Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

Pesquisas Agrárias e Ambientais

DOI: https://doi.org/10.31413/nativa.v10i1.12996 ISSN: 2318-7670

Ontogenetic age and inoculation methods for the

in vitro

establishment

Eucalyptus pilularis

Smith

Maria Lopes Martins AVELAR1*, Bruno Alves MOSCARDINI1, Denys Matheus Santana Costa SOUZA1,

Letícia Vaz MOLINARI1, Douglas Santos GONÇALVES1, Júlio Cezar Tannure FARIA1,

Gilvano Ebling BRONDANI1*

1Laboratory of In Vitro Culture of Forest Species, Department of Forestry Sciences, Federal University of Lavras, Lavras, MG, Brazil.

*E-mail: maria.lma@hotmail.com; gilvano.brondani@ufla.br

(ORCID: 0000-0001-6790-685X; 0000-0002-8223-4905; 0000-0003-4256-7163; 0000-0002-2543-4628;

0000-0003-2580-8463; 0000-0001-7081-3726; 0000-0001-8640-5719)

Recebido em 15/09/2021; Aceito em 14/02/2022; Publicado em 14/03/2022.

ABSTRACT: We aimed to evaluate the in vitro establishment of nodal segments of Eucalyptus pilularis Smith

considering two origins of tissues (Or1 - epicormic shoots collected from pruned branches of selected adult

trees; Or2 - shoots collected from seminal mini-stumps) and four inoculation methods (Me1 - culture medium

supplemented with 0.5 g L-1 activated charcoal; Me2 - culture medium supplemented with 800 mg L-1 PVP30;

Me3 - exposure to light for 30 days; Me4 - exposure to dark for 7 days). At 30 days after the in vitro inoculation

of tissues, there was no establishment of tissues from epicormic shoots (Or1). Or2 resulted in lower percentages

of tissue oxidation and contamination by microorganisms, in addition to having presented establishment and

formation of shoots. Me1 resulted in a lower mean tissue oxidation, although it differed statistically only from

Me4. An origin of the tissues of ontogenetic age was a determining factor for the successful in vitro establishment

of E. pilularis. The use of the Or2 origin and the Me1, Me2, and Me3 methods are recommended to reduce

phenolic oxidation of tissues in the in vitro establishment.

Keywords: antioxidant agents; exposure to light; epicormic shoots; tissue oxidation.

Idade ontogenética e métodos de inoculação para o estabelecimento

in vitro

Eucalyptus pilularis

Smith

RESUMO: Objetivou-se avaliar o estabelecimento in vitro de segmentos nodais de Eucalyptus pilularis

considerando duas origens de tecidos (Or1 - brotos epicórmicos coletados em galhos podados de matrizes

adultas e Or2 - brotos coletados de minicepas seminais) e quatro métodos de inoculação (Me1 - meio de cultura

suplementado com 0,5 g L-1 de carvão ativado, Me2 - meio de cultura suplementado com 800 mg L-1 de PVP30,

Me3 - exposição à luminosidade por trinta dias e Me4 - exposição em ambiente com ausência de luminosidade

por sete dias). Aos 30 dias após a inoculação in vitro dos tecidos, constatou-se que não houve estabelecimento

de tecidos oriundos da Or1. A utilização de segmentos nodais provenientes da Or2 resultou em menores

percentuais de oxidação e de contaminação por microrganismos, além de ter apresentado estabelecimento e

emissão de brotos. O uso do Me1 resultou em menor média de oxidação, embora tenha diferido estatisticamente

somente do Me4. A origem dos tecidos associada à idade ontogenética foi um fator determinante para o sucesso

do estabelecimento in vitro de E. pilularis. Recomenda-se a utilização da Or2 e dos métodos Me1, Me2 e Me3 a

fim de reduzir a oxidação fenólica dos tecidos durante o estabelecimento in vitro.

Palavras-chave: agentes antioxidantes; exposição à luminosidade; brotos epicórmicos; oxidação de tecidos.

1. INTRODUCTION

Eucalyptus pilularis Smith is a tree species that stands out

in Australia for its rapid growth and its excellent-quality wood

(MOURA, 2001). It is found mainly in the coastal plains and

mountainous coastal areas of the state of New South Wales

to the south of Queensland (latitudes between 25°50’ and

37°50’ (FONSECA et al., 2010).

In Brazil, despite the potential that the species has for

sawmilling and laminating, especially in the south-eastern

region of the country (Moura et al. 1980), there are few

planted areas of it (CASTELLANO et al., 2013), and studies

on its forestry and vegetative propagation are still scarce.

However, genetic variation is observed within the species, so

it is possible to explore this variation to obtain superior

genotypes and, later, rescue them for cloning in order to

create artificial forests (SILVA et al., 2015).

Several techniques have been used for the vegetative

propagation of eucalypts species, including

micropropagation, which allows the in vitro conservation of

germplasm, the acceleration of breeding programmes

through mass production of selected genotypes, clonal

cleaning, and the possibility of rejuvenating and

reinvigorating tissues of selected plants in the adult phase

(WENDLING et al., 2014). The success of in vitro culture

depends on several factors, including those related to the

tissue, the degree of juvenility of the vegetative propagule

(ontogenetic age), the level of contamination control, and the

vigour (physiological age) of the plant from which the tissues

Avelar et al.

Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

originate (WENDLING et al., 2014; BACCARIN et al.,

2015; OLIVEIRA et al., 2015). In vitro establishment is one

of the limiting steps of micropropagation, which seeks to

obtain contamination-free tissues to continue with the other

stages through the induction of new meristems (TRUEMAN

et al., 2018). Phenolic oxidation is another recurrent

challenge to in vitro propagation, especially of woody species,

and may reduce growth and development or even kill the

tissue, thus preventing or hindering establishment

(OLIVEIRA et al., 2015; BACCARIN et al., 2015).

The use of antioxidants in the culture medium, such as

activated charcoal and polyvinylpyrrolidone (PVP), and

different incubation conditions regarding lighting are ways to

reduce tissue oxidation and contamination by

microorganisms, making a successful in vitro establishment

more likely (FAGUNDES et al., 2017; LENCINA et al.;

2018; GOLLE et al., 2020). However, there is a need to

define the ideal asepsis conditions for each species according

to the origin of the tissues.

Thus, the objective of the present study was to evaluate

the in vitro establishment of nodal segments of Eucalyptus

pilularis considering two tissue origins and four inoculation

methods.

2. MATERIALS AND METHODS

2.1. Study site and experimental material

The experiment was conducted at the Forest Nursery and

the Laboratory of In Vitro Culture of Forest Species, both

belonging to the Department of Forestry Sciences of the

Federal University of Lavras (UFLA), Lavras, Minas Gerais,

Brazil.

The experimental materials used to obtain the explants

(1.5-cm-long nodal segments containing an axillary bud and

no leaves) were derived from two tissue sources: Or1 -

epicormic shoots collected from pruned branches of 46-year-

old Eucalyptus pilularis mother plants (Figure 1A); and Or2 -

shoots collected from 1-year-old seminal mini-stumps

established in a mini-garden (Figure 1B), both from a test of

Eucalyptus and Corymbia species, established in 1974, at the

UFLA Forest Nursery (IPEF, 1984).

Figure 1. Flowchart of the process for obtaining propagules of different origins, and the different inoculation methods used for the in vitro

establishment of nodal segments of Eucalyptus pilularis.

Figura 1. Fluxograma do processo para obtenção de propágulos de diferentes origens e diferentes métodos de inoculação utilizados para o

estabelecimento in vitro de segmentos nodais de Eucalyptus pilularis.

2.2. Collection and preparation of branches for inducing

the formation of epicormic shoots

The mother plants used for pruning the branches were

selected based on visual criteria. We preferred the straightest

stem possible, free of pathogen attacks, and with branches

located in the lower portion of the canopy to minimize the

effects of tissue maturity and facilitate the cutting and

collection of branches. At the end of September 2019, the

branches were sectioned to 0.50 m length and placed in a

climate-controlled greenhouse with relative air humidity

higher than 80% and air temperature between 20 °C and 35

°C. The plants were humidified by an intermittent

nebulization system with a high-pressure and low-flow

nebulizer, automatically controlled by a humidistat. The

branches were arranged vertically in polyethylene pots (5 L)

that were filled with washed sand, without fertilization, to

induce the formation of epicormic shoots (Figure 1A). After

40 days of permanence of the branches in a greenhouse,

Ontogenetic age and inoculation methods for the in vitro establishment of Eucalyptus pilularis Smith

Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

epicormic shoots of 3 to 5 cm were collected, immersed in

autoclaved deionized water, and transported to the

laboratory.

2.3. Collection of shoots from seminal mini-stumps

The mini-stumps were obtained from seedlings produced

from seeds that were collected from the test of Eucalyptus and

Corymbia species located in the Forest Nursery of the Federal

University of Lavras (IPEF, 1984) and were established in a

seminal mini-garden under a semi-hydroponic system in

raised beds filled with sand at the Forest Nursery of the

Federal University of Lavras (UFLA), Lavras, Minas Gerais.

Shoots were collected from the mini-stumps and were used

to prepare explants for in vitro establishment.

The nutrient solution, composed of calcium nitrate (0.920

g L-1), potassium chloride (0.240 g L-1), potassium nitrate

(0.140 g L-1), monoammonium phosphate (0.096 g L-1),

magnesium sulfate (0.364 g L-1), water-soluble iron (0.040 g

L-1), boric acid (2,800 mg L-1), zinc sulfate (0.480 mg L- 1),

manganese sulfate (1,120 mg L-1), copper sulfate (0.100 mg

L-1), and sodium molybdate (0.040 mg L-1), was applied by

dripping, four times a day, at a total daily flow rate of 4 L m-

2. The electrical conductivity of the nutrient solution was kept

at approximately 2 mS m-2.

Shoots 10 cm in length were collected from the mini-

stumps in a seminal mini-garden (Figure 1B) 15 days after

pruning performed below the terminal meristem, aiming to

break the apical dominance and form axillary shoots. Then,

they were immersed in autoclaved deionized water and

transported to the laboratory.

2.4.

In vitro

establishment

For both the pruned branches and the seminal mini-

stumps, 48 h before the collection of shoots, a dimethyl 4,4'-

(o-phenylene)bis(3-thioallophanate) fungicide was applied at

a concentration of 0.5 g L-1. From the shoots collected (Or1

and Or2), the nodal segments were standardized to two

axillary buds, without leaves and 1.5 cm length. The nodal

segments (explants) were washed under running water for 5

min and then immersed in 70% alcohol solution (v/v) for 30

s with constant agitation inside a horizontal laminar flow

hood. Then they were immersed in NaOCl solution (1.00-

1.25% active chlorine) for 15 min. After each immersion in

alcohol or NaOCl, the nodal segments were washed three

times with autoclaved deionized water. Then they were

immersed in Orthocide 500® fungicide solution (50% captan

as the active ingredient; 0.5 g L-1) before inoculation. After

asepsis, the explants were inoculated vertically under aseptic

conditions in 15 cm × 2.5 cm glass test tubes containing 10

mL of WPM culture medium (LLOYD; MCCOWN, 1981).

The time from the collection of shoots under field conditions

to inoculation in culture medium was less than 2 hours.

During collection, transport, and the intervals between

disinfection and inoculation, the explants were kept

immersed in autoclaved deionized water to avoid dehydration

and preserve the turgidity of the tissues.

The culture medium was supplemented with 20 g L-1

sucrose and 6 g L-1 agar and was prepared using deionized

water. The pH of the culture medium was adjusted to 5.8 ±

0.05 with NaOH (0.1 M) and HCl (0.1 M), before autoclaving

and adding agar. Autoclaving of the culture medium was

performed at a temperature of 127 °C and pressure of 1.5 kgf

cm-2 for 20 min.

After inoculation, the explants were kept in a growth

room at 24 °C ± 1 °C under a 16-h photoperiod and 40 μmol

m-2 s-1 irradiance (quantified by a LI-250A Light Metre

radiometer, LI-COR®).

2.5. Experimental design and evaluations

The experiment was conducted in a completely

randomized design with a 2 × 4 factorial arrangement. Two

tissue origins were tested (Or1 - epicormic shoots collected

from pruned branches of 46-year-old mother plants; Or2 -

shoots collected from 1-year-old seminal mini-stumps) and

four inoculation methods (Me1 - culture medium

supplemented with 0.5 g L-1 of activated charcoal; Me2 -

culture medium supplemented with 800 mg L-1 PVP30; Me3

- exposure to light for 30 days; Me4 - exposure to dark for 7

days). At 30 days after inoculation, the percentage of tissue

oxidation, percentage of unresponsive explants (explants

with green colour and no oxidation, but absence of bud and

shoot formation), rate of fungal and/or bacterial

contamination, establishment percentage (explants free of

contamination and oxidation and that formed shoots), and

the number of shoots formed per explant were calculated

(Figure 2A-D).

Figure 2. Details of the characteristics evaluated during the in vitro

establishment of explants (nodal segments) from shoots of E.

pilularis seminal mini-stumps. A) Explant representing oxidized

tissue; B) Unresponsive explant; C) Contaminated explant; D)

Explant in vitro established. Bar = 1 cm.

Figura 2. Detalhes das características avaliadas durante o

estabelecimento in vitro de explantes (segmentos nodais)

provenientes de brotações de minicepas seminais de Eucalyptus

pilularis. A) Explante oxidado; B) Explante não-responsivo; C)

Explante contaminado; D) Explante estabelecido in vitro. Barra = 1

cm.

2.6. Data analysis

The analyses were performed in R Core Team software

(2018). The variables that did not have a normal distribution

according to the Shapiro-Wilk test (p > 0.05) but did not

show homogeneity of variances according to the Bartlett test

(p > 0.05) were arcsin-transformed. The means of the

treatments were subjected to analysis of variance (ANOVA,

p < 0.05) and compared by the Duncan test (p < 0.05).

3. RESULTS

The ontogeny of the tissues and the inoculation methods

tested in the in vitro establishment of nodal segments of

Eucalyptus pilularis influenced the morphophysiological

responses of the tissues in the evaluated characteristics

(Figure 3). The two inputs showed no interaction.

Comparing phenolic oxidation between the two tissue

origins, the shoots from seminal mini-stumps (Or2) had the

lowest means (27.9%), differing statistically from epicormic

shoots (66.2%) (Figure 3A). Regarding the inoculation

methods, incubation for 7 days in the dark (Me4) resulted in

significantly higher means of tissue oxidation (67.7%) than

the other methods (Figure 3B).

Avelar et al.

Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

The rate of unresponsive explants (Figure 3C) was

affected only by inoculation method. Exposure to the dark

for 7 days (Me4) and the presence of PVP30 in the culture

medium (Me2) resulted in the lowest means (26.5%), differing

statistically from Me3 (55.9%).

The origin of the tissues significantly affected in fungal

and/or bacterial contamination, in vitro establishment, and

number of shoots per explant. The inoculation of Or2 shoots

resulted in a lower mean contamination rate (54.4%) (Figure

3D) and higher mean establishment percentage (33.8%)

(Figure 3E) and mean number of shoots per explant (0.6

shoots) (Figure 3F). It was not possible to establish nodal

segments from epicormic shoots with high ontogenetic age

(Or1).

Figure 3. Characteristics evaluated in the in vitro establishment of nodal segments of Eucalyptus pilularis from two tissue sources (Or1 and

Or2) and subjected to four inoculation methods (Me1, Me2, Me3, and Me4). A) Oxidation (%) as a function of the origin of the tissues; B)

Oxidation (%) as a function of inoculation method; C) Unresponsive explants (%) as a function of inoculation method; D) Fungal and/or

bacterial contamination (%) as a function of tissue origin; E) In vitro establishment (%) as a function of tissue origin; F) Number of shoots

per explant as a function of the origin of the explants. The lowercase letters (a, b) above the bars represent significant differences between

treatments according to the Duncan test at 5% significance.

Figura 3. Características avaliadas no estabelecimento in vitro de segmentos nodais de Eucalyptus pilularis provenientes de duas origens de

tecidos (Or1 e Or2) e submetidos a quatro métodos de inoculação methods (Me1, Me2, Me3, e Me4). A) Oxidação (%) em função da origem

dos tecidos; B) Oxidação (%) em função do método de inoculação; C) Explantes não-responsivos (%) em função do método de inoculação;

D) Contaminação fúngica e/ou bacteriana (%) em função da origem dos tecidos; E) Estabelecimento in vitro (%) em função da origem dos

tecidos; F) Número de brotos por explante em função da origem dos explantes. As letras minúsculas (a, b) acima das barras representam

diferenças significativas entre os tratamentos de acordo com o teste de Duncan a 5% de significância.

4. DISCUSSION

The use of antioxidants, such as PVP and activated

charcoal, is one of the methods indicated for the control of

tissue oxidation, especially in woody species (AHMAD et al.,

2013). In this context, the use of activated charcoal (Me1) or

PVP30 (Me2) in the culture medium or exposure to light for

Ontogenetic age and inoculation methods for the in vitro establishment of Eucalyptus pilularis Smith

Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

30 days (Me3) reduced the oxidation of E. pilularis tissues. The

adsorption of phenolic compounds and toxic substances by

activated charcoal during the in vitro culture of woody species

has the advantage of reducing tissue oxidation, which can

improve and regulate the in vitro growth under certain

conditions (FAGUNDES et al., 2017; LENCINA et al.,

2018), corroborating the results of the present study.

However, few studies have reported its effects according to

ontogenetic age. Here, tissue with a more advanced

ontogenetic age probably exuded more phenolic compounds

in the culture medium, which may have favoured oxidation,

since younger explants are less prone to oxidation than more

mature explants (Paiva; Paiva, 2001).

PVP is a polyamide that prevents oxidation and

polymerization of phenolic compounds and is selective for

this type of substance (ZHOU et al., 2010). The use of the

antioxidant effectively reduced phenolic oxidation in E.

pilularis, as also observed by Silveira et al. (2016) in Calophyllum

brasiliense (Cambess.). However, Sartor et al. (2013) observed

that the use of PVP as an antioxidant for Dalbergia nigra (Vell.)

led to greater oxidation of explants. These differences show

that the responses found for a given trait are influenced by

the antioxidant agent and by the species studied.

Miranda et al. (2019), studying the in vitro establishment

of Eremanthus incanus, observed that PVP and activated

charcoal had non-significant effects on the oxidation

percentage of the explants, and values of 50-60% were

observed, which were higher than those found in the present

study for E. pilularis. In Rubus idaeus L., there was an increase

in oxidation in the cultivars as the dose of activated charcoal

in the culture medium increased (FAGUNDES et al., 2017).

The increase in the concentration of an antioxidant can be

harmful by adsorbing other substances from the nutrient

medium, causing undesirable effects on in vitro culture

(GALDIANO-JÚNIOR et al., 2010). Some studies report

the efficiency of supplementation of the culture medium with

activated charcoal and PVP or the combination of both in

reducing phenolic oxidation, as observed in nodal segments

of Eugenia pyriformis (ASSIS et al., 2017) and Psidium guajava

(AGUILAR et al., 2016), corroborating the results found for

E. pilularis.

Alfenas et al. (2009) indicate that, in addition to the use

of antioxidants, incubation of explants in the dark for 5-7

days may reduce oxidation. According to Termignoni (2005),

plants with high tissue oxidation should be kept in the dark

immediately after inoculation in the culture medium, where

they should remain for one week before being exposed to

light. The absence of light during the culture period of

Eugenia involucrata DC. reduced phenolic oxidation and

favoured the development of callogenic structures (GOLLE

et al., 2020). However, the inoculation method of exposure

of the tissues to the dark for 7 days (Me4) was not efficient in

reducing the phenolic oxidation in the tissues of nodal

segments, resulting in the highest percentage of oxidation

(67.6%), corroborating the results observed by Miranda et al.

(2019) in Eremanthus incanus.

Although Me4 resulted in the lowest percentages of

unresponsive explants (26.5%), i.e., explants that showed

green colour, absence of oxidation, but absence of bud and

shoot formation, this does not imply that the other explants

produced buds and did not oxidize, given that the treatment

generated many oxidized explants. Therefore, the high

percentages of phenolic oxidation in the group left in the

dark for 7 days may have resulted in tissue death.

Regarding the contamination percentages, Fagundes et al.

(2017) observed that the largest losses due to fungal

contamination in Rubus idaeus L. were found in culture

medium without the addition of activated charcoal, but the

use of antioxidant doses did not promote significant

differences. Regarding the number of shoots, in Apuleia

leiocarpa, activated charcoal increased the length of shoots and

the number of microcuttings per shoot and per micro-stump

(LENCINA et al., 2018).

Here, in E. pilularis, the inoculation method did not

influence the in vitro responses of contamination, in vitro

establishment, or number of shoots per explant. However,

the differences found between the two tissue origins for these

traits may be related to the degree of tissue juvenility, the

origin of the explants, and the physiological and

phytosanitary state of the mother plant (WENDLING et al.,

2014; OLIVEIRA et al., 2015; BACCARIN et al., 2015).

The mother plant exerts great influence on the exudation

of phenolic compounds by the in vitro tissues, which is

dependent on the genotype, the development phase of the

plant, and the season of the year in which the tissues are

collected (WERNER et al., 2009). The ontogenetic age of the

tissues can be altered by ex vitro and in vitro methods that

accelerate or delay maturation or induce juvenility

(WENDLING et al., 2014) and the nutritional, water,

phytosanitary, and light conditions under which the plants are

grown may alter the physiological age of the tissues

(WENDLING et al., 2014).

In this context, the complete reversal of maturation that

occurs through meiosis, gametogenesis, and formation of the

zygotic embryo, induction of juvenility in mature clones by

cultural treatments and reduction of ontogenetic age

characterize tissue rejuvenation (WENDLING et al., 2014).

On the other hand, the reduction in physiological age, i.e., the

increase in tissue vigour, characterizes reinvigoration

(WENDLING et al., 2014). The ideal management of a mini-

garden in terms of mineral nutrition, for example, can make

propagules more predisposed to rooting, as observed by

Lopes et al. (2016) for Eucalyptus urophylla. A plant with a

balanced nutritional status generates propagules with

carbohydrates, auxins, and metabolic compounds that are

essential for the initiation of the rhizogenic process and

formation of adventitious roots (CUNHA et al., 2009),

components that may also favour in vitro establishment.

Explants from seminal mini-stumps have a younger

ontogenetic age (higher tissue juvenility) than explants from

epicormic shoots of 46-year-old trees (lower tissue juvenility)

according to the tissue maturation gradient (ontogenetic age)

(BACCARIN et al., 2015; OLIVEIRA et al., 2015). In

addition, the phytosanitary, nutritional, water, and light

conditions (physiological age) to which mini-stumps are

subjected can be more easily controlled than the natural

conditions, which are a source of microorganisms to which

trees in the field are exposed. Thus, the inoculation of

explants from mini-stumps can favour the vigour of tissues

as linked to physiological age, enabling reinvigoration

(WENDLING et al., 2014), a lesser release of phenolic

compounds as linked to ontogenetic age, the ability to form

shoots, and lower percentages of fungal and/or bacterial

contamination, as observed in the present study. For this

reason, the in vitro establishment of explants from E. pilularis

epicormic shoots may have been compromised by the high

percentages of contamination by microorganisms and tissue

oxidation when compared to explants originated from shoots

Avelar et al.

Nativa, Sinop, v. 10, n. 1, p. 40-46, 2022.

of seminal mini-stumps. This result indicates that the

juvenility factor should be considered for in vitro

establishment when cloning selected trees by

micropropagation.

However, the temperatures and rains accumulated during

the pruning of branches and in the collection of epicormic

shoots may have influenced the ability to emit shoots and the

in vitro establishment of tissues (Oliveira et al., 2015).

According to Avelar et al. (2020), Eucalyptus pilularis emitted

the highest number of total epicomic shoots (219) at 45 days

in the greenhouse and resulted in one of the highest

percentages of in vitro establishment (60%). Differences in

seasonality at the time of installation of the experiments may

have influenced the emission of shoots on the branches and

the morphophysiological responses in the in vitro

establishment, however other combined factors may have

induced the results found in the present study.

5. CONCLUSION

The use of nodal segments collected from shoots of E.

pilularis (Or2) seminal mini-stumps showed the best results

because they had less tissue oxidation and fungal and/or

bacterial contamination, in addition to favouring the in vitro

establishment of tissues. Supplementation of the culture

medium with activated charcoal (Me1) or PVP30 (Me2) or

exposure to light for 30 days (Me3) was effective at reducing

the oxidation of E. pilularis tissues.

6. ACKNOWLEDGEMENT

We thank the National Counsel of Technological and

Scientific Development (CNPq), Coordination for

Improvement of Higher Education Personnel (CAPES) and

Research Support Foundation of the State of Minas Gerais

(FAPEMIG) for the financial support and scholarship.

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