Optimizing the LED Light Spectrum for Enhanced Seed Germination of Lettuce cv. ‘Lollo Bionda’ in Controlled-Environment Agriculture

Abstract

Light is crucial in controlled-environment agriculture (CEA), affecting germination, growth, and overall plant quality. Here, we explored the optimization of various LED light spectra on the germination traits such as germination percentage, mean germination time, germination index, vigor index, and early seedling growth of ‘Lollo Bionda’ lettuce seedlings in a plant factory. A completely randomized design was implemented, involving three replications. LED lamps with different spectral compositions—red (R, peak at 656 nm), red/blue (3:1 ratio, R:B, peak at 656 nm), blue (B, peak at 450 nm), and white (400–700 nm)—were utilized in this study. The combination of red and blue LED lights, along with monochromatic red and blue treatments, significantly enhanced germination traits and early seedling growth compared to white and ambient lighting. The combined spectrum resulted in the highest seedling emergence, the longest shoot and root lengths, and the highest fresh weight. These findings underscore the potential of the LED technology to improve germination efficiency and enhance seedling quality in CEA. Future studies should refine multispectral LED strategies by examining factors such as light intensity and photoperiod, while also elucidating the molecular pathways involved in light-driven germination and early development in lettuce.

1. Introduction

The 21st century faces extraordinary environmental challenges, particularly the threats of global warming and climate change to ecosystems, economies, and human well-being. Increased greenhouse gas emissions have contributed to rising global temperatures, more frequent extreme weather events, higher sea levels, and disrupted ecosystems [1,2]. These changes are particularly alarming given the projected growth of the global population, which is expected to reach nearly 10 billion by 2050, with 70% residing in urban areas [2,3]. This demographic shift, evolving dietary preferences, and rapid urbanization will pressure agricultural systems to meet escalating food demands. The growing human population poses a significant challenge for future strategies for sustainable development worldwide and their long-term success [4]. Agriculture, while essential for food production, contributes to and is a victim of climate change. Concurrently, climate change contributes to rising global temperatures, increased atmospheric CO₂ concentrations, and more frequent extreme weather events such as heat waves—factors that profoundly affect horticultural crops. Furthermore, shifting temperature and humidity patterns are intensifying abiotic disorders in horticulture. Elevated temperatures can accelerate plant development and trigger premature ripening, potentially reducing the nutritional quality of specialty crops through alterations in sugar, acid, and antioxidant levels. Thus, climate-driven changes threaten yield and quality, emphasizing the urgent need for adaptive strategies to maintain resilient horticultural systems [5]. Studies have shown that the relationship between intensive greenhouse horticulture and climate change is bidirectional: while agricultural activities, such as fossil fuel consumption, contribute to climate change, the resulting climatic shifts, including droughts and heat waves, disrupt greenhouse growing conditions. These changes present serious challenges for protected cultivation systems, necessitating rapid adaptation. As a result, greenhouse vegetable producers face increasing pressure to implement sustainable practices that balance productivity with environmental stewardship [6]. Meanwhile, public awareness of global warming has increased significantly, making it a subject of widespread debate. Discussions often evoke strong opinions, with divergent interpretations of the causes and effects fueling polarized perspectives [7]. At the same time, climate change exacerbates water scarcity, soil degradation, and the spread of pests and diseases—factors that collectively undermine crop productivity and global food security [4]. Thus, sustainable agricultural practices and innovative solutions are necessary to ensure food security while mitigating environmental impacts [5].

Controlled-environment agriculture (CEA) has emerged as a promising approach to address challenges in sustainable food production [8]. Urban vertical farming exemplifies this innovation, using advanced technologies to optimize crop growth in climate-controlled indoor spaces. These systems lower the environmental impact, enable year-round yields, and reduce reliance on fossil fuels and long-distance transport [9]. Lettuce, a fast-growing leafy vegetable sensitive to environmental conditions, is particularly well-suited for vertical farming [10,11]. Precise control of light, temperature, and nutrients enables consistent, high-quality yields with lower resource input. Specifically, red and blue LED lighting enhances photosynthesis and morphological development, improving overall system efficiency. CEA reduces the risks of traditional open-field farming and enhances resource use efficiency, conserving water and land while improving yield and nutritional quality [12,13]. As urbanization increases the demand for fresh, local produce, CEA offers a sustainable solution by shortening food supply chains and ensuring reliable access to nutritious vegetables [14]. Balancing environmental parameters such as light, temperature, humidity, oxygen, and carbon dioxide is crucial for seed germination and early growth. However, the thresholds beyond which further optimization offers no added benefit remain unclear. Identifying these limits requires systematic, standardized, and scalable CEA studies across diverse crop species [15].

A critical stage in lettuce production is seed germination, which is heavily influenced by light conditions. Recent studies demonstrate that combining specific light wavelengths can enhance germination, underscoring the need for cultivar-specific light strategies in lettuce production [6,7]. Bączek-Kwinta and Michałek [16] reported that a high proportion of red light in the spectrum is essential for seed germination and seedling establishment. Research findings (e.g., [6,17,18]) provide a foundation for understanding how specific light conditions, including red/blue ratios and light intensity, influence early growth parameters such as hypocotyl length, leaf area, and root development. Additionally, insights from research on other crops, such as stevia [19], pea, melon [20], and pepper [21], further underscore the potential of the LED technology to enhance germination and seedling growth across diverse plant species. Compared to white LED light, colored LED light improves germination and development by promoting faster radicle protrusion, a higher germination speed index, longer total seedling length, and increased primary root length [22]. Studies also suggest that ‘Lollo Rosa’ and ‘Lollo Bionda’ lettuce cultivars exhibit better development, including germination period and number of leaves, under LED lighting compared to neon lights [23]. Additionally, a combination of red and blue LED lights plays a critical role in lettuce seedling production when optimal growing conditions are maintained in greenhouse environments [24].

Lettuce, a photoblastic plant, exhibits varying germination responses to light quality, intensity, and photoperiod [25,26]. Advances in the LED technology have revolutionized plant light research, allowing for precise control over spectral composition and developing tailored light recipes to optimize germination and early seedling growth [27]. Studies have demonstrated that specific wavelengths, such as red (660 nm) and far-red (730 nm) lights, play crucial roles in regulating seed dormancy, germination rates, and seedling vigor through phytochrome-mediated signaling pathways [28,29]. Blue light (450 nm) has also been shown to influence germination and early growth, though its effects are often cultivar-specific [28]. Temperatures above 30 °C may delay or inhibit germination in most commercial lettuce cultivars. Ethylene enhances lettuce seed germination at high temperatures [30]. Research showed that four days were required for the germination of ‘Lollo Bionda’ lettuce seeds, with a maximum germination rate of 93.5%, under greenhouse conditions with daytime temperatures of 21 °C and nighttime temperatures of 19 °C [31]. Due to high summer temperatures and limited electricity and water supply, greenhouses are often unsuitable for cultivating leafy vegetables such as lettuce in hot regions. As a result, the production of these crops is declining sharply in warmer parts of the world, even as demand for lettuce continues to rise. Given these challenges, adopting innovative methods to reduce water and energy consumption in leafy vegetable production is essential. Moreover, purchasing sprouted seedlings from other producers increases transportation losses and initial costs. To address this, every producer and greenhouse should be equipped to grow high-quality, long-lasting lettuce seedlings. By cultivating healthy seedlings in controlled growth chambers, producers worldwide (in tropical or cold regions) can significantly reduce costs while maintaining stable, year-round production for maximum profitability.

Increased germination rate and seedling production are critical stages in plant factories for cultivating fresh vegetables. Robust seedlings determine overall success, accelerated maturity, and efficient transplant readiness, ultimately enhancing productivity. However, despite growing evidence on the influence of specific light spectra on early plant development, a clear understanding of how distinct LED spectral compositions—particularly red, blue, and their combinations—affect germination efficiency, seedling vigor, and early growth of ‘Lollo Bionda’ lettuce under tightly controlled plant factory conditions remains limited. Moreover, the existing studies often lack comprehensive comparative analyses under standardized environmental parameters.

To address this gap, we investigated the effects of monochromatic and combined light spectra. We hypothesized that the combined red and blue LED treatment would result in superior germination performance and seedling vigor compared to monochromatic and white light treatments, due to the synergistic effects of red light on phytochrome activation and of blue light on cryptochrome-mediated processes. Refining LED lighting strategies further offers practical insights tailored to lettuce production, enhancing early-stage crop performance in sustainable indoor farming systems.

2. Materials and Methods

2.1. Plant Materials, Growth Conditions, and Experimental Setup

This experiment was conducted in the greenhouse of Vali-e-Asr University in 2024. Seeds of lettuce cv. ‘Lollo Bionda’ were purchased from Sepahan Rooyesh Co, Isfahan (Iran). The seeds were sown into the seed tray filled with a fine perlite medium.

2.2. LED Tubes and Light Treatments

In this study, lettuce plants were grown under 24 W LED lamps (supplied by Parto Roshd Novin Ltd. Co., Tehran, Iran) with varying spectral compositions: red (R, peak at 656 nm), red/blue (3:1 ratio, R:B, peak at 656 nm), blue (B, peak at 450 nm), and white (400–700 nm). The LED lamps were positioned 30 cm above the plant canopy to ensure consistent and optimal photosynthetically active radiation (PAR) of 215 ± 5.5 μmol m⁻² s⁻¹ for all experimental groups. A 16 h light and 8 h dark photoperiod was maintained to simulate ideal growing conditions. The cultivation layers were fully enclosed with reflective aluminum foil to eliminate external light contamination and maintain a controlled environment. The growth chamber’s relative humidity was carefully regulated at 75 ± 5%, and a ventilation system was implemented to manage CO₂ levels and prevent excessive heat buildup. Blower–suction fans were strategically placed on both sides of the chamber to generate a horizontal airflow of 0.3–0.5 m/s across the plant surfaces. This airflow facilitated efficient CO₂ and humidity distribution at the stomata and enhanced the overall photosynthetic efficiency, transpiration rates, and plant growth. To further optimize the experimental setup, temperature and CO₂ levels were continuously monitored, ensuring stable conditions throughout the study. The spectral characteristics and technical details of the LEDs used in this experiment are comprehensively outlined in

Table 1. Characteristics of the LEDs used in this experiment.

Manufacturer	CRI (Color Rendering Index)	No. of LEDs	Light Coverage Area	Power Consumption	Lens Type	Input Voltage	DC Voltage	Output Current	Output Frequency
Iran Grow Light	90%	24	40 × 100 cm	24 × 3 W	90°	AC 100–260 V	54–84 V	600 mA ± 5%	50/60 Hz

and Figure 1 and Figure 2, providing an appropriate reference for the light conditions applied.

2.3. Measurement of Germination Parameters

After sowing lettuce seeds in the seed tray, seed germination parameters were examined over 15 days, and the method for calculating each parameter is presented below.

2.3.1. Germination Percentage (GP)

GP = (Number of germinated seeds/Total number of seeds) × 100

2.3.2. Mean Germination Time (MGT)

$$\mathrm{M}\mathrm{G}\mathrm{T}={\displaystyle \frac{\sum (\mathrm{n}\times \mathrm{t})}{\sum \mathrm{n}}}$$

where n = number of seeds germinated on day t.

2.3.3. Germination Index (GI)

$$\mathrm{GI}=\sum \left({\displaystyle \frac{\mathrm{G}\mathrm{t}}{\mathrm{T}\mathrm{t}}}\right)$$

where Gt = number of seeds germinated on day t, and Tt = number of days since sowing.

2.3.4. Seedling Vigor Assessment

Root and shoot length: the length of the radicle (root) and the hypocotyl (shoot) was measured after 15 days.

2.3.5. Vigor Index (VI)

VI = Germination Percentage × (Root Length + Shoot Length)

2.3.6. Seedling Emergence

Three replicates of 100 seeds per treatment were placed in 200-cell trays filled with perlite as the growing medium. The trays were kept in a plant factory set to ~24 ± 1 °C. Emergence percentages were noted at 8 and 15 days after planting, where emergence was characterized by the cotyledons breaking through the perlite. Additionally, seedlings with fully developed cotyledons were counted. The proportion of healthy seedlings was assessed 20 days post-planting, classified as viable plants [32].

2.3.7. Shoot and Root Length and Fresh Mass

The length of the lettuce seedlings was measured using a ruler, while the fresh weight of the shoots and roots was recorded with a scale accurate to 0.0001 g.

2.4. Statistical Analysis

This study followed a completely randomized design with three replications. Data analysis was conducted using SAS software version 9.4 (SAS Institute, Cary, NC, USA). A two-way ANOVA was conducted for the statistical analysis, with Duncan’s multiple range test serving as a post hoc assessment to pinpoint significant differences between the group means. Differences were considered significant at p ≤ 0.05. Multivariate analyses of variance and heatmap diagrams were executed using (XLSTAT PREMIUM 2022 software (Addinsoft, New York, NY, USA). The results were presented as the mean values ± standard errors (SE) of the means. A correlation plot and a biplot of the principal component analysis (PCA) were drawn with Origin Pro software version 2024.

3. Results

3.1. Effect of the LED Light Spectrum on Germination Traits

The present study showed that light positively affected the germination characteristics of lettuce seeds of cv. ‘Lollo Bionda’ using a combination of red and blue lights to improve the germination characteristics. In addition, the highest GP (Figure 3A) and GI (Figure 3B) were observed for the combination of red and blue LED lights. Red and blue lighting also increased the germination characteristics of lettuce seeds compared to white light. The lowest mean germination time (Figure 3C) was also observed for the combination of red and blue light treatment, but seeds treated with white light germinated for a longer time.

3.2. Vigor Index

The vigor index in this study was affected by different LED light spectra. The results also indicated that on the 7th and 10th days, using a combination of red and blue lights caused a significant increase in the vigor index. Still, no significant difference was observed between red and blue lights alone, and the lowest vigor index was observed in the control treatment (white light) (Figure 4A).

3.3. Percentage of Emerged Seedlings

Different LED light spectra affected the percentage of emerged seedlings. In this study, the highest number of emerged seedlings was observed in the combination of red and blue light treatments, and there was no significant difference between red and monochromatic blue lights for this trait (Figure 4B).

3.4. Shoot and Root Lengths

Shoot and root lengths were significantly influenced by LED light spectra, such that the combination of red and blue lights, and monochromatic blue and red lights increased the shoot length compared to the white light treatment. However, the root length showed the most significant increase in the combination of red and blue light treatments. Still, the other light treatments did not have a statistically significant difference from each other (Figure 5A,B).

3.5. Fresh Weights of Shoots and Roots

Different LED light spectra affected the fresh weight of the shoots and roots. Combining red and blue lights and monochromatic red and blue light treatments increased the fresh weight of lettuce seedlings’ shoots and roots (Figure 6A,B).

3.6. Correlation, Heatmap, and Multivariate Analyses

The correlation plot (Figure 7) shows the correlation between germination and early growth parameters of lettuce cv. ‘Lollo Bionda’ under different LED light spectrum treatments. The germination and early growth traits were significantly correlated, such that increasing germination traits corresponded to enhanced early growth in lettuce seedlings under LED light treatment conditions in the controlled environment of the plant factory.

The heatmap illustrates the distribution of measured variables across treatments using a color gradient—from red (low values) to green (high values). Among the parameters, GP, VI, emerged seedlings, wet weight of shoots, wet weight of roots, and shoot length proved to be the most responsive indicators of treatment effects in lettuce under LED lighting. Furthermore, the heatmap effectively grouped the treatments based on their influence on plant traits, yielding two distinct clusters for the employed treatments. As shown in Figure 5, the treatments with almost white, red, and blue LED lights were clustered together, while a combination of red and blue LED lights formed another group (Figure 8).

Principal component analysis (PCA) was applied to explore the multidimensional relationships among the measured traits. This technique reduced the complexity of the dataset by summarizing variation in nine parameters across the four LED light treatments. In the resulting ordination plot, the length of each vector indicates the relative contribution of a given variable to the principal components. At the same time, its orientation reflects the direction and strength of association within the PCA axes. The corresponding values were 100% under LED light treatments (Figure 9).

4. Discussion

4.1. Seed Germination Traits

This study highlights the strong influence of red and blue LED spectra—both monochromatic and combined—on enhancing seed germination traits in ‘Lollo Bionda’ lettuce. The results align with previous research demonstrating that red and blue light are critical for plant development, as they align with the peak absorption ranges of the key photoreceptors such as phototropin, phytochromes, and cryptochromes [33]. In addition, our findings provide novel evidence under standardized plant factory conditions, most likely due to the synergistic stimulation of enzymatic activity and gene expression involved in early metabolic processes, suggesting a refined light strategy for improving germination efficiency in CEA [11,32].

Red light has been shown to improve seed germination and promote plant growth regulators, leading to increased cell division, elongation, and overall development [34,35]. Similarly, blue light enhances photosynthesis and biomass production, supporting plant growth [36]. In this study, the combination of red and blue lights significantly boosted germination traits and early growth of lettuce seedlings (Figure 3A–C), with notable improvements in root architecture, particularly in root hair volume. Root hairs are crucial for water and nutrient absorption, directly influencing seedling growth rates [37]. The observed increase in shoot and root length under red, blue, and their monochromatic light treatments (Figure 5A,B) is consistent with findings in other crops, such as pea and melon, where red light exposure led to increased fresh weight and root and shoot elongation [20]. These findings suggest that combining red and blue lights effectively fosters strong seedling development, offering a promising method for optimizing growth in controlled environments, such as plant factories.

The role of phytochromes in mediating responses to red light is well-documented. Phytochromes, particularly PhyB, are central in regulating seed germination under red and far-red light conditions [38]. Red light induces the conversion of phytochromes to their active form (Pfr), enhancing seed germination by regulating the balance of gibberellins (GA) and abscisic acid (ABA), which are the key hormones in germination control [39]. In contrast, far-red light can reverse this process, converting Pfr back to its inactive form (Pr). This dynamic interplay between red and far-red light underscores the importance of optimizing light spectra to maximize germination efficiency. Research has shown that phytochrome A (phyA) plays a key role in mediating seed germination responses under blue light, with both wild-type and phyA mutant lines exhibiting reduced germination rates under these conditions [40]. Interestingly, the same study reported that blue light triggered a similar germination response in phyB mutants as white light did in wild-type, phyA, and phyB backgrounds, suggesting a prominent regulatory function of phyA under blue light exposure. In contrast, red light was observed to stimulate germination primarily through the activation of phytochromes that enhance gibberellin biosynthesis, a hormone known to facilitate the germination process [41]. Critical research found that the lowest mean germination time, highest germination rate, most extended radicle length, hypocotyl length, number of lateral roots, and fresh weight of radicles were observed under red LED light in motherwort. Still, a combination of red and blue resulted in the largest shoot and root weight [42].

4.2. Average Time for Germination

The mean germination time is a key indicator of seed vigor, with shorter values reflecting faster emergence and higher vigor [43]. In this study, the red and blue light combination produced the shortest MGT, indicating enhanced vigor and more rapid germination. This aligns with previous findings showing that high-vigor seeds germinate quickly and yield more uniform seedlings [44]. The elevated vigor index under red and blue lights further supports the positive impact of these spectra on seed quality and performance.

4.3. Seed Vigor

Seed vigor is a multifaceted trait influenced by genetic, environmental, and maternal factors [45]. It encompasses germination rate, uniformity, and the ability to withstand unfavorable conditions, all critical for successful crop establishment [46]. In this study, the red and blue light combination improved germination rates and enhanced seedling emergence and uniformity (Figure 3A), which are essential for optimizing stand establishment in direct-seeded crops [47]. The higher rate of seedling emergence under red and blue light treatments (Figure 4B) is attributed to enhanced photosynthetic efficiency and improved coordination of photosynthetic pigments, as Rahman et al. [7] stated. These findings suggest that red and blue light treatments can significantly enhance early plant development, leading to better crop performance and increased marketable yields [48]. The significant increase in seedling emergence percentage, speed, and value under R light was observed in balsam (Impatiens balsamina), zinnia (Zinnia elegans), petunia (Petunia × hybrida), and verbena (Verbena aubletia) plants [49]. Red LED light can increase photosynthetic pigments, especially chlorophyll a and b, and carotenoids [50,51,52].

4.4. Early Growth

The blue LED light significantly enhanced the effect of red light on normal photosynthesis by mediating the activity of photosystems and the capacity for photosynthetic electron transport [53]. Meanwhile, red and blue lights have a higher quantum yield of CO₂ assimilation (QY, moles of CO₂ assimilated per mole of photons) than other light spectra [54]. In addition, it has been determined that monochromatic R light can reduce the CO₂ assimilation amount, and monochromatic blue light treatment causes lower chlorophyll concentration and net photosynthetic rate in the coriander plant [55]. Therefore, the combination of red and blue lights, considering the role of each in increasing photosynthetic pigmentation and photosynthetic electron transfer capacity, can significantly ensure increased growth and development of lettuce plants after germination under controlled conditions in a plant factory. The increase in chloroplasts in plants treated with a combination of red and blue lights likely contributes to the growth and photosynthetic activity in lettuce seedlings under controlled conditions, as noted by Lee et al. in their study on Ulmus pumila with red/blue LED treatment [56]. In this way, the energy needed for plant organs, including leaves and roots, is provided by increasing the light absorption by photosynthetic pigments and converting it into carbohydrates. In this way, plant growth will increase significantly under optimal LED light use. It is reported that a combination of red and blue can improve carbohydrate accumulation and pigment ratio in strawberry plants [57]. In addition, Zare et al. [58] indicated that a combination of red/blue and GA3 applications improved growth, photosynthetic capacity, and pigment contents in African violet.

Figure 7 reveals a tight coupling between germination traits and early seedling development in Lactuca sativa cv. ‘Lollo Bionda’ under distinct LED light regimens. Elevated GP, GI, and VI were strongly associated with increased shoot/root elongation (SL/RL) and fresh biomass (WS/WR), suggesting that robust germination underpins superior seedling establishment. This mirrors seed physiology principles, where rapid germination often precedes vigorous growth [59,60]. Conversely, prolonged MGT correlated inversely with seedling vigor and biomass accumulation, implying that delayed germination may hinder early photosynthetic activity—a critical factor for resource efficiency in CEA [61,62].

These findings underscore how light-mediated seed responses shape early developmental trajectories. Photoreceptor-driven signaling pathways (e.g., phytochromes and cryptochromes) likely modulate germination and seedling growth [63,64], with LED spectra fine-tuning physiological outcomes. The observed trait correlations imply that spectral optimization can trigger synergistic effects, such as enhanced chlorophyll production and photosynthetic performance, ultimately boosting biomass [65,66]. Consequently, precision lighting strategies are crucial for improving early vigor and yield in CEA [67], especially for leafy crops such as Lactuca sativa, where early-stage performance dictates marketability and nutrient density.

Figure 8 utilizes a heatmap to illustrate the spectral influences on germination and growth parameters. Treatments clustered into two dominant groups, reflecting shared physiological responses to specific light qualities, consistent with studies demonstrating spectrum-dependent plant responses [68,69]. Notably, white/red/blue LED blends elicited distinct effects compared to red–blue mixtures, highlighting divergent outcomes between polychromatic and narrow-bandwidth lighting. Parameters such as GP, VI, and SL exhibited pronounced treatment-dependent variation, reaffirming light quality as a key modulator of early growth.

Figure 9′s PCA biplot distills multivariate variability across LED spectra. The spatial segregation of monochromatic and red–blue combination treatments reinforces spectrum-specific physiological adaptations [65], while high loadings for VI and GP align with their established roles in vigor assessment [60]. By complementing correlation and heatmap analyses, PCA advances the precision of LED regimen design for CEA [67].

5. Conclusions

Unlike many previous studies, this work provides a direct comparison of monochromatic and combined spectra under standardized plant factory conditions, offering practical insights specific to lettuce cultivation. The combination of red and blue LED lights significantly enhanced seed germination traits, seedling vigor, and early growth in ‘Lollo Bionda’ lettuce. These spectra synergize the key physiological processes, promoting uniform and robust development. Future research should investigate specific parameters, including light intensity thresholds, photoperiod variations, and their interactions, to better understand long-term impacts on yield, quality, and the molecular mechanisms of light-mediated germination. Optimizing light conditions can improve crop performance and sustainability in CEA.