Growth regulators induced shoot regeneration and volatile compound production in Lippia rotundifolia Cham., a threatened medicinal plant

Industrial Crops & Products 137 (2019) 401–409

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Growth regulators induced shoot regeneration and volatile compound production in Lippia rotundifolia Cham., a threatened medicinal plant

T

Bety Shiue de Hsiea, Ana Izabela Sales Buenoa, Suzan Kelly Vilela Bertoluccib, ⁎ Alexandre Alves de Carvalhoa, Ernane Ronie Martinsc, Jose Eduardo Brasil Pereira Pintoa, a

Laboratory of Plant Tissue Culture and Medicinal Plants, Department of Agriculture, Federal University of Lavras, 37200-000, Lavras, Brazil Laboratory of Phytochemistry and Medicinal Plants, Department of Agriculture, Federal University of Lavras, 37200-000, Lavras, Brazil c Laboratory of Medicinal and Aromatic Plants, Institute of Agrarian Sciences, Federal University of Minas Gerais, Montes Claros, Brazil b

ARTICLE INFO

ABSTRACT

Keywords: Chemical compounds Medicinal plants Micropropagation Verbenaceae

Lippia rotundifolia Cham. is an endemic and endangered plant found in the savanna that is aromatic and rich in monoterpenes. The aims of the study were to evaluate the influence of plant growth regulators on the growth and chemical analysis of L. rotundifolia by headspace/GC–MS. Nodal segments (1.0 cm) were cultured on basal Murashige & Skoog (MS) medium supplemented with the following amounts of growth regulators: 0.0, 2.22, 6.66, 11.10, and 15.54 μM 6-benzylaminopurine (BAP) combined with 0.0, 2.68, and 5.36 μM naphthaleneacetic acid (NAA) or 0.0, 2.27, 4.54, 6.81, and 9.08 μM thidiazuron (TDZ) combined with 0.0, 2.68, and 5.36 μM NAA. The most effective concentrations were 11.10 μM BAP + 2.68 μM NAA, which induced 11 shoots explant−1, and 15.54 μM BAP + 2.68 μM NAA, with 11.6 shoots explant−1. The TDZ at a concentration of 6.81 μM with 2.68 μM NAA resulted in 20 shoots explant−1, 9.08 μM TDZ without NAA in 22.8 shoots explant−1, and 9.08 μM TDZ + 2.68 μM NAA in 18.1 shoots explant−1. Rooting took place on MS medium free of plant growth regulators, and acclimatization was successful (95%). The influence of plant growth regulators on the fraction of volatile compounds appeared to be quite variable. The cytokinin concentration was found to significantly influence the production of volatile compounds in L. rotundifolia shoot cultures. These findings indicate that shoots regenerated from nodal segments on MS medium containing higher concentrations of BAP and NAA have increased myrcenone and limonene. Ocimenone contents increased only at low concentrations of BAP and NAA.

1. Introduction

vegetative propagation, the percentage of rooting and survival of plantlets are low. Lippia rotundifolia is considered a critically threatened medicinal plant in its native habitat (Meira et al., 2017). Therefore, before this species goes extinct, conservation measures are necessary. Micropropagation is an alternative method for large multiplication and germplasm conservation of endangered species such as L. rotundifolia. Thus, the importance of further studies on this species is emphasized (Pimenta et al., 2007; Salimena and Silva, 2009). Furthermore, there is just one report on the in vitro propagation of L. rotundifolia (Resende et al., 2015). Currently, biotechnology application to medicinal plants is widespread, aiming to provide better plant materials. Among the in vitro cultivation techniques, micropropagation is a method of large-scale vegetative propagation for species of economic interest, including medicinal plants with pharmacological value. Although this technique is very costly, its use is justified due to the rising demand from the pharmaceutical industry for indexed, virus-free plants with high

Lippia rotundifolia Cham. (Verbenaceae) is an aromatic shrubby plant with a height of 0.5–2 m that is native and endemic to the campos rupestres found in the “Cadeia do Espinhaço” in the state of Minas Gerais, Brazil. The species is characterized by persistent leaves and coriaceous lamina and a corymb inflorescence with pink flowers (Fig. 1A–C). The leaves have glandular trichomes, which are rich in monoterpenes and popularly used to make “tea of pedestrian” (“chá-depedestre”). The literature shows that this species is used mostly for the treatment of respiratory disorders and for its sedative and antimicrobial activities (Leitão et al., 2008). In local communities, it is used for relaxing baths and hot footbaths. The major compounds limonene, myrcene, and myrtenal are among the volatile compounds found in L. rotundifolia (Leitão et al., 2008). Agronomic studies on the species report low seed germination potential, reaching a maximum of 40% when subjected to different GA3 concentrations. Regarding conventional

⁎

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

https://doi.org/10.1016/j.indcrop.2019.05.050 Received 12 February 2019; Received in revised form 16 May 2019; Accepted 17 May 2019 Available online 23 May 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. In vitro propagation of Lippia rotundifolia: (A) source plant; (B) persistent leaves with a coriaceous; and (C) inflorescence with pink flowers. L. rotundifolia plantlet cultured in vitro in MS medium supplemented with different BAP and NAA concentrations for 45 days: (D) 0.0 μM BAP combined with 0.0, 2.68, and 5.36 μM NAA; (E) 2.22 μM BAP combined with 0.0, 2.68, and 5.36 μM NAA; (F) 6.66 μM BAP combined with 0.0, 2.68, and 5.36 μM NAA; (G) 11.10 μM BAP combined with 0.0, 2.68, and 5.36 μM NAA; and (H) 15.54 μM BAP combined with 0.0, 2.68, and 5.36 μM NAA. (I) Plantlet rooting without PGR. (J) Acclimatized plant.

phytosanitary and physiological quality, as well as with the ability to synthesize secondary metabolites (Morais et al., 2012). Because of this high demand, it is quite common to use the interaction of auxins and cytokinins in the differentiation process. Some studies related to in vitro micropropagation of species from the genus Lippia showed positive results in the production of multiple shoots, and the most commonly used regulators were the cytokinin BAP (6-benzylaminopurine) and auxin NAA (naphthaleneacetic acid) in MS medium (Asmar et al., 2012; Blank et al., 2008; Luz et al., 2014; Marinho et al., 2011; Resende et al., 2015). Several authors report the need to supplement the culture medium, combining auxins and cytokinins to ensure efficiency in the micropropagation of different medicinal plants; for instance, studies have been conducted with Lippia alba (Mill.) (Asmar et al., 2012), Erythrina velutina (Willd.) (Costa et al., 2010), Pfaffia glomerata (Spreng.)

(Flores et al., 2009), Stryphnodendron adstringens (Mart.) Coville (Nicioli et al., 2008), Calendula officinalis L. (Victoria et al., 2012), and Lippia gracilis (Lazzarini et al., 2019). Plant growth regulators (PRGs) can influence essential oil production through effects on plant growth, essential oil biosynthesis, and the number of oil storage structures (Castilho et al., 2019; Monfort et al., 2018; Rout et al., 2000; Sharafzadeh and Zare, 2011). The use of growth regulators in tissue culture can also stimulate the accumulation of metabolites in some cases as well as increase essential oil production (Fracaro and Echeverrigaray, 2001; Lazzarini et al., 2019; Monfort et al., 2018; Sangwan et al., 2001). For instance, there was a great increase in nerol and geraniol in in vitro Melissa officinalis culture for 60 days when auxin and cytokinin supplements were used (Silva et al., 2005). Castilho et al. (2019) evaluated the effect of cytokinins on 402

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volatile constituents of Lippia origanoides and concluded that, through plant tissue culture, it is possible to optimize the synthesis of terpenes. Although research on in vitro propagation has been conducted for the genus Lippia, for L. rotundifolia, just one study has been reported due to its restricted accessibility. It is important to develop suitable micropropagation techniques for rapid mass propagation and germplasm conservation of threatened medicinal plants for the prevention of genetic erosion. The present study attempted to establish reliable in vitro propagation using nodal explants for large-scale production and germplasm conservation of this plant and corroborated the volatile composition of L. rotundifolia in vitro. Here, we report the effects of cytokinins (6-benzylaminopurine (BAP) and thidiazuron (TDZ)) with αnaphthaleneacetic acid (NAA) on shoot proliferation and chemical analysis of volatile compounds by headspace/GC–MS in shoots from Lippia rotundifolia Cham.

2.3. In vitro rooting Regenerated shoots approximately 1.5 cm in length isolated from the treatments were used for in vitro rooting. Shoots were separated and inoculated into solidified MS medium with 0.6% (w/v) agar without growth regulators. 2.4. Acclimatization The plantlets with 45 days old were washed with water and planted in trays containing commercial substrate in a greenhouse under black net (50%) to ensure a mild temperature and high humidity. 2.5. Analysis of volatile compounds by headspace GC–MS

2. Materials and methods

For the analysis of volatile compounds, dried leaves of L. rotundifolia from the BAP + NAA and TDZ + NAA treatments were used and gathered into a composite sample. The samples, comprising 100 mg of dried leaves in triplicate, were added to 20 mL vials sealed with a PTFE/ silicone septum. The static headspace technique was used for the extraction of the volatile fraction of L. rotundifolia. A CombiPAL Autosampler System (CTC Analytic AG, Switzerland) automatic headspace extractor coupled to a GC–MS (gas chromatograph/mass spectrometer) system was used in the analyses. The following parameters were set after the operating conditions were optimized: a sample incubation temperature of 110 °C for 30 min and syringe temperature of 120 °C. The injection volume was 500 μL in the steam phase, and it was injected in split mode at a ratio of 20:1. The volatile fraction was analyzed in a headspace GC–MS system on an Agilent® 7890A gas chromatograph coupled with an Agilent® 5975C Series MSD (Agilent Technologies, California, USA) mass selective detector operated by electron impact ionization at 70 eV in scan mode at a speed of 1.0 scan/ s, with a material acquisition interval of 40–400 m/z. An HP-5MS fused silica capillary column (30 m length x 0.25 mm internal diameter x0.25 μm film thickness) (California, USA) was used. Helium gas was used as the carrier gas with a flow rate of 1.0 mL/min; the injector and transfer line temperatures for the MS were maintained at 230 and 240 °C, respectively. The initial oven temperature was 60 °C, followed by a temperature ramp of 3 °C/min to 230 °C, followed by a ramp of 10 °C/min to 250 °C, which was held isothermally for 1 min. The contents of the constituents present in the volatile fraction were expressed as the normalized area percentage of the chromatographic peaks. Volatile fraction constituents were identified by comparing their linear retention indices relative to the coinjection of a standard solution of nalkanes (C9-C18, Sigma-Aldrich®, St. Louis, USA) and by comparing mass spectra from the NIST library and Adams (2017) database. The retention index was calculated using the equation proposed by Van den Dool and Kratz (1963), and the retention indices reported by Adams (2017) were consulted for the assignments.

2.1. General conditions of the experiments and establishment of in vitro culture Scions from Lippia rotundifolia Cham, approximately 15 cm in size, were collected in São Gonçalo do Rio das Pedras, a savanna area in the state of Minas Gerais, Brazil. The scions were acclimatized in the medicinal herb garden of the Federal University of Lavras, MG, Brazil. Exsiccates were deposited in the PAMG herbarium of the Agricultural Research Agency of the state of Minas Gerais (EPAMIG) under voucher 58027 identified by Dr. Andréia Fonseca Silva. Axillary buds were collected from 3-month-old plants, washed in running tap water for 30 min and immersed in a bleach solution (1% active sodium hypochlorite) for 20 min under stirring. The explants (1 cm size) were rinsed four times in sterile distilled water and inoculated in test tubes (25 x 150 mm) with plastic caps containing 15 mL of MS (Murashige and Skoog, 1962) medium. Culture medium free of growth regulators, supplemented with 30 g L−1 sucrose and 6 g L−1 agar (Himedia®, Type I) and with a pH of 5.7 ± 0.1 adjusted with NaOH and HCl (0.1 and 0.5 N, respectively) was autoclaved (15 min, 121 °C) and used to establish the explants. After inoculation, the tubes were kept in a growth room with cold white fluorescent lamps (Osram™, Brazil) and a light intensity of 39 μmol m-2 s−1, with a 16 h photoperiod and a temperature of 26 ± 1 °C. 2.2. Multiple shoot regeneration and biomass production Nodal segments (1 cm) from cultures established in vitro were inoculated in test tubes (25 x 150 mm) containing 15 mL of MS medium with or without supplementation with growth regulators and maintained in a growth room under the conditions previously described. The explants were incubated in medium supplemented with BAP and NAA. The experimental design was completely randomized (CRD), using BAP at 0.0, 2.22, 6.66, 11.10, and 15.54 μM concentrations combined with NAA at 0.0, 2.68, and 5.36 μM concentrations, totaling 15 treatments with four replicates, where each replicate consisted of 10 tubes containing one segment each, totaling 40 nodal segments per treatment. In the experiment testing TDZ and NAA, the experimental design was also a CRD, using TDZ at 0.0, 2.27, 4.54, 6.81, and 9.08 μM concentrations combined with NAA at 0.0, 2.68, and 5.36 μM concentrations, totaling 15 treatments with four replicates, where each replicate consisted of 10 tubes containing one segment each, totaling 40 nodal segments per treatment. Experiments were repeated twice, and each series consisted of 40 explants. The following data were evaluated after 45 days: shoot number (SN), shoot length (SL), percentage of hyperhydricity (Hyp.), percentage of callus (Callus), root percentage (Root), shoot dry weight (SDW), leaf dry weight (LDW), root dry weight (RDW), and total dry weight (TDW). The drying of plant material was performed in a convection oven at 36 ± 2 °C for approximately 48 h until constant weight was reached.

2.6. Statistical analysis The data were submitted to analyses of variance, and the averages were compared by the Scott-Knott test at 5% probability using Sisvar® software (Ferreira, 2000). Principal component analysis (PCA) was used to study the major compounds of the essential oil in relation to different concentrations of growth regulators. Each variable (i.e., percentage of an identified compound of the total oil composition) was subtracted by the variable mean; this process ensured that all results would be interpretable in terms of variation from the mean. Statistica® software version 13.3 (StatSoft, Tulsa, USA) was used for these statistical analyses. 403

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Fig. 1G, H). However, when cultures supplemented with TDZ were evaluated, the combination of TDZ and NAA generated a larger number of shoots than the BAP and NAA treatment. The largest number of shoots per explant was obtained at concentrations of 6.81 μM TDZ + 2.68 μM NAA (20 shoots explant−1), 9.08 μM TDZ without NAA (22.8 shoots explant−1), and 9.08 μM TDZ + 2.68 μM NAA (18.1 shoots explant−1) (Fig. 2A–C, Table 2). Several authors have successfully used BAP to stimulate shoot proliferation in explants from the Verbenaceae family (Balaraju et al., 2008; Braga et al., 2012; Peixoto et al., 2006). Asmar et al. (2012), working with Lippia alba, found a larger number of shoots (4 shoots explant−1) using 6.66 μM BAP. In Lippia sidoides, there was no significant difference between supplementation with BAP and the control, even at concentrations up to 17.76 μM, showing that the regulatory action may vary according to the species (Blank et al., 2008). It is noteworthy that no study in the literature reported the effect of TDZ in Lippia species since the use of BAP is common in species from Verbenaceae. However, from the results observed in this study, it can be seen that TDZ is very efficient at increasing shoot proliferation, showing approximately twice the efficiency of BAP at lower concentrations. This work reveal that at concentrations above 11.10 μM BAP, the shoot length (SL) was reduced, ranging from 1.16 to 1.95 cm (Table 1), and at concentrations above 6.81 μM TDZ, the SL was reduced from 0.76 to 1.15 cm (Table 2). An inhibitory effect of BAP on shoot length was also reported for the in vitro multiplication of L. alba (Asmar et al., 2012) and L. rotundifolia (Resende et al., 2015). Explants showed hyperhydricity symptoms in the presence of BAP at a concentration above 6.66 μM (Table 1) and in the medium with TDZ at a concentration above 6.81 μM (Table 2). However, when these multiple shoots were subcultured in medium without PGRs, the hyperhydricity disappeared. Resende et al. (2015) also observed a high percentage of hyperhydricity, a low number of shoots (2.6 shoots explant−1), and callus formation in plants grown in medium supplemented with BAP, especially at the highest concentration (0.31 μM BAP). The presence of callius in the base of the explants was observed at concentrations above 6.66 μM BAP, regardless of whether BAP was combined with NAA (Table 1). This species was sensitive to callus formation at the base of the explant, even in medium supplemented with low concentrations of PGRs. Resende et al. (2015) used lower concentrations of BAP, from 0.31 to 0.98 μM, and observed callus formation. Usually, culture medium supplemented with higher concentrations of cytokinins inhibits root formation. The rooting percentage decreased drastically at concentrations above 6.66 μM BAP and 4.54 μM TDZ with or without the addition of NAA. Similar results were found by Resende et al. (2015) when BAP was used. With an increase in cytokinin concentration, there is higher investment in shoots and a smaller investment in root formation. Dry weights varied with supplementation of PGRs in the culture medium. Concentrations of BAP above 6.66 μM in combination with 2.68 μM NAA resulted in a higher SDW (14–17 mg plant−1). In contrast, LDW and RDW increased with lower concentrations of BAP (Table 1). The results indicated that the medium supplemented with TDZ increased dry weights in relation to BAP. Higher leaf (23.7–41.6 mg plant−1), shoot (10.8–24.5 mg plant−1), and total dry (40.6–66.0 mg plant−1) weights occurred at concentrations of TDZ above 6.81 μM with or without the addition of NAA (Table 2). TDZ has been shown to be biologically more active than BAP and is required at low concentrations of less than 10 μM in basal medium for in vitro propagation. TDZ efficiency in cell multiplication may be greater than that of other cytokinins because TDZ is a strong inhibitor of cytokinin oxidase and acts by modulating the endogenous hormone level. This mechanism occurs due to the increased activity of the acid phosphatase enzyme that makes endogenous cytokinins more active (Ribeiro et al., 2010). Normally, this mechanism is responsible for the reduction in shoot growth, hyperhydricity and very narrow abnormal leaves at high concentrations (Huetteman and Preece, 1993; Malik et al., 2010; Mondal

Fig. 2. Lippia rotundifolia plantlets cultured in vitro in MS medium supplemented with different TDZ and NAA concentrations for 45 days: (A) 0.0 μM NAA combined with 0.0, 2.27, 4.54, 6.81 and 9.08 μM TDZ; (B) 2.68 μM NAA combined with 0.0, 2.27, 4.54, 6.81 and 9.08 μM TDZ; and (C) 5.36 μM NAA combined with 0.0, 2.27, 4.54, 6.81 and 9.08 μM TDZ.

3. Results and discussion 3.1. Multiple shoot regeneration and biomass production In this work, different concentrations of BAP and TDZ in combination with NAA were evaluated in terms of L. rotundifolia multiple shoot proliferation. The results showed that cytokinin concentrations increased shoot multiplication (Figs. 1D–H; 2 A–C). Significant differences in the in vitro regeneration of multiple shoots were observed in nodal segments when different PGR concentrations were used. Using nodal segments as explants, the most effective treatments were 11.10 μM BAP + 2.68 μM NAA, which induced 11 shoots explant−1, and 15.54 μM BAP + 2.68 μM NAA, with 11.6 shoots explant−1 (Table 1, 404

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Table 1 Growth of Lippia rotundifolia plantlets derived from nodal segments cultivated in vitro and supplemented with BAP + NAA at different concentrations after 45 days. Concentrations (μM) BAP

NAA

0.0 0.0 0.0 2.22 2.22 2.22 6.66 6.66 6.66 11.10 11.10 11.10 15.54 15.54 15.54

0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36

Shoot Number

Shoot Length (cm)

Hypa (%)

Callus (%)

Roots (%)

Shoot Dry Weight (mg plant−1)

Leaf Dry Weight (mg plant−1)

Root Dry Weight (mg plant−1)

Total Dry Weight (mg plant−1)

2.0c 2.1c 2.0c 2.1c 2.1c 1.9c 6.6b 6.3b 6.4b 8.1b 11.0a 6.8b 9.3a 11.6a 10.1a

6.74a 5.84b 6.91a 5.09b 6.00b 6.13b 2.05c 3.27c 2.42c 1.81d 1.71d 1.95d 1.28d 1.31d 1.16d

0.0c 0.0c 0.0c 0.0c 0.0c 0.0c 44b 63b 63b 50b 75a 81a 100a 88a 100a

0.0c 0.0c 0.0c 0.0c 13b 0.0c 100a 100a 100a 100a 100a 100a 100a 100a 100a

100a 100a 100a 100a 94b 100a 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c 0.0c

9.2b 7.5b 9.1b 7.6b 9.6b 7.6b 10.0b 17.0a 11.0b 11.0b 15.0a 9.4b 9.9b 14.0a 9.3b

17.4a 18.1a 21.2a 18.5a 18.7a 16.8a 13.0b 17.8a 12.9b 14.3b 16.3a 10.1c 9.1c 12.0b 7.3c

1.8d 2.8c 4.2a 1.4e 2.6c 3.4b 0.0f 0.0f 0.0f 0.0f 0.0f 0.0f 0.0f 0.0f 0.0f

28.3b 28.3b 34.5a 27.5b 30.9a 27.8b 23.0c 34.7a 23.8c 25.7b 31.6a 19.5c 18.9c 25.7b 16.6c

Means followed by the same letter in the columns do not differ from each other, Scott-Knott test, p ≤ 0.05. a Hyperhydricity. Table 2 Growth of Lippia rotundifolia plantlets derived from nodal segments cultivated in vitro and supplemented with TDZ + NAA at different concentrations after 45 days. Concentrations (μM) TDZ

NAA

0.0 0.0 0.0 2.27 2.27 2.27 4.54 4.54 4.54 6.81 6.81 6.81 9.08 9.08 9.08

0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36 0.0 2.68 5.36

Shoot Number

Shoot Length (cm)

Hypa (%)

Callus (%)

Roots (%)

Shoot Dry Weight (mg plant−1)

Leaf Dry Weight (mg plant−1)

Root Dry Weight (mg plant−1)

Total Dry Weight (mg plant−1)

3.5c 2.4c 2.8c 4.7c 4.0c 3.1c 4.2c 3.8c 3.6c 12.8b 20.0a 15.6b 22.8a 18.1a 14.7b

4.10c 5.33b 6.44a 3.07d 4.46c 4.40c 3.18d 4.88b 4.47c 0.79e 1.15e 0.95e 0.91e 1.04e 0.76e

0.0d 0.0d 0.0d 69b 31c 0.0d 44c 19c 0.0d 63b 94a 100a 94a 100a 100a

0.0c 0.0c 0.0c 88a 50b 56b 100a 75b 81a 100a 100a 100a 100a 100a 100a

100a 100a 94a 19c 38b 31b 9d 0.0d 19c 0.0d 0.0d 0.0d 0.0d 0.0d 0.0d

13.6b 10.1b 15.0b 13.9b 19.7a 16.5b 12.9b 20.2a 17.3a 10.8b 22.1a 19.4a 21.6a 24.5a 19.2a

22.4b 21.5b 25.1b 27.8a 24.8b 20.6b 18.4b 23.0b 20.0b 29.8a 34.1a 31.2a 29.6a 41.6a 23.7b

1.12b 2.11ª 2.23ª 0.07d 0.49c 0.63c 0.32c 0.00d 0.15d 0.00d 0.00d 0.00d 0.00d 0.00d 0.00d

36.0b 33.7b 42.2b 41.8b 44.8b 37.5b 32.2b 43.3b 37.6b 40.6b 56.2a 50.6a 51.2a 66.0a 42.8b

Means followed by the same letter in the columns do not differ from each other, Scott-Knott test, p ≤ 0.05. a Hyperhydricity.

et al., 1998). The occurrence of hyperhydricity has been reported in several species with the use of TDZ (Ivanova and Van Staden, 2011; Sunagawa et al., 2007) and for other medicinal plants, such as Arnebia euchroma (Royle) Johnston (Malik et al., 2010) and Bacopa monnieri (L.) Wettst. (Tiwari et al., 2001). The use of cytokinins in culture medium may promote hyperhydricity; however, this may be difficult to reverse when the regulator becomes necessary for the micropropagation of species (Palma et al., 2011). In the results obtained with the use of BAP and TDZ to multiply L. rotundifolia, the high hyperhydricity percentage could be explained by the viability of these shoots; in this way, finding new ways to minimize hyperhydricity would further improve cytokinin use. In a supplementary experiment (data not shown), just BAP at 2.22, 3.33, 4.44, and 6.66 μM concentrations was used to obtain greater proliferation of shoots and a reduction in hyperhydricity. At concentrations of 3.33 and 4.44 μM BAP, 10 shoots explant−1 were induced; nevertheless, 28% of the shoots showed hyperhydricity symptoms and basal callus formation (12%).

culture (Fig. 1I). It was important to keep expenses down since the overall cost of in vitro propagation is normally higher than that of convention propagation methods. The shoot micropropagation system in this study could be an important step towards helping build a program for the reintroduction of L. rotundifolia into its natural habitats (Fig. 1J). 3.3. Analyses of volatile compounds by headspace GC–MS Analysis of volatile compounds of L. rotundifolia leaves from plantlets in medium supplemented with BAP + NAA by headspace GC–MS showed qualitative and quantitative differences. Fourteen (14) chemical constituents were detected, accounting for more than 92% of the total chemical composition (Table 3). Among these compounds, the sum of the four most representative compounds in the volatile fraction from leaves ranged from 78.52 to 82.97%, including myrcene, limonene, myrcenone, and Z-ocimenone. In this study, we observed an 18% decrease in myrcene as the BAP concentration increased. This PGR seemed to interfere negatively with myrcene because the highest myrcene content (19.73%) was found in the cultures grown in medium without growth regulators. However, limonene and myrcenone

3.2. In vitro rooting and acclimatization of regenerated plants Rooting was observed in all shoots in PGR-free medium after 4 wk in 405

406

16.30 ± 0.24c 13.28 ± 0.04c 1.24 ± 0.01e 0.21 ± 0.01a 36.42 ± 1.48a 1.17 ± 0.03b 0.46 ± 0.04d 15.28 ± 0.51c 0.75 ± 0.01f 2.16 ± 0.19d 0.49 ± 0.08b 1.27 ± 0.21c 0.16 ± 0.04c 2.99 ± 0.45d 92.18

BAP 6.66 NAA 2.68

μM

15.63 ± 0.09c 15.52 ± 0.59b 1.40 ± 0.06d 0.17 ± 0.01b 33.70 ± 2.22a 1.13 ± 0.06b 0.46 ± 0.03d 15.62 ± 0.81c 1.10 ± 0.12d 2.55 ± 0.06b 0.59 ± 0.09a 1.51 ± 0.15b 0.19 ± 0.02c 3.12 ± 0.32d 92.69

BAP 6.66 NAA 5.36 15.94 ± 0.19c 14.16 ± 0.02c 1.63 ± 0.02a 0.00 ± 0.00c 34.80 ± 2.39a 1.23 ± 0.12a 0.41 ± 0.07e 16.43 ± 1.46b 1.04 ± 0.17d 2.37 ± 0.09c 0.55 ± 0.12a 1.09 ± 0.15d 0.15 ± 0.02c 2.65 ± 0.34d 92.45

BAP 11.10 NAA 0.0

1.44 ± 0.06c 0.18 ± 0.01c 3.74 ± 0.17c 93.91

18.04 ± 0.64b 14.02 ± 0.58c 1.13 ± 0.01f 0.21 ± 0.03a 33.74 ± 0.24a 1.06 ± 0.02c 0.55 ± 0.03c 16.27 ± 0.17b 1.01 ± 0.06d 2.12 ± 0.06d 0.40 ± 0.02c

BAP 0.0 NAA 2.68

16.86 ± 0.85c 15.84 ± 0.66b 1.57 ± 0.04b 0.00 ± 0.00c 31.48 ± 0.39b 1.33 ± 0.01a 0.41 ± 0.02e 16.83 ± 0.70b 1.23 ± 0.02c 2.44 ± 0.13c 0.55 ± 0.08a 1.11 ± 0.21d 0.16 ± 0.02c 2.83 ± 0.22d 92.64

BAP 11.10 NAA 2.68

1.68 ± 0.18b 0.22 ± 0.03b 4.23 ± 0.52b 94.43

17.74 ± 0.78b 14.14 ± 0.59c 1.13 ± 0.02f 0.21 ± 0.02a 33.10 ± 2.18a 1.04 ± 0.01c 0.54 ± 0.02c 16.70 ± 0.25b 1.02 ± 0.05d 2.30 ± 0.16c 0.38 ± 0.00c

BAP 0.0 NAA 5.36

Means followed by the same letter in the rows, do not differ from each other, Scott-Knott test, p ≤ 0.05. a Relative retention index n-alkane series (C8–C18) HP-5MS column in order of elution.

991 1027 1100 1102 1149 1153 1189 1231 1240 1300 1365 1417 1450 1500 Total (%)

RIa

1.51 ± 0.04b 0.20 ± 0.01c 3.42 ± 0.09c 94.29

19.73 ± 0.37a 15.51 ± 0.17b 1.01 ± 0.02g 0.20 ± 0.01a 31.26 ± 1.38b 1.15 ± 0.13b 0.48 ± 0.01d 16.47 ± 0.17b 1.06 ± 0.03d 1.91 ± 0.03e 0.38 ± 0.01c

Control

μM

Content (%) ± Standard deviation

Content (%) ± Standard deviation

Myrcene Limonene Linalool Isomyrcene Myrcenone trans-tagetone α –Terpineol Z-ocimenone E-ocimenone Tridecane Piperitenone oxide β-Caryophyllene α -humulene Pentadecane

991 1027 1100 1102 1149 1153 1189 1231 1240 1300 1365

1417 1450 1500 Total (%)

Compounds

RIa

16.28 ± 0.20c 17.00 ± 0.05a 1.38 ± 0.03d 0.20 ± 0.01a 30.88 ± 1.50b 1.12 ± 0.05b 0.47 ± 0.03d 15.60 ± 0.60c 0.96 ± 0.10d 2.63 ± 0.13b 0.68 ± 0.08a 1.66 ± 0.15b 0.23 ± 0.01b 3.33 ± 0.15c 92.42

BAP 11.10 NAA 5.36

1.79 ± 0.08a 0.23 ± 0.01b 4.84 ± 0.06a 94.23

17.93 ± 0.06b 11.77 ± 0.90d 1.08 ± 0.04f 0.21 ± 0.05a 33.26 ± 0.52a 1.12 ± 0.03b 0.59 ± 0.02b 17.50 ± 0.39a 1.19 ± 0.05c 2.35 ± 0.00c 0.37 ± 0.02c

BAP 2.22 NAA 0.0

Table 3 Compound content (%) in plantlets of L. rotundifolia cultivated in vitro and supplemented with different BAP + NAA concentrations after 45 days.

16.10 ± 0.12c 18.23 ± 0.97a 1.45 ± 0.05c 0.17 ± 0.03b 32.31 ± 1.56a 1.04 ± 0.07c 0.34 ± 0.01f 14.56 ± 0.21d 1.02 ± 0.03d 2.58 ± 0.13b 0.47 ± 0.05b 1.53 ± 0.06b 0.19 ± 0.02c 2.70 ± 0.34d 92.69

BAP 15.54 NAA 0.0

1.92 ± 0.06a 0.26 ± 0.00a 5.09 ± 0.17a 93.91

17.02 ± 0.48c 10.71 ± 0.39d 1.10 ± 0.01f 0.22 ± 0.03a 33.52 ± 1.69a 1.14 ± 0.05b 0.64 ± 0.00a 18.14 ± 0.52a 1.36 ± 0.06b 2.41 ± 0.01c 0.38 ± 0.08c

BAP 2.22 NAA 2.68

15.42 ± 0.20c 16.79 ± 0.70a 1.57 ± 0.06b 0.00 ± 0.00c 35.36 ± 1.32a 0.99 ± 0.07c 0.43 ± 0.03e 14.43 ± 0.33d 0.91 ± 0.05e 2.33 ± 0.15c 0.61 ± 0.10a 1.17 ± 0.08d 0.15 ± 0.01c 2.66 ± 0.30d 92.82

BAP 15.54 NAA 2.68

1.68 ± 0.10b 0.21 ± 0.02b 5.08 ± 0.05a 93.91

17.26 ± 0.56b 11.14 ± 0.13d 1.13 ± 0.01f 0.21 ± 0.01a 32.89 ± 0.15a 1.14 ± 0.00b 0.59 ± 0.02b 18.10 ± 0.13a 1.50 ± 0.02a 2.59 ± 0.06b 0.39 ± 0.00c

BAP 2.22 NAA 5.36

15.89 ± 0.80c 17.74 ± 0.66a 1.56 ± 0.05b 0.00 ± 0.00c 34.40 ± 1.20a 1.02 ± 0.08c 0.40 ± 0.02e 14.52 ± 0.40d 0.92 ± 0.05e 2.36 ± 0.12c 0.48 ± 0.08b 1.32 ± 0.10c 0.17 ± 0.02c 2.95 ± 0.32d 93.73

BAP 15.54 NAA 5.36

1.99 ± 0.28a 0.27 ± 0.06a 3.78 ± 0.42c 92.41

17.51 ± 1.90b 17.22 ± 1.63a 1.47 ± 0.03c 0.00 ± 0.00c 27.80 ± 2.49c 1.17 ± 0.03b 0.42 ± 0.01e 15.99 ± 0.92c 1.30 ± 0.00c 2.88 ± 0.19a 0.61 ± 0.02a

BAP 6.66 NAA 0.0

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Industrial Crops & Products 137 (2019) 401–409

407

19.79 ± 0.68a 15.13 ± 0.80b 0.98 ± 0.02b 0.29 ± 0.01a 29.46 ± 1.24e 1.21 ± 0.16a 0.60 ± 0.03a 16.00 ± 0.21a 0.96 ± 0.10a 2.15 ± 0.08a 0.35 ± 0.02b 2.16 ± 0.16a 0.29 ± 0.03a 3.72 ± 0.08a 93.09

TDZ 4.54 NAA 2.68

μM

13.77 ± 0.61c 15.96 ± 0.80b 1.24 ± 0.03a 0.00 ± 0.00c 38.80 ± 3.23c 0.76 ± 0.30b 0.50 ± 0.02c 13.76 ± 0.87c 0.65 ± 0.08b 2.05 ± 0.21a 0.31 ± 0.02b 1.64 ± 0.31b 0.22 ± 0.05b 3.58 ± 0.46a 93.24

TDZ 4.54 NAA 5.36 13.38 ± 0.43c 15.76 ± 0.25b 1.00 ± 0.32b 0.00 ± 0.00c 41.75 ± 1.10b 0.68 ± 0.25b 0.49 ± 0.04c 12.69 ± 0.40d 0.67 ± 0.04b 1.92 ± 0.17b 0.36 ± 0.02b 1.64 ± 0.20b 0.00 ± 0.00d 3.57 ± 0.50a 93.91

TDZ 6.81 NAA 0.0

1.44 ± 0.06c 0.18 ± 0.01c 3.74 ± 0.17a 93.91

18.04 ± 0.64b 14.02 ± 0.58c 1.13 ± 0.01a 0.21 ± 0.03b 33.74 ± 0.24d 1.06 ± 0.02a 0.55 ± 0.03b 16.27 ± 0.17a 1.01 ± 0.06a 2.12 ± 0.06a 0.40 ± 0.02a

TDZ 0.0 NAA 2.68

19.42 ± 0.19a 12.75 ± 0.21d 1.04 ± 0.02b 0.22 ± 0.02b 34.70 ± 0.48d 1.17 ± 0.12a 0.61 ± 0.01a 16.31 ± 0.02a 1.02 ± 0.05a 1.89 ± 0.03b 0.18 ± 0.02d 1.41 ± 0.03c 0.18 ± 0.01c 3.33 ± 0.11b 94.23

TDZ 6.81 NAA 2.68

1.68 ± 0.18b 0.22 ± 0.03b 4.23 ± 0.52a 94.43

17.74 ± 0.78b 14.14 ± 0.59c 1.13 ± 0.02a 0.21 ± 0.02b 33.10 ± 2.18d 1.04 ± 0.01a 0.54 ± 0.02b 16.70 ± 0.25a 1.02 ± 0.05a 2.30 ± 0.16a 0.38 ± 0.00a

TDZ 0.0 NAA 5.36

Means followed by the same letter in the rows, do not differ from each other, Scott-Knott test, p ≤ 0.05. a Relative retention index n-alkane series (C8–C18) HP-5MS column in order of elution.

991 1027 1100 1102 1149 1153 1189 1231 1240 1300 1365 1417 1450 1500 Total (%)

RIa

1.51 ± 0.04c 0.20 ± 0.01b 3.42 ± 0.09b 94.29

19.73 ± 0.37a 15.51 ± 0.17b 1.01 ± 0.02b 0.20 ± 0.01b 31.26 ± 1.38e 1.15 ± 0.13a 0.48 ± 0.01c 16.47 ± 0.17a 1.06 ± 0.03a 1.91 ± 0.03b 0.38 ± 0.01a

Control

μM

Content (%) ± Standard deviation

Content (%) ± Standard deviation

Myrcene Limonene Linalool Isomyrcene Myrcenone trans-tagetone α –Terpineol Z-ocimenone E-ocimenone Tridecane Piperitenone oxide β-Caryophyllene α -humulene Pentadecane

991 1027 1100 1102 1149 1153 1189 1231 1240 1300 1365

1417 1450 1500 Total (%)

Compounds

RIa

18.67 ± 1.19b 13.45 ± 0.39c 1.00 ± 0.05b 0.28 ± 0.04a 33.35 ± 1.27d 1.08 ± 0.09a 0.67 ± 0.03a 16.05 ± 0.89a 0.97 ± 0.12a 1.81 ± 0.17b 0.22 ± 0.03c 1.82 ± 0.17b 0.25 ± 0.02a 3.28 ± 0.21b 92.9

TDZ 6.81 NAA 5.36

1.12 ± 0.26d 0.00 ± 0.00d 3.03 ± 0.53b 94.61

14.13 ± 0.36c 17.71 ± 0.61a 1.25 ± 0.06a 0.00 ± 0.00c 37.91 ± 1.94c 0.89 ± 0.25a 0.43 ± 0.02d 14.98 ± 0.59b 0.94 ± 0.08a 1.91 ± 0.25b 0.31 ± 0.04b

TDZ 2.27 NAA 0.0

Table 4 Compound content (%) in plantlets of L. rotundifolia cultivated in vitro and supplemented with different TDZ + NAA concentrations after 45 days.

19.14 ± 1.49a 16.25 ± 0.78b 0.97 ± 0.05b 0.26 ± 0.07a 30.24 ± 1.50e 0.98 ± 0.14a 0.63 ± 0.04a 14.90 ± 0.54b 1.10 ± 0.04a 2.21 ± 0.24a 0.23 ± 0.03c 2.06 ± 0.42a 0.28 ± 0.06a 3.97 ± 0.64a 93.22

TDZ 9.08 NAA 0.0

1.02 ± 0.04d 0.00 ± 0.00d 2.87 ± 0.25b 90.39

13.69 ± 0.78c 15.28 ± 0.93b 1.19 ± 0.10a 0.00 ± 0.00c 39.07 ± 3.56c 0.60 ± 0.07b 0.46 ± 0.06d 13.16 ± 1.12c 0.78 ± 0.06b 1.93 ± 0.14b 0.34 ± 0.03b

TDZ 2.27 NAA 2.68

13.09 ± 1.40c 14.03 ± 1.27c 0.88 ± 0.15c 0.00 ± 0.00c 41.72 ± 1.94b 0.72 ± 0.26b 0.44 ± 0.03d 13.59 ± 0.54c 0.70 ± 0.12b 1.77 ± 0.01b 0.41 ± 0.03a 1.38 ± 0.25c 0.00 ± 0.00d 2.96 ± 0.15b 91.69

TDZ 9.08 NAA 2.68

1.19 ± 0.19d 0.15 ± 0.03c 3.10 ± 0.28b 94.78

18.99 ± 0.27a 12.78 ± 0.26d 1.09 ± 0.02a 0.21 ± 0.01b 36.13 ± 0.19d 1.08 ± 0.14a 0.56 ± 0.02b 16.39 ± 0.32a 0.97 ± 0.07a 1.79 ± 0.10b 0.35 ± 0.02b

TDZ 2.27 NAA 5.36

11.09 ± 0.12d 13.88 ± 0.71c 0.73 ± 0.00c 0.00 ± 0.00c 45.57 ± 0.75a 0.48 ± 0.04b 0.42 ± 0.06d 11.83 ± 0.44d 0.53 ± 0.02c 1.92 ± 0.17b 0.00 ± 0.00e 1.96 ± 0.24a 0.00 ± 0.00d 3.60 ± 0.50a 92.01

TDZ 9.08 NAA 5.36

1.72 ± 0.17b 0.22 ± 0.03b 2.92 ± 0.06b 94.24

20.40 ± 0.61a 15.99 ± 0.11b 1.00 ± 0.03b 0.29 ± 0.02a 32.11 ± 0.64e 0.95 ± 0.21a 0.63 ± 0.04a 15.06 ± 0.48b 0.85 ± 0.04a 1.77 ± 0.09b 0.33 ± 0.04b

TDZ 4.54 NAA 0.0

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increased overall as the BAP concentration increased (Table 3). PGRs can influence metabolism via the activation or inhibition of several metabolic pathway enzymes in the synthesis of essential oils and secondary metabolites (Coste et al., 2011; Gonçalves and Romano, 2013; Sharafzadeh and Zare, 2011; Victoria et al., 2012). Changes in essential oil production and in vitro chemical composition may occur because of growth regulators. For example, auxins combined with cytokinins have been reported to increase the production of estragole synthesis in shoot cultures of Ocimum basilicum and linalool synthesis in the presence of auxin isolates (Monfort et al., 2018). Santos-Gomes and FernandesFerreira (2003) observed an increase in the essential oil of Salvia officinalis in medium with kinetin. Affonso et al. (2009) reported that plants of Thymus vulgaris in medium supplemented with indole-3-acetic acid (IAA) experienced increased volatile compound levels. Several factors of in vitro culture, such as the ontogenetic stage, photoperiod, temperature, alternative membrane system, type and concentration of PGRs, type of medium and salt concentration of the medium, can influence the content and quality of volatile compounds (Alvarenga et al., 2015; Figueiredo et al., 2008; Monfort et al., 2018; Prins et al., 2010;

Silva et al., 2017). The other compounds were also influenced as PGRs. Linalool, isomyrcene, trans-tagetone, α-terpineol, E-ocimenone, piperitenone oxide, β-caryophyllene, and α-humulene showed values below 2%. The tridecane compound showed values varying between 1.91 and 2.88%. The pentadecane contents were higher in plantlets grown in culture medium supplemented with lower PGRs (Table 3). The present study showed that changes in the amount of TDZ supplemented in culture medium can influence the volatile fraction content. The sum of the four most representative compounds in the volatile fraction from leaves ranged from 80.53 to 84.73%, including myrcene, limonene, myrcenone, and Z-ocimenone. In this study observed a 45.70% increase in myrcenone at 9.08 μM of TDZ and 5.36 μM of NAA compared with control. These PGRs did not appear to interfere with Zocimenone, tridecane, and pentadecane (Table 4). 3.4. Comparison of constituents of essential oil with PCA The influence of PGRs on volatile compounds appears to be quite variable. To understand the influence of PGRs on volatile compounds, a PCA was used to distinguish the compound differences occurring in the microplants. The data obtained from the scores (a) and loadings (b) of the PCA provide a conceptual overview of the treatments by explaining a total of 85.19% of the variance in BAP + NAA. The level of variation associated with principal component 1 was 59.16%, whereas principal component 2 explained 26.03% of the variation (Fig. 3A). Two separate clusters were observed: those with higher (above 6.66 μM) and lower (below 2.22 μM) BAP concentrations. The higher BAP cluster is located on the positive side of principal component 1 (Fig. 3A). The analysis of loadings (Fig. 3B) permitted the observation that concentrations above 6.66 μM of BAP, alone or in combination with NAA, positively influenced the myrcenone and limonene contents in this study. The plantlets cultivated in medium without growth regulators or with 2.22 μM of BAP alone or BAP and NAA combinations had higher content of myrcene and ocimenone. The analysis of loadings indicated that myrcene had a positive correlation with ocimenone. These results suggest that BAP and NAA influence the pathway of monoterpene synthesis in L. rotundifolia cultures. This study showed that the content of the compounds can be significantly changed in L. rotundifolia cultures using an in vitro propagation system with supplementation of growth regulators at different concentrations. 4. Conclusions The present study demonstrates that L. rotundifolia can be successfully propagated in vitro using nodal segment explants. TDZ was most effective for shoot proliferation and biomass production. The best results for shoot induction were obtained with 22.8 and 9.08 μM TDZ without NAA. Rooting of shoots can be achieved using MS medium without PGRs. The cytokinin concentration was found to significantly influence the volatile fraction synthesis in L. rotundifolia shoot cultures. These findings indicate that shoots regenerated from nodal segments on MS medium containing higher concentrations of BAP and NAA can increase myrcenone and limonene. Further research is needed to evaluate whether the production of the volatile compounds in L. rotundifolia shoots depends on the type and concentration of auxin combined with other cytokinins. 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).

Fig. 3. Scores (A) and loadings (B) of the principal component analysis (PCA) of the matrix of correlations built using data from growth regulator effects on major constituents of L. rotundifolia. 408

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