Introduction

Plants utilize multiple photoreceptors to perceive various wavelengths, which subsequently trigger physiological responses (^{Zhang et al., 2018}). Plants not only respond to light intensity but also to its duration and quality. These factors and their variations can continuously modify plants physiology, promoting the developed of different mechanisms for constant adjust to varying conditions (^{Zhang & Folta, 2012}).

In strawberry (Fragaria ananassa), propagation occurs through stolons, and seeds, depending on the purpose for which the seedlings are required (^{Ashrafuzzaman et al., 2013}; ^{Kang et al., 2020}; ^{Zheng, He et al., 2019}). Strawberry seeds are germinated in controlled conditions such as humidity, temperature, soil, and recently, artificial light is used to enhance the quality of seedlings formed (^{Mireles Arriaga et al., 2020}; ^{Tewolde et al., 2016}). The use of artificial light in seedling production deserves special attention due to the benefits obtained, including disease tolerance, greater vigor, and resistant to handling stress during transplantation (^{Misu et al., 2018}).

Previously utilized light sources included metal halide fluorescent lamps, high-pressure sodium lamps, and incandescent lamps, but Light Emitting Diodes (LEDs) offer several advantages (^{Samuolienė et al., 2010}). LEDs have been employed as an alternative source of artificial lighting due to their low energy consumption, spectral emission control, long durability, reduced size, less heat generation, high plant effectiveness, greater plants proximity, as well as emission of a specific wavelength to improve confined plant cultivation (^{Singh et al., 2015}).

The blue and red lights exert strong effects on plant growth and metabolism and have been utilized to regulate fruit and vegetable quality. Blue light is absorbed by phototropins and cryptochromes, which are involved in plant morphogenesis, stomatal opening, photosynthetic leaf function, high chlorophylls levels generation, leaf expansion, stem elongation, phototropism, transition to flowering. In Arabidopsis, proteins involved in phototropism include phot1, phot2, and PKS1 to PKS4, while cryptochromes are represented by proteins cry1 and cry2. Red light affects flowering initiation, leaf expansion, seed germination, and flowering induction; phytochromes, red, and far-red sensing pigments, are synthetized in an inactive form and convert to an active form after absorbing red light (^{Costa Galvão & Fankhauser, 2015}; ^{Naznin et al., 2016}; ^{Zhao et al., 2020}). Different combinations of wavelengths have varying beneficial effects on plant development, depend on each species (^{Olle & Viršile, 2013}).

The strawberry crop requires a lower amount of light compared to other horticultural crops such as tomato and melon (100-300 mmol m^-2 s^-1) (^{Yoshida et al. 2016}). Additionally, strawberry plants require less space and can be grow in multiple layers. In Mexico, the strawberry crop covered a planted area of 9,342 ha in 2020, with a total production of 425,007 t, of which 52 % was destined for exportation, mainly to the United States of America (^{Servicio de Información Agroalimentaria y Pesquera (SIAP), 2021}). Seedling production with artificial light treatments could be an alternative to generate vigorous plants. The aim of the present study was to evaluate strawberry seedlings formation under different intensities of violet, blue, and red LED light.

Materials and methods

Results

Morphological and physiological measurements subjected to ANOVA, and Tukey (0.05) tests were performed, as showing in Table 1. Highly significant differences were observed in germination percentage (GE) (p<0.01). Greater germination percentages were observed under higher intensities of all light treatments. However, the greatest average reduction of 19.2 % was identified under red light.

Highly significant differences were identified in root length (RL) (p<0.01), with blue and red lights at their lower intensities being the ones that mostly limited root growth. Compared to white light at its lower intensity, the reductions in RL under blue and red lights were 22.3 % and 33.1 %, respectively. A highly significant positive correlation of 0.83 (p<0.01) was identified between RL and GE.

Highly significant differences in steam length (SL) (p<0.01) were also identified. There was an increase in SL under blue (2.06 cm), red (2.08 cm), and even violet (2.02 cm) lights at their lower intensities.

Regarding hypocotyl diameter (HD), highly significant differences (p<0.01) were observed, with smaller diameters presented at lower intensities under violet, blue, and red lights, measuring 1.20 mm, 1.14 mm, and 1.02 mm, respectively. The greatest average reduction rate of 42.1 % was observed under red light. The respond of SL and HD indicated that low light intensity induced elongation and thinning as seedlings searched for a better light source, eventually leading to the constriction of conducting vessels. This was confirmed by the highly significant negative correlation of -0.80 (p<0.01) between both variables.

For biomass (BM) and chlorophyll area (CA), highly significant differences (p<0.01) were identified. Higher values were observed under light treatments at high intensities, except for red light and low-intensity blue light. Similar results were identified for BM and CA, which were confirmed by a highly significant positive correlation of 0.85 (p<0.01).

In the case of chlorophyll area (CA) (p<0.01), seedling under the violet light at high intensity presented the highest value of 25.9 mm², followed by white light at high, medium, and low intensities, with 23.3 mm², 23.0 mm², and 21.2 mm², respectively. Under blue light at high intensity, the CA was 22.9 mm²; which is noteworthy as this light treatment had lower energy consumption, an important characteristic for larger-scale applications.

For the Lab color space components (p<0.01), the highest luminosity (L) values were observed at the highest intensity levels of white, violet, blue, and red light, with values of 75.3, 75.2, 71.2, and 70.5, respectively. Cotyledons under blue and red lights were 7.7 % and 9.9 % less luminous, respectively. In the case of a (green/red), all values were negative, particularly under blue and red lights, where values became less negative as light intensity was decreased. Violet light exhibited the least negative values. In b (blue to yellow), the highest values were observed under white light, while under red light, there was a tendency for values to increase as light intensity decreased, with an average rate of 7.9 %. This indicated that red light caused cotyledons to turn yellow (etiolated) at higher rate.

Regarding chlorophyll total (Chlt) (p<0.01), the highest concentrations were observed under the highest intensities of white, violet, blue, and red lights, with values of 45.7, 44.9, 40.8, and 39.5 mg mL^-1, respectively. Each light treatment showed a reduction in Chlt as light intensity decreased, with blue and red lights exhibiting the greates reduction rates, averaging 41.6 % and 48.7 %, respectively. A highly significant positive correlation of 0.91 (p<0.01) was identified between Chlt and L.

Highly significant differences (p<0.01) were identified in chlorophyll a (Chla). The highest concentrations were determined under blue and red lights, with 28.1 and 27.0 mg mL^-1, respectively. However, under the same light treatments, the greatest reduction rates were observed as light intensity decreased, averaging 48.6 % and 40.3 %, respectively.

In the case of chlorophyll b (Chlb), highly significant differences were identified (p<0.01). The highest concentrations were determined under white and violet lights. The proportions of Chla and Chlb varied depending on the light treatments and their intensities (Figure 1). It should be noted that Chlb variation not only affected Chtl values but also the luminosity of the cotyledons area, as highly significant positive correlations were identified between Chlb and Chlt of 0.90 (p<0.01), and between Chlb and L of 0.91 (p<0.01).

Figure 1 <of chlorophyll a and  c (%) under a) violet, b) blue, c) red, and d) white lights in strawberry (Fragaria ananassa) cultivar San Andreas. Laboratory of Plant Genetic, Department of Agronomy, Universidad de Guanajuato, Mexico, 2020. 

In the determination of proline concentration (PR) (p<0.01), higher concentrations were observed at lower intensities of violet, blue, and red lights, measuring 3.81, 4.02, and 4.12 µg mL^-1, respectively. Notably, for red light at medium intensity, PR was 3.62 µg mL^-1, representing the highest proline concentration at this intensity level. Highly significant negative correlations (p<0.01) of PR with GE, RL, CA, L, BM, Chlt, and Chla were identified, with correlation coefficients of -0.78, -0.74, -0.86, -0.83, -0.95, -0.89, and -0.85, respectively.

Regarding antioxidant activity measurement, highly significant differences (p<0.01) were observed in DPPH and ABTS determinations (Table 2). The responses mirrored those seen in chlorophyll concentration, with lower remaining percentages of both radicals observed under medium-intensity red light compared to lower intensity. For DPPH, the values were 53.1 % and 77.1 %, while for ABTS, they were 38.0 % and 57.7 %, respectively. A highly significant positive correlation of 0.97 (p<0.01) was identified between DPPH and ABTS determinations.

Discussion

The results of the morphological variables exhibited statistical differences based on the type of light and its intensity. In terms of red light, our findings align with those of ^{Porras Mechán et al. (2020)}, who observed that seeds treated with red light displayed higher germination percentage, compared to those treated with blue light, as noted in Campomanesia lineatifolia. Similarly, ^{Paniagua-Pardo et al. (2015)} reported a 25 % increase in germination rates in Brassica oleracea seeds when exposed to high-intensity red light for 12 hours. Photoblastic seeds, which respond to light presence, exhibit enhanced germination, mediated by phytochrome, pigment, or photoreceptor that trigger gene expression changes (^{Demotes-Mainard et al., 2016}).

Contrary to the findings in the present study, ^{Simlat et al. (2016)} observed a 26.7 % increase in germination in Stevia rebaudiana Beroni seeds exposed to blue light. Interestingly, ^{Hinojosa-Dávalos et al. (2019)} found that broccoli seeds treated with blue light had higher germinations rates, whereas those treated with red light exhibited lower rates. Additionally, ^{Jacobsen et al. (2013)} reported a significant relationship between coleorhiza and root emergence in Triticum aestivum Sunstate seeds after exposure to bright light. Clearly, the wavelength of light plays a pivotal role in germination, albeit with species-specific effects.

The morphological findings in the present study echo those of ^{Spaninks et al. (2020)}, who investigated Arabidopsis thaliana seedlings and the tomato cultivar Moneymaker under red, blue, and white LED lights. Their results demonstrated that red and blue lights induced alterations in root growth, with blue light resulting in the shortest roots length compared to red and white lights. Moreover, ^{Simlat et al. (2016)} observed stimulated root elongation in stevia plants with under red light treatment compared to control (white light) at 20 °C, while at 25 °C, treatments of red, white, and white + red light showed no statistically significant differences.

Hypocotyl length responses to different intensities of blue and red light were examined in arugula and mustard plants, revealing those plants exposed to low light intensity displayed longer hypocotyl as part of their shade-avoidance response, without exhibiting etiolation. Similar experiments conducted with Arabidopsis and tomato seedlings grown in vitro under white, red, and blue lights demonstrated that the highest hypocotyl growth occurred under blue light (^{Spaninks et al., 2020}).

Regarding Cotyledon area and biomass, it appears that red light may not be suitable for obtaining strawberry seedling development. ^{Casierra-Posada et al. (2011)} evaluated strawberry stolons under different light conditions and found that the lowest biomass accumulation occurred with red light treatment. Conversely, ^{Zheng, Ji, et al. (2019)} observed biomass accumulation in strawberries under high-intensity light. A negative correlation of -0.650 (p>0.01) between photosynthesis rate and maximum efficiency of PSII was observed under a shade conditions, indicating that CO₂ assimilation may be affected by light deficiency, leading to reduce dry matter formation (^{Choi et al., 2016}).

The etiolation effect, characterized by hypocotyl elongation and lack of plastid pigmentation, occurs due to the absence light, as plastid development is only activated in the presence of light by phytochromes (^{Kusnetsov et al., 2020}). Gibberellic acid (GA) plays a crucial role in germination, cell elongation, hypocotyl elongation, and expression of GA2ox and GA3ox genes during germination in many plant species (^{Çayan et al., 2021}). Phytochromes perceive light information, including intensity and duration, and regulate processes such as germination, flowering, and senescence.

Germination processes are regulated by two key growth regulators: abscisic acid (ABA) and gibberellic acid (GA). ABA is associated with seed dormancy under unfavorable conditions, while GA promotes seed germination in favorable environments. However, upon emergence, cotyledons begin to open, iniciating chlorophyll biosynthesis, chloroplast development, and autotrophic growth (re-etiolation). Phytochromes play a crucial role in both etiolation and re-etiolation processes (^{Tripathi et al., 2019}). Cryptochromes mediate the blue light regulation of GA levels, influencing GA-induced photomorphogenesis and reducing hypocotyl elongation (^{Tsuchida-Mayama et al., 2010}; ^{Zhao et al., 2020}).

The color of cotyledons may have been altered due to the synthesis of different pigments in response to light treatments. This is supported by the highly significant differences (p<0.01) observed in the Chroma (C) and Hue (H) indices (Table 1), which were primarily influenced by variations in b. A highly significant negative correlation was identified between C and b (-0.77, p<0.01), while a highly significant positive correlation was found between H and b (0.98, p<0.01). Colorimetric analyses can be valuable for comparing color values under different conditions and observing changes in response to LED radiation, aiding in the development of indicators for assessing strawberry health status (^{Garcia-Noguera et al., 2014}).

In experiments with Rhafanus officinale plants exposed to red light, higher concentrations of Chlt, Chla and Chlb were reported compared to results obtained in the present study (^{AbdElgader et al., 2015}). Similarly, an experiment with Lycopersicum seedlings found higher Chlb content in red light treatment compared to white light. However, contrary to the present study results, these experiments reported higher Chlb concentration in white light than in red light. Additionally, the study found that monochromatic treatment stimulated seed germination at the beginning of the lighting treatment and resulted in taller tomato seedlings with less root biomass (^{Izzo et al., 2020}). In another study, strawberry plants treated with LED lighting exhibited inhibited flower bud initiation due to prolonged light exposure (^{Hidaka et al., 2014}).

Based on these findings, monochromatic red light appears unsuitable for the formation of strawberry, as indicated by the increase in proline concentration (PR), which serves as an indicator of various sources of abiotic stress such as light (^{Mireles Arriaga et al., 2020}). Significant differences (p<0.01) were observed in chlorophyll concentrations, with the most notable variations occurring under red light. Specifically, there was 14.9 % increase between high and medium intensities, followed by a 38.8 % reduction between the medium and low intensities.

The data suggests that under medium-intensity red light, the concentration of phenolic compounds increased in response to light stress, whereas under low intensity, seedlings were unable to maintain the antioxidant response. In contrast, under blue light, there was a 9.4 % increase in chlorophyll between high and medium intensities, with only a 1.6 % reduction under low intensity. This indicates that red light induced higher levels of light stress on the seedlings, while blue light even favored certain morphological and physiological characteristics.

Low light intensity adversely affects dry matter accumulation and reduces plant yield, as plants experience stress due to photon deficiency, resulting in abnormal growth patterns (^{Banerjee & Roychoudhury, 2016}). Conversely, excessive light intensity can also induce stress, leading to severe photoinactivation or photodamage on proteins in the photosystems, such as damage to the D1 protein (PSII) and the large rubisco subunit, as well as a decrease in PSI peptides. High levels of blue light can activate biochemical and physiological processes, resulting in the accumulation of flavonoids in leaves to acclimatize the plant to UV stress (^{Huché-Thélier et al., 2016}).

Studies have shown that the content of antioxidants, such a anthocyanin, in strawberry fruits can be influenced by exposure to different colored plastic films. For instance, ^{Miao et al. (2016)} reported higher antioxidant content in fruits exposed to white and red film. Similarly, ^{Manivannan et al. (2017)} found that in Dianthus caryophyllus plants, red light primarily increased the antioxidant activity of enzymes like superoxide dismutase, guaiac peroxidase, ascorbate peroxidase, and catalase. Additionally, ^{Zhang and Xie (2022)} demonstrated that Brassica rapa treated with red-violet LED light exhibited delayed postharvest senescence and maintained higher levels of antioxidant enzymes.

However, experiments involving blue-violet light has shown contrasting results. ^{Taulavuori et al. (2018)} observed low biomass accumulation in three species but noted a greater accumulation of secondary metabolites compared to the control (^{Taulavuori et al., 2018}). ^{Huang et al. (2020)} found that exposure of Vicia faba to water stress with violet illumination led to decreased activities of antioxidant enzymes compare to white light, except under high levels of violet light where the effect was similar to white light.

On the other hand, blue light has been shown to promote plant growth, increase nutrient absorption, and enhance crop quality. ^{Wang et al. (2018)} demonstrated that different light wavelengths can be used to control the physiology of crops, with blue-violet light promoting protein synthesis followed by carbohydrate synthesis underred-orange light. Similarly, ^{Liu et al. (2022)} reported that blue light not only promotes plant growth but also enhances nutrient uptake, yield, and overall crop quality (^{Liu, et al. 2022}).

Conclusion

Aside from white light, the most effective treatments for strawberry seedlings formation were violet light at high and medium intensities, along with blue light at high intensity. Regardless of treatment, the germination rate was 60 % under high light intensities; without these treatments, it averaged 51.9 %. Under these conditions, seedlings did not elongate or thin out, had cotyledons with the largest surface area, and exhibited the highest chlorophyll concentrations. The energy consumption of these treatments was, on average, 31.2 % lower than that of their corresponding white light treatments. In contrast, red light was unsuitable for seedling formation due to its high induction of light stress.