Optimal cytokinin/auxin balance for indirect shoot organogenesis of Eucalyptus cloeziana and production of ex vitro rooted micro-cuttings

Leandro Silva de Oliveira·Gilvano Ebling Brondani·Letícia Vaz Molinari·Rafaella Zanetti Dias·Gustavo Leal Teixeira·Ant?nio Natal Gon?alves·Marcílio de Almeida

Abstract Shoot organogenesis is critical for the shortening of long breeding cycles and circumvent the barrier of cloning mature Eucalyptus cloeziana trees. It enables large-scale production of plants from transformed tissues.This study evaluates the effect of α-naphthaleneacetic acid(NAA)，thidiazuron (TDZ) and benzylaminopurine (BAP)on the organogenesis of E. cloeziana from hypocotyls and cotyledonary leaves.In the induction stage，hypocotyls and cotyledonary leaves were established in a Murashige and Skoog (MS) culture medium supplemented with NAA or TDZ.Callus tissues were cultivated in a MS culture medium containing only BAP or different concentrations of BAP/NAA in the differentiation stage.Adventitious buds were multiplied in vitro and elongated in a WPM culture medium supplemented with 0.89 μM BAP and 0.05 μM NAA.Cotyledonary leaves exhibited the best in vitro regeneration.The induction of adventitious buds occurred only in calluses induced from TDZ.In the differentiation stage，4.4 μM BAP treatment promoted an increase of adventitious bud regeneration.Micro-cuttings from regenerated shoots were acclimatized and rooted ex vitro in mini-incubators.The results confirm the establishment of an efficient protocol for the in vitro regeneration of E.cloeziana by indirect organogenesis，providing new insights regarding cloning of this species.

Keywords Micropropagation·Recalcitrant species·Plant growth regulator·Explants source

Introduction

The combination of biotechnology and tree breeding may contribute to the increase of biomass productivity ofEucalyptusspp.(Baccarin et al.2015;Oliveira et al.2015;Brondani et al.2018).However，breeding programs aim for genetic gains inEucalyptusdue to their long vegetative periods and the need for clonal tests in commercial plantations(Wendling et al.2014a，2014b;Baccarin et al.2015).

Gene insertion via genetic transformation is a valuable tool for incorporating new traits of economic interest in genotypes ofEucalyptusin short periods of time (Dibax et al.2005).The potential gains involve better productivity and wood quality，and pest and disease resistance (Bandyopadhyay et al.1999;Girijashankar 2011).Therefore，the development of an in vitro regeneration protocol is essential to produceEucalyptustransgenic plants for field plantations(Silva et al.2013，2015).

Organogenesis and somatic embryogenesis are useful strategies for cloning desirable genetic materials for largescale production (Ribas et al.2000;Fernando et al.2016).The organogenesis of severalEucalyptusspecies (Barrueto Cid et al.1999;Nugent et al.2001;Dibax et al.2005;Zorz et al.2020)，and somatic embryogenesis (Pinto et al.2008)，have been extensively studied.However，there have been no reports for the in vitro regeneration of the Gympie messmate，Eucalyptus cloezianaF.Muell.

Several factors are involved in the in vitro regeneration ofEucalyptusspp.the explant，and the proportion and concentration of plant growth regulators being the most important ones (Hajari et al.2006).The type of explants is one of the primary reasons for plant morphogenesis success (Bandyopadhyay et al.1999;Rani et al.2003).Zygotic embryos(Ribas et al.2000)，cotyledons (Bandyopadhyay et al.1999)，hypocotyls (Azmi et al.1997;Bandyopadhyay et al.1999)，apexes (Glocke et al.2006)，and nodal segments (Brondani et al.2012a;Girijashankar，2012) have been used for in vitro regeneration ofEucalyptusspecies.The choice for explants relies on the availability and morphogenic response capacity and the biological，chemical，or physical stimuli during in vitro culture.Explants in more dedifferentiated stages show higher potential for the regeneration of adventitious buds.

The culture medium supplemented with plant growth regulators has important implications for morphogenic events.The hormonal balance established by growth regulators leads to the dedifferentiation and redifferentiation of cells from explants，promoting the formation and development of adventitious buds (Almeida et al.2012).Cytokinins and auxins are the main plant growth regulators for cell dedifferentiation，leading trans-differentiation to an alternative route，forming new structures such as adventitious buds (Almeida et al.2015).

Our study aimed to test the effects of NAA and TDZ growth regulators on in vitro induction of organogenic callus from hypocotyls and cotyledonary leaves ofE.cloeziana.BAP and NAA were applied for the differentiation of adventitious buds and for the establishment a protocol for in vitro regeneration ofE.cloeziana.

Material and methods

Seeds were collected from 26-year-old trees ofE.cloeziana，comprising open-pollinated progenies from 25 trees selected in Gympie，Queensland，Australia (26°18’ S，152°18’ E，altitude 600 m) by the Commonwealth Scientific and Industrial Research Organization (CSIRO).The breeding population was installed at the Experimental Station of the Department of Forest Sciences，“Luiz de Queiroz” Superior Agricultural School (ESALQ)，University of S?o Paulo (USP)，Anhembi，S?o Paulo State (22°47’ S，48°09’ W，altitude 500 m).

In vitro disinfection and germination of seeds

Seeds were soaked in distilled water for 12 h and then washed in running water for five minutes.Disinfection was performed by rinsing for 30 s in 70% ethanol followed by 15 min in sodium hypochlorite (2.0%-2.5% of active chlorine，v/v) with the supplement of a drop of Tween-20 (0.01%v/v).Seeds were washed six times in sterilized and deionized water.

Seeds were germinated in clear glass flasks (?=5.0 cm and height=8.0 cm) on in vitro aseptic conditions.Each flask contained 30 mL of MS culture medium (Murashige and Skoog 1962)，with half of the original concentration of each salt supplemented with sucrose at 15 g L-1and agar at 4 g L-1.At this stage，30 containers had 25 seeds each.

Induction of in vitro organogenic culture

Seedlings collected on the 20thday after radicle emergence were the source for the explants.Hypocotyl segments 0.5 cm and expanded cotyledonary leaves were aseptically excised and inoculated in clear glass containers (diameter 5 cm and height 8 cm) containing 30 mL of MS medium.The containers were filled with 0.1 g L-1of calcium pantothenate，0.1 g L-1of biotin，0.8 g L-1of polyvinylpyrrolidone (PVP)，30 g L-1of sucrose，and 4.0 g L-1of agar.Cotyledonary leaves were inoculated with their lower surface in contact with the culture medium.Containers were kept in a dark growth room.Plant growth regulators，α-naphthaleneacetic acid (NAA) and thidiazuron (TDZ) were added separately to the basic medium at different concentrations for the induction of adventitious buds (Table 1).

Table 1 Concentrations of NAA and TDZ growth regulators for organogenesis induction in hypocotyls and cotyledonary leaf explants of Eucalyptus cloeziana

A visual evaluation of callogenesis，oxidation and rhizogenesis of the explants was performed after 30 days，with a grading scale of three levels，1=low，2=moderate，and 3=high intensity.The lowest level indicated the presence of callogenesis only at the base of the explant，the callus had low phenolic exudation，a whitish tone and the formation of one adventitious root.The moderate level had intense callogenesis at the base of the explant，the phenolic exudation of the callus was yellowish and two or three adventitious roots had formed.The highest level had intense callogenesis on the entire surface of the explant，with darkened phenolic exudation from the callus and the formation of more than four adventitious roots.

The experiment was carried out in random blocks with a three-way factor arrangement (2×5×2)，testing two types of explants (hypocotyl and cotyledonary leaves)，five plant growth regulator concentrations (0.05，0.54，1.34，2.69，and 5.37 μM NAA or 0.05，0.45，1.14，2.27，and 4.54 μM TDZ) and two types of plant growth regulators (NAA and TDZ).The tests were performed with five replicates and 10 explants per replicate.

Data were subjected to the Shapiro-Wilk and Lilliefors(p＜0.05) tests，as well as Hartley’s test (p＜0.05).Overall effectiveness of treatments was assessed by ANOVA(p＜0.05).The data from qualitative factors were compared by Tukey’s test (p＜0.05) in Statistica 9.0 (StatSoft Inc 2009).

Development of in vitro adventitious buds

After the induction stage，the explants were transferred to a MS culture medium with the same composition as the previous stage supplemented with 2.0 mg L-1of activated charcoal and different concentrations of NAA and 6-benzylaminopurine (BAP):T1 (2.22 μM BAP);T2 (4.44 μM BAP);T3(2.22 μM BAP+2.69 μM NAA);T4 (2.69 μM NAA)，and T5 (5.37 μM NAA).The explants were then sub-cultivated and transferred after 30 days to the same culture medium for nutritional renovation.

After 60 days of in vitro culture (two subcultures)，adventitious bud development was tested for regeneration frequency，and evaluation of the morphogenetic pathway and ergastic substances based on histological and histochemical tests.Tissue samples were collected from ten hypocotyls and cotyledonary leaf segments from bud differentiation cultures.The samples were then submitted to specific histochemical tests for the detection of polysaccharides，starch，polyphenols and proteins.

The samples were fixed in a Karnovsky fixative solution (Karnovsky 1965) and later dehydrated in a standard ethyl-alcohol series at increasing concentrations.A hydroxymethyl methacrylate resin (Historesin，Leica?，Heidelberg，Germany) was added to the samples according to the manufacturer’s instructions.The blocks containing callus samples were longitudinally sectioned (adventitious buds) to a 0.5 cm depth with steel type C razors coupled with a manual microtome.

Histological sections were stained with periodic acid-Schiffand naphthol blue-black (Fisher 1968) and preserved in histological layers with synthetic resin (Entellan?).In positive reactions，polysaccharides from cell walls，cytoplasm and amyloplasts were stained pink，phenolic compounds were stained orange by periodic acid-Schiff，and proteins were stained blue by naphthol blue-black.The histological layers were analyzed and photographed through a light microscope (Zeiss-Jenemed 2?)and in a Micronal stereomicroscope，with images captured on the same scale with a Samsung?camera (SDC-313).

The experiment was designed in a randomized twoway factor arrangement (2×4) with two types of explants(hypocotyl and cotyledonary leaves) and five treatments with plant growth regulators (2.22 and 4.44 μM BAP only or 2.22:2.69 μM and 4.44:2.69 μM BAP:NAA)，using four replicates and eight explants per replicate.

In vitro multiplication and elongation

Adventitious buds from TDZ treatments (0.45，1.14，2.27，and 4.54 μM)，regenerated in the bud differentiation stage in T2 (4.44 μM BAP)，were transferred to clear glass containers (diameter 5 cm and height 8 cm) containing 40 mL WPM (Lloyd and McCown 1980) multiplication culture medium supplemented with BAP at 0.89 μM，NAA at 0.05 μM，sucrose at 30 g L-1，and agar at 4.0 g L-1.

Adventitious buds were transferred every 30 days to a new culture medium with the same formulation as previous.In each sub-culture，explants were detached from shoot clusters，i.e.，adventitious bud agglomerate，standardized with five 5-mm buds with normal vegetative vigor).Therefore，the number of shoot clusters produced per explant in each sub-culture stage was determined.

The multiplication rate of adventitious buds was determined at the end of each sub-culture stage，based on the following calculation:

where MR (%)=relative multiplication rate;SC1=initial number of shoot clusters;SC2=final number of shoot clusters;T1=final time;T2=initial time.

The experiment was arranged in randomized blocks with four treatments and five replicates，and four explants per replicate.

Ex vitro rooting

In vitro elongated shoots (＞ 1.5 cm) were transferred to a cell tray，12 cm3per cell，in a rectangular plastic container(47 cm×17 cm×3 cm) and covered with a polyethylene bag for the maintenance of high humidity as in a mini-incubator.

Shoots that developed adventitious roots，i.e.，microplantlets，were transferred to 55 cm3plastic tubes after 30 days.Trays and tubes were kept in mini-incubators and irrigated every day.At this stage，the micro-plantlets were submitted to an acclimatization step in a greenhouse covered with 50% black mesh，with weekly irrigations and nutrient solutions for the full acclimatization period.The microplantlets were then transferred to outdoor conditions where they were daily irrigated until development was completed.Micropropagated plants 15 cm long and completely developed ones were observed after 120 days.

Culture medium preparation and experimental conditions

Culture media were prepared with deionized water，pH adjusted to 5.8 (± 0.05) using NaOH (0.1 M) and HCl(0.1 M)，prior to agar mixing and sterilizing.They were autoclaved at 121°C and 98.06 kPa for 15 min.

During in vitro culture，the explants were maintained in a culture room at 25 (± 2°C) with a 12-h photoperiod and 40 μmol m-2s-1of irradiance.

Results

Induction stage

The results revealed different rates of oxidation for each type of explant，hypocotyl and cotyledonary leaves (Figs.1 and 2).The severity of oxidation varied according plant growth regulator concentrations.Cotyledonary explants showed a reduction in number with severe oxidation as TDZ concentration increased (Fig.1 a).Most of the explants treated with TDZ showed moderate oxidation (Fig.1 b).Conversely，high to moderate oxidation of explants from hypocotyls increased in concentrations higher than 1.34 μM NAA (Fig.2 a)，while the frequency of low oxidized ones decreased.The frequency of highly oxidized explants increased as TDZ concentration increased (Fig.2 b).

Fig.1 Percent oxidation of Eucalyptus cloeziana cotyledonary leaves in the organogenesis induction stage after 30 days of in vitro culture.a NAA concentration，and b TDZ concentration;bars=standard deviation;averages followed by the same letter，lower and uppercase，do not differ by Tukey’s test (p＜0.05);lowercase letters compare plant growth regulator concentration and uppercase letters different scales of callogenesis

Fig.2 Percent oxidation of Eucalyptus cloeziana hypocotyl leaves in the organogenesis induction stage after 30 days of in vitro culture.a NAA concentration，b TDZ concentration;bars=standard deviation;averages followed by the same letter，lower and uppercase，do not differ by Tukey’s test (p＜0.05);lowercase letters compare plant growth regulator concentration，uppercase letters compare different scales of callogenesis

Callogenesis was more frequent in TDZ-treated explants than those treated with NAA.Low frequency of callogenesis was observed in cotyledonary leaves in concentrations as high as 2.69 μM of NAA (Fig.3a).Moderate callogenesis was found for 0.05 μM TDZ.However，induction of callogenesis was higher for other concentrations of TDZ(Fig.3 b).

Fig.3 Percent callogenesis of Eucalyptus cloeziana cotyledonary leaves in the organogenesis induction stage after 30 days of in vitro culture.a NAA concentration，b TDZ concentration;bars=standard deviation，averages followed by the same lower and uppercase letters do not differ by Tukey’s test (p＜0.05).Lowercase letters compare plant growth regulator concentration，uppercase letters compare different scales of callogenesis

Conversely，callogenesis was induced at low concentrations of NAA (Fig.4 a).The results were similar to hypocotyls cultivated in the induction medium supplemented with different concentrations of TDZ.However，there was a slight increase in the percent of hypocotyl callogenesis from the level considered moderate，mainly for the 2.27 μM TDZ concentration，although the treatments did not differ statistically.(Fig.4 b).

Fig.4 Percent callogenesis of Eucalyptus cloeziana hypocotyl leaves in the organogenesis induction stage after 30 days of in vitro culture.a NAA concentration，b TDZ concentration;bars=standard deviation，averages followed by the same lower and uppercase letters do not differ by the Tukey’s test (p＜0.05);lowercase letters compare plant growth regulator concentrations and uppercase letters compare different scales of callogenesis

The beginning of callogenesis was observed after 10 days of in vitro culture.This was noticed in all treatments independent of the plant growth regulators in the induction of the organogenic culture (Fig.5 a，b).Typically，each organogenic callus was friable and a white-yellowish color (Fig.5 c).

Rhizogenesis was present in callus from hypocotyls and cotyledonary leaves，with the highest concentrations of NAA (2.69 and 5.37 μM).For treatments with 0.05 μM TDZ，adventitious roots were formed only on cotyledonary leaves (Fig.5 d).No adventitious buds were observed after the induction treatments;however，this stage was crucial for regeneration in the following stages.

Differentiation stage

The regeneration of adventitious buds inE.cloezianawas detected in callus induced with treatments with TDZ，as in the previous stage.With treatments，anthocyanins were observed in sections of the induced callus.(Fig.5 e).Calluses induced from NAA，when sub-cultivated in differentiation culture media，were highly oxidated (Fig.5 f).

In the differentiation stage，the regeneration of adventitious buds occurred only in calluses induced from hypocotyls and cotyledonary leaves in concentrations higher than 0.05 μM TDZ.The development of adventitious bud regeneration was better in the culture medium supplemented with 4.44 μM BAP (T1) from callus induced on cotyledonary leaves (Table 2).Adventitious bud regeneration in hypocotyl-induced callus was taken from the same treatment，but in lower frequencies (Table 2).Organogenesis of adventitious buds was only noted in callus induced from cotyledonary leaves (Fig.5 g).

Table 2 Regeneration frequency (%) of adventitious buds in callus induced from hypocotyls (Hyp) and cotyledonary leaves (Cot)of Eucalyptus cloeziana in the differentiation stage in treatments with BAP and NAA after 60 days of in vitro subculture

Samples were collected from organogenic calluses with moderate differentiation after 60 days of culture.Meristematic areas were observed in longitudinal cuts in the petiole region of cotyledonary leaves (Fig.6 a) and contained small，isodiametric cells undifferentiated from the callus(Fig.6 b).The development of meristematic centers was only identified in the second sub-culture and in a differentiation culture medium.Abundant cell proliferation occurred from meristematic cells with long intercellular spaces without differentiation in meristematic regions.The emergence of lumps from meristematic development was observed during the differentiation stage (Fig.6 b).Accumulation of phenol was observed (red-orange tone)，mainly in epidermic cells of the organogenic callus (Fig.6 b，c).

Indirect adventitious organogenesis from callus induced from cotyledonary leaves was observed by the vascular connection between adventitious buds and the vascular tissue of the explant (Fig.6 d).

Fig.6 Callus induced in cotyledonary leaves of Eucalyptus cloeziana:a meristem cells(arrows) in the superficial layer of the callus; b meristem developing in the callus surface (arrow); c development of adventitious buds in callus(arrow); d longitudinal cut of adventitious shoots underlying the capsular connection among maternal tissue (arrows).Bars=50 μm

Multiplication and elongation of adventitious buds

For efficiency，micropropagation relies on a high proliferation rate during the multiplication stage.The regenerated adventitious buds were transferred to the multiplication culture medium for increasing of micro-cuttings.There was an increase in the multiplication rate of the micro-cuttings during subculturing (Fig.5 h).The best performances were from TDZ 2.27，0.45 and 1.14 μM，with percentages of 35%，28%，and 26%，respectively，(Fig.7).

Fig.7 Multiplication rates (%) of micro-cuttings collected from adventitious buds induced in callus of Eucalyptus cloeziana cotyledonary leaves in treatments with TDZ over five sub-cultures

Ex vitro rooting and acclimatization of shoots

Overall，70% ofex vitrorooting were collected from shoots grown in mini-incubators for 30 days (F i g.5 i).The adventitious roots showed normal vigor and standard morphological structure，with numerous secondary roots.Rooted micro-cuttings had survival rates over 90% in the last stages of acclimatization in a shade-house at 45 days and under outdoor conditions (fully exposed to natural light) at 90 days (Fig.5 j，k).The acclimatization of elongated shoots withex vitrorooting ofE.cloezianawas performed in a mini-incubator.

Fig.5 Stages of indirect organogenesis，late rooting，and acclimatization of adventitious buds of Eucalyptus cloeziana.a Hypocotyls in the induction stage.Bar=1 cm; b Cotyledonary leaves in the induction stage.Bar=1 cm; c Details of calli in the TDZ treatment during the induction stage.Bar=50 μm; d Rhizogenesis (arrows) in cotyledonary leaves in the induction stage.Bar=0.1 cm; e Anthocyanin presence (white arrows) and roots (yellow arrows) in calluses of cotyledon leaves in the induction stage.Bar=50 μm; fOxidation in calluses induced in cotyledonary leaves.Bar=1 cm;g Adventitious buds in cotyledonary leaves.Bar=1 cm; h Micro-cuttings formed from adventitious buds in vitro multiplication.Bar=1 cm; i Micro-plant from micro-cutting rooted in a mini-incubator.Bar=1 cm; j Microplant rooted and transplanted to a tube.Bar=1 cm; k Rooted microplant 120 days after acclimatization.Bar=2 cm

Discussion

Effects of explants and plant growth regulators

Our results conf rim the morphogenic potential of cotyledonary leaves ofE.cloeziana，highlighting the effect of plant growth regulators in the regeneration of the species.Plant growth regulators are essential for in vitro morphogenesis，considering the regulation and balance of their interaction with naturally produced plant hormones (Wendling et al.2014a，2014b).The interaction among plant growth regulators often depends on the plant species and the tissues used for in vitro culture (Coenen and Lomax 1998;Salla et al.2018).Choosing the plant growth regulators that best suit organogenesis is therefore the first step to be optimized(Silva et al.2019).

Auxins and cytokinins are the major group of growth regulators involved in plant morphogenesis (Almeida et al.2015).In this study，differences among TDZ and NAA treatments during the induction stage were observed.The best results with explant callogenesis with low oxidation were observed in treatments with TDZ，regardless of the concentration.TDZ is a plant growth regulator with activities like cytokinins，acting on many morphogenic responses (Guo et al.2013)，including stimulating regeneration of adventitious shoots in various woody species (Ipekci and Gozukirmizi 2003).Specifically，forEucalyptus，TDZ has been used in callus induction of the hybridEucalyptus grandis×E.urophylla(Barrueto Cid et al.1999;Alves et al.2004)，the organogenesis ofE.globulusLabill.(Azmi et al.1997) and somatic embryogenesis ofE.microthecaF.Muell.(Mamaghani et al.2009).

One of the primary purposes of this study was to verify the type of explant that showed low oxidation and high organogenic potential from the selected tissues.Regardless of the explant，identical organogenic routes were found.Initially，the development of callus was from the superficial layers of the explants，followed by the formation of adventitious buds.

Over the course of the experiment，there was a gradual increase in the intensity of explant oxidation (Fig.5 F).The presence of oxidative substances in the organogenic induction media was apparently one of the major limiting factors for the success of in vitro bud regeneration ofE.cloeziana.Oxidative substances in the culture medium are the result of the oxidation of phenolic compounds which act as markers of plant growth regulators，cellular differentiation，and organogenesis (Silva et al.2019).The accumulation of such substances in the culture media however，might alter the induction of organogenic structures.In the organogenesis ofE.cloeziana，oxidation occurred in almost all explants with the three treatments in the differentiation of adventitious buds (F i g.5 i).The reduction of subculture periods and the addition of antioxidants to the culture medium can contribute to the reduction of phenol oxidation (Jones and Saxena 2013).Thus，the culture medium was supplemented with activated charcoal in the differentiation stage，which consequently resulted in a reduction in oxidation severity.

Callus formed in the treatments with TDZ had a reddish pigmentation suggesting anthocyanins，mostly in callus from cotyledonary leaves (Fig.5 e).Various studies have reported the presence，synthesis，and accumulation of anthocyanins，as well as its correlation with the in vitro development of cells and callus in plants (Mathur et al.2010).The correlation between the presence of anthocyanins and organogenesis has been reported forE.camaldulensisDehnh.(Dibax et al.2005)，E.gunniiHook.(Hervé et al.2001) andE.urophylla×E.grandis(Alves et al.2004).

In this study，rhizogenesis was successfully induced at the highest concentrations of NAA in callus from hypocotyls and cotyledonary leaves (Fig.5 d).The absorption of exogenous auxin possibly may have raised endogenous levels of auxin within cells and redirected the induction of adventitious roots.High endogenous auxin content in leaves is an obstacle for the in vitro regeneration of adventitious buds(Sanikhani et al.2006).

The regeneration of adventitious buds was not synchronized;the results were heterogeneous formation and growth from the explants (Fig.5 g).The differences in the in vitro regeneration of the same explant might be an implication of the genetic variation among the seeds (Tibok et al.1995).As shown in Figs.3 and 4，the best performance of callogenesis in both cotyledonary and hypocotyl leaves ofE.cloezianawas with the supplementation of TDZ to the culture medium.A residual effect of TDZ used in this induction stage might explain this result.TDZ can induce the formation of adventitious buds and somatic embryos at higher frequencies than with auxin-cytokinin combinations (Asghar et al.2013).Nevertheless，TDZ is resistant to oxidases，thereby a stable and biologically active molecule found in lower concentrations than in cytokinins (Mok et al.1987;Vinoth and Ravindhran 2018).A similar effect was found forEucalyptus salignaSm.，E.smithiiR.T.Baker，E.macarthuriiDeane et Maiden andE.macarthurii×E.grandisHill ex Maiden cultivated in a culture medium containing 0.45 and 0.90 μM TDZ (Le Roux and Van-Staden 1991).

The differences in the organogenic potential of the explants may be associated with alterations in endogenous levels of phytohormones and the cell types responsible for the induction of adventitious buds.Similar results regarding the response of the explant type to organogenesis were reported forE.grandis×E.urophylla(Barrueto Cid et al.1999).Calluses were induced at the end of hypocotyls and on the basal region of cotyledon leaf petioles.It is possibly related to the direct exposure of the explant’s vascular cambium cells to the culture medium，as it is a high-responsive tissue to adventitious bud regeneration (Hajari et al.2 006;Micock and Watt 2012).Similarly，higher rates of adventitious bud induction ofE.camaldulensisoccurred at the base of cotyledon leaf petioles (Dibax et al.2005) and from callus induced in the proximal region to petioles ofE.gunniileaves(Hervé et al.2001).

Differentiation and formation of adventitious buds occurred in treatments where BAP was the only growth regulator added to the culture medium，or when it was combined with higher NAA concentrations (Table 2).Such results emphasize the positive effect of BAP in the shoot organogenesis ofE.cloezianain the differentiation stage(Fig.5 g).Organogenesis from leaf explants was observed from the combination of auxins and cytokinins in various species (Sanikhani et al.2006).The balance among auxins and cytokinins，with a higher concentration of the latter，seems to play a crucial role in regeneration (Dibax et al.2005).InE.globulus，higher regeneration frequency was observed in treatments that combined BAP and TDZ (Azmi et al.1997)，and the use of BAP for bud differentiation was also reported forEucalyptus tereticornis(Prakash and Gurumurthi 2005;Aggarwal et al.2010) andE.camaldulensis(Dibax et al.2005).

Histological analyses were carried out in callus developed from cotyledonary leaves for the confirmation of the ontogenic origin of adventitious buds.Through histological cuts，cells with reduced isodiametric size and high nucleus/plasma ratios were observed，allowing for their distinction from undifferentiated calli cells (Fig.6 b，c).Cell clusters with the characteristics define meristematic regions or centers (Thorpe and Kumar 1993)，which show intense mitotic activity responsible for callus cellular differentiation (Dobrowolska et al.2017).

Buds were differentiated from superficial cell layers of the explants in regions with callus formation (Fig.6 a，b，c).Cells from the superficial layer of the callus showed high meristematic activity which turned into meristems，followed by their differentiation into adventitious buds (Fig.6 b，c).Meristems originated from periclinal and anticlinal divisions of epidermic and sub-epidermic callus cells.The initial organization of meristems into other tissues was evident from the histological cuts from the most external set to the central region (Fig.6 c).

Histological analyses showed that the shoots in explants from cotyledonary leaves were connected to the vascular system where they originated (Fig.6 d).These results corroborate with Almeida et al.(2012) who highlighted the influence of combinations of exogenously applied plant growth regulators (NAA/BAP and NAA/TDZ) on the activities of pro-cambium cells，which can act on different morphogenic pathways to establish mature cells.

Multiplication and elongation of adventitious buds

Our results show that the culture of adventitious buds in WPM medium supplemented with 0.89 μM BAP increased their multiplication and elongation.Elongated buds，able to be acclimatized，were produced from the 5thsub-culture in the culture medium of the multiplication stage.

Adventitious buds were multiplied through the proliferation of axillary shoots (Fig.5 h).The plant growth hormone balance between BAP (0.89 μM) and NAA (0.05 μM) was efficient in promoting the multiplication and elongation of adventitious buds with WPM culture medium.BAP corresponds to the most used cytokinin in culture media for bud multiplication ofEucalyptusspecies (Alves et al.2004).However，a variable performance was observed in the multiplication rates of micro-cuttings according to the TDZ concentration in the previous induction stage (Fig.7).

The lower rate of multiplication of buds regenerated from callus induced with 4.54 μM TDZ is possibly related to the inhibitory effect of this growth regulator.Shoots from this treatment were not elongated or exhibited significant callus formation at the base of the explants.The inhibitory effect of TDZ during in vitro elongation has been reported in woody species (Pruski et al.2005).TDZ stimulates the biosynthesis of cytokinins，increasing endogenous levels in tissues (Ru?i? and Vujovi? 2008).Therefore，treatment with 4.54 μM TDZ possibly promoted the maintenance of high levels of endogenous cytokinins in shoots，which suggests an inadequate hormone balance for in vitro multiplication and elongation.

Ex vitro rooting of buds and acclimatization

It is worth noting that micropropagation is limited by the high death rates of material when transferred toex vitroconditions (Silva et al.2019).Therefore，the results of adventitious rooting and acclimatization of micro-cuttings ofE.cloezianain the mini-incubators were adequate (F i g.5 i，j，k).The possibility of combiningex vitrorooting and shoot acclimatization has important implications for micropropagation，mainly due to cost reduction and in vitro root suppression (Baccarin et al.2015;Oliveira et al.2015;Brondani et al.2018;Silva et al.2019).The methods show the viability of the protocol in obtaining physiologically active adventitious roots (Wendling et al.2014a，2014b).

Theex vitroacclimatization of micro propagated plants is carried out progressively，increasing radiance and maintaining high relative humidity following transplanting，with a gradual reduction up to the completion of the hardening stage (Silva et al.2019).The acclimatization of the shoots is a critical stage，most likely due to their fragile features，to obtain a high survival.Mini-incubators created an adequate microenvironment for the reduction of water losses by micro-cuttings.Moreover，they promoted the adaptation of the micro-cuttings to autotrophic growth conditions and induced adventitious rooting (Brondani et al.2012b，2018).

The efficiency of callus induction from hypocotyls and cotyledons has been reported forEucalyptusspecies (Dibax et al.2 005;Silva et al.2015;Salla et al.2018).However，our results are the first report on the indirect organogenesis ofE.cloeziana.These results represent a new perspective to overcome the propagation problem of adult genotypes for this species，considered recalcitrant in adventitious rooting via mini-cuttings (Oliveira et al.2015;Zorz et al.2020)，which impedes the advancement of its breeding program.In addition，these results represent an important contribution towards understanding the organogenic pathway inEucalyptusspecies.In the present study，it was clear that greater success in the regeneration of adventitious shoots occurs when using optimal concentrations of growth regulators and at least two stages to obtain organogenic cultures (induction and differentiation stages).Therefore，the results also open up prospects for overcoming obstacles to the production of clonal seedlings of the species，and provide possibilities for using other biotechnological techniques such as genetic transformation.Our protocol could be used for the regeneration stage of genetically engineered plants of the species.Lastly，this research can help to the preservation of trees of great genetic value becauseE.cloezianais endemic to Queensland，Australia.

In conclusion，(1) the cotyledonary leaves were the most responsive explants，producing the highest number of adventitious buds;(2) in vitro regeneration of adventitious buds was only for callus induced by TDZ treatment in cotyledonary leaves;(3) the treatment with 4.44 μM BAP promoted an increase of adventitious buds from cotyledonary leaves;(4) in vitro multiplication in the WPM culture media supplemented with 0.89 μM BAP and 0.05 μM NAA ensured high multiplication rates;and，(5) acclimatization andex vitrorooting of adventitious buds regenerated from cotyledonary leaves were successfully produced in mini-incubators and later in the shade-house.

AcknowledgementsThe authors are grateful to CAPES (Coordination for the Improvement of Higher Personnel，Brazil) for their scholarship，CNPq (National Counsel of Technological and Scientific Development，Brazil) and IPEF (Forestry Science and Research Institute，Brazil) for technical support.