In vitro ESTABLISHMENT OF Eucalyptus AND Corymbia SPECIES FROM EPICORMIC SHOOTS

The importance of Eucalyptus and Corymbia has been evident in forestry programs, mainly due to their adaptation to various environmental conditions and the multiple products that can be obtained. As the selection and cloning of superior individuals are usually performed in adulthood, the development and adaptation of the vegetative propagation techniques that enable the rescue and in vitro establishment of species are necessary. Thus, the present study aimed to induce epicormic shoots from pruned branches of adult trees and to promote the in vitro establishment of nine species: six of Eucalyptus and three of Corymbia. The material used to obtain the explants came from the selection of two 44-year-old mother plants of each species, from species, and provenance tests. The number of buds and shoots of each branch was evaluated at 15, 25, 35, and 45 days after the beginning of the experiment in a greenhouse. According to the results, the induction of epicormic shoots from pruned branches was considered a viable and effi cient technique to obtain propagule sources from adult mother plants of all species, presenting a gradual increase in the evaluated characteristics over time. Also, in vitro culture is an eff ective alternative to improve the propagation of the evaluated species, with an establishment of up to 62.5%. However, for Corymbia citriodora (Hook.) K.D. Hill & L.A.S Johnson and Eucalyptus tereticornis Smith it is necessary to develop further studies to enable the in vitro establishment.


INTRODUCTION
In the mid-1970s, eucalypts species and provenance tests were implemented in Brazil for testing potential species for industrial applications, aimed at possibly forming future forest plantations. The implementation of these tests was carried out through the 'Projeto de Desenvolvimento e Pesquisa Florestal (PRODEPEF)', together with the 'Instituto Brasileiro de Desenvolvimento Florestal (IBDF)' and 'Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)' (IPEF, 1984).
One of the last remaining and well-preserved test sites is in Lavras, Minas Gerais, in the Forest Nursery, Department of Forestry Sciences, Federal University of Lavras (UFLA), where seedlings produced from seeds from diff erent regions of Australia were planted, comprising twenty-seven species in total, of the genera Eucalyptus and Corymbia.
The cloning of superior individuals, which aims to increase the homogeneity and productivity of stands or species conservation, involves steps that begin with the selection of genotypes, followed by the selection of vegetative propagation techniques. In most cases, this selection is only performed in the adult stage, where vegetative propagation has shown certain limitations, such as reduced rooting ability of cuttings due to the advanced maturity of plant tissues, which is linked to ontogenetic age (Wendling et al., 2014).
Several propagation techniques have been adopted to promote tissue rejuvenation and/or juvenile shoot induction in adult plants, such as girdling (Hartmann et al., 2011), cutting (Trueman et al., 2013), mini-cutting (Brondani et al., 2012a), coppicing (Xavier et al., 2013), epicormic shoot induction  and micropropagation (Brondani et al., 2012b;Brondani et al., 2018;Souza et al., 2019a). These techniques can be used to rescue and clone selected trees (Baccarin et al., 2015). Some of these techniques become infeasible due to the impossibility of slaughter or girdling of the mother plants in species and provenance tests. Thus, the induction of epicormic shoots from pruned branches of selected mother plants provides a feasible alternative for obtaining juvenile shoots Baccarin et al., 2015;Oliveira et al., 2015). These buds originate from latent axillary buds, called epicormic buds, are used to reconstitute the plant crown  and can contribute to the propagation and multiplication of selected trees.
Thus, the objective of this study was to induce epicormic shoots from pruned branches of adult trees of species and provenance test and promote the in vitro establishment of nine species: six from the genus Eucalyptus and three from the genus Corymbia.

Study site and experimental material
The experiments were conducted at the Forest Nursery and Laboratory of In Vitro Culture of Forest Species, belonging to the Department of Forestry Sciences of the Federal University of Lavras (UFLA), located in the municipality of Lavras, Minas Gerais (MG) state, Brazil (21°14'S latitude; 44°59'W longitude and 919 m altitude).
The material used to obtain the explants (nodal segments) originated from two 44-year-old mother plants of each species (Table 1)

Collection and preparation of branches for the induction of epicormic shoots
The mother plants were selected based on visual criteria, ensuring that the trunk was as straight as possible, free of pathogen attacks and had branches in the lower portion of the crown to minimize the eff ects of ontogenetic age (Almeida et al., 2007) and to facilitate the cutting and collection of the branches ( Figure 1A).
The branches were sectioned into lengths of approximately 50 cm and placed in a greenhouse with controlled relative humidity and temperature (RH > 80%; temperature between 20 and 35°C) and irrigation by an intermittent misting system with high-pressure and low-fl ow nozzles, controlled automatically by a humidistat. These branches were placed vertically in polyethylene pots (5 L), which were fi lled with washed sand, without fertilization, for induction of epicormic shoots, for 45 days since collection, performed on the same day for all species ( Figure 1B).
The experiment was arranged in a randomized block design, with six species of Eucalyptus and three species of Corymbia (i.e., nine species in total), with six replicates composed of plots with two branches per pot, totalling 108 branches. Total number of epicormic buds and the total number of shoots per branch at 15, 25, 35 and 45 days after installation of the experiment were evaluated.

In vitro establishment
Shoots ( Figure 1G) were collected after the branches stayed for 45 days in the greenhouse, being applied dimethyl 4,4'-(o-phenylene) bis (3-thioallophanate) fungicide 48 h before the shoot collection (i.e., concentration of 0.5 g L -1 ). Epicormic shoots with 4 to 5 cm of length were collected, immersed in autoclaved deionized water and transported to the laboratory.
The nodal segments (standardized with two axillary buds, without leaves, and 2 to 3 cm of length) were used as explants ( Figure 1H); these explants were washed in running water for 5 min. Subsequently, they were immersed in 70% alcohol solution (v/v) for 30 s with constant agitation inside a horizontal laminar fl ow hood. They were then immersed in Clarix ® NaOCl solution (1.00-1.50% of active chlorine) for 10 min. The nodal segments were washed in autoclaved deionized water three times after immersion in alcohol and NaOCl and inoculated vertically under aseptic conditions in test tubes (15.0 cm × 2.5 cm) containing 10 mL of MS culture medium (Murashige and Skoog, 1962). Throughout the entire process, the equipment used was disinfected with 70% alcohol solution (v/v).
The time from explant collection in the fi eld until inoculation in culture medium was less than 2 h. During the collection, transport and intervals between disinfection and inoculation, the explants were kept immersed in autoclaved deionized water to prevent dehydration.
The culture medium was supplemented with 30 g L -1 of sucrose (Synth Ltda) and 6 g L -1 of agar (Merck S.A.), without adding of plant growth regulators. The culture medium was prepared using deionized water, and the pH was adjusted to 5.80 ± 0.05 with NaOH (0.1 M) and HCl (0.1 M) before autoclaving and the addition of the agar. The culture medium was autoclaved at 127ºC and 1.5 kgf cm -2 of pressure for 20 min.
After inoculation, the explants were kept in a growth room at 24 ± 1°C, a photoperiod of 16 h and irradiance of 40 μmol m -2 s -1 (quantifi ed by radiometer, LI-COR ® , LI-250A Light Metre) for 35 days.
Regarding the in vitro establishment ( Figure  1K), the experiment was performed in a completely randomized design, with nine species constituting the treatments with forty replicates composed of plots with one explant per test tube. At 35 days after inoculation for in vitro establishment, the following characteristics were evaluated: mean percentage of fungal and/or bacterial manifestation (i.e., in vitro contamination), oxidation of tissues, non-responsive explants (i.e., explants that showed green coloration and absence of oxidation, however absence of bud and/or shoot emission), in vitro establishment (i.e., explants with absence of contamination, oxidation and which bud and/or shoot emission), length of shoots (> 0.5 cm) and mean number of shoots per explant (>0.5 cm).

Data analysis
The analyses were performed using R Core Team software (2018), using the ExpDes package, version 1.1.2 (Ferreira et al., 2013). The data collected from the treatments were used to perform the polynomial regression analysis of the variables evaluated according to the evaluation times for each species in a greenhouse, and to perform statistical analysis (ANOVA) and Tukey's test at 5% of probability for in vitro establishment of explants.

Induction of epicormic buds and shoots
The data observed between species of Eucalyptus and Corymbia for the number of buds and shoots in relation to the time of evaluation were better adjusted in polynomial curves of the second degree, in which they presented higher coeffi cient of determination (R 2 ) ( Figure 2).
The number of buds (Figure 2), i.e. protruding meristems observed in the branches, from which the shoots were emitted, E. camaldulensis had the highest values (39 and 70 buds at 15 and 25 days in a greenhouse, respectively). However, in the fi rst evaluation, at 15 days, E. cloeziana, C. citriodora, C. maculata, C. torelliana and E. tereticornis did not show epicormic buds. Subsequent evaluations resulted in a higher number of buds for E. grandis (80 and 87 buds at 35 and 45 days, respectively) and lower values for E. tereticornis (7 and 10 buds at 35 and 45 days, respectively) ( Figure 2).
Among the species evaluated, regarding the sprouting rate (Figure 3), it was observed that, on the fi rst evaluation (15 days), E. grandis had the highest number of shoots (54 shoots), while E. cloeziana, C. citriodora, C. maculata, C. torelliana and E. tereticornis did not show epicormic shoots. The largest number of shoots (159, 188 and 219 shoots at 25, 35 and 45 days, respectively) was observed for E. pilularis, with the lowest values observed for E. tereticornis in all evaluations, resulting in 14 shoots at 45 days. Throughout the experiment, a gradual increase was observed for the number of epicormic buds and shoots in relation to the diff erent evaluation times (15,25,35 and 45 days after the beginning of the experiment).

In vitro establishment
At 35 days after inoculation in culture medium, the species exhibited signifi cant diff erences for percentages of contamination, with the lowest mean (20.0%) being observed for E. grandis ( Figure 4A), which diff ered statistically from the species. On the other hand, E. tereticornis and C. citriodora reached, on average, 75.0% of contamination ( Figure 4A), being one of the factors that limited the in vitro establishment, however they did not diff erentiate from E. cloeziana, E. microcorys and C. maculata.
Regarding phenolic oxidation in the tissues, the lowest means, 5 and 8%, were observed, respectively, for E. pilularis and E. microcorys ( Figure 4B), which diff ered signifi cantly from the other species.
The amount of non-responsive explants in vitro (explants that showed green coloration and no oxidation, however absence of bud and shoot emission - Figure  1C) was statistically similar for E. camaldulensis, C. citriodora and E. tereticornis, in which all the explants were responsive, and for E. pilularis and E. grandis, in which both presented 5.0% of non-responsive explants.
The numbers of shoots induced from the explants ( Figure 4E) in E. grandis (mean of 2.2 shoots), E. cloeziana (mean of 2.4 shoots), E. microcorys (mean of 1.9 shoots) and C. torelliana (mean of 2.5 shoots) were the highest observed, showing a signifi cant diff erence from the other species. For the length of the shoots ( Figure 4F), C. torelliana had the highest values (mean of 0.95 cm), diff ering statistically only for species that could not be in vitro established.

Induction of epicormic buds and shoots
The induction of epicormic buds and shoots from pruned branches was considered a viable technique for all species studied, showing a gradual increase in the characteristics evaluated over time. Several     similar studies have indicated the effi ciency of this rescue method, including plants of Ilex paraguariensis with 19-year-old , Eucalyptus benthamii with 13-year-old (Baccarin et al., 2015) and Eucalyptus cloeziana with 26-year-old . Wendling et al. (2013) used branches from adult individuals of Ilex paraguariensis to induce epicormic shoots and observed the emission of six shoots per pruned branch at 50 days. Nascimento et al. (2019) and Duarte et al. (2019), also working with Ilex paraguariensis, observed that the emission of epicormic shoots was effi cient in the propagation of the species. In addition, Maggioni et al. (2020) reported the use of epicormic shoots as a way to promote higher rooting rates in Araucaria angustifolia.
According to Wendling et al. (2013) and Pires et al. (2013), the use of this technique is especially important for germplasm conservation and selection of superior individuals, for which rescue is normally performed in the adult stage when propagation may be limited due to ontogenetic age. The physiological principle for the induction of epicormic buds and shoots in pruned branches is related to the alteration of the balance in plant growth regulation (e.g., auxin and cytokinin), favoring the bud and shoot induction .
The vegetative propagation of adult trees can be facilitated by the epicormic shoots induction, where tissues with a higher degree of juvenility can be obtained (Hartmann et al., 2011;Oliveira et al., 2015). However, in adulthood, vegetative propagation has shown limitations, such as reduced of adventitious rooting due to the advanced maturity of plant tissues (Wendling et al., 2014).
At 35 days after in vitro inoculation of Eucalyptus and Corymbia tissues, variations were observed through epicormic shoots. According to Trueman et al. (2018) the success of micropropagation is dependent on only a few explants forming shoots free of contamination, because the onset of in vitro propagation is a limiting phase.
However, when a large amount of micropropagated plants is required, higher rates of explants with shoots can be necessary to rapidly increase the number of clones produced. The diff erent percentages of in vitro contamination demonstrate that the metabolic pathway of microorganisms may be regulated by the genotype, through diff erential genetic expression in carotenoid biosynthesis and hyphal aggregation (Postemsky and Curvetto, 2016).
As for phenolic oxidation, the percentages were considered low, despite the fact that these are mother plants with advanced ontogenetic age, from which higher oxidation values were expected. Phenolic oxidation has been a problem associated with the micropropagation of woody species, as reported in several studies with E. cloeziana , E. benthamii ( Brondani et al., 2012b;Baccarin et al., 2015), Eucalyptus dunnii ( Navroski et al., 2014), Corymbia citriodora × Corymbia torelliana and Corymbia torelliana × Corymbia citriodora (Souza et al., 2019b), which reported similar response.
This allows advancement to the multiplication phase, showing the importance of high induction rates. In E. cloeziana, Oliveira et al. (2015) obtained, on average, 51.2% explants with shoots. For Eucalyptus urophylla × E. globulus, Borges et al. (2012) observed 95.0% of explants with shoots. Thus, diff erent results are observed according to the genotype and the culture conditions.
The use of epicormic shoots as a source of explants for micropropagation allows the rejuvenation/ reinvigoration of tissues from mother plants in the fi eld, which are usually found in the adult stage, when the tree tissues already show a certain level of maturity ( Trueman et al., 2018) In this context, epicormic shoots is an eff ective alternative for vegetative propagation of adult trees through in vitro culture.

CONCLUSION
The epicormic shoots from branches pruned is an effi cient technique for the rescue of eucalypts mature trees with 44-year-old.
In vitro culture is an alternative technique to improve the vegetative propagation of eucalypts adult trees, with establishment of 62.5%, except for C. citriodora and E. tereticornis, which need further studies to enable in vitro establishment.

ACKNOWLEDGMENTS
We thank the National Council for Scientifi c and Technological Development, Brazil (Conselho Nacional de Desenvolvimento Científi co e Tecnológico -CNPq), Coordination for Improvement of Higher Education Personnel, Brazil (