Letícia Vaz Molinari·Denys Matheus Santana Costa Souza·Maria Lopes Martins Avelar·Sérgio Bruno Fernandes·Douglas Santos Gonçalves·Júlio Cézar Tannure Faria·Dulcineia de Carvalho·Gilvano Ebling Brondani

Abstract Hybrid combinations of Eucalyptus have increased due to expansion of plantations into unconventional areas and to the search for higher quality timber.However,most of these species have difficulties surviving in vitro cultivation.Active chlorine and sealing systems are often used to reduce contamination and increase gas exchange.The aim of the present study is to evaluate the establishment,multiplication,elongation and adventitious rooting of E.grandis × E.urophylla.Two clones (C1 and C2) and four active chlorine concentrations (0.000%,0.001%,0.003%,and 0.005%) were tested in the establishment and multiplication phases.Three sealing forms (W/M,1/M and 3/M) and the same four active chlorine concentrations were applied to the elongation phase.Two luminosities (dark and light)and three sealings (W/M,1/M and 3/M) were tested during adventitious rooting.Active chlorine concentration of 0.005% led to the lowest fungal contamination rate and to the highest in vitro establishment.Active chlorine concentration of 0.003% resulted in the greatest length and highest number of shoots per explant in the multiplication phase.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was maintained in comparison with those grown in an autoclave.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.

Keywords Asepsis·Cloning·Gas exchange·In vitro propagation

Introduction

Eucalyptusis the most cultivated genus by the forestry sector and has been widely studied to maximize plant production by using its species and hybrid combinations.Eucalyptus grandisHill (ex.Maiden) hybridization withEucalyptus urophyllaS.T.Blake has led to excellent wood quality and growth outcomes,as well as to efficient and easy vegetative propagation (Trueman et al.2018).

Micropropagation based on the proliferation of axillary buds has been recommended as an alternative to the rejuvenation/invigoration of selected hybrids ofEucalyptus(Brondani et al.2018).The number of research reports on the micropropagation ofE.grandis,E.urophyllaand their hybrids has increased in recent years following the adoption of in vitro introduction (Keret et al.2018),multiplication(Trueman et al.2018),elongation (Gallo et al.2017),adventitious rooting and acclimatization processes (Brondani et al.2018).

The need for basic studies to maximize microstumps production is clear,since these are essential for identifying strategies to overcome the limiting factors in each of the assessed processes.Several technologies have been proposed to automate the micropropagation process,including adjustments in multiplication protocols,chemical sterilization to reduce contamination,adoption of gas exchange systems presenting porous membranes,and provision of the most efficient physical environment for the system,particularly light sources (Brondani et al.2018;Batista et al.2018).

Reducing culture medium contamination is one of the main challenges in plant propagation.It is important to sterilize growth media to avoid contamination sources and eliminate microorganisms that hinder in vitro plant development and growth (Medjemem et al.2016).Recent studies of chemical sterilization based on active chlorine application have assessed ways to reduce contamination in micropropagation (Sawant and Tawar 2011;Brondani et al.2013;Furlan et al.2018;Gavilan et al.2018).However,sterilizing agents added to nutrient media can cause phytotoxicity in plants due to tissue oxidation and growth inhibition (Salles et al.2017).

Increased gas exchange in ventilation systems enhances in vitro plant growth and development (Pawowska et al.2018).These systems create a more favorable culture environment when they maintain adequate concentrations of CO2concentration is adequate,which increases photoassimilation and changes the density of stomata (Saldanha et al.2012;Martins et al.2015;Souza et al.2019).

The aim of the present study was to evaluate the micropropagation ofE.grandis×E.urophyllaclones to:(1) supplement the culture medium with active chlorine on in vitro establishment,multiplication and elongation;(2) the effect of porous membranes on in vitro elongation and adventitious rooting;and (3) luminosity in the adventitious rooting phase of these clones.

Materials and methods

Study site and experimental material

Experiments were conducted in the Laboratory of In Vitro Culture of Forest Species,Department of Forestry Sciences,Federal University of Lavras (UFLA),Lavras,Minas Gerais State,Brazil.Ministumps from twoE.grandis×E.urophyllaclones (both produced at ‘Viveiro Esteio Produção de Mudas’,localized in São João Del Rei,Minas Gerais State,Brazil) provided the tissues for producing explants.

The ministumps were established in 2015 in a clonal mini-garden system consisting of a channel filled with medium sand.Semi-hydroponic system was used to grow mini-stumps,based on nutrient solution (Table 1) application four times a day by means of dripping at total flow of 4 L m−2.

Table 1 Composition of the nutritive solution for fertigation of E.grandis × E.urophylla clone

Shoot collection and explant preparation

Long shoot nodal segments (3–4 cm) were collected twenty days after ministump apex pruning.The third and fourth terminal nodes were prepared as explants,which were subsequently immersed in autoclaved deionized water and transported to the laboratory for the introduction phase.

In vitro establishment of nodal segments

The nodal segments were washed five times in running water and immersed in fungicide solution containing 2.4 g L−1of Orthocide 500® (Captan 50% was used as active ingredient)for 15 min.Next,they were washed five times in autoclaved deionized water and immersed in 70% (v/v) alcohol solution for 30 s under constant agitation in horizontal laminar flow chamber and,then,immersed in 1% (v/v) Clarix®NaOCl solution for 15 min.Finally,the nodal segments were washed five times in autoclaved deionized water and the explants were prepared and vertically inoculated under aseptic conditions in test tubes (25 mm × 150 mm) filled with 10 mL of MS culture medium (Murashige and Skoog 1962).

Explants were kept immersed in autoclaved deionized water after collection,during transport,and between the disinfestation and inoculation steps to prevent dehydration.The culture medium was supplemented with 30 g L−1of sucrose(Synth Ltd) and 6 g L−1of agar (Merck S.A.),without using plant growth regulator.

The experiment followed a completely randomized design(DIC),employing a 2 × 4 factorial arrangement:twoE.grandis×E.urophylla(C1 and C2) clones and four active chlorine concentrations [0.000%;0.001%;0.003% and 0.005%(v/v) Clarix®] were tested with forty replications (each plot had one explant).Active chlorine application in the culture medium was carried out after autoclaving in a laminar flow chamber.

In vitro contamination (i.e.,fungal and/or bacterial),oxidation,establishment and number of shoots per explant(＞ 0.5 cm) were measured at 30 days after inoculation.The culture media were prepared in deionized water and pH was adjusted to 5.8 ± 0.05.Autoclaving was carried out at 127ºC,at pressure of 1.5 kgf cm−2,for 20 min.Explants were stored in a growth room at 25 °C ± 1 °C under a 16-h light photoperiod and irradiance of 40 μmol m−2s−1(quantified by radiometer,LI-COR®,LI-250A Light Meter).

In vitro multiplication of shoots

Shoots (± 0.5 cm long) produced in the in vitro establishment phase were subcultured in test tubes (25 mm × 150 mm)filled with 10 mL of basic WPM culture medium (Lloyd and McCown 1980) supplemented with 0.05 mg L−1of α-naphthaleneacetic acid (NAA,Sigma®),0.5 mg L−1of6-benzylaminopurine (BAP,Sigma®),20 g L−1of sucrose and 6 g L−1of agar.

The experiment followed a completely randomized design (DIC),at 2 × 4 factorial arrangement,twoE.grandis×E.urophylla(C1 and C2) clones and four active chlorine concentrations [0.000%;0.001%;0.003% and 0.005%(v/v)] were tested,with forty replications (each plot had one explant per replication).Active chlorine application in the culture medium was carried out after autoclaving in laminar flow chamber.

Oxidation and chlorosis were evaluated every 30 days,based on the grade scale proposed by Oliveira et al.(2016)(Fig.1 a–f):shoots length ＞ 0.5 cm and mean number of shoots per explant ＞ 0.5 cm.

Fig.1 Chlorosis,oxidation and vigor evaluations based on the scale of notes of E.grandis × E.urophylla clones.a 1=Nil:without chlorosis.b 2=Medium:moderate chlorosis in explant leaves.c 3=High:complete chlorosis in the leaves.d 1=Null:no oxidation.e 2=Medium:moderate oxidation at the base of the explant (grayish culture medium.f 3=High:complete shoot oxidation (blackish culture medium).g 1=Great:shoot induction and active growth,without apparent nutritional deficiency.h 2=Good:shoot induction and reduced growth.i 3=Low:little shoot development and/or senescence and death).Bar=0.5 cm

In vitro elongation of shoots

Shoots produced in the in vitro multiplication phase were standardized at 0.5 cm long and inoculated in glass flasks(250 mL capacity) under aseptic conditions.They were grown in flasks filled with 50 mL of basic WPM culture medium,supplemented with 0.5 mg L−1NAA,0.05 mg L−1BAP,20 g L−1sucrose and 6 g L−1of agar,for 30 days.

The experiment followed a completely randomized design (DIC) with a 3 × 4 factorial arrangement–three sealing forms were tested:rigid polypropylene cap without membrane (W/M),polypropylene cap with 1.0 cm hole in it (1/M),polypropylene cap with three 1.0 cm holes each (3/M);as well as four active chlorine concentrations[0.000%;0.001%;0.003% and 0.005% (v/v),with thirty replications (each plot had one explant per replication).Active chlorine application in the culture medium was carried out after autoclaving in laminar flow chamber.

Explant oxidation and vigor (scale of notes) were evaluated 30 days after inoculation in the in vitro elongation phase:shoot length ＞ 0.5 cm,mean number of shoots per explant ＞ 0.5 cm,photosynthetic pigments and leaf anatomy.

In vitro adventitious rooting of shoots

Shoots produced in the in vitro elongation phase were standardized at 2.0 cm long and inoculated in glass flasks(250 mL capacity) under aseptic conditions.They were grown in flasks filled with 50 mL of basic WPM culture medium,supplemented with 0.05 mg L−1BAP,0.2 mg L−1NAA,0.2 mg L−1of indole-3-butyric acid (IBA,Sigma®),20 g L−1of glucose (Synth Ltda) and 6 g L−1of agar,for 45 days (Brondani et al.2012).

The experiment followed a completely randomized design (DIC) with a 2 × 3 factorial arrangement,two luminosity forms (dark and light) and three sealing forms:rigid polypropylene cap without membrane (W/M),polypropylene cap with a 1.0 cm hole in it (1/M),polypropylene cap with three 1.0 cm holes each (3/M) were tested,with thirty replications (each plot had one explant per replication).

The length (cm) and diameter of the largest root (mm),the mean number of roots per explant and rooting rate were recorded 45 days after inoculation in the in vitro adventitious rooting phase.

Stomatal density

Stomatal analysis was carried out through epidermis digestion.Young leaves were clarified in 50% sodium hypochlorite commercial solution after being washed in distilled water and stained with safranin.Slides were mounted in 50%aqueous glycerin (Strasburger 1924);coverslip edges were sealed with enamel.

Slides were photographed using a digital camera (Canon A-630) coupled to a light microscope (Olympus CBB)–40 × objective lens.Anatomical characters were recorded.Three points were sampled in each plant,the recorded images showed 0.755 × 0.576 in dimension and area of 0.43488 mm2.The number of stomata in each sample unit was counted in the Image J software to calculate the stomatal density (DE).Data were recorded in electronic spreadsheet based on the formula:DE=number of stomata mm−2.

Data analysis

Analyses were processed in R software,version 3.0.3,Exp-Des package,version 1.1.2 (Ferreira et al.2013).Means calculated for the treatments were used in the statistical analyses and adjusted to the regression equations.Variables that did not show normal distribution before the Shapiro–Wilk test (at 5% significance level) were transformed into arcsin.Tukey test (at 5% significance level) was applied to the significant variables.

Results

Effect of active chlorine on in vitro establishment

Clones responded differently to our experimental conditions.C1 and C2 clone explant contamination rates at 30 days were similar (Fig.2 a).Based on the second-degree polynomial regression curve,greater active chlorine concentration led to lower explant contamination (Fig.2 b).

C1 clone recorded the lowest mean oxidized explant rate(6.8%) (Fig.2 c),lower than that of C2 clone.The best chemical sterilization results were observed in culture medium without active chlorine,since it did not show oxidized explants (Fig.2 d).

Mean in vitro establishment rates forE.grandis×E.urophyllaclones were statistically similar (C1:71.6% and C2:65.0%) (Fig.2 e).Increased application of active chlorine (0.0%,0.001%,0.003%,and 0.005%) led to increased in vitro establishment rates at 63.3%,66.6%,66.6%,and 76.6%,respectively (Fig.2 f).

The clone and active chlorine showed significant interaction in the number of shoots per explant on in vitro establishment.C1 clone stood out for the highestnumber of shoots per explant (1.13 shoots) at concentration of 0.003% (Fig.2 g).Regression curves showed second-degree polynomial behavior due to correlation between active-chlorine concentration and clones.However,numbers of shoots per explant peaked between active chlorine concentrations of 0.003% and 0.005%(Fig.2 g).

Fig.2 Features observed for in vitro establishment as function of different active chlorine concentrations(0.000%;0.001%;0.003% and 0.005%) and E.grandis Hill(ex.Maiden) × E.urophylla S.T.Blake clones (C1 and C2).a and b:Contamination rate,c and d:Shoot oxidation rate,e and f:Rate of in vitro establishment,g:Number of shoots per explant.Means followed by the same letter did not differ in Tukey’s test at 5% significance level

Effect of active chlorine on in vitro multiplication

Shoot length,chlorosis and explant oxidation features differed by clone and active chlorine concentration without significant interaction.Numbers of shoots per explant varied differently for clone and active chlorine concentration.Regression curves showed second-degree polynomial behavior.

According to the scale of notes represented in Fig.1 a,d,C1 clone had lowest mean chlorosis (1.0) (Fig.3 a) and mean oxidation (1.2) (Fig.3 c).Lower the active chlorine concentrations yielded greater chlorosis (Fig.3 b) and oxidation (Fig.3 d).

C1 and C2 clones had similar mean shoot lengths(Fig.3 d).The tallest mean shoot height (147 cm) was recorded for active chlorine concentration of 0.001%(Fig.3 e).The critical point for shoot length ranged between active chlorine concentrations of 0.001 and 0.003% (Fig.3 e).High chlorine concentrations [0.005% v/v)] can cause phytotoxicity in explants,which can be seen in the visual analysis of leaf yellowing.

Number of shoots per explant differed by clone and active chlorine concentration.C1 and 0.003% active chlorine yielded the highest mean number of shoots at 5.4 (Fig.3 g).The multiplication of axillary buds and the initiation of shoots in culture medium supplemented with chlorine after subculture showed that this effect resulted from the active chlorine addition to the culture medium.

Fig.3 In vitro multiplication features recorded for different active-chlorine concentrations(0.000%,0.001%,0.003%,and 0.005%) and E.grandis Hill(ex.Maiden) × E.urophylla S.T.Blake clones (C1 and C2).a and b:Shoot chlorosis,c and d:Shoot oxidation,e and f:Shoot length,g:Number of shoots per explant.Means followed by the same letter did not differ in Tukey’s test at 5% significance level

Active chlorine and seal form effects on in vitro elongation

In vitro shoot oxidation,vigor and mean length observed in the elongation showed no interaction between factors ‘sealing forms’ and ‘active chlorine’.These factors did,however,affect number of shoots per explant.

Greatest mean oxidation and lowest vigor were recorded for the cap treatment 1/M (2.4 and 2.21,respectively),the closest being the scale of notes 2 (Fig.1 e,h).The W/M treatment led to lower oxidation and higher vigor (Fig.4 a,c).Treatment 1/M yielded the longest shoots (3.69 cm) (Fig.4 e)and greatest stomatal density (134.9 mm2) (Fig.4 g),both means differing from the W/M treatment.

According to the scale of notes 1 represented in (Fig.1 c,g),the treatment with 0.000% active chlorine concentration recorded lowest mean oxidation rate (1.37) (Fig.4 b) and greatest mean vigor (1.41) (Fig.4 d).

According to the high-shoot polynomial regression curve,the longest shoot length (3.75 cm) (Fig.4 f) and greatest stomatal density (129.03 mm2,on average) (Fig.4 h) were recorded at concentration of 0.003%.Stomata were identified on both sides of the epidermis,characteristic of amphistomatic leaves (Fig.5 a).There were more stomata when porous membranes were used,possibly due to greater availability of CO2.In contrast,stomatal density was lower at higher concentrations of active chlorine (Fig.5 b–e).

Fig.5 Stomatal density of E.grandis × E.urophylla in vitro cultivated with 1/M and different active chlorine concentrations.a Copy of the sheet used to count the stomata.b Abaxial face of the leaf grown without active chlorine.c Abaxial face of the leaf grown with 0.001% active chlorine.d Abaxial face of the leaf grown with 0.003%active chlorine.e Abaxial face of the leaf grown with 0.005% active chlorine

Treatments with natural ventilation yielded more shoots per explant:3/M with 0.000% produced 11.12 shoots;1/M with 0.001% produced 9.75 shoots;1/M with 0.003% produced 10.37 shoots;and 1/M with 0.005% produced 10.06 shoots (Fig.4 i).

Fig.4 In vitro elongation as a function of different active chlorine concentrations (0.000%,0.001%,0.003%,and 0.005%)and sealing forms (W/M,1/M e 3/M).a and b show shoot oxidation;c and d show shoot vigor;e and fshow shoot length;g and h show stomata density;i shows number of shoots per explant.Means followed by the same letter did not differ in Tukey’s test at 5% significance level

Light source and sealing shape effect on in vitro adventitious rooting

Root length,number of roots per explant and rooting rate all differed by light source and sealing-form.Root diameter showed no interaction of these factors.

In vitro adventitious rooting results showed the longest roots (5.29 cm and 4.32 cm) (Fig.6 a),number of roots per explant (4.9 roots and 4.9 roots) (Fig.6 b) and rooting (73.0%and 68.0%) (Fig.6 e) means,which were very close in treatments carried out in the dark and under light.

The greatest root diameter results were recorded for treatments under fluorescent light source (0.48 mm,on average)(Fig.6 c) and 1/M (0.47 mm,on average) (Fig.6 d),but there was not significant difference between treatments.

Fig.6 In vitro adventitious rooting features observed as function of different light sources (dark and light) and sealing forms (W/M,1/M and 3/M).a:Root length,b:Number of roots per explant,c and d:Root diameter,e:Rooting rate;a,b and e:lowercase letters represent statistical differences among different sealing forms in the same treatment(light source);uppercase letters represent statistical differences among different light sources in the same treatment (sealing forms).Means followed by the same letter did not differ in the Tukey’s test at 5% significance level

Discussion

Active chlorine effect on in vitro establishment

Explant contamination decreased when active chlorine was added to the culture medium.This process is related to microbial metabolic pathways,since active chlorine use leads to chemical sterilization and regulates fungal photoresponse by means of differential genetic expressions (Postemsky and Curvetto 2016).

The excessive use of sterilizing agents,such as active chlorine,as well as of nutrients,can cause phytotoxicity in plants due to tissue darkening and in vitro growth inhibition(Salles et al.2017).Thus,active chlorine can have either positive or negative effects,depending on the adopted concentrations and assessed species.

In vitro establishment yielded results similar to those observed for otherEucalyptusspecies and for their hydroxides.E.urophylla×E.globulushybrid yielded 95.0% of explants with buds (Borges et al.2011).Oliveira et al.(2016) found 51.2% ofEucalyptus cloezianaexplants with buds.High contamination rates are mainly related to material origins or tissue exposure to greenhouse environmentswith high temperature and humidity.These conditions can contribute to the emergence of pathogens and make it difficult to achieve disinfection.Our results are similar to those reported by Brondani et al.(2018),who recorded high rates of in vitro establishment for protected clonal mini-garden explants.They attributed this to the type of protection that enabled greater control over environmental and nutritional conditions.Thus,tissue establishment during the in vitro introduction phase assumingly depends on both plant material (genotype) and adopted

cultivation conditions,such as chemical sterilization in culture medium.

Active chlorine effect on in vitro multiplication

According to Trueman et al.(2018),it is necessary to have few explants with contamination-free shoots in order to accomplish successful micropropagation,because the beginning of in vitro propagation is the main limiting phase.Thus,it is possible to go on with the in vitro multiplication phase,and it highlights the importance of reaching high bud-induction rates.

The highest chlorosis rates were achieved under the highest chlorine concentrations (0.003% and 0.005%),and this outcome suggests that chlorine can have phytotoxic effect on plant shoots.Borges et al.(2011) assessedEucalyptusand reported that some clones were recalcitrant during the in vitro multiplication phase,since they presented high oxidation and necrosis,conditions that limited their cultivation.The occurrence of phenolic oxidation is one of the main limiting factors for explant regeneration (Tisarum et al.2018);therefore,strategies aimed at overcoming or reducing them are relevant.

Active chlorine addition to culture medium can increase the adsorption of inhibitory substances,as well as reduce the number of microorganisms competing for water and mineral salts,and this process can favor plant development (Salles et al.2017).Using alternative raw materials in the culture medium is becoming a matter of great interest,mainly due to their lower price and high efficiency (Medjemem et al.2016).

Active chlorine and seal forms effect on in vitro elongation

The assessed features presented different responses to different sealing forms and active chlorine concentrations used inE.grandis×E.urophyllamicropropagation,at the 30th day of culture (Fig.7).

Fig.7 Shoots of E.grandis × E.urophylla clones in vitro cultivated at different active chlorine concentrations.a–d:30 days after in vitro introduction.e–h:In vitro multiplication.i–l:In vitro elongation.a,e and i:Active chlorine (0.000%).b,fand j:Active chlorine (0.001%).c,g and k:Active chlorine (0.003%).d,h and l:Active chlorine(0.005%).Bar=0.5 cm

In vitro conditions are stressful for plant growth;high CO2concentrations can cause oxidation (Tisarum et al.2018).Using porous membranes on in vitro propagation allows gas exchange between flasks’ external and internal atmosphere due to natural ventilation,as long as the CO2concentration is adequate,which results in increased growth(Martins et al.2015).Batista et al.(2017) found longer shoot length and larger number of leaves per explant in twoCapsicum annumvarieties subjected to a membrane system than in plants subjected to conventional system.

When porous membranes are used,it is possible associating stomatal density with increased photosynthesis caused by high CO2availability.Increased stomatal density and improved in vitro plant development were observed inPlectranthus amboinicusdue to the use of membranes in the containers (Silva et al.2017).

The highest oxidation rates were recorded under the highest sodium hypochlorite concentrations.Brondani et al.(2018) reported that oxidation results from the release of phenolic compounds in culture medium,which can beevidenced by culture medium darkening.Phenolic oxidation has been associated with the micropropagation of woody species,mainly in studies withE.cloeziana(Oliveira et al.2015);C.citriodora×C.torellianaandC.torelliana×C.citriodora(Souza et al.2018).

Light source and sealing shape effects on in vitro adventitious rooting

Using mixotrophic systems to increase in vitro CO2supply to plants can increase plant growth,improve physiological features and facilitate plant acclimatization to ex vitro conditions by promoting the development of the photosynthetic apparatus,as well as of the root system (Saldanha et al.2012;Souza et al.2019).Using a membrane inLippia gracilisSchauer explants resulted in optimized root system development in comparison to the conventional system (Lazzarini et al.2019).

Several environmental factors can influence in vitro morphogenetics,among them:light,temperature,oxidizing agents,growth regulators,among others (Saldanha et al.2012;Batista et al.2018;Souza et al.2019).Smirnakou et al.(2016) used fluorescent lamp light sources inAbies borisii-regisMattf.explants and recorded the best root system development results.The in vitro ventilation system allowed longer root length and diameter ofVaccinium ashei(Hung et al.2016).

Conclusions

The use of chemical sterilization is a suitable practice for in vitro culture.Active chlorine can be used for sterilization with efficiency similar to that of autoclaving.There were no phytotoxicity problems and the quality of plants grown in an environment with active chlorine was similar to that of plants grown in an autoclave environment.The increase in gas exchange in ventilation systems had a positive impact on the in vitro growth and development of plants.