Enhancing Growth and Reducing Contamination in Coelogyne pandurata Culture: A Smart IoT-Controlled Temporary Immersion System

Enhancing Growth and Reducing Contamination in Coelogyne pandurata Culture: A Smart IoT-Controlled Temporary Immersion System | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing Growth and Reducing Contamination in Coelogyne pandurata Culture: A Smart IoT-Controlled Temporary Immersion System Irma Jamaluddin, Rinaldi Sjahril, Feranita Haring, Elkawakib Syam'un, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8251105/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract The integration of Internet of Things (IoT) technology into a Temporary Immersion System (TIS) was investigated for the micropropagation of the endangered black orchid, Coelogyne pandurata. The study aimed to optimize the photoperiod and immersion frequency for enhanced growth and reduced contamination. Plantlets were subjected to four photoperiods (12, 16, 20, and 24 h light) and three immersion frequencies (4, 6, and 8 times/day for 2 min each). The IoT-controlled TIS effectively regulated environmental parameters, reducing contamination from 100% to 10%. Continuous illumination (24 h) significantly increased shoot multiplication, while moderate immersion (4 times/day) produced the best balance of biomass accumulation, chlorophyll content, and minimal hyperhydricity. Shorter photoperiods favored root elongation, indicating distinct photomorphogenic responses between shoot and root tissues. Principal Component Analysis confirmed the joint influence of photoperiod and immersion frequency on plantlet vigor, contributing to uniform and reproducible growth. Pigment analysis revealed that moderate photoperiods (12–16 h) and immersion frequencies enhanced chlorophyll and carotenoid accumulation. Stomatal density and size were also affected, with extended illumination and frequent immersion promoting higher densities of smaller stomata. The IoT-integrated TIS offers an efficient, scalable, and automated platform for the conservation and large-scale propagation of C. pandurata, demonstrating the potential of smart biotechnological systems to support sustainable ex situ conservation of endangered orchids. Coelogyne pandurata Temporary Immersion System Internet of Things Micropropagation Photoperiod Immersion frequency Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Black orchid ( Coelogyne pandurata ), an orchid species endemic to Borneo, has been classified as endangered because of habitat loss and overexploitation of its population. This orchid is distinguished by its large, bright green petals and sepals along with a deep black labellum or lip, which serves as a distinctive and primary attraction (Hartati et al., 2017). The uniqueness and rarity of black orchids have rendered them a target for excessive exploitation, which, in conjunction with the destruction of their natural habitat, has led to a significant decline in the wild population (Kartiman et al., 2025). This raises serious concerns regarding the survival of this species and underscores the urgency for effective conservation and propagation efforts to ensure its survival. In vitro culture is a viable alternative; however, traditional semi-solid media are limited in terms of their scalability and efficiency. Conventional propagation methods are inadequate to meet the conservation requirements of this species. The Temporary Immersion System (TIS) is a promising technology that enhances nutrient uptake, reduces hyperhydricity, and improves plantlet quality compared with conventional systems (Etienne & Berthouly, 2002). TIS has been successfully applied to various crops and ornamentals, including pineapples (Escalona et al., 1999), orchid fields(Leyva-Ovalle et al., 2020; Hallaç et al., 2024), and other tropical species. The temporary immersion bioreactor system demonstrated efficacy for orchid propagation. Numerous studies have established a positive correlation between immersion duration and light period. Bozkurt et al. (2023) indicated that the duration of immersion in a TIS significantly influenced plant propagation outcomes. The frequency and duration of immersion can affect the success of rooting and incidence of hyperhydricity (Etienne & Berthouly, 2002). For example, adjusting the immersion interval can alleviate the adverse effects of hyperhydricity, which is a common issue in plant tissue culture. Furthermore, Etienne and Berthouly (2002) the immersion frequency is a critical factor influencing the success of in vitro propagation. Leyva-Ovalle et al. (2020) concluded that nutrient immersion for 2 min every 4 h resulted in the highest number of shoots in Guarianthe skinneri orchids. Variations in photoperiod can significantly influence the regulation of photosensitive pigments within enzymes involved in sucrose metabolism, thereby enhancing enzymatic activity and leading to substantial accumulation of photosynthates in juvenile plants. Xu et al. (2020) reported that a 16-hour photoperiod in Cunninghamia lanceolata plants enhances the capacity of plantlet leaves in in vitro culture to capture, convert, and transfer light energy. Elevated chlorophyll levels indicate an increased capacity for light adaptation, improved electron transfer within chlorophyll, and increased activity of the Calvin cycle enzymes. Optimal light duration can reduce culture time, increase rooting rates, and enhance leaf and root number (Marques et al., 2021). Kaladharan et al. (2024) observed that a light period of 16 h, followed by 8 h of darkness, yielded optimal results for Coelogyne mossiase shoots. (Uma et al., 2021) demonstrated that immersion for 2 min every 6 h was effective for banana shoot and root development. Furthermore, continuous lighting for 24 h per day was applied to one of the racks as several studies have underscored the benefits of continuous lighting for plant growth (Smirnov et al., 2022). Technology in agriculture is developing rapidly, especially in automation and the use of Internet of Things (IoT) systems in agriculture. Automated systems such as climate control in greenhouses, irrigation, and fertigation have been shown to increase agricultural yields (Skripko & Skripko, 2017). The IoT in agriculture enables the precise monitoring and control of temperature, light, and humidity (Dalina & Sobejana, 2019). TIS technology connected to IoT opens new opportunities for preserving and propagating rare plants, such as black orchids. This system optimizes growth conditions in the laboratory and is expected to improve the success rate of black orchid propagation in the future. Using this technology, researchers and farmers can create a more controlled and efficient environment to support the recovery of black orchids from the wild. The integration of TIS and IoT not only increases propagation yields but also accelerates the conservation and recovery of black orchids in their natural habitats. Although TIS provide significant advantages, contamination and technical limitations often hinder their implementation. Optimizing culture conditions, particularly immersion frequency and photoperiod, is crucial for maximizing growth and ensuring reproducibility of results. Photoperiods regulate photosynthetic efficiency and morphogenesis, with balanced light–dark cycles being more beneficial than continuous light exposure (Smirnov et al., 2022). The present study aimed to evaluate the effects of immersion frequency and photoperiod on the in vitro propagation of black orchids using a TIS and to address the challenges of contamination control and equipment modification. Materials and Methods 2.1 Plant Material In vitro-derived black orchid ( C. pandurata ) plantlets were used as explants. 2.2 Experimental Design A factorial design (3 × 4) with immersion frequency (F) and photoperiod (P) was applied, resulting in 12 treatment combinations with four replicates each (total of 48 experimental units): · Immersion frequency (F): F1 = six times/day, F2 = four times/day, F3 = eight times/day (2 minutes each). · Photoperiod (P): P1 = 12h light/12h dark, P2 = 16h light/8h dark, P3 = 20h light/4h dark, P4 = 24h light. 2.3 Data Collection After 30 days, the following growth parameters were recorded: number of shoots, plant height (cm), number of leaves, number of leaf, leaf length (cm), fresh weight (g), root length (cm), hyperhydricity, leaf color, chlorophyll, and stomata. 2.4 Contamination Management Sterilization was conducted in three stages: (1) conventional sterilization of vessels and media; (2) double sterilization (media poured into sterilized vessels under laminar airflow and re-sterilization); and (3) TIS component modification (central tube and culture basket). Contamination rates were monitored weekly. 2.5 Statistical Analysis Data were subjected to two-way Analysis of Variance (ANOVA) (P < 0.05). Non-significant results were further examined using boxplots to visualize trends. Principal Component Analysis (PCA) was conducted using ClustVis (biit.cs.ut.ee/clustvis/), based on ln(x)-independent, row-centered, and unit variance–scaled data with arcsine-transformed survival proportions (Alive_Trans) of Mets. Results 3.1 Modification of Internet of Things Design for Temporary Immersion System and Automatic Lighting System Figure 1 shows the design of a TIS bioreactor for plant tissue culture by our research partner, the Nabila Cultura Laboratory in Bogor, Indonesia. This bioreactor has two main parts: a lower section for storing the nutrient medium, and an upper section for the culture space. The culture space was a 1 L transparent bottle with a plastic cap. A silicone hose connected the lower and upper sections, and a 0.22 µm Millipore filter maintained sterility in the medium- and gas-exchange areas. The system features CO 2 gas injection at a volume of 0.1 vvm and a timer to regulate the frequency of plantlet immersion in the liquid medium. The hardware included a water pump for medium circulation, and an air pump for supplying CO2 and fresh air (Fig. 1 B). The lighting was set automatically with four settings: P1 = 12 h light, 12 h dark; P2 = 16 h light, 8 h dark; P3 = 20 h light, 4 h dark; and P4 = 24 h light, 0 h dark. The Real-Time Clock (RTC) sensor precisely tracked time. The relay connects the Arduino Uno to the actuator system. The Arduino Uno managed these settings (Fig. 1 A) and sent information to the relay to control the electric current. The IoT-based TIS developed in the laboratory was equipped with a DHT22 sensor to detect temperature and humidity, a Lux sensor to detect light intensity, and a vibration sensor to detect vibrations when the TIS was immersed in plantlets. Vibration occurred when air entered the TIS (code 1: TIS immersed; code 0: TIS not immersed). This information was sent to NodeMCU ESP8266 and displayed on an LCD and the Blynk IoT application on an Android phone (Fig. 1 C), showing the temperature ( Suhu ), humidity ( Kelembapan ), lux ( LUX ), and vibration ( Vibration ). 3.2 Contamination Control Initial trials showed contamination rates of 100% by day 30. Double sterilization reduced contamination to approximately 30%, whereas component modifications further decreased it to approximately 10% (Fig. 2 ), indicating a reduction in contamination levels following the applied sterilization and equipment modifications. 3.3 Modification of Temporary Immersion System Equipment Remedial measures were implemented to reduce contamination. The first stage focused on sterilization techniques, media formulation, procedures for transferring plantlets into the TIS vessel, and adjustments to TIS components, such as the central tube, filter, and culture basket. The second stage involved multi-level sterilization: sterilizing the TIS equipment separately, pouring media into the already sterilized TIS equipment under LAF, and re-sterilizing the equipment and media. The results of the second stage show a 30% reduction in contamination (Fig. 3 ). The third stage involved multilevel sterilization and equipment modification with the TIS supplier partner, including replacement of the central tube and culture basket models (Fig. 3 ). These improvements successfully addressed contamination and technical issues related to inadequate pumping of the media. The results of the third-stage modifications showed a 10% reduction in contamination. 3.4 Shoot Multiplication The increase in shoot number of Coelogyne pandurata plantlets at 30 days was influenced by both photoperiod (P) and immersion frequency (F). Among the photoperiod treatments, continuous light exposure (P₄ = 24 h light) produced the highest average number of shoots (4.01), significantly greater than P₃ (20 h light; 2.85). The lowest shoot formation occurred under P , indicating reduced proliferation under shorter dark intervals. Across the immersion frequencies, F₂ (four immersions/day) yielded the highest mean (4.20), which was significantly different from F₁ (six immersions/day; 3.15) and F₃ (eight immersions/day; 3.01). No significant interaction was found between photoperiod and immersion frequency, indicating that the effects of these factors on shoot number were statistically independent under the tested conditions. Table 1 Average increase in shoots of Black Orchid ( Coelogyne pandurata ) plantlets at 30 days after culture under different photoperiods (P) and immersion frequencies (F) in a Temporary Immersion System. F 1 F 2 F 3 Average p q DMRT P 1 3.3 4.26 2.96 3.51 ab 2 0.79 P 2 3.2 3.98 3.18 3.45 ab 3 0.83 P 3 2.68 3.58 2.28 2.85 b 4 0.86 P 4 3.44 4.98 3.62 4.01 a Average 3.15 q 4.20 p 3.01 q 3.455 p 2 3 q DMRT 0.68 0.72 Note: Values represent the means of four replicates. Means followed by different letters within the same column or row differ significantly according to Duncan’s Multiple Range Test (DMRT) at p ≤ 0.05. p = number of photoperiod levels; q DMRT = critical value for Duncan’s test. 3.5 Plant Height (Cm) The average plant height of Coelogyne pandurata after 30 days of culture ranged from 0.80 to 1.13 cm across the various combinations of photoperiod and immersion frequency. Among the photoperiods, the highest mean height (1.01 cm) was observed under P₁ (12 h light/12 h dark), followed by P₄ (24 h light; 0.96 cm). The shortest plantlets occurred under P₃ (20 h light/4 h dark; 0.88 cm). Immersion frequency significantly affected plant height, where F₃ (eight immersions/day) produced the tallest plantlets (1.06 cm), while F₁ and F₂ showed lower averages (0.88 cm and 0.89 cm, respectively). No significant interaction was detected between the photoperiod and immersion frequency. Table 2 Average height of Black Orchid ( Coelogyne pandurata ) plantlets at 30 days after culture under different photoperiods (P) and immersion frequencies (F) in a Temporary Immersion System. F 1 F 2 F 3 Average P 1 0.92 0.97 1.13 1.01 P 2 0.86 0.90 1.03 0.93 P 3 0.83 0.80 1.02 0.88 P 4 0.89 0.92 1.07 0.96 Average 0.88 b 0.89 b 1.06 a 0.95 p 2 3 q DMRT 0.12 0.13 Note: Values represent the means of four replicates. Means followed by different letters within the same column or row differ significantly according to Duncan’s Multiple Range Test (DMRT) at p ≤ 0.05. p = number of photoperiod levels; q DMRT = critical value for Duncan’s test. 3.6 Increase in Number (Units) and Leaf Length (Cm) The boxplots in Fig. 4 illustrate the effects of photoperiod (P) and immersion frequency (F) on the average number (a) and length (b) of leaves in Coelogyne pandurata ​plantlets after one month of culture in the TIS. The number of leaves per plantlet showed broad variation across treatments, with median values generally ranging from 10 to 20 leaves and several outliers exceeding 30. The treatments P₄F₂ (24 h light, four immersions/day) and P₃F₂ (12 h light, four immersions/day) displayed higher medians and wider ranges, showing higher median leaf numbers under these treatment combinations. In contrast, P₂F₁ and P₃F₃ tended to produce fewer leaves with narrower ranges. For leaf length, the average values ranged from approximately 1 cm to 5 cm. The treatments P₄F₃ (24 h light, eight immersions/day) and P₂F₂ (16 h light, four immersions/day) exhibited slightly greater medians, although overall differences among treatments were relatively small. This indicated that leaf elongation was less responsive to photoperiod and immersion frequency than leaf initiation. 3.7 Average Fresh Weight of Plantlets (Gram) Figure 5 presents the effect of photoperiod (P) and immersion frequency (F) on the average fresh weight of Coelogyne pandurata plantlets after one month of culture in the TIS. The average fresh weight ranged from 0.19 g to 0.41 g across treatments. The highest value (0.41 g) occurred under P₂F₂ (16h light / 8h dark with four immersions per day), followed by P₄F₃ (24h light with eight immersions/day, 0.37 g) and P₃F₂ (20h light with four immersions/day, 0.35 g). The lowest biomass values (0.19 g) were observed in P₁F₃ and P₃F₃, indicating that excessive immersion frequency suppressed fresh weight accumulation. Across photoperiods, plantlets exposed to P₂ (16 h light) and P₄ (24 h light) produced higher mean weights than those under shorter illumination (P₁ = 12 h L/12 h D; P₃ = 20 h L/4 h D). Meanwhile, the lowest immersion frequency (F₂, four times/day) resulted in a higher biomass in most photoperiod combinations. 3.8 Average Root Length (cm) Table 3 presents the average root length of black orchids 30 days after planting (DAP) under different light exposure durations (P) and soaking frequencies (F). The results were measured in centimeters (cm). This experiment investigated four light exposure levels (P1–P4) and three soaking frequency levels (F 1 –F 3 ). The data showed that different combinations of these treatments had varying effects on the root length. The results showed that the P 1 treatment yielded the longest average root length (1.00 cm), which was significantly different from the P 2 and P 3 treatments. Soaking frequency also influenced root growth, with F 3 showing the highest average root length (0.99 cm) across all light-exposure treatments. The results of the ANOVA indicated that factor P (F = 8.07 > F critical = 2.80, P-value = 0.02 0.05). P1 treatment (12 h light (L), 12 h dark (D)) was the best for increasing plantlet growth, whereas P 1 (24 h L, h D) also showed good potential. The F 3 treatment (eight times/day) was the best for increasing the root growth of Black Orchid plantlets. This indicates that both the duration of light exposure and soaking frequency play important roles in the root development of black orchids, with potential implications for optimizing cultivation practices for orchid propagation and growth. Table 3 Average root length of Coelogyne pandurata plantlets at 30 days after planting under different combinations of photoperiod (P) and immersion frequency (F) in a temporary immersion system (cm). F 1 F 2 F 3 Average p q DMRT P 1 0.868 0.874 1.250 1.00 a 2 0.11 P 2 0.736 0.714 0.796 0.75 c 3 0.12 P 3 0.750 0.740 0.932 0.80b c 4 0.12 P 4 0.824 0.830 1.068 0.89a b Average 0.79 q 0.78 q 0.99 p 0.87 p 2 3 q DMRT 0.10 0.10 Note : Numbers in each column followed by the same lowercase letter are not significantly different according to Duncan’s Multiple Range Test (DMRT) at α = 0.05. P: Photoperiod - P₁ = 12h light/12h dark; P₂ = 16h light/8h dark; P₃ = 20h light/4h dark; P₄ = 24 h continuous light. F: Immersion frequency - F₁ = six times/day; F₂ = four times/day; F₃ = eight times/day, with each immersion lasting 2 min. p : number of means; q DMRT : critical value of Duncan’s Multiple Range Test. 3.9 Hyperhydricity Percentages (Units) Figure 6 shows that the degree of hyperhydricity varied among treatments combining different photoperiods (P) and immersion frequencies (F) after one month of culture. The highest average hyperhydricity score (8) was recorded in P₁F₁ (12 h light / 12 h dark; six immersions/day), followed by P₁F₃ = 6 and P₂F₁ = 4. In contrast, the lowest hyperhydricity level (1) occurred under P₁F₂ (12 h light / 12 h dark; four immersions/day), indicating that the reduction in immersion frequency mitigated tissue vitrification. Generally, increasing the immersion frequency led to greater hyperhydricity, whereas extended light exposure (P₃–P₄) tended to decrease the severity. The microscopic observations shown in Fig. 7 visually confirm these findings. Plantlets subjected to frequent immersions (F₃) exhibited typical hyperhydric symptoms such as translucent and brittle leaves, swollen stems, and impaired chlorophyll development (e.g., P₁F₃, P₂F₃, P₃F₃). Conversely, plantlets cultured under fewer immersions (F₂) or longer photoperiods (P₃–P₄) maintained firmer, greener leaves and more normal morphology, with substantially reduced tissue waterlogging (e.g., P₁F₂, P₄F₃). 3.10 Leaf Color Observation Based on RHS Colour Chart Figure 8 shows the leaf color variations of Coelogyne pandurata plantlets grown under different photoperiod (P) and immersion frequency (F) combinations in the TIS. Leaf color was classified using the RHS Color Chart codes 143 A–C, representing shades from strong yellow-green to light yellow-green. Quantitative scoring (1 = light/pale; 2 = moderate; 3 = dark green) revealed that the RHS scores ranged from 1 to 3, with higher scores indicating greater greenness and chlorophyll accumulation. Across all photoperiods, shorter light exposures (P₁ = 12 hours and P₂ = 16 hours) resulted in more intense green color (total score of 8 each) compared to longer photoperiods (P₃ = 20 hours and P₄ = 24 hours with total score of 7 each). Regarding immersion, plantlets cultured at F₁ (six immersions/day) showed more stable and darker green leaves, with the highest total score of 11. In contrast, frequent immersions (F₃ = eight times/day with a total score of 9) resulted in a paler color for the P₄F₃ combination. 3.11​ Principal Component Analysis PCA was performed to summarize the multivariate growth response of Coelogyne pandurata plantlets cultured under different photoperiod and immersion frequency combinations in the TIS. The first two principal components, PC1 (94.3%) and PC2 (5.6%), cumulatively explained 99.9% of the total variance among the treatments (Fig. 9 ). PC1 represents the overall plantlet vigor, integrating the parameters related to shoot multiplication, root elongation, plant height, and fresh biomass. PC2 reflects secondary morphological and physiological differences, including variations in leaf elongation and hyperhydricity. The PCA biplot revealed that most treatments clustered tightly on the positive side of PC1, indicating uniform and vigorous growth performance among TIS-grown plantlets. Only treatment P 1 F 1 appeared slightly separated along the negative axis of PC1, suggesting a lower overall growth rate, possibly due to a shorter photoperiod or reduced immersion frequency. These results indicate that the combination of photoperiod and immersion frequency primarily influences the magnitude of growth responses rather than inducing distinct growth patterns, reflecting a generally consistent growth performance of TIS-grown plantlets under the tested conditions. 3.12​ Effects of Photoperiod and Immersion Frequency on Pigment Accumulation Pigment accumulation varied significantly across photoperiod × immersion treatments (Fig. 4 ; Table 2 ). Total chlorophyll ranged from 6.19 to 23.42 µg mL⁻¹, whereas carotenoid concentrations ranged from non-detectable to 6.29 µg mL⁻¹. The highest chlorophyll a, chlorophyll b, and total chlorophyll levels were recorded in the P1F1 treatment (16.63, 6.80, and 23.42 µg mL⁻¹, respectively), representing a nearly two-fold increase compared with the control (12.06 µg mL⁻¹). Treatments P 2 F 2 and P 2 F 3 also showed consistently elevated pigment levels, indicating that moderate photoperiods (12–16 h light) combined with moderate immersion frequencies supported chlorophyll biosynthesis. Carotenoid accumulation followed a similar pattern. P 1 F 1 produced the highest carotenoid concentration (6.29 µg mL⁻¹), followed by P 2 F 2 (5.08 µg mL⁻¹) and P 2 F 3 (4.03 µg mL⁻¹). In contrast, longer photoperiods (P 3 –P 4 ) resulted in markedly lower carotenoid levels (1.20–3.92 µg mL⁻¹). The P 4 F 1 treatment produced a negative calculated value after correction for chlorophyll interference; this value was therefore classified as “not detected (ND)”. Overall, shorter photoperiods coupled with moderate immersion frequencies produced the highest pigment contents, whereas continuous or extended photoperiods reduced both chlorophyll and carotenoid accumulation. 3.13 Effects of Photoperiod and Immersion Frequency on Stomatal Density and Size in In-Vitro Plantlets Stomatal density differed markedly among treatments, ranging from 42 to 117 stomata mm⁻² (Fig. 11 , left panel). The highest densities were recorded in P 3 F 2 (117 mm⁻²) and P 1 F 3 (108 mm⁻²), whereas P 2 F 1 –P 2 F 3 consistently produced the lowest densities (42–88 mm⁻²). Morning and afternoon measurements showed similar patterns, indicating that treatment effects were stable across time. Stomatal size also varied considerably across treatments (Fig. X, right panel). Afternoon stomatal length ranged from 1.3 to 4.8 µm, with the largest stomata observed in P 1 F 1 , P 2 F 2 , and P 4 F 3 , whereas morning values ranged from 2.4 to 3.9 µm. Stomatal width showed a similar pattern, with afternoon values between 0.8 and 3.5 µm and morning values ranging from 1.6 to 2.9 µm. Treatments with extended photoperiods (P 3 and P 4 ) generally produced moderate-to-large stomata, while short photoperiods (P 1 and P 2 ) yielded more variable structures. Discussion The integration of the Internet of Things (IoT) into plant tissue culture laboratories represents a transformative advancement in environmental regulation, automation, and micropropagation efficiency optimization. IoT-based systems enable real-time monitoring and precise control of critical parameters such as photoperiod, vibration, temperature, humidity, and light intensity, all of which are vital for promoting optimal plant growth and improving culture yields. As illustrated in Fig. 1 , the environmental data recorded in this study demonstrated the effectiveness of the IoT system, with incubation room conditions maintained at 26.9°C, 37.8% humidity, and 1,534 lux light intensity, while vibration data confirmed the operational status of the TIS between irrigated and non-irrigated states. These findings indicate that the IoT framework effectively supported the growth of the Coelogyne pandurata and advanced innovation in automated culture systems. (Rahayu 2024 ) reported that a well-designed bioreactor equipped with an automatic control system facilitates laboratory operations, reduces manual labor, and ensures precision in the micropropagation of black orchids. However, as noted by Harun et al. ( 2019 ), the rapid emergence of new technologies challenges researchers to continuously update their technical skills and fully harness their potential. By integrating sensors and automated control modules, laboratories can minimize labor requirements, increase operational efficiency, and maintain stable environmental conditions tailored to the physiological needs of cultured plants. In the present study, the IoT-integrated TIS combined real-time sensors (DHT22, Lux, and vibration) with programmable microcontrollers (Arduino Uno and NodeMCU ESP8266) to regulate and monitor key environmental parameters, including temperature, humidity, light intensity, and immersion cycles, which are essential for the successful micropropagation of C. pandurata . This approach aligns with the growing trend toward smart bioreactor systems that ensure controlled microenvironments, while reducing manual intervention and the risk of contamination. Similar IoT-based control frameworks have proven effective in automating plant tissue culture systems, minimizing human errors, and maintaining consistent growth conditions (Dalina & Sobejana, 2019 ; Dhanaraju et al., 2022 ). The DHT22 sensors enabled precise monitoring of temperature and humidity, whereas automated immersion cycles allowed the dynamic adjustment of growth conditions, which is a critical feature for tropical orchids that are highly sensitive to microclimatic fluctuations. Furthermore, the incorporation of a real-time clock (RTC) and a Blynk mobile interface enhanced accessibility and responsiveness by allowing wireless data transmission through the NodeMCU ESP8266 module. This capability enables researchers to remotely monitor and modify culture parameters, ensuring uninterrupted 24-hour operation and early detection of potential system failures. Such remote supervision is particularly beneficial for delicate in vitro systems, such as TIS, where continuous oversight improves reproducibility and reduces contamination rates, as previously demonstrated by Rahayu ( 2024) in C. pandurata . Automation of photoperiod control through integrated light sensors and relay systems has further enabled the accurate simulation of natural light cycles, which are essential for photosynthetic and morphogenic regulation. Stable photoperiod regimes enhance chlorophyll biosynthesis, photosystem performance, and carbon assimilation, ultimately improving plant vigor and morphology (Smirnov et al., 2022 ; Xu et al., 2020 ). This synchronization between artificial illumination and environmental control mitigates photoperiod-induced stress and ensures consistent growth. Additionally, vibration sensing serves as an indirect indicator of immersion events, verifying the correct operation of the air pump and medium circulation system. This innovation addresses one of the persistent challenges in conventional TIS setups: maintaining a consistent immersion frequency (Etienne & Berthouly, 2002 ), by providing real-time validation of mechanical performance. Collectively, the integration of these IoT components establishes a feedback-driven, data-based monitoring system that enhances the experimental precision, reproducibility, and scalability. The IoT-integrated TIS developed in this study exemplifies a next-generation platform for plant micropropagation, combining biotechnology and digital technology to enable the automated, precise, and sustainable in vitro propagation of endangered species, such as the black orchid ( Coelogyne pandurata ). Figure 2 shows the trend of contamination in the TIS, where improvements have been made to reduce it. Initially, improvements focused on sterilization techniques, media formulation, procedures for transferring plantlets into TIS vessels, and adjustments to TIS components such as the central tube, filter, and culture basket. In the second stage, layered sterilization was implemented: separate sterilization of the TIS equipment, pouring media into the sterile TIS equipment under the LAF, and re-sterilization of the equipment and media. The results of the second stage showed a 30% reduction in the contamination. In the third stage, in addition to layered sterilization, the equipment was modified in collaboration with the TIS supplier partner by changing the model of the central tube and culture basket (Fig. 3 ). This improvement addressed contamination and technical issues such as the improper pumping of liquid media. The results of the third-stage modifications showed a reduction in contamination of up to 10%. These results are in line with the research of Leyva-Ovalle et al. ( 2020 ) on the importance of sterilization management and TIS system design, and also support Rahayu's (2024) report on TIS development in black orchids. Other contamination factors included the culture equipment, plantlets, planting room air, plant species, research personnel, and frequently visited rooms. According to Klayraung et al. ( 2017 ), bacterial contamination is higher in frequently visited areas, which indicates a relationship between human activity and microbial density. Every step in plant tissue culture must be carefully considered to prevent bacterial and fungal contamination. As shown in Table 1 , the average number of shoots ranged from 2.28 to 4.98. Among the photoperiod treatments, P₄ (24 h continuous light) produced the highest mean number of shoots (4.01), which was significantly greater than P₃ (20 h light/4 h dark), which resulted in the lowest mean (2.85). This indicates that prolonged illumination enhances shoot proliferation in Coelogyne pandurata , possibly by maintaining uninterrupted photosynthetic and metabolic activities that support cell division and organogenesis. Continuous light exposure significantly enhances shoot proliferation in plants by optimizing photomorphogenic responses and photosynthesis. Studies have shown that controlling light characteristics, like photoperiod, is crucial for increasing shoot numbers in plant tissue cultures (Cavallaro et al., 2022 ). In date palms, high light intensities decreased shoot bud proliferation, underscoring the need for precise light management (Meziani et al., 2015 ). Light also plays a vital role in plant regeneration, affecting processes such as shoot regeneration and root formation (Han et al., 2025 ). These findings align with those on Coelogyne pandurata, where continuous light supports photosynthetic and metabolic activities necessary for organogenesis. Across immersion frequencies, F₂ (four times per day) yielded the highest average number of shoots (4.20 p), while F₁ (six times per day) and F₃ (eight times per day) produced fewer shoots (3.15 q and 3.01 q, respectively). Moderate immersion appears to be optimal for balancing nutrient absorption and aeration, without inducing hyperhydricity or hypoxia. Excessive immersion (F₃) likely caused oxygen limitation in the medium and suppressed shoot development (Preil, 2005 ). Plantlet height also varied among the treatments (Table 2 ). The tallest plantlets were observed under F₃ (1.06 cm), significantly higher than those in F₁ and F₂ (0.88–0.89 cm). Increased immersion frequency may enhance nutrient uptake and reduce desiccation, thus supporting elongation growth (Etienne & Berthouly, 2002 ). However, photoperiod effects on height were less pronounced; the average height ranged narrowly between 0.88 cm and 1.01 cm, with no significant differences. This suggests that plant height in C. pandurata may be more responsive to immersion frequency than photoperiod length during the early growth phase. No significant interaction between photoperiod and immersion frequency was observed for shoot number or height, indicating that their effects were largely independent. Nonetheless, the best overall growth response was recorded under the combination of continuous light (P₄) with the lower immersion frequency (F₂, four times per day), which produced the highest shoot number (4.98 units) and good shoot elongation (0.92 cm). This finding supports previous reports that continuous illumination enhances morphogenesis when combined with adequately spaced immersion cycles that prevent oversoaking stress (Kozai et al., 2020 ). These results demonstrate that in Coelogyne pandurata , 24 h light photoperiod and moderate immersion (four times per day, 2 min each) are the most favorable conditions for early shoot multiplication and balanced plantlet growth in the TIS culture system. Leaf formation in C. pandurata was strongly influenced by both light duration and immersion frequency. The higher leaf numbers observed under continuous illumination (P₄) reflect enhanced photosynthetic activity and carbohydrate accumulation, which provide sufficient energy for organogenesis and leaf initiation. Continuous illumination significantly enhanced leaf formation by increasing the photosynthetic activity and carbohydrate accumulation. For example, in tomato seedlings, prolonged light exposure leads to increased photosynthesis and chlorophyll content under long photoperiods (Wu et al., 2024 ). Similarly, a study of Mentha species found that longer photoperiods resulted in higher concentrations of essential oils, reflecting increased metabolic activity (Fahlén et al., 1997 ). Moreover, research on Mesembryanthemum crystallinum showed that longer photoperiods positively influenced yield and antioxidant capacity, indicating increased biomass production through enhanced photosynthesis (Chen et al., 2024 ). These studies illustrate the benefits of extended light duration on leaf formation and overall plant growth. The superior performance of treatments with four immersions per day (F₂) indicated that a lower immersion frequency created a favorable balance between nutrient uptake and aeration. Less frequent immersion may prevent hypoxia and tissue waterlogging, which can limit growth or cause hyperhydricity under excessive immersion (Preil, 2005 ). Similar findings were reported by (Etienne & Berthouly, 2002 ), who noted that properly spaced immersion cycles enhanced shoot and leaf differentiation in TIS. Although longer photoperiods promoted leaf formation, the leaf elongation was more variable. Treatments with more frequent immersions (e.g., F₃) showed slightly longer leaves, likely due to increased water absorption and turgor pressure that encourage elongation. However, frequent immersion may also cause partial vitrification and reduce the photosynthetic efficiency (Kozai et al., 2020 ). These results demonstrate that C. pandurata plantlets achieve optimal leaf growth under 24 h continuous illumination combined with four immersions per day (P₄F₂). This combination promotes vigorous leaf initiation while maintaining a healthy tissue structure, making it an effective condition for early morphogenesis in TIS cultures. The average fresh weight of plantlets demonstrated that both photoperiod length and immersion frequency affected the biomass accumulation of C. pandurata plantlets in the TIS. The highest fresh weight under P₂F₂ (16 h light, four immersions/day) indicated that a moderate photoperiod combined with less frequent immersion created optimal physiological conditions for nutrient uptake and gas exchange. The finding that photoperiod length and immersion frequency affect biomass accumulation in C. pandurata plantlets is supported by several studies in different species. A study on celery cultivars found that a 12 h/12 h photoperiod stimulates growth, suggesting that optimal light conditions enhance nutrient uptake and photosynthesis (Chu et al., 2023 ). Research on in vitro plant regeneration using LED lighting has highlighted the significant influence of light characteristics, including photoperiod, on optimizing growth environments (Dutta Gupta & Agarwal, 2017 ). Additionally, studies on microalgae, such as Chlorella vulgaris and Scenedesmus obliquus, have shown that varying photoperiods significantly alter biomass and metabolomic profiles, indicating that precise photoperiod management can optimize biomass production (Amini Khoeyi et al., 2012 ; Vendruscolo et al., 2019 ). These studies emphasize the universal role of light in enhancing biomass production and optimizing plant tissue culture systems. However, continuous light (P₄) and frequent immersion (F₃ = eight times/day) may cause excessive hydration and oxygen limitation, reducing tissue dry matter despite high metabolic activity (Preil, 2005 ). The superior response at F₂ (four times/day) suggests that less frequent immersion provided sufficient nutrient contact while maintaining better aeration and preventing hyperhydricity, similar to the findings reported by (Etienne & Berthouly, 2002 ) in other orchids and tropical species. Overall, C. pandurata plantlets achieved the best biomass accumulation under 16 h light/8 h dark with four immersions per day (P₂F₂), confirming that both the light regime and immersion timing must be carefully balanced to optimize TIS performance. Root growth in C. pandurata was influenced by both photoperiod and immersion frequency, although no interaction effects were detected. The longer roots under frequent immersion (F₃) suggested that increased nutrient contact and hydration promoted root elongation. Frequent immersion enhances medium absorption, thereby improving the availability of mineral nutrients and growth regulators essential for root initiation and elongation (Etienne & Berthouly, 2002 ). However, excessively frequent cycles may also risk partial hypoxia if gas exchange is insufficient; thus, the benefit observed here may be specific to the early growth stages when roots remain short and metabolically active. Regarding photoperiod, short-day exposure (12 h L/12 h D) favored root elongation, whereas extended or continuous illumination (16–24 h) tended to reduce root length. Research indicates that shorter photoperiods (12 h light/12 h dark) favor root elongation, whereas extended exposure (16–24 h) can inhibit it. Studies on hydroponically grown cucumbers have revealed that longer photoperiods increase phytotoxic root exudates and inhibit growth (Pramanik et al., 2000 ). Similarly, systems such as D-Root have shown that light exposure can reduce root length and induce early lateral root emergence, disrupting typical growth patterns(Silva-Navas et al., 2015 ). These findings highlight the complex effects of photoperiods on root development in various species. The results indicate that short photoperiod (P₁) combined with frequent immersion (F₃) provides favorable conditions for root elongation of C. pandurata in TIS. These conditions likely balance water and nutrient uptake, while maintaining sufficient aeration to support active root metabolism and elongation. The results demonstrated that immersion frequency strongly influenced the incidence of hyperhydricity in C. pandurata plantlets. Excessive immersion (six–eight times/day) likely caused prolonged exposure of tissues to the liquid medium, leading to reduced gas exchange, oxygen limitation, and accumulation of ethylene in the culture vessels. These conditions promote water saturation and cell wall loosening, which are characteristics of hyperhydric tissues (Preil, 2005 ). In contrast, the lower immersion frequency (F₂ = four times/day) provided better aeration, allowing tissues to recover between immersions and prevent excessive hydration, which is consistent with earlier findings in Dendrobium and Ananas species (Etienne & Berthouly, 2002 ). The photoperiod effect was also evident; shorter illumination (12 h light) intensified hyperhydricity, whereas extended or continuous light (20–24 h) reduced it. Prolonged illumination promotes higher photosynthetic activity and transpiration, enhancing cell wall lignification and gas exchange, which counteracts hyperhydric conditions (Kozai et al., 2020 ). Thus, the lowest symptom expression under P₄ (24 h light) and moderate immersion (F₂) reflected an improved physiological balance between hydration and ventilation. Overall, C. pandurata plantlets grown under continuous light (P₄) with four immersions per day (F₂) exhibited the healthiest morphology, with minimal hyperhydricity. These findings emphasize the importance of optimizing both the illumination duration and immersion regime to maintain tissue quality and prevent physiological disorders during temporary immersion culture. Leaf color variation in Coelogyne pandurata reflects differences in chlorophyll content and physiological adaptation under varying illumination and immersion conditions. The observation that shorter photoperiods (P₁ = 12 h and P₂ = 16 h) produced darker green leaves suggests that a moderate duration of light exposure favors balanced chlorophyll synthesis and prevents photooxidative stress. Extended or continuous illumination (P₃ = 20 h and P₄ = 24 h) may lead to excessive light energy that exceeds the plantlets’ photosynthetic capacity, causing partial chlorophyll degradation or photoinhibition. The connection between photoperiod and chlorophyll synthesis has been observed in studies that examine the effect of light duration on plants. For example, rice mutants exhibit reduced chlorophyll content and increased oxidative stress under altered light cycles, indicating that proper light duration is crucial for balanced chlorophyll synthesis (Li et al., 2019 ). Salicylic acid application in plants mimics the effects of prolonged light by reducing chlorophyll and enhancing photosystem efficiency (Moustakas et al., 2022 ). Under high-light conditions, chlorophyll accumulation is delayed when mitochondrial respiration is inhibited (Zhang et al., 2016 ). The xanthophyll cycle protects against phototoxidative stress by dissipating excess light energy (Latowski et al., 2011 ). These studies support the notion that moderate photoperiods aid chlorophyll synthesis, whereas prolonged exposure risks degradation and photoinhibition. The influence of the immersion frequency was also evident. The six immersions per day (F₁) treatment resulted in the most stable and darker green coloration, indicating that this frequency provided an optimal balance between nutrient absorption and gas exchange. Adequate, but not excessive, immersion maintains sufficient oxygen availability, thereby supporting chloroplast development and photosynthetic efficiency. In contrast, the most frequent immersion (F₃ = eight times/day) produced paler leaves, particularly under continuous illumination (P₄F₃), likely due to tissue overhydration, reduced oxygen diffusion, and early symptoms of hyperhydric stress (Preil, 2005 ). These findings are consistent with previous reports in orchids and other tropical species, where properly timed immersion cycles combined with moderate light exposure improved leaf greenness and chlorophyll retention (Etienne & Berthouly, 2002 ; Kozai et al., 2020 ). The combination of short-to-moderate photoperiods (12–16 h light) with six immersions per day (F₁) supported optimal chlorophyll accumulation, resulting in darker green, physiologically healthier leaves of C. pandurata in the temporary immersion system. PCA clearly separated plantlet responses according to the combined influence of photoperiod and immersion frequency, indicating that these two factors jointly regulate the morphophysiological performance of Coelogyne pandurata cultured in TIS. The dominance of PC1 (94.3%) suggests that most of the variation in the dataset was attributed to differences in overall plant vigor, which integrated shoot proliferation, leaf development, and biomass accumulation. This pattern implies that photoperiod duration and immersion timing predominantly modulate photosynthetic efficiency and nutrient uptake, which is consistent with earlier reports on orchids grown in liquid systems (Lorenzo & Tapia y Figueroa, 2025; Ramírez-Mosqueda & Cruz-Cruz, 2024 ). The tight clustering of most treatments on the positive side of PC1 reflects physiological stability across immersion regimes, suggesting that TIS provides a balanced gaseous exchange and sufficient nutrient supply for uniform growth. The slight separation of P1F1 along the negative PC1 axis may indicate a stress response under suboptimal light exposure, resulting in a lower chlorophyll content and reduced shoot elongation. Similar findings were reported by (Escalona et al., 2003 ), who observed that short photoperiods limited carbohydrate accumulation and delayed organogenesis in pineapples cultured under temporary immersion. PCA confirmed that while minor quantitative variations existed among treatments, the TIS environment successfully minimized physiological heterogeneity, leading to consistent morphogenesis and survival of C. pandurata plantlets. This stability highlights the reliability of the TIS for mass propagation and supports its application for large-scale conservation of black orchids under controlled micropropagation conditions. Pigment profiles in Coelogyne pandurata were strongly influenced by the interaction between photoperiod and immersion frequency. The highest chlorophyll and carotenoid contents were obtained under moderate photoperiods (12–16 h) combined with moderate immersion frequency (P1F 1 and P 2 F 2 ). These conditions likely promoted optimal plastid differentiation by improving aeration, reducing hyperhydricity, and maintaining balanced CO₂ and light availability within the TIS vessels. Similar enhancements of chlorophyll accumulation under optimized TIS settings have been reported in Dendrobium , Phalaenopsis , and Vanilla cultures (Ramírez-Mosqueda & Cruz-Cruz, 2024 ; Tokuhara & Mii, 2003 ). Carotenoid accumulation followed the same pattern, with the highest concentrations recorded in P 1 F 1 and P 2 F 2 . Carotenoids function as key photoprotective pigments, dissipating excess excitation energy and quenching reactive oxygen species (ROS). Increased carotenoid levels, therefore, suggest that these immersion–photoperiod combinations established a more favorable light environment, minimizing photooxidative damage. Carotenoid biosynthesis is strongly light-regulated, with photoreceptors and light-responsive transcription factors activating key biosynthetic genes when plants are exposed to appropriate light conditions. Together, evidence from molecular studies and in vitro orchids shows that balanced light quality and intensity enhance carotenoid accumulation, improving photoprotection and reducing photooxidative stress (Pizarro & Stange, 2009 ; Shin et al., 2008 ; Toledo-Ortiz et al., 2010 ). Conversely, prolonged photoperiods (20–24 h) led to sharp declines in both chlorophyll and carotenoid content. Excessive or continuous illumination is well known to induce photoinhibition, disrupt thylakoid structure, inhibit chlorophyll biosynthesis, and accelerate pigment degradation (Powles, 2003 ). Shade-adapted species, including many orchids, are susceptible to high light loads during in vitro culture (Benkov et al., 2019 ; Kitao et al., 2000 , 2006 ). Under such conditions, chlorophyll degradation often surpasses synthesis, and carotenoids are rapidly consumed as antioxidants, leading to the low pigment values observed in P 3 and P 4 treatments. The “not detected” carotenoid value in P4F1 likely indicates severe photobleaching and ROS accumulation, a phenomenon frequently reported when light exposure exceeds the physiological tolerance of in-vitro plantlets. These findings underscore the importance of balancing light duration with immersion dynamics. Photoperiod serves as a primary regulator of pigment metabolism and chloroplast development (Batista et al., 2018 ), while immersion frequency modulates hydration, nutrient exposure, and gaseous exchange within TIS. The superior performance of P 1 F 1 (12 h light, 6 immersions/day) demonstrates that synchronizing moderate light exposure with optimal immersion cycles creates conditions that maximize plastid differentiation and pigment biosynthesis in C. pandurata . The combined evidence suggests that optimizing both photoperiod and immersion frequency is essential for enhancing pigment quality in orchids propagated through temporary immersion systems. This insight is valuable for refining in vitro protocols aimed at producing physiologically robust plantlets with strong photosynthetic capacity. Differences in stomatal density across treatments suggest that photoperiod and immersion frequency influence epidermal development under in vitro conditions. Higher densities observed in treatments such as P3F2 and P1F3 indicate that extended illumination combined with appropriate immersion frequency may promote epidermal cell division and stomatal initiation. These observations are consistent with previous reports indicating that prolonged light exposure and water availability can modulate cellular and hormonal pathways associated with stomatal development, including transcriptional and signaling networks involving SPCH, auxin activity, and light-responsive factors (J.-H. Lee et al., 2017 ; S. B. Lee et al., 2014 ; MacAlister et al., 2007 ). In contrast, the consistently lower stomatal density observed under P2 treatments may reflect suboptimal morphogenic signaling under intermediate photoperiods. The similarity between morning and afternoon density values confirms that stomatal density is an anatomical trait fixed during leaf expansion. Minor morning–afternoon differences are more likely due to variations in leaf hydration and turgor affecting visibility during microscopy rather than true physiological changes. This understanding is consistent with findings highlighting stomatal density as a stable trait shaped by genetic and developmental factors rather than short-term physiological adjustments (Liu et al., 2023 ; Loranger & Shipley, 2010 ; Tian et al., 2016 ). Thus, treatment effects—not diurnal patterns—are the primary drivers of the observed differences. Stomatal size responses provide an additional layer of insight. Treatments producing higher stomatal density often exhibited smaller stomata, reflecting a classic inverse relationship well documented in developmental plant physiology (Franks & Beerling, 2009 ; Gj et al., 2014 ). A high density of small stomata is often associated with faster pore dynamics and improved responsiveness under fluctuating environments—an advantageous anatomical configuration for plants transitioning from in vitro to ex vitro conditions. Conversely, treatments such as P 1 F 1 and P2F2, which produced larger stomata despite moderate densities, may reflect altered water relations or partial hyperhydricity, conditions known to cause enlarged guard cells due to excessive tissue hydration (Debergh et al., 1992 ; Kevers et al., 1984 ). Overall, the combined patterns of stomatal density and size highlight that optimizing photoperiod and immersion frequency in Temporary Immersion Systems not only affects shoot vigor but also shapes leaf anatomical traits crucial for acclimatization. Treatments such as P3F2, which produced high densities of moderately sized stomata, may support more efficient gas exchange and better water-use control. Combined evidence shows that photoperiod and immersion frequency in Temporary Immersion Systems (TIS) shape stomatal density–size patterns that influence shoot vigor and leaf anatomical traits essential for acclimatization, as demonstrated by studies reporting that the SETIS™ bioreactor increases shoot proliferation, stomatal index, and stomatal closure (Ramírez-Mosqueda & Bello-Bello, 2021 ) and that modulation of immersion frequency enhances stomatal index, chlorophyll content, and shoot survival—thereby improving gas exchange efficiency and water-use regulation critical for post-culture establishment (Mancilla-Álvarez et al., 2024 ). Limitations of the Study This study was conducted under controlled in vitro conditions using a single orchid species ( Coelogyne pandurata ) and a culture duration of 30 days. Therefore, the observed effects of photoperiod and immersion frequency primarily reflect short-term responses during early growth stages and may not directly represent long-term development or ex vitro acclimatization performance. In addition, the Temporary Immersion System and IoT configuration evaluated in this study were implemented at a laboratory scale. Although the system demonstrated stable environmental regulation and reduced contamination under the tested conditions, further investigation is required to assess its robustness, economic feasibility, and scalability for commercial or large-scale conservation applications. Finally, the present study focused on morphological, physiological, and anatomical parameters without evaluating post-culture survival or field establishment. Future research integrating acclimatization trials and extended culture periods would be valuable to confirm the broader applicability of the proposed system. Conclusion The integration of Internet of Things (IoT) technology into a Temporary Immersion System (TIS) improved the control of environmental parameters and supported consistent in vitro growth of the endangered black orchid, Coelogyne pandurata . The IoT-controlled TIS enabled precise regulation of photoperiod and immersion cycles and was associated with a reduction in contamination levels from 100% to approximately 10% under the modified sterilization and equipment conditions applied in this study. Among the treatments tested, continuous illumination (24 h light) resulted in higher shoot multiplication, whereas a moderate immersion frequency (four times per day) provided a favorable balance between biomass accumulation, chlorophyll content, and reduced hyperhydricity. In contrast, shorter photoperiods promoted greater root elongation, indicating differential photomorphogenic responses between shoot and root tissues. Multivariate analysis further indicated that photoperiod and immersion frequency jointly influenced overall plantlet vigor and growth uniformity under the tested in vitro conditions. Overall, the results suggest that an IoT-integrated TIS can provide a controlled and automated platform for optimizing in vitro propagation conditions of C. pandurata at the laboratory scale. This approach shows potential for supporting ex situ conservation efforts of endangered orchids, although further studies are required to evaluate long-term performance, acclimatization success, and scalability beyond controlled in vitro environments. Declarations 7. Acknowledgments 8. Compliance and Ethical Statements 8.1 Ethics Statement This study did not involve human participants, human data, or animal subjects. All experimental procedures were conducted on plant materials ( C. pandurata ) in accordance with the institution's biosafety and ethical guidelines. 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Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in A rabidopsis . Plant, Cell & Environment , 39 (1), 12–25. https://doi.org/10.1111/pce.12438 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 09 Mar, 2026 Reviews received at journal 03 Mar, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviewers agreed at journal 27 Feb, 2026 Reviews received at journal 12 Feb, 2026 Reviewers agreed at journal 05 Feb, 2026 Reviewers invited by journal 05 Feb, 2026 Submission checks completed at journal 21 Jan, 2026 First submitted to journal 20 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8251105","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":586344359,"identity":"ef42ba9d-1e44-4088-b67a-7de8f3c51f75","order_by":0,"name":"Irma Jamaluddin","email":"data:image/png;base64,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","orcid":"","institution":"Hasanuddin University","correspondingAuthor":true,"prefix":"","firstName":"Irma","middleName":"","lastName":"Jamaluddin","suffix":""},{"id":586344360,"identity":"40ee1c04-96d0-4e75-8e1b-451a8b8e89f6","order_by":1,"name":"Rinaldi Sjahril","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Rinaldi","middleName":"","lastName":"Sjahril","suffix":""},{"id":586344361,"identity":"41663ad3-9c8a-4e6f-ac76-abfb52c15efc","order_by":2,"name":"Feranita Haring","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Feranita","middleName":"","lastName":"Haring","suffix":""},{"id":586344362,"identity":"0e92d378-8496-4c7c-a9f7-67d02bf558c4","order_by":3,"name":"Elkawakib Syam'un","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Elkawakib","middleName":"","lastName":"Syam'un","suffix":""},{"id":586344363,"identity":"523cd3d6-8466-444c-8f47-4ba59432d7db","order_by":4,"name":"Novianti Sampepadang","email":"","orcid":"","institution":"Hasanuddin University","correspondingAuthor":false,"prefix":"","firstName":"Novianti","middleName":"","lastName":"Sampepadang","suffix":""}],"badges":[],"createdAt":"2025-12-01 13:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8251105/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8251105/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102296930,"identity":"1d57b7fc-e196-4b48-a6ae-e73f92aaab40","added_by":"auto","created_at":"2026-02-10 10:22:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":712808,"visible":true,"origin":"","legend":"\u003cp\u003eA: Photoperiod and IoT Hardware, B: Temporary Immersion Control System, and C: Blynk IoT Display via Mobile Phone\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/d0ae9f1070fc9d51a8eb42ea.png"},{"id":102296947,"identity":"049b4d62-a5ee-4b86-8647-e121c575ecfb","added_by":"auto","created_at":"2026-02-10 10:23:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148853,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage of TIS contamination levels at each stage.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/955d18cc8aedba0708066754.png"},{"id":102188940,"identity":"ccb996a4-9a1f-470f-be58-6c821744a8e5","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":727907,"visible":true,"origin":"","legend":"\u003cp\u003eModification of the TIS components, consisting of a central tube and culture basket, by Nabila Cultura Laboratory. A: old TIS components and B: new/modified TIS components.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/fcc80413250dcbbac52ced89.png"},{"id":102188941,"identity":"1ecee778-e9ee-4b9a-a24d-625a1b9787ce","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":143354,"visible":true,"origin":"","legend":"\u003cp\u003eBoxplot of the average number (a) and length (b) of planlet leaves under various combinations of photoperiod (P) and immersion frequency (F), one month after planting. The first factor was the duration of illumination (P): P1 = 12h light (L)/12h dark (D); P2 = 16 L/8 D; P3 = 20 L/4 D; and P4 = 24 L/0 D (P). The second factor was the frequency of immersion (F): F1 = 6 times/day, F2 = 4 times/day, and F3 = 8 times/day, with each immersion lasting for 2 min.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/212ba87939612b6b25f34200.png"},{"id":102188939,"identity":"c1417214-f82b-43a4-b025-3a4ae73f5088","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":86772,"visible":true,"origin":"","legend":"\u003cp\u003eAverage fresh weight of \u003cem\u003eCoelogyne pandurata\u003c/em\u003eplantlets at 30 days after planting under different combinations of photoperiod (P) and immersion frequency (F) in a temporary immersion system. The first factor was the photoperiod: P₁ = 12h light / 12h dark, P₂ = 16h light / 8h dark, P₃ = 20h light / 4h dark, and P₄ = 24h continuous light. The second factor was the immersion frequency: F₁ = six times/day, F₂ = four times/day, and F₃ = eight times/day, each immersion lasting 2 minutes. Error bars indicate variation among replicates.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/e0a8edda91664259e3373bb2.png"},{"id":102296951,"identity":"c96bc3f4-7a16-44bd-8250-e2a7772d3ba6","added_by":"auto","created_at":"2026-02-10 10:23:15","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":85004,"visible":true,"origin":"","legend":"\u003cp\u003eThe average hyperhydricity level in each treatment was observed 1 month after planting. The first factor was the duration of light exposure (P): P\u003csub\u003e1\u003c/sub\u003e = 12h light (L), 12h dark (D); P\u003csub\u003e2\u003c/sub\u003e = 16L, 8D; P3 = 20L, 4D; and P\u003csub\u003e4\u003c/sub\u003e = 24L, 0D. The second factor was the frequency of immersion (F): F\u003csub\u003e1\u003c/sub\u003e= 4 times/day; F\u003csub\u003e2\u003c/sub\u003e= 6 times/day; F\u003csub\u003e3\u003c/sub\u003e= 8 times/day, with an immersion duration of 2 min each.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/8119ea7b4373cad9b05e6530.png"},{"id":102188945,"identity":"95521e00-2333-47f9-8689-b7493c8ac3a4","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2341965,"visible":true,"origin":"","legend":"\u003cp\u003eMicroscopic observation of hyperhydricity symptoms in Coelogyne pandurata plantlets under different treatment combinations (P\u003csub\u003e1\u003c/sub\u003e–P\u003csub\u003e4\u003c/sub\u003e) and immersion frequencies (F\u003csub\u003e1\u003c/sub\u003e–F\u003csub\u003e3\u003c/sub\u003e\u003cem\u003e).\u003c/em\u003e\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/f3ed9ea8df35f9a28ec8f4de.png"},{"id":102296944,"identity":"214d33cc-bf53-430b-b711-eab7c7363d10","added_by":"auto","created_at":"2026-02-10 10:23:10","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1991331,"visible":true,"origin":"","legend":"\u003cp\u003eLeaf color variation of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets grown in the Temporary Immersion System under different immersion frequencies (F₁–F₃) and photoperiods (P₁–P₄). The color was determined using the RHS Color Chart, predominantly categorized as 143 Strong Yellow Green (A–C) and 144 Yellow Green A. Visual differences among treatments indicate the influence of immersion and light duration on chlorophyll accumulation and leaf greenness of the plants.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/aaa0add0159baa096d07df46.png"},{"id":102296830,"identity":"78656ba8-96f0-4e49-bff8-143c08815f49","added_by":"auto","created_at":"2026-02-10 10:22:04","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":33373,"visible":true,"origin":"","legend":"\u003cp\u003ePCA of growth parameters of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets cultured under different photoperiod and immersion frequency treatments in the Temporary Immersion System.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/e37311c7dec0f8c3535d92b8.png"},{"id":102188942,"identity":"2f85515b-a9e5-4d91-ae90-233a2e60e588","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":84809,"visible":true,"origin":"","legend":"\u003cp\u003eTotal chlorophyll and carotenoid contents of plantlets cultured under different photoperiod × immersion frequency treatments. Blue bars indicate total chlorophyll (µg mL⁻¹), while orange bars represent carotenoid concentrations (µg mL⁻¹). Pigment quantification was performed spectrophotometrically, and values represent mean concentrations for each treatment.\u003c/p\u003e","description":"","filename":"10.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/6a1d5a081d7a0f8ebac9994b.png"},{"id":102188946,"identity":"ea0560b4-3da4-4de7-95c7-822da056fd30","added_by":"auto","created_at":"2026-02-09 08:51:38","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":285970,"visible":true,"origin":"","legend":"\u003cp\u003eStomatal density (left panel) and stomatal size (right panel) of plantlets subjected to different photoperiod × immersion frequency combinations. The bar chart on the left shows stomatal density (no. mm⁻²) measured in the afternoon and morning based on a microscopic field of view of 0.12 mm². The right panel displays stomatal length (upper graph) and stomatal width (lower graph) measured in the afternoon (orange) and morning (blue). Each bar represents the mean value obtained for each treatment.\u003c/p\u003e","description":"","filename":"11.png","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/e815609f5cd7d36c0db45751.png"},{"id":102397244,"identity":"2d18965c-6a2b-4dbb-9bb9-69df71b98dba","added_by":"auto","created_at":"2026-02-11 10:12:16","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11698848,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8251105/v1/fc7a48b2-5bb2-4f5d-af44-a0532b600004.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing Growth and Reducing Contamination in Coelogyne pandurata Culture: A Smart IoT-Controlled Temporary Immersion System","fulltext":[{"header":"Introduction","content":"\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Black orchid (\u003cem\u003eCoelogyne pandurata\u003c/em\u003e), an orchid species endemic to Borneo, has been classified as endangered because of habitat loss and overexploitation of its population. This orchid is distinguished by its large, bright green petals and sepals along with a deep black labellum or lip, which serves as a distinctive and primary attraction (Hartati et al., 2017). The uniqueness and rarity of black orchids have rendered them a target for excessive exploitation, which, in conjunction with the destruction of their natural habitat, has led to a significant decline in the wild population (Kartiman et al., 2025). This raises serious concerns regarding the survival of this species and underscores the urgency for effective conservation and propagation efforts to ensure its survival. In vitro culture is a viable alternative; however, traditional semi-solid media are limited in terms of their scalability and efficiency. Conventional propagation methods are inadequate to meet the conservation requirements of this species. The Temporary Immersion System (TIS) is a promising technology that enhances nutrient uptake, reduces hyperhydricity, and improves plantlet quality compared with conventional systems (Etienne \u0026amp; Berthouly, 2002). TIS has been successfully applied to various crops and ornamentals, including pineapples (Escalona et al., 1999), orchid fields(Leyva-Ovalle et al., 2020; Halla\u0026ccedil; et al., 2024), and other tropical species.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The temporary immersion bioreactor system demonstrated efficacy for orchid propagation. Numerous studies have established a positive correlation between immersion duration and light period. Bozkurt et al. (2023) indicated that the duration of immersion in a TIS significantly influenced plant propagation outcomes. The frequency and duration of immersion can affect the success of rooting and incidence of hyperhydricity (Etienne \u0026amp; Berthouly, 2002). For example, adjusting the immersion interval can alleviate the adverse effects of hyperhydricity, which is a common issue in plant tissue culture. Furthermore, Etienne and Berthouly (2002) the immersion frequency is a critical factor influencing the success of in vitro propagation. Leyva-Ovalle et al. (2020) concluded that nutrient immersion for 2 min every 4 h resulted in the highest number of shoots in Guarianthe skinneri orchids.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Variations in photoperiod can significantly influence the regulation of photosensitive pigments within enzymes involved in sucrose metabolism, thereby enhancing enzymatic activity and leading to substantial accumulation of photosynthates in juvenile plants. Xu et al. (2020) reported that a 16-hour photoperiod in Cunninghamia lanceolata plants enhances the capacity of plantlet leaves in in vitro culture to capture, convert, and transfer light energy. Elevated chlorophyll levels indicate an increased capacity for light adaptation, improved electron transfer within chlorophyll, and increased activity of the Calvin cycle enzymes. Optimal light duration can reduce culture time, increase rooting rates, and enhance leaf and root number (Marques et al., 2021). Kaladharan et al. (2024) observed that a light period of 16 h, followed by 8 h of darkness, yielded optimal results for \u003cem\u003eCoelogyne\u003c/em\u003e mossiase shoots. (Uma et al., 2021) demonstrated that immersion for 2 min every 6 h was effective for banana shoot and root development. Furthermore, continuous lighting for 24 h per day was applied to one of the racks as several studies have underscored the benefits of continuous lighting for plant growth (Smirnov et al., 2022).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Technology in agriculture is developing rapidly, especially in automation and the use of Internet of Things (IoT) systems in agriculture. Automated systems such as climate control in greenhouses, irrigation, and fertigation have been shown to increase agricultural yields (Skripko \u0026amp; Skripko, 2017). The IoT in agriculture enables the precise monitoring and control of temperature, light, and humidity (Dalina \u0026amp; Sobejana, 2019). TIS technology connected to IoT opens new opportunities for preserving and propagating rare plants, such as black orchids. This system optimizes growth conditions in the laboratory and is expected to improve the success rate of black orchid propagation in the future. Using this technology, researchers and farmers can create a more controlled and efficient environment to support the recovery of black orchids from the wild. The integration of TIS and IoT not only increases propagation yields but also accelerates the conservation and recovery of black orchids in their natural habitats.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Although TIS provide significant advantages, contamination and technical limitations often hinder their implementation. Optimizing culture conditions, particularly immersion frequency and photoperiod, is crucial for maximizing growth and ensuring reproducibility of results. Photoperiods regulate photosynthetic efficiency and morphogenesis, with balanced light\u0026ndash;dark cycles being more beneficial than continuous light exposure (Smirnov et al., 2022). The present study aimed to evaluate the effects of immersion frequency and photoperiod on the in vitro propagation of black orchids using a TIS and to address the challenges of contamination control and equipment modification.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003ch2\u003e2.1 Plant Material\u003c/h2\u003e\n\u003cp\u003eIn vitro-derived black orchid (\u003cem\u003eC. pandurata\u003c/em\u003e) plantlets were used as explants.\u003c/p\u003e\n\u003ch2\u003e2.2 Experimental Design\u003c/h2\u003e\n\u003cp\u003eA factorial design (3 \u0026times; 4) with immersion frequency (F) and photoperiod (P) was applied, resulting in 12 treatment combinations with four replicates each (total of 48 experimental units):\u003c/p\u003e\n\u003cp\u003e\u0026middot; Immersion frequency (F): F1 = six times/day, F2 = four times/day, F3 = eight times/day (2 minutes each).\u003c/p\u003e\n\u003cp\u003e\u0026middot; Photoperiod (P): P1 = 12h light/12h dark, P2 = 16h light/8h dark, P3 = 20h light/4h dark, P4 = 24h light.\u003c/p\u003e\n\u003ch2\u003e2.3 Data Collection\u003c/h2\u003e\n\u003cp\u003eAfter 30 days, the following growth parameters were recorded: number of shoots, plant height (cm), number of leaves, number of leaf, leaf length (cm), fresh weight (g), root length (cm), hyperhydricity, leaf color, chlorophyll, and stomata.\u003c/p\u003e\n\u003ch2\u003e2.4 Contamination Management\u003c/h2\u003e\n\u003cp\u003eSterilization was conducted in three stages: (1) conventional sterilization of vessels and media; (2) double sterilization (media poured into sterilized vessels under laminar airflow and re-sterilization); and (3) TIS component modification (central tube and culture basket). Contamination rates were monitored weekly.\u003c/p\u003e\n\u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e\n\u003cp\u003eData were subjected to two-way Analysis of Variance (ANOVA) (P \u0026lt; 0.05). Non-significant results were further examined using boxplots to visualize trends. Principal Component Analysis (PCA) was conducted using ClustVis (biit.cs.ut.ee/clustvis/), based on ln(x)-independent, row-centered, and unit variance\u0026ndash;scaled data with arcsine-transformed survival proportions (Alive_Trans) of Mets.\u003c/p\u003e\n"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Modification of Internet of Things Design for Temporary Immersion System and Automatic Lighting System\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the design of a TIS bioreactor for plant tissue culture by our research partner, the Nabila Cultura Laboratory in Bogor, Indonesia. This bioreactor has two main parts: a lower section for storing the nutrient medium, and an upper section for the culture space. The culture space was a 1 L transparent bottle with a plastic cap. A silicone hose connected the lower and upper sections, and a 0.22 \u0026micro;m Millipore filter maintained sterility in the medium- and gas-exchange areas. The system features CO\u003csub\u003e2\u003c/sub\u003e gas injection at a volume of 0.1 vvm and a timer to regulate the frequency of plantlet immersion in the liquid medium. The hardware included a water pump for medium circulation, and an air pump for supplying CO2 and fresh air (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eThe lighting was set automatically with four settings: P1\u0026thinsp;=\u0026thinsp;12 h light, 12 h dark; P2\u0026thinsp;=\u0026thinsp;16 h light, 8 h dark; P3\u0026thinsp;=\u0026thinsp;20 h light, 4 h dark; and P4\u0026thinsp;=\u0026thinsp;24 h light, 0 h dark. The Real-Time Clock (RTC) sensor precisely tracked time. The relay connects the Arduino Uno to the actuator system. The Arduino Uno managed these settings (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and sent information to the relay to control the electric current.\u003c/p\u003e \u003cp\u003eThe IoT-based TIS developed in the laboratory was equipped with a DHT22 sensor to detect temperature and humidity, a Lux sensor to detect light intensity, and a vibration sensor to detect vibrations when the TIS was immersed in plantlets. Vibration occurred when air entered the TIS (code 1: TIS immersed; code 0: TIS not immersed). This information was sent to NodeMCU ESP8266 and displayed on an LCD and the Blynk IoT application on an Android phone (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), showing the temperature (\u003cem\u003eSuhu\u003c/em\u003e), humidity (\u003cem\u003eKelembapan\u003c/em\u003e), lux (\u003cem\u003eLUX\u003c/em\u003e), and vibration (\u003cem\u003eVibration\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Contamination Control\u003c/h2\u003e \u003cp\u003eInitial trials showed contamination rates of 100% by day 30. Double sterilization reduced contamination to approximately 30%, whereas component modifications further decreased it to approximately 10% (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), indicating a reduction in contamination levels following the applied sterilization and equipment modifications.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Modification of Temporary Immersion System Equipment\u003c/h2\u003e \u003cp\u003eRemedial measures were implemented to reduce contamination. The first stage focused on sterilization techniques, media formulation, procedures for transferring plantlets into the TIS vessel, and adjustments to TIS components, such as the central tube, filter, and culture basket. The second stage involved multi-level sterilization: sterilizing the TIS equipment separately, pouring media into the already sterilized TIS equipment under LAF, and re-sterilizing the equipment and media. The results of the second stage show a 30% reduction in contamination (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe third stage involved multilevel sterilization and equipment modification with the TIS supplier partner, including replacement of the central tube and culture basket models (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). These improvements successfully addressed contamination and technical issues related to inadequate pumping of the media. The results of the third-stage modifications showed a 10% reduction in contamination.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Shoot Multiplication\u003c/h2\u003e \u003cp\u003eThe increase in shoot number of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets at 30 days was influenced by both photoperiod (P) and immersion frequency (F). Among the photoperiod treatments, continuous light exposure (P₄ = 24 h light) produced the highest average number of shoots (4.01), significantly greater than P₃ (20 h light; 2.85). The lowest shoot formation occurred under P\u0026thinsp;\u0026lt;\u0026thinsp;ls \u0026gt;, indicating reduced proliferation under shorter dark intervals. Across the immersion frequencies, F₂ (four immersions/day) yielded the highest mean (4.20), which was significantly different from F₁ (six immersions/day; 3.15) and F₃ (eight immersions/day; 3.01). No significant interaction was found between photoperiod and immersion frequency, indicating that the effects of these factors on shoot number were statistically independent under the tested conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage increase in shoots of Black Orchid (\u003cem\u003eCoelogyne pandurata\u003c/em\u003e) plantlets at 30 days after culture under different photoperiods (P) and immersion frequencies (F) in a Temporary Immersion System.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e DMRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.51\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.45\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.85\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e4.01\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3.15\u003csup\u003eq\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e4.20\u003c/b\u003e\u003csup\u003e\u003cb\u003ep\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.01\u003csup\u003eq\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.455\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e DMRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003eNote: Values represent the means of four replicates. Means followed by different letters within the same column or row differ significantly according to Duncan\u0026rsquo;s Multiple Range Test (DMRT) at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;number of photoperiod levels; \u003cem\u003eq\u003c/em\u003e DMRT\u0026thinsp;=\u0026thinsp;critical value for Duncan\u0026rsquo;s test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Plant Height (Cm)\u003c/h2\u003e \u003cp\u003eThe average plant height of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e after 30 days of culture ranged from 0.80 to 1.13 cm across the various combinations of photoperiod and immersion frequency. Among the photoperiods, the highest mean height (1.01 cm) was observed under P₁ (12 h light/12 h dark), followed by P₄ (24 h light; 0.96 cm). The shortest plantlets occurred under P₃ (20 h light/4 h dark; 0.88 cm). Immersion frequency significantly affected plant height, where F₃ (eight immersions/day) produced the tallest plantlets (1.06 cm), while F₁ and F₂ showed lower averages (0.88 cm and 0.89 cm, respectively). No significant interaction was detected between the photoperiod and immersion frequency.\u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage height of Black Orchid (\u003cem\u003eCoelogyne pandurata\u003c/em\u003e) plantlets at 30 days after culture under different photoperiods (P) and immersion frequencies (F) in a Temporary Immersion System.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.97\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.96\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.88\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.89\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e1.06\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e DMRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Values represent the means of four replicates. Means followed by different letters within the same column or row differ significantly according to Duncan\u0026rsquo;s Multiple Range Test (DMRT) at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05. \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;number of photoperiod levels; \u003cem\u003eq\u003c/em\u003e DMRT\u0026thinsp;=\u0026thinsp;critical value for Duncan\u0026rsquo;s test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Increase in Number (Units) and Leaf Length (Cm)\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe boxplots in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e illustrate the effects of photoperiod (P) and immersion frequency (F) on the average number (a) and length (b) of leaves in \u003cem\u003eCoelogyne pandurata\u003c/em\u003e ​plantlets after one month of culture in the TIS. The number of leaves per plantlet showed broad variation across treatments, with median values generally ranging from 10 to 20 leaves and several outliers exceeding 30. The treatments P₄F₂ (24 h light, four immersions/day) and P₃F₂ (12 h light, four immersions/day) displayed higher medians and wider ranges, showing higher median leaf numbers under these treatment combinations. In contrast, P₂F₁ and P₃F₃ tended to produce fewer leaves with narrower ranges. For leaf length, the average values ranged from approximately 1 cm to 5 cm. The treatments P₄F₃ (24 h light, eight immersions/day) and P₂F₂ (16 h light, four immersions/day) exhibited slightly greater medians, although overall differences among treatments were relatively small. This indicated that leaf elongation was less responsive to photoperiod and immersion frequency than leaf initiation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.7 Average Fresh Weight of Plantlets (Gram)\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents the effect of photoperiod (P) and immersion frequency (F) on the average fresh weight of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets after one month of culture in the TIS.\u003c/p\u003e \u003cp\u003eThe average fresh weight ranged from 0.19 g to 0.41 g across treatments. The highest value (0.41 g) occurred under P₂F₂ (16h light / 8h dark with four immersions per day), followed by P₄F₃ (24h light with eight immersions/day, 0.37 g) and P₃F₂ (20h light with four immersions/day, 0.35 g). The lowest biomass values (0.19 g) were observed in P₁F₃ and P₃F₃, indicating that excessive immersion frequency suppressed fresh weight accumulation.\u003c/p\u003e \u003cp\u003eAcross photoperiods, plantlets exposed to P₂ (16 h light) and P₄ (24 h light) produced higher mean weights than those under shorter illumination (P₁ = 12 h L/12 h D; P₃ = 20 h L/4 h D). Meanwhile, the lowest immersion frequency (F₂, four times/day) resulted in a higher biomass in most photoperiod combinations.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.8 Average Root Length (cm)\u003c/h2\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the average root length of black orchids 30 days after planting (DAP) under different light exposure durations (P) and soaking frequencies (F). The results were measured in centimeters (cm). This experiment investigated four light exposure levels (P1\u0026ndash;P4) and three soaking frequency levels (F\u003csub\u003e1\u003c/sub\u003e\u0026ndash;F\u003csub\u003e3\u003c/sub\u003e). The data showed that different combinations of these treatments had varying effects on the root length.\u003c/p\u003e \u003cp\u003eThe results showed that the P\u003csub\u003e1\u003c/sub\u003e treatment yielded the longest average root length (1.00 cm), which was significantly different from the P\u003csub\u003e2\u003c/sub\u003e and P\u003csub\u003e3\u003c/sub\u003e treatments. Soaking frequency also influenced root growth, with F\u003csub\u003e3\u003c/sub\u003e showing the highest average root length (0.99 cm) across all light-exposure treatments. The results of the ANOVA indicated that factor P (F\u0026thinsp;=\u0026thinsp;8.07\u0026thinsp;\u0026gt;\u0026thinsp;F critical\u0026thinsp;=\u0026thinsp;2.80, P-value\u0026thinsp;=\u0026thinsp;0.02\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and factor F had significant effects on plant height. The P \u0026times; F interaction did not have a significant effect (P-value\u0026thinsp;=\u0026thinsp;0.30\u0026thinsp;\u0026gt;\u0026thinsp;0.05). P1 treatment (12 h light (L), 12 h dark (D)) was the best for increasing plantlet growth, whereas P\u003csub\u003e1\u003c/sub\u003e (24 h L, h D) also showed good potential. The F\u003csub\u003e3\u003c/sub\u003e treatment (eight times/day) was the best for increasing the root growth of Black Orchid plantlets.\u003c/p\u003e \u003cp\u003eThis indicates that both the duration of light exposure and soaking frequency play important roles in the root development of black orchids, with potential implications for optimizing cultivation practices for orchid propagation and growth.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAverage root length of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets at 30 days after planting under different combinations of photoperiod (P) and immersion frequency (F) in a temporary immersion system (cm).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eF\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eF\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eF\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e DMRT\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.868\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.874\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e1.00\u003c/b\u003e\u003csup\u003e\u003cb\u003ea\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.736\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.714\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.796\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.75\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.740\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.932\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.80b\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.824\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.830\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.068\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.89a\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.79\u003csup\u003eq\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.78\u003csup\u003eq\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e0.99\u003c/b\u003e\u003csup\u003e\u003cb\u003ep\u003c/b\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eq\u003c/em\u003e DMRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cem\u003eNote\u003c/em\u003e: Numbers in each column followed by the same lowercase letter are not significantly different according to Duncan\u0026rsquo;s Multiple Range Test (DMRT) at α\u0026thinsp;=\u0026thinsp;0.05. P: Photoperiod - P₁ = 12h light/12h dark; P₂ = 16h light/8h dark; P₃ = 20h light/4h dark; P₄ = 24 h continuous light. F: Immersion frequency - F₁ = six times/day; F₂ = four times/day; F₃ = eight times/day, with each immersion lasting 2 min. \u003cem\u003ep\u003c/em\u003e: number of means; \u003cem\u003eq DMRT\u003c/em\u003e: critical value of Duncan\u0026rsquo;s Multiple Range Test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.9 Hyperhydricity Percentages (Units)\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows that the degree of hyperhydricity varied among treatments combining different photoperiods (P) and immersion frequencies (F) after one month of culture. The highest average hyperhydricity score (8) was recorded in P₁F₁ (12 h light / 12 h dark; six immersions/day), followed by P₁F₃ = 6 and P₂F₁ = 4. In contrast, the lowest hyperhydricity level (1) occurred under P₁F₂ (12 h light / 12 h dark; four immersions/day), indicating that the reduction in immersion frequency mitigated tissue vitrification. Generally, increasing the immersion frequency led to greater hyperhydricity, whereas extended light exposure (P₃\u0026ndash;P₄) tended to decrease the severity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe microscopic observations shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e visually confirm these findings. Plantlets subjected to frequent immersions (F₃) exhibited typical hyperhydric symptoms such as translucent and brittle leaves, swollen stems, and impaired chlorophyll development (e.g., P₁F₃, P₂F₃, P₃F₃). Conversely, plantlets cultured under fewer immersions (F₂) or longer photoperiods (P₃\u0026ndash;P₄) maintained firmer, greener leaves and more normal morphology, with substantially reduced tissue waterlogging (e.g., P₁F₂, P₄F₃).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.10 Leaf Color Observation Based on RHS Colour Chart\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e shows the leaf color variations of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets grown under different photoperiod (P) and immersion frequency (F) combinations in the TIS. Leaf color was classified using the RHS Color Chart codes 143 A\u0026ndash;C, representing shades from strong yellow-green to light yellow-green. Quantitative scoring (1\u0026thinsp;=\u0026thinsp;light/pale; 2\u0026thinsp;=\u0026thinsp;moderate; 3\u0026thinsp;=\u0026thinsp;dark green) revealed that the RHS scores ranged from 1 to 3, with higher scores indicating greater greenness and chlorophyll accumulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAcross all photoperiods, shorter light exposures (P₁ = 12 hours and P₂ = 16 hours) resulted in more intense green color (total score of 8 each) compared to longer photoperiods (P₃ = 20 hours and P₄ = 24 hours with total score of 7 each). Regarding immersion, plantlets cultured at F₁ (six immersions/day) showed more stable and darker green leaves, with the highest total score of 11. In contrast, frequent immersions (F₃ = eight times/day with a total score of 9) resulted in a paler color for the P₄F₃ combination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.11​ Principal Component Analysis\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCA was performed to summarize the multivariate growth response of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e plantlets cultured under different photoperiod and immersion frequency combinations in the TIS. The first two principal components, PC1 (94.3%) and PC2 (5.6%), cumulatively explained 99.9% of the total variance among the treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). PC1 represents the overall plantlet vigor, integrating the parameters related to shoot multiplication, root elongation, plant height, and fresh biomass. PC2 reflects secondary morphological and physiological differences, including variations in leaf elongation and hyperhydricity.\u003c/p\u003e \u003cp\u003eThe PCA biplot revealed that most treatments clustered tightly on the positive side of PC1, indicating uniform and vigorous growth performance among TIS-grown plantlets. Only treatment P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e appeared slightly separated along the negative axis of PC1, suggesting a lower overall growth rate, possibly due to a shorter photoperiod or reduced immersion frequency. These results indicate that the combination of photoperiod and immersion frequency primarily influences the magnitude of growth responses rather than inducing distinct growth patterns, reflecting a generally consistent growth performance of TIS-grown plantlets under the tested conditions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.12​ Effects of Photoperiod and Immersion Frequency on Pigment Accumulation\u003c/h2\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePigment accumulation varied significantly across photoperiod \u0026times; immersion treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e; Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Total chlorophyll ranged from 6.19 to 23.42 \u0026micro;g mL⁻\u0026sup1;, whereas carotenoid concentrations ranged from non-detectable to 6.29 \u0026micro;g mL⁻\u0026sup1;. The highest chlorophyll a, chlorophyll b, and total chlorophyll levels were recorded in the P1F1 treatment (16.63, 6.80, and 23.42 \u0026micro;g mL⁻\u0026sup1;, respectively), representing a nearly two-fold increase compared with the control (12.06 \u0026micro;g mL⁻\u0026sup1;). Treatments P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e and P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e also showed consistently elevated pigment levels, indicating that moderate photoperiods (12\u0026ndash;16 h light) combined with moderate immersion frequencies supported chlorophyll biosynthesis. Carotenoid accumulation followed a similar pattern. P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e produced the highest carotenoid concentration (6.29 \u0026micro;g mL⁻\u0026sup1;), followed by P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e (5.08 \u0026micro;g mL⁻\u0026sup1;) and P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e (4.03 \u0026micro;g mL⁻\u0026sup1;). In contrast, longer photoperiods (P\u003csub\u003e3\u003c/sub\u003e\u0026ndash;P\u003csub\u003e4\u003c/sub\u003e) resulted in markedly lower carotenoid levels (1.20\u0026ndash;3.92 \u0026micro;g mL⁻\u0026sup1;). The P\u003csub\u003e4\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e treatment produced a negative calculated value after correction for chlorophyll interference; this value was therefore classified as \u0026ldquo;not detected (ND)\u0026rdquo;. Overall, shorter photoperiods coupled with moderate immersion frequencies produced the highest pigment contents, whereas continuous or extended photoperiods reduced both chlorophyll and carotenoid accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.13 Effects of Photoperiod and Immersion Frequency on Stomatal Density and Size in \u003cem\u003eIn-Vitro\u003c/em\u003e Plantlets\u003c/h2\u003e \u003cp\u003eStomatal density differed markedly among treatments, ranging from 42 to 117 stomata mm⁻\u0026sup2; (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e, left panel). The highest densities were recorded in P\u003csub\u003e3\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e (117 mm⁻\u0026sup2;) and P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e (108 mm⁻\u0026sup2;), whereas P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e\u0026ndash;P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e consistently produced the lowest densities (42\u0026ndash;88 mm⁻\u0026sup2;). Morning and afternoon measurements showed similar patterns, indicating that treatment effects were stable across time.\u003c/p\u003e \u003cp\u003eStomatal size also varied considerably across treatments (Fig. X, right panel). Afternoon stomatal length ranged from 1.3 to 4.8 \u0026micro;m, with the largest stomata observed in P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e, P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e, and P\u003csub\u003e4\u003c/sub\u003eF\u003csub\u003e3\u003c/sub\u003e, whereas morning values ranged from 2.4 to 3.9 \u0026micro;m. Stomatal width showed a similar pattern, with afternoon values between 0.8 and 3.5 \u0026micro;m and morning values ranging from 1.6 to 2.9 \u0026micro;m. Treatments with extended photoperiods (P\u003csub\u003e3\u003c/sub\u003e and P\u003csub\u003e4\u003c/sub\u003e) generally produced moderate-to-large stomata, while short photoperiods (P\u003csub\u003e1\u003c/sub\u003e and P\u003csub\u003e2\u003c/sub\u003e) yielded more variable structures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe integration of the Internet of Things (IoT) into plant tissue culture laboratories represents a transformative advancement in environmental regulation, automation, and micropropagation efficiency optimization. IoT-based systems enable real-time monitoring and precise control of critical parameters such as photoperiod, vibration, temperature, humidity, and light intensity, all of which are vital for promoting optimal plant growth and improving culture yields. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the environmental data recorded in this study demonstrated the effectiveness of the IoT system, with incubation room conditions maintained at 26.9\u0026deg;C, 37.8% humidity, and 1,534 lux light intensity, while vibration data confirmed the operational status of the TIS between irrigated and non-irrigated states. These findings indicate that the IoT framework effectively supported the growth of \u003cem\u003ethe Coelogyne pandurata\u003c/em\u003e and advanced innovation in automated culture systems. (Rahayu \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) reported that a well-designed bioreactor equipped with an automatic control system facilitates laboratory operations, reduces manual labor, and ensures precision in the micropropagation of black orchids. However, as noted by Harun et al. (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the rapid emergence of new technologies challenges researchers to continuously update their technical skills and fully harness their potential. By integrating sensors and automated control modules, laboratories can minimize labor requirements, increase operational efficiency, and maintain stable environmental conditions tailored to the physiological needs of cultured plants.\u003c/p\u003e \u003cp\u003eIn the present study, the IoT-integrated TIS combined real-time sensors (DHT22, Lux, and vibration) with programmable microcontrollers (Arduino Uno and NodeMCU ESP8266) to regulate and monitor key environmental parameters, including temperature, humidity, light intensity, and immersion cycles, which are essential for the successful micropropagation of \u003cem\u003eC. pandurata\u003c/em\u003e. This approach aligns with the growing trend toward smart bioreactor systems that ensure controlled microenvironments, while reducing manual intervention and the risk of contamination. Similar IoT-based control frameworks have proven effective in automating plant tissue culture systems, minimizing human errors, and maintaining consistent growth conditions (Dalina \u0026amp; Sobejana, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dhanaraju et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The DHT22 sensors enabled precise monitoring of temperature and humidity, whereas automated immersion cycles allowed the dynamic adjustment of growth conditions, which is a critical feature for tropical orchids that are highly sensitive to microclimatic fluctuations.\u003c/p\u003e \u003cp\u003eFurthermore, the incorporation of a real-time clock (RTC) and a Blynk mobile interface enhanced accessibility and responsiveness by allowing wireless data transmission through the NodeMCU ESP8266 module. This capability enables researchers to remotely monitor and modify culture parameters, ensuring uninterrupted 24-hour operation and early detection of potential system failures. Such remote supervision is particularly beneficial for delicate in vitro systems, such as TIS, where continuous oversight improves reproducibility and reduces contamination rates, as previously demonstrated by Rahayu ( 2024) in \u003cem\u003eC. pandurata\u003c/em\u003e. Automation of photoperiod control through integrated light sensors and relay systems has further enabled the accurate simulation of natural light cycles, which are essential for photosynthetic and morphogenic regulation. Stable photoperiod regimes enhance chlorophyll biosynthesis, photosystem performance, and carbon assimilation, ultimately improving plant vigor and morphology (Smirnov et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This synchronization between artificial illumination and environmental control mitigates photoperiod-induced stress and ensures consistent growth.\u003c/p\u003e \u003cp\u003eAdditionally, vibration sensing serves as an indirect indicator of immersion events, verifying the correct operation of the air pump and medium circulation system. This innovation addresses one of the persistent challenges in conventional TIS setups: maintaining a consistent immersion frequency (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), by providing real-time validation of mechanical performance. Collectively, the integration of these IoT components establishes a feedback-driven, data-based monitoring system that enhances the experimental precision, reproducibility, and scalability. The IoT-integrated TIS developed in this study exemplifies a next-generation platform for plant micropropagation, combining biotechnology and digital technology to enable the automated, precise, and sustainable in vitro propagation of endangered species, such as the black orchid (\u003cem\u003eCoelogyne pandurata\u003c/em\u003e).\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the trend of contamination in the TIS, where improvements have been made to reduce it. Initially, improvements focused on sterilization techniques, media formulation, procedures for transferring plantlets into TIS vessels, and adjustments to TIS components such as the central tube, filter, and culture basket. In the second stage, layered sterilization was implemented: separate sterilization of the TIS equipment, pouring media into the sterile TIS equipment under the LAF, and re-sterilization of the equipment and media. The results of the second stage showed a 30% reduction in the contamination. In the third stage, in addition to layered sterilization, the equipment was modified in collaboration with the TIS supplier partner by changing the model of the central tube and culture basket (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This improvement addressed contamination and technical issues such as the improper pumping of liquid media. The results of the third-stage modifications showed a reduction in contamination of up to 10%. These results are in line with the research of Leyva-Ovalle et al. (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) on the importance of sterilization management and TIS system design, and also support Rahayu's (2024) report on TIS development in black orchids. Other contamination factors included the culture equipment, plantlets, planting room air, plant species, research personnel, and frequently visited rooms. According to Klayraung et al. (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), bacterial contamination is higher in frequently visited areas, which indicates a relationship between human activity and microbial density. Every step in plant tissue culture must be carefully considered to prevent bacterial and fungal contamination.\u003c/p\u003e \u003cp\u003eAs shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, the average number of shoots ranged from 2.28 to 4.98. Among the photoperiod treatments, P₄ (24 h continuous light) produced the highest mean number of shoots (4.01), which was significantly greater than P₃ (20 h light/4 h dark), which resulted in the lowest mean (2.85). This indicates that prolonged illumination enhances shoot proliferation in \u003cem\u003eCoelogyne pandurata\u003c/em\u003e, possibly by maintaining uninterrupted photosynthetic and metabolic activities that support cell division and organogenesis. Continuous light exposure significantly enhances shoot proliferation in plants by optimizing photomorphogenic responses and photosynthesis. Studies have shown that controlling light characteristics, like photoperiod, is crucial for increasing shoot numbers in plant tissue cultures (Cavallaro et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In date palms, high light intensities decreased shoot bud proliferation, underscoring the need for precise light management (Meziani et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Light also plays a vital role in plant regeneration, affecting processes such as shoot regeneration and root formation (Han et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). These findings align with those on Coelogyne pandurata, where continuous light supports photosynthetic and metabolic activities necessary for organogenesis. Across immersion frequencies, F₂ (four times per day) yielded the highest average number of shoots (4.20 p), while F₁ (six times per day) and F₃ (eight times per day) produced fewer shoots (3.15 q and 3.01 q, respectively). Moderate immersion appears to be optimal for balancing nutrient absorption and aeration, without inducing hyperhydricity or hypoxia. Excessive immersion (F₃) likely caused oxygen limitation in the medium and suppressed shoot development (Preil, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePlantlet height also varied among the treatments (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The tallest plantlets were observed under F₃ (1.06 cm), significantly higher than those in F₁ and F₂ (0.88\u0026ndash;0.89 cm). Increased immersion frequency may enhance nutrient uptake and reduce desiccation, thus supporting elongation growth (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, photoperiod effects on height were less pronounced; the average height ranged narrowly between 0.88 cm and 1.01 cm, with no significant differences. This suggests that plant height in \u003cem\u003eC. pandurata\u003c/em\u003e may be more responsive to immersion frequency than photoperiod length during the early growth phase.\u003c/p\u003e \u003cp\u003eNo significant interaction between photoperiod and immersion frequency was observed for shoot number or height, indicating that their effects were largely independent. Nonetheless, the best overall growth response was recorded under the combination of continuous light (P₄) with the lower immersion frequency (F₂, four times per day), which produced the highest shoot number (4.98 units) and good shoot elongation (0.92 cm). This finding supports previous reports that continuous illumination enhances morphogenesis when combined with adequately spaced immersion cycles that prevent oversoaking stress (Kozai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results demonstrate that in \u003cem\u003eCoelogyne pandurata\u003c/em\u003e, 24 h light photoperiod and moderate immersion (four times per day, 2 min each) are the most favorable conditions for early shoot multiplication and balanced plantlet growth in the TIS culture system.\u003c/p\u003e \u003cp\u003eLeaf formation in \u003cem\u003eC. pandurata\u003c/em\u003e was strongly influenced by both light duration and immersion frequency. The higher leaf numbers observed under continuous illumination (P₄) reflect enhanced photosynthetic activity and carbohydrate accumulation, which provide sufficient energy for organogenesis and leaf initiation. Continuous illumination significantly enhanced leaf formation by increasing the photosynthetic activity and carbohydrate accumulation. For example, in tomato seedlings, prolonged light exposure leads to increased photosynthesis and chlorophyll content under long photoperiods (Wu et al., \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Similarly, a study of Mentha species found that longer photoperiods resulted in higher concentrations of essential oils, reflecting increased metabolic activity (Fahl\u0026eacute;n et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Moreover, research on Mesembryanthemum crystallinum showed that longer photoperiods positively influenced yield and antioxidant capacity, indicating increased biomass production through enhanced photosynthesis (Chen et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). These studies illustrate the benefits of extended light duration on leaf formation and overall plant growth. The superior performance of treatments with four immersions per day (F₂) indicated that a lower immersion frequency created a favorable balance between nutrient uptake and aeration. Less frequent immersion may prevent hypoxia and tissue waterlogging, which can limit growth or cause hyperhydricity under excessive immersion (Preil, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Similar findings were reported by (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), who noted that properly spaced immersion cycles enhanced shoot and leaf differentiation in TIS.\u003c/p\u003e \u003cp\u003eAlthough longer photoperiods promoted leaf formation, the leaf elongation was more variable. Treatments with more frequent immersions (e.g., F₃) showed slightly longer leaves, likely due to increased water absorption and turgor pressure that encourage elongation. However, frequent immersion may also cause partial vitrification and reduce the photosynthetic efficiency (Kozai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These results demonstrate that \u003cem\u003eC. pandurata\u003c/em\u003e plantlets achieve optimal leaf growth under 24 h continuous illumination combined with four immersions per day (P₄F₂). This combination promotes vigorous leaf initiation while maintaining a healthy tissue structure, making it an effective condition for early morphogenesis in TIS cultures.\u003c/p\u003e \u003cp\u003eThe average fresh weight of plantlets demonstrated that both photoperiod length and immersion frequency affected the biomass accumulation of \u003cem\u003eC. pandurata\u003c/em\u003e plantlets in the TIS. The highest fresh weight under P₂F₂ (16 h light, four immersions/day) indicated that a moderate photoperiod combined with less frequent immersion created optimal physiological conditions for nutrient uptake and gas exchange. The finding that photoperiod length and immersion frequency affect biomass accumulation in \u003cem\u003eC. pandurata\u003c/em\u003e plantlets is supported by several studies in different species. A study on celery cultivars found that a 12 h/12 h photoperiod stimulates growth, suggesting that optimal light conditions enhance nutrient uptake and photosynthesis (Chu et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Research on in vitro plant regeneration using LED lighting has highlighted the significant influence of light characteristics, including photoperiod, on optimizing growth environments (Dutta Gupta \u0026amp; Agarwal, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, studies on microalgae, such as Chlorella vulgaris and Scenedesmus obliquus, have shown that varying photoperiods significantly alter biomass and metabolomic profiles, indicating that precise photoperiod management can optimize biomass production (Amini Khoeyi et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Vendruscolo et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These studies emphasize the universal role of light in enhancing biomass production and optimizing plant tissue culture systems.\u003c/p\u003e \u003cp\u003eHowever, continuous light (P₄) and frequent immersion (F₃ = eight times/day) may cause excessive hydration and oxygen limitation, reducing tissue dry matter despite high metabolic activity (Preil, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The superior response at F₂ (four times/day) suggests that less frequent immersion provided sufficient nutrient contact while maintaining better aeration and preventing hyperhydricity, similar to the findings reported by (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e) in other orchids and tropical species. Overall, \u003cem\u003eC. pandurata\u003c/em\u003e plantlets achieved the best biomass accumulation under 16 h light/8 h dark with four immersions per day (P₂F₂), confirming that both the light regime and immersion timing must be carefully balanced to optimize TIS performance.\u003c/p\u003e \u003cp\u003eRoot growth in \u003cem\u003eC. pandurata\u003c/em\u003e was influenced by both photoperiod and immersion frequency, although no interaction effects were detected. The longer roots under frequent immersion (F₃) suggested that increased nutrient contact and hydration promoted root elongation. Frequent immersion enhances medium absorption, thereby improving the availability of mineral nutrients and growth regulators essential for root initiation and elongation (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). However, excessively frequent cycles may also risk partial hypoxia if gas exchange is insufficient; thus, the benefit observed here may be specific to the early growth stages when roots remain short and metabolically active.\u003c/p\u003e \u003cp\u003eRegarding photoperiod, short-day exposure (12 h L/12 h D) favored root elongation, whereas extended or continuous illumination (16\u0026ndash;24 h) tended to reduce root length. Research indicates that shorter photoperiods (12 h light/12 h dark) favor root elongation, whereas extended exposure (16\u0026ndash;24 h) can inhibit it. Studies on hydroponically grown cucumbers have revealed that longer photoperiods increase phytotoxic root exudates and inhibit growth (Pramanik et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Similarly, systems such as D-Root have shown that light exposure can reduce root length and induce early lateral root emergence, disrupting typical growth patterns(Silva-Navas et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). These findings highlight the complex effects of photoperiods on root development in various species. The results indicate that short photoperiod (P₁) combined with frequent immersion (F₃) provides favorable conditions for root elongation of \u003cem\u003eC. pandurata\u003c/em\u003e in TIS. These conditions likely balance water and nutrient uptake, while maintaining sufficient aeration to support active root metabolism and elongation.\u003c/p\u003e \u003cp\u003eThe results demonstrated that immersion frequency strongly influenced the incidence of hyperhydricity in \u003cem\u003eC. pandurata\u003c/em\u003e plantlets. Excessive immersion (six\u0026ndash;eight times/day) likely caused prolonged exposure of tissues to the liquid medium, leading to reduced gas exchange, oxygen limitation, and accumulation of ethylene in the culture vessels. These conditions promote water saturation and cell wall loosening, which are characteristics of hyperhydric tissues (Preil, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In contrast, the lower immersion frequency (F₂ = four times/day) provided better aeration, allowing tissues to recover between immersions and prevent excessive hydration, which is consistent with earlier findings in \u003cem\u003eDendrobium\u003c/em\u003e and \u003cem\u003eAnanas\u003c/em\u003e species (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). The photoperiod effect was also evident; shorter illumination (12 h light) intensified hyperhydricity, whereas extended or continuous light (20\u0026ndash;24 h) reduced it. Prolonged illumination promotes higher photosynthetic activity and transpiration, enhancing cell wall lignification and gas exchange, which counteracts hyperhydric conditions (Kozai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Thus, the lowest symptom expression under P₄ (24 h light) and moderate immersion (F₂) reflected an improved physiological balance between hydration and ventilation. Overall, \u003cem\u003eC. pandurata\u003c/em\u003e plantlets grown under continuous light (P₄) with four immersions per day (F₂) exhibited the healthiest morphology, with minimal hyperhydricity. These findings emphasize the importance of optimizing both the illumination duration and immersion regime to maintain tissue quality and prevent physiological disorders during temporary immersion culture.\u003c/p\u003e \u003cp\u003eLeaf color variation in \u003cem\u003eCoelogyne pandurata\u003c/em\u003e reflects differences in chlorophyll content and physiological adaptation under varying illumination and immersion conditions. The observation that shorter photoperiods (P₁ = 12 h and P₂ = 16 h) produced darker green leaves suggests that a moderate duration of light exposure favors balanced chlorophyll synthesis and prevents photooxidative stress. Extended or continuous illumination (P₃ = 20 h and P₄ = 24 h) may lead to excessive light energy that exceeds the plantlets\u0026rsquo; photosynthetic capacity, causing partial chlorophyll degradation or photoinhibition. The connection between photoperiod and chlorophyll synthesis has been observed in studies that examine the effect of light duration on plants. For example, rice mutants exhibit reduced chlorophyll content and increased oxidative stress under altered light cycles, indicating that proper light duration is crucial for balanced chlorophyll synthesis (Li et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Salicylic acid application in plants mimics the effects of prolonged light by reducing chlorophyll and enhancing photosystem efficiency (Moustakas et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Under high-light conditions, chlorophyll accumulation is delayed when mitochondrial respiration is inhibited (Zhang et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The xanthophyll cycle protects against phototoxidative stress by dissipating excess light energy (Latowski et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These studies support the notion that moderate photoperiods aid chlorophyll synthesis, whereas prolonged exposure risks degradation and photoinhibition.\u003c/p\u003e \u003cp\u003eThe influence of the immersion frequency was also evident. The six immersions per day (F₁) treatment resulted in the most stable and darker green coloration, indicating that this frequency provided an optimal balance between nutrient absorption and gas exchange. Adequate, but not excessive, immersion maintains sufficient oxygen availability, thereby supporting chloroplast development and photosynthetic efficiency. In contrast, the most frequent immersion (F₃ = eight times/day) produced paler leaves, particularly under continuous illumination (P₄F₃), likely due to tissue overhydration, reduced oxygen diffusion, and early symptoms of hyperhydric stress (Preil, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). These findings are consistent with previous reports in orchids and other tropical species, where properly timed immersion cycles combined with moderate light exposure improved leaf greenness and chlorophyll retention (Etienne \u0026amp; Berthouly, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Kozai et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The combination of short-to-moderate photoperiods (12\u0026ndash;16 h light) with six immersions per day (F₁) supported optimal chlorophyll accumulation, resulting in darker green, physiologically healthier leaves of \u003cem\u003eC. pandurata\u003c/em\u003e in the temporary immersion system.\u003c/p\u003e \u003cp\u003ePCA clearly separated plantlet responses according to the combined influence of photoperiod and immersion frequency, indicating that these two factors jointly regulate the morphophysiological performance of \u003cem\u003eCoelogyne pandurata\u003c/em\u003e cultured in TIS. The dominance of PC1 (94.3%) suggests that most of the variation in the dataset was attributed to differences in overall plant vigor, which integrated shoot proliferation, leaf development, and biomass accumulation. This pattern implies that photoperiod duration and immersion timing predominantly modulate photosynthetic efficiency and nutrient uptake, which is consistent with earlier reports on orchids grown in liquid systems (Lorenzo \u0026amp; Tapia y Figueroa, 2025; Ram\u0026iacute;rez-Mosqueda \u0026amp; Cruz-Cruz, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe tight clustering of most treatments on the positive side of PC1 reflects physiological stability across immersion regimes, suggesting that TIS provides a balanced gaseous exchange and sufficient nutrient supply for uniform growth. The slight separation of P1F1 along the negative PC1 axis may indicate a stress response under suboptimal light exposure, resulting in a lower chlorophyll content and reduced shoot elongation. Similar findings were reported by (Escalona et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), who observed that short photoperiods limited carbohydrate accumulation and delayed organogenesis in pineapples cultured under temporary immersion. PCA confirmed that while minor quantitative variations existed among treatments, the TIS environment successfully minimized physiological heterogeneity, leading to consistent morphogenesis and survival of \u003cem\u003eC. pandurata\u003c/em\u003e plantlets. This stability highlights the reliability of the TIS for mass propagation and supports its application for large-scale conservation of black orchids under controlled micropropagation conditions.\u003c/p\u003e \u003cp\u003ePigment profiles in \u003cem\u003eCoelogyne pandurata\u003c/em\u003e were strongly influenced by the interaction between photoperiod and immersion frequency. The highest chlorophyll and carotenoid contents were obtained under moderate photoperiods (12\u0026ndash;16 h) combined with moderate immersion frequency (P1F\u003csub\u003e1\u003c/sub\u003e and P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e). These conditions likely promoted optimal plastid differentiation by improving aeration, reducing hyperhydricity, and maintaining balanced CO₂ and light availability within the TIS vessels. Similar enhancements of chlorophyll accumulation under optimized TIS settings have been reported in \u003cem\u003eDendrobium\u003c/em\u003e, \u003cem\u003ePhalaenopsis\u003c/em\u003e, and \u003cem\u003eVanilla\u003c/em\u003e cultures (Ram\u0026iacute;rez-Mosqueda \u0026amp; Cruz-Cruz, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Tokuhara \u0026amp; Mii, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCarotenoid accumulation followed the same pattern, with the highest concentrations recorded in P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e and P\u003csub\u003e2\u003c/sub\u003eF\u003csub\u003e2\u003c/sub\u003e. Carotenoids function as key photoprotective pigments, dissipating excess excitation energy and quenching reactive oxygen species (ROS). Increased carotenoid levels, therefore, suggest that these immersion\u0026ndash;photoperiod combinations established a more favorable light environment, minimizing photooxidative damage. Carotenoid biosynthesis is strongly light-regulated, with photoreceptors and light-responsive transcription factors activating key biosynthetic genes when plants are exposed to appropriate light conditions. Together, evidence from molecular studies and in vitro orchids shows that balanced light quality and intensity enhance carotenoid accumulation, improving photoprotection and reducing photooxidative stress (Pizarro \u0026amp; Stange, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Shin et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Toledo-Ortiz et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConversely, prolonged photoperiods (20\u0026ndash;24 h) led to sharp declines in both chlorophyll and carotenoid content. Excessive or continuous illumination is well known to induce photoinhibition, disrupt thylakoid structure, inhibit chlorophyll biosynthesis, and accelerate pigment degradation (Powles, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Shade-adapted species, including many orchids, are susceptible to high light loads during in vitro culture (Benkov et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Kitao et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Under such conditions, chlorophyll degradation often surpasses synthesis, and carotenoids are rapidly consumed as antioxidants, leading to the low pigment values observed in P\u003csub\u003e3\u003c/sub\u003e and P\u003csub\u003e4\u003c/sub\u003e treatments. The \u0026ldquo;not detected\u0026rdquo; carotenoid value in P4F1 likely indicates severe photobleaching and ROS accumulation, a phenomenon frequently reported when light exposure exceeds the physiological tolerance of in-vitro plantlets.\u003c/p\u003e \u003cp\u003eThese findings underscore the importance of balancing light duration with immersion dynamics. Photoperiod serves as a primary regulator of pigment metabolism and chloroplast development (Batista et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), while immersion frequency modulates hydration, nutrient exposure, and gaseous exchange within TIS. The superior performance of P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e (12 h light, 6 immersions/day) demonstrates that synchronizing moderate light exposure with optimal immersion cycles creates conditions that maximize plastid differentiation and pigment biosynthesis in \u003cem\u003eC. pandurata\u003c/em\u003e. The combined evidence suggests that optimizing both photoperiod and immersion frequency is essential for enhancing pigment quality in orchids propagated through temporary immersion systems. This insight is valuable for refining in vitro protocols aimed at producing physiologically robust plantlets with strong photosynthetic capacity.\u003c/p\u003e \u003cp\u003eDifferences in stomatal density across treatments suggest that photoperiod and immersion frequency influence epidermal development under in vitro conditions. Higher densities observed in treatments such as P3F2 and P1F3 indicate that extended illumination combined with appropriate immersion frequency may promote epidermal cell division and stomatal initiation. These observations are consistent with previous reports indicating that prolonged light exposure and water availability can modulate cellular and hormonal pathways associated with stomatal development, including transcriptional and signaling networks involving SPCH, auxin activity, and light-responsive factors (J.-H. Lee et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; S. B. Lee et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; MacAlister et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). In contrast, the consistently lower stomatal density observed under P2 treatments may reflect suboptimal morphogenic signaling under intermediate photoperiods.\u003c/p\u003e \u003cp\u003eThe similarity between morning and afternoon density values confirms that stomatal density is an anatomical trait fixed during leaf expansion. Minor morning\u0026ndash;afternoon differences are more likely due to variations in leaf hydration and turgor affecting visibility during microscopy rather than true physiological changes. This understanding is consistent with findings highlighting stomatal density as a stable trait shaped by genetic and developmental factors rather than short-term physiological adjustments (Liu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Loranger \u0026amp; Shipley, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Tian et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Thus, treatment effects\u0026mdash;not diurnal patterns\u0026mdash;are the primary drivers of the observed differences.\u003c/p\u003e \u003cp\u003eStomatal size responses provide an additional layer of insight. Treatments producing higher stomatal density often exhibited smaller stomata, reflecting a classic inverse relationship well documented in developmental plant physiology (Franks \u0026amp; Beerling, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gj et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). A high density of small stomata is often associated with faster pore dynamics and improved responsiveness under fluctuating environments\u0026mdash;an advantageous anatomical configuration for plants transitioning from in vitro to ex vitro conditions. Conversely, treatments such as P\u003csub\u003e1\u003c/sub\u003eF\u003csub\u003e1\u003c/sub\u003e and P2F2, which produced larger stomata despite moderate densities, may reflect altered water relations or partial hyperhydricity, conditions known to cause enlarged guard cells due to excessive tissue hydration (Debergh et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1992\u003c/span\u003e; Kevers et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). Overall, the combined patterns of stomatal density and size highlight that optimizing photoperiod and immersion frequency in Temporary Immersion Systems not only affects shoot vigor but also shapes leaf anatomical traits crucial for acclimatization. Treatments such as P3F2, which produced high densities of moderately sized stomata, may support more efficient gas exchange and better water-use control. Combined evidence shows that photoperiod and immersion frequency in Temporary Immersion Systems (TIS) shape stomatal density\u0026ndash;size patterns that influence shoot vigor and leaf anatomical traits essential for acclimatization, as demonstrated by studies reporting that the SETIS\u0026trade; bioreactor increases shoot proliferation, stomatal index, and stomatal closure (Ram\u0026iacute;rez-Mosqueda \u0026amp; Bello-Bello, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and that modulation of immersion frequency enhances stomatal index, chlorophyll content, and shoot survival\u0026mdash;thereby improving gas exchange efficiency and water-use regulation critical for post-culture establishment (Mancilla-\u0026Aacute;lvarez et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e"},{"header":"Limitations of the Study","content":"\u003cp\u003eThis study was conducted under controlled in vitro conditions using a single orchid species (\u003cem\u003eCoelogyne pandurata\u003c/em\u003e) and a culture duration of 30 days. Therefore, the observed effects of photoperiod and immersion frequency primarily reflect short-term responses during early growth stages and may not directly represent long-term development or ex vitro acclimatization performance.\u003c/p\u003e \u003cp\u003eIn addition, the Temporary Immersion System and IoT configuration evaluated in this study were implemented at a laboratory scale. Although the system demonstrated stable environmental regulation and reduced contamination under the tested conditions, further investigation is required to assess its robustness, economic feasibility, and scalability for commercial or large-scale conservation applications.\u003c/p\u003e \u003cp\u003eFinally, the present study focused on morphological, physiological, and anatomical parameters without evaluating post-culture survival or field establishment. Future research integrating acclimatization trials and extended culture periods would be valuable to confirm the broader applicability of the proposed system.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe integration of Internet of Things (IoT) technology into a Temporary Immersion System (TIS) improved the control of environmental parameters and supported consistent in vitro growth of the endangered black orchid, \u003cem\u003eCoelogyne pandurata\u003c/em\u003e. The IoT-controlled TIS enabled precise regulation of photoperiod and immersion cycles and was associated with a reduction in contamination levels from 100% to approximately 10% under the modified sterilization and equipment conditions applied in this study.\u003c/p\u003e \u003cp\u003eAmong the treatments tested, continuous illumination (24 h light) resulted in higher shoot multiplication, whereas a moderate immersion frequency (four times per day) provided a favorable balance between biomass accumulation, chlorophyll content, and reduced hyperhydricity. In contrast, shorter photoperiods promoted greater root elongation, indicating differential photomorphogenic responses between shoot and root tissues. Multivariate analysis further indicated that photoperiod and immersion frequency jointly influenced overall plantlet vigor and growth uniformity under the tested in vitro conditions.\u003c/p\u003e \u003cp\u003eOverall, the results suggest that an IoT-integrated TIS can provide a controlled and automated platform for optimizing in vitro propagation conditions of \u003cem\u003eC. pandurata\u003c/em\u003e at the laboratory scale. This approach shows potential for supporting ex situ conservation efforts of endangered orchids, although further studies are required to evaluate long-term performance, acclimatization success, and scalability beyond controlled in vitro environments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e7. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Acknowledgments \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;8. \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Compliance and Ethical Statements\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e8.1 Ethics Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve human participants, human data, or animal subjects. All experimental procedures were conducted on plant materials (\u003cem\u003eC. pandurata\u003c/em\u003e) in accordance with the institution\u0026apos;s biosafety and ethical guidelines. This study complied with the national and international standards for the ethical use of plant materials in scientific research.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e8.2 Data Availability Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e8.3 Conflict of Interest Statement\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing financial interests or personal relationships that could have influenced the work reported in this paper. Collaboration with Nabila Cultura Laboratory was limited to equipment modification and did not influence the study design, data analysis, or interpretation.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAmini Khoeyi, Z., Seyfabadi, J., \u0026amp; Ramezanpour, Z. (2012). 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Light intensity affects chlorophyll synthesis during greening process by metabolite signal from mitochondrial alternative oxidase in \u003cem\u003eA rabidopsis\u003c/em\u003e. \u003cem\u003ePlant, Cell \u0026amp; Environment\u003c/em\u003e, \u003cem\u003e39\u003c/em\u003e(1), 12\u0026ndash;25. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/pce.12438\u003c/span\u003e\u003cspan address=\"10.1111/pce.12438\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-the-saudi-society-of-agricultural-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of the Saudi Society of Agricultural Sciences](https://link.springer.com/journal/44447)","snPcode":"44447","submissionUrl":"https://submission.springernature.com/new-submission/44447/3","title":"Journal of the Saudi Society of Agricultural Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Coelogyne pandurata, Temporary Immersion System, Internet of Things, Micropropagation, Photoperiod, Immersion frequency","lastPublishedDoi":"10.21203/rs.3.rs-8251105/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8251105/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe integration of Internet of Things (IoT) technology into a Temporary Immersion System (TIS) was investigated for the micropropagation of the endangered black orchid, Coelogyne pandurata. The study aimed to optimize the photoperiod and immersion frequency for enhanced growth and reduced contamination. Plantlets were subjected to four photoperiods (12, 16, 20, and 24 h light) and three immersion frequencies (4, 6, and 8 times/day for 2 min each). The IoT-controlled TIS effectively regulated environmental parameters, reducing contamination from 100% to 10%. Continuous illumination (24 h) significantly increased shoot multiplication, while moderate immersion (4 times/day) produced the best balance of biomass accumulation, chlorophyll content, and minimal hyperhydricity. Shorter photoperiods favored root elongation, indicating distinct photomorphogenic responses between shoot and root tissues. Principal Component Analysis confirmed the joint influence of photoperiod and immersion frequency on plantlet vigor, contributing to uniform and reproducible growth. Pigment analysis revealed that moderate photoperiods (12\u0026ndash;16 h) and immersion frequencies enhanced chlorophyll and carotenoid accumulation. Stomatal density and size were also affected, with extended illumination and frequent immersion promoting higher densities of smaller stomata. The IoT-integrated TIS offers an efficient, scalable, and automated platform for the conservation and large-scale propagation of C. pandurata, demonstrating the potential of smart biotechnological systems to support sustainable ex situ conservation of endangered orchids.\u003c/p\u003e","manuscriptTitle":"Enhancing Growth and Reducing Contamination in Coelogyne pandurata Culture: A Smart IoT-Controlled Temporary Immersion System","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 08:51:28","doi":"10.21203/rs.3.rs-8251105/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-09T09:42:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-03T18:12:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"6315092619608348514561188059290002517","date":"2026-02-27T07:49:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"71615434174304330847453455438770333330","date":"2026-02-27T07:30:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-12T20:06:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"50999804048509463924008501421673533147","date":"2026-02-05T12:12:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-05T08:46:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T05:45:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of the Saudi Society of Agricultural Sciences","date":"2026-01-21T04:23:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-the-saudi-society-of-agricultural-sciences","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Journal of the Saudi Society of Agricultural Sciences](https://link.springer.com/journal/44447)","snPcode":"44447","submissionUrl":"https://submission.springernature.com/new-submission/44447/3","title":"Journal of the Saudi Society of Agricultural Sciences","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Open","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"748db458-4348-450c-a35b-e694a7cb0271","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T16:09:57+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 08:51:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8251105","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8251105","identity":"rs-8251105","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}