Pinus Spp. Somatic Embryo Conversion Under High Temperature: Eﬀect on the Morphological and Physiological Characteristics of Plantlets

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Article Pinus spp. Somatic Embryo Conversion under High Temperature: Eﬀect on the Morphological and Physiological Characteristics of Plantlets

Antonia Maiara Marques do Nascimento 1 , Priscila Alves Barroso 2 , Naysa Flavia Ferreira do Nascimento 3, Tomás Goicoa 4,5 , María Dolores Ugarte 4,5 , Itziar Aurora Montalbán 1,* and Paloma Moncaleán 1,*

1 Departamento de Ciencias Forestales, Neiker-BRTA, 01080 Arkaute, Spain; [email protected] 2 Departamento de Agronomía, Campus Professora Cinobelina Elvas, Universidade Federal do Piauí, Bom Jesus, Piauí 64900-000, Brazil; [email protected] 3 Departamento de Fitotecnia e Ciências Ambientais, Universidade Federal da Paraíba, Areia, Paraíba 58397-000, Brazil; [email protected] 4 Departamento de Estadística, Informática y Matemáticas, Universidad Pública de Navarra, 31006 Pamplona, Spain; [email protected] (T.G.); [email protected] (M.D.U.) 5 INMAT2, Universidad Pública de Navarra, 31006 Pamplona, Spain * Correspondence: [email protected] (I.A.M.); [email protected] (P.M.); Tel.: +34-637-423-096 (I.A.M.); +34-636-184-806 (P.M.) Received: 20 October 2020; Accepted: 5 November 2020; Published: 8 November 2020

Abstract: Climatic variations in the current environmental scenario require plants with tolerance to sudden changes in temperature and a decrease in water availability. Accordingly, this tolerance will enable successful plantations and the maintenance of natural and planted forests. Consequently, in the last two decades, drought tolerance and high temperatures in conifers have been an important target for morphological, physiological, and epigenetic studies. Based on this, our research team has optimized different stages of somatic embryogenesis (SE) in Pinus spp. improving the success of the process. Through this method, we can obtain a large amount of clonal material and then analyze the somatic plants under different conditions ex vitro. The analysis of the morphological and physiological parameters in somatic embryos (ses) and plants with different tolerances to abiotic stress can provide us with valuable information about the mechanisms used by plants to survive under adverse environmental conditions. Thus, the objective of this work was to evaluate the influence of high temperatures (23, 40, 50, and 60 ◦C, after 12 weeks, 90, 30, 5 min, respectively) on the morphology of somatic embryos obtained from Pinus radiata D.Don (Radiata pine) and Pinus halepensis Mill. (Aleppo pine). In addition, we carried out a physiological evaluation of the somatic plants of P. radiata submitted to heat and water stress in a greenhouse. We observed that the number of somatic embryos was not affected by maturation temperatures in both species. Likewise, P. radiata plants obtained from these somatic embryos survived drought and heat stress in the greenhouse. In addition, plants originating from embryonal masses (EMs) subjected to high maturation temperature (40 and 60 ◦C) had a significant increase in gs and E. Therefore, it is possible to modulate the characteristics of somatic plants produced by the manipulation of environmental conditions during the process of SE.

Keywords: abiotic stress; embryonal masses; Pinus halepensis; Pinus radiata; somatic embryogenesis; somatic plantlets; tolerance

Forests 2020, 11, 1181; doi:10.3390/f11111181 www.mdpi.com/journal/forests Forests 2020, 11, 1181 2 of 14

1. Introduction In the current climate change scenario, research is needed to enable plants to have greater water use efficiency in different environmental conditions [1] and to develop drought tolerance [2] and thermotolerance [3]. This thermotolerance can be achieved by changing the conditions in the different stages of somatic embryogenesis (SE) without it being necessary to enforce any change in the DNA, but only by allowing the formation of epigenetic memory [4,5]. Pinus halepensis is an important forest tree for reforestation in Mediterranean regions, because it has adapted to drought conditions with high temperatures [6,7]. On the other hand, Pinus radiata D. Don is a forest tree of economic importance due to the capacity it has for intensive use of its wood [8]. Preliminary studies with P. radiata species have reported that the high heat and drought tolerance in environments with climate variation is dependent on the ecotype [2,9]. The same observation has been made with Basque Country/Spain ecotypes, which are more sensitive to water stress compared to ecotypes from other parts of the world [2,9]. SE is a biotechnological tool which consists in the dedifferentiation of somatic cells and their subsequent cell re-differentiation in somatic embryos (ses), through genetic reprogramming [10]. Based on the morphogenetic response, SE is widely used to obtain large amounts of cloned material from elite material in response to an external stress stimulus [11–17]. In recent years, several studies have reported the influence of high temperature in the induction of SE in Pinus spp. [18–21]. In this regard, the effect of different temperatures applied in the initiation [4,19–21] and maturation [4,20,22,23] stages of SE process has been reported at 18, 23, or 28 ◦C. Recently, our research group has reported that somatic plants of P. radiata coming from EMs initiated at different temperatures have a different behavior in the greenhouse under water stress conditions [24]. Taking into account the abovementioned studies, in this work, we used higher maturation temperatures than those described in previous studies with P.radiata and P.halepensis. Thus, the objective of this work was to evaluate the influence of high temperatures (23 (control temperature), 40, 50, and 60 ◦C, after 12 weeks, 90, 30, 5 min, respectively) applied during the maturation stage of the SE in terms of both quantity and morphology of the ses obtained of P. radiata and P. halepensis. Moreover, we carried out a physiological evaluation of the obtained P. radiata plantlets subjected to heat and water stress in the greenhouse in order to test the possible improvement related to heat and water stress tolerance.

2. Materials and Methods

2.1. Experiment I EMs obtained from immature female cones of P. radiata, were collected from four mother trees in a seed orchard established by Neiker-BRTA in Deba (Spain) and P. halepensis cones were collected from five mother trees in Berantevilla (Spain). The immature seeds were extracted and surface sterilized following Montalbán et al. [25]. Seed coats were removed and intact megagametophytes excised out aseptically were initiated and proliferated following the protocol described by Montalbán et al. for P. radiata [25] and for P. halepensis [26]. For P.radiata SE, the basal medium was Embryo Development Medium (EDM) (Duchefa Biochemie, Amsterdam, Netherlands) [27]. The maturation of EMs was performed following the protocol described by Montalbán et al. [28]. The EMs were briefly suspended in liquid basal medium devoiding plant growth regulators and then filtered on a filter paper in a Büchner funnel. Each filter paper, with 0.08 g of EMs, was placed in the corresponding maturation media. The basal medium was supplemented 1 1 ® with 60 g L− of sucrose, 9 g L− of Gelrite (Duchefa Biochemie, Amsterdam, Netherlands), 60 µM of abscisic acid (ABA), and the amino acid mixture of EDM medium used for initiation and proliferation 1 1 1 1 (550 mg L− of L-glutamine, 525 mg L− of asparagine, 175 mg L− of arginine, 19.75 mg L− of citrulline, 1 1 1 1 19 mg L− of ornithine, 13.75 mg L− of lysine, 10 mg L− of alanine, and 8.75 mg L− of proline) [27]. ForestsForests 20202020,, 1110,, 1181x FOR PEER REVIEW 3 of 1416

For P. halepensis SE, the basal medium used was DCR medium (Duchefa Biochemie, Amsterdam, Netherlands)For P. halepensis [29] supplementedSE, the basal with medium 75 µM used of wasABA, DCR 60 mediumg L−1 of sucrose, (Duchefa 9 g Biochemie, L−1 of Gelrite Amsterdam,® and the 1 1 ® Netherlands)amino acid mixture [29] supplemented of the EDM medium. with 75 µTheM ofmaturati ABA, 60on gof L the− of 0.08 sucrose, g of EMs/ 9 g Lplate− of was Gelrite carriedand out the as aminodescribed acid above. mixture of the EDM medium. The maturation of the 0.08 g of EMs/ plate was carried out as describedThe cultures above. were kept at different maturation temperatures (MT) (23 (control temperature), 40, 50, andThe 60 cultures °C) during were keptdifferent at different incubation maturation periods temperatures (12 weeks, 90, (MT) 30, (235 min, (control respectively). temperature), Based 40, 50,on andprevious 60 ◦C) studies, during we different observed incubation that extreme periods temper (12 weeks,atures 90, cannot 30, 5 min,be applied respectively). over extended Based on periods previous as studies,the ECLs we are observed killed. Once that the extreme different temperatures treatments cannot had finished, be applied all cultures over extended were kept periods in darkness as the ECLs at 23 are°C. killed. Once the different treatments had finished, all cultures were kept in darkness at 23 ◦C. Germination and and acclimatization acclimatization of of cotyledonary cotyledonary ses ses were were performed performed according according to Montalbán to Montalb andán andMoncaleán Moncale [30].án [30]. Therefore, four four different diﬀerent temperatures temperatures were were tested tested in insixsix established established cell celllines lines (ECLs) (ECLs) (R2, R9, (R2, R16, R9, R16,R49, R130, R49, R130,and R138 and for R138 P. radiata for P. and radiata H2, andH23, H2, H48, H23, H60, H48, H153, H60, and H153,H204 for and P. H204halepensis for),P. in halepensis a factorial), indesign a factorial with eight design repetitions with eight (plates) repetitions per treatment (plates) and per ECL. treatment After and16 weeks ECL. from After the 16 beginning weeks from of thethe beginningexperiments, of the experiments,number of normal the number mature ofsomatic normal embryos mature (NNE) somatic (for embryos P. radiata (NNE) and (forP. halepensisP. radiata, andFigureP. halepensis1a,b, respectively), Figure1a,b, and respectively)abnormal somatic and abnormalembryos (NAE) somatic for embryos P. radiata (NAE) and P. forhalepensisP. radiata, Figureand P.1c,d, halepensis respectively), Figure per1c,d, 0.08 respectively) g of EMs were per count 0.08ed. g ofNAE EMs displayed were counted. abnormal NAE morphology, displayed manifested abnormal morphology,by precocious manifested germination, by precociousand in some germination, cases by a lack and of in cotyledons some cases [28] by. aAdditionally, lack of cotyledons the length [28]. Additionally,(LE) and width the (WE) length of (LE)960 NNE and widthwas measured. (WE) of 960After NNE two was months measured. in germination After two medium, months the in germinationpercentage of medium, the germination the percentage for the NNE of the was germination calculated. for the NNE was calculated.

(a) P. radiata (c) P. radiata

(b) P. halepensis (d) P. halepensis

Figure 1.1. Somatic embryos showing distinct morphologies:morphologies: ( a) normal somatic embryo (NNE)(NNE) forfor PinusPinus radiata D.Don; (b)) normalnormal somaticsomatic embryoembryo (NNE)(NNE) forfor Pinus halepensis Mill.;Mill.; (c) abnormal somatic embryo (NAE)(NAE) forfor P.P. radiataradiata;(; (dd)) abnormalabnormal somaticsomatic embryoembryo (NAE) (NAE) for forP. P. halepensis halepensis,, bar bar= =2 2mm. mm.

2.2. Experiment II Six-month-old radiata pine plants (Figure2 2)) werewere analyzedanalyzed inin OctoberOctober 2019.2019. TwentyTwenty plantsplants werewere used per MT;MT; oneone fourthfourth ofof eacheach MTMT werewere randomlyrandomly selectedselected andand submittedsubmitted toto thethe followingfollowing fourfour treatments:treatments: greenhouse greenhouse temperature temperature (GT) (GT) at at23 23°C ◦andC and under under an irrigation an irrigation rate of rate three of threetimes timesper week per week(UI); GT (UI); at GT 23 at°C 23 and◦C andwithout without irrigation irrigation (NI); (NI); GT GTat 40 at 40°C◦ Cand and UI; UI; GT GT at at 40 40 °C◦C and and NI. IrrigationIrrigation conditions were maintained for for two two weeks, weeks, an andd GT GT at at 40 40 °C◦C two two hours/day hours/day for for five ﬁve days. days.

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ForestsSummarizing,2020, 11, 1181 in this analysis 16 treatments were considered for each species (four MT × two4 ofGT 14 × two irrigation condition) in a factorial with five repetitions (plants), totaling 80 plants analyzed.

Figure 2.2.Pinus Pinus radiata radiataD.Don D.Don plantlets plantlets obtained obtained from embryonal from embryonal masses maturated masses atmaturated high temperatures at high temperaturesduring the hardening during the stage hardening in the greenhouse, stage in the bar greenhouse,= 8 cm. bar = 8 cm.

Summarizing, in this analysis 16 treatments were considered for each species (four MT two After two weeks from the beginning of the greenhouse experiments, plantlets from the greenhouse× GT two irrigation condition) in a factorial with ﬁve repetitions (plants), totaling 80 plants analyzed. with× a temperature at 23 °C with or without irrigation or with a greenhouse temperature at 40 °C with or withoutAfter twoirrigation, weeks started from the to beginning present external of the greenhousesymptoms of experiments, drought stress plantlets such as from needle the greenhouseepinasty or withapical a curvature temperature [9]. at Subsequently, 23 ◦C with or the without leaf water irrigation status or and with gas a greenhouseexchange parameters temperature were at 40measured.◦C with or without irrigation, started to present external symptoms of drought stress such as needle epinasty or The water potential (Ψleaf, MPa) of one needle per plant, was measured at predawn using a Scholander chamberapical curvature (Skye SKPM [9]. Subsequently, 1400) and the the pressure-equilibration leaf water status and technique gas exchange [31]. parameters were measured. The waterThe response potential of ( Ψinstantleaf, MPa) net ofphotosynthesis one needle per (A plant,N, µmol was CO measured2 m−2s−1), stomatal at predawn conductance using a Scholander (gs, mmol Hchamber2O m−2s− (Skye1), and SKPM instant 1400) leaf andtranspiration the pressure-equilibration (E, mmol H2O m− technique2s−1) were quantified [31]. at midday with a LI- 2 1 6400XTThe Portable response Photosynthesis of instant net System photosynthesis (Li-Cor Bios (Aciences)N, µmol equipped CO2 m with− s− the), stomatal 6400-05 Clear conductance Conifer 2 1 2 1 (Chambergs, mmol (Li-Cor H2O m Biosciences).− s− ), and instant leaf transpiration (E, mmol H2O m− s− ) were quantiﬁed at midday with a LI-6400XT Portable Photosynthesis System (Li-Cor Biosciences) equipped with the 2.3.6400-05 Statistical Clear Analysis Conifer Chamber (Li-Cor Biosciences).

2.3. StatisticalFor experiment Analysis I, ECLs were considered as a block in the model to decrease variability. Deviance analysis was performed with the Χ2 test (p < 0.05) to assess the effect of temperature on the parameters For experiment I, ECLs were considered as a block in the model to decrease variability. Deviance studied. According to the data distribution, Poisson (NNE and NAE) or Gamma (LE and WE) analysis was performed with the X2 test (p < 0.05) to assess the effect of temperature on the parameters distribution for P. radiata, and Poisson (NNE and NAE) and normal (LE and WE) were used for P. studied. According to the data distribution, Poisson (NNE and NAE) or Gamma (LE and WE) halepensis. The data were analyzed using R software®, version 3.6.1. [32] using the general linear distribution for P. radiata, and Poisson (NNE and NAE) and normal (LE and WE) were used for model (glm) function. P. halepensis. The data were analyzed using R software®, version 3.6.1. [32] using the general linear To assess the effect of maturation temperature on the percentage of germinated NNE and the model (glm) function. percentage of surviving plants, a logistic regression and the corresponding analysis of deviance was To assess the effect of maturation temperature on the percentage of germinated NNE and the conducted. The cell line was included in the model to cope with variability. When required, a percentage of surviving plants, a logistic regression and the corresponding analysis of deviance quasibinomial family was considered to deal with overdispersion. was conducted. The cell line was included in the model to cope with variability. When required, For experiment II, an analysis of variance was conducted to assess the effects of MT, greenhouse a quasibinomial family was considered to deal with overdispersion. temperature (GT), and irrigation condition (I) on water potential, instant net photosynthesis, stomatal For experiment II, an analysis of variance was conducted to assess the effects of MT, greenhouse conductance, and instant leaf transpiration. Full models with the complete interaction MT × GT × I temperature (GT), and irrigation condition (I) on water potential, instant net photosynthesis, stomatal were fitted. conductance, and instant leaf transpiration. Full models with the complete interaction MT GT I After the analysis of variance was conducted, differences in means between treatment× × were fitted. combinations were assessed using the Tukey post-hoc test (α = 0.05) adjusted for multiple After the analysis of variance was conducted, differences in means between treatment combinations comparisons. were assessed using the Tukey post-hoc test (α = 0.05) adjusted for multiple comparisons.

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3. Results 3. Results

3.1.3.1. Experiment Experiment I I

3.1.1.3.1.1. P.P. radiata radiata TheThe application application of of high high temperatures temperatures caused caused stat statisticallyistically significant significant differences differences for for the the NNE NNE and and NAENAE (Table (Table 11).). However, thethe applicationapplication ofof highhigh temperaturetemperature waswas notnot statisticallystatistically significantsignificant forfor thethe LELE and and WE WE (Table (Table 11).).

TableTable 1. AnalysisAnalysis of of deviance deviance for for the the effect eﬀect of of differe diﬀerentnt maturation temperatures temperatures in in the the number number of of normalnormal (NNE) (NNE) and and abnormal abnormal somatic somatic embryos embryos (NAE) (NAE) per per 0.08 0.08 g of g embryonal of embryonal masses; masses; the length the length (LE- mm)(LE-mm) and width and width of normal of normal embryos embryos (WE-mm) (WE-mm) of Pinus of Pinus radiata radiata D.Don.D.Don.

NNENNE NAE NAE LE LE WE WE Source df Source df 2 2 2 2 xxTest2 Test p-Valuep-Value x xTest2 Test p-Valuep-Value x Testx2Test p-Valuep-Value x Testx2 Test p-Valuep-Value T 3 34.69 ≤0.05 * 27.56 ≤0.05 * 0.04 >0.05 ns 0.09 >0.05 ns T 3 34.69 0.05 * 27.56 0.05 * 0.04 >0.05 ns 0.09 >0.05 ns * Significant differences≤ at p ≤ 0.05; ns nonsignificant;≤ df—degrees of freedom. * Significant differences at p 0.05; ns nonsignificant; df—degrees of freedom. ≤ There was a tendency to increase the NNE (Figure 3a) and the NAE (Figure 3a) from MT 23 °C There was a tendency to increase the NNE (Figure3a) and the NAE (Figure3a) from MT 23 C to to MT 50 °C, and the NNE decreased significantly at 60 °C. ◦ MT 50 ◦C, and the NNE decreased significantly at 60 ◦C.

25 3.5 a a a a a 3 20 a a b 2.5 15 2 1 1 1 1 1 1 1.5 10 1 2 1 5 0.5 Number of somatic g embryos/0.08

0 0 Characterization of somatic embryos (mm) embryossomatic Characterization of 23°C 40°C 50°C 60°C 23°C 40°C 50°C 60°C Temperature of maturation Temperature of maturation

Number of normal embryos Number of abnormal embryos Length of embryo Width of embryo

(a) (b)

FigureFigure 3. 3. SomaticSomatic embryos embryos obtained obtained per per 0.08 0.08 g g of of embryo embryonalnal masses masses submitted submitted in in different diﬀerent maturation maturation

temperaturestemperatures (23, (23, 40, 40, 50, 50, and and 60 60 °C,◦C, after after 12 12 weeks, weeks, 90, 90, 30, 30, 5 5 min, min, respectively). respectively). ( (aa)) Number Number of of normal normal andand abnormal abnormal somatic somatic embryos; embryos; ( (bb)) the the length length and and width width of of PinusPinus radiata radiata D.DonD.Don normal normal embryos. embryos. DifferentDifferent letters or numbers show significan significantt differences differences by thethe Tukey–KramerTukey–Kramer testtest ((pp ≤ 0.05). ≤ AlthoughAlthough nonsignificant nonsignificant differen differencesces were were found found between between MT MT for for LE LE and and WE WE (Table (Table 11),), aa similar trendtrend was was observed observed for for NNE NNE and and NAE. NAE. The The highest highest LE LE and and WE WE were were recorded recorded in in ses ses from from MT MT 40 40 (3.03(3.03 mm mm for for LE LE and and 1.49 1.49 for for WE) and 23 °C;◦C; the 50 °C◦C treatment showed intermediate values values and and treatmenttreatment at at 60 60 °C◦C presented presented the the lowest lowest (2.92 (2.92 mm mm for for LE LE and and 1.42 1.42 mm mm for for WE). WE). We We also also observed observed that that thethe longest longest embryos embryos were were also also the the widest widest (Figure (Figure 33b).b). TheThe application ofof highhigh temperaturetemperature was was not not statistically statistically significant significant for for the the germination germination of ses of andses andthe survivalthe survival percentage percentage of somatic of somatic plants plants in the in the greenhouse greenhouse (44%, (44%, 51%, 51%, 46%, 46%, and and 51% 51% for for 23, 23, 40, 40, 50, 50,and and 60 ◦ C,60 respectively).°C, respectively). However, However, plantlets plantlets were obtained were obtained from all from the tested all the maturation tested maturation treatments treatments(63%, 55%, (63%, 60%, and55%, 63% 60%, from and temperatures 63% from temperatures 23, 40, 50, and23, 40, 60 ◦50,C, respectively)and 60 °C, respectively) (Figure4a,b) (Figure and a 4a,b)total and of 614 a total plantlets of 614 were plantlets acclimated were acclimated in a greenhouse in a greenhouse (Figure4c). (Figure 4c).

Forests 2020, 11, 1181 6 of 14 Forests 2020, 10, x FOR PEER REVIEW 6 of 16

(a) (b) (c)

FigureFigure 4. 4. GerminationGermination and and acclimatizatio acclimatizationn of of somatic somatic plantlets plantlets from from P.P. radiata radiata obtainedobtained from from different different embryogenicembryogenic cell cell lines lines and and maturation maturation temperatures. temperatures. (a (a) )Plantlets Plantlets after after two two months months in in germination germination medium,medium, bar = 1 cm; (b) plantletsplantlets afterafter three three months months cultivated cultivated in in the the germination germination medium, medium, bar bar= 2 = cm; 2 cm;(c) plantlets(c) plantlets derived derived from from normal normal cotyledonary cotyledonary somatic somatic embryos embryos growing growing in the greenhouse,in the greenhouse, bar = 2 bar cm. = 2 cm. 3.1.2. P. halepensis 3.1.2. MaturationP. halepensis temperature affected significantly the NNE and LE. On the contrary, MT was not statisticallyMaturation significant temperature for the affected NAE and significantly WE (Table2 ).the NNE and LE. On the contrary, MT was not statistically significant for the NAE and WE (Table 2). Table 2. Analysis of deviance for the effect of different maturation temperatures in the number of Tablenormal 2. (NNE)Analysis and of aberrantdeviance somaticfor the effect embryos of differe (NAE)nt permaturation 0.08 g of temperatures embryonal masses; in the number the length of Pinus halepensis normal(LE-mm) (NNE) and widthand aberrant of normal somatic embryos embryos (WE-mm) (NAE) of per 0.08 g of embryonalMill. masses; the length (LE- mm) and width of normalNNE embryos (WE-mm) of NAE Pinus halepensis Mill.. LE WE Source df x2 TestNNE p-Value x2 TestNAE p-Value x2 Test LE p-Value x2 Test WE p-Value Source df T 3x2 Test 21.15 p-Value0.05 *x2 Test 0.98 p-Value>0.05 ns x2 Test2.44 p-Value0.05 *x2 Test 0.17 p-Value>0.05 ns ≤ ns ≤ ns T 3 *21.15 Significant ≤ di0.05fferences* at0.98p 0.05; ns>0.05nonsignificant; 2.44 df—degrees ≤0.05 of* freedom. 0.17 >0.05 ≤ * Significant differences at p ≤ 0.05; ns nonsignificant; df—degrees of freedom. Unlike in radiata pine, the increase in the MT from 23 to 40 ◦C did not promote an increase in NNE,Unlike but a in decrease radiata in pine, their the production increase in was the observed MT from (Figure 23 to 540a). °C On did the not other promote hand, an no increase significant in NNE,differences but aForests weredecrease 2020 found, 10, xin FOR their between PEER productionREVIEW the control was MT observed (23 ◦C) (Figure and temperatures 5a). On the ofother 50 or hand, 60 ◦C 7no of (Figure 16significant 5a). differences were found between the control MT (23 °C) and temperatures of 50 or 60 °C (Figure 5a). 14 3 a ab 12 a ab b 2.5 a a 10 b 2

8 1.5 6 1 1 11 1 1 4 1 1 1

Number of somatic 0.08 g embryos/ 0.5 2 Characterization of somatic embryos (mm) embryos somatic Characterization of

0 0 23°C 40°C 50°C 60°C 23°C 40°C 50°C 60°C Temperature of maturation Temperature of maturation

Number of normal embryos Number of abnormal embryos Length of embryos Width of embryos

(a) (b)

Figure 5. SomaticFigure 5. Somatic embryos embryos obtained obtained per per 0.08 0.08 g g ofof embryo embryonalnal masses masses submitted submitted in different in di maturationfferent maturation temperatures (23, 40, 50, and 60 °C, after 12 weeks, 90, 30, 5 min, respectively). (a) Number of normal temperatures (23, 40, 50, and 60 C, after 12 weeks, 90, 30, 5 min, respectively). (a) Number of normal and abnormal somatic embryos;◦ (b) the length and width of normal embryos from Pinus halepensis and abnormalMill. somaticDifferent letters embryos; or numbers (b) the show length significant andwidth differences of normal according embryos to the Tukey–Kramer from Pinus test halepensis (p Mill. Different letters≤ 0.05). or numbers show significant differences according to the Tukey–Kramer test (p 0.05). ≤ Although no significant differences were detected between MT for the NAE (Table 2) (Figure 5a), the MT 50 °C promoted a significant increase in LE when compared to 60 °C, whereas MT 23 and 40 °C led to intermediate values (Figure 5b). A similar tendency was observed in WE, but in this case no significant differences were found between MT (Figure 5b). In total, 262 viable plantlets were obtained in this experiment (Figure 6c). The application of high temperature was not statistically significant for the germination percentage of ses and the survival of somatic plants in the greenhouse (22%, 30%, 24%, and 34% for 23, 40, 50, and 60 °C, respectively). Furthermore, in all ECLs and MTs, a high acclimatization rate was obtained (91.82%, 92.61%, 98.08%, and 88.91% from temperatures of 23, 40, 50, and 60 °C, respectively).

(a) (b) (c)

Figure 6. Germination and acclimatization of somatic plantlets from Pinus halepensis Mill. obtained from different embryogenic cell lines and maturation temperatures. (a) Somatic plantlets after two months in germination medium, bar = 1 cm; (b) plantlets cultivated after three months in the germination medium, bar = 2 cm; (c) plantlets derived from normal cotyledonary somatic embryos growing in the greenhouse, bar = 5 cm.

Forests 2020, 10, x FOR PEER REVIEW 7 of 16

14 3 a ab 12 a ab b 2.5 a a 10 b 2

8 1.5 6 1 1 11 1 1 4 1 1 1

Number of somatic 0.08 g embryos/ 0.5 2 Characterization of somatic embryos (mm) embryos somatic Characterization of

0 0 23°C 40°C 50°C 60°C 23°C 40°C 50°C 60°C Temperature of maturation Temperature of maturation

Number of normal embryos Number of abnormal embryos Length of embryos Width of embryos

(a) (b)

Figure 5. Somatic embryos obtained per 0.08 g of embryonal masses submitted in different maturation temperatures (23, 40, 50, and 60 °C, after 12 weeks, 90, 30, 5 min, respectively). (a) Number of normal Forests 2020,and11, 1181abnormal somatic embryos; (b) the length and width of normal embryos from Pinus halepensis 7 of 14 Mill. Different letters or numbers show significant differences according to the Tukey–Kramer test (p ≤ 0.05). Although no significant differences were detected between MT for the NAE (Table2) (Figure5a), the MT 50Although◦C promoted no significant a significant differences increase were in LEdetect whened between compared MT to for 60 the◦C, NAE whereas (Table MT 2) 23(Figure and 40 ◦C led to5a), intermediate the MT 50 °C values promoted (Figure a significant5b). A similarincrease tendencyin LE when was compared observed to 60 in°C, WE, whereas but inMT this 23 and case no significant40 °C led diff toerences intermediate were foundvalues (Figure between 5b). MT A simi (Figurelar tendency5b). was observed in WE, but in this case no significant differences were found between MT (Figure 5b). In total, 262 viable plantlets were obtained in this experiment (Figure6c). The application of high In total, 262 viable plantlets were obtained in this experiment (Figure 6c). The application of high temperaturetemperature was was not not statistically statistically significant significant for for the the germination germination percentage percentage of ses of and ses the and survival the survival of of somaticsomatic plants plants in in the the greenhousegreenhouse (22%, (22%, 30%, 30%, 24%, 24%, and and 34% 34% forfor 23, 23,40, 40,50, 50,andand 60 °C, 60 respectively).◦C, respectively). Furthermore,Furthermore, in all in ECLsall ECLs and and MTs, MTs, a a high high acclimatization acclimatization rate rate was was obtained obtained (91.82%, (91.82%, 92.61%, 92.61%, 98.08%, 98.08%, and 88.91%and 88.91% from from temperatures temperatures of of 23, 23, 40, 40, 50, 50, and and 6060 ◦°C,C, respectively). respectively).

Forests 2020, 10, x FOR PEER REVIEW 8 of 16 (a) (b) (c) 3.2. Experiment II FigureFigure 6. Germination 6. Germination and and acclimatization acclimatizatio ofn somaticof somatic plantlets plantlets from fromPinus Pinus halepensis halepensisMill. Mill. obtainedobtained from diﬀerentfrom embryogenic different embryogenic cell lines cell and lines maturation and maturation temperatures. temperatures. (a) Somatic (a) Somatic plantlets plantlets after after two two months Water Potential and Gas Exchange Parameters in germinationmonths in germination medium, bar medium,= 1 cm; bar (b )= plantlets1 cm; (b) cultivated plantlets cultivated after three after months three inmonths the germination in the germination medium, bar = 2 cm; (c) plantlets derived from normal cotyledonary somatic embryos medium,Plants bar coming= 2 cm; from (c) plantlets all treatments derived fromapplied normal during cotyledonary the maturation somatic stage embryos survived growing after in the growing in the greenhouse, bar = 5 cm. greenhouse,drought and bar thermic= 5 cm. stress in the greenhouse (Figure 7). Significant differences were observed for the effect of MT on the initial and final evaluation of water potential in plants subjected to different 3.2. Experiment stress conditions II (Table 3); but in the first evaluation no significant differences were observed for gas exchange parameters (Table 3). Water PotentialStatistically and Gassignificant Exchange differences Parameters were also found for GT for the final assessment of water

potential (Table 3). At the end of the experiment, water potential in plants at GT of 23 °C was Plants coming from all treatments applied during the maturation stage survived after the drought significantly lower (−0.3 MPa) than Ψleaf final in plants at GT of 40 °C (−0.26 MPa) by Student’s t-test and thermic(p < 0.05). stress This implies in the greenhousethat plants originating (Figure7 from). Significant EMs subjected di ff erencesto different were MTs observed had a tolerance for the to e ffect of MTheat on stress the initialin the andgreenhouse, final evaluation as plants exposed of water to potentialGT of 40 °C in were plants more subjected hydrated to compared different to stress conditionsplants (Tableexposed3); butto GT in of the 23 first °C. evaluationThe irrigation no significantregime did dinotff erencesshow signific wereant observed differences for gas forexchange this parametersparameter. (Table 3).

(a) (b) (c)

FigureFigure 7. Pinus 7. Pinus radiata radiataD.Don D.Don somatic somatic plants: plants: (a) showing needle needle epinasty epinasty and and apical apical curvature curvature at at greenhousegreenhouse temperature temperature of 40of ◦40C °C under under irrigation irrigation or or nonirrigated nonirrigated (left and and righ right,t, respectively), respectively), bar bar = = 6 cm; (6b )cm; plants (b) plants not irrigated not irrigated submitted submitted to to 40 40 and and 23 23 ◦°CC in greenhouse greenhouse (left (left and and right, right, respectively), respectively), bar = 3 cm; (c) plants not irrigated submitted to 23 and 40 °C in greenhouse (left and right, bar = 3 cm; (c) plants not irrigated submitted to 23 and 40 ◦C in greenhouse (left and right, respectively), afterrespectively), rewatering, barafter= rewatering,6 cm. bar = 6 cm.

Table 3. Analysis of variance (ANOVA) for the effect of different maturation temperatures (MT) and

greenhouse temperatures (GT) on the initial and final water potential (Ψleaf initial and Ψleaf final, respectively) in plants of Pinus radiata D.Don under irrigation and/or no irrigation conditions (I).

ANOVA Effect df Ψleaf initial MT 3 ≤0.05 * Effect df Ψleaf initial MT 3 ≤0.05 * GT 1 ≤0.05 * I 1 >0.05 ns MT × GT 3 >0.05 ns MT × I 3 >0.05 ns GT × I 1 >0.05 ns MT × GT × I 3 >0.05 ns * Significant differences at p ≤ 0.05; ns nonsignificant at p ≤ 0.05; df—degrees of freedom.

Forests 2020, 11, 1181 8 of 14

Table 3. Analysis of variance (ANOVA) for the effect of different maturation temperatures (MT) and greenhouse temperatures (GT) on the initial and final water potential (Ψleaf initial and Ψleaf final, respectively) in plants of Pinus radiata D.Don under irrigation and/or no irrigation conditions (I).

ANOVA

Effect df Ψleaf initial MT 3 0.05 * ≤ Effect df Ψleaf initial MT 3 0.05 * ≤ GT 1 0.05 * ≤ I 1 >0.05 ns MT GT 3 >0.05 ns × MT I 3 >0.05 ns × GT I 1 >0.05 ns × MT GT I 3 >0.05 ns × × * Significant differences at p 0.05; ns nonsignificant at p 0.05; df—degrees of freedom. ≤ ≤ Statistically significant differences were also found for GT for the final assessment of water potential (Table3). At the end of the experiment, water potential in plants at GT of 23 ◦C was significantly lower ( 0.3 MPa) than Ψ final in plants at GT of 40 C( 0.26 MPa) by Student’s t-test − leaf ◦ − (p < 0.05). This implies that plants originating from EMs subjected to different MTs had a tolerance to heat stress in the greenhouse, as plants exposed to GT of 40 ◦C were more hydrated compared to plants exposed to GT of 23 ◦C. The irrigation regime did not show significant differences for this parameter. A decrease in water potential was observed in plants from ECLs matured in MTs of 23 ◦C (Figure8a). Similarly, the same pattern of decline was observed when assessing water potential after Forestswater 2020 and, 10,heat x FOR stress PEER REVIEW conditions, which in turn was less than the initial water potential (Figure9 of8 16a). For this characteristic, the data fit the model with a determination coefficient above 98% (R2 = 0.98). A decrease in water potential was observed in plants from ECLs matured in MTs of 23 °C (Figure In the first evaluation, there were no significant statistical differences for AN, gs, and E (Table4). 8a). Similarly, the same pattern of decline was observed when assessing water potential after water By contrast, in the second evaluation, the AN presented significant statistical differences for the effects andMT, heat MT stressGT, conditions, GT I, and which in the in triple turn interactionwas less than MT the GTinitialI water (Table potential5). Stomatal (Figure conductance 8a). For this was × × × × 2 characteristic,statistically significant the data fit for the all model the factors with anda determination instant transpiration coefficient was above statistically 98% (R significant = 0.98). for all the factors assessed, except MT I and the triple interaction (Table5). ×

MT (°C) 0 102030405060 -0.0 16

14 -0.1 Ψ final = 0.0003x2 - 0.0357x + 1.1447 leaf a ) 12 R² = 0.9822 a -1 a s -0.2 -2 10 m 2 a a 8 (MPa) -0.3 a leaf mol CO Ψ

μ 6 (

-0.4 N

A UI 23°C = 0.0066x2 - 0.5146x + 19.884 NI 23°C = 0.0016x2 - 0.192x + 17.683 Ψ 2 4 b leaf initial = 0.0003x - 0.0318x + 1.0442 R² = 0.4262 R² = 0.3496 R² = 0.9797 2 -0.5 2 NI 40°C = 0.0118x - 0.8118x + 20.226 2 UI 40°C = -0.0141x + 1.245x - 12.572 b R² = 0.9922 R² = 0.78 -0.6 0 0204060 MT (°C) Ψleaf initial Ψleaf final 23°C UI 23°C NI 40°C UI NI 40°C

(a) (b)

Cont. 0.50 Figure 8. 9 a UI 40°C= -0.0002x2 + 0.0229x - 0.2032 R² = 0.9608 8 GT 40°C= 0.0909x + 2.479 R² = 0.9487 0.40 b NI 40°C= 0.0004x2 - 0.0231x + 0.4591 7

) R² = 0.8991 b ) -1 -1 s s -2 6 -2 0.30 c

O m c 2 O m 5 2 c c 4 0.20 (mmol H

s 3 g E (mmol H E (mmol 0.10 2 NI 23°C= 6E-05x2 - 0.0053x + 0.2852 UI 23°C= 0.0001x2 - 0.009x + 0.3386 GT 23°C= 0.0018x2 - 0.1521x + 7.0429 R² = 0.2335 R² = 0.4914 1 R² = 0.3488 0.00 0 0 102030405060 0 102030405060 MT (°C) MT (°C)

23°C UI 23°C NI 40°C UI 40°C NI GT 23°C GT 40°C

Figure 8. Water potentials and gas exchange parameters in plants with greenhouse temperature (23 or 40 °C) under (UI) irrigation or no irrigation (NI) conditions (I) obtained from EMs of Pinus radiata D.Don submitted to different maturation temperatures (MT). (a) Leaf water potentials (Ψleaf) initial

and final (applying of these treatments), (b) instantaneous net photosynthesis (AN, µmol CO2 m−2 s−1), (c) stomatal conductance (gs, mmol H2O m−2 s−1), and (d) instant transpiration (E, mmol H2O m−2 s−1). Different letters show significant differences by the Tukey–Kramer test (p ≤ 0.05).

In the first evaluation, there were no significant statistical differences for AN, gs, and E (Table 4). By contrast, in the second evaluation, the AN presented significant statistical differences for the effects MT, MT × GT, GT × I, and in the triple interaction MT × GT × I (Table 5). Stomatal conductance was

Forests 2020, 10, x FOR PEER REVIEW 9 of 16

A decrease in water potential was observed in plants from ECLs matured in MTs of 23 °C (Figure 8a). Similarly, the same pattern of decline was observed when assessing water potential after water and heat stress conditions, which in turn was less than the initial water potential (Figure 8a). For this characteristic, the data fit the model with a determination coefficient above 98% (R2 = 0.98).

MT (°C) 0 102030405060 -0.0 16

14 -0.1 Ψ final = 0.0003x2 - 0.0357x + 1.1447 leaf a ) 12 R² = 0.9822 a -1 a s -0.2 -2 10 m 2 a a 8 (MPa) -0.3 a leaf mol CO Ψ

μ 6 (

-0.4 N

Forests 2020, 11, 1181 9 of 14 (a) (b)

0.50 9 a UI 40°C= -0.0002x2 + 0.0229x - 0.2032 R² = 0.9608 8 GT 40°C= 0.0909x + 2.479 R² = 0.9487 0.40 b NI 40°C= 0.0004x2 - 0.0231x + 0.4591 7

) R² = 0.8991 b ) -1 -1 s s -2 6 -2 0.30 c

O m c 2 O m 5 2 c c 4 0.20 (mmol H

23°C UI 23°C NI 40°C UI 40°C NI GT 23°C GT 40°C

FigureFigure 8. Water8. Water potentials potentials and and gas gas exchange exchange parameters parameters in in plants plants with with greenhouse greenhouse temperature temperature (23(23 or

40 or◦C) 40 under °C) under (UI) (UI) irrigation irrigation or noor no irrigation irrigation (NI) (NI) conditions conditions(I) (I) obtainedobtained from EMs EMs of of PinusPinus radiata radiata Ψ D.DonD.Don submitted submitted to to di ffdifferenterent maturation maturation temperatures temperatures (MT).(MT). (a) LeafLeaf water potentials potentials ( (Ψleafleaf) initial) initial −2 2−1 1 andand final final (applying (applying of of these these treatments), treatments), (b ()b instantaneous) instantaneous net net photosynthesis photosynthesis ( A(ANN,, µµmolmol CO2 mm− s s),− ), (c) stomatal conductance (gs, mmol H2O m2−2 s−11), and (d) instant transpiration (E, mmol H2O m−2 2s−1). 1 (c) stomatal conductance (gs, mmol H2O m− s− ), and (d) instant transpiration (E, mmol H2O m− s− ). DiffDifferenterent letters letters show show significant significant di diffefferencesrences by by the the Tukey–Kramer Tukey–Kramer testtest ((pp ≤ 0.05).0.05). ≤

TableIn the 4. ANOVA first evaluation, for the e thereﬀect of were diﬀerent no significant maturation statistical temperatures differences (MT) infor the AN instantaneous, gs, and E (Table net 4). 2 1 2 1 Byphotosynthesis contrast, in the (A secondN, µmol evaluation, CO2 m− s the− ), A stomatalN presented conductance significant (gs ,statistical mmol H2 differencesO m− s− ), andfor the instant effects 2 1 MT,transpiration MT × GT, (E,GT mmol× I, and H2 Oin mthe− striple− ) in interaction plants of Pinus MT radiata × GT ×D.Don. I (Table 5). Stomatal conductance was

ANOVA Gas Exchange Initial

Eﬀect df AN gs E

MT 3 >0.01 ns >0.01 ns >0.01 ns ns nonsigniﬁcant at p 0.05; df—degrees of freedom. ≤

Table 5. ANOVA for the eﬀect of diﬀerent maturation temperatures (MT), greenhouse temperature (GT), and irrigation or no irrigation conditions (I) in the instantaneous net photosynthesis (AN, µmol 2 1 2 1 CO2 m− s− ), stomatal conductance (gs, mmol H2O m− s− ), and instant transpiration (E, mmol H2O 2 1 m− s− ) in plants of Pinus radiata D.Don.

ANOVA Gas Exchange Final

Effect df AN gs E MT 3 0.01 ** 0.001 *** 0.001 *** ≤ ≤ ≤ GT 1 >0.01 ns 0.001 *** 0.001 *** ≤ ≤ I 1 >0.01 ns 0.01 ** 0.01 ** ≤ ≤ MT GT 3 0.05 * 0.001 *** 0.001 *** × ≤ ≤ ≤ MT I 3 >0.01 ns 0.01 ** >0.01 ns × ≤ GT I 1 0.01 ** 0.001 *** 0.001 *** × ≤ ≤ ≤ MT GT I 3 0.01 ** 0.01 ** >0.01 ns × × ≤ ≤ *; **; *** Significant differences at p 0.05, p 0.01, or p 0.001, respectively; ns nonsignificant at p 0.01; df—degrees of freedom. ≤ ≤ ≤ ≤ As we can see in Table6, the relative e ffect of heat and water stress did not induce statistically significant differences in instantaneous liquid photosynthesis between any treatments, except those plants originating from ECLs submitted to maturation temperatures of 23 and 50 ◦C (Figure8b). Forests 2020, 11, 1181 10 of 14

Table 6. Eﬀect of diﬀerent maturation temperatures (MT-◦C), greenhouse temperature (GT-◦C), and irrigation (1) or no irrigation (2) conditions (I) on the instantaneous net photosynthesis (AN, µmol 2 1 2 1 CO2 m− s− ), stomatal conductance (gs, mmol H2O m− s− ) in plants of Pinus radiata D.Don.

Effect (MT-GT-I) AN gs 40-40-1 14.89 a 0.38 b,c 60-40-2 14.79 a 0.49 a 50-40-1 14.04 a 0.48 a,b 40-23-2 13.97 a 0.19 c,d 23-23-2 13.82 a 0.19 c,d 60-23-1 13.46 a 0.18 c,d 60-23-2 12.52 a 0.19 c,d 60-40-1 11.40 a 0.45 a,b 40-23-1 11.35 a 0.17 c,d 23-23-1 11.24 a 0.18 c,d 50-23-2 10.24 a 0.13 d 50-23-1 8.78 a 0.12 d 23-40-1 8.54 a 0.23 c,d 40-40-2 8.39 a 0.21 c,d 23-40-2 7.44 b 0.12 d 50-40-2 6.90 b 0.21 c,d Different letters within a column show significant differences in the means observed by Tukey–Kramer’s post hoc test (p 0.05). ≤ However, plants have greater variability with statistically significant differences in stomatal conductance (Table6). In addition, the plants that maintained higher stomatal conductance compared to those under stress conditions or otherwise, were plants originating from ECLs exposed to high 2 1 maturation temperatures (0.49 mmol H2O m− s− by 60 ◦C) (Figure8c) (Table6). At the end of the experiment, under high temperature conditions in a greenhouse, we observed a significantly increase in the instant transpiration in plants originating from ECLs subjected to MT at 60 ◦C, followed by 50 and 40 ◦C. We also observed that this increase was greater under conditions of heat stress than in nonstressed plants (Figure8d). This parameter also showed statistically significant differences in plants growing in the greenhouse at 40 ◦C under irrigation, with an increase in transpiration in this cultivation condition (Table7).

Table 7. Eﬀect of diﬀerent greenhouse temperatures (GT, ◦C) and irrigation (UI) or no irrigation (NI) 2 1 conditions (I) in the instant transpiration (E, mmol H2O m− s− ) in plants of Pinus radiata D.Don.

Effect (GT (◦C)-I) E 40-UI 7.19 a 40-NI 5.61 b 23-NI 4.26 b,c 23-UI 3.96 c Different letters show significant differences in the means observed by Tukey–Kramer’s post hoc test (p 0.05). ≤ 4. Discussion The formation of somatic embryos is affected by several factors, such the genetic background and the cultivation conditions [33,34]. In this study, the same ECLs (five for each species), were subjected to different maturation treatments, in order to try to minimize the genetic impact on the results obtained. In our experiments we observed that, in both species, embryos were formed with normal and abnormal morphologies. Merino et al. [35] in Scots pine attributed the formation of ses with normal and abnormal morphology to the difference of transcripts in embryogenic cells. However, the increase in temperature did not significantly promote the formation of abnormal embryos. In this work we observed that the NNE was not affected by high temperatures applied at the beginning of maturation stage in P. radiata, except in P. halepensis in which we observed a reduction in Forests 2020, 11, 1181 11 of 14

NNE in at 50 ◦C. Kvaalen and Johnsen [22] and García-Mendiguren et al. [18], when working with Picea abies and P. radiata, respectively, also reported an increase in the number of embryos obtained when high temperatures were applied in the initiation stage. In contrast, Arrillaga et al. [20], in proliferation and maturation stages Pinus pinaster SE showed that control temperature (23 ◦C) provoked the best results in terms of the number of embryos developed. Castander-Olarieta et al. [21] in P. radiata showed that somatic embryos originating from EMs initiated at 50 ◦C for 30 min had a higher LE. This is in agreement with our results for Aleppo pine where the longest embryos were found at this temperature. The germination mechanism of cotyledonary somatic embryos is complex and an alternative in this case are studies with post-maturation treatments [36], because in P. halepensis (22%, 30%, 24%, and 34% for 23, 40, 50, and 60 ◦C, respectively) we observed a low percentage rate of germination compared to P. radiata (44%, 51%, 46%, and 51% for 23, 40, 50, and 60 ◦C, respectively). Stresses applied to EMs in late SE stages, besides inducing different responses between species of conifers, can also guarantee an improvement in the germination of cotyledonary somatic embryos [37]. The germination rate changes according to species and treatments. This result was also described in other conifer species [20,38,39]. In this work, we observed that the high MTs did not affect either the germination of ses or the acclimatization of plants in both species of Pinus. The conditions of cultivation during the maturation stage in the SE are crucial for the regeneration of the somatic embryos in quality plantlets with an ex vitro survival capacity [40]. We obtained the highest acclimatization ( 88.91%) independently of the line or temperature for P. halepensis in relation ≤ to P. radiata (ranging from 55.84% to 63.47% for temperature). The maturation of EMs in ses and their subsequent conversion to plants, in both species, prove the tolerance of these EMs to high temperatures. The pressures exerted by high temperatures and drought affect numerous defense mechanisms in plants [41], such as changes in photosynthetic machinery [42]. This can happen at an embryogenic level with the formation of an epigenetic memory [4] even at the plant level. In this work, we observed that the different MTs applied to EMs promoted ses without significant physical anomalies. However, P. radiata plants originated from these treatments responded physiologically in different ways (Figure8) when they were subjected to drought and heat stress in the greenhouse. Taiz and Zeiger [43] reported that water stress significantly affects stomatal conductance more than photosynthesis, corroborating our results (Table6). In addition, we also observed that E was affected by heat and water stress (Figure8d and Table7). It is possible that plants originating from ECLs subjected to high temperatures in the maturation stage have developed epigenetic mechanisms that allowed a better response to stress [44], since the plants maintained or adapted their water and gas exchange potential to adverse temperature conditions and water stress to which they were subjected (Figure8). In this work, plants from all MT submitted to GT of 23 ◦C showed a similar behavior for gs and E. However, when subjected to heat stress (40 ◦C), plants originating from EMs subjected to high MTs (40 and 60 ◦C) had a significant increase in gs and E, reinforcing once again the possibility of “priming” in ECLs subjected to high temperatures. In natural conditions, similar behavior is observed in the apical meristem of plants, which is the center of the morphogenesis of the aerial part and is in constant cell division. The environmental restrictions, for example, heat and drought, perceived by these meristematic regions, trigger changes in the epigenetic state of the plants, developing a memory which, when under stress conditions again, enables the plants to have better tolerance [45]. In conclusion, it is possible to modulate the tolerance to stress by applying high temperatures during the final stages of the embryogenic process. Future experiments will be carried out to analyze methylation, physiological and biochemical aspects in one-year old plants to assess whether the plant characteristics endure over time. Forests 2020, 11, 1181 12 of 14

5. Conclusions P. radiata and P. halepensis EMs produced ses at high maturation temperatures. High maturation temperatures compared to the control temperature (23 ◦C) did not aﬀect the morphological characteristics of the embryos obtained, except for the LE in both species, and WE in P. halepensis. The plants obtained from these somatic embryos survived drought and heat stresses in the greenhouse. Moreover, plants originated from EMs submitted to a maturation temperature of 40 and 60 ◦C, presented better adaptation to drought and heat stress based on the water potential and gas exchange parameters analyzed in this experiment. Studies will be carried out to characterize possible epigenetic marks in the obtained plants caused by heat stress applied during the embryogenic process and drought and heat stress applied in plants in greenhouses.

Author Contributions: P.M. and I.A.M. conceived and planned the experiments. A.M.M.d.N. performed the experiments. P.A.B., N.F.F.d.N., T.G. and M.D.U. carried out the statistical analyses. A.M.M.d.N. wrote the manuscript and all authors provided critical feedback and helped shape the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by MINECO (Spanish Government) project (AGL2016-76143-C4-3R), CYTED (P117RT0522), and MINECO (BES-2017-081249, “Ayudas para contratos predoctorales para la formación de doctores”). MULTIFOREVER (Project MULTIFOREVER) is supported under the umbrella of ERA-NET cofund Forest Value by ANR (FR), FNR (DE), MINCyT (AR), MINECO-AEI (ES), MMM (FI), and VINNOVA (SE). Forest value has received funding from the European Union’s Horizon 2020 research and innovation programmed under agreement No 773324. Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

ABA, abscisic acid; AN, instant net photosynthesis; E, instant leaf transpiration; ECLs, established cell lines; EDM, Embryo Development Medium; EMs, embryonal masses; gS, stomatal conductance; GT, greenhouse’s temperature; I, irrigation condition; LE, length of somatic embryo; MT, maturation temperatures; NAE, abnormal somatic embryos; NI, without irrigation; NNE, number of normal mature somatic embryos; SE, somatic embryogenesis; ses, somatic embryos; UI, under irrigation; WE, width of somatic embryo; Ψleaf, water potential.

References

1. Pareek, A.; Dhankher, O.P.; Foyer, C.H. Mitigating the impact of climate change on plant productivity and ecosystem sustainability. J. Exp. Bot. 2020, 71, 451–456. [CrossRef][PubMed] 2. De Diego, N.; Saiz-Fernández, I.; Rodríguez, J.L.; Pérez-Alfocea, P.; Sampedro, M.C.; Barrio, R.J.; Lacuesta, M.; Moncaleán, P. Metabolites and hormones are involved in the intraspecific variability of drought hardening in radiata pine. J. Plant Physiol. 2015, 188, 64–71. [CrossRef][PubMed] 3. Priya, M.; Sharma, L.; Kaur, R.; Bindumadhava, H.; Nair, R.M.; Siddique, K.H.M.; Nayyar, H. Gaba (γ-aminobutyric acid), as a thermo-protectant, to improve the reproductive function of heat-stressed mungbean plants. Sci. Rep. 2019, 9, 7788. [CrossRef][PubMed] 4. Yakovlev, I.A.; Carneros, E.; Lee, Y.; Olsen, J.E.; Fossdal, C.G. Transcriptional profiling of epigenetic regulators in somatic embryos during temperature induced formation of an epigenetic memory in norway spruce. Planta 2016, 243, 1237–1249. [CrossRef][PubMed] 5. Taïbi, K.; del Campo, A.D.; Vilagrosa, A.; Bellés, J.M.; López-Gresa, M.P.; Pla, D.; Calvete, J.J.; López-Nicolás, J.M.; Mulet, J.M. Drought tolerance in Pinus halepensis seed sources as identified by distinctive physiological and molecular markers. Front. Plant Sci. 2017, 8, 8. [CrossRef] 6. Gazol, A.; Ribas, M.; Gutiérrez, E.; Camarero, J.J. Aleppo pine forests from across Spain show drought-induced growth decline and partial recovery. Agric. For. Meteorol. 2017, 232, 186–194. [CrossRef] 7. Manrique-Alba, À.; Beguería, S.; Molina, A.J.; González-Sanchis, M.; Tomàs-Burguera, M.; del Campo, A.D.; Colangelo, M.; Camarero, J.J. Long-term thinning effects on tree growth, drought response and water use efficiency at two aleppo pine plantations in Spain. Sci. Total Environ. 2020, 728, 138536. [CrossRef] 8. Fuentes-Sepúlveda, R.; García-Herrera, C.; Vasco, D.A.; Salinas-Lira, C.; Ananías, R.A. Heat characterization of pinus radiata wood vacuum-impregnated with octadecane. Energies 2020, 13, 942. [CrossRef] Forests 2020, 11, 1181 13 of 14

9. De Diego, N.; Pérez-Alfocea, F.; Cantero, E.; Lacuesta, M.; Moncaleán, P. Physiological response to drought in radiata pine: Phytohormone implication at leaf level. Tree Physiol. 2012, 32, 435–449. [CrossRef] 10. Fehér, A. Somatic embryogenesis—Stress-induced remodeling of plant cell fate. Biochim. Biophys. Acta (BBA) Gene Regul. Mech. 2015, 1849, 385–402. [CrossRef][PubMed] 11. Castander-Olarieta, A.; Pereira, C.; Montalbán, I.A.; Canhoto, J.; Moncaleán, P. Stress modulation in Pinus spp. Somatic embryogenesis as model for climate change mitigation: Stress is not always a problem. In Agricultural, Forestry and Bioindustry Biotechnology and Biodiscovery; Chong, P.A., Newman, D.J., Steinmacher, D.A., Eds.; Springer International Publishing: Cham, Switzerland, 2020; pp. 117–130. 12. Raza, G.; Singh, M.B.; Bhalla, P.L. Somatic embryogenesis and plant regeneration from commercial soybean cultivars. Plants 2020, 9, 38. [CrossRef] 13. Almeida, R.F.; Meira, F.S.; Gomes, H.T.; Balzon, T.A.; Bartos, P.M.C.; Meira, R.d.O.; da Cunha, R.N.V.; Lopes, R.; Mehta, A.; Scherwinski-Pereira, J.E. Capacity for somatic embryogenesis of adult oil palm genitors (Elaeis guineensis, var. Pisifera) from immature leaf tissues. S. Afr. J. Bot. 2020, 131, 229–239. 14. Arnholdt-Schmitt, B.; Ragonezi, C.; Cardoso, H. Do mitochondria play a central role in stress-induced somatic embryogenesis? In Vitro Embryogenesis in Higher Plants; Germana, M.A., Lambardi, M., Eds.; Springer New York: New York, NY, USA, 2016; pp. 87–100. 15. Pires, R.; Cardoso, H.; Ribeiro, A.; Peixe, A.; Cordeiro, A. Somatic embryogenesis from mature embryos of Olea europaea l. Cv. ‘Galega vulgar’ and long-term management of calli morphogenic capacity. Plants 2020, 9, 758. [CrossRef] 16. Gao, F.; Peng, C.; Wang, H.; Shen, H.; Yang, L. Selection of culture conditions for callus induction and proliferation by somatic embryogenesis of Pinus koraiensis. J. For. Res. 2020.[CrossRef] 17. Maruyama, T.E.; Ueno, S.; Hirayama, S.; Kaneeda, T.; Moriguchi, Y. Somatic embryogenesis and plant regeneration from sugi (japanese cedar, Cryptomeria japonica d. Don, Cupressaceae) seed families by marker assisted selection for the male sterility allele ms1. Plants 2020, 9, 1029. [CrossRef] 18. García-Mendiguren, O.; Montalbán, I.A.; Goicoa, T.; Ugarte, M.D.; Moncaleán, P. Environmental conditions at the initial stages of Pinus radiata somatic embryogenesis affect the production of somatic embryos. Trees 2016, 30, 949–958. [CrossRef] 19. Pereira, C.; Montalbán, I.A.; Goicoa, T.; Ugarte, M.D.; Correia, S.; Canhoto, J.M.; Moncaleán, P. Short communication: The effect of changing temperature and agar concentration at proliferation stage in the final success of aleppo pine somatic embryogenesis. For. Syst. 2018, 26, eSC05. [CrossRef] 20. Arrillaga, I.; Morcillo, M.; Zanón, I.; Lario, F.; Segura, J.; Sales, E. New approaches to optimize somatic embryogenesis in maritime pine. Front. Plant Sci. 2019, 10, 138. [CrossRef] 21. Castander-Olarieta, A.; Montalbán, I.A.; De Medeiros Oliveira, E.; Dell’Aversana, E.; D’Amelia, L.; Carillo, P.; Steiner, N.; Fraga, H.P.D.F.; Guerra, M.P.; Goicoa, T.; et al. Effect of heat stress on tissue ultrastructure and metabolite profiles during initiation of radiata pine somatic embryogenesis. Front. Plant Sci. 2019, 9, 2004. [CrossRef] 22. Kvaalen, H.; Johnsen, Ø. Timing of bud set in Picea abies is regulated by a memory of temperature during zygotic and somatic embryogenesis. New Phytol. 2008, 177, 49–59. [CrossRef] 23. Moncaleán, P.; García-Mendiguren, O.; Novák, O.; Strnad, M.; Goicoa, T.; Ugarte, M.D.; Montalbán, I.A. Temperature and water availability during maturation affect the cytokinins and auxins profile of radiata pine somatic embryos. Front. Plant Sci. 2018, 9, 1898. [CrossRef][PubMed] 24. Castander-Olarieta, A.; Moncaleán, P.; Pereira, C.; Pˇenˇcík, A.; Petˇrík, I.; Pavlović,I.; Novák, O.; Strnad, M.; Goicoa, T.; Ugarte, M.D.; et al. Cytokinins are involved in drought tolerance of Pinus radiata plants originating from embryonal masses induced at high temperatures. Tree Physiol. 2020.[CrossRef] 25. Montalbán, I.A.; De Diego, N.; Moncaleán, P. Enhancing initiation and proliferation in radiata pine (Pinus radiata d. Don) somatic embryogenesis through seed family screening, zygotic embryo staging and media adjustments. Acta Physiol. Plant. 2012, 34, 451–460. [CrossRef] 26. Montalbán, I.A.; Setién-Olarra, A.; Hargreaves, C.L.; Moncaleán, P. Somatic embryogenesis in Pinus halepensis mill.: An important ecological species from the mediterranean forest. Trees 2013, 27, 1339–1351. [CrossRef] 27. Walter, C.; Find, J.I.; Grace, L.J. Somatic embryogenesis and genetic transformation in Pinus radiata. In Protocol for Somatic Embryogenesis in Woody Plants; Jain, S.M., Gupta, P.K., Eds.; Springer Netherlands: Dordrecht, The Netherlands, 2005; pp. 11–24. 28. Montalbán, I.A.; De Diego, N.; Moncaleán, P. Bottlenecks in Pinus radiata somatic embryogenesis: Improving maturation and germination. Trees 2010, 24, 1061–1071. [CrossRef] Forests 2020, 11, 1181 14 of 14

29. Gupta, P.K.; Durzan, D.J. Shoot multiplication from mature trees of douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep. 1985, 4, 177–179. [CrossRef] 30. Montalbán, I.A.; Moncaleán, P. Rooting of Pinus radiata somatic embryos: Factors involved in the success of the process. J. For. Res. 2019, 30, 65–71. [CrossRef] 31. Scholander, P.F.; Bradstreet, E.D.; Hemmingsen, E.A.; Hammel, H.T. Sap pressure in vascular plants. Science 1965, 148, 339. [CrossRef] 32. R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for Statistical Computing: Vienna, Austria, 2017. 33. Breton, D.; Harvengt, L.; Trontin, J.-F.; Bouvet, A.; Favre, J.-M. High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine. In Vitro Cell. Dev. Biol. Plant 2005, 41, 494. [CrossRef] 34. Lelu-Walter, M.-A.; Bernier-Cardou, M.; Klimaszewska, K. Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (ait.). Plant Cell Rep. 2006, 25, 767–776. [CrossRef] 35. Merino, I.; Abrahamsson, M.; Larsson, E.; von Arnold, S. Identification of molecular processes that differ among scots pine somatic embryogenic cell lines leading to the development of normal or abnormal cotyledonary embryos. Tree Genet. Genomes 2018, 14, 34. [CrossRef] 36. Maruyama, T.E.; Hosoi, Y.Post-maturation treatment improves and synchronizes somatic embryo germination of three species of japanese pines. Plant Cell Tissue Organ Cult. (PCTOC) 2012, 110, 45–52. [CrossRef] 37. Stasolla, C.; Yeung, E.C. Recent advances in conifer somatic embryogenesis: Improving somatic embryo quality. Plant Cell Tissue Organ Cult. 2003, 74, 15–35. [CrossRef] 38. Niskanen, A.-M.; Lu, J.; Seitz, S.; Keinonen, K.; Von Weissenberg, K.; Pappinen, A. Effect of parent genotype on somatic embryogenesis in scots pine (Pinus sylvestris). Tree Physiol. 2004, 24, 1259–1265. [CrossRef] [PubMed] 39. Carneros, E.; Celestino, C.; Klimaszewska, K.; Park, Y.S.; Toribio, M.; Bonga, J.M. Plant regeneration in stone pine (Pinus pinea l.) by somatic embryogenesis. Plant Cell Tissue Organ Cult. (PCTOC) 2009, 98, 165–178. [CrossRef] 40. Yang, F.; Xia, X.-R.; Ke, X.; Ye, J.; Zhu, L.-H. Somatic embryogenesis in slash pine (Pinus elliottii engelm): Improving initiation of embryogenic tissues and maturation of somatic embryos. Plant Cell Tissue Organ Cult. (PCTOC) 2020, 143, 159–171. [CrossRef] 41. Gratkowska-Zmuda, D.M.; Kubala, S.; Sarnowska, E.; Cwiek, P.; Oksinska, P.; Steciuk, J.; Rolicka, A.T.; Zaborowska, M.; Bucior, E.; Maassen, A.; et al. The swi/snf atp-dependent chromatin remodeling complex in arabidopsis responds to environmental changes in temperature-dependent manner. Int. J. Mol. Sci. 2020, 21, 762. [CrossRef][PubMed] 42. Johnová, P.; Skalák, J.; Saiz-Fernández, I.; Brzobohatý, B. Plant responses to ambient temperature fluctuations and water-limiting conditions: A proteome-wide perspective. Biochim. Biophys. Acta (BBA) Proteins Proteom. 2016, 1864, 916–931. [CrossRef] 43. Taiz, L.; Zeiger, E. Stress physiology (Chapter 25). In Plant Physiology; Sinauer Associates Inc.: Sunderland, MA, USA, 2010; pp. 591–623. 44. Correia, B.; Valledor, L.; Meijón, M.; Rodriguez, J.L.; Dias, M.C.; Santos, C.; Cañal, M.J.; Rodriguez, R.; Pinto, G. Is the interplay between epigenetic markers related to the acclimation of cork oak plants to high temperatures? PLoS ONE 2013, 8, e53543. [CrossRef] 45. Sow, M.D.; Allona, I.; Ambroise, C.; Conde, D.; Fichot, R.; Gribkova, S.; Jorge, V.; Le-Provost, G.; Pâques, L.; Plomion, C.; et al. Chapter twelve—Epigenetics in forest trees: State of the art and potential implications for breeding and management in a context of climate change. In Advances in Botanical Research; Mirouze, M., Bucher, E., Gallusci, P., Eds.; Academic Press: Cambridge, MA, USA, 2018; Volume 88, pp. 387–453.

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