Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

Paloma Moncaleán; Olatz García-Mendiguren; Ondrej Novák; Miroslav Strnad; Tomás Goicoa; María D. Ugarte; Itziar A. Montalbán

doi:10.3389/fpls.2018.01898

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

Moncaleán, Paloma;García-Mendiguren, Olatz;Novák, Ondrej;Strnad, Miroslav;Goicoa, Tomás;Ugarte, María D.;Montalbán, Itziar A.; 2018-12-20 00:00:00 fpls-09-01898 December 18, 2018 Time: 19:6 # 1 ORIGINAL RESEARCH published: 20 December 2018 doi: 10.3389/fpls.2018.01898 Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Proﬁle of Radiata Pine Somatic Embryos 1 1 2 2 Paloma Moncaleán , Olatz García-Mendiguren , Ondrej Novák , Miroslav Strnad , 3 3 1 Tomás Goicoa , María D. Ugarte and Itziar A. Montalbán 1 2 Department of Forestry Science, NEIKER-Tecnalia, Arkaute, Spain, Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Institute of Experimental Botany CAS, Palacký University Olomouc, Olomouc, Czechia, Department of Statistics, Computer Science and Mathematics, Universidad Pública de Navarra, Pamplona, Spain Edited by: Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate Munetaka Sugiyama, The University of Tokyo, Japan the physical and chemical conditions (water availability) along the process and to study Reviewed by: their effect in the ﬁnal success in terms of quantity of somatic embryos produced. In Glória Catarina Pinto, the last years, our research team has been focused on the study of different aspects University of Aveiro, Portugal of the SE in Pinus spp. One of the main aspects affecting SE is the composition of Tsuyoshi E. Maruyama, Forestry and Forest Products culture media; in this sense, phytohormones play one of the most crucial roles in this Research Institute, Japan propagation system. Many studies in conifers have shown that different stages of SE and *Correspondence: somatic embryo development are correlated with distinct endogenous phytohormone Itziar A. Montalbán [email protected] proﬁles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on Specialty section: this knowledge, the aim of this study was to test the effect of different temperatures This article was submitted to Plant Development and EvoDevo, (18, 23, and 28 C) and gelling agent concentrations (8, 9, and 10 gL ) during the a section of the journal maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, Frontiers in Plant Science phytohormone proﬁle of somatic embryos developed was evaluated. In this sense, Received: 24 September 2018 the highest gellan gum concentration led to signiﬁcantly lower water availability. At this Accepted: 07 December 2018 Published: 20 December 2018 gellan gum concentration and 23 C a signiﬁcantly higher number of somatic embryos Citation: was obtained and the overall success of the process increased with respect to other Moncaleán P, treatments assayed. The somatic embryos produced in these conditions showed the García-Mendiguren O, Novák O, Strnad M, Goicoa T, Ugarte MD and highest concentration of iP-type cytokinins and total ribosides. Although, the different Montalbán IA (2018) Temperature conditions applied during maturation of somatic embryos led to different hormonal and Water Availability During proﬁles, they did not affect the ex vitro survival of the resulting somatic plants, where Maturation Affect the Cytokinins and Auxins Proﬁle of Radiata Pine no signiﬁcant differences were observed. Somatic Embryos. Front. Plant Sci. 9:1898. Keywords: cytokinins, embryonal masses, embryogenic cell lines, indol-3-acetic acid, Pinus radiata, somatic doi: 10.3389/fpls.2018.01898 embryogenesis Frontiers in Plant Science | www.frontiersin.org 1 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 2 Moncaleán et al. Phytohormones in Pine Somatic Embryos Moncaleán et al., 2003) and gymnosperms (Moncaleán et al., INTRODUCTION 2005; Montalbán et al., 2012). On the other hand, the role of The use of vegetative propagation in forestry is the fastest, indole-3- acetic acid (IAA) and CKs in the SE process have been most ﬂexible and eﬀective way to produce enough genetically analyzed in cotton (Zeng et al., 2007; Jin et al., 2014) and wheat improved material to meet future wood demands (Lelu-Walter (Hess and Carman, 1998; Jiménez and Bangerth, 2001) and fern et al., 2013). Somatic embryogenesis (SE) is currently considered (Grzyb et al., 2017). However, up to know a deep analysis of one of the successful biotechnological techniques for mass endogenous cytokinins and IAA concentration in SE process in propagation, enabling multi-varietal forestry (Park et al., 2016) pines has not been determined. In this sense, and as it has been and for cryopreservation of embryonal masses (EMs) from elite described previously, IAA and other phytohormones have an genotypes (Corredoira et al., 2006). important role in embryo diﬀerentiation events (Silveira et al., In the last years, several improvements in diﬀerent aspects 2004; Vondrakova et al., 2018); thus, a better understanding of of Pinus spp. SE process, such as initiation (Montalbán et al., the phytohormone proﬁle in somatic embryos under diﬀerent 2012), maturation (Montalbán et al., 2010) and germination environmental conditions could enable us to manipulate this (Montalbán and Moncaleán, 2018) have been carried out stage of SE to increase its eﬃciency. Taking into account all the in our laboratory. Traditionally, the adjustments have been above mentioned studies, the main objective of this study was focused on modiﬁcations of basal medium components (Salajova the evaluation of how the environment of cultures (gellan gum and Salaj, 2005; Zhang et al., 2007), exposure to diﬀerent concentration and temperature) along maturation stage aﬀects types and concentrations of exogenous plant growth regulators the success of radiata pine SE processes as well as to analyze its (Choudhury et al., 2008) and the use of diﬀerent initial explants eﬀect in CK and IAA endogenous levels. (Hargreaves et al., 2017). One of the main bottlenecks of the SE in conifers is the progression from immature embryogenic cultures into MATERIALS AND METHODS mature cotyledonary embryos able to develop well-growing plants (Harvengt, 2005). Several external stimuli, such as Maturation Experiment plant growth regulators (Ayil-Gutiérrez et al., 2013), osmotic Plant Material agents (Kikuchi et al., 2006), nutritional components (Pen ˇ cík ˇ Ten embryogenic cell lines (ECLs) from four open-pollinated et al., 2015), amongst others, have been recognized as essential Pinus radiata D. Don trees were selected for the experiments. The factors in determining both the hormone biosynthesis and trees belonged to a seed orchard established by Neiker-Tecnalia 0 0 0 0 the developmental fate of explant cells (Grzyb et al., 2017). in Deba (Spain; latitude: 43 16 59 N, longitude: 2 17 59 W, In conifers, maturation is stimulated by reduction of water elevation: 50 m). The ECLs were initiated and proliferated in availability by means of raising the osmoticum of the medium clumps in standard conditions following Montalbán et al. (2012) (Montalbán et al., 2010) or the concentration of gelling (Figure 1). agent (Klimaszewska and Smith, 1997). Modifying gellan Maturation Procedure gum concentration in maturation media has been studied in a few species of angiosperms (Márquez-Martín et al., The basal medium used in all phases of SE process was 2011) and gymnosperms (Morel et al., 2014); these authors Embryo Development Medium (EDM, Walter et al., 2005). reported an improvement in the development and maturation For initiation and proliferation 30 gL sucrose, 4.5 mM 2,4- of somatic embryos (Se) of P. pinaster by increasing gellan dichlorophenoxyacetic acid and 2.7 mM benzyladenine (BA) were 1 1 R gum concentration, pointing out that the eﬀect of gellan gum added. As gelling agent, 3 gL and 4.5 gL Gelrite were used concentration on plant response is provoked by changes of for initiation and proliferation, respectively. The pH was adjusted water availability in the culture medium. In our laboratory, we to 5.7 prior to sterilization at 121 C for 20 min. After autoclaving, demonstrated in previous studies that the agar concentration ﬁlter-sterilized solutions (pH 5.7) of the following amino acids and the temperature had a signiﬁcant eﬀect on initiation step of (Walter et al., 2005) were added to partially cooled medium prior radiata pine SE; and in the case of temperature, in the subsequent to dispensing into Petri dishes (90 mm 20 mm): 550 mgL 1 1 phases of the process (García-Mendiguren et al., 2016). As far L-glutamine, 525 mgL L-asparagine, 175 mgL L-arginine, 1 1 1 as we know, the eﬀect of diﬀerent temperatures in maturation 19.75 mgL L-citrulline, 19 mgL L-ornithine, 13.75 mgL 1 1 step has not been tested in Pinus spp., only in some angiosperms L-lysine, 10 mgL L-alanine, and 8.75 mgL L-proline. All (Wang et al., 2014). chemical products were purchased from Duchefa. The active cytokinin (CK) pool is regulated during Three weeks before the start of maturation experiment, EMs development by biosynthesis, uptake from extracellular (Figure 2A) were suspended in liquid growth regulators-free sources, metabolic interconversions, inactivation, degradation, basal medium in 50 mL centrifuge tubes; then the suspension was and transport (Kakimoto, 2003). The relative abundance of poured onto a ﬁlter paper disk (Whatman No. 2) in a Büchner diﬀerent CKs can vary greatly between plant species, tissues funnel. The ﬁlter papers with 250 mg of EMs each were laid and developmental stages, and depends on the environmental on proliferation media. Maturation of ECLs was carried out conditions (Frébort et al., 2011). Some studies have examined the following the protocol described by Montalbán et al. (2010). eﬀect of varying the culture atmosphere on endogenous CK levels Brieﬂy, EMs were suspended in liquid growth regulators-free after organogenesis in angiosperms (Jiménez and Bangerth, 2001; basal medium and ﬁltered as described above. The ﬁlter papers Frontiers in Plant Science | www.frontiersin.org 2 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 3 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 2 | Pinus radiata embryonal masses (A), mature somatic embryos (B), germinated somatic planlets (C), and acclimatized somatic plants (D). embryonic root caps pointing downward, were titled vertically FIGURE 1 | Scheme of the experimental design of maturation experiment. at an angle of approximately 45 , and cultured at 23 1 C Cultures were stored at three different temperatures: 18 C (circle), 23 C 2 1 (square), or 28 C (hexagon) and at three different agar concentrations (inside under 16 h photoperiod at 100 mmolm s provided by cool circles, squares, and hexagons). white ﬂuorescent tubes (TFL 58 W/33; Philips, France). Plantlets were subcultured to culture vessels with medium of the same composition after 6 weeks. with 80 mg of EMs each were laid on maturation media. The basal After a germination period of 14–16 weeks, plantlets were media used were the same as described before supplemented transferred to sterile peat:perlite (7:3, v/v) and acclimatized in a 60 gL sucrose and the amino acid mixture used for initiation greenhouse with environmental control where the humidity was and proliferation of the EMs. Also, 60 mM abscisic acid (ABA, progressively decreased from 99 to 70% during the ﬁrst month. purchased from Olchemim) was added. In order to increase or reduce the water availability of the medium, the amount of Gelrite in the maturation medium was Water Availability Determination 1 1 increased (10 gL ) or reduced (8 gL ), respectively, respect to The water availability of maturation media (EDM) under the standard conditions (9 gL ). The cultures were kept under diﬀerent maturation conditions (three gellan gum concentrations diﬀerent temperatures, 5 C above (28 C) and below (18 C) the and three temperatures) was determined by weighing and placing standard (23 C). Thus, nine diﬀerent treatments were tested, one autoclaved ﬁlter paper disk (Whatman No. 2) on the surface carrying out the other SE steps under standard conditions of the medium following Klimaszewska et al. (2000). The Petri (Figure 1). Summarizing, the whole experiment comprised nine dishes were incubated under the same conditions as those used diﬀerent treatments, 10 ECLs and four replicates per ECL and for maturation for 15 weeks. Then, the ﬁlter paper disks were maturation condition (a total of 360 maturation plates). All the weighed and the amount of water (g) absorbed was calculated. cultures were kept in darkness. Four replicates per maturation condition were used. Germination Procedure After 15–18 weeks, Se were transferred to germination medium. Extraction, Puriﬁcation and This medium was half strength macronutrients LP (Quoirin Quantiﬁcation of Endogenous Cytokinins and Lepoivre, 1977, modiﬁed by Aitken-Christie et al., 1988) Plant Material supplemented with 2 gL activated charcoal (AC, Sigma- Aldrich) and 9.5 gL Difco Agar granulated (Becton & Somatic embryos (Figure 2B) obtained from EMs maturated Dickinson). In those ECLs and maturation conditions producing at 18, 23, and 28 C in EDM medium solidiﬁed with 8, 9, and Se, three to four Petri dishes, and 20 Se per Petri dish, with the 10 gL (Figure 1). Frontiers in Plant Science | www.frontiersin.org 3 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 4 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 3 | Water availability (g) on EDM medium (A) stored at three different temperatures (18, 23, and 28 C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L ). Different letters show signiﬁcant differences at a signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test. Speed-Vac concentrator and dissolved in 30 ml of mobile phase Quantitative Analysis of CKs and IAA by Liquid prior to UHPLC-MS/MS analyses. Chromatography-Single Quadrupole MS Mass analysis was carried out using an Acquity UPLC System The 41 CKs and auxins analyzed were the following: cis-Zeatin (Waters, Milford, MA, United States), and a triple-quadrupole (cZ), cis-Zeatin riboside (cZR), cis-Zeatin O-glucoside (cZOG), TM mass spectrometer Xevo TQ-S MS (Waters MS Technologies, cis-Zeatin-9-glucoside (cZ9G), cis-Zeatin riboside O-glucoside Manchester, United Kingdom). Quantiﬁcation was achieved (cZROG), cis-Zeatin riboside-5 -monophosphate (cZMP), by monitoring protonated precursors and the appropriate trans-Zeatin (tZ), trans-Zeatin riboside (tZR), trans-Zeatin product ions. Multiple reaction monitoring transitions as well O-glucoside (tZOG), trans-Zeatin-7-glucoside (tZ7G), trans- as chromatographic and tandem mass spectrometry (MS) Zeatin-9-glucoside (tZ9G), trans-Zeatin riboside O-glucoside conditions were selected according to the method described by (tZROG), trans-Zeatin riboside-5 -monophosphate (tZMP), Novák et al. (2008, 2012). All MS data were processed using Dihydrozeatin (DHZ), Dihydrozeatin riboside (DHZR), TM TM the MassLynx software with TargetLynx program (version Dihydrozeatin O-glucoside (DHZOG), Dihydrozeatin-7- 4.2. Waters, Milford, MA, United States), and compounds were glucoside (DHZ7G), Dihydrozeatin-9-glucoside (DHZ9G), quantiﬁed by standard isotope dilution analysis (Rittenberg and Dihydrozeatin riboside O-glucoside (DHZROG), Dihydrozeatin 0 6 Foster, 1940). riboside-5 -monophosphate (DHZMP), N -Isopentenyladenine 6 6 (iP), N -Isopentenyladenosine (iPR), N -Isopentenyladenine- 7-glucoside (iP7G), N -Isopentenyladenine-9-glucoside Data Collection and Statistical Analysis 6 0 (iP9G), N -Isopentenyladenosine-5 -monophosphate The ECLs subjected to diﬀerent maturation conditions that had 6 6 (iPMP), N -Benzyladenine (BA), N -Benzyladenosine (BAR), produced Se were registered and the percentage of maturation 6 6 0 N -Benzyladenine-9-glucoside (BA9G), N -benzyladenosine-5 - and the number of Se per gram of EM (fresh weight) were monophosphate (BARMP), ortho-Topolin (oT), ortho-Topolin riboside (oTR), ortho-Topolin-9-glucoside (oT9G), meta-Topolin (mT), meta-Topolin riboside (mTR), meta- Topolin-9-glucoside (mT9G), para-Topolin (pT), para-Topolin riboside (pTR). Indol-3-acetic acid (IAA), 2-oxindole-3-acetic acid (oxIAA), IAA-aspartate (IAA-Asp), and IAA-glutamate (IAA-Glu). Three replicates of 3 mg of Se were analyzed according to the protocol described by Svacinov ˇ á et al. (2012) and Pen ˇ cík ˇ et al. (2018) for cytokinin and auxin proﬁling, respectively, using miniaturized puriﬁcation (pipette tip solid-phase extraction). Samples were extracted with the addition of stable isotope-labeled internal standards (each at 0.5 pmol per sample) and transferred after extraction onto Stage Tips and puriﬁed according to the aforementioned protocol, consisting of C18, SDB-RPS, and FIGURE 4 | Maturation percentages (%) in P. radiata for embryogenic cell lines Cation-SR sorbents for cytokinins or C18 and SDB-XC sorbents cultured at three different temperatures (18, 23, and 28 C) and three gellan for IAA and IAA conjugates. Eluates were collected into new gum concentrations (8, 9, and 10 g L ). clean microcentrifuge tubes and evaporated to dryness in a Frontiers in Plant Science | www.frontiersin.org 4 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 5 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 5 | Number of P. radiata somatic embryos per gram of embryogenic tissue (fresh weight) maturated on EDM medium (A) at three different temperatures (18, 23, and 28 C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L ). Different letters show signiﬁcant differences at a signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test. calculated. The number of somatic embryos from diﬀerent Signiﬁcant diﬀerences were found among the levels of gellan maturation conditions that had germinated was recorded and gum and temperature although the interaction between the germination percentages were calculated (Figure 2C). A logistic tested variables was not signiﬁcant. In this sense, the highest regression and the corresponding analysis of deviance were temperature analyzed (28 C) led to a signiﬁcantly lower water conducted to evaluate the eﬀect of temperature and gellan availability than the other temperatures tested (Figure 3A). gum concentration on diﬀerent stages of plant conversion for EDM medium supplemented with 10 gL gelrite showed also P. radiata. signiﬁcantly lower water availability than those supplemented Then, the success rate (plants able to be planted in ex vitro with 8 or 9 gL (Figure 3B). conditions) was evaluated in plantlets coming from ECLs Maturation percentages were 100% in all treatments except in cultured under diﬀerent maturation treatments and it was ECLs cultured at 23 and 28 C in culture media solidiﬁed with 1 1 calculated with respect to standard conditions (23 C and 9 gL 8 gL gelrite (90 and 60%, respectively) and in those cultured at gellan gum, considered as 100% for maturation rate, number of 28 C with 10 gL gelrite (80%, Figure 4). Se per gram of EM and germination percentage). When the analysis of deviance for the number of P. radiata After 14 weeks under ex vitro conditions (Figure 2D), the Se was evaluated, statistical diﬀerences were found for the survival was calculated. To evaluate the eﬀect of the maturation treatments, an analysis of variance (ANOVA) was carried out. For water availability analyses, a two-way analysis of variance (ANOVA) was carried out to assess the eﬀect of temperature and gellan gum concentration on the water availability of the media. When signiﬁcant diﬀerences between the levels of the two variables were found, the Tukey HSD post hoc test was carried out to ﬁnd out which levels were statistically diﬀerent. Data for the diﬀerent CK types and metabolites, for IAA contents and the ratio of IAA: active CKs were evaluated using a two-way ANOVA to identify diﬀerences between treatments. Multiple comparisons were performed using Tukey’s post hoc test. FIGURE 6 | Germination percentages (%) of P. radiata somatic embryos RESULTS maturated on EDM medium supplemented with three different gellan gum concentrations (8, 9, and 10 g L ) and stored at three different temperatures (18, 23, and 28 C). Different letters show signiﬁcant differences at a Maturation Experiment signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with Detailed information on the statistical analyses performed is the Tukey HSD post hoc test. given as Supplementary Material. Frontiers in Plant Science | www.frontiersin.org 5 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 6 Moncaleán et al. Phytohormones in Pine Somatic Embryos diﬀerent temperatures and gellan gum concentrations tested highest temperature with the standard concentration of gelrite but no interaction between them was found. The Tukey HSD (9 gL , Figure 8A). The levels of DHZ were signiﬁcantly higher post hoc test revealed signiﬁcant diﬀerences between the three in somatic embryos cultured at 18 and 23 C with the lowest temperatures tested; being 23 and 28 C the temperatures that Gelrite concentration in the culture media (8 gL , Figure 8B). produced the highest and the lowest number of Se (1036 The iP concentration was the highest in Se developed in a culture 1 1 and 374 Seg EM, respectively, Figure 5A). Moreover, ECLs media solidiﬁed with 10 gL gelrite at 28 C (Figure 8C). maturated in culture media with the lowest water availability Signiﬁcant diﬀerences in CK ribosides (Figure 9) were found (10 gL ) produced a signiﬁcantly higher number of somatic among the levels of gellan gum but no signiﬁcant diﬀerences embryos (Figure 5B). were observed among the levels of temperature for tZR, cZR, and Regarding germination percentage, signiﬁcant interaction DHZR; however, a signiﬁcant interaction between temperature between temperature, and gellan gum concentration at and agar was observed for all CK ribosides analyzed. tZR levels maturation was observed. The highest germination rates, increased signiﬁcantly when the gelrite concentration increase above 90%, were obtained when EMs were maturated at 18 and from 9 to 10 in somatic embryos cultured at 18 and 23 C 23 C, whereas maturation at 28 C led to lower germination (Figure 9B). The highest DHZR concentration was found in rates, particularly when it was combined with 9 or 10 gL gellan Se cultured in presence of the lowest concentration of gelrite gum (74 and 64%, respectively, Figure 6). (8 gL ) at 18 C (Figure 9C), the DHZR levels obtained this The eﬀect of physical and chemical modiﬁcation of culture treatment did not present diﬀerences with those at standard media in the success of the conversion from EMs to plants is temperature (23 C) regardless the gellan gum concentration. shown in Figure 7. An improvement in plant conversion was iPR levels were signiﬁcantly higher in Se cultured at standard observed when the gellan gum concentration was increased at all temperature in presence of the highest gelrite concentration 1 1 temperatures tested. In this sense, 23 C and 10 gL showed the (10 gL ) (Figure 9D). best results improving by 132% the plant conversion process. The levels of tZRMP, cZRMP, DHZRMP, and iPRMP were Somatic plants coming from diﬀerent treatments during under detection limit in all the samples analyzed. Also, tZOG, maturation stage did not show signiﬁcant diﬀerences in tZROG, cZROG, and DHZROG were under the limit of ex vitro survival after 14 weeks growing in the greenhouse detection. Signiﬁcant diﬀerences in CK O- glucosides were found (Supplementary Material). However, highest rates were found in among the levels of gellan gum and temperature (except for plants coming from EMs maturated at 28 C independently of the DHZOG), and for the interaction between the two variables. The gellam gum concentration. Survival percentage increased parallel CK O-glucosides were represented by a low amount of cZOG and to the increment of temperature during the maturation stage. DHZOG. In this sense, Se maturated at the highest temperature and gelling agent concentration showed the highest levels of Endogenous Phytohormone Analyses cZOG (Figure 10A). On the contrary, DHZOG concentration Detailed information on the contents of the 41 CKs and auxins in was the highest in Se developed at the lowest temperature and all samples analyzed is provided as Supplementary Material. gelrite concentration in the culture media (Figure 10B). Signiﬁcant diﬀerences in CK bases were found among the Regarding the isoprenoid CK type, signiﬁcant diﬀerences in levels of temperature, gellan gum (except for cZ) and the CK types were found among the levels of temperature (except interaction between the tested variables. In all the Se analyzed for cZ), gellan gum and the interaction between the tested the appearance of tZ was under the limit of detection. The variables. The tZ-type CK concentration was the highest in Se concentration of cZ was the highest in Se maturated at the developed at 18 and 23 C with the highest concentration of FIGURE 7 | Success rate (%) in the process of plant conversion in P. radiata embryogenic cell lines maturated at three different temperatures (18, 23, and 28 C) and 1 1 three gellan gum concentrations (8, 9, and 10 g L ) with respect to standard maturation conditions (23 C and 9 gL gellan gum, considered as 100% of success). Frontiers in Plant Science | www.frontiersin.org 6 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 7 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 8 | Endogenous levels (pmol g DW) of the cytokinin bases in FIGURE 9 | Endogenous levels (pmol g DW) of the cytokinin ribosides in somatic embryos maturated at different temperatures in culture media somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. cZ (A), DHZ (B), and iP solidiﬁed with different concentrations of gellan gum. cis-zeatin riboside (cZR) (C) concentrations. Different letters indicate signiﬁcant differences among (A), trans-zeatin riboside (tZR) (B), dihydrozeatin riboside (DHZR) (C), and treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square N6-isopentenyladenosine (iPR) (D) concentrations. Different letters indicate continuous line (23 C), and square discontinuous line (18 C). signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). gelling agent and at 28 C with the standard concentration of gelrite (Figure 11A). The lowest cZ-type CK concentration was observed in treatments at 18 and 23 C with the standard gelrite values for this group were obtained in Se cultured at standard concentration (Figure 11B). The concentration of DHZ-type temperature (23 C) and the highest gelrite concentration CKs was signiﬁcantly higher in Se developed at 18 and 23 C (Figure 12B). O-glucosides were signiﬁcantly higher in Se in culture media solidiﬁed with 8 gL of gelrite (Figure 11C). cultured at the highest temperature and gelrite concentration On the contrary, iP-types endogenous concentration was (Figure 12C). signiﬁcantly higher in Se cultured with the highest concentration Figure 13 shows the relative abundance of isoprenoid CKs of gelling agent (10 gL ) at standard temperature (23 C) than in Se by treatment. CK ribosides are the predominant group of Se from other treatments (Figure 11D). cytokinins respect to bases and O-glucosides and a decrease Analyzing the results by groups, CK nucleotides and in the bases content is observed parallel to an increase of N -glucosides were under the limit of detection. Signiﬁcant gelrite concentration and temperature. Although the presence diﬀerences in CK functional groups were found among the levels of aromatic type CKs (N -benzyladenine, meta-topolin, ortho- of temperature, gellan gum and the interaction between the tested topolin, and para-topolin) and their metabolites was also variables. A signiﬁcantly higher concentration of bases was found analyzed, theirs levels were under the limit of detection. in Se developed at 18 and 23 C in culture media solidiﬁed with Taking a general look at the results presented, it was observed a the lowest gelrite concentration (Figure 12A). Ribosides was similar trend for CK concentration in Se cultured at 18 C y 23 C the CK group with the most remarkable presence, the highest when the agar concentration increased from 8 to 9 gL , except Frontiers in Plant Science | www.frontiersin.org 7 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 8 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 10 | Endogenous levels (pmol g DW) of the O-glucosides in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. cis-zeatin O-glucoside (cZOG) (A), dihydrozeatin O-glucoside (DHZOG) (B) concentrations. Different letters indicate signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). iPR, DHZR and cZOG. When considering the results by CK- type or CK-metabolite this trend was also observed in all cases. At 28 C, an increase of bases and ribosides concentration was observed from 8 to 9 gL ; in the case of iP, iPR, and cZOG their concentration continued increasing signiﬁcantly with gellan gum concentration (10 gL ). The IAA, 2-oxindole-3-acetic acid (oxIAA), and IAA- glutamate levels showed the highest levels in Se developed at the highest temperature with diﬀerent concentrations of gelrite (Figures 14A–C). Moreover, Se cultured at 28 C in media FIGURE 11 | Endogenous levels (pmol g DW) of different isoprenoid with the highest gelrite concentration showed signiﬁcantly cytokinin types in somatic embryos maturated at different temperatures in higher levels of oxIAA and IAA-glutamate (Figures 14B,C). culture media solidiﬁed with different concentrations of gellan gum. cZ-type (A), tZ-type (B), DHZ-type (C), and iP-type (D) concentrations. Different IAA-aspartate levels were under the limit of detection in all letters indicate signiﬁcant differences among treatments by Tukey’s post hoc samples analyzed. When relation between IAA and active test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square CKs (basesCribosides) was evaluated, we could observe discontinuous line (18 C). low values in Se developed at standard temperature (23 C) independently of the gelrite concentration in the culture media (Figure 15). To this respect, in Douglas-ﬁr, a reduction in water availability of the culture media provoked an increase in Se quality (Lelu- Walter et al., 2018) probably because it has been described that DISCUSSION the reduction in water availability leads to obtain embryos with The inductive conditions which allow diﬀerentiated somatic low water content similar to their zygotic counterparts (Morel cells to develop into competent dediﬀerentiated cells can be et al., 2014). achieved by the addition of speciﬁc phytohormones, and/or by The culture of embryogenic calli in media with an increased exposing the tissue to diﬀerent stress factors (Leljak-Levanic ˇ gelling agent concentration is a common procedure for the et al., 2016). Moreover, water relations between the embryo and production of high quality mature embryos (Krajnákov ˇ á et al., its environment play a regulatory role in embryo development, 2009; Troch et al., 2009; Buendía-González et al., 2012). Our particularly during maturation (from Márquez-Martín et al., results in P. radiata are in agreement with those obtained in 2011) and for this reason, the water availability of maturation P. strobus where the best maturation results were obtained media was determined in our experiments. Radiata pine somatic increasing gellan gum concentration due to a shift in the embryos cultured in EDM medium supplemented with 10 gL developmental program of the culture, from proliferation of cells and stored at 28 C grew in an environment with the lowest and early somatic embryos to the production of embryos at water availability in comparison to the other conditions assayed. advanced stages (Klimaszewska et al., 2000). Frontiers in Plant Science | www.frontiersin.org 8 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 9 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 13 | Relative abundances (expressed in percentage) of endogenous isoprenoid cytokinins in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Bases (black bar), Ribosides (gray bar), and O-glucosides (white bar). Pereira et al., 2016) as well as in proliferation stage of P. halepensis (Pereira et al., 2017) was studied and we found that the highest temperature seemed to produce a selective pressure as pointed by Fehér (2015); In this regard, Bonga et al. (2010) suggested that reducing or increasing temperatures may improve initiation and proliferation since temperature stress may promote cellular reprogramming. Our results are in agreement with de Almeida et al. (2014) where temperature had a signiﬁcant inﬂuence on the direct SE capacity in coﬀee. Regarding gellan gum concentration, some reports suggested 1 that increasing the concentration of a gelling agent from 4 to FIGURE 12 | Endogenous levels (pmol g DW) of the cytokinin groups in 1 1 8 gL (Teyssier et al., 2011) or from 4 to 9 gL (Morel somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Cytokinin Bases (A), et al., 2014) promoted the maturation of the Se of some Ribosides (B), and O-glucosides (C) concentrations. Different letters indicate conifers. In this sense, Garin et al. (2000) demonstrated that signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). the phytagel concentration was critical for the maturation Triangle (28 C), square continuous line (23 C), and square discontinuous line of Se of ﬁve tested ECLs of Pinus strobus. The increase (18 C). of phytagel concentration in the maturation media allowed obtaining well-developed Se capable of germination in various species of pines (Klimaszewska et al., 2009) and hybrid larch Respect to the number of embryos developed, the temperature (Lelu-Walter and Pâques, 2009) probably due to the action and gellan gum concentration aﬀected signiﬁcantly the number in endogenous ABA modulation in mature somatic embryos of Se per gram of EM. In this sense the temperature (28 C) (Morel et al., 2014). In summary, our results agree with and the agar concentration (10 gL ) that provoked the lowest abovementioned studies pointing that increasing gelling agent water availability led to the lowest and the highest number of concentration is a good strategy to improve the Se yield in radiata Se, respectively. For this reason, it seems that water availability pine. was not related to the success of the process at this stage. In this The highest germination rates (above 90%) were obtained sense, there are two studies suggesting that under sub-optimal in Se coming from EMs maturated at 18 and 23 C, whereas water availability, plants may take to a premature drought maturation at 28 C led to lower germination rates, particularly stress response and arrest or drastically reduce growth due to when it was combined with 9 or 10 gL gellan gum. Our the eﬀect on source–sink relationships through preferentially results are not in agreement to Prewein et al. (2004) when inhibiting nutrient acquisition processes more and earlier than they described that desiccation of the embryos due to decreased necessary (Skirycz et al., 2011). It is clear that the water water availability in maturation media improves the germination availability registered in our experiments is not sub-optimal frequency, probably by reducing the endogenous ABA level or for the maturation of EMs. In our laboratory, the eﬀect of by changing the sensitivity to ABA (Jiménez, 2005). Unlike diﬀerent physical and chemical conditions at the initiation stage observed by Morel et al. (2014) in maritime pine, we found no of P. radiata and P. halepensis (García-Mendiguren et al., 2016; diﬀerences in germination rates with increasing concentration of Frontiers in Plant Science | www.frontiersin.org 9 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 10 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 15 | Indole-3-acetic acid/Cytokinin bases and ribosides ration in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). were obtained in somatic plants coming from EMs maturated at 28 C. There are only a few reports describing how the contents of diﬀerent endogenous hormones are altered by diﬀerent FIGURE 14 | Endogenous levels (pmol g DW) of indole-3-acetic acid (IAA) environmental conditions. The relative abundance of diﬀerent (A), IAA-glutamate (IAA-Glu) (B), 2-oxindole-3-acetic acid (oxIAA) (C) in CKs can vary greatly between plant species, tissues and somatic embryos IAA-glutamate (IAA-Glu), 2-oxindole-3-acetic acid (oxIAA) in developmental stages, and depends on the environmental somatic embryos maturated at different temperatures in culture media conditions (Frébort et al., 2011). In our experiments, the solidiﬁed with different concentrations of gellan gum. Different letters indicate prevailing isoforms of CKs were cis-isomers of Z and ZR signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line according with Zur et al. (2015) in microspore embryogenesis (18 C). in triticale. Based on studies with Arabidopsis, cis-isomers were regarded as CK derivatives without any or with low biological activity. Further studies with non-model plants, like pines, agar, however, this could be due to diﬀerences in the range of showed that cis-isomers can be the dominant form of CK concentrations used by these authors (4 and 9 gL Phytagel) and in speciﬁc plant organs and/or stages of development (Emery us (8, 9, and 10 gL Gelrite). et al., 1998: Vyroubalová et al., 2009; Kudo et al., 2012). An improvement in plant conversion when the gellan gum To this respect, Gajdošová et al. (2011) proposed that cZ concentration was increased at all temperatures tested was can be qualiﬁed as a regulator of CK responses in plants observed. However, 23 C showed the best results improving under growth-limiting conditions. Our results only showed by 132% the plant conversion process. Summarizing, increasing signiﬁcantly diﬀerences in cZ in Se cultured with 9 gL the gellan gum concentration in the maturation stage improved of gelrite at the highest temperature (28 C) with the others the SE process in all radiata pine cultures independently of analyzed. the temperature of storage obtaining the best results when Highest amount of bases, the active forms of cytokinins somatic embryos were submitted to maturation process at control (Klemš et al., 2011), were found in Se developed under 18 and temperature (23 C). So, higher temperature and gellan gum 23 C and low gellan gum concentration, treatments showing the concentration applied at maturation stage did not result in an highest water availability. So, our results are in agreement to other increment of the eﬃciency of SE process like it was observed authors describing that a decrease in water availability could be when this stressful condition was applied at initiation stage related to the rapidly utilization of CK bases and tightly regulated (García-Mendiguren et al., 2016). Moreover, ex vitro survival did by mechanisms that prevent their accumulation at high levels not show signiﬁcant diﬀerences although the highest percentages (Moncaleán et al., 2005; Stirk et al., 2005). Frontiers in Plant Science | www.frontiersin.org 10 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 11 Moncaleán et al. Phytohormones in Pine Somatic Embryos CK ribosides, mainly represented by cZR and iPR, were regulate several developmental processes and their ability to higher in the treatment that led to the highest somatic embryo crosstalk makes them ideal candidates for mediating stress- production and success rate (23 C and 10 gL ). In terms adaptation responses according with Bielach et al. (2017) as of metabolic groups, ribosides were the predominant group of well as initiating various signal transduction pathways (Kohli cytokinins and its percentage respect to bases and O-glucosides et al., 2013). In this regard, comparing the endogenous levels increased in parallel with the decrease of water availability. of IAA, Z, ZR, iP, and iPR in several embryogenic habituated Nevertheless, in peach seeds, phosphates and ribosides of the callus lines of Asparagus oﬃcinalis, no correlation between DHZ and iP-type are the predominant CKs and functions embryogenic potential and endogenous hormone levels could be like embryo formation and growth, division of endosperm found (Limanton-Grevet et al., 2000). These and other ﬁndings primary nucleus, endosperm cellularization and endosperm cell demonstrate that there is no direct correlation in endogenous division have been attributed to them (Arnau et al., 1999). hormone content and embryogenic competence of diﬀerent So, opposite to Pacheco de Freitas et al. (2016) active CK genotypes, or between competent and non-competent genotypes, levels seem to be important during the later stages of embryo which suggests the involvement of additional, hormone-related development and maturation showing large amounts of these internal mechanisms for embryogenic competence regulation phytohormones in treatments with high production of mature and regeneration ability in vitro (Leljak-Levanic ˇ et al., 2016). embryos. However, from our results, it seems clear that low levels in the CK O-glucosides (reversible forms of storage), particularly relation between IAA and CK basesCribosides are related to cZOG, were highest in those embryos coming from EMs highest success of SE process in radiata pine. maturated in the most stressful conditions (28 C and 10 gL Further studies increasing the temperatures during shorter gelrite, lowest water availability). However, CK O-glucosides were periods of time can help us to go further in the understanding found in minute amounts (below 4 pmolg DW), for instance of the mechanisms involved in SE in Pinus radiata as well as the Montalbán et al. (2011) reported O-glucoside concentrations role of phytohormones in this process. above 240 pmolg DW in P. radiata vegetative buds. Low content of IAA and iP were obtained in Se treated with the highest gelrite concentration at the standard temperature AUTHOR CONTRIBUTIONS (23 C). In this sense, in cotton a high content of both groups of phytohormones were related to the rediﬀerentiation stage that PM and IM conceived and planned the experiments. PM, OG-M, leads to embryogenesis induction (Zeng et al., 2007). Somleva and IM carried out maturation experiments. OG-M, ON, and MS et al. (1995) and Sáenz et al. (2003) have found an inverse carried out phytohormone analyses. IM, MU, and TG carried out relationship between cytokinins and the embryogenic response; the statistical analyses. All authors provided critical feedback and in this sense, the treatment that provoked the highest success helped to shape the research, analyses, and manuscript. in the SE process (23 C, 10 gL gelrite) showed a low level of IAA/basesCribosides CKs relation. So it seems that IAA can play an important role in radiata pine SE maturation FUNDING process; this conclusion is in agreement to the postulation that auxin metabolism (biosynthesis, conjugation, degradation), This research was funded by MINECO (Spanish Government) intercellular transport, and signaling is crucial in coordinating the project (AGL2016-76143-C4-3R), CYTED (P117RT0522), and morphogenesis and development of plant reproductive organs, DECO (Basque Government). The Ministry of Education, Youth including the formation of embryos (Benková et al., 2003; and Sports of the Czechia provided support for this project via Bernardi et al., 2012). The accumulation of endogenous IAA in the National Program for Sustainability I (LO1204). response to abiotic stress may have a mediating role during SE (Pen ˇ cík ˇ et al., 2015; Nic-Can et al., 2016). It seems that the diﬀerences found in speciﬁc CKs analyzed SUPPLEMENTARY MATERIAL did not provoke changes in maturation rates. Our results suggest that none of phytohormones found acts alone in the acquisition The Supplementary Material for this article can be found online of embryogenic maturation capacity; it seems that the dynamic at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01898/ and complementary actions of the auxin and cytokinin pathways full#supplementary-material Arnau, J. A., Tadeo, F. R., Guerri, J., and Primo-Millo, E. (1999). 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Changes in water status and proline and abscisic acid concentrations in developing somatic embryos of Conﬂict of Interest Statement: The authors declare that the research was pedunculate oak (Quercus robur) during maturation and germination. Tree conducted in the absence of any commercial or ﬁnancial relationships that could Physiol. 24, 1251–1257. doi: 10.1093/treephys/24.11.1251 be construed as a potential conﬂict of interest. Quoirin, M., and Lepoivre, P. (1977). Études des milieu adaptés aux cultures in vitro de Prunus. Acta Hortic. 78, 437–442. doi: 10.17660/ActaHortic.1977.78.54 Copyright © 2018 Moncaleán, García-Mendiguren, Novák, Strnad, Goicoa, Ugarte Rittenberg, D., and Foster, L. (1940). A new procedure for quantitative analysis by and Montalbán. This is an open-access article distributed under the terms of isotope dilution, with application to the determination of amino acids and fatty the Creative Commons Attribution License (CC BY). The use, distribution or acids. J. Biol. Chem. 133, 727–744. reproduction in other forums is permitted, provided the original author(s) and the Sáenz, L., Jones, L. H., Oropeza, C., Vlácil, ˇ D., and Strnad, M. (2003). Endogenous copyright owner(s) are credited and that the original publication in this journal isoprenoid and aromatic cytokinins in diﬀerent plant parts of Cocos nucifera is cited, in accordance with accepted academic practice. No use, distribution or (L.). Plant Growth Regul. 39, 205–215. doi: 10.1023/A:1022851012878 reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 13 December 2018 | Volume 9 | Article 1898 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Plant Science Unpaywall http://www.deepdyve.com/lp/unpaywall/temperature-and-water-availability-during-maturation-affect-the-60i2kClcim

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Abstract

fpls-09-01898 December 18, 2018 Time: 19:6 # 1 ORIGINAL RESEARCH published: 20 December 2018 doi: 10.3389/fpls.2018.01898 Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Proﬁle of Radiata Pine Somatic Embryos 1 1 2 2 Paloma Moncaleán , Olatz García-Mendiguren , Ondrej Novák , Miroslav Strnad , 3 3 1 Tomás Goicoa , María D. Ugarte and Itziar A. Montalbán 1 2 Department of Forestry Science, NEIKER-Tecnalia, Arkaute, Spain, Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Institute of Experimental Botany CAS, Palacký University Olomouc, Olomouc, Czechia, Department of Statistics, Computer Science and Mathematics, Universidad Pública de Navarra, Pamplona, Spain Edited by: Somatic embryogenesis (SE) provides us a potent biotechnological tool to manipulate Munetaka Sugiyama, The University of Tokyo, Japan the physical and chemical conditions (water availability) along the process and to study Reviewed by: their effect in the ﬁnal success in terms of quantity of somatic embryos produced. In Glória Catarina Pinto, the last years, our research team has been focused on the study of different aspects University of Aveiro, Portugal of the SE in Pinus spp. One of the main aspects affecting SE is the composition of Tsuyoshi E. Maruyama, Forestry and Forest Products culture media; in this sense, phytohormones play one of the most crucial roles in this Research Institute, Japan propagation system. Many studies in conifers have shown that different stages of SE and *Correspondence: somatic embryo development are correlated with distinct endogenous phytohormone Itziar A. Montalbán [email protected] proﬁles under the stress conditions needed for the process (i.e., cytokinins play a regulatory role in stress signaling, which it is essential for radiata pine SE). Based on Specialty section: this knowledge, the aim of this study was to test the effect of different temperatures This article was submitted to Plant Development and EvoDevo, (18, 23, and 28 C) and gelling agent concentrations (8, 9, and 10 gL ) during the a section of the journal maturation stage of Pinus radiata SE in maturation and germination rates. Parallel, Frontiers in Plant Science phytohormone proﬁle of somatic embryos developed was evaluated. In this sense, Received: 24 September 2018 the highest gellan gum concentration led to signiﬁcantly lower water availability. At this Accepted: 07 December 2018 Published: 20 December 2018 gellan gum concentration and 23 C a signiﬁcantly higher number of somatic embryos Citation: was obtained and the overall success of the process increased with respect to other Moncaleán P, treatments assayed. The somatic embryos produced in these conditions showed the García-Mendiguren O, Novák O, Strnad M, Goicoa T, Ugarte MD and highest concentration of iP-type cytokinins and total ribosides. Although, the different Montalbán IA (2018) Temperature conditions applied during maturation of somatic embryos led to different hormonal and Water Availability During proﬁles, they did not affect the ex vitro survival of the resulting somatic plants, where Maturation Affect the Cytokinins and Auxins Proﬁle of Radiata Pine no signiﬁcant differences were observed. Somatic Embryos. Front. Plant Sci. 9:1898. Keywords: cytokinins, embryonal masses, embryogenic cell lines, indol-3-acetic acid, Pinus radiata, somatic doi: 10.3389/fpls.2018.01898 embryogenesis Frontiers in Plant Science | www.frontiersin.org 1 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 2 Moncaleán et al. Phytohormones in Pine Somatic Embryos Moncaleán et al., 2003) and gymnosperms (Moncaleán et al., INTRODUCTION 2005; Montalbán et al., 2012). On the other hand, the role of The use of vegetative propagation in forestry is the fastest, indole-3- acetic acid (IAA) and CKs in the SE process have been most ﬂexible and eﬀective way to produce enough genetically analyzed in cotton (Zeng et al., 2007; Jin et al., 2014) and wheat improved material to meet future wood demands (Lelu-Walter (Hess and Carman, 1998; Jiménez and Bangerth, 2001) and fern et al., 2013). Somatic embryogenesis (SE) is currently considered (Grzyb et al., 2017). However, up to know a deep analysis of one of the successful biotechnological techniques for mass endogenous cytokinins and IAA concentration in SE process in propagation, enabling multi-varietal forestry (Park et al., 2016) pines has not been determined. In this sense, and as it has been and for cryopreservation of embryonal masses (EMs) from elite described previously, IAA and other phytohormones have an genotypes (Corredoira et al., 2006). important role in embryo diﬀerentiation events (Silveira et al., In the last years, several improvements in diﬀerent aspects 2004; Vondrakova et al., 2018); thus, a better understanding of of Pinus spp. SE process, such as initiation (Montalbán et al., the phytohormone proﬁle in somatic embryos under diﬀerent 2012), maturation (Montalbán et al., 2010) and germination environmental conditions could enable us to manipulate this (Montalbán and Moncaleán, 2018) have been carried out stage of SE to increase its eﬃciency. Taking into account all the in our laboratory. Traditionally, the adjustments have been above mentioned studies, the main objective of this study was focused on modiﬁcations of basal medium components (Salajova the evaluation of how the environment of cultures (gellan gum and Salaj, 2005; Zhang et al., 2007), exposure to diﬀerent concentration and temperature) along maturation stage aﬀects types and concentrations of exogenous plant growth regulators the success of radiata pine SE processes as well as to analyze its (Choudhury et al., 2008) and the use of diﬀerent initial explants eﬀect in CK and IAA endogenous levels. (Hargreaves et al., 2017). One of the main bottlenecks of the SE in conifers is the progression from immature embryogenic cultures into MATERIALS AND METHODS mature cotyledonary embryos able to develop well-growing plants (Harvengt, 2005). Several external stimuli, such as Maturation Experiment plant growth regulators (Ayil-Gutiérrez et al., 2013), osmotic Plant Material agents (Kikuchi et al., 2006), nutritional components (Pen ˇ cík ˇ Ten embryogenic cell lines (ECLs) from four open-pollinated et al., 2015), amongst others, have been recognized as essential Pinus radiata D. Don trees were selected for the experiments. The factors in determining both the hormone biosynthesis and trees belonged to a seed orchard established by Neiker-Tecnalia 0 0 0 0 the developmental fate of explant cells (Grzyb et al., 2017). in Deba (Spain; latitude: 43 16 59 N, longitude: 2 17 59 W, In conifers, maturation is stimulated by reduction of water elevation: 50 m). The ECLs were initiated and proliferated in availability by means of raising the osmoticum of the medium clumps in standard conditions following Montalbán et al. (2012) (Montalbán et al., 2010) or the concentration of gelling (Figure 1). agent (Klimaszewska and Smith, 1997). Modifying gellan Maturation Procedure gum concentration in maturation media has been studied in a few species of angiosperms (Márquez-Martín et al., The basal medium used in all phases of SE process was 2011) and gymnosperms (Morel et al., 2014); these authors Embryo Development Medium (EDM, Walter et al., 2005). reported an improvement in the development and maturation For initiation and proliferation 30 gL sucrose, 4.5 mM 2,4- of somatic embryos (Se) of P. pinaster by increasing gellan dichlorophenoxyacetic acid and 2.7 mM benzyladenine (BA) were 1 1 R gum concentration, pointing out that the eﬀect of gellan gum added. As gelling agent, 3 gL and 4.5 gL Gelrite were used concentration on plant response is provoked by changes of for initiation and proliferation, respectively. The pH was adjusted water availability in the culture medium. In our laboratory, we to 5.7 prior to sterilization at 121 C for 20 min. After autoclaving, demonstrated in previous studies that the agar concentration ﬁlter-sterilized solutions (pH 5.7) of the following amino acids and the temperature had a signiﬁcant eﬀect on initiation step of (Walter et al., 2005) were added to partially cooled medium prior radiata pine SE; and in the case of temperature, in the subsequent to dispensing into Petri dishes (90 mm 20 mm): 550 mgL 1 1 phases of the process (García-Mendiguren et al., 2016). As far L-glutamine, 525 mgL L-asparagine, 175 mgL L-arginine, 1 1 1 as we know, the eﬀect of diﬀerent temperatures in maturation 19.75 mgL L-citrulline, 19 mgL L-ornithine, 13.75 mgL 1 1 step has not been tested in Pinus spp., only in some angiosperms L-lysine, 10 mgL L-alanine, and 8.75 mgL L-proline. All (Wang et al., 2014). chemical products were purchased from Duchefa. The active cytokinin (CK) pool is regulated during Three weeks before the start of maturation experiment, EMs development by biosynthesis, uptake from extracellular (Figure 2A) were suspended in liquid growth regulators-free sources, metabolic interconversions, inactivation, degradation, basal medium in 50 mL centrifuge tubes; then the suspension was and transport (Kakimoto, 2003). The relative abundance of poured onto a ﬁlter paper disk (Whatman No. 2) in a Büchner diﬀerent CKs can vary greatly between plant species, tissues funnel. The ﬁlter papers with 250 mg of EMs each were laid and developmental stages, and depends on the environmental on proliferation media. Maturation of ECLs was carried out conditions (Frébort et al., 2011). Some studies have examined the following the protocol described by Montalbán et al. (2010). eﬀect of varying the culture atmosphere on endogenous CK levels Brieﬂy, EMs were suspended in liquid growth regulators-free after organogenesis in angiosperms (Jiménez and Bangerth, 2001; basal medium and ﬁltered as described above. The ﬁlter papers Frontiers in Plant Science | www.frontiersin.org 2 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 3 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 2 | Pinus radiata embryonal masses (A), mature somatic embryos (B), germinated somatic planlets (C), and acclimatized somatic plants (D). embryonic root caps pointing downward, were titled vertically FIGURE 1 | Scheme of the experimental design of maturation experiment. at an angle of approximately 45 , and cultured at 23 1 C Cultures were stored at three different temperatures: 18 C (circle), 23 C 2 1 (square), or 28 C (hexagon) and at three different agar concentrations (inside under 16 h photoperiod at 100 mmolm s provided by cool circles, squares, and hexagons). white ﬂuorescent tubes (TFL 58 W/33; Philips, France). Plantlets were subcultured to culture vessels with medium of the same composition after 6 weeks. with 80 mg of EMs each were laid on maturation media. The basal After a germination period of 14–16 weeks, plantlets were media used were the same as described before supplemented transferred to sterile peat:perlite (7:3, v/v) and acclimatized in a 60 gL sucrose and the amino acid mixture used for initiation greenhouse with environmental control where the humidity was and proliferation of the EMs. Also, 60 mM abscisic acid (ABA, progressively decreased from 99 to 70% during the ﬁrst month. purchased from Olchemim) was added. In order to increase or reduce the water availability of the medium, the amount of Gelrite in the maturation medium was Water Availability Determination 1 1 increased (10 gL ) or reduced (8 gL ), respectively, respect to The water availability of maturation media (EDM) under the standard conditions (9 gL ). The cultures were kept under diﬀerent maturation conditions (three gellan gum concentrations diﬀerent temperatures, 5 C above (28 C) and below (18 C) the and three temperatures) was determined by weighing and placing standard (23 C). Thus, nine diﬀerent treatments were tested, one autoclaved ﬁlter paper disk (Whatman No. 2) on the surface carrying out the other SE steps under standard conditions of the medium following Klimaszewska et al. (2000). The Petri (Figure 1). Summarizing, the whole experiment comprised nine dishes were incubated under the same conditions as those used diﬀerent treatments, 10 ECLs and four replicates per ECL and for maturation for 15 weeks. Then, the ﬁlter paper disks were maturation condition (a total of 360 maturation plates). All the weighed and the amount of water (g) absorbed was calculated. cultures were kept in darkness. Four replicates per maturation condition were used. Germination Procedure After 15–18 weeks, Se were transferred to germination medium. Extraction, Puriﬁcation and This medium was half strength macronutrients LP (Quoirin Quantiﬁcation of Endogenous Cytokinins and Lepoivre, 1977, modiﬁed by Aitken-Christie et al., 1988) Plant Material supplemented with 2 gL activated charcoal (AC, Sigma- Aldrich) and 9.5 gL Difco Agar granulated (Becton & Somatic embryos (Figure 2B) obtained from EMs maturated Dickinson). In those ECLs and maturation conditions producing at 18, 23, and 28 C in EDM medium solidiﬁed with 8, 9, and Se, three to four Petri dishes, and 20 Se per Petri dish, with the 10 gL (Figure 1). Frontiers in Plant Science | www.frontiersin.org 3 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 4 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 3 | Water availability (g) on EDM medium (A) stored at three different temperatures (18, 23, and 28 C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L ). Different letters show signiﬁcant differences at a signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test. Speed-Vac concentrator and dissolved in 30 ml of mobile phase Quantitative Analysis of CKs and IAA by Liquid prior to UHPLC-MS/MS analyses. Chromatography-Single Quadrupole MS Mass analysis was carried out using an Acquity UPLC System The 41 CKs and auxins analyzed were the following: cis-Zeatin (Waters, Milford, MA, United States), and a triple-quadrupole (cZ), cis-Zeatin riboside (cZR), cis-Zeatin O-glucoside (cZOG), TM mass spectrometer Xevo TQ-S MS (Waters MS Technologies, cis-Zeatin-9-glucoside (cZ9G), cis-Zeatin riboside O-glucoside Manchester, United Kingdom). Quantiﬁcation was achieved (cZROG), cis-Zeatin riboside-5 -monophosphate (cZMP), by monitoring protonated precursors and the appropriate trans-Zeatin (tZ), trans-Zeatin riboside (tZR), trans-Zeatin product ions. Multiple reaction monitoring transitions as well O-glucoside (tZOG), trans-Zeatin-7-glucoside (tZ7G), trans- as chromatographic and tandem mass spectrometry (MS) Zeatin-9-glucoside (tZ9G), trans-Zeatin riboside O-glucoside conditions were selected according to the method described by (tZROG), trans-Zeatin riboside-5 -monophosphate (tZMP), Novák et al. (2008, 2012). All MS data were processed using Dihydrozeatin (DHZ), Dihydrozeatin riboside (DHZR), TM TM the MassLynx software with TargetLynx program (version Dihydrozeatin O-glucoside (DHZOG), Dihydrozeatin-7- 4.2. Waters, Milford, MA, United States), and compounds were glucoside (DHZ7G), Dihydrozeatin-9-glucoside (DHZ9G), quantiﬁed by standard isotope dilution analysis (Rittenberg and Dihydrozeatin riboside O-glucoside (DHZROG), Dihydrozeatin 0 6 Foster, 1940). riboside-5 -monophosphate (DHZMP), N -Isopentenyladenine 6 6 (iP), N -Isopentenyladenosine (iPR), N -Isopentenyladenine- 7-glucoside (iP7G), N -Isopentenyladenine-9-glucoside Data Collection and Statistical Analysis 6 0 (iP9G), N -Isopentenyladenosine-5 -monophosphate The ECLs subjected to diﬀerent maturation conditions that had 6 6 (iPMP), N -Benzyladenine (BA), N -Benzyladenosine (BAR), produced Se were registered and the percentage of maturation 6 6 0 N -Benzyladenine-9-glucoside (BA9G), N -benzyladenosine-5 - and the number of Se per gram of EM (fresh weight) were monophosphate (BARMP), ortho-Topolin (oT), ortho-Topolin riboside (oTR), ortho-Topolin-9-glucoside (oT9G), meta-Topolin (mT), meta-Topolin riboside (mTR), meta- Topolin-9-glucoside (mT9G), para-Topolin (pT), para-Topolin riboside (pTR). Indol-3-acetic acid (IAA), 2-oxindole-3-acetic acid (oxIAA), IAA-aspartate (IAA-Asp), and IAA-glutamate (IAA-Glu). Three replicates of 3 mg of Se were analyzed according to the protocol described by Svacinov ˇ á et al. (2012) and Pen ˇ cík ˇ et al. (2018) for cytokinin and auxin proﬁling, respectively, using miniaturized puriﬁcation (pipette tip solid-phase extraction). Samples were extracted with the addition of stable isotope-labeled internal standards (each at 0.5 pmol per sample) and transferred after extraction onto Stage Tips and puriﬁed according to the aforementioned protocol, consisting of C18, SDB-RPS, and FIGURE 4 | Maturation percentages (%) in P. radiata for embryogenic cell lines Cation-SR sorbents for cytokinins or C18 and SDB-XC sorbents cultured at three different temperatures (18, 23, and 28 C) and three gellan for IAA and IAA conjugates. Eluates were collected into new gum concentrations (8, 9, and 10 g L ). clean microcentrifuge tubes and evaporated to dryness in a Frontiers in Plant Science | www.frontiersin.org 4 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 5 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 5 | Number of P. radiata somatic embryos per gram of embryogenic tissue (fresh weight) maturated on EDM medium (A) at three different temperatures (18, 23, and 28 C), (B) supplemented with three different gellan gum concentrations (8, 9, and 10 g L ). Different letters show signiﬁcant differences at a signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with the Tukey HSD post hoc test. calculated. The number of somatic embryos from diﬀerent Signiﬁcant diﬀerences were found among the levels of gellan maturation conditions that had germinated was recorded and gum and temperature although the interaction between the germination percentages were calculated (Figure 2C). A logistic tested variables was not signiﬁcant. In this sense, the highest regression and the corresponding analysis of deviance were temperature analyzed (28 C) led to a signiﬁcantly lower water conducted to evaluate the eﬀect of temperature and gellan availability than the other temperatures tested (Figure 3A). gum concentration on diﬀerent stages of plant conversion for EDM medium supplemented with 10 gL gelrite showed also P. radiata. signiﬁcantly lower water availability than those supplemented Then, the success rate (plants able to be planted in ex vitro with 8 or 9 gL (Figure 3B). conditions) was evaluated in plantlets coming from ECLs Maturation percentages were 100% in all treatments except in cultured under diﬀerent maturation treatments and it was ECLs cultured at 23 and 28 C in culture media solidiﬁed with 1 1 calculated with respect to standard conditions (23 C and 9 gL 8 gL gelrite (90 and 60%, respectively) and in those cultured at gellan gum, considered as 100% for maturation rate, number of 28 C with 10 gL gelrite (80%, Figure 4). Se per gram of EM and germination percentage). When the analysis of deviance for the number of P. radiata After 14 weeks under ex vitro conditions (Figure 2D), the Se was evaluated, statistical diﬀerences were found for the survival was calculated. To evaluate the eﬀect of the maturation treatments, an analysis of variance (ANOVA) was carried out. For water availability analyses, a two-way analysis of variance (ANOVA) was carried out to assess the eﬀect of temperature and gellan gum concentration on the water availability of the media. When signiﬁcant diﬀerences between the levels of the two variables were found, the Tukey HSD post hoc test was carried out to ﬁnd out which levels were statistically diﬀerent. Data for the diﬀerent CK types and metabolites, for IAA contents and the ratio of IAA: active CKs were evaluated using a two-way ANOVA to identify diﬀerences between treatments. Multiple comparisons were performed using Tukey’s post hoc test. FIGURE 6 | Germination percentages (%) of P. radiata somatic embryos RESULTS maturated on EDM medium supplemented with three different gellan gum concentrations (8, 9, and 10 g L ) and stored at three different temperatures (18, 23, and 28 C). Different letters show signiﬁcant differences at a Maturation Experiment signiﬁcance level of a = 0.05. Pairwise comparisons have been assessed with Detailed information on the statistical analyses performed is the Tukey HSD post hoc test. given as Supplementary Material. Frontiers in Plant Science | www.frontiersin.org 5 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 6 Moncaleán et al. Phytohormones in Pine Somatic Embryos diﬀerent temperatures and gellan gum concentrations tested highest temperature with the standard concentration of gelrite but no interaction between them was found. The Tukey HSD (9 gL , Figure 8A). The levels of DHZ were signiﬁcantly higher post hoc test revealed signiﬁcant diﬀerences between the three in somatic embryos cultured at 18 and 23 C with the lowest temperatures tested; being 23 and 28 C the temperatures that Gelrite concentration in the culture media (8 gL , Figure 8B). produced the highest and the lowest number of Se (1036 The iP concentration was the highest in Se developed in a culture 1 1 and 374 Seg EM, respectively, Figure 5A). Moreover, ECLs media solidiﬁed with 10 gL gelrite at 28 C (Figure 8C). maturated in culture media with the lowest water availability Signiﬁcant diﬀerences in CK ribosides (Figure 9) were found (10 gL ) produced a signiﬁcantly higher number of somatic among the levels of gellan gum but no signiﬁcant diﬀerences embryos (Figure 5B). were observed among the levels of temperature for tZR, cZR, and Regarding germination percentage, signiﬁcant interaction DHZR; however, a signiﬁcant interaction between temperature between temperature, and gellan gum concentration at and agar was observed for all CK ribosides analyzed. tZR levels maturation was observed. The highest germination rates, increased signiﬁcantly when the gelrite concentration increase above 90%, were obtained when EMs were maturated at 18 and from 9 to 10 in somatic embryos cultured at 18 and 23 C 23 C, whereas maturation at 28 C led to lower germination (Figure 9B). The highest DHZR concentration was found in rates, particularly when it was combined with 9 or 10 gL gellan Se cultured in presence of the lowest concentration of gelrite gum (74 and 64%, respectively, Figure 6). (8 gL ) at 18 C (Figure 9C), the DHZR levels obtained this The eﬀect of physical and chemical modiﬁcation of culture treatment did not present diﬀerences with those at standard media in the success of the conversion from EMs to plants is temperature (23 C) regardless the gellan gum concentration. shown in Figure 7. An improvement in plant conversion was iPR levels were signiﬁcantly higher in Se cultured at standard observed when the gellan gum concentration was increased at all temperature in presence of the highest gelrite concentration 1 1 temperatures tested. In this sense, 23 C and 10 gL showed the (10 gL ) (Figure 9D). best results improving by 132% the plant conversion process. The levels of tZRMP, cZRMP, DHZRMP, and iPRMP were Somatic plants coming from diﬀerent treatments during under detection limit in all the samples analyzed. Also, tZOG, maturation stage did not show signiﬁcant diﬀerences in tZROG, cZROG, and DHZROG were under the limit of ex vitro survival after 14 weeks growing in the greenhouse detection. Signiﬁcant diﬀerences in CK O- glucosides were found (Supplementary Material). However, highest rates were found in among the levels of gellan gum and temperature (except for plants coming from EMs maturated at 28 C independently of the DHZOG), and for the interaction between the two variables. The gellam gum concentration. Survival percentage increased parallel CK O-glucosides were represented by a low amount of cZOG and to the increment of temperature during the maturation stage. DHZOG. In this sense, Se maturated at the highest temperature and gelling agent concentration showed the highest levels of Endogenous Phytohormone Analyses cZOG (Figure 10A). On the contrary, DHZOG concentration Detailed information on the contents of the 41 CKs and auxins in was the highest in Se developed at the lowest temperature and all samples analyzed is provided as Supplementary Material. gelrite concentration in the culture media (Figure 10B). Signiﬁcant diﬀerences in CK bases were found among the Regarding the isoprenoid CK type, signiﬁcant diﬀerences in levels of temperature, gellan gum (except for cZ) and the CK types were found among the levels of temperature (except interaction between the tested variables. In all the Se analyzed for cZ), gellan gum and the interaction between the tested the appearance of tZ was under the limit of detection. The variables. The tZ-type CK concentration was the highest in Se concentration of cZ was the highest in Se maturated at the developed at 18 and 23 C with the highest concentration of FIGURE 7 | Success rate (%) in the process of plant conversion in P. radiata embryogenic cell lines maturated at three different temperatures (18, 23, and 28 C) and 1 1 three gellan gum concentrations (8, 9, and 10 g L ) with respect to standard maturation conditions (23 C and 9 gL gellan gum, considered as 100% of success). Frontiers in Plant Science | www.frontiersin.org 6 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 7 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 8 | Endogenous levels (pmol g DW) of the cytokinin bases in FIGURE 9 | Endogenous levels (pmol g DW) of the cytokinin ribosides in somatic embryos maturated at different temperatures in culture media somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. cZ (A), DHZ (B), and iP solidiﬁed with different concentrations of gellan gum. cis-zeatin riboside (cZR) (C) concentrations. Different letters indicate signiﬁcant differences among (A), trans-zeatin riboside (tZR) (B), dihydrozeatin riboside (DHZR) (C), and treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square N6-isopentenyladenosine (iPR) (D) concentrations. Different letters indicate continuous line (23 C), and square discontinuous line (18 C). signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). gelling agent and at 28 C with the standard concentration of gelrite (Figure 11A). The lowest cZ-type CK concentration was observed in treatments at 18 and 23 C with the standard gelrite values for this group were obtained in Se cultured at standard concentration (Figure 11B). The concentration of DHZ-type temperature (23 C) and the highest gelrite concentration CKs was signiﬁcantly higher in Se developed at 18 and 23 C (Figure 12B). O-glucosides were signiﬁcantly higher in Se in culture media solidiﬁed with 8 gL of gelrite (Figure 11C). cultured at the highest temperature and gelrite concentration On the contrary, iP-types endogenous concentration was (Figure 12C). signiﬁcantly higher in Se cultured with the highest concentration Figure 13 shows the relative abundance of isoprenoid CKs of gelling agent (10 gL ) at standard temperature (23 C) than in Se by treatment. CK ribosides are the predominant group of Se from other treatments (Figure 11D). cytokinins respect to bases and O-glucosides and a decrease Analyzing the results by groups, CK nucleotides and in the bases content is observed parallel to an increase of N -glucosides were under the limit of detection. Signiﬁcant gelrite concentration and temperature. Although the presence diﬀerences in CK functional groups were found among the levels of aromatic type CKs (N -benzyladenine, meta-topolin, ortho- of temperature, gellan gum and the interaction between the tested topolin, and para-topolin) and their metabolites was also variables. A signiﬁcantly higher concentration of bases was found analyzed, theirs levels were under the limit of detection. in Se developed at 18 and 23 C in culture media solidiﬁed with Taking a general look at the results presented, it was observed a the lowest gelrite concentration (Figure 12A). Ribosides was similar trend for CK concentration in Se cultured at 18 C y 23 C the CK group with the most remarkable presence, the highest when the agar concentration increased from 8 to 9 gL , except Frontiers in Plant Science | www.frontiersin.org 7 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 8 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 10 | Endogenous levels (pmol g DW) of the O-glucosides in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. cis-zeatin O-glucoside (cZOG) (A), dihydrozeatin O-glucoside (DHZOG) (B) concentrations. Different letters indicate signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). iPR, DHZR and cZOG. When considering the results by CK- type or CK-metabolite this trend was also observed in all cases. At 28 C, an increase of bases and ribosides concentration was observed from 8 to 9 gL ; in the case of iP, iPR, and cZOG their concentration continued increasing signiﬁcantly with gellan gum concentration (10 gL ). The IAA, 2-oxindole-3-acetic acid (oxIAA), and IAA- glutamate levels showed the highest levels in Se developed at the highest temperature with diﬀerent concentrations of gelrite (Figures 14A–C). Moreover, Se cultured at 28 C in media FIGURE 11 | Endogenous levels (pmol g DW) of different isoprenoid with the highest gelrite concentration showed signiﬁcantly cytokinin types in somatic embryos maturated at different temperatures in higher levels of oxIAA and IAA-glutamate (Figures 14B,C). culture media solidiﬁed with different concentrations of gellan gum. cZ-type (A), tZ-type (B), DHZ-type (C), and iP-type (D) concentrations. Different IAA-aspartate levels were under the limit of detection in all letters indicate signiﬁcant differences among treatments by Tukey’s post hoc samples analyzed. When relation between IAA and active test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square CKs (basesCribosides) was evaluated, we could observe discontinuous line (18 C). low values in Se developed at standard temperature (23 C) independently of the gelrite concentration in the culture media (Figure 15). To this respect, in Douglas-ﬁr, a reduction in water availability of the culture media provoked an increase in Se quality (Lelu- Walter et al., 2018) probably because it has been described that DISCUSSION the reduction in water availability leads to obtain embryos with The inductive conditions which allow diﬀerentiated somatic low water content similar to their zygotic counterparts (Morel cells to develop into competent dediﬀerentiated cells can be et al., 2014). achieved by the addition of speciﬁc phytohormones, and/or by The culture of embryogenic calli in media with an increased exposing the tissue to diﬀerent stress factors (Leljak-Levanic ˇ gelling agent concentration is a common procedure for the et al., 2016). Moreover, water relations between the embryo and production of high quality mature embryos (Krajnákov ˇ á et al., its environment play a regulatory role in embryo development, 2009; Troch et al., 2009; Buendía-González et al., 2012). Our particularly during maturation (from Márquez-Martín et al., results in P. radiata are in agreement with those obtained in 2011) and for this reason, the water availability of maturation P. strobus where the best maturation results were obtained media was determined in our experiments. Radiata pine somatic increasing gellan gum concentration due to a shift in the embryos cultured in EDM medium supplemented with 10 gL developmental program of the culture, from proliferation of cells and stored at 28 C grew in an environment with the lowest and early somatic embryos to the production of embryos at water availability in comparison to the other conditions assayed. advanced stages (Klimaszewska et al., 2000). Frontiers in Plant Science | www.frontiersin.org 8 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 9 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 13 | Relative abundances (expressed in percentage) of endogenous isoprenoid cytokinins in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Bases (black bar), Ribosides (gray bar), and O-glucosides (white bar). Pereira et al., 2016) as well as in proliferation stage of P. halepensis (Pereira et al., 2017) was studied and we found that the highest temperature seemed to produce a selective pressure as pointed by Fehér (2015); In this regard, Bonga et al. (2010) suggested that reducing or increasing temperatures may improve initiation and proliferation since temperature stress may promote cellular reprogramming. Our results are in agreement with de Almeida et al. (2014) where temperature had a signiﬁcant inﬂuence on the direct SE capacity in coﬀee. Regarding gellan gum concentration, some reports suggested 1 that increasing the concentration of a gelling agent from 4 to FIGURE 12 | Endogenous levels (pmol g DW) of the cytokinin groups in 1 1 8 gL (Teyssier et al., 2011) or from 4 to 9 gL (Morel somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Cytokinin Bases (A), et al., 2014) promoted the maturation of the Se of some Ribosides (B), and O-glucosides (C) concentrations. Different letters indicate conifers. In this sense, Garin et al. (2000) demonstrated that signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). the phytagel concentration was critical for the maturation Triangle (28 C), square continuous line (23 C), and square discontinuous line of Se of ﬁve tested ECLs of Pinus strobus. The increase (18 C). of phytagel concentration in the maturation media allowed obtaining well-developed Se capable of germination in various species of pines (Klimaszewska et al., 2009) and hybrid larch Respect to the number of embryos developed, the temperature (Lelu-Walter and Pâques, 2009) probably due to the action and gellan gum concentration aﬀected signiﬁcantly the number in endogenous ABA modulation in mature somatic embryos of Se per gram of EM. In this sense the temperature (28 C) (Morel et al., 2014). In summary, our results agree with and the agar concentration (10 gL ) that provoked the lowest abovementioned studies pointing that increasing gelling agent water availability led to the lowest and the highest number of concentration is a good strategy to improve the Se yield in radiata Se, respectively. For this reason, it seems that water availability pine. was not related to the success of the process at this stage. In this The highest germination rates (above 90%) were obtained sense, there are two studies suggesting that under sub-optimal in Se coming from EMs maturated at 18 and 23 C, whereas water availability, plants may take to a premature drought maturation at 28 C led to lower germination rates, particularly stress response and arrest or drastically reduce growth due to when it was combined with 9 or 10 gL gellan gum. Our the eﬀect on source–sink relationships through preferentially results are not in agreement to Prewein et al. (2004) when inhibiting nutrient acquisition processes more and earlier than they described that desiccation of the embryos due to decreased necessary (Skirycz et al., 2011). It is clear that the water water availability in maturation media improves the germination availability registered in our experiments is not sub-optimal frequency, probably by reducing the endogenous ABA level or for the maturation of EMs. In our laboratory, the eﬀect of by changing the sensitivity to ABA (Jiménez, 2005). Unlike diﬀerent physical and chemical conditions at the initiation stage observed by Morel et al. (2014) in maritime pine, we found no of P. radiata and P. halepensis (García-Mendiguren et al., 2016; diﬀerences in germination rates with increasing concentration of Frontiers in Plant Science | www.frontiersin.org 9 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 10 Moncaleán et al. Phytohormones in Pine Somatic Embryos FIGURE 15 | Indole-3-acetic acid/Cytokinin bases and ribosides ration in somatic embryos maturated at different temperatures in culture media solidiﬁed with different concentrations of gellan gum. Triangle (28 C), square continuous line (23 C), and square discontinuous line (18 C). were obtained in somatic plants coming from EMs maturated at 28 C. There are only a few reports describing how the contents of diﬀerent endogenous hormones are altered by diﬀerent FIGURE 14 | Endogenous levels (pmol g DW) of indole-3-acetic acid (IAA) environmental conditions. The relative abundance of diﬀerent (A), IAA-glutamate (IAA-Glu) (B), 2-oxindole-3-acetic acid (oxIAA) (C) in CKs can vary greatly between plant species, tissues and somatic embryos IAA-glutamate (IAA-Glu), 2-oxindole-3-acetic acid (oxIAA) in developmental stages, and depends on the environmental somatic embryos maturated at different temperatures in culture media conditions (Frébort et al., 2011). In our experiments, the solidiﬁed with different concentrations of gellan gum. Different letters indicate prevailing isoforms of CKs were cis-isomers of Z and ZR signiﬁcant differences among treatments by Tukey’s post hoc test (a = 0.05). Triangle (28 C), square continuous line (23 C), and square discontinuous line according with Zur et al. (2015) in microspore embryogenesis (18 C). in triticale. Based on studies with Arabidopsis, cis-isomers were regarded as CK derivatives without any or with low biological activity. Further studies with non-model plants, like pines, agar, however, this could be due to diﬀerences in the range of showed that cis-isomers can be the dominant form of CK concentrations used by these authors (4 and 9 gL Phytagel) and in speciﬁc plant organs and/or stages of development (Emery us (8, 9, and 10 gL Gelrite). et al., 1998: Vyroubalová et al., 2009; Kudo et al., 2012). An improvement in plant conversion when the gellan gum To this respect, Gajdošová et al. (2011) proposed that cZ concentration was increased at all temperatures tested was can be qualiﬁed as a regulator of CK responses in plants observed. However, 23 C showed the best results improving under growth-limiting conditions. Our results only showed by 132% the plant conversion process. Summarizing, increasing signiﬁcantly diﬀerences in cZ in Se cultured with 9 gL the gellan gum concentration in the maturation stage improved of gelrite at the highest temperature (28 C) with the others the SE process in all radiata pine cultures independently of analyzed. the temperature of storage obtaining the best results when Highest amount of bases, the active forms of cytokinins somatic embryos were submitted to maturation process at control (Klemš et al., 2011), were found in Se developed under 18 and temperature (23 C). So, higher temperature and gellan gum 23 C and low gellan gum concentration, treatments showing the concentration applied at maturation stage did not result in an highest water availability. So, our results are in agreement to other increment of the eﬃciency of SE process like it was observed authors describing that a decrease in water availability could be when this stressful condition was applied at initiation stage related to the rapidly utilization of CK bases and tightly regulated (García-Mendiguren et al., 2016). Moreover, ex vitro survival did by mechanisms that prevent their accumulation at high levels not show signiﬁcant diﬀerences although the highest percentages (Moncaleán et al., 2005; Stirk et al., 2005). Frontiers in Plant Science | www.frontiersin.org 10 December 2018 | Volume 9 | Article 1898 fpls-09-01898 December 18, 2018 Time: 19:6 # 11 Moncaleán et al. Phytohormones in Pine Somatic Embryos CK ribosides, mainly represented by cZR and iPR, were regulate several developmental processes and their ability to higher in the treatment that led to the highest somatic embryo crosstalk makes them ideal candidates for mediating stress- production and success rate (23 C and 10 gL ). In terms adaptation responses according with Bielach et al. (2017) as of metabolic groups, ribosides were the predominant group of well as initiating various signal transduction pathways (Kohli cytokinins and its percentage respect to bases and O-glucosides et al., 2013). In this regard, comparing the endogenous levels increased in parallel with the decrease of water availability. of IAA, Z, ZR, iP, and iPR in several embryogenic habituated Nevertheless, in peach seeds, phosphates and ribosides of the callus lines of Asparagus oﬃcinalis, no correlation between DHZ and iP-type are the predominant CKs and functions embryogenic potential and endogenous hormone levels could be like embryo formation and growth, division of endosperm found (Limanton-Grevet et al., 2000). These and other ﬁndings primary nucleus, endosperm cellularization and endosperm cell demonstrate that there is no direct correlation in endogenous division have been attributed to them (Arnau et al., 1999). hormone content and embryogenic competence of diﬀerent So, opposite to Pacheco de Freitas et al. (2016) active CK genotypes, or between competent and non-competent genotypes, levels seem to be important during the later stages of embryo which suggests the involvement of additional, hormone-related development and maturation showing large amounts of these internal mechanisms for embryogenic competence regulation phytohormones in treatments with high production of mature and regeneration ability in vitro (Leljak-Levanic ˇ et al., 2016). embryos. However, from our results, it seems clear that low levels in the CK O-glucosides (reversible forms of storage), particularly relation between IAA and CK basesCribosides are related to cZOG, were highest in those embryos coming from EMs highest success of SE process in radiata pine. maturated in the most stressful conditions (28 C and 10 gL Further studies increasing the temperatures during shorter gelrite, lowest water availability). However, CK O-glucosides were periods of time can help us to go further in the understanding found in minute amounts (below 4 pmolg DW), for instance of the mechanisms involved in SE in Pinus radiata as well as the Montalbán et al. (2011) reported O-glucoside concentrations role of phytohormones in this process. above 240 pmolg DW in P. radiata vegetative buds. Low content of IAA and iP were obtained in Se treated with the highest gelrite concentration at the standard temperature AUTHOR CONTRIBUTIONS (23 C). In this sense, in cotton a high content of both groups of phytohormones were related to the rediﬀerentiation stage that PM and IM conceived and planned the experiments. PM, OG-M, leads to embryogenesis induction (Zeng et al., 2007). Somleva and IM carried out maturation experiments. OG-M, ON, and MS et al. (1995) and Sáenz et al. (2003) have found an inverse carried out phytohormone analyses. IM, MU, and TG carried out relationship between cytokinins and the embryogenic response; the statistical analyses. All authors provided critical feedback and in this sense, the treatment that provoked the highest success helped to shape the research, analyses, and manuscript. in the SE process (23 C, 10 gL gelrite) showed a low level of IAA/basesCribosides CKs relation. So it seems that IAA can play an important role in radiata pine SE maturation FUNDING process; this conclusion is in agreement to the postulation that auxin metabolism (biosynthesis, conjugation, degradation), This research was funded by MINECO (Spanish Government) intercellular transport, and signaling is crucial in coordinating the project (AGL2016-76143-C4-3R), CYTED (P117RT0522), and morphogenesis and development of plant reproductive organs, DECO (Basque Government). The Ministry of Education, Youth including the formation of embryos (Benková et al., 2003; and Sports of the Czechia provided support for this project via Bernardi et al., 2012). The accumulation of endogenous IAA in the National Program for Sustainability I (LO1204). response to abiotic stress may have a mediating role during SE (Pen ˇ cík ˇ et al., 2015; Nic-Can et al., 2016). It seems that the diﬀerences found in speciﬁc CKs analyzed SUPPLEMENTARY MATERIAL did not provoke changes in maturation rates. Our results suggest that none of phytohormones found acts alone in the acquisition The Supplementary Material for this article can be found online of embryogenic maturation capacity; it seems that the dynamic at: https://www.frontiersin.org/articles/10.3389/fpls.2018.01898/ and complementary actions of the auxin and cytokinin pathways full#supplementary-material Arnau, J. A., Tadeo, F. R., Guerri, J., and Primo-Millo, E. (1999). 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Changes in water status and proline and abscisic acid concentrations in developing somatic embryos of Conﬂict of Interest Statement: The authors declare that the research was pedunculate oak (Quercus robur) during maturation and germination. Tree conducted in the absence of any commercial or ﬁnancial relationships that could Physiol. 24, 1251–1257. doi: 10.1093/treephys/24.11.1251 be construed as a potential conﬂict of interest. Quoirin, M., and Lepoivre, P. (1977). Études des milieu adaptés aux cultures in vitro de Prunus. Acta Hortic. 78, 437–442. doi: 10.17660/ActaHortic.1977.78.54 Copyright © 2018 Moncaleán, García-Mendiguren, Novák, Strnad, Goicoa, Ugarte Rittenberg, D., and Foster, L. (1940). A new procedure for quantitative analysis by and Montalbán. This is an open-access article distributed under the terms of isotope dilution, with application to the determination of amino acids and fatty the Creative Commons Attribution License (CC BY). The use, distribution or acids. J. Biol. Chem. 133, 727–744. reproduction in other forums is permitted, provided the original author(s) and the Sáenz, L., Jones, L. H., Oropeza, C., Vlácil, ˇ D., and Strnad, M. (2003). Endogenous copyright owner(s) are credited and that the original publication in this journal isoprenoid and aromatic cytokinins in diﬀerent plant parts of Cocos nucifera is cited, in accordance with accepted academic practice. No use, distribution or (L.). Plant Growth Regul. 39, 205–215. doi: 10.1023/A:1022851012878 reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 13 December 2018 | Volume 9 | Article 1898

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Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

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Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

Temperature and Water Availability During Maturation Affect the Cytokinins and Auxins Profile of Radiata Pine Somatic Embryos

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