore, under the R and B combination treatments, every value-aside from the chlorophyll a/b ratio-was higher than it was for R or B light alone.These findings indicate that an optimal combination of red and blue LED light had a positive impact on the synthesis of chlorophyll.
The measurement of various parameters related to chlorophyll fluorescence serves as a direct and widely used indicator for assessing the efficiency of photosynthesis and the impact of environmental stress [38].These parameters offer valuable insights into how light energy is absorbed, utilized, transferred, and dissipated by PSI and primarily PSII.The light quality is the main cause of photochemical processes, by which photosystems II and I (PSII and PSI) convert solar energy to chemical energy [3].The assessment of plant health and photosynthetic performance can be achieved by measuring the minimum and maximum photochemical quantum yields, commonly referred to as Fv/Fo and Fv/Fm.These parameters serve as indicators for the efficiency of photosynthesis and electron transfer activity in plants [43].The low values of Fv/Fo and Fv/Fm observed in the Dendrobium plantlets cultivated under fluorescent light (CK) suggested that these plantlets experienced a minor level of photoinhibition, the lower activity and photochemical efficiency of PSII were observed, indicating a decrease in the quantum yield of PSII photochemistry and potential disruption or damage to the photosynthetic system.Conversely, the LED light treatments resulted in significant increases in Fv/Fo and Fv/Fm values, with the highest values achieved under the 7R:3B treatment.The Fv/Fm ratio is commonly used as an indicator of Chlorophyll fluorescence (regular value 0.74-0.85),while a decrease in this ratio suggests impaired PSII photochemistry and potential harm to the photosynthetic system.Although displaying higher values and a wider dynamic range than Fv/Fm, the Fv/Fo ratio conveys similar core information [44].The qP parameter measures the photosynthetic efficiency of PSII during light adaptation and represents the ratio of open PSII reaction centers [45] and reached the highest under the 7R:3B light regimes in this article, means the plantlets of Dendrobium hybrid 'Shuijing' reached its peak photosynthetic capacity.Similarly, the non-photochemical quenching coefficient (NPQ) signifies the fraction of light energy absorbed by the pigment in the PSII reaction center antenna that is unable to participate in photosynthetic electron transport, but instead dissipates excessive light energy through thermal processes, which reflects the photoprotective capacity of plants.
NPQ reached the highest level under 8R:2B in this paper, demonstrating that the most efficient use of light energy was achieved.Additionally, ETR II , which quantifies the rate at which electrons are transferred in photosynthesis [43], was highest under the 7R:3B light regime.Furthermore, the aforementioned alterations may suggest an enhancement in PSII function and an increase in excitation energy from the reaction center under optimal light conditions, both of which would contribute to the improvement of photosynthetic efficiency.This enhanced photosynthetic efficiency could potentially account for the increased leaf and root count, elevated root activity, as well as greater fresh and dry weights observed in plantlets subjected to the 7R:3B light treatment.
The process of photosynthesis is influenced by factors such as the characteristics of light, including its quality, intensity, and duration.Specifically, chlorophyll pigments have a higher affinity for blue and red light when absorbing light energy during photosynthesis [43].Therefore, any alterations in the light spectrum can elicit corresponding modifications in photosynthetic performance, thereby further impacting the growth and development of plants.Our investigation revealed that tissue-cultured plantlets exhibited variations in growth, physiological functions, biochemical processes, and antioxidant capacity metrics depending on the specific type of light utilized.More frequent occurrence of improved growth characteristics was observed under a ratio of 7 parts red light to 3 parts blue light, including enhanced root activities, increased root length, leaf number, fresh weight, and dry weight, as well as higher numbers of roots and longer leaves.The optimal combination of red and blue light for promoting the in vitro growth of Dendrobium plantlets was found to be 7 parts red light to 3 parts blue light.Furthermore, we observed a significant correlation between the growth of Dendrobium plantlets and the chlorophyll content, as well as chlorophyll fluorescence in their leaves across different light qualities.These indices indicated a significant correlation between NPQ and leaf length, while chlorophyll b showed a significant correlation with both root and leaf numbers.Additionally, the levels of chlorophyll a and chlorophyll a/b displayed a noteworthy positive correlation with root length, and PSII was significantly correlated with fresh and dry weights.This could be the case because higher plants have a suite of light signal transduction and light-receiving systems that are able to recognize changes in light quality and respond appropriately to support healthy plant growth under the right conditions.The combination of the principal component analysis (PCA) results revealed that a light quality ratio of 7:3, which achieved the highest score, was determined to be the optimal condition for promoting growth and development in Dendrobium hybrid 'Shuijing' test-tube seedlings.

Conclusions
In this study, we observed significant effects of light quality on the growth, physiology, biochemistry, chlorophyll content, and chlorophyll fluorescence of in vitro-cultured Dendrobium hybrid plantlets.A proper combination of red (R) and blue (B) wavelengths from LED sources was found to enhance plantlets growth and photosynthesis compared to monochromatic red or blue light.Discreet combinations of red and blue light resulted in larger plants with increased nutrient content, antioxidant capacity, and photosynthetic activity in Dendrobium plantlets.Based on a combination of principal component analysis and comprehensive scores for different ratios of red to blue LED light, we conclude that a 7:3 ratio exhibited suitable plant photomorphogenesis for controlled environment cultivation.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/horticulturae10080774/s1.Table S1: Growth index of Dendrobium officinale hybrid 'Shuijing' seedlings under different red and blue LED light.Table S2: The content of SS, SP and FP in Dendrobium officinale hybrid 'Shuijing' under different red and blue LED light.Table S3: Antioxidant enzyme activities in leaves of Dendrobium officinale hybrid in vitro culture under different red and blue LED light.Table S4: Chlorophyll content in leaves of Dendrobium officinale hybrid in vitro culture under different red and blue LED light.Table S5: Six parameters of chlorophyll fluorescence of Dendrobium officinale hybrid 'Shuijing' seedlings under different red and blue LED light.

Figure 2 .
Figure 2. Growth of Dendrobium hybrid 'Shuijing' plants grown in isolation under different light conditions: the plant height (A), leaf length (B), leaf number (C), fresh weight (D), dry weight (E), root length (F), root number (G), root activity (H).Results are presented as mean values ± standard deviation (n = 10).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.

Figure 4 .
Figure 4. Contents of SS (A), SP (B), and FP (C) in leaves of in vitro-cultured Dendrobium hybrid 'Shuijing' plantlets grown under varying ratios of red and blue LED wavelengths.Results are presented as mean values ± standard deviation (n = 3).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.

Figure 4 .
Figure 4. Contents of SS (A), SP (B), and FP (C) in leaves of in vitro-cultured Dendrobium hybrid 'Shuijing' plantlets grown under varying ratios of red and blue LED wavelengths.Results are presented as mean values ± standard deviation (n = 3).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.

Figure 5 .
Figure 5. Activities of antioxidant enzymes MDA (A), POD (B), APX (C), SOD (D), and CAT (E) in leaves of in vitro-cultured Dendrobium hybrid plantlets under different red and blue LED lighting.Results are presented as mean values ± standard deviation (n = 3).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.

Figure 5 . 18 Figure 6 .
Figure 5. Activities of antioxidant enzymes MDA (A), POD (B), APX (C), SOD (D), and CAT (E) in leaves of in vitro-cultured Dendrobium hybrid plantlets under different red and blue LED lighting.Results are presented as mean values ± standard deviation (n = 3).Different superscript letters (a-f) within columns mean statistically significant differences at p < 0.05.Horticulturae 2024, 10, x FOR PEER REVIEW 9 of 18

Figure 6 .
Figure 6.The levels of chlorophyll and carotenoid contents were measured in leaves of Dendrobium hybrid plantlets cultured in vitro under different ratios of red and blue LED wavelengths.chlorophyll a (A), chlorophyll b (B), total chlorophyll (C) carotenoid (D), chlorophyll a/b (E).Results are presented as mean values ± standard deviation (n = 3).a-e different superscript letters within columns mean statistically significant differences at p < 0.05.

Figure 8 .
Figure 8. Principal component analysis biplot of 27 indices of Dendrobium hybrid plantlets cultured under different combinations of red and blue LED light.(A) Biplot illustrating PCA results.(B) Overall scores for the first four principal components across different red and blue LED light combinations.

Figure 8 .
Figure 8. Principal component analysis biplot of 27 indices of Dendrobium hybrid plantlets cultured under different combinations of red and blue LED light.(A) Biplot illustrating PCA results.(B) Overall scores for the first four principal components across different red and blue LED light combinations.
Table S5 lists the specific values of fluorescence of chlorophyll in each treatment group.

Table 1 .
Physiological and biochemical parameters, as well as chlorophyll fluorescence, were incorporated into linear regression models.

Table 2 .
Displays the rotated component matrix derived from the PCA.