Growth regulators affect the dry weight production, carvacrol and thymol content of Lippia gracilis Schauer

Industrial Crops & Products 129 (2019) 35–44

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Growth regulators affect the dry weight production, carvacrol and thymol content of Lippia gracilis Schauer

T

Luiz Eduardo Santos Lazzarini, Suzan Kelly Vilela Bertolucci, Alexandre Alves de Carvalho, Alexsandro Carvalho Santiago, Fernanda Ventorim Pacheco, Maria Mariana Ferreira Célio, ⁎ José Eduardo Brasil Pereira Pinto Department of Agriculture, Federal University of Lavras, Lavras, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Alecrim de tabuleiro Auxins Cytokinins Micropropagation

The essential oil from Lippia gracilis Schauer (Verbenaceae) leaves are rich in thymol and carvacrol with antibactericidal and antinflammatory action. The aim of the present study was to evaluate the effect of growth regulators and their combinations on shoot proliferation, growth, and volatile fraction analysis of Lippia gracilis in vitro. In the first experiment, five different concentrations of 6-benzylaminopurine (BAP) were used: 0.0, 2.22, 3.33, 4.44, and 5.55 μM and three of naphthaleneacetic acid (NAA): 0.0, 2.68, and 5.36 μM, in a factorial design. In the second, the treatments consisted in combination of BAP + TDZ at concentration (μM): 0 + 0, 1.11 + 1.13, 1.33 + 0.91, 0.88 + 1.36, 0.43 + 1.81, and 1.78 + 0.46. At 30 days of culture in ½ Murashige and Skoog (MS) medium, the growth and volatile constituents were evaluated. Growth regulators significantly influenced in vitro growth of L. gracilis. Growth data for the first experiment were predicted from the three-dimensional (3D) response surface of the different variables analyzed. In the second experiment, the presence of growth regulators reduced shoots and root length in all combinations. The combinations among regulators (BAP and TDZ) stimulated the number of shoots per explant, but BAP and NAA induced higher shoot number. Variation in the number, content, and profile of volatile compounds were also observed under the influence of growth regulators. The major constituents ρ-cimene, γ-terpinene, thymol, carvacrol, and E-caryophyllene were identified, independent of the experimental conditions. However, the use of growth regulators significantly reduced carvacrol and thymol levels.

1. Introduction

molluscicide, larvicide, antinociceptive, and antinflammatory action (Ferraz et al., 2013). The propagation of L. gracilis by seed becomes impracticable due to the reduced size of seeds, precluding their collection and manipulation, and resulting in low rooting percentage of cuttings (Santos et al., 2016). Therefore, the use of in vitro propagation methods is an alternative. Moreover, this technique may allow standardizing the raw material in aromatic species from the micropropagation of clones with high essential oil content and levels of their major constituents. The totipotency of plant cells allows them being readily used for in vitro propagation or cell culture development (Davies and Deroles, 2014). In vitro propagation supplies a lot of plantlets, species preservation, and plants free from diseases (Silva et al., 2017). The use of plant growth regulators (PGRs) in the culture medium overcomes possible endogenous deficiencies and improves in vitro culture conditions. The concentrations and type of regulator added to the

The genus Lippia occurs mostly in Brazil and has more than 70% of all species already catalogued. A lot of these species are found in endemic regions of Brazil where these areas are considered in a risk extinction due to human action and present in low densities (Souza et al., 2017). Currently, this genus has approximately 200 species being approximately 120 species occurring in Brazil. Lippia gracilis Schauer (Verbenaceae), locally known as ‘alecrim de tabuleiro’, is a typical plant from the northeastern semiarid region, characterized by being a small, deciduous, branched shrub with a brittle stem, up to 2 m height and its propagation occurs by thinner stem cuttings (Lorenzi and Matos, 2002). It is used in traditional medicine for treatment of colds, coughs, sinusitis, bronchitis, and headache, besides used externally to treat skin infections, burns, wounds, and ulcers (dos Santos et al., 2016). The leaves from this species are rich in essential oil with antibactericidal,

⁎

Corresponding author. E-mail address: [email protected]fla.br (J.E.B. Pereira Pinto).

https://doi.org/10.1016/j.indcrop.2018.11.070 Received 11 October 2018; Received in revised form 28 November 2018; Accepted 30 November 2018 0926-6690/ © 2018 Elsevier B.V. All rights reserved.

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Fig. 1. Analysis of in vitro growth response of Lippia gracilis cultured in MS medium with half the salt concentration supplemented for 30 days with different doses of BAP x NAA: (A) Shoot Length (SL); (B) Root Length (RL); (C) Number of Shoots (NS).

cultured in vitro, better results of micropropagation rate and number of shoots were obtained in the absence of BAP and NAA, which formed calluses when added to the medium (de Oliveira et al., 2016). MS medium supplemented with 2 mg L−1 of BAP and 1 mg L−1 of TDZ enhanced results for induction of multiple shoots in Boucerosia umbellata cultured in vitro (Susheela et al., 2016). It has done measure of volatile compounds in vitro to provide information if the PGRs alter the composition. So the results of this study can be applied in vivo cultivation of this medicinal plant. Application of PGRs effect growth and production of essential oil of medicinal plants (Khan et al., 2015; Sangwan et al., 2001). Thus, the aim of the present study was to evaluate the effect of different growth regulators (BAP, NAA and TDZ) and their combinations on shoot proliferation, growth, and analysis of the volatile compounds of Lippia gracilis cultured in vitro.

medium influence the growth and development of most cultures under these conditions (Hartmann et al., 2002). PGRs are synthetic chemicals with action similar to plant hormones, which act on plant metabolism causing physiological responses in plants (Lamas, 2001). They act as chemical signals acting on the growth regulation and development of plants by binding to receptors and triggering a series of cellular changes that affect the development of organs or tissues (Rodrigues et al., 2003). The most used PGRs in tissue culture are the auxins and cytokinins, and the balance between these substances highly influences the differentiation of shoot, roots, and callus formation. Cytokinins act in the processes of cell division and differentiation, whereas auxins control cell growth and elongation, especially in the formation of lateral and adventitious roots (Taiz and Zeiger, 2013). The 6-benzylaminopurine (BAP) is the most commonly used cytokinin, being effective in micropropagation of several woody species. Naphthaleneacetic acid (NAA) is the most used auxin in combinations with cytokinins for induction and elongation of shoots and rooting (de Oliveira et al., 2016). Thidiazuron (TDZ) has similar effects on both cytokinins and auxins, being considered a bioregulator highly efficient for plant morphogenesis in tissue culture (Diengngan and Murthy, 2014). In vitro culture, several research with different concentrations and combinations of growth regulators have been used in the culture media aiming to meet the relevant needs for the referred species, thus stimulating responses as growth, elongation and micropropagation of shoot. The use of 2.5 mg L−1 of BAP with 1.0 mg L−1 of NAA promoted a higher micropropagation rate in Heliconia latispatha (de Jesus Rodrigues et al., 2016). However, regarding the Hancornia speciosa

2. Material and methods 2.1. In vitro establishment of plant material The plants were collected from Tomar do Geru, Sergipe State, Brazil (11°19′16.7″ S; 37°55′09.2″ W) and cultivated in 10 L pots in the greenhouse. The plant material was herborized and the exsiccate was deposited in the PAMG herbarium of the Agricultural research agency of the state of Minas Gerais (EPAMIG) under registration PAMG 57859. After one year, these plants underwent drastic pruning, followed by fertilization with ammonium sulfate and constant irrigation, in order to stimulate regrowth and collection of plant material for in vitro 36

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Fig. 2. Analysis of in vitro growth response of Lippia gracilis cultured in MS medium with half the salt concentration supplemented for 30 days with different doses of BAP x NAA: (A) Shoot dry weight (SDW); (B) Leaf dry weight (LDW); (C) Root dry weight (RDW); (D) Callus dry weight (CDW); (E) Total dry weight without callus (TDWwoC); (F) Total dry weight with Callus (TDWwC).

15 min. In aseptic laminar flow cabinet, the explants (1 cm) were inoculated in test tubes (150 × 25 mm) containing 15 mL of MS medium (Murashige and Skoog, 1962) with half concentration of salts supplemented with 30 g L− 1 sucrose, 6 g L−1 agar, pH 5.7 ± 0.1 and autoclaved (121 °C, 20 min at 1.1 atm). After inoculation, the tubes were placed in a growth room with fluorescent lamps and intensity of 32 μmol m−2 s−1, 16 h of light photoperiod, and temperature of

establishment. Two doses of fungicide and systemic bactericide, Kazumin, were applied at seven and two days prior to the material collection both in field (mother plants) and in controlled environment at the dosage of 3 mL L−1 (0.06 g of active ingredient) of water, aiming to reduce in vitro contamination. The apical and nodal segments were washed in running water for 30 min and immersed in sodium hypochlorite solution (1.25% active chlorine) under constant stirring for 37

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Fig. 3. Visual aspect of Lippia gracilis plantlets at 30 days cultured in vitro under different doses of BAP and NAA.

25 ± 1 °C. After 30 days, the plantlets were propagated in flasks containing 40 mL of MS medium with half concentration of salts and kept under identical light and temperature conditions until sufficient material was obtained for the experiments.

and without callus (mg).

2.2. Growth of explant culture supplemented with BAP x NAA

Nodal segments (1 cm) from cultures established in vitro were inoculated in test tubes containing 15 mL of MS medium with half concentration of salts supplemented with 30 g L−1 sucrose, 6 g L−1 agar (Himedia®, type I), and pH 5.7 ± 0.1, added with different combinations between the growth regulators BAP and TDZ. The test tubes with culture medium were autoclaved at 121 °C for 20 min at 1.1 atm. Six different combinations of BAP (Sigma-Aldrich®) and TDZ (SigmaAldrich®) were used so that the final concentration of growth regulators in the medium did not exceed 2.24 μM. The treatments consisted of in combination of BAP + TDZ at concentration (μM): 0 + 0, 1.11 + 1.13, 1.33 + 0.91, 0.88 + 1.36, 0.43 + 1.81, and 1.78 + 0.46, totaling six treatments cultured in growth rooms with 16 h light photoperiod at 25 ± 1 °C. Four replicates were used per treatment with four explants per replicate in a completely randomized design (CRD). After 30 days, the plantlets were evaluated as previously described for the experiment of BAP x NAA.

2.3. Growth of explant culture supplemented with BAP x TDZ

Nodal segments (1 cm) from cultures established in vitro were inoculated in test tubes containing 15 mL of MS medium with half concentration of salts supplemented with 30 g L−1 sucrose, 6 g L−1 agar (Himedia®, type I), and pH 5.7 ± 0.1, added with different combinations between the growth regulators BAP and NAA. The test tubes containing culture medium were autoclaved at 121 °C for 20 min at 1.1 atm. Five different concentrations of BAP (0.0, 2.22, 3.33, 4.44, and 5.55 μM) and three concentrations of NAA (0.0, 2.68 and 5.36 μM), totaling 15 treatments cultured in growth rooms with 16 h of light photoperiod at 25 ± 1 °C. After 30 days, the growth and analysis of the volatile fraction were evaluated. Four replicates per treatment were used with three explants per replicate in a completely randomized design (CRD) in a factorial design (5 × 3). The obtained plantlets were evaluated in nine parameters: shoot length (cm), root length (cm), number of shoots, and leaf, shoot, root, callus, and total dry weight with 38

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5.56 98.67 4.88 99.18 5.36 98.43 3.44 99.34 4.69 99.12 4.91 98.02 2.56 98.74

HP-5MS column retention index in elution order. SD: standard deviation (n = 3).

Fig. 4. Principal component analysis (PCA) in matrix correlation constructed using data for four major compounds under different growth regulator (BAP x NAA) doses of Lippia gracilis. In the score plot, the numerical values refer to the dosages of growth regulators (μM) used in the experiments.

2.4. Chemical analysis by headspace GC/MS Samples of L. gracilis leaves were collected from plantlets at 30 days of culture in vitro. The plant material was dried in a convection oven at 40 °C for 72 h and 100 mg of dried leaves were conditioned in 20 mL headspace vials, sealed with PTFE/silicone septa. In order to extract the volatile fraction of L. gracilis, the static headspace technique was employed using a headspace extract/sampler CombiPAL Autosampler System (CTC Analytic AG, Switzerland) coupled to the GC/MS system. The operating conditions were: sample incubation temperature of 110 °C for 30 min, syringe temperature at 120 °C, and 500 u L of the vapor phase were injected into the chromatographic system. Chemical analyses were performed on an Agilent® 7890 A gas chromatograph system coupled to an Agilent® MSD 5975C mass selective detector (Agilent Technologies, California, USA), operated by electronic impact ionization at 70 eV in scan mode at a speed of 1.0 scan/s, with a mass acquisition interval of 40–400 m/z. An HP-5 ms fused silica capillary column (30 m long × 0.25 mm internal diameter × 0.25 μm film thickness) (California, USA) was used. The helium gas was used as carrier gas with flow of 1.0 mL/min. The

a

5.96 97.74 4.12 98.75 4.61 99.10

2.95 99.04

3.37 98.61

3.55 98.51

2.74 98.59

2.63 98.21

23.52 5.76 46.28 21.74 6.30 45.72 21.79 6.45 45.30 20.89 6.51 46.27 21.92 6.58 45.01 23.17 6.07 41.96 20.91 7.40 44.50 23.13 6.20 43.33 19.21 7.64 51.60 17.06 8.16 56.97

19.96 7.37 52.06

20.41 7.29 45.22

20.82 6.72 43.81

21.61 6.87 41.28

20.96 7.27 42.48

0.38 0.27 0.28 0.25 0.28 0.19 0.18 0.22 0.27 0.50

0.28

0.22

0.19

0.18

0.19

1.78 0.38 2.84 2.45 0.34 9.38 1.94 0.39 2.99 2.76 0.35 11.84 1.78 0.38 3.03 2.65 0.34 11.07 1.83 0.34 3.06 3.03 0.37 13.35 1.92 0.39 2.99 2.90 0.37 12.07 1.97 0.41 3.38 2.99 0.39 12.58 2.15 0.35 3.28 3.15 0.39 13.87 2.06 0.45 3.09 2.56 0.34 10.40 1.46 0.26 2.38 2.33 0.30 9.18

Monoterpene hydrocarbon α-thujene 925 α-pinene 932 myrcene 991 α-terpinene 1016 limonene 1027 γ-terpinene 1057 Oxygenated monoterpenes cis-sabinene hydrate 1065 Aromatic monoterpenes ρ-cymene 1023 thymol 1293 carvacrol 1302 Sesquiterpene hydrocarbon E-Caryophyllene 1418 Total (%)

1.11 0.20 1.91 1.70 0.32 6.56

1.54 0.25 2.55 2.38 0.32 9.38

1.83 0.33 3.15 3.13 0.39 13.27

2.08 0.37 3.16 3.28 0.39 14.14

2.13 0.36 3.42 3.61 0.43 15.96

2.21 0.34 3.37 3.42 0.41 14.93

3.33 + 5.36 3.33 + 2.68 3.33 + 0 2.22 + 5.36 2.22 + 2.68 2.22 + 0 0 + 5.36 0+0 0 + 2.68 Content (%) ± SD

BAP + ANA (μM) RIa Compounds

Table 1 Volatile chemical composition of L. gracilis plantlets grown in vitro for 30 days under different combinations of BAP + ANA.

4.44 + 0

4.44 + 2.68

4.44 + 5.36

5.55 + 0

5.55 + 2.68

5.55 + 5.36

L.E. Santos Lazzarini et al.

39

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Table 2 Mean shoot length (SL), number of leaf (NL), number of shoot (NS), root length (RL), shoot dry weight (SDW), leaf dry weight (LDW), root dry weight (RDW), callus dry weight (CDW), total dry weight without callus (TDWwoC), and total dry weight with callus (TDWwC) of Lippia gracilis cultivated in MS medium with half the salt concentration, supplemented for 30 days with different doses of BAP x TDZ. Treatments BAP + TDZ (μM)

SL (cm)

0+0 1.11 + 1.13 1.33 + 0.91 0.88 + 1.36 0.43 + 1.81 1.78 + 0.46

4.07 1.33 1.37 1.77 1.84 1.33

a

NL

aa c c b b c

18.95 30.58 27.39 17.14 24.59 16.03

NS

c a ab c b c

2.00 3.71 3.14 1.81 2.76 1.96

RL (cm) c a ab c b c

2.92 0.91 0.60 0.55 1.58 0.00

SDW (mg) a c c c b d

3.52 2.72 2.81 2.86 3.36 1.99

a ab ab ab a b

LDW (mg)

RDW (mg)

11.75 a 6.73 c 8.63 b 8.34 b 8.17 b 6.50 c

3.77 0.34 0.11 0.16 0.54 0.00

a c c c c c

CDW (mg)

TDWwoC (mg)

TDWwC (mg)

0.00 e 58.84 b 70.32 a 45.70 c 43.56 c 25.11 d

19.04 a 9.79 bc 11.55 b 11.36 b 12.07 b 8.49 c

19.04 68.63 81.86 57.06 55.63 33.60

c b a c c d

The mean values followed by the same letter within a column do not differ, according to the Tukey test at 5% significance level.

The constituents were identified by comparing their retention indices relative to the co-injection of a standard solution of n-alkanes (C8C20, Sigma-Aldrich®, St. Louis, USA) and by comparing mass spectra from the library (NIST, 2008) and the literature (Adams, 2017) database. The retention index was calculated using the equation proposed by Van Den Dool and Kratz (1963) and the (Adams, 2017) literature retention indices were consulted for the assignments.

2.5. Statistical analysis In the experiment BAP x NAA, the central composite rotational design (CCRD) was used to investigate the dose effects of the independent factors "NAA" and "BAP". The complete design consisted of 60 experimental runs, including four repetitions of the center points. The analysis of response surface plots (3D) with the effect of different doses of growth regulators were plotted using the Statistica® software, version 13.3 (StatSoft - Tulsa, USA). The data from BAP x TDZ experiment were submitted to ANOVA by the F test (p < 0.05). After checking the significance of variables by the F test, the means were compared by the Tukey’s multiple range test at 5% probability. Standardized data of three replicate were subjected to multivariate analysis, i. e., principal component analysis (PCA), using the same program for the assessment of chemical composition diversity of L. gracilis.

Fig. 5. Visual aspect of Lippia gracilis plantlets at 30 days cultured in vitro under different doses of BAP + TDZ.

injector temperatures and the transfer line to the MS were maintained at 240 °C. The initial temperature of the oven was 50 °C, followed by a temperature ramp of 3 °C/min up to 240 °C and finished with a ramp of 10 °C/min up to 280 °C. The injection was performed in split mode at 1:50 injection ratio. Analyses were performed in triplicate, and the concentrations of present constituents were expressed by the relative percentage area of chromatographic peaks.

Table 3 Volatile chemical composition of L. gracilis plantlets grown in vitro for 30 days under different combinations of BAP + TDZ. Compounds

Monoterpene hydrocarbon α-thujene α-pinene myrcene α-terpinene limonene γ-terpinene Oxygenated monoterpenes cis-sabinene hydrate terpinen-4-ol Aromatic monoterpenes ρ-cymene thymol carvacrol Sesquiterpene hydrocarbon E–caryophyllene Total (%) a

RIa

BAP + TDZ (μM) 0+0 Content (%) ± SD

1.11 + 1.13

1.33 + 0.91

0.88 + 1.36

0.43 + 1.81

1.78 + 0.46

925 932 991 1016 1027 1057

1.85 ± 0.11 nd 2.77 ± 0.16 2.59 ± 0.13 nd 11.35 ± 0.40

2.66 ± 0.12 0.47 ± 0.03 3.86 ± 0.15 3.42 ± 0.13 0.47 ± 0.01 14.71 ± 0.66

2.64 ± 0.15 0.43 ± 0.02 3.79 ± 0.09 3.59 ± 0.12 0.46 ± 0.03 16.27 ± 0.47

2.57 ± 0.06 0.40 ± 0.01 3.79 ± 0.09 3.94 ± 0.11 0.48 ± 0.01 19.23 ± 0.78

2.72 ± 0.06 0.43 ± 0.01 3.88 ± 0.09 3.75 ± 0.08 0.50 ± 0.02 16.30 ± 0.51

2.42 ± 0.06 0.39 ± 0.02 3.90 ± 0.10 4.10 ± 0.03 0.48 ± 0.00 21.10 ± 0.36

1065 1183

nd nd

nd 0.29 ± 0.01

0.32 ± 0.02 0.27 ± 0.03

0.27 ± 0.00 0.23 ± 0.04

0.28 ± 0.00 nd

0.20 ± 0.05 nd

1023 1293 1302

18.14 ± 1.14 8.55 ± 0.17 49.75 ± 1.26

22.49 ± 0.81 6.94 ± 0.29 37.45 ± 1.83

21.90 ± 0.66 6.84 ± 0.11 37.19 ± 0.40

21.26 ± 0.47 6.60 ± 0.14 36.45 ± 0.90

22.89 ± 0.56 7.02 ± 0.09 37.33 ± 0.83

21.25 ± 0.17 6.06 ± 0.14 35.70 ± 0.81

1418

4.27 ± 0.31 99.24

5.25 ± 0.09 98.01

4.50 ± 0.46 98.20

3.47 ± 0.09 98.70

3.65 ± 0.09 98.75

3.14 ± 0.16 98.74

HP-5MS column retention index in elution order. nd: not detected. SD: standard deviation (n = 3). 40

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weight (CDW), total dry weight with callus (TDWwC), and without callus (TDWwoC). The response surface plots illustrate the magnitude of growth regulator response (BAP and NAA) with growth parameters (SL, NS, RL, SDW, LDW, RDW, CDW, TDWwoC, and TDWwC). The highest increasing rates imply that the response value highly influenced the growth parameters in the NS variable; however, all analyzed variables generally presented decreasing responses for the different analyzed combinations. It is observed that the highest values were found under low BAP dosages, regardless of NAA for the variables SL and RL (Fig. 1A and B) and SDW, LDW, RDW, CDW, TDWwoC, and TDWwC (Fig. 2A–F). Auxins promote cell division, elongation, and differentiation, besides being responsible for apical dominance in plants (Taiz and Zeiger, 2013), as well as root induction and growth (Rossi and Sartoretto, 2013). Therefore, such superiority was expected in these parameters (SL and RL) for the treatments in which the NAA dosage was higher than BAP. All parameters were reduced, except for NS, CDW, and TDWwC, inasmuch as the BAP dosage increased. Cytokinins are plant hormones responsible for the control of cell division and differentiation and formation of buds. They also act on the breakdown of apical dominance, consequently reducing the plant length (Taiz and Zeiger, 2013), corroborating with data observed in the present study, which showed reduction for these parameters inasmuch as concentrations of BAP increased (Fig. 1A). A higher number of shoots were found in the highest dose of BAP x NAA. The response surface methodology (RSM) predicted 8–10 shoots per explant in higher dose of BAP and NAA (Fig. 1C). Auxin and cytokinins are two types of PGRs that are frequently used to induce plant morphogenesis, being used together in order to stimulate shoots (Sjahril et al., 2016). It is worth mentioning that the ideal levels of growth regulators to stimulate the highest number of shoots is a species-specific response, and should be analyzed on a case-by-case basis. According to Colombo et al. (2010), the use and combination of PGRs in the in vitro culture are needed in order to compensate deficiencies of endogenous hormone levels, since explants are isolated from plantlets that meet these needs. Once there is an increase in the number of shoots with increasing concentrations of BAP and NAA, the importance of establishing the adequate proportion of auxin and cytokinin that seems to be beneficial for the production of multiple shoots is highlighted (de Jesus Rodrigues et al., 2016). All combinations of growth regulators evaluated in this experiment induced callus in the explant base (Fig. 3). According to the response surface analysis, the combination of BAP and NAA resulted in the greatest effect on callus dry weight (CDW) (Fig. 2D). The similar figure (Fig. 2F) was obtained for total dry weight with callus (TDWwC). These results can be explained because the average callus (91.34 mg) showed higher compared with leaves (16.75 mg), shoots (7.82 mg), and root (5.08 mg) dry weight. It can be observed that total dry weight without callus (TDWwoC) shows a behavior similar than the other variables (Fig. 2A–C), where there was a greater accumulation of dry weight under low dosages of BAP (Fig. 2E). Similar results were found by Andrade et al. (2017), which evaluated different doses of BAP and NAA in vitro culture of Hyptis suaveolens and found callus formation in all tested combinations, whereas it was not found callus formation in the control treatment, without the presence of regulators. The combination of 2.68 μM of NAA and 2.22 μM of BAP in the MS medium promoted higher shoot number and shoot length for Chrysanthemum morifolium (Sjahril et al., 2016). For Caesalpinia pyramidalis, the use of growth regulators, BAP and NAA reduced the number of shoots, number of leaves, shoot length, and shoot dry weight (dos Santos et al., 2016).

Fig. 6. Principal component analysis (PCA) in matrix correlation constructed using data for four major compounds under different growth regulator (BAP x TDZ) doses of Lippia gracilis. In the score plot, the numerical values refer to the dosages of growth regulators (μM) used in the experiments.

3. Results and discussion 3.1. Growth of explant culture supplemented with BAP x NAA The response surface methodology (RSM) is symmetric of second order and consists of two parts (factorial), with one or more center points, and the axial. RSM is a method used to design experiments empirically, evaluate variable effects and optimize growth conditions. Orthogonal design methods cannot measure the interactions among variables, but RSM can overwhelmed these disabilities (Liu et al., 2015). Custódio et al. (2000) showed that the RSM is efficient in the analysis of experiments in factorial design when the referred factors are quantitative. Three-dimensional (3D) response surface was used to predict the growth data. In Figs. 1 and 2, the response surface shows the effect of different combinations of BAP and NAA growth regulators on shoot length (SL), number of shoots (NS), root length (RL), shoot dry weight (SDW), leaf dry weight (LDW), root dry weight (RDW), callus dry 41

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supplemented with growth regulator inhibited shoot elongation, leaf number, and root length (Table 2) and induced callus formation in the base of nodal segments (Fig. 5). According to Sjahril et al. (2016), different concentrations of growth regulators can inhibit plant growth or even be toxic to plantlets, corroborating with the results found in the present study, in which there was reduced growth with different combinations of regulators. These same authors suggest that the interaction of these regulators added to the medium and those produced endogenously determine the speed and direction of the culture development. According to Diengngan and Murthy (2014), TDZ is more efficient in shoot regeneration, but its effects depend not only on its concentration in the culture medium but it is also a species-specific response. Cytokinins may stimulate the production of multiple shoots up to certain concentrations, inasmuch as they influence the cell division and the dormancy breakdown of axillary buds, inhibited by apical dominance (Monfort et al., 2012). The data found in the present study may have been hampered by the fact that the different combinations of regulators used favored the callogenesis, directly affecting in the development of shoots. BAP was more efficient than TDZ for shoot development and for number of leaves in Simmondsia chinesis, whereas TDZ had a greater participation in callogenesis in relation to BAP (Taha et al., 2016). Amali et al. (2014) used a combination of BAP (4.44 μM) and TDZ (4.54 μM) found synergistic effect of these regulators on the shoot proliferation of Sorghum bicolor. For Boucerosia umbellata cultured in vitro with 8.88 μM BAP and 4.54 μM TDZ showed maximum response for the induction of multiple shoots and longer shoot length in relation to other combinations of tested regulators (Susheela et al., 2016). The BAP + TDZ supplementation for shoot proliferation generally resulted in a significant reduction in shoot weight (Table 2), and the medium without supplementation of growth regulators promoted greater accumulation of dry weight. In this species, it was observed that even low concentrations of growth regulator induce callus formation in the base of nodal segment. The total concentration of BAP + TDZ did not exceed 2.24 μM. Analyzing the total dry weight with the calluses, the medium without RL was lower due to the great contribution of callus formation in the media supplemented with BAP + TDZ (Table 2). Without considering the callus dry weight, the medium without RL accumulated higher total dry weight (Table 2). Similar results were found by Dos Santos Silva et al. (2013), which evaluated the addition of different growth regulators in the in vitro culture of Caesalpinia pyramidalis and found higher averages for shoot dry weight in the medium without growth regulator. Annona glabra showed greater accumulation of dry weight in in vitro culture when BAP was used in relation to TDZ (Oliveira et al., 2007). Grzegorczyk-Karolak et al. (2015) evaluated the effect of different cytokinins in the in vitro culture of Scutellaria alpina and reported greater effectiveness in shoot proliferation and biomass production using BAP in relation to TDZ.

3.2. Chemical analysis of the volatile fraction supplemented with BAP x NAA The different combinations of growth regulators, BAP and NAA, affected the volatile chemical composition of Lippia gracilis plantlets (Table 1). The chemical composition consisted of monoterpenes (91.78–96.09%) and sesquiterpenes (2.63–5.96%). Moreover, 11.80–25.91% of monoterpenes were hydrocarbons, 0.18 - 0.50% were oxygenated monoterpenes, and 69.76–82.19% were aromatic monoterpenes. The identified sesquiterpene (E- caryophyllene) belonged only to the hydrocarbon type. The major constituents were γ-terpinene, ρcymene, thymol, and carvacrol, regardless of the culture conditions, totaling from 83.06 to 88.77% of the total chemical composition. The effects of PGRs on the production of secondary metabolites are variable. Changes can only be observed in the yield or content of these compounds. Growth regulators influence plant growth and development, affect physiological, and biochemical processes, and can even regulate genes. Thus, there are several ways in which the use of these compounds can affect the production of secondary metabolites (Prins et al., 2010). Ontogeny, rate of photosynthesis, photoperiod, light quality, climate changes, nutrition, moisture, salinity, temperature, storage structures, and PGRs are factors that qualitatively and quantitatively affect the production of secondary metabolites (Sangwan et al., 2001). It is observed a fitting of 97.32% (PC1 + PC2) by the principal component analysis (PCA) of the four major chemical compounds (Fig. 4). Principal components (PCs) divided the treatments into three groups, where it is possible to observe that L. gracilis accumulated higher content of carvacrol and thymol when cultured in medium without growth regulator supplementation. The explant cultured in medium with BAP dosages above 4.44 μM showed higher content of ρcymene, and below 3.33 μM, higher content of γ-terpinene (Fig. 4). The loading analysis showed that carvacrol and thymol negatively correlated with γ-terpinene and ρ-cymene. The data confirm that there is a conversion from one compound to another. According to Crocoll (2011) and Poulose and Croteau (1978), these constituents are precursors of carvacrol, and there was probably greater conversion of these elements under these culture conditions, i.e., without growth regulator. On the other hand, inasmuch as the regulators were being used, the total content of the hydrocarbon monoterpenes increased, mainly due to the increase in γ-terpinene levels. Several research report that essential oil production and in vitro chemical composition of tissues are influenced by PGRs. Santos-Gomes and Fernandes-Ferreira (2003) reported the culture medium with kinetin increased essential oil of Salvia officinalis. Thymus vulgaris cultured in vitro significantly increased the volatile compounds, including thymol, by 315% (Affonso et al., 2009). Monfort et al. (2018) showed that the medium supplemented with auxin and cytokinin increase the compound estragole and auxin alone increase linalool synthesis in Ocimum basilicum.

3.4. Chemical analysis of the volatile fraction supplemented with BAP and TDZ

3.3. Growth of explant culture supplemented with BAP x TDZ In order to test whether different PGRs compositions in the propagation medium influence the proliferation of L. gracilis shoots, nodal segments obtained from in vitro plantlets were transferred to half concentration of MS supplemented with different combinations of BAP + TDZ with concentration of both not more than 2.24 μM. After 30 days in culture, the response was evaluated (Table 2). The results showed that media supplemented with BAP + TDZ were more effective in promoting the development of shoots than those without growth regulator. The medium containing BAP + TDZ produced multiple shoots. The maximum number of multiple shoots (3.71) was obtained in the medium containing 1.11 BAP + 1.13μM TDZ (Table 2). The induction of multiple shoots was not observed in the medium without growth regulator, only the growth of axillary buds. The medium

The different combinations of growth regulators, BAP and TDZ, affected the volatile chemical composition of Lippia gracilis plantlets (Table 3). The chemical composition consisted of monoterpenes (92.76 to 95.60%) and sesquiterpene hydrocarbons (3.14 to 5.25%). The monoterpene hydrocarbons were represented by 18.56 to 32.39%, 0.29 to 0.59% oxygenates and 63.01 to 76.44% aromatics. The sesquiterpene hydrocarbon was represented by the unique presence of E-caryophyllene. The major constituents were γ-terpinene, ρ-cymene, thymol, and carvacrol, regardless of the culture conditions, totaling from 81.58 to 87.79% of the total chemical composition. These constituents were already observed by other authors for the studied species (Cruz et al., 2013; dos Santos et al., 2016; Ferraz et al., 2013; Neto 42

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et al., 2010). It is observed a fitting of 99.25% (PC1 + PC2) by the principal component analysis (PCA) of the four major chemical compounds (Fig. 6). It is possible to observe that L. gracilis accumulated higher content of carvacrol and thymol when cultured in medium without growth regulator supplementation. The explant cultured in medium with higher BAP (1.78 μM) and lower TDZ (0.46 μM) showed higher γterpinene content. In contrast, higher TDZ (1.81μM) and lower BAP (0.43 μM) showed higher ρ-cymene accumulation. Principal component analysis (PCA) showed that carvacrol negatively correlated with γ-terpinene. The data confirm that there is a conversion from γ-terpinene to carvacrol, as reported by Crocoll (2011). Thymol negatively correlated with ρ-cymene. Preliminary studies have reported the influence of plant hormones on the volatile fraction of some species. The use of growth regulators significantly affected the production of secondary metabolites in the in vitro culture of Aconitum violaceum (Rawat et al., 2013) and Ocimum basilicum (Monfort et al., 2018). Different types and concentrations of cytokinins significantly affected the accumulation of secondary metabolites with antioxidant action in Scutellaria alpina cultured in vitro (Grzegorczyk-Karolak et al., 2015). The use of TDZ in the culture medium reduced the levels of bioactive compounds of Hypericum hirsutum and Hypericum maculatum cultured in vitro (Coste et al., 2011).

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4. Conclusions The research findings this study suggest that PGRs can affect growth and production of volatile compounds of L. gracilis cultured in vitro. This species is very sensitive to induce callogenesis in the medium supplemented even with low concentration of PGRs. A higher number of shoots were found in the highest dose of BAP x NAA. The response surface methodology predicted 8–10 shoots per explant in higher dose of BAP (5.55 μM) and NAA (5.36 μM) and average 3.71 shoots was obtained with 1.11μM BAP + 1.13μM TDZ. The present study demonstrates that L. gracilis can be successfully propagated in vitro using nodal segment explants and multiple shoots. There is the tendency to have higher carvacrol and thymol contents in the medium without PGRs. Acknowledgements This study was financed in parts by National Council for Scientific and Technological Development (CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico), the Minas Gerais State Research Foundation (FAPEMIG - Fundação de Pesquisa do Estado de Minas Gerais), and the Coordination for the Improvement of Higher Education Personnel (CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – Brasil (CAPES – Finance Code 001). References Adams, R.P., 2017. Identification of Essential Oil Components by Gas chromatography/ mass Spectrometry. 5 Online Ed. Texensis Publishing. Affonso, V.R., Bizzo, H.R., Lage, C.L.S., Sato, A., 2009. Influence of growth regulators in biomass production and volatile profile of in vitro plantlets of Thymus vulgaris L. J. Agric. Food Chem. 57, 6392–6395. Amali, P., Ramakrishnan, M., Kingsley, S., Ignacimuthu, S., 2014. Direct regeneration potential of Sorghum bicolor (L.) Moench under the influence of plant growth regulators. Plant Cell Biotechnol. Mol. Biol. 15, 118–126. Andrade, H.B., Braga, A.F., Bertolucci, S.K.V., Hsie, B.S., Silva, S.T., Pinto, J.E.B.P., 2017. Effect of plant growth regulators, light intensity and LED on growth and volatile compound of Hyptis suaveolens (L.) Poit in vitro plantlets. Acta Hortic. 277–284. Colombo, L.A., Assis, A.Md., de Faria, R.T., Roberto, S.R., 2010. Estabelecimento de protocolo para multiplicação in vitro de Bastão do-imperador (Etlingera elatior) Jack RM Sm. Acta Sci. Agron 32. Coste, A., Vlase, L., Halmagyi, A., Deliu, C., Coldea, G., 2011. Effects of plant growth regulators and elicitors on production of secondary metabolites in shoot cultures of Hypericum hirsutum and Hypericum maculatum. Plant Cell Tissue Organ Cult. 106, 279–288. Crocoll, C., 2011. Biosynthesis of the Phenolic Monoterpenes, Thymol and Carvacrol, by Terpene Synthases and Cytochrome P450s in Oregano and Thyme. Dissertation, der

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