Introduction

Lippia alba (Mill.) N. E. Brown (Verbenaceae) is a small shrub native to South America. This species produces essential oils with analgesic, anti-inflammatory, anticonvulsant, antifungal, and myorelaxant properties (Aguiar et al. 2008; Hennebelle et al. 2008; Carmona et al. 2013; Oliveira et al. 2014). L. alba produces essential oils at different stages of leaf development and can be categorized into different chemotypes named after the major component identified, such as the monoterpenes linalool, citral, and carvone (Pandeló et al. 2012).

Recent studies have shown how different portions of the visible region of electromagnetic radiation affect several metabolic pathways in plants. Blue light (450–495 nm), red (620–750 nm), far-red (750–850 nm), and even green light (495–570 nm) play specific roles in plant morphogenesis and its regulation (Golovatskaya and Karnachuk 2015; Wang et al. 2015; Zienkiewicz et al. 2015). In vitro culture is a helpful tool to better understand these relationships because of the relative ease in manipulating light quality, irradiance, and photoperiod (Sáez et al. 2013).

Studies in several species have shown that in vitro development and metabolic pathways can be manipulated by changing light quality. Spectral quality also affects leaf anatomy, with greatest effects during the leaf blade expansion process (Sims and Pearcy 1992; Saebo et al. 1995; Schuerger et al. 1997).

The use of light-emitting diode (LED) lamps in plant tissue culture seems to be advantageous over the use of classic fluorescent tubes, since LEDs can more efficiently provide light in particular points of the light spectrum (Gupta and Jatothu 2013). Because of this, in recent years LEDs have been the predominant light source used in growth chambers and bioreactors to improve in vitro plant development (Yeh and Chung 2009). In addition, LED panels with controlled spectral peaks can provide specific spectral patterns leading to desired physiological responses (Gupta and Jatothu 2013).

Plants of chrysanthemum (Dendranthema grandiflorum Kitam. ‘Cheonsu’), for example, grown in vitro under red and far-red LEDs had greater stem elongation than those grown under white fluorescent lamps (Kim et al. 2004). However, the blue component interacts with the red and far-red components, inhibiting the formation of roots (Kurilčik et al. 2008). A mixture of blue, red, and far-red light allowed greater elongation of internodes in salvia (Salvia splendens cv. Red Vista) and marigold (Tagetes erecta L. cv. Orange Boy) (Heo et al. 2006). In strawberry, the best growth rate of plantlets was achieved with a balance of 70 % red and 30 % blue LEDs (Nhut et al. 2003).

Spectral quality of light also regulates secondary metabolism. Changes in light quality and quantity in yarrow (Achillea millefolium L.) led to variation in the number, content, and profile of volatile constituents (Alvarenga et al. 2015). The accumulation of anthocyanin was enhanced in Perilla frutescens var. acuta Kudo with an 8:1:1 mixture of red, blue, and white LEDs (Park et al. 2013). Calluses of Stevia rebaudiana, cultured under green and red lights, exhibited increased reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH)-radical scavenging activity than those cultured under white light (Ahmad et al. 2016).

Although previous studies have shown the influence of light quality on plant physiology and morphology, certain effects are difficult to quantify because they are species specific. Thus, spectral-dependent plant responses should be analyzed for each species of interest (Massa et al. 2008; Poudel et al. 2008).

In a review of the literature, no data were found on the effect of light quality on growth and essential oil production in the genus Lippia. This is of special interest in L. alba because of its economic importance. The present study describes how the quality of the light spectrum affects growth and the qualitative production of essential oils in three L. alba chemotypes.

Materials and methods

Plant material

Three chemotypes of L. alba (Table 1) were obtained from the Department of Biology, Federal University of Juiz de Fora (UFJF, Juiz de Fora, MG, Brazil), and from Embrapa Genetic Resources and Biotechnology (Cenargen, Brasília, DF, Brazil). In vitro plantlets were subcultivated on a monthly basis, on MS-based medium devoid of growth regulators, in the Plant Tissue Culture Laboratory at the Institute of Applied Biotechnology for Agriculture (BIOAGRO, Federal University of Viçosa, MG, Brazil).

Table 1 Ploidy levels and major components of essential oils of Lippia alba chemotypes used in the experiment
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Light quality effect

Hypocotyl segments (1-cm length) were transferred to 500-mL glass flasks closed with polypropylene lids with two 10 mm-orifices each covered with a Fluoropore hydrophobic membrane with 0.45-μm-diameter pore size (PTFE; MilliSeal® AVS-045 Air Vent, Tokyo, Japan). The explants were inoculated onto a culture medium containing MS salts and vitamins (Murashige and Skoog 1962), 30 g L−1 sucrose, 100 mg L−1 myo-inositol (Sigma-Aldrich® Co, St. Louis, MO), and 6.5 g L−1 granulated agar (Merck®, Darmstadt, Germany). The pH was adjusted to 5.70 ± 0.01, and the medium was sterilized by autoclaving at 121°C and 108 kPa for 20 min. These flasks were kept in a growth room environment at 25°C and a 16-h photoperiod.

In order to compare the effects of light quality on oil production and other plant characteristics, each of the three chemotypes was tested with three different light sources—two fluorescent bulbs (HO Sylvania T12, 110 W, São Paulo, Brazil), two white LED bulbs (SMD 100, 18 W, Vilux®, Vitória, ES, Brazil), and two blue/red LED bulbs (LabPAR LL-HR/DB-480, 11.6 W) (LabLumens®, Carapicuíba, SP, Brazil)—for a total of nine treatments (Table 2). The irradiance was standardized at 41 μmol m−2 s−1 via a light meter (LI-250A, LI-COR® Inc., Lincoln, NE) and the absorption spectra at the bench were recorded over a wavelength range of 200 to 800 nm with a spectroradiometer and Ocean Optics Spectra-Suite data acquisition software system (Ocean Optics®, Dunedin, FL) (Fig. 1).

Table 2 Lippia alba chemotype—light source combinations used in the experiment
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Fig. 1
figure 1

Light spectra of the three treatments evaluated in this study: (A) fluorescent lamps (HO Sylvania T12, 110 W), (B) white LEDs (Vilux® SMD 100, 18 W), (C) blue/red LEDs (LabPAR LL-HR/DB-480, 11.6 W).

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After 40 d of cultivation, qualitative analysis of essential oil profiles and quantitative analysis of growth parameters (plant height, fresh, and dry weight, total chlorophyll, and total carotenoids) were performed.

Extraction of essential oils

For each culture flask, all of the fresh leaves and stems were harvested and transferred to a 500-mL round-bottom flask, and 200 mL of water was added. Hydrodistillation was performed for 2 h using a modified Clevenger apparatus. The distillate was then collected and centrifuged at 1100×g for 5 min. The essential oil was removed with a Pasteur pipette, transferred to a glass bottle wrapped with aluminum foil, and stored at 4°C (Agência Nacional de Vigilância Sanitária 2010).

Qualitative analyses of essential oils

Qualitative analyses were carried out in a Shimadzu CG-17A gas chromatograph (Shimadzu Inc., Kyoto, Japan) coupled to a QP5000 mass spectrometer (GC-MS; Shimadzu Inc.), under the following operational conditions: DB5 fused silica capillary column; helium carrier gas; injector temperature 250°C; column temperature 50°C for 2 min, followed by an increase of 4°C min−1 until 200°C; initial column pressure 100.2 kPa; and split ratio 1:10. The injected sample volume was 1 μL (dichloromethane 1 % [v/v] solution). Under the same conditions as the samples, a series of standard hydrocarbons was injected (C9H20 … C26H54) (Mjos et al. 2006). The obtained spectra were compared with the data bank of the Wiley 229 mass spectral library using Kovat’s index, calculated for each component according to Adams (2007).

Statistical analysis

The experiments were carried out as a 3 × 3 factorial design (three chemotypes and three light conditions) with 12 replicates, each replicate composed of one flask with eight plantlets. Growth and physiological parameter data were submitted to analysis of variance followed by the Scott & Knott test (Scott and Knott 1974) at a significance level of 5 %. The experiments were performed twice to validate the data.

The essential oil data were subjected to multivariate analysis of variance (MANOVA) and canonical discriminant analysis (CDA), combining chemotypes and light qualities, in order to create linear transformations of the response variables (oil data) into successive canonical variables with maximal separation among those combinations (Yeater et al. 2015). A biplot graph was generated to assess multivariate differences among the combinations of chemotypes and light qualities and to examine the interrelationships among the variables and these combinations in a two-dimensional plane. The Candisc package (Friendly and Fox 2013) in the software R (R Core Team 2014) was used for the CDA.

Results

There was no significant effect of the tested light qualities on plant height, which differed among chemotypes. Plant height was greatest for BGEN-02 under all light conditions (Table 3).

Table 3 Growth and physiological parameters of Lippia alba in vitro cultures as affected by light condition and chemotype, after 40 d of cultivation
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The greatest mass accumulation (on both fresh- and dry weight bases) in BGEN-01 and BGEN-02 was achieved with blue/red LEDs, whereas for BGEN-42 the opposite was observed: greatest mass accumulation occurred with fluorescent tubes and white LEDs (Table 3). A high rate of leaf abscission was also observed in BGEN-42:B/R.

Chlorophyll and carotenoid contents were higher in BGEN-42 than in the other chemotypes under all light conditions. Comparing within BGEN-42 treatments, the highest levels of photosynthetic pigments were obtained with blue/red LEDs (Table 3).

The treatments were separated into groups using CDA analysis (Fig. 2). With the first canonical variable (Can1), nearly 85 % of the variability between treatments was explained. This variability was mainly due to differences in amounts of eucalyptol and linalool, which were negatively correlated with the levels of nerolidol, carvone, and alpha-bisabolol (Table 4). The BGEN-01:F treatment had the highest levels of eucalyptol and linalool and the lowest level of carvone.

Fig. 2
figure 2

Biplot containing the mean scores and 95 % confidence ellipses for three chemotypes of Lippia alba grown under different light conditions. Axes represent the first two canonical variables (Can1 and Can 2). Blue arrows and text show the original response variables (oil content) as related to the first two canonical variables.

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Table 4 Loadings of original variables (essential oil components) on the first two canonical variables for three chemotypes of Lippia alba grown under three different light conditions
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The second canonical variable (Can2) showed different behaviors. BGEN-01:F accumulated more benzyl alcohol, but less spathulenol, than BGEN-01:W and BGEN-01:B/R; BGEN-02:B/R and BGEN-02:W were similar, with values close to the overall mean; and BGEN-02:F, BGEN-42:B/R, BGEN-42:F, and BGEN-42:W showed the lowest levels of eucalyptol and linalool and the highest level of carvone.

Discussion

The present data showed that differences in light quality led to different growth responses among chemotypes of L. alba. A similar genotypic variation within the same species was also demonstrated by Kurepin et al. (2015), in which two ecotypes of Stellaria longipes responded differently to a low ratio of red to far-red light.

The higher mass and photosynthetic pigment levels in plants grown under blue/red LEDs support the fact that there is a greater use of light in these regions of the visible spectrum. Higher photosynthetic rates can be achieved when a leaf is illuminated with light in the red region (600–680 nm) than in others (Kalaji et al. 2014); these wavelengths stimulate PSII activity (Zienkiewicz et al. 2015). In addition, wavelengths in the blue range (around 480 nm) induce the strongest preferential excitation of PSII (Hogewoning et al. 2012). For BGEN-42, the blue/red LEDs did not increase the plant biomass. This can be explained by the high rate of leaf abscission that occurred in the BGEN-42 plants growing under blue and red light. Hoffman et al. (2015) also reported that red and blue LEDs modified the timing of some stress responses, such as leaf yellowing and chilling injury.

The effectiveness of combining blue and red light was also demonstrated by Lin et al. (2011), wherein either blue or red and blue LEDs significantly promoted the production of Dendrobium officinale shoots and increased the accumulation of shoot dry matter in vitro. Blue light during growth is required for normal photosynthetic functioning and mediates leaf responses (Hogewoning et al. 2012).

Photosynthetic pigment levels were higher under blue/red LEDs, which is in agreement with the results of biomass accumulation. It is generally acknowledged that a decrease in photosynthetic pigments is related to reduction in leaf photosynthesis and consequently leads to poor photosynthetic performance (Soussi et al. 1998; Braun et al. 2006). Similar results were observed for Dendranthema grandiflorum, in which net photosynthetic rate was consistent with plantlet growth, with the highest rate of plantlet growth being under red and blue LEDs (Kim et al. 2004).

In the present study, the different light qualities induced changes in essential oil patterns in L. alba, even changing the major constituents. In yarrow (Achillea millefolium), the amount and composition of the volatile compounds varied with light intensity and quality (Alvarenga et al. 2015). Yu et al. (2005) reported that the accumulation of ginsenoside was highest in cultures grown under fluorescent light, emphasizing how the responses of secondary metabolite production to light source are species dependent. However, in the present study of L. alba, genotype seemed to be a more important factor than light quality for explaining the effects of different treatments on essential oil profile. Viccini et al. (2014) demonstrated that these differences among chemotypes are correlated with plant DNA content. BGEN-01, BGEN-02, and BGEN-42 are respectively triploid, diploid, and hexaploid (Table 1), and these accessions with varied ploidy levels differed in essential oil profile as well as in distinct major components.

Conclusions

Alteration of light quality during the cultivation of L. alba led to changes in the qualitative pattern of essential oils. A deeper understanding of how this regulation works at the genetic or epigenetic level may enable production of essential oils of greater economic and industrial interest. As such, the present data open new possibilities to explore the production of essential oil components and also to elucidate the pathway of synthesis of those substances.