Benzyladenine Effects On Polyamine Contents And Proteomic Profiles During In Vitro Shoot Development And On Ex Vitro Rooting In Dalbergia Nigra (Vell.) Allemão Ex Benth. (Fabaceae)

Benzyladenine Effects On Polyamine Contents And Proteomic Profiles During In Vitro Shoot Development And On Ex Vitro Rooting In Dalbergia Nigra (Vell.) Allemão Ex Benth. 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Allemão Ex Benth. (Fabaceae) Lidia dos Santos Pessanha, Victor Paulo Mesquita Aragão, Tadeu dos Reis de Oliveira, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-1419531/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Dalbergia nigra is an endangered species from the Brazilian Atlantic Rainforest, and in vitro propagation can be applied for the conservation of this species. The aim of this study was to establish in vitro propagation and ex vitro rooting in D. nigra and evaluate the alterations in polyamines (PAs) and protein profiles during shoot development . The effect of MS and WPM culture media on in vitro germination was tested (%). For shoot induction, explants of apical and cotyledonary nodal segments from 45-day-old seedlings were inoculated in WPM culture medium supplemented with benzyladenine (BA; 0, 2.5 and 5 μM). Shoots obtained in vitro without and with 2.5 μM BA were rooted ex vitro with different concentrations (0, 100 and 500 μM) of indole-3-butyric acid (IBA). The best growth of seedlings was obtained in WPM culture medium. Treatment with 2.5 μM BA significantly increased the length of shoots by increasing free putrescine contents and the accumulation of proteins associated with shoot elongation, such as aspartate aminotransferase, elongation factor, calreticulin-3, and cell division cycle protein 48. Ex vitro rooting was obtained in all treatments of IBA, and the use of auxin was not necessary. The BA used for shoot multiplication significantly affected rooting, reducing the induction and number of roots but increasing the length of roots. This study showed the relevance of cytokinin, PAs and proteomic profiles on in vitro shoot development, as well as the auxin and cytokinin balance on ex vitro rooting in D. nigra. Adventitious rooting Auxin Cytokinin In vitro propagation Plant proteomics Polyamines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Message Micropropagation of Dalbergia nigra (Fabaceae) Introduction Several native wood species from the Atlantic rainforest are endangered, including Dalbergia nigra (Vell.) Allemão ex Benth, commonly known as Jacarandá-da-Bahia. Due to the intense exploitation of woody plants and the lack of reforestation programs, this species has been included as vulnerable on the Red List of the International Union for Conservation of Nature (IUCN 2021 ). Appropriate biotechnological and sustainable conservation strategies for many woody species need further research and development, and in vitro propagation technologies can be applied for conservation, with a great going to a global economic and ecological impact on sustaining tropical forest woody biodiversity (Pijut et al. 2012 ). Biotechnological tools, such as micropropagation, have been applied in studies aiming at the propagation of woody species (Fermino-Junior and Scherwinski-Pereira 2012 ; Perveen et al. 2013 ). Micropropagation is the commercially efficient propagation of species in a short period of time (Gupta et al. 2014 ), enabling clonal production and conservation of germplasm (Giri et al. 2004 ; Shukla et al. 2008 ; Kodym and Leeb 2019 ). Knowledge of the biochemical and molecular aspects of in vitro morphogenesis, such as the cellular mechanisms involved in the growth and development of these species, is necessary for the propagation and preservation of threatened species with difficulties in propagation by conventional methods (Pijut et al. 2007 ; Dias et al. 2012 ; Stuepp et al. 2018 ). The steps of in vitro propagation involve manipulation of the type of explants as well as the components of the culture medium, such as plant growth regulators (PGRs), to achieve optimal conditions for shoot multiplication and root induction (Bunn et al. 2011 ). Among the PGRs, cytokinins and auxins are the most commonly used in plant tissue cultures for shoot and root development (Phillips and Garda 2019 ). In the propagation of woody species, benzyladenine (BA) is the cytokinin most commonly used to promote the development of axillary buds, breaking apical dominance and stimulating shoot proliferation (Giri et al. 2004 ; Pijut et al. 2012 ). Studies have shown the relationship of PGRs with other compounds, such as polyamines (PAs), in shoot development (Aragão et al. 2016 ; Aragão et al. 2017b ). PAs are low molecular weight, aliphatic, polycationic compounds with positively charged nitrogen atoms naturally occurring in plants (Baron and Stasolla 2008 ). These compounds can interact with negatively charged macromolecules, such as DNA, RNA, phospholipids, cell wall components, and proteins (Baron and Stasolla 2008 ). Thus, PAs are essential for various physiological and developmental processes in plants (Santa-Catarina et al. 2007 ; Dutra et al. 2013 ), including shoot development (Aragão et al. 2017b ; Lerin et al. 2019 ; Oliveira et al. 2020 ). Changes in endogenous PA contents induced by exogenous addition, especially putrescine (Put), demonstrated its relevance for shoot development in C. fissilis (Aragão et al. 2017b ). In addition to PAs, specific proteins have been candidate markers associated with morphogenic competence during in vitro plant morphogenesis (Reis et al. 2016 ; Heringer et al. 2018 ). Proteomic studies revealed the involvement of multiple proteins with specific functions in competence for the in vitro development of shoots (Mitrović et al. 2012 ; Ghosh and Pal 2013 ). Changes in the accumulation of some proteins involved mainly in metabolic and cellular processes, such as cell division, during in vitro shoot development induced or not induced by exogenous putrescine (Put) demonstrated an important relationship of specific proteins with shoot development in C. fissilis (Aragão et al. 2016 ; Aragão et al. 2017b ). In addition to shoot development, the formation of adventitious roots is an essential step of in vitro plant propagation. Auxin plays an essential role in rooting, and indole-3-butyric acid (IBA) is the most commonly used due to its higher root-inducing capacity and greater stability to light (Pacurar et al. 2014 ). IBA use is well documented during in vitro and ex vitro rooting in several woody species (Pijut et al. 2012 ). Ex vitro rooting has the advantage of lower cost and saves time, reducing the cost of a micropropagation protocol up to 70% compared to in vitro rooting(Yan et al. 2009 ; Ranaweera et al. 2013 ; Patel et al. 2014 ). In addition, another advantage of ex vitro rooting is that plantlets do not need additional acclimatization, exhibiting good root development and improved plantlet survival compared to in vitro rooting (Yan et al. 2009 ; Gupta et al. 2014 ). Studies on the in vitro propagation of D. nigra have not yet been developed. In this sense, the establishment of in vitro propagation for this species can contribute to conservation programs and the repositioning of impacted areas. In addition, biochemical and molecular approaches can improve the knowledge of in vitro morphogenesis competence. Thus, the aim of this work was to establish the in vitro propagation and ex vitro rooting of D. nigra and evaluate the alterations in PA contents and protein profiles during shoot development. Materials And Methods Plant material Mature seeds obtained from Caiçara Comércio de Sementes LTDA located in Brejo Alegre, SP, Brazil (21°10'S and 50°10'W) were used for in vitro germination. Forty-five-day-old seedlings in vitro germinated were used as the source of apical and cotyledonary nodal explants for the shoot development experiments. Forty-five-day-old micropropagated shoots were used for the ex vitro rooting experiments. Effect of plant culture medium on in vitro seed germination For in vitro germination, seeds were surface disinfected according to Santa-Catarina et al. (2001), with modifications. First, seeds were washed with 250 mL distilled water, followed by immersion in 70% ethanol for 1 min and incubation in 2.5% sodium hypochlorite solution supplemented with the fungicide Derosal ® 500 SC (Bayer; São Paulo, Brazil; active ingredient carbendazim 500 g L -1 ; 200 µL of commercial solution per liter of water) for 30 min. Seeds were washed five times for 10 min each in sterile distilled water in a flow chamber. After disinfection, seeds were transferred to Murashige and Skoog (MS; Phytotechnology Lab, Overland Park, USA) (Murashige and Skoog 1962) and Woody Plant Medium (WPM; Phytotechnology) (Lloyd and McCown 1981) culture media, both supplemented with 20 g L -1 sucrose (Synth; São Paulo, Brazil) and 2 g L -1 Phytagel ® (Sigma–Aldrich, St. Louis, USA). The pH of the culture medium was adjusted to 5.7 before the use of Phytagel and autoclaved at 121 °C for 15 min. Then, seeds were incubated in a 16-h photoperiod at a light intensity of 55 μmol m -2 s -1 and a temperature of 25 ± 2 °C. The germination (%) and morphology of seedlings were analyzed after 30 days of incubation. Each treatment consisted of five repetitions, with 20 seeds of each repetition. Effect of explant, plant culture medium and BA concentration on shoot development Forty-five-day-old seedlings germinated in vitro were used as the source of explants. Apical and cotyledonary nodal segments (± 2 cm) were isolated from seedlings and cultured on MS and/or WPM culture medium supplemented with 20 g L -1 sucrose, 2 g L -1 Phytagel ® and different concentrations (0, 2.5 and 5 µM) of BA (Sigma–Aldrich). The pH of the culture medium was adjusted to 5.7 and autoclaved at 121 °C for 15 min. The explants were transferred to the culture medium with different treatments and maintained at a 16-h photoperiod under a light intensity of 55 μmol m -2 s -1 and a temperature of 25 ± 2 °C. Eight repetitions per treatment were used, with four explants of each repetition. The induction (%), number of shoots per explant and length of the first and second shoots were analyzed after 45 days of incubation. Samples were collected for PA and proteomic analyses and maintained at -80 °C until analysis. Effect of IBA on ex vitro rooting of shoots and plant acclimatization Shoots from apical and cotyledonary nodal segments (± 2 cm) cultured in WPM culture medium supplemented without and with 2.5 µM BA were used for shoot rooting. Shoots containing the apical meristem and leaves were treated for 30 s with different concentrations (0, 100 and 500 μM) of IBA (Sigma–Aldrich). Then, shoots were transferred to 50 mL plastic pots containing a mixture of PlantMax substrate (DDL Agroindustria, Paulínia, Brazil) with vermiculite (2:1; v/v). The shoots were maintained in plastic trays covered with a plastic film to maintain the high humidity needed for root development during ex vitro rooting and acclimatization. These plastic trays were kept in the growth room under a 16-h photoperiod, a light intensity of 55 μmol m -2 s -1 , and a temperature of 25 ± 2 °C. After 30 days, the humidity was gradually reduced until 40 days, when rooted shoots were considered acclimatized. The induction of rooting (%), number of roots initiated per shoot and root length were recorded after 45 days. Each treatment consisted of eight repetitions, with four shoots in each repetition. Free PA determination Determination of free PAs was performed according to Santa-Catarina et al. (2006) using samples of shoots from apical and cotyledonary nodal segments with 45 days of incubation without (control) and with 2.5 µM BA in WPM medium. Samples (200 mg fresh matter – FM – each, in triplicate) were ground in 1.2 mL of 5% perchloric acid (Merck Millipore, Darmstadt, Germany). After 1 h of incubation at 4 °C, the samples were centrifuged for 20 min at 20,000×g at 4 °C. The supernatant was collected, and free PAs were determined directly from the supernatant by derivatization with dansyl chloride (Merck Millipore) and identified by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) using a 5-μm C18 reverse-phase column (Shimadzu Shin-pack CLC ODS). The HPLC column gradient was created by adding increasing volumes of absolute acetonitrile (Merck Millipore) to a 10% aqueous acetonitrile solution with the pH adjusted to 3.5 with hydrochloric acid (Merck Millipore). The absolute acetonitrile concentration was maintained at 65% for the first 10 min, increased from 65 to 100% between 10 and 13 min, and maintained at 100% between 13 and 21 min. The mobile phase was added at a flow rate of 1 mL min −1 and 40 °C. The PA concentration was determined using a fluorescence detector at 340 nm (excitation) and 510 nm (emission). The peak areas and retention times of the samples were measured through comparisons with the standard Pas putrescine (Put), spermidine (Spd), and spermine (Spm) (Sigma–Aldrich). Protein extraction and digestion Proteins were extracted from samples (three biological triplicates, 300 mg FM per sample) of shoots from apical and cotyledonary nodal explants grown without (0 µM - control) and with 2.5 µM BA at 45 days of incubation in WPM medium. Proteins were extracted using the trichloroacetic acid (TCA)/acetone method with modifications (Damerval et al. 1986). Initially, the samples were pulverized in liquid nitrogen using a ceramic mortar and pestle. The resulting powder was resuspended in 1 mL of chilled extraction buffer containing 10% (w/v) TCA (Sigma) in acetone with 20 mM dithiothreitol (DTT; GE Healthcare, Piscataway, USA) and vortexed for 5 min at 8 °C. Next, the samples were kept at −20 °C for 1 h before centrifugation at 16,000 x g for 30 min at 4 °C. The resulting pellets were washed three times with cold acetone plus 20 mM DTT and centrifuged for 5 min each time. The pellets were air dried and resuspended in buffer containing 7 M urea (GE Healthcare), 2 M thiourea (GE Healthcare), 2% Triton X-100 (GE Healthcare), 1% DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma–Aldrich), and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), vortexed for 30 min at 8 °C, and centrifuged for 20 min at 16,000 x g at 4 °C. The supernatants were collected, and the protein concentrations were determined using a 2-D Quant Kit (GE Healthcare). Before the trypsin digestion step, protein samples were precipitated using the methanol/chloroform methodology (Nanjo et al. 2012). After protein precipitation, the samples were resuspended in 7 M urea/2 M thiourea solution. Aliquots of 100 µg of protein were subjected to tryptic digestion using the filter-aided sample preparation (FASP) methodology (Reis et al. 2021). Next, the peptides were resuspended in 100 μL solution containing 95% 50 mM ammonium bicarbonate, 5% acetonitrile and 0.1% formic acid and quantified by A205 nm protein and peptide methodology using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). The samples were transferred to Total Recovery Vials (Waters) for mass spectrometry analysis. Mass spectrometry analysis Mass spectrometry was performed using a nanoAcquity UPLC connected to a Q-TOF SYNAPT G2-Si instrument (Waters, Manchester, UK) according to Passamani et al. (2018). Runs consisted of three biological replicates of 1 µg of peptide samples. During separation, samples were loaded onto the nanoAcquity UPLC M-Class Symmetry C18 5 μm trap column (180 μm × 20 mm) at 5 μL.min -1 for 3 min and then onto the nanoAcquity M-Class HSS T3 1.8 μm analytical reversed-phase column (75 μm × 150 mm) at 400 nL.min -1 , with a column temperature of 45 °C. For peptide elution, a binary gradient was used, with mobile phase A consisting of water (Tedia, Fairfield, Ohio, USA) and 0.1% formic acid (Sigma–Aldrich) and mobile phase B consisting of acetonitrile (Sigma Aldrich) and 0.1% formic acid. The gradient elution started at 7% B, then ramped from 7% B to 40% B until 91.12 min, then ramped again from 40% B to 99.9% B until 92.72 min, then remained at 99.9% until 106.00 min, then decreased to 7% B until 106.1 min, and finally remained at 7% B until the end of experiment at 120 min. Mass spectrometry was performed in positive and resolution mode (V mode), 35,000 full widths at half maximum, with ion mobility separation (IMS), and in data-independent acquisition mode (HDMS E ). The ion mobility wave was set to a velocity of 600 m s -1 , and helium and IMS gas flows were 180 and 90 mL min −1 , respectively. The transfer collision energy ramped from 19 to 55 V in high-energy mode; the cone and capillary voltages were 30 and 2750 V, respectively; and the source temperature was 70 °C. Regarding the time of flight (TOF) parameters, the scan time was set to 0.5 s in continuum mode with a mass range of 50 to 2000 Da. Human [Glu1]-fibrinopeptide B at 100 fmol.μL -1 was used as an external calibrant, and lock mass acquisition was performed every 30 s. Mass spectra were acquired by MassLynx v4.0 software. Proteomics data analysis Spectra processing and database search conditions were performed using ProteinLynx Global Server (PLGS) software v.3.0.2 (Waters). The PLGS was processed by the following parameters: Apex3D of 150 counts for low-energy threshold, 50 counts for elevated-energy threshold, and 750 counts for intensity threshold; two missed cleavages; minimum fragment ions per peptide equal to three; minimum fragment ions per protein equal to seven; minimum peptides per protein equal to two; fixed modifications of carbamidomethyl (C) and variable modifications of oxidation (M) and phosphoryl (STY); default false discovery rate (FDR) was set to a maximum of 1%. We used the Arachis hypogaea protein databank from UniProtKB (http://www.unipr ot.org) for protein identification, as it is the largest databank with proximity to D. nigra . Label-free quantification analysis was performed using ISOQuant workflow software v.1.7 (Distler et al. 2014). Briefly, the following parameters were used to identify proteins: FDR 1%, a peptide score greater than six, a minimum peptide length of six amino acids, and at least two peptides per protein were considered for label-free quantitation using the TOP3 approach, followed by the multidimensional normalized process within ISOQuant. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al. 2022) partner repository with the dataset identifier PXD031999. To ensure the quality of the results after data processing, only the proteins that were either present or absent (for unique proteins) in all three biological replicates were considered for differential accumulation analysis. Data were analyzed using Student’s t test (two-tailed). Proteins with ANOVA ( P < 0.05) were considered up-accumulated if the log 2 value of the fold change (FC) was greater than 0.60 and down-accumulated if the log 2 value of the FC was less than -0.60. Functional annotations were performed using OmicsBox software 1.0.34 and UniProtKB (http://www.uniprot.org). Data analyses The experimental design was completely randomized. Data were analyzed using analysis of variance ( P < 0.05 ) followed by the Student-Newman–Keuls (SNK) test using the R Environment (R Core Team 2014). Results Effects of plant culture medium on in vitro germination and seedling development Both culture media tested showed no significant difference on germination, resulting in similar percentages for MS (80%) and WPM (81%) (Fig. 1a). However, the seedlings from WPM culture medium showed well-developed leaves (Fig. 1c), while those obtained in the MS culture medium showed more senescence of leaves (Fig. 1b). Effects of explants , plant culture media and BA on shoot development Due to the difference in length between the first (Figs. 2a and 2b) and second shoots (Figs. 2c and 2d) developed, they were measured separately. BA addition promoted the elongation of the first shoot developed from both types of explants, the apical and cotyledonary nodal segments, in both culture media, MS (Fig. 2a) and WPM (Fig. 2b), compared to shoots obtained without BA (control). Moreover, no significant difference was observed between the two types of explants in the same BA concentration and same culture medium or between the two culture media used (Figs. 2a and 2b). As BA is essential for shoot growth and there were no significant differences in the length of shoots between 2.5 and 5 µM BA in either culture medium, the two types of explants can be used for in vitro propagation of D. nigra , considering the elongation of the first shoot developed. The second shoot showed lower elongation (Figs. 2d and 2e) than the first shoot (Figs. 2a and 2b). In MS culture medium, the BA concentration increased the length of the second shoot from cotyledonary nodal segments compared to the control, while no significant effects were observed in shoots from apical nodal segments (Fig. 2c). In WPM culture medium, the 2.5 μM BA treatment showed lower elongation of second shoots from cotyledonary nodal segments compared to the control and 5 μM BA (Fig. 2d). There were no significant differences in the number of shoots between the two types of explants at each BA concentration (0 or 2.5 µM) or in either culture medium, MS (Fig. 2e) or WPM (Fig. 2f), except for apical explants in the control treatment (without BA) in MS culture medium, which showed a significantly lower number of shoots (Fig. 2e). On the other hand, no significant differences were observed for the number of shoots from apical and cotyledonary nodal segments incubated with WPM culture medium (Fig. 2f). For shoot induction, no significant differences were observed in explant type, culture medium or BA concentration, showing 100% shoot induction in all treatments (data not shown). Effect of BA and explant type on endogenous PA contents during shoot development Free PAs were quantified in 45-day-old shoots from apical and cotyledonary nodal segments incubated without and with 2.5 µM BA and WPM culture medium (Fig. 3). Free Put content was significantly higher when shoots were grown in 2.5 µM BA compared to the control in both types of explants, being significantly higher in shoots from apical nodal segments compared to cotyledonary nodal segments (Fig. 3a). Higher free Spd content was observed in shoots from apical explants grown in 2.5 µM BA, differing statistically from shoots in the control treatment (Fig. 3b). Moreover, a higher free-Spm content was observed in shoots from cotyledonary nodal segments in the 2.5 µM BA treatment than in the control (Fig. 3c). The content of total free PAs was significantly higher in shoots from both types of explants incubated in 2.5 µM BA than in shoots from the control treatment (Fig. 3d). Effect of BA and the type of explant on the proteomic profile during shoot development Proteomic analysis was performed comparing the effects of BA (by the comparisons BA2.5_Apical/BA0_Apical and BA2.5_Cotyledonary/BA0_Cotyledoenary) and the type of explant (by the comparisons BA2.5_Cotyledonary/BA2.5_Apical and BA0_Cotyledonary/BA0_Apical) on shoot development. A total of 1232 proteins were identified (Supplementary Table 1). Among the DAPs, some proteins were highlighted according to their relevance to cell division and shoot growth. Comparing the BA concentrations (0 and 2.5 µM) in shoots obtained from apical nodal segments (BA2.5_Apical/BA0_Apical), a total of 292 proteins were differentially accumulated (DAPs), and 923 were unchanged. Among the DAPs, 93 proteins were up- and 158 down-accumulated, with 13 unique in shoots grown under BA2.5_Apical and 28 unique in shoots under BA0_Apical (Fig. 4a; Supplementary table 1). In the comparison of BA concentration treatment in shoots from cotyledonary nodal segments (BA2.5_Cotyledonary/BA0_Cotyledonary), a total of 374 proteins were DAPs, and 846 were unchanged. Among the DAPs, 179 were up- and 158 were down-accumulated. In addition, 18 proteins were unique in shoots from cotyledonary nodal segments incubated with 2.5 µM BA (BA2.5_Cotyledonary), and 19 proteins were unique in shoots from cotyledonary nodal segments without BA (BA0_Cotyledonary) (Fig. 4b; Supplementary table 1). Among these proteins, some were up-accumulated in shoots from both types of explants (apical and cotyledonary nodal segments) incubated with 2.5 BA compared to shoots without BA (comparisons BA2.5_Cotyledonary/BA0_Cotyledonary and BA2.5_Apical/BA0_Apical), as the pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH7-like (A0A444YGB9), calreticulin-3 (A0A445A993), aspartate aminotransferase 1 (A0A445B4C1), protein elongation factor 1-alpha (A0A444Y7Y0) and cell division control protein 48 homolog D (A0A444Z3W1) (Supplementary Table 1). In addition, the proteins phosphoenolpyruvate carboxylase 2 (A0A445BXQ0), phosphoribosylamine-glycine ligase isoform X1 (A0A445E7J0), FT-interacting protein 3 (A0A444ZYV6), dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit (A0A445A8R7), and 60S ribosomal protein L35a-1 (A0A444ZT56) were up-accumulated in shoots from cotyledonary nodal segments incubated in 2.5 µM BA compared to shoots without BA (comparison BA2.5_Cotyledonary/BA0_Cotyledonary) (Supplementary Table 1). The effect of explant type at the same BA concentration on the protein profile was evaluated. Comparing cotyledonary shoots from apical nodal segments incubated at 2.5 µM BA (comparison BA2.5_Cotyledonary/BA2.5_Apical), 242 proteins were DAPs, and 973 were unchanged. Among the DAPs, 154 were up- and 46 were down-accumulated, with 28 proteins unique to shoots under the BA2.5_Cotyledonary treatment and 14 proteins unique to shoots under the BA2.5_Apical treatment (Fig. 4c). Comparing the shoots from cotyledonary to apical nodal segments incubated without BA (comparison BA0_Cotyledonary/BA0_Apical), a total of 231 proteins were DAP, and 997 were unchanged (Fig. 4d). Among the DAPs, 89 were up- and 90 down-accumulated, presenting 26 unique proteins in shoots from cotyledonary nodal segments without BA (BA0_Cotyledonary treatment) and 26 unique proteins in shoots from apical nodal segments without BA (BA0_Apical treatment) (Supplementary table 1). Among the DAPs, the malate dehydrogenase 2, peroxisomal (A0A445DRP9), ATP synthase CF1 beta subunit (A0A445AH22) proteins were up-accumulated in shoots from cotyledonary nodal segments compared to those from apical segments at both BA concentrations. The bifunctional dTDP-4-dehydrorhamnose 3,5-epimerase/dTDP-4-dehydrorhamnose reductase (A0A444Z945) protein was unique in D. nigra shoots from BA-treated cotyledonary nodal segments compared to shoots without BA (BA2.5_Cotyledonary/BA0_Cotyledonary) and shoots from BA-treated apical nodal segments (BA2.5_Cotyledonary/BA2.5_Apical). Effect of IBA on ex vitro rooting of shoots and acclimatization The ex vitro root induction of shoots was not significantly affected by IBA concentrations or types of explants (Fig. 5). However, the induction of roots was significantly affected by BA supplementation on the culture medium of shoot development, with the induction of roots being significantly higher in shoots grown in culture medium without BA (Fig. 5a) compared to 2.5 µM BA (Fig. 5b). The number of roots was also significantly affected by the BA concentration used on shoot multiplication, being significantly higher in shoots multiplied without BA (Fig. 5c) compared to BA (Fig. 5d). The length of shoots was significantly affected by BA treatment during shoot multiplication and was significantly higher in shoots from cotyledonary nodal explants grown with BA (Figs. 5e and 5f). On the other hand, the length of roots was not significantly affected by IBA concentrations used in shoots from either type of explant used (Figs. 5e and 5f). Discussion The establishment of the best culture medium for in vitro seed germination is relevant to obtaining explants for micropropagation. In addition to no significant differences in seed germination (Fig. 1a), the WPM culture medium resulted in seedlings with better growth (Fig. 1c). In Cariniana legalis , the WPM culture medium improved the percentage of in vitro seed germination, which was significantly higher than that of MS culture medium (Aragão et al. 2017a). The WPM culture medium has only 25% of the concentrations of nitrate and ammonium ions present in MS culture medium, in addition to more potassium and a high level of sulfate ions, which are widely used for micropropagation of woody species (Hazubska-Przybył 2019). The lower concentrations of total nitrogen and ammonium in WPM culture medium reduce the possibility of toxicity to ammonium, which can contribute to the development of seedlings in some woody species (Phillips and Garda 2019), as observed in D. nigra in the present work. For shoot development, BA is the most common cytokinin used for the proliferation of axillary buds in many plant species (Sahai and Shahzad 2013), including several trees, such as Santalum album (Mujib 2005), Cedrela fissilis (Aragão et al. 2016; Aragão et al,. 2017b), Azadirachta excelsa (Foan and Othman 2006), Sapium sebiferum and Calophyllum brasiliensis (Stein et al. 2017). The structural stability of BA and the ability of plant cells to easily assimilate make this cytokinin an efficient promoter of plant development (Ahmad et al. 2013). Our results showed that BA addition was essential to increase the length of shoots from both types of explants (apical and cotyledonary nodal segments) and culture media (MS and WPM) (Fig. 2) in D. nigra . The use of BA also promoted longer shoots in Juglans nigra (Stevens and Pijut 2018) and Rauvolfia tetraphylla (Hussain et al. 2018). This promotion in the length of BA-induced shoots may be associated with the effects of cytokinins in the control of cell division, providing greater growth and development (Wybouw and De Rybel 2019). In addition to the positive effects of BA on shoot length, this cytokinin showed no effects on shoot induction (%) or the number of shoots per explant (Fig. 2) in D. nigra . Similarly, no significant effects of BA on the number of shoots were observed for C. legalis (Aragão et al. 2017a). On the other hand, in Dalbergia sisso , the use of 4.4 µM BA provided a greater number of shoots compared to the control (Sahu et al. 2014). These results show that the in vitro morphogenic response induced by BA is intrinsic to the species, and this response may be different even within species of the same genus as that observed between D. nigra and D. sissoo . In addition to cytokinins, PAs are involved in plant growth and development, as they can act in various physiological processes, such as the promotion of cell division, differentiation, and elongation, which are essential to embryo development, seed germination, rhizogenesis and shoot development in woody plants (Santa-Catarina et al. 2006; Kusano et al. 2008; Pieruzzi et al. 2011; Aragão et al. 2016; Lerin et al. 2019). The higher content of free Put (Fig. 3a) in shoots from cotyledonary and apical nodal segments incubated with BA was correlated with the higher length of shoots in D. nigra (Figs. 2c and 2d). Similar results were observed during in vitro shoot development in other species, such as Bixa orellana (Parimalan et al. 2011) and C. fissilis (Aragão et al. 2016; Oliveira et al. 2020). A high content of Put was shown to be directly related to cell cycle progression at the G1/S transition, stimulating the synthesis of proteins such as tubulins, which contribute to cell growth (Tiburcio et al. 2014). Cross-talk among PAs and other plant hormones, such as cytokinin, has been proposed, as BA can affect PA metabolism and thereby their homeostasis by changing the expression of the genes responsible for PA biosynthesis, catabolism, or both (Ahanger et al. 2020). Therefore, the modulation of endogenous PA contents is relevant for shoot elongation in D. nigra. Comparative proteomics is an important tool for the comprehension of physiological and molecular processes during in vitro morphogenesis, as it is possible to compare DAPs under different treatments (Heringer et al. 2018). This approach was applied in the present work to comprehend the effects of BA and the type of explant (apical and cotyledonary nodal segments) (Fig. 4) on protein accumulation during shoot development in D. nigra . The accumulation of some proteins was significantly affected by BA addition in the shoots of D. nigra from both types of explants (Supplementary Table 1), such as the up-accumulation of the factor ATP-dependent RNA helicase DEAH7 (A0A444YGB9). This protein is involved in the expression of genes related to auxin-mediated development, such as the apical-basal standardization of embryonic development and vascular development in Arabidopsis (Tsugeki and Terada 2015). Thus, the accumulation of this protein under BA treatment can improve shoot elongation in D. nigra , probably interacting with auxin metabolism, as this protein is related to auxin polar transport. Auxin polar transport is essential for cell elongation of the embryo scutellum owing to auxin-induced cell acidification and elongation to the plasma membrane, enabling growth (Chen et al. 2010). Calreticulin is a molecular calcium-binding chaperone that promotes folding, oligomeric assembly and quality control in the endoplasmic reticulum through the calreticulin/calnexin cycle. Calcium is an important stabilizing agent in the control of plant cell metabolism, playing a role in the structure and permeability of cell membranes, cell division and elongation, translocation of carbohydrates and nitrogen metabolism, presenting a direct effect on plant growth (Ahmad et al. 2016). In this sense, the increase in the accumulation of calreticulin-3 (A0A445A993) protein BA induced in shoots from both types of explants may be related to calcium and other compounds important for shoot elongation observed in D. nigra. In addition, calreticulin proteins are able to transiently interact with almost all monoglucosylated glycoproteins necessary for the accumulation of elongation factor receptors (Ahmad et al. 2016). In the present work, the up-accumulation of elongation factor 1-alpha (A0A444Y7Y0) protein in shoots from both types of explants under BA treatment compared to shoots grown without BA (Supplementary Table 1) could be relevant for the best shoot development in D. nigra . This protein works as a promoter of GTP-dependent binding of aminoacyl-tRNA to ribosome sites during protein biosynthesis, an important process in growth and development (White et al. 2019). In addition, the up-accumulation of protein cell division cycle protein 48 homolog (A0A444Z3W1) in shoots from both types of explants could be relevant for the higher shoot elongation BA-induced in D. nigra . This protein is directly related to cell division, cytokinesis and growth processes in plants (Rancour et al. 2002). Nitrogen (N) is essential to carbon skeletons for the biosynthesis of the primary amino acids glutamine and glutamate, which serve as N donors for the biosynthesis of major N compounds in plants, including other amino acids, nucleic acid bases, PAs, and chlorophyll (de la Torre et al. 2014). The aspartate aminotransferase protein is important for aspartate biosynthesis and plays a key role in the metabolic regulation of carbon and nitrogen metabolism in all organisms (Cánovas et al. 2007). The induced gene silencing of aspartate aminotransferase in Nicotiana benthamiana causes a reduction in growth and chlorosis symptoms and decreases the levels of chlorophyll and lignin (de la Torre et al. 2014). Moreover, aspartate aminotransferase activity was involved in biomass increments in Brassica napus (McAllister et al. 2016). The up-accumulation of aspartate aminotransferase 1 (A0A445B4C1) protein in shoots from both types of explants grown under BA treatment (Supplementary Table 1) may be important for the increase in biomass due to the higher length of shoots in D. nigra induced by BA. The protein phosphoribosylamine-glycine ligase is associated with N assimilation in bacterial nitrogen fixation (Resendis-Antonio et al. 2011), and the accumulation of this protein (A0A445E7J0) in shoots from BA-treated cotyledonary nodal segments can promote the elongation of D. nigra shoots, altering nitrogen metabolism. In addition to nitrogen, carbohydrate metabolism is essential for energy supply in plants. The phosphoenolpyruvate carboxylase protein is an important cytosolic enzyme situated at a crucial branch point of plant carbohydrate metabolism (Scholl et al. 2020). Phosphoenolpyruvate carboxylase 2 also fulfils essential nonphotosynthetic functions, particularly the replenishment of tricarboxylic acid (TCA) cycle intermediates consumed during biosynthesis and N assimilation (Scholl et al. 2020). Some developmental or metabolic processes that require these organic acids will benefit from increased carbon flux through the phosphoenolpyruvate carboxylase 2 reaction (Willick et al. 2019). Thus, the up-accumulation of phosphoenolpyruvate carboxylase 2 (A0A445BXQ0) could be relevant for the elongation of shoots from BA-treated cotyledonary nodal segments compared to those without BA in D. nigra (Supplementary Table 1). Another up-accumulated protein in shoots from cotyledonary nodal explants incubated with BA compared to those without BA was the FT-interacting protein (A0A444ZYV6) (Supplementary Table 1). This protein plays an essential role in mediating the proliferation and differentiation of shoot stem cells in Arabidopsis (Liu et al. 2018). FT-interacting protein prevents intracellular trafficking of a key regulator, SHOOTMERISTEMLESS, to the plasma membrane in cells in the peripheral shoot meristem region. This facilitates SHOOTMERISTEMLESS recycling to the nucleus to maintain stem cells and accelerates stem cell differentiation (Liu et al. 2018). In this sense, these proteins may be interesting to shed light on BA signaling for the promotion of higher shoot elongation in D. nigra . In addition, the dolichyl-diphosphooligosaccharide glycosyltransferase protein is known to be involved in protein glycosylation and protein modification and participates in biological processes relevant for plant growth and development, such as mechanisms controlling the assembly of cell wall polymers, protein N-linked glycosylation through asparagine and cell growth (Lerouxel et al. 2005). An increase in the accumulation of dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit (A0A445A8R7) protein in shoots from cotyledonary nodal segments BA-treated can be related to cytokinin promotion of the higher elongation of shoots in D. nigra . Citrate synthase mitochondrial (mtCS) (A0A445EVE9 and A0A445CF35) was another protein up-accumulated only in shoots from BA-treated cotyledonary nodal segments. The citrate synthase mitochondrial (mtCS) protein has an enhanced ability to excrete citric acid, and the overexpression of mtCS in carrot cells results in better cell growth than that in wild-type cells (Koyama et al. 1999). It appears that the overexpression of citrate synthase in Arabidopsis improves growth in phosphorus-limited soils due to the increased excretion of citrate from the roots (Koyama et al. 2000). This evidence may suggest that the increase in the accumulation of citrate synthase mitochondrial (mtCS) (A0A445EVE9 and A0A445CF35) proteins in shoots from cotyledonary explants treated with BA can regulate oxidative metabolism, promoting the elongation of shoots in D. nigra . Another up-accumulated protein, the phosphoribosylamine-glycine ligase chloroplastic (A0A445E7J0), is involved in enzymes in the de novo purine biosynthesis pathway (Zhang et al. 2018). Plants can degrade purines, and the final products glyoxylate and ammonia are recovered to synthesize organic molecules for new growth (Amarante et al. 2006). The increase in accumulation of this protein may be involved in the biosynthesis of organic molecules necessary for the higher growth of D. nigra shoots from cotyledonary nodal segments incubated with BA. The 60S ribosomal protein L35a-3 (A0A444ZT56) observed in D. nigra shoots from cotyledonary nodal segments treated with BA (Supplementary Table 1) is a structural constituent of ribosomes and has cytoplasmic translation and ribosomal large subunit biogenesis as a biological function (Xiao et al. 2019). The eukaryotic ribosome is a complex structure composed of several ribosomal RNAs and ribosomal proteins (r-proteins) (Taylor et al. 2009), which are responsible for protein synthesis necessary for cell growth, division, and development (Barakat et al. 2001). It has been shown that genetic defects in ribosomal components, such as a reduction in the levels of individual r-proteins, can induce deleterious effects on the development of plants (Barakat et al. 2001). Thus, a higher accumulation of 60S ribosomal protein L35a-3 (A0A444ZT56) could be relevant to maintaining higher levels of r-proteins and, consequently, higher elongation of D. nigra shoots from cotyledonary nodal segments incubated with BA. The bifunctional dTDP-4-desidrorhamnose 3,5-epimerase/dTDP-4-desidrorhamnose reductase (A0A444Z945) protein was unique in D. nigra shoots from BA-treated cotyledonary nodal segments compared to shoots without BA (BA2.5_Cotyledonary/BA0_Cotyledonary) and to shoots from BA-treated apical nodal segments (BA2.5_Cotyledonary/BA2.5_Apical). This protein is involved in dTDP-L-rhamnose biosynthesis, which is part of carbohydrate metabolism (Watt et al. 2004). The analysis of sugar composition and the study of gene expression at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under specific tissue regulation (Martinez et al. 2012). In addition, this protein is also present in the cell wall organization process, which can be an interesting factor associated with the differential elongation of D. nigra shoots incubated with BA. Some proteins identified were associated with the type of explant, being more accumulated in shoots from cotyledonary nodal segments compared to apical segments, both with (BA2.5_Cotyledonary/BA2.5_apical comparison) or without BA (BA0_cotyledonary/BA0_apical comparison) (Supplementary Table 1). The malate dehydrogenase 2 protein catalyzes a reversible NAD-dependent dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments (Tomaz et al. 2010) and is also required for the maintenance of photosynthetic rates under photorespiratory conditions (Cousins et al. 2008). ATP synthase subunit beta, chloroplastic (A0A445AH22), can be found in the plasma membrane of eubacteria, in thylakoids of chloroplasts and in the inner mitochondrial membrane of eukaryotic cells (Mulkidjanian et al. 2009). Loss of ATP synthase assembly defective in the β subunit results in mitochondria deprived of cristae structures, and when ATP2 is silenced, cells show a peculiar organization of thylakoid stacks in the chloroplast with a reduced number of lamellae compared to wild-type, harming plant development (Lapaille et al. 2010). The up-accumulation of dehydrogenase 2 (peroxisome) (A0A445DRP9) and ATP synthase subunit beta and chloroplastic (A0A445AH22) in shoots of D. nigra from cotyledonary nodal segments treated or not with BA shows that this type of explant better regulates redox homeostasis between organelle compartments and ATP biosynthesis, an essential process for growth. Rooting is a critical phase of in vitro propagation, and overcoming this phase can ensure the success of the process (Zeng et al. 2019). Usually, exogenous auxins are necessary to promote root induction in some species, as observed for Malus domestica rootstocks (Meng et al. 2019) and Populus alba (Zeng et al. 2019). Dalbergia nigra has the possibility of propagation using the cutting method, however, reaching rates below 50%. In our work, ex vitro rooting was efficient for plantlet production using shoots from both types of explants, with no necessity of IBA use (Figs. 5a and 5b). IBA was also not necessary for root induction or the number of roots in Prunus persica and Prunus davidiana (Zhou et al. 2010). Thus, it was possible to reach a rate higher than 80% rooting, showing better results when compared to the cutting technique used in the species. Moreover, the balance between auxins and cytokinins is important for root induction (Růžička et al. 2009; Jing and Strader 2019), and differences in rooting may occur due to the accumulation of cytokinins in plant tissues (Da Costa et al. 2013). In the present work, a comparison between shoots multiplied in culture medium without (control) and with 2.5 µM BA was performed to analyze whether shoot multiplication in BA concentrations affects the induction of roots. The use of 2.5 µM BA is essential for shoot elongation; however, the treatments with cytokinin significantly affected the root induction and number of roots in D. nigra compared to the treatment without BA (Figs. 5c and 5d). In this way, we can infer that the balance between auxins and cytokinins influences D. nigra shoot rooting. This balance adjustment was also considered important in Ceropegia bulbosa , where different concentrations of cytokinin (BA) and auxins, such as naphthalene acetic acid (NAA) and IBA, were tested, demonstrating the importance of crosstalk among hormones (Phulwaria et al. 2013). Unlike in Albizia lebbeck , the use of 250 µM IBA promoted the largest number and longer length of roots from shoots grown under concentrations of the cytokinin thidiazuron (Perveen et al. 2013), showing that endogenous hormones present in the explant have an important role in plant organogenesis (Pal et al. 2012; Zeng et al. 2019). Thus, we can infer that the results obtained for rooting depend on the species and concentrations of PGRs for root induction, as well as those used in the shoot multiplication step. Conclusions The WPM culture medium promoted the best seedling growth. The addition of BA is necessary for longer shoot lengths for both types of explants. BA addition promoted an increase in endogenous Put content, which induced the higher growth of shoots in both explants. Some proteins involved in central metabolism, redox homeostasis, maintenance of photosynthetic rates and carbon flow during photorespiration conditions were differentially accumulated in shoots from cotyledonary nodal explants with and without BA and are important for the growth of these shoots. Ex vitro rooting of shoots can be performed without IBA in both types of explants. This work enabled the production of seedlings that were directed to an ecological reserve. Furthermore, the first results demonstrated the involvement of PAs and proteomic profiles in the development of D. nigra shoots. Abbreviations BA benzyladenine CDC48 Cell division cycle protein 48 homolog DAPs Differentially accumulated proteins DTT Dithiothreitol FDR False discovery rate IBA Indole-3-butyric acid MS Murashige and Skoog mtCS Citrate synthase mitochondrial PA Polyamines PGRs Plant growth regulators Put Putrescine TCA Trichloroacetic acid Spd Spermidine Spm Spermine WPM Woody Plant Medium Declarations Acknowledgments The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for funding. This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001. LSP, TRO, VPMA and KRS are thankful for the scholarship funding provided by FAPERJ. Author contributions LPS and CSC conceived the study, designed the experiments and wrote the manuscript. LSP was responsible for the in vitro culture of shoots and ex vitro rooting experiments and performed the statistical analyses. LSP and VPMA were responsible for PA analyses. LSP, TRO and VS were responsible for the proteomic analyses. All the authors read and approved the final manuscript. Funding This research was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (444453/2014-8; 309303/2019-2) and the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E26/202.969/2016; E26/202.533/2019). This study was also financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001. Data availability The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031999. All identified proteins are available in the supplementary material. Code availability PXD031999. Financial interests The authors declare they have no financial interests. Conflicts of interest/Competing interests The authors have no conflicts of interest to declare that are relevant to the content of this article. References Ahanger MA, Aziz U, Alsahli A, Alyemeni MN, Ahmad P (2020) Combined kinetin and spermidine treatments ameliorate growth and photosynthetic inhibition in Vigna angularis by up-regulating antioxidant and nitrogen metabolism under cadmium stress. Biomolecules 10:147. https://doi.org/10.3390/biom10010147 Ahmad N, Javed SB, Khan MI, Anis M (2013) Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia : an important medicinal plant. Acta Physiol Plant 35:2493–2500. https://doi.org/10.1007/s11738-013-1285-y Ahmad P, Abdel Latef AA, Abd_Allah EF, Hashem A, Sarwat M, Anjum NA, Gucel S (2016) Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmium-exposed chickpea ( Cicer arietinum L.). Front Plant Sci 7. https://doi.org/10.3389/fpls.2016.00513 Amarante L, Lima JD, Sodek L (2006) Growth and stress conditions cause similar changes in xylem amino acids for different legume species. Environ Exp Bot 58:123–129. https://doi.org/10.1016/j.envexpbot.2005.07.002 Aragão VPM, Navarro BV, Silva AT, Silveira V, Santa-Catarina C (2017a) Micropropagation of Cariniana legalis (Martius) O. Kuntze, an endangered hardwood tree from the Brazilian Atlantic Forest. Plant Cell Culture & Micropropagation 13:41–50 Aragão VPM, Reis RS, Silveira V, Santa-Catarina C (2017b) Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in Cedrela fissilis Vellozo (Meliaceae). Plant Cell Tiss Org Cult 130:495–505. https://doi.org/10.1007/s11240-017-1239-y Aragão VPM, Ribeiro YRS, Reis RS, Macedo AF, Floh EIS, Silveira V, Santa-Catarina C (2016) In vitro organogenesis of Cedrela fissilis Vell. (Meliaceae): the involvement of endogenous polyamines and carbohydrates on shoot development. Plant Cell Tiss Org Cult 124:611–620. https://doi.org/10.1007/s11240-015-0919-8 Barakat A, Szick-Miranda K, Chang I-F, Guyot R, Blanc G, Cooke R, Delseny M, Bailey-Serres J (2001) The organization of cytoplasmic ribosomal protein genes in the Arabidopsis genome. Plant Physiol 127:398–415. https://doi.org/10.1104/pp.010265 Baron K, Stasolla C (2008) The role of polyamines during in vivo and in vitro development. In Vitro Cell Dev Biol Plant 44:384–395. https://doi.org/10.1007/s11627-008-9176-4 Bunn E, Turner SR, Dixon KW (2011) Biotechnology for saving rare and threatened flora in a biodiversity hotspot. In Vitro Cell Dev Biol Plant 47:188–200. https://doi.org/10.1007/s11627-011-9340-0 Cánovas FM, Avila C, Cantón FR, Cañas RA, de la Torre F (2007) Ammonium assimilation and amino acid metabolism in conifers. J Exp Bot 58:2307–2318. https://doi.org/10.1093/jxb/erm051 Chen D, Ren Y, Deng Y, Zhao J (2010) Auxin polar transport is essential for the development of zygote and embryo in Nicotiana tabacum L. and correlated with ABP1 and PM H + -ATPase activities. J Exp Bot 61:1853–1867. https://doi.org/10.1093/jxb/erq056 Cousins AB, Pracharoenwattana I, Zhou W, Smith SM, Badger MR (2008) Peroxisomal malate dehydrogenase is not essential for photorespiration in Arabidopsis but its absence causes an increase in the stoichiometry of photorespiratory CO 2 release. Plant Physiol 148:786–795. https://doi.org/10.1104/pp.108.122622 Da Costa C, De Almeida M, Ruedell C, Schwambach J, Maraschin F, Fett-Neto A (2013) When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings. Front Plant Sci 4. https://doi.org/10.3389/fpls.2013.00133 Damerval C, De Vienne D, Zivy M, Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat‐seedling proteins. Electrophoresis 7:52–54 de la Torre F, Cañas RA, Pascual MB, Avila C, Cánovas FM (2014) Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants. J Exp Bot 65:5527–5534. https://doi.org/10.1093/jxb/eru240 Dias PC, Oliveira LS, Xavier A, Wendling I (2012) Estaquia e miniestaquia de espécies florestais lenhosas do Brasil. Pesquisa Florestal Brasileira 32:453. https://doi.org/10.4336/2012.pfb.32.72.453 Distler U, Kuharev J, Navarro P, Levin Y, Schild H, Tenzer S (2014) Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Meth 11:167–170. https://doi.org/10.1038/nmeth.2767 Dutra NT, Silveira V, Azevedo IG, Gomes-Neto LR, Facanha AR, Steiner N, Guerra MP, Floh EIS, Santa-Catarina C (2013) Polyamines affect the cellular growth and structure of pro-embryogenic masses in Araucaria angustifolia embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines. Physiol Plant 148:121–132. https://doi.org/10.1111/j.1399-3054.2012.01695.x Fermino-Junior PCP, Scherwinski-Pereira JE (2012) Germinação e propagação in vitro de cerejeira ( Amburana acreana (Ducke) A.C.Smith - Fabaceae). Cienc Florest 22:1–9. https://doi.org/10.5902/198050985074 Foan CC, Othman RY (2006) In vitro direct shoot organogenesis and regeneration of plantlets from leaf explants of Sentang ( Azadirachta excelsa ). Biotechnology 5:337–340. https://doi.org/10.3923/biotech.2006.337.340 Ghosh S, Pal A (2013) Proteomic analysis of cotyledonary explants during shoot organogenesis in Vigna radiata . Plant Cell Tiss Org Cult 115:55–68. https://doi.org/10.1007/s11240-013-0340-0 Giri CC, Shyamkumar B, Anjaneyulu C (2004) Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees 18:115–135. https://doi.org/10.1007/s00468-003-0287-6 Gupta AK, Harish, Rai MK, Phulwaria M, Agarwal T, Shekhawat NS (2014) In vitro propagation, encapsulation, and genetic fidelity analysis of Terminalia arjuna : a cardioprotective medicinal tree. Appl Biochem Biotechnol 173:1481–1494. https://doi.org/10.1007/s12010-014-0920-4 Hazubska-Przybył T (2019) Propagation of juniper species by plant tissue culture: A mini-review. Forests 10:1028. https://doi.org/10.3390/f10111028 Heringer AS, Santa-Catarina C, Silveira V (2018) Insights from proteomic studies into plant somatic embryogenesis. Proteomics 18:e1700265. https://doi.org/10.1002/pmic.201700265 Hussain SA, Ahmad N, Anis M (2018) Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in Rauvolfia tetraphylla (L.). Rend Lincei Scienze Fis e Naturali 29:109–115. https://doi.org/10.1007/s12210-018-0667-x IUCN (2021) The IUCN Red List of Threatened Species. Version 2021-3. International Union for Conservation of Nature. http://www.iucnredlist.org . Accessed 31 Jan 2021 Jing H, Strader LC (2019) Interplay of auxin and cytokinin in lateral root development. Int J Mol Sci 20:486 Kodym A, Leeb CJ (2019) Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis. Plant Cell Tiss Org Cult 138:399–402. https://doi.org/10.1007/s11240-019-01635-1 Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D (2000) Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol 41:1030–1037. https://doi.org/10.1093/pcp/pcd029 Koyama H, Takita E, Kawamura A, Hara T, Shibata D (1999) Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant Cell Physiol 40:482–488. https://doi.org/10.1093/oxfordjournals.pcp.a029568 Kusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines: essential factors for growth and survival. Planta 228:367–381. https://doi.org/10.1007/s00425-008-0772-7 Lapaille M, Thiry M, Perez E, González-Halphen D, Remacle C, Cardol P (2010) Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in Chlamydomonas . Biochim et Biophys Acta (BBA) - Bioenergetics 1797:1533–1539. https://doi.org/10.1016/j.bbabio.2010.04.013 Lerin J, Aragao VPM, Reis RS, Silveira V, Santa-Catarina C (2019) Proteomic profile and polyamine contents are modulated by light source to promote in vitro shoot development in Cariniana legalis (Martius) O.Kuntze (Lecythidaceae). Plant Cell Tiss Org Cult 137:329–342. https://doi.org/10.1007/s11240-019-01574-x Lerouxel O, Mouille G, Andème-Onzighi C, Bruyant M-P, Séveno M, Loutelier-Bourhis C, Driouich A, Höfte H, Lerouge P (2005) Mutants in DEFECTIVE GLYCOSYLATION, an Arabidopsis homolog of an oligosaccharyltransferase complex subunit, show protein underglycosylation and defects in cell differentiation and growth. Plant J 42:455–468. https://doi.org/10.1111/j.1365-313X.2005.02392.x Liu L, Li C, Song S, Teo ZWN, Shen L, Wang Y, Jackson D, Yu H (2018) FTIP-Dependent STM Trafficking Regulates Shoot Meristem Development in Arabidopsis . Cell Rep 23:1879–1890. https://doi.org/10.1016/j.celrep.2018.04.033 Lloyd G, McCown B (1981) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia , by use of shoot-tip culture. Combined Proceedings, International Plant Propagators' Society 30:421–427 Martinez V, Ingwers M, Smith J, Glushka J, Yang T, Bar-Peled M (2012) Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana . J Biol Chem 287:879–892. https://doi.org/10.1074/jbc.M111.287367 McAllister CH, Wolansky M, Good AG (2016) The impact on nitrogen-efficient phenotypes when aspartate aminotransferase is expressed tissue-specifically in Brassica napus . New Negatives in Plant Science 3–4:1–9. https://doi.org/10.1016/j.neps.2016.03.001 Meng Y, Xing L, Li K, Wei Y, Wang H, Mao J, Dong F, Ma D, Zhang Z, Han M, Zhao C, Tahir MM, Zhang D (2019) Genome-wide identification, characterization and expression analysis of novel long non-coding RNAs that mediate IBA-induced adventitious root formation in apple rootstocks. Plant Growth Regul 87:287–302. https://doi.org/10.1007/s10725-018-0470-9 Mitrović A, Janošević D, Budimir S, Pristov JB (2012) Changes in antioxidative enzymes activities during Tacitus bellus direct shoot organogenesis. Biol Plant 56:357–361 Mujib A (2005) In vitro regeneration of sandal ( Santalum album L.) from leaves. Turk J Bot 29:63–67 Mulkidjanian AY, Galperin MY, Koonin EV (2009) Co-evolution of primordial membranes and membrane proteins. Trends Biochem Sci 34:206–215. https://doi.org/10.1016/j.tibs.2009.01.005 Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x Nanjo Y, Skultety L, Uvackova L, Klubicova K, Hajduch M, Komatsu S (2012) Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings. J Proteome Res 11:372–385. https://doi.org/10.1021/pr200701y Oliveira TR, Aragão VPM, Moharana KC, Fedosejevs E, do Amaral FP, Sousa KR, Thelen JJ, Venâncio TM, Silveira V, Santa-Catarina C (2020) Light spectra affect the in vitro shoot development of Cedrela fissilis Vell. (Meliaceae) by changing the protein profile and polyamine contents. Biochimica et Biophysica Acta (BBA) -. Proteins and Proteomics 1868:140529. https://doi.org/10.1016/j.bbapap.2020.140529 Pacurar DI, Perrone I, Bellini C (2014) Auxin is a central player in the hormone cross-talks that control adventitious rooting. Physiol Plant 151:83–96. https://doi.org/10.1111/ppl.12171 Pal AK, Acharya K, Ahuja PS (2012) Endogenous auxin level is a critical determinant for in vitro adventitious shoot regeneration in potato ( Solanum tuberosum L.). J Plant Biochem Biotechnol 21:205–212. https://doi.org/10.1007/s13562-011-0092-z Parimalan R, Giridhar P, Ravishankar G (2011) Enhanced shoot organogenesis in Bixa orellana L. in the presence of putrescine and silver nitrate. Plant Cell Tiss Org Cult 105:285–290. https://doi.org/10.1007/s11240-010-9865-7 Passamani LZ, Bertolazi AA, Ramos AC, Santa-Catarina C, Thelen JJ, Silveira V (2018) Embryogenic competence acquisition in sugarcane callus is associated with differential H + pump abundance and activity. J Proteome Res 17:2767–2779. https://doi.org/10.1021/acs.jproteome.8b00213 Patel AK, Phulwaria M, Rai MK, Gupta AK, Shekhawat S, Shekhawat NS (2014) In vitro propagation and ex vitro rooting of Caralluma edulis (Edgew.) Benth. & Hook. f.: an endemic and endangered edible plant species of the Thar Desert. Sci Hortic 165:175–180. https://doi.org/10.1016/j.scienta.2013.10.039 Perez-Riverol Y, Bai J, Bandla C, García-Seisdedos D, Hewapathirana S, Kamatchinathan S, Kundu DJ, Prakash A, Frericks-Zipper A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizcaíno JA (2022) The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res 50:D543–d552. https://doi.org/10.1093/nar/gkab1038 Perveen S, Anis M, Aref IM (2013) In vitro plant regeneration of Albizia lebbeck (L.) from seed explants. For Syst 22:241–248. https://doi.org/10.5424/fs/2013222-03261 Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. In Vitro Cell Dev Biol Plant 55:242–257. https://doi.org/10.1007/s11627-019-09983-5 Phulwaria M, Shekhawat NS, Rathore JS, Singh RP (2013) An efficient in vitro regeneration and ex vitro rooting of Ceropegia bulbosa Roxb. - A threatened and pharmaceutical important plant of Indian Thar Desert. Ind Crops Prod 42:25–29. https://doi.org/10.1016/j.indcrop.2012.05.013 Pieruzzi FP, Dias LLC, Balbuena TS, Santa-Catarina C, dos Santos ALW, Floh EIS (2011) Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm). Ann Bot 108:337–345. https://doi.org/10.1093/aob/mcr133 Pijut PM, Beasley RR, Lawson SS, Palla KJ, Stevens ME, Wang Y (2012) In vitro propagation of tropical hardwood tree species - a review (2001–2011). Propag Ornam Plants 12:25–51 Pijut PM, Woeste KE, Vengadesan G, Michler CH (2007) Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications. In Vitro Cell Dev Biol Plant 43:283–303. https://doi.org/10.1007/s11627-007-9026-9 R Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria Ranaweera KK, Gunasekara MTK, Eeswara JP (2013) Ex vitro rooting: A low cost micropropagation technique for Tea ( Camellia sinensis (L.) O. Kuntz) hybrids. Sci Hortic 155:8–14. https://doi.org/10.1016/j.scienta.2013.03.001 Rancour DM, Dickey CE, Park S, Bednarek SY (2002) Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants. Plant Physiol 130:1241–1253. https://doi.org/10.1104/pp.011742 Reis RS, de Moura Vale E, Heringer AS, Santa-Catarina C, Silveira V (2016) Putrescine induces somatic embryo development and proteomic changes in embryogenic callus of sugarcane. J Proteom 130:170–179. https://doi.org/10.1016/j.jprot.2015.09.029 Reis RS, Vale EM, Sousa KR, Santa-Catarina C, Silveira V (2021) Pretreatment free of 2,4-dichlorophenoxyacetic acid improves the differentiation of sugarcane somatic embryos by affecting the hormonal balance and the accumulation of reserves. Plant Cell Tiss Org Cult 145:101–115. https://doi.org/10.1007/s11240-020-01995-z Resendis-Antonio O, Hernández M, Salazar E, Contreras S, Batallar GM, Mora Y, Encarnación S (2011) Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling. BMC Syst Biol 5:120. https://doi.org/10.1186/1752-0509-5-120 Růžička K, Šimášková M, Duclercq J, Petrášek J, Zažímalová E, Simon S, Friml J, Montagu MCEV, Benková E (2009) Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc Natl Acad Sci U S A 106:4284–4289. https://doi.org/10.1073/pnas.0900060106 Sahai A, Shahzad A (2013) High frequency in vitro regeneration system for conservation of Coleus forskohlii : a threatened medicinal herb. Acta Physiol Plant 35:473–481. https://doi.org/10.1007/s11738-012-1090-z Sahu J, Khan S, Sahu RK, Roy A (2014) Micropropagation of Dalbergia sissoo Roxb. through tissue culture technique. Pak J Biol Sci 17:597–600. https://doi.org/10.3923/pjbs.2014.597.600 Santa-Catarina C, Maciel SdC, Pedrotti EL (2001) Germinação in vitro e embriogênese somática a partir de embriões imaturos de canela sassafrás ( Ocotea odorifera Mez). Rev Bras Bot 24:501–510. https://doi.org/10.1590/S0100-84042001000500004 Santa-Catarina C, Silveira V, Balbuena TS, Viana AM, Estelita MEM, Handro W, Floh EIS (2006) IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of Ocotea catharinensis . Plant Growth Regul 49:237–247. https://doi.org/10.1007/s10725-006-9129-z Santa-Catarina C, Silveira V, Scherer GFE, Floh EIS (2007) Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of Ocotea catharinensis . Plant Cell Tissue and Organ Culture 90:93–101. https://doi.org/10.1007/s11240-007-9259-7 Scholl J, Dengler L, Bader L, Forchhammer K (2020) Phosphoenolpyruvate carboxylase from the cyanobacterium Synechocystis sp. PCC 6803 is under global metabolic control by PII signaling. Mol Microbiol 114:292–307. https://doi.org/10.1111/mmi.14512 Shukla S, Shukla SK, Mishra SK (2008) In vitro plant regeneration from seedling explants of Stereospermum personatum D.C.: a medicinal tree. Trees 23:409. https://doi.org/10.1007/s00468-008-0290-z Stein VC, Ferreira TR, Rossato M, Macedo BF, da Silva FF, Paiva R, Paiva LV (2017) Establishment and in vitro multiplication of Calophyllum brasiliensis . Acta Hortic 1155:149–156. https://doi.org/10.17660/ActaHortic.2017.1155.20 Stevens ME, Pijut PM (2018) Rapid in vitro shoot multiplication of the recalcitrant species Juglans nigra L. Vitro Cell Dev Biol Plant 54:309–317. https://doi.org/10.1007/s11627-018-9892-3 Stuepp CA, Wendling I, Xavier A, Zuffellato-Ribas KC (2018) Vegetative propagation and application of clonal forestry in Brazilian native tree species. Pesqu Agropecu Bras 53:985–1002. https://doi.org/10.1590/S0100-204X2018000900002 Taylor DJ, Devkota B, Huang AD, Topf M, Narayanan E, Sali A, Harvey SC, Frank J (2009) Comprehensive molecular structure of the eukaryotic ribosome. Structure 17:1591–1604. https://doi.org/10.1016/j.str.2009.09.015 Tiburcio AF, Altabella T, Bitrián M, Alcázar R (2014) The roles of polyamines during the lifespan of plants: from development to stress. Planta 240:1–18. https://doi.org/10.1007/s00425-014-2055-9 Tomaz T, Bagard M, Pracharoenwattana I, Lindén P, Lee CP, Carroll AJ, Ströher E, Smith SM, Gardeström P, Millar AH (2010) Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in Arabidopsis . Plant Physiol 154:1143–1157. https://doi.org/10.1104/pp.110.161612 Tsugeki R, Terada S (2015) The Arabidopsis ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development. Plant Signal Behav 10:e1074369. https://doi.org/10.1080/15592324.2015.1074369 Watt G, Leoff C, Harper AD, Bar-Peled M (2004) A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis . Plant Physiol 134:1337–1346. https://doi.org/10.1104/pp.103.037192 White JT, Cato T, Deramchi N, Gabunilas J, Roy KR, Wang C, Chanfreau GF, Clarke SG (2019) Protein methylation and translation: Role of lysine modification on the function of yeast elongation factor 1A. Biochemistry 58:4997–5010. https://doi.org/10.1021/acs.biochem.9b00818 Willick IR, Plaxton WC, Lolle SJ, Macfie SM (2019) Transcriptional and post-translational upregulation of phosphoenolpyruvate carboxylase in Arabidopsis thaliana (L. Heynh) under cadmium stress. Environ Exp Bot 164:29–39. https://doi.org/10.1016/j.envexpbot.2019.04.018 Wybouw B, De Rybel B (2019) Cytokinin - A Developing Story. Trends Plant Sci 24:177–185. https://doi.org/10.1016/j.tplants.2018.10.012 Xiao R, Li L, Ma Y (2019) A label-free proteomic approach differentiates between conventional and organic rice. J Food Compos Anal 80:51–61. https://doi.org/10.1016/j.jfca.2019.04.004 Yan H, Liang C, Yang L, Li Y (2009) In vitro and ex vitro rooting of Siratia grosvenorii , a traditional medicinal plant. Acta Physiol Plant 32:115. https://doi.org/10.1007/s11738-009-0386-0 Zeng Q, Han Z, Kang X (2019) Adventitious shoot regeneration from leaf, petiole and root explants in triploid ( Populus alba × P. glandulosa )× P. tomentosa . Plant Cell Tiss Org Cult 138:121–130. https://doi.org/10.1007/s11240-019-01608-4 Zhang T, Feng P, Li Y, Yu P, Yu G, Sang X, Ling Y, Zeng X, Li Y, Huang J, Zhang T, Zhao F, Wang N, Zhang C, Yang Z, Wu R, He G (2018) VIRESCENT-ALBINO LEAF 1 regulates leaf colour development and cell division in rice. J Exp Bot 69:4791–4804. https://doi.org/10.1093/jxb/ery250 Zhou H, Li M, Zhao X, Fan X, Guo A (2010) Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ ( Prunus persica × P. davidiana ). Plant Cell Tiss Org Cult 101:79–87. https://doi.org/10.1007/s11240-010-9666-z Supplementary Files TableS1.xlsx Supplementary Table 1 Complete list of all identified proteins in 45-day-old shoots of Dalbergia nigra obtained from apical and cotyledonary nodal segments used as explants and the benziladenine (BA) concentrations, comparing the effect of BA concentration in the same explant (BA2.5_Apical/BA0_Apical and BA2.5_Cotyledonary/BA0_Cotyledonary comparisons) and the effect of explant (Apical and Cotyledonary) on the same BA concentration (BA2.5_Cotyledonary/BA2.5_Apical and BA0_Cotyledonary/BA0_Apical comparisons). Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 15 Mar, 2022 Reviewers invited by journal 14 Mar, 2022 Editor assigned by journal 08 Mar, 2022 First submitted to journal 04 Mar, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1419531","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":90681975,"identity":"04b2bd52-b7f0-49b8-b173-0c33bfbfea05","order_by":0,"name":"Lidia dos Santos Pessanha","email":"","orcid":"","institution":"Universidade Estadual do Norte Fluminense: Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":false,"prefix":"","firstName":"Lidia","middleName":"dos Santos","lastName":"Pessanha","suffix":""},{"id":90681976,"identity":"65522491-50b7-4af0-a596-91763a54bd7d","order_by":1,"name":"Victor Paulo Mesquita Aragão","email":"","orcid":"","institution":"Universidade Estadual do Norte Fluminense: Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":false,"prefix":"","firstName":"Victor","middleName":"Paulo Mesquita","lastName":"Aragão","suffix":""},{"id":90681977,"identity":"c06a813a-00a7-4cf4-ae93-b164539562c6","order_by":2,"name":"Tadeu dos Reis de Oliveira","email":"","orcid":"","institution":"Universidade Estadual do Norte Fluminense: Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":false,"prefix":"","firstName":"Tadeu","middleName":"dos Reis","lastName":"de Oliveira","suffix":""},{"id":90681978,"identity":"92393a31-2625-4669-b8e0-42ee4285e562","order_by":3,"name":"Kariane Rodrigues de Sousa","email":"","orcid":"","institution":"Universidade Estadual do Norte Fluminense: Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":false,"prefix":"","firstName":"Kariane","middleName":"Rodrigues","lastName":"de Sousa","suffix":""},{"id":90681979,"identity":"f2e8f4fb-accb-4853-b0a1-b972f26baeab","order_by":4,"name":"Vanildo Silveira","email":"","orcid":"","institution":"Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":false,"prefix":"","firstName":"Vanildo","middleName":"","lastName":"Silveira","suffix":""},{"id":90681980,"identity":"1d612ae2-e8ef-45d0-a7bb-3bf1cee8dade","order_by":5,"name":"Claudete Santa-Catarina","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYDACdiDmAWJ+EKeC4QARWpjBWgwYJBuAjDMkaTE4QKwW/mbmZxJvKv7IGd9IPvbgAMOdfIJaJA6zmUnOOWNgbHYjLR1o0zPLBkJaDJgZzKR52wwSt93IMZP+wHDYgKAtBszs36R5/xnUb56R/03iAHFaeIC2NBgkGEjksBGnReIwT7HlnGPGhjPOPDOTOGDwjLAW/vb2jTfe1MjJ87cnP5M4UHGHsBZ0d5KqYRSMglEwCkYBVgAAoMc5RO420zkAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-1669-660X","institution":"Universidade Estadual do Norte Fluminense Darcy Ribeiro","correspondingAuthor":true,"prefix":"","firstName":"Claudete","middleName":"","lastName":"Santa-Catarina","suffix":""}],"badges":[],"createdAt":"2022-03-04 14:41:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1419531/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1419531/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":19289992,"identity":"d3886c1d-2c23-4b58-87c5-c64a568ac6a9","added_by":"auto","created_at":"2022-03-16 15:08:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":552821,"visible":true,"origin":"","legend":"\u003cp\u003eGermination (%) of \u003cem\u003eDalbergia nigra\u003c/em\u003e seeds after 30 days of incubation in MS and WPM culture media (a) and morphological aspects of 30-day-old seedlings germinated in MS (b) and WPM (c) culture media. (n = 5; coefficient of variation = 13.18%). Bars in figures b and c = 1\u0026nbsp;cm.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/37f6b80913f3fd4d4a19fcd7.png"},{"id":19289743,"identity":"260eef6e-7be0-447a-b789-155417c1e01b","added_by":"auto","created_at":"2022-03-16 15:05:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":229832,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of shoots (a and b) and length (cm) of first (c and d) and second (e and f) shoots obtained from apical and cotyledonary nodal segments of \u003cem\u003eDalbergia nigra\u003c/em\u003e after 45 days of incubation in MS (a, c, and e) and WPM (b, d and f) culture media. Means followed by different letters are significantly different (P \u0026lt; 0.05) according to the SNK test. Capital letters denote significant differences comparing the same type of explant (apical and cotyledonary) at different BA concentrations (0, 2.5 and 5 µM). Lowercase letters denote significant differences between the two types of explants (apical and cotyledonary) at the same BA concentration (0, 2.5 or 5 µM). Asterisks (*) denote significant differences comparing the MS and WPM culture media for apical nodal segment explants at the same BA concentration (0, 2.5 or 5 µM). CV = Coefficient of Variation. (n = 8; CV of shoot number = 21.13%; CV of length of first shoot = 29.82%; CV of length of second shoot = 66.4%).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/30abc7246e846d2ed61bdcf6.png"},{"id":19290164,"identity":"d6ceaca0-b249-490d-94aa-a8d418777902","added_by":"auto","created_at":"2022-03-16 15:11:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":163374,"visible":true,"origin":"","legend":"\u003cp\u003eFree Put (a), Spd (b) and Spm (c) contents (µg.g\u003csup\u003e-1\u003c/sup\u003e FM) in shoots obtained from apical and cotyledonary nodal segments of \u003cem\u003eDalbergia nigra\u003c/em\u003e at 45 days of incubation on WPM culture medium without and with 2.5 µM BA. Means followed by different letters are significantly different (P \u0026lt; 0.05) according to the SNK test. Capital letters denote significant differences between the same type of explant (apical or cotyledonary) at different BA concentrations (0 and 2.5 µM). Lowercase letters denote significant differences between different types of explants (apical and cotyledonary) at the same BA concentration (0 or 2.5 µM). CV = Coefficient of Variation. (n = 3; CV of Put = 8.33%, CV of Spd = 10.99%, CV of Spm = 15.16%, CV of total free PAs = 7.57%).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/44549ed274da821843dcf8d3.png"},{"id":19289991,"identity":"d2ed7e85-2879-44c8-94e7-040ab5a8fca7","added_by":"auto","created_at":"2022-03-16 15:08:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1186441,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially accumulated proteins (DAPs) in 45-day-old shoots of \u003cem\u003eDalbergia nigra\u003c/em\u003e obtained from apical and cotyledonary nodal segments used as explants in the following comparisons: BA2.5_Apical/BA0_Apical (a), BA2.5_Cotyledonary/BA0_Cotyledonary (b), BA2.5_Cotyledonary/BA2.5_Apical (c) and BA0_Cotyledonary/BA0_Apical (d).\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/a7923af3dd1a2bef80ecebef.png"},{"id":19289744,"identity":"a2ae9cc8-fa15-49cd-9ca7-b3b79a32ffde","added_by":"auto","created_at":"2022-03-16 15:05:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":250243,"visible":true,"origin":"","legend":"\u003cp\u003eRoot induction (a, b), root number (c, d) and root length (cm) (e, f) in shoots from two types of explants (apical and cotyledonary nodal segments) of \u003cem\u003eDalbergia nigra\u003c/em\u003e obtained in WPM culture medium without (0 µM) and with 2.5 µM BA at 45 days in acclimatization. Capital letters denote significant differences comparing the same type of explant (shoots from apical or cotyledonary nodal segments) at different IBA concentrations. Lowercase letters denote significant differences comparing shoots from the two types of explants (apical and cotyledonary nodal segments) at the same IBA concentration. Asterisks (*) denote significant differences for shoots from apical and cotyledonary nodal segments comparing the BA treatment (0 and 2.5 µM) at each IBA concentration. (n = 8; CV of root induction = 27.4%; CV of root number = 24.92%; CV of root length = 24.4%)\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/9d4536436998dd7df3f7a6e9.png"},{"id":19290167,"identity":"f2030062-d988-4b45-bda8-62692dc5f9be","added_by":"auto","created_at":"2022-03-16 15:12:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":482139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/2f62efae-07d0-40bf-968d-1912fefc97b6.pdf"},{"id":19289748,"identity":"ff426d4a-c9ac-409c-85fe-b829f082045a","added_by":"auto","created_at":"2022-03-16 15:05:55","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":615206,"visible":true,"origin":"","legend":"\u003cp\u003e\u0026nbsp;\u003cstrong\u003eSupplementary Table 1\u003c/strong\u003e Complete list of all identified proteins in 45-day-old shoots of \u003cem\u003eDalbergia nigra\u003c/em\u003e obtained from apical and cotyledonary nodal segments used as explants and the benziladenine (BA) concentrations, comparing the effect of BA concentration in the same explant (BA2.5_Apical/BA0_Apical and BA2.5_Cotyledonary/BA0_Cotyledonary comparisons) and the effect of explant (Apical and Cotyledonary) on the same BA concentration (BA2.5_Cotyledonary/BA2.5_Apical and BA0_Cotyledonary/BA0_Apical comparisons).\u003c/p\u003e","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-1419531/v1/8cbbaa1be58c81972799ad90.xlsx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eBenzyladenine Effects On Polyamine Contents And Proteomic Profiles During \u003cem\u003eIn Vitro\u003c/em\u003e Shoot Development And On \u003cem\u003eEx Vitro\u003c/em\u003e Rooting In \u003cem\u003eDalbergia Nigra\u003c/em\u003e (Vell.) Allemão Ex Benth. (Fabaceae)\u003c/p\u003e","fulltext":[{"header":"Key Message ","content":"\u003cp\u003eMicropropagation of \u003cem\u003eDalbergia nigra\u003c/em\u003e (Fabaceae)\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eSeveral native wood species from the Atlantic rainforest are endangered, including \u003cem\u003eDalbergia nigra\u003c/em\u003e (Vell.) Allem\u0026atilde;o ex Benth, commonly known as Jacarand\u0026aacute;-da-Bahia. Due to the intense exploitation of woody plants and the lack of reforestation programs, this species has been included as vulnerable on the Red List of the International Union for Conservation of Nature (IUCN \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Appropriate biotechnological and sustainable conservation strategies for many woody species need further research and development, and \u003cem\u003ein vitro\u003c/em\u003e propagation technologies can be applied for conservation, with a great going to a global economic and ecological impact on sustaining tropical forest woody biodiversity (Pijut et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiotechnological tools, such as micropropagation, have been applied in studies aiming at the propagation of woody species (Fermino-Junior and Scherwinski-Pereira \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Perveen et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Micropropagation is the commercially efficient propagation of species in a short period of time (Gupta et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), enabling clonal production and conservation of germplasm (Giri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Shukla et al. \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Kodym and Leeb \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Knowledge of the biochemical and molecular aspects of \u003cem\u003ein vitro\u003c/em\u003e morphogenesis, such as the cellular mechanisms involved in the growth and development of these species, is necessary for the propagation and preservation of threatened species with difficulties in propagation by conventional methods (Pijut et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dias et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Stuepp et al. \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe steps of \u003cem\u003ein vitro\u003c/em\u003e propagation involve manipulation of the type of explants as well as the components of the culture medium, such as plant growth regulators (PGRs), to achieve optimal conditions for shoot multiplication and root induction (Bunn et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Among the PGRs, cytokinins and auxins are the most commonly used in plant tissue cultures for shoot and root development (Phillips and Garda \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In the propagation of woody species, benzyladenine (BA) is the cytokinin most commonly used to promote the development of axillary buds, breaking apical dominance and stimulating shoot proliferation (Giri et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Pijut et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Studies have shown the relationship of PGRs with other compounds, such as polyamines (PAs), in shoot development (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePAs are low molecular weight, aliphatic, polycationic compounds with positively charged nitrogen atoms naturally occurring in plants (Baron and Stasolla \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). These compounds can interact with negatively charged macromolecules, such as DNA, RNA, phospholipids, cell wall components, and proteins (Baron and Stasolla \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Thus, PAs are essential for various physiological and developmental processes in plants (Santa-Catarina et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Dutra et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), including shoot development (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e; Lerin et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Oliveira et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Changes in endogenous PA contents induced by exogenous addition, especially putrescine (Put), demonstrated its relevance for shoot development in \u003cem\u003eC. fissilis\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to PAs, specific proteins have been candidate markers associated with morphogenic competence during \u003cem\u003ein vitro\u003c/em\u003e plant morphogenesis (Reis et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Heringer et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Proteomic studies revealed the involvement of multiple proteins with specific functions in competence for the \u003cem\u003ein vitro\u003c/em\u003e development of shoots (Mitrović et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Ghosh and Pal \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Changes in the accumulation of some proteins involved mainly in metabolic and cellular processes, such as cell division, during \u003cem\u003ein vitro\u003c/em\u003e shoot development induced or not induced by exogenous putrescine (Put) demonstrated an important relationship of specific proteins with shoot development in \u003cem\u003eC. fissilis\u003c/em\u003e (Arag\u0026atilde;o et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Arag\u0026atilde;o et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017b\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition to shoot development, the formation of adventitious roots is an essential step of \u003cem\u003ein vitro\u003c/em\u003e plant propagation. Auxin plays an essential role in rooting, and indole-3-butyric acid (IBA) is the most commonly used due to its higher root-inducing capacity and greater stability to light (Pacurar et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). IBA use is well documented during \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003eex vitro\u003c/em\u003e rooting in several woody species (Pijut et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003eEx vitro\u003c/em\u003e rooting has the advantage of lower cost and saves time, reducing the cost of a micropropagation protocol up to 70% compared to \u003cem\u003ein vitro\u003c/em\u003e rooting(Yan et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ranaweera et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Patel et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In addition, another advantage of \u003cem\u003eex vitro\u003c/em\u003e rooting is that plantlets do not need additional acclimatization, exhibiting good root development and improved plantlet survival compared to \u003cem\u003ein vitro\u003c/em\u003e rooting (Yan et al. \u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Gupta et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eStudies on the \u003cem\u003ein vitro\u003c/em\u003e propagation of \u003cem\u003eD. nigra\u003c/em\u003e have not yet been developed. In this sense, the establishment of \u003cem\u003ein vitro\u003c/em\u003e propagation for this species can contribute to conservation programs and the repositioning of impacted areas. In addition, biochemical and molecular approaches can improve the knowledge of \u003cem\u003ein vitro\u003c/em\u003e morphogenesis competence. Thus, the aim of this work was to establish the \u003cem\u003ein vitro\u003c/em\u003e propagation and \u003cem\u003eex vitro\u003c/em\u003e rooting of \u003cem\u003eD. nigra\u003c/em\u003e and evaluate the alterations in PA contents and protein profiles during shoot development.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature seeds obtained from Cai\u0026ccedil;ara Com\u0026eacute;rcio de Sementes LTDA located in Brejo Alegre, SP, Brazil (21\u0026deg;10\u0026apos;S and 50\u0026deg;10\u0026apos;W) were used for in vitro germination. Forty-five-day-old seedlings in vitro germinated were used as the source of apical and cotyledonary nodal explants for the shoot development experiments. Forty-five-day-old micropropagated shoots were used for the ex vitro rooting experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of plant culture medium on in vitro seed germination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor in vitro germination, seeds were surface\u0026nbsp;disinfected according to\u0026nbsp;Santa-Catarina et al. (2001), with modifications. First, seeds were washed with 250 mL distilled water, followed by immersion in\u0026nbsp;70%\u0026nbsp;ethanol for 1 min\u0026nbsp;and\u0026nbsp;incubation in 2.5%\u0026nbsp;sodium hypochlorite solution supplemented with the fungicide Derosal\u003csup\u003e\u0026reg;\u003c/sup\u003e 500 SC (Bayer; S\u0026atilde;o Paulo, Brazil; active ingredient carbendazim 500 g L\u003csup\u003e-1\u003c/sup\u003e; 200 \u0026micro;L of commercial solution per liter of water) for 30 min. Seeds were washed five times for 10 min each in sterile distilled water\u0026nbsp;in\u0026nbsp;a flow chamber. After disinfection, seeds were transferred to Murashige and Skoog (MS; Phytotechnology Lab, Overland Park, USA)\u0026nbsp;(Murashige and Skoog 1962)\u0026nbsp;and Woody Plant Medium (WPM; Phytotechnology)\u0026nbsp;(Lloyd and McCown 1981)\u0026nbsp;culture media, both supplemented with 20 g L\u003csup\u003e-1\u003c/sup\u003e sucrose (Synth; S\u0026atilde;o Paulo, Brazil) and 2 g L\u003csup\u003e-1\u003c/sup\u003e Phytagel\u003csup\u003e\u0026reg;\u003c/sup\u003e (Sigma\u0026ndash;Aldrich, St. Louis, USA). The pH of the culture medium was adjusted to 5.7 before the use of Phytagel and autoclaved at 121 \u0026deg;C for 15 min. \u0026nbsp;Then, seeds were incubated in a 16-h photoperiod at a light intensity of 55 \u0026mu;mol m\u003csup\u003e-2\u0026nbsp;\u003c/sup\u003es\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003eand\u0026nbsp;a\u0026nbsp;temperature of\u0026nbsp;25 \u0026plusmn; 2 \u0026deg;C. The germination\u0026nbsp;(%) and morphology of seedlings were analyzed after 30 days of incubation. Each treatment consisted of five repetitions,\u0026nbsp;with\u0026nbsp;20 seeds of each repetition.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of explant, plant culture medium and BA concentration on shoot development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eForty-five-day-old seedlings germinated \u003cem\u003ein vitro\u003c/em\u003e were used as the source of explants. Apical and cotyledonary nodal segments (\u0026plusmn; 2 cm) were isolated from seedlings and cultured on MS and/or WPM culture medium supplemented with 20 g L\u003csup\u003e-1\u003c/sup\u003e sucrose, 2 g L\u003csup\u003e-1\u003c/sup\u003e Phytagel\u003csup\u003e\u0026reg;\u003c/sup\u003e and different concentrations (0, 2.5 and 5 \u0026micro;M) of BA (Sigma\u0026ndash;Aldrich). The pH of the culture medium was adjusted to 5.7 and autoclaved at 121 \u0026deg;C for 15 min. The explants were transferred to the culture medium with different treatments and maintained at a 16-h photoperiod under a light intensity of 55 \u0026mu;mol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e and a temperature of 25 \u0026plusmn; 2 \u0026deg;C. Eight repetitions per treatment were used, with four explants of each repetition. The induction (%), number of shoots per explant and length of the first and second shoots were analyzed after 45 days of incubation. Samples were collected for PA and proteomic analyses and maintained at -80 \u0026deg;C until analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of IBA on ex vitro rooting of shoots and plant acclimatization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShoots from apical and cotyledonary nodal segments (\u0026plusmn; 2 cm) cultured in WPM culture medium supplemented without and with 2.5 \u0026micro;M BA were used for shoot rooting. Shoots containing the apical meristem and leaves were treated for 30 s with different concentrations (0, 100 and 500 \u0026mu;M) of IBA (Sigma\u0026ndash;Aldrich). Then, shoots were transferred to 50 mL plastic pots containing a mixture of PlantMax substrate (DDL Agroindustria, Paul\u0026iacute;nia, Brazil) with vermiculite (2:1; v/v). The shoots were maintained in plastic trays covered with a plastic film to maintain the high humidity needed for root development during \u003cem\u003eex vitro\u003c/em\u003e rooting and acclimatization. These plastic trays were kept in the growth room under a 16-h photoperiod, a light intensity of 55 \u0026mu;mol m\u003csup\u003e-2\u003c/sup\u003e s\u003csup\u003e-1\u003c/sup\u003e, and\u0026nbsp;a\u0026nbsp;temperature of 25 \u0026plusmn; 2 \u0026deg;C. After 30 days, the humidity was gradually reduced until 40 days, when rooted shoots were considered acclimatized. The induction of rooting (%), number of roots initiated per shoot and root length were recorded after 45 days. Each treatment consisted of eight repetitions, with four shoots in each repetition.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFree PA determination\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDetermination of free PAs was performed according to\u0026nbsp;Santa-Catarina et al. (2006)\u0026nbsp;using samples of shoots from apical and cotyledonary nodal segments with 45 days of incubation without (control) and with 2.5 \u0026micro;M BA in WPM medium. Samples (200 mg fresh matter \u0026ndash; FM \u0026ndash; each, in triplicate) were\u0026nbsp;ground\u0026nbsp;in 1.2 mL of 5% perchloric acid (Merck Millipore, Darmstadt, Germany). After 1 h of incubation at 4 \u0026deg;C, the samples were centrifuged for 20 min at 20,000\u0026times;g at 4 \u0026deg;C. The supernatant was collected, and free\u0026nbsp;PAs were determined directly from the supernatant by derivatization with dansyl chloride (Merck Millipore) and identified by high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan) using a 5-\u0026mu;m C18 reverse-phase column (Shimadzu Shin-pack CLC ODS). The HPLC column gradient was created by adding increasing volumes of absolute acetonitrile (Merck Millipore) to a 10% aqueous acetonitrile solution with the pH adjusted to 3.5 with hydrochloric acid (Merck Millipore). The absolute acetonitrile concentration was maintained at 65% for the first 10 min, increased from 65 to 100% between 10 and 13 min, and maintained at 100% between 13 and 21 min. The mobile phase was added at a flow rate of 1 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e and 40 \u0026deg;C. The PA concentration was determined using a fluorescence detector at 340 nm (excitation) and 510 nm (emission). The peak areas and retention times of the samples were measured through comparisons with the standard Pas putrescine (Put), spermidine (Spd), and spermine (Spm) (Sigma\u0026ndash;Aldrich).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein extraction and digestion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted from samples (three biological triplicates, 300 mg FM per sample) of shoots from apical and cotyledonary nodal explants grown without (0 \u0026micro;M - control) and with 2.5 \u0026micro;M BA at 45 days of incubation in WPM medium. Proteins were extracted using the trichloroacetic acid (TCA)/acetone method with modifications (Damerval et al. 1986). Initially, the samples were pulverized in liquid nitrogen using a ceramic mortar and pestle. The resulting powder was resuspended in 1 mL of chilled extraction buffer containing 10% (w/v) TCA (Sigma) in acetone with 20 mM dithiothreitol (DTT; GE Healthcare, Piscataway, USA) and vortexed for 5 min at 8 \u0026deg;C. Next, the samples were kept at \u0026minus;20 \u0026deg;C for 1 h before centrifugation at 16,000 x \u003cem\u003eg\u003c/em\u003e for 30 min at 4 \u0026deg;C. The resulting pellets were washed three times with cold acetone plus 20 mM DTT and centrifuged for 5 min each time. The pellets were air dried and resuspended in buffer containing 7 M urea (GE Healthcare), 2 M thiourea (GE Healthcare), 2% Triton X-100 (GE Healthcare), 1% DTT, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma\u0026ndash;Aldrich), and complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany), vortexed for 30 min at 8 \u0026deg;C, and centrifuged for 20 min at 16,000 x \u003cem\u003eg\u003c/em\u003e at 4 \u0026deg;C. The supernatants were collected, and the protein concentrations were determined using a 2-D Quant Kit (GE Healthcare).\u003c/p\u003e\n\u003cp\u003eBefore the trypsin digestion step, protein samples were precipitated using the methanol/chloroform methodology (Nanjo et al. 2012). After protein precipitation, the samples were resuspended in 7 M urea/2 M thiourea solution. Aliquots of 100 \u0026micro;g of protein were subjected to tryptic digestion using the filter-aided sample preparation (FASP) methodology (Reis et al. 2021). Next, the peptides were resuspended in 100 \u0026mu;L solution containing 95% 50 mM ammonium bicarbonate, 5% acetonitrile and 0.1% formic acid and quantified by A205 nm protein and peptide methodology using a NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific). The samples were transferred to Total Recovery Vials (Waters) for mass spectrometry analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMass spectrometry analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMass spectrometry was performed using a nanoAcquity UPLC connected to a Q-TOF SYNAPT G2-Si instrument (Waters, Manchester, UK) according to\u0026nbsp;Passamani et al. (2018). Runs consisted of three biological replicates of 1 \u0026micro;g of peptide samples. During separation, samples were loaded onto the nanoAcquity UPLC M-Class Symmetry C18 5 \u0026mu;m trap column (180 \u0026mu;m \u0026times; 20 mm) at 5 \u0026mu;L.min\u003csup\u003e-1\u003c/sup\u003e for 3 min and then onto the nanoAcquity M-Class HSS T3 1.8 \u0026mu;m analytical reversed-phase column (75 \u0026mu;m \u0026times; 150 mm) at 400 nL.min\u003csup\u003e-1\u003c/sup\u003e, with a column temperature of 45 \u0026deg;C. For peptide elution, a binary gradient was used, with mobile phase A consisting of water (Tedia, Fairfield, Ohio, USA) and 0.1% formic acid (Sigma\u0026ndash;Aldrich) and mobile phase B consisting of acetonitrile (Sigma Aldrich) and 0.1% formic acid. The gradient elution started at 7% B, then ramped from 7% B to 40% B until 91.12 min, then ramped again from 40% B to 99.9% B until 92.72 min, then remained at 99.9% until 106.00 min, then decreased to 7% B until 106.1 min, and finally remained at 7% B until the end of experiment at 120 min. Mass spectrometry was performed in positive and resolution mode (V mode), 35,000 full widths at half maximum, with ion mobility separation (IMS), and in data-independent acquisition mode (HDMS\u003csup\u003eE\u003c/sup\u003e). The ion mobility wave was set to a velocity of 600 m s\u003csup\u003e-1\u003c/sup\u003e\u003csup\u003e,\u003c/sup\u003e and helium and IMS gas flows were 180 and 90 mL min\u003csup\u003e\u0026minus;1\u003c/sup\u003e, respectively. The transfer collision energy ramped from 19 to 55 V in high-energy mode; the cone and capillary voltages were 30 and 2750 V, respectively; and the source temperature was 70 \u0026deg;C. Regarding\u0026nbsp;the\u0026nbsp;time of flight (TOF) parameters, the scan time was set to 0.5 s in continuum mode with a mass range of 50 to 2000 Da.\u0026nbsp;Human\u0026nbsp;[Glu1]-fibrinopeptide B at 100 fmol.\u0026mu;L\u003csup\u003e-1\u003c/sup\u003e was used as an external calibrant, and lock mass acquisition was performed every 30 s. Mass spectra were acquired by MassLynx v4.0 software.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProteomics data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpectra processing and database search conditions were performed using ProteinLynx Global Server (PLGS) software v.3.0.2 (Waters). The PLGS was processed by the following parameters: Apex3D of 150 counts for low-energy threshold, 50 counts for elevated-energy threshold, and 750 counts for intensity threshold; two missed cleavages; minimum fragment ions per peptide equal to three; minimum fragment ions per protein equal to seven; minimum peptides per protein equal to two; fixed modifications of carbamidomethyl (C) and variable modifications of oxidation (M) and phosphoryl (STY); default false discovery rate (FDR) was set to a maximum of 1%. We used the \u003cem\u003eArachis hypogaea\u003c/em\u003e protein databank from UniProtKB (http://www.unipr ot.org) for protein identification, as it is the largest databank with proximity to \u003cem\u003eD. nigra\u003c/em\u003e. Label-free quantification analysis was performed using ISOQuant workflow software v.1.7\u0026nbsp;(Distler et al. 2014). Briefly, the following parameters were used to identify proteins:\u0026nbsp;FDR 1%, a peptide score greater than six, a minimum peptide length of six amino acids, and at least two peptides per protein were considered for label-free quantitation using the TOP3 approach, followed by the multidimensional normalized process within ISOQuant. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE\u0026nbsp;(Perez-Riverol et al. 2022)\u0026nbsp;partner repository with the dataset identifier PXD031999.\u0026nbsp;To ensure the quality of the results after data processing, only the proteins that were either present or absent (for unique proteins) in all three biological replicates were considered for differential accumulation analysis. Data were analyzed using Student\u0026rsquo;s t\u0026nbsp;test (two-tailed). Proteins with ANOVA (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05) were considered up-accumulated if the log\u003csub\u003e2\u003c/sub\u003e value of the fold change (FC) was greater than 0.60 and down-accumulated if the log\u003csub\u003e2\u003c/sub\u003e value of the FC was less than -0.60. Functional annotations were performed using OmicsBox software 1.0.34 and UniProtKB (http://www.uniprot.org).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental design was completely randomized. Data were analyzed using analysis of variance (\u003cem\u003eP \u0026lt; 0.05\u003c/em\u003e) followed by the Student-Newman\u0026ndash;Keuls (SNK) test using the R Environment (R Core Team 2014).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of plant culture medium on in vitro germination and seedling development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth culture media tested showed no significant difference on germination, resulting in similar percentages for MS (80%) and WPM (81%) (Fig. 1a). However, the seedlings from WPM culture medium showed well-developed leaves (Fig. 1c), while those obtained in the MS culture medium showed more senescence of leaves (Fig. 1b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexplants\u003c/strong\u003e\u003cstrong\u003e, plant culture media and BA on shoot development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the difference in length between the first (Figs. 2a and 2b) and second shoots (Figs. 2c and 2d) developed, they were measured separately. BA addition promoted the elongation of the first shoot developed from both types of explants, the apical and cotyledonary nodal segments, in both culture media, MS (Fig. 2a) and WPM (Fig. 2b), compared to shoots obtained without BA (control). Moreover, no significant difference was observed between the two types of explants in the same BA concentration and same culture medium or between the two culture media used (Figs. 2a and 2b). As BA is essential for shoot growth and there were no significant differences in the length of shoots between 2.5 and 5 \u0026micro;M BA in either culture medium, the two types of explants can be used for \u003cem\u003ein vitro\u003c/em\u003e propagation of \u003cem\u003eD. nigra\u003c/em\u003e, considering the elongation of\u0026nbsp;the\u0026nbsp;first shoot developed.\u0026nbsp;The second shoot showed lower elongation (Figs. 2d and 2e)\u0026nbsp;than\u0026nbsp;the first\u0026nbsp;shoot\u0026nbsp;(Figs. 2a and 2b).\u0026nbsp;In\u0026nbsp;MS culture medium, the BA concentration increased the length of\u0026nbsp;the\u0026nbsp;second shoot from cotyledonary nodal segments compared to\u0026nbsp;the\u0026nbsp;control, while no significant effects were observed in shoots from apical nodal segments (Fig. 2c).\u0026nbsp;In\u0026nbsp;WPM culture medium, the 2.5 \u0026mu;M BA treatment showed lower elongation of second shoots from cotyledonary nodal segments compared to\u0026nbsp;the\u0026nbsp;control and 5 \u0026mu;M BA (Fig. 2d).\u003c/p\u003e\n\u003cp\u003eThere were no significant differences in the number of shoots between the two types of explants at each BA concentration (0 or 2.5 \u0026micro;M) or in either culture medium, MS (Fig. 2e) or WPM (Fig. 2f), except for apical explants in the control treatment (without BA) in MS culture medium, which showed a significantly lower number of shoots (Fig. 2e). On the other hand, no significant differences were observed for the number of shoots from apical and cotyledonary nodal segments incubated with WPM culture medium (Fig. 2f). For shoot induction, no significant differences were observed in explant type, culture medium or BA concentration, showing 100% shoot induction in all treatments (data not shown).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of BA and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eexplant\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etype on endogenous\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ePA\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;contents during shoot development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFree PAs\u0026nbsp;were quantified in 45-day-old shoots from apical and cotyledonary nodal segments incubated without and with 2.5 \u0026micro;M BA and WPM culture medium (Fig. 3).\u0026nbsp;Free\u0026nbsp;Put content was significantly higher when shoots were grown in 2.5 \u0026micro;M BA compared to\u0026nbsp;the\u0026nbsp;control in both\u0026nbsp;types\u0026nbsp;of explants, being\u0026nbsp;significantly\u0026nbsp;higher in shoots from apical nodal segments compared to cotyledonary nodal segments (Fig. 3a). Higher free\u0026nbsp;Spd content was observed in shoots from apical explants grown in 2.5 \u0026micro;M BA, differing statistically from shoots in\u0026nbsp;the\u0026nbsp;control treatment (Fig. 3b). Moreover, a higher free-Spm content was observed in shoots from cotyledonary nodal segments in\u0026nbsp;the\u0026nbsp;2.5 \u0026micro;M BA treatment\u0026nbsp;than in the\u0026nbsp;control (Fig. 3c). The content of total free\u0026nbsp;PAs was significantly higher in shoots from both\u0026nbsp;types\u0026nbsp;of explants incubated in 2.5 \u0026micro;M BA\u0026nbsp;than in\u0026nbsp;shoots from\u0026nbsp;the\u0026nbsp;control treatment (Fig. 3d).\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of BA and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ethe\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etype of explant on\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;the\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;proteomic profile during shoot development\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteomic analysis was performed comparing the effects of BA (by the comparisons BA2.5_Apical/BA0_Apical and BA2.5_Cotyledonary/BA0_Cotyledoenary) and\u0026nbsp;the\u0026nbsp;type of explant (by the comparisons BA2.5_Cotyledonary/BA2.5_Apical and BA0_Cotyledonary/BA0_Apical) on shoot development. A total of 1232 proteins were identified (Supplementary\u0026nbsp;Table\u0026nbsp;1). Among the DAPs, some proteins were highlighted according to their relevance\u0026nbsp;to\u0026nbsp;cell division and\u0026nbsp;shoot\u0026nbsp;growth.\u003c/p\u003e\n\u003cp\u003eComparing the BA concentrations (0 and 2.5 \u0026micro;M) in shoots obtained from apical nodal segments (BA2.5_Apical/BA0_Apical),\u0026nbsp;a total of 292 proteins were differentially accumulated (DAPs),\u0026nbsp;and 923\u0026nbsp;were\u0026nbsp;unchanged. Among the DAPs, 93 proteins were up- and 158 down-accumulated, with 13 unique in shoots grown under BA2.5_Apical and 28 unique in shoots under BA0_Apical (Fig. 4a; Supplementary table 1).\u0026nbsp;In the\u0026nbsp;comparison of BA\u0026nbsp;concentration\u0026nbsp;treatment in shoots from cotyledonary nodal\u0026nbsp;segments\u0026nbsp;(BA2.5_Cotyledonary/BA0_Cotyledonary), a total of 374 proteins were DAPs,\u0026nbsp;and 846\u0026nbsp;were\u0026nbsp;unchanged. Among the DAPs, 179 were up- and 158 were down-accumulated. In addition, 18 proteins were unique in shoots from cotyledonary nodal segments incubated with 2.5 \u0026micro;M\u0026nbsp;BA (BA2.5_Cotyledonary),\u0026nbsp;and 19 proteins were unique in shoots from cotyledonary nodal segments without BA (BA0_Cotyledonary) (Fig. 4b; Supplementary table 1).\u003c/p\u003e\n\u003cp\u003eAmong these proteins, some were up-accumulated in shoots from both\u0026nbsp;types\u0026nbsp;of explants (apical and cotyledonary nodal segments) incubated with 2.5 BA compared to shoots without BA (comparisons BA2.5_Cotyledonary/BA0_Cotyledonary and BA2.5_Apical/BA0_Apical), as the pre-mRNA-splicing factor ATP-dependent RNA helicase DEAH7-like (A0A444YGB9), calreticulin-3 (A0A445A993), aspartate aminotransferase 1 (A0A445B4C1), protein elongation factor 1-alpha (A0A444Y7Y0) and cell division control protein 48 homolog D (A0A444Z3W1) (Supplementary Table 1). In addition, the proteins phosphoenolpyruvate carboxylase 2 (A0A445BXQ0), phosphoribosylamine-glycine ligase isoform X1 (A0A445E7J0), FT-interacting protein 3 (A0A444ZYV6), dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit (A0A445A8R7), and 60S ribosomal protein L35a-1 (A0A444ZT56) were up-accumulated in shoots from cotyledonary nodal segments incubated in 2.5 \u0026micro;M BA compared to shoots without BA (comparison BA2.5_Cotyledonary/BA0_Cotyledonary) (Supplementary Table 1).\u003c/p\u003e\n\u003cp\u003eThe effect of explant type at the same BA concentration on the protein profile was evaluated. Comparing cotyledonary shoots from apical nodal segments incubated at 2.5 \u0026micro;M BA (comparison BA2.5_Cotyledonary/BA2.5_Apical), 242 proteins were DAPs, and 973 were unchanged. Among the DAPs, 154 were up- and 46 were down-accumulated, with 28 proteins unique to shoots under the BA2.5_Cotyledonary treatment and 14 proteins unique to shoots under the BA2.5_Apical treatment (Fig. 4c). Comparing the shoots from cotyledonary to apical nodal segments incubated without BA (comparison BA0_Cotyledonary/BA0_Apical), a total of 231 proteins were DAP, and 997 were unchanged (Fig. 4d). Among the DAPs, 89 were up- and 90 down-accumulated, presenting 26 unique proteins in shoots from cotyledonary nodal segments without BA (BA0_Cotyledonary treatment) and 26 unique proteins in shoots from apical nodal segments without BA (BA0_Apical treatment) (Supplementary table 1). Among the DAPs, the malate dehydrogenase 2, peroxisomal (A0A445DRP9), ATP synthase CF1 beta subunit (A0A445AH22) proteins were up-accumulated in shoots from cotyledonary nodal segments compared to those from apical segments at both BA concentrations. The bifunctional dTDP-4-dehydrorhamnose 3,5-epimerase/dTDP-4-dehydrorhamnose reductase (A0A444Z945) protein was unique in \u003cem\u003eD. nigra\u003c/em\u003e shoots from BA-treated cotyledonary nodal segments compared to shoots without BA (BA2.5_Cotyledonary/BA0_Cotyledonary) and shoots from BA-treated apical nodal segments (BA2.5_Cotyledonary/BA2.5_Apical). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of IBA on \u003cem\u003eex vitro\u003c/em\u003e rooting of shoots and acclimatization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ex vitro root induction of shoots was not significantly affected by IBA concentrations or types of explants (Fig. 5). However, the induction of roots was significantly affected by BA supplementation on the culture medium of shoot development, with the induction of roots being significantly higher in shoots grown in culture medium without BA (Fig. 5a) compared to 2.5 \u0026micro;M BA (Fig. 5b). The number of roots was also significantly affected by the BA concentration used on shoot multiplication, being significantly higher in shoots multiplied without BA (Fig. 5c) compared to BA (Fig. 5d). The length of shoots was significantly affected by BA treatment during shoot multiplication and was significantly higher in shoots from cotyledonary nodal explants grown with BA (Figs. 5e and 5f). On the other hand, the length of roots was not significantly affected by IBA concentrations used in shoots from either type of explant used (Figs. 5e and 5f).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe establishment of the best culture medium for \u003cem\u003ein vitro\u003c/em\u003e seed germination is relevant to\u0026nbsp;obtaining\u0026nbsp;explants for micropropagation.\u0026nbsp;In addition to\u0026nbsp;no significant differences in seed germination (Fig. 1a), the WPM culture medium resulted in seedlings with better growth (Fig. 1c). In \u003cem\u003eCariniana legalis\u003c/em\u003e, the WPM culture medium improved the percentage of \u003cem\u003ein vitro\u003c/em\u003e seed germination,\u0026nbsp;which was\u0026nbsp;significantly higher\u0026nbsp;than that of\u0026nbsp;MS culture medium\u0026nbsp;(Arag\u0026atilde;o et al. 2017a). The WPM culture medium has only 25% of the concentrations of nitrate and ammonium ions present in MS culture medium, in addition to more potassium and a high level of sulfate ions,\u0026nbsp;which are\u0026nbsp;widely used for micropropagation of woody species\u0026nbsp;(Hazubska-Przybył 2019). The\u0026nbsp;lower\u0026nbsp;concentrations of total nitrogen and ammonium in WPM culture medium\u0026nbsp;reduce\u0026nbsp;the possibility of toxicity to ammonium, which can contribute to the development of seedlings in some woody species\u0026nbsp;(Phillips and Garda 2019), as observed in \u003cem\u003eD. nigra\u003c/em\u003e in the present work.\u003c/p\u003e\n\u003cp\u003eFor shoot development, BA is the most\u0026nbsp;common\u0026nbsp;cytokinin used for\u0026nbsp;the\u0026nbsp;proliferation of axillary buds in many plant species\u0026nbsp;(Sahai and Shahzad 2013), including several trees, such as \u003cem\u003eSantalum album\u0026nbsp;\u003c/em\u003e(Mujib 2005),\u003cem\u003e\u0026nbsp;Cedrela fissilis\u003c/em\u003e (Arag\u0026atilde;o et al. 2016; Arag\u0026atilde;o et al,. 2017b), \u003cem\u003eAzadirachta excelsa\u003c/em\u003e (Foan and Othman 2006), \u003cem\u003eSapium sebiferum\u0026nbsp;\u003c/em\u003eand \u003cem\u003eCalophyllum brasiliensis\u003c/em\u003e (Stein et al. 2017). The structural stability of BA and the ability of plant cells to easily assimilate make this cytokinin\u0026nbsp;an efficient promoter of plant development\u0026nbsp;(Ahmad et al. 2013). Our results showed that BA addition was essential to increase the length of shoots from both types of explants (apical and cotyledonary nodal segments) and culture media (MS and WPM) (Fig. 2) in \u003cem\u003eD. nigra\u003c/em\u003e. The use of BA also promoted longer shoots in \u003cem\u003eJuglans nigra\u003c/em\u003e (Stevens and Pijut 2018)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eRauvolfia tetraphylla\u003c/em\u003e (Hussain et al. 2018). This promotion in the length of BA-induced\u0026nbsp;shoots\u0026nbsp;may be associated with\u0026nbsp;the\u0026nbsp;effects of cytokinins in the control of cell division, providing greater growth and development\u0026nbsp;(Wybouw and De Rybel 2019).\u0026nbsp;In addition to\u0026nbsp;the positive effects of BA on shoot length, this cytokinin showed no effects on shoot induction (%)\u0026nbsp;or the\u0026nbsp;number of shoots per explant (Fig. 2) in \u003cem\u003eD. nigra\u003c/em\u003e. Similarly, no significant effects of BA on\u0026nbsp;the\u0026nbsp;number of shoots\u0026nbsp;were\u0026nbsp;observed for \u003cem\u003eC. legalis\u003c/em\u003e (Arag\u0026atilde;o et al. 2017a). On the other hand, in \u003cem\u003eDalbergia sisso\u003c/em\u003e, the use of 4.4 \u0026micro;M BA provided a greater number of shoots compared to the control\u0026nbsp;(Sahu et al. 2014). These results show that the \u003cem\u003ein vitro\u003c/em\u003e morphogenic response\u0026nbsp;induced by\u0026nbsp;BA is intrinsic to the species, and this response may be different even within species of the same genus as that observed between \u003cem\u003eD. nigra\u003c/em\u003e and \u003cem\u003eD. sissoo\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition to cytokinins, PAs are involved in plant growth and development,\u0026nbsp;as\u0026nbsp;they can act in various physiological processes,\u0026nbsp;such as\u0026nbsp;the\u0026nbsp;promotion of cell division, differentiation, and elongation,\u0026nbsp;which are\u0026nbsp;essential to embryo development, seed germination, rhizogenesis and shoot development in woody plants\u0026nbsp;(Santa-Catarina et al. 2006; Kusano et al. 2008; Pieruzzi et al. 2011; Arag\u0026atilde;o et al. 2016; Lerin et al. 2019). The higher content of free Put (Fig. 3a) in shoots from cotyledonary and apical nodal segments incubated with BA was correlated with the higher length of shoots in \u003cem\u003eD. nigra\u003c/em\u003e (Figs. 2c and\u0026nbsp;2d). Similar results\u0026nbsp;were\u0026nbsp;observed during \u003cem\u003ein vitro\u003c/em\u003e shoot development in other species, such as \u003cem\u003eBixa orellana\u003c/em\u003e (Parimalan et al. 2011)\u0026nbsp;and \u003cem\u003eC. fissilis\u003c/em\u003e (Arag\u0026atilde;o et al. 2016; Oliveira et al. 2020). A high content of Put was shown\u0026nbsp;to\u0026nbsp;be\u0026nbsp;directly related\u0026nbsp;to\u0026nbsp;cell cycle progression at the G1/S transition, stimulating\u0026nbsp;the\u0026nbsp;synthesis of\u0026nbsp;proteins\u0026nbsp;such as tubulins, which contribute to cell growth\u0026nbsp;(Tiburcio et al. 2014). Cross-talk among PAs and other plant hormones, such as cytokinin, has been proposed,\u0026nbsp;as\u0026nbsp;BA can affect PA metabolism and thereby their homeostasis by changing the expression of the genes responsible for PA biosynthesis, catabolism, or both\u0026nbsp;(Ahanger et al. 2020). Therefore, the modulation of endogenous PA contents is relevant for shoot elongation in \u003cem\u003eD. nigra.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eComparative proteomics is an important tool for the comprehension of\u0026nbsp;physiological\u0026nbsp;and molecular processes during \u003cem\u003ein vitro\u003c/em\u003e morphogenesis,\u0026nbsp;as\u0026nbsp;it is possible to compare DAPs under different treatments\u0026nbsp;(Heringer et al. 2018). This approach was applied in the present work to comprehend the effects of BA and the type of explant (apical and cotyledonary nodal segments) (Fig. 4) on protein accumulation during shoot development in \u003cem\u003eD. nigra\u003c/em\u003e. The accumulation of some proteins was significantly affected by BA addition in\u0026nbsp;the\u0026nbsp;shoots of \u003cem\u003eD. nigra\u003c/em\u003e from both\u0026nbsp;types\u0026nbsp;of\u0026nbsp;explants\u0026nbsp;(Supplementary Table 1), such as the up-accumulation of\u0026nbsp;the\u0026nbsp;factor ATP-dependent RNA helicase DEAH7 (A0A444YGB9). This protein is involved in the expression of genes related to auxin-mediated development, such as the apical-basal standardization of embryonic development and vascular development in\u003cem\u003e\u0026nbsp;Arabidopsis\u0026nbsp;\u003c/em\u003e(Tsugeki and Terada 2015). Thus, the accumulation of this protein under BA treatment can improve shoot elongation in \u003cem\u003eD. nigra\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e probably interacting with auxin metabolism,\u0026nbsp;as this protein is related to auxin polar transport.\u0026nbsp;Auxin\u0026nbsp;polar transport is essential for cell elongation of the embryo scutellum owing to auxin-induced cell acidification and elongation to the plasma membrane,\u0026nbsp;enabling growth\u0026nbsp;(Chen et al. 2010).\u003c/p\u003e\n\u003cp\u003eCalreticulin\u0026nbsp;is a molecular calcium-binding chaperone that promotes folding, oligomeric assembly and quality control in the endoplasmic reticulum through the calreticulin/calnexin cycle. Calcium is an important stabilizing agent in the control of plant cell metabolism, playing a role in the structure and permeability of cell membranes, cell division and elongation, translocation of carbohydrates and nitrogen metabolism, presenting\u0026nbsp;a\u0026nbsp;direct effect on plant growth\u0026nbsp;(Ahmad et al. 2016). In this sense, the increase in the accumulation of calreticulin-3 (A0A445A993) protein BA\u0026nbsp;induced in shoots from both\u0026nbsp;types\u0026nbsp;of explants may be related\u0026nbsp;to\u0026nbsp;calcium and\u0026nbsp;other\u0026nbsp;compounds important for shoot elongation observed in \u003cem\u003eD. nigra.\u003c/em\u003e In addition, calreticulin proteins are able to transiently interact with almost all monoglucosylated glycoproteins necessary for the accumulation of elongation factor\u0026nbsp;receptors\u0026nbsp;(Ahmad et al. 2016).\u003c/p\u003e\n\u003cp\u003eIn the present work, the up-accumulation of elongation factor 1-alpha (A0A444Y7Y0) protein in shoots from both\u0026nbsp;types\u0026nbsp;of explants under BA treatment compared to shoots grown without BA (Supplementary Table 1) could be relevant for the best shoot development in \u003cem\u003eD. nigra\u003c/em\u003e. This protein works\u0026nbsp;as\u0026nbsp;a promoter of GTP-dependent binding of aminoacyl-tRNA to ribosome\u0026nbsp;sites\u0026nbsp;during protein biosynthesis, an important process\u0026nbsp;in\u0026nbsp;growth and development\u0026nbsp;(White et al. 2019). In addition, the up-accumulation of protein cell division cycle protein 48 homolog (A0A444Z3W1) in shoots from both\u0026nbsp;types\u0026nbsp;of explants could be relevant for the higher shoot elongation BA-induced in \u003cem\u003eD. nigra\u003c/em\u003e. This protein is\u0026nbsp;directly\u0026nbsp;related to cell division, cytokinesis and growth processes in plants\u0026nbsp;(Rancour et al. 2002).\u003c/p\u003e\n\u003cp\u003eNitrogen (N) is essential\u0026nbsp;to\u0026nbsp;carbon skeletons for the biosynthesis of the primary amino acids glutamine and glutamate, which serve as N donors for the biosynthesis of major N compounds in plants, including other amino acids, nucleic acid bases, PAs, and\u0026nbsp;chlorophyll\u0026nbsp;(de la Torre et al. 2014). The\u0026nbsp;aspartate aminotransferase protein is important\u0026nbsp;for\u0026nbsp;aspartate biosynthesis and plays a key role in the metabolic regulation of carbon and nitrogen metabolism in all organisms\u0026nbsp;(C\u0026aacute;novas et al. 2007). The induced gene silencing of aspartate aminotransferase in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e causes a\u0026nbsp;reduction in growth\u0026nbsp;and\u0026nbsp;chlorosis symptoms and\u0026nbsp;decreases\u0026nbsp;the levels of chlorophyll and lignin\u0026nbsp;(de la Torre et al. 2014). Moreover, aspartate aminotransferase activity was involved\u0026nbsp;in\u0026nbsp;biomass\u0026nbsp;increments\u0026nbsp;in \u003cem\u003eBrassica napus\u003c/em\u003e (McAllister et al. 2016). The up-accumulation of aspartate aminotransferase 1 (A0A445B4C1) protein in shoots from both\u0026nbsp;types\u0026nbsp;of\u0026nbsp;explants\u0026nbsp;grown under BA treatment (Supplementary Table 1) may be important for the\u0026nbsp;increase\u0026nbsp;in biomass due to the higher length of shoots in \u003cem\u003eD. nigra\u0026nbsp;\u003c/em\u003einduced by BA.\u003c/p\u003e\n\u003cp\u003eThe protein phosphoribosylamine-glycine ligase is associated with N\u0026nbsp;assimilation in bacterial nitrogen fixation\u0026nbsp;(Resendis-Antonio et al. 2011), and the accumulation of this protein (A0A445E7J0) in shoots from\u0026nbsp;BA-treated\u0026nbsp;cotyledonary nodal segments can promote the elongation of \u003cem\u003eD. nigra\u003c/em\u003e shoots,\u0026nbsp;altering nitrogen metabolism. In addition to nitrogen, carbohydrate metabolism is essential for energy supply in plants. The phosphoenolpyruvate carboxylase protein is an important cytosolic enzyme situated at a crucial branch point of plant carbohydrate metabolism\u0026nbsp;(Scholl et al. 2020). Phosphoenolpyruvate carboxylase 2 also\u0026nbsp;fulfils\u0026nbsp;essential\u0026nbsp;nonphotosynthetic\u0026nbsp;functions, particularly the replenishment of tricarboxylic acid (TCA) cycle intermediates consumed during biosynthesis and N\u0026nbsp;assimilation\u0026nbsp;(Scholl et al. 2020). Some developmental or metabolic\u0026nbsp;processes\u0026nbsp;that\u0026nbsp;require\u0026nbsp;these organic acids will benefit from increased carbon flux through the phosphoenolpyruvate carboxylase 2 reaction\u0026nbsp;(Willick et al. 2019). Thus, the up-accumulation of phosphoenolpyruvate carboxylase 2 (A0A445BXQ0) could be relevant for\u0026nbsp;the\u0026nbsp;elongation of shoots from\u0026nbsp;BA-treated\u0026nbsp;cotyledonary nodal segments compared to\u0026nbsp;those\u0026nbsp;without BA in \u003cem\u003eD. nigra\u0026nbsp;\u003c/em\u003e(Supplementary Table 1). Another up-accumulated protein in shoots from cotyledonary nodal explants incubated with BA compared to those without BA was the FT-interacting protein (A0A444ZYV6) (Supplementary Table 1). This protein\u0026nbsp;plays\u0026nbsp;an essential role in mediating\u0026nbsp;the\u0026nbsp;proliferation and differentiation of shoot stem cells in \u003cem\u003eArabidopsis\u003c/em\u003e (Liu et al. 2018). FT-interacting protein\u0026nbsp;prevents\u0026nbsp;intracellular trafficking of a key regulator, SHOOTMERISTEMLESS, to the plasma membrane in cells in the peripheral shoot meristem region. This facilitates SHOOTMERISTEMLESS recycling to the nucleus to maintain stem cells and accelerates stem cell differentiation\u0026nbsp;(Liu et al. 2018). In this sense, these proteins may be interesting to\u0026nbsp;shed\u0026nbsp;light on BA signaling for the promotion of higher shoot elongation in \u003cem\u003eD. nigra\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eIn addition, the dolichyl-diphosphooligosaccharide glycosyltransferase protein is known to be involved\u0026nbsp;in\u0026nbsp;protein glycosylation and protein modification and participates in biological processes relevant for plant growth and development, such as mechanisms controlling the assembly of cell wall polymers, protein N-linked glycosylation through asparagine and cell\u0026nbsp;growth\u0026nbsp;(Lerouxel et al. 2005). An increase in the accumulation of dolichyl-diphosphooligosaccharide-protein glycosyltransferase 48 kDa subunit (A0A445A8R7) protein in shoots from cotyledonary nodal segments BA-treated can be related to cytokinin promotion\u0026nbsp;of\u0026nbsp;the higher elongation of\u0026nbsp;shoots\u0026nbsp;in \u003cem\u003eD. nigra\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eCitrate\u0026nbsp;synthase mitochondrial (mtCS) (A0A445EVE9 and A0A445CF35) was\u0026nbsp;another\u0026nbsp;protein up-accumulated only in shoots from\u0026nbsp;BA-treated\u0026nbsp;cotyledonary nodal segments.\u0026nbsp;The citrate synthase mitochondrial (mtCS) protein has an enhanced ability to excrete citric acid,\u0026nbsp;and\u0026nbsp;the overexpression of mtCS in carrot cells results in better cell growth than\u0026nbsp;that in\u0026nbsp;wild-type cells\u0026nbsp;(Koyama et al. 1999). It appears that the overexpression of citrate synthase in \u003cem\u003eArabidopsis\u003c/em\u003e improves growth in phosphorus-limited soils due to the increased excretion of citrate from the roots\u0026nbsp;(Koyama et al. 2000).\u0026nbsp;This evidence\u0026nbsp;may suggest that the increase in\u0026nbsp;the\u0026nbsp;accumulation of citrate synthase mitochondrial (mtCS) (A0A445EVE9 and A0A445CF35) proteins in shoots from cotyledonary explants\u0026nbsp;treated with BA\u0026nbsp;can regulate oxidative metabolism, promoting the elongation of shoots in \u003cem\u003eD. nigra\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003eAnother\u0026nbsp;up-accumulated protein, the phosphoribosylamine-glycine ligase chloroplastic (A0A445E7J0), is involved\u0026nbsp;in\u0026nbsp;enzymes\u0026nbsp;in the \u003cem\u003ede novo\u0026nbsp;\u003c/em\u003epurine biosynthesis pathway\u0026nbsp;(Zhang et al. 2018). Plants can degrade purines,\u0026nbsp;and the final products glyoxylate and ammonia are recovered to synthesize organic molecules for new growth\u0026nbsp;(Amarante et al. 2006). The increase\u0026nbsp;in\u0026nbsp;accumulation of this protein\u0026nbsp;may be\u0026nbsp;involved\u0026nbsp;in the\u0026nbsp;biosynthesis of organic molecules necessary\u0026nbsp;for\u0026nbsp;the higher growth of \u003cem\u003eD. nigra\u003c/em\u003e shoots from cotyledonary nodal segments incubated with BA. The 60S ribosomal protein L35a-3 (A0A444ZT56) observed in \u003cem\u003eD. nigra\u003c/em\u003e shoots from cotyledonary nodal segments treated with BA (Supplementary Table 1) is a structural constituent of\u0026nbsp;ribosomes\u0026nbsp;and has\u0026nbsp;cytoplasmic translation and ribosomal large subunit biogenesis\u0026nbsp;as a biological function\u0026nbsp;(Xiao et al. 2019). The eukaryotic ribosome is a complex structure composed of several ribosomal RNAs and ribosomal proteins (r-proteins)\u0026nbsp;(Taylor et al. 2009), which are responsible for protein synthesis necessary for cell growth, division, and development\u0026nbsp;(Barakat et al. 2001). It has been shown that genetic defects in ribosomal components, such as\u0026nbsp;a\u0026nbsp;reduction\u0026nbsp;in the\u0026nbsp;levels of individual r-proteins, can induce deleterious effects on the development of plants\u0026nbsp;(Barakat et al. 2001). Thus, a higher accumulation of 60S ribosomal protein L35a-3 (A0A444ZT56) could be relevant to\u0026nbsp;maintaining\u0026nbsp;higher levels of r-proteins and,\u0026nbsp;consequently, higher elongation of \u003cem\u003eD. nigra\u003c/em\u003e shoots from cotyledonary nodal segments incubated with BA.\u003c/p\u003e\n\u003cp\u003eThe\u0026nbsp;bifunctional\u0026nbsp;dTDP-4-desidrorhamnose 3,5-epimerase/dTDP-4-desidrorhamnose reductase (A0A444Z945) protein was unique in \u003cem\u003eD. nigra\u003c/em\u003e shoots from\u0026nbsp;BA-treated\u0026nbsp;cotyledonary nodal segments compared to shoots without BA (BA2.5_Cotyledonary/BA0_Cotyledonary) and to shoots from\u0026nbsp;BA-treated\u0026nbsp;apical nodal segments (BA2.5_Cotyledonary/BA2.5_Apical).\u0026nbsp;This protein is involved in dTDP-L-rhamnose biosynthesis, which is part of carbohydrate metabolism\u0026nbsp;(Watt et al. 2004). The analysis of sugar composition and the study of gene expression at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under specific tissue regulation\u0026nbsp;(Martinez et al. 2012).\u0026nbsp;In addition, this protein is also present in the cell wall organization process, which can be an interesting factor associated with the differential elongation of \u003cem\u003eD. nigra\u003c/em\u003e shoots incubated with BA.\u003c/p\u003e\n\u003cp\u003eSome proteins identified were associated with the type of explant, being more accumulated in shoots from cotyledonary nodal segments compared to apical\u0026nbsp;segments, both with (BA2.5_Cotyledonary/BA2.5_apical comparison) or without BA (BA0_cotyledonary/BA0_apical comparison) (Supplementary Table 1). The malate dehydrogenase 2 protein\u0026nbsp;catalyzes\u0026nbsp;a reversible NAD-dependent dehydrogenase reaction involved in central metabolism and redox homeostasis between organelle compartments\u0026nbsp;(Tomaz et al. 2010)\u0026nbsp;and is also required for\u0026nbsp;the\u0026nbsp;maintenance of photosynthetic rates under photorespiratory conditions\u0026nbsp;(Cousins et al. 2008). ATP synthase subunit beta, chloroplastic (A0A445AH22),\u0026nbsp;can be found in the plasma membrane of eubacteria, in thylakoids of\u0026nbsp;chloroplasts\u0026nbsp;and in the inner mitochondrial membrane of eukaryotic cells\u0026nbsp;(Mulkidjanian et al. 2009).\u0026nbsp;Loss\u0026nbsp;of ATP synthase assembly defective in\u0026nbsp;the\u0026nbsp;\u0026beta; subunit results in mitochondria deprived of cristae structures,\u0026nbsp;and when ATP2\u0026nbsp;is\u0026nbsp;silenced,\u0026nbsp;cells show a peculiar organization of thylakoid stacks in the chloroplast with a reduced number of lamellae compared to wild-type,\u0026nbsp;harming plant development\u0026nbsp;(Lapaille et al. 2010). The up-accumulation of dehydrogenase 2 (peroxisome) (A0A445DRP9) and ATP synthase subunit beta\u0026nbsp;and\u0026nbsp;chloroplastic (A0A445AH22) in shoots of \u003cem\u003eD. nigra\u003c/em\u003e from cotyledonary nodal segments treated or not with BA shows that this type of explant better\u0026nbsp;regulates\u0026nbsp;redox homeostasis between organelle compartments and ATP biosynthesis, an essential process for growth.\u003c/p\u003e\n\u003cp\u003eRooting is a critical phase of \u003cem\u003ein vitro\u003c/em\u003e propagation,\u0026nbsp;and\u0026nbsp;overcoming\u0026nbsp;this phase can\u0026nbsp;ensure\u0026nbsp;the\u0026nbsp;success\u0026nbsp;of\u0026nbsp;the\u0026nbsp;process\u0026nbsp;(Zeng et al. 2019). Usually,\u0026nbsp;exogenous auxins are necessary to promote root induction in some species, as observed for \u003cem\u003eMalus domestica\u003c/em\u003e rootstocks\u0026nbsp;(Meng et al. 2019)\u0026nbsp;and \u003cem\u003ePopulus alba\u003c/em\u003e (Zeng et al. 2019). \u003cem\u003eDalbergia nigra\u003c/em\u003e has the possibility of propagation using the cutting method, however, reaching rates below 50%. In our work, \u003cem\u003eex vitro\u003c/em\u003e rooting was efficient for\u0026nbsp;plantlet\u0026nbsp;production using shoots from both\u0026nbsp;types\u0026nbsp;of explants, with no necessity of IBA use (Figs. 5a and 5b).\u0026nbsp;IBA was also not necessary for root induction\u0026nbsp;or the\u0026nbsp;number of\u0026nbsp;roots\u0026nbsp;in \u003cem\u003ePrunus persica\u0026nbsp;\u003c/em\u003eand \u003cem\u003ePrunus davidiana\u003c/em\u003e (Zhou et al. 2010).\u0026nbsp;Thus, it was possible to reach a rate higher than 80% rooting, showing better results when compared to the cutting technique used in the species.\u003c/p\u003e\n\u003cp\u003eMoreover, the balance between auxins and cytokinins is important for root induction\u0026nbsp;(Růžička et al. 2009; Jing and Strader 2019), and differences in rooting may occur due to the accumulation of cytokinins in plant tissues\u0026nbsp;(Da Costa et al. 2013). In the present work, a comparison between shoots multiplied in culture medium without (control) and with 2.5 \u0026micro;M BA was performed to analyze\u0026nbsp;whether\u0026nbsp;shoot multiplication in BA concentrations affects the induction of\u0026nbsp;roots. The use of 2.5 \u0026micro;M BA is essential for shoot elongation;\u0026nbsp;however,\u0026nbsp;the treatments with cytokinin significantly\u0026nbsp;affected\u0026nbsp;the root induction and number of roots in \u003cem\u003eD. nigra\u003c/em\u003e compared to\u0026nbsp;the\u0026nbsp;treatment without BA (Figs. 5c and 5d). In this way, we can infer that the balance between auxins and cytokinins influences \u003cem\u003eD. nigra\u0026nbsp;\u003c/em\u003eshoot rooting. This balance adjustment was also considered important in \u003cem\u003eCeropegia bulbosa\u003c/em\u003e, where different concentrations of cytokinin (BA) and auxins,\u0026nbsp;such as naphthalene acetic acid (NAA) and IBA,\u0026nbsp;were tested, demonstrating the importance of crosstalk among hormones\u0026nbsp;(Phulwaria et al. 2013). Unlike in \u003cem\u003eAlbizia lebbeck\u003c/em\u003e\u003cem\u003e,\u003c/em\u003e the use of 250 \u0026micro;M IBA promoted the largest number and longer length of roots from shoots grown under concentrations of the cytokinin thidiazuron (Perveen et al. 2013), showing that endogenous hormones present in the explant have an important role in plant organogenesis (Pal et al. 2012; Zeng et al. 2019). Thus, we can infer that the results obtained for rooting depend on the species and concentrations of PGRs for root induction, as well as those used in the shoot multiplication step.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe WPM culture medium promoted the best seedling growth. The addition of BA is necessary\u0026nbsp;for\u0026nbsp;longer shoot lengths for both types of explants. BA addition promoted an increase in endogenous Put content, which induced the higher growth of shoots in both explants. Some proteins involved\u0026nbsp;in\u0026nbsp;central metabolism, redox homeostasis, maintenance of photosynthetic rates and carbon flow during photorespiration conditions were differentially accumulated in shoots from cotyledonary nodal\u0026nbsp;explants\u0026nbsp;with and without BA and are important for the growth of these shoots. \u003cem\u003eEx vitro\u003c/em\u003e rooting of shoots can be performed without IBA in both types of explants. This work enabled the production of seedlings that were directed to an ecological reserve. Furthermore,\u0026nbsp;the\u0026nbsp;first results\u0026nbsp;demonstrated\u0026nbsp;the involvement of PAs and proteomic\u0026nbsp;profiles\u0026nbsp;in the development of \u003cem\u003eD. nigra\u003c/em\u003e shoots.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;benzyladenine\u003c/p\u003e\n\u003cp\u003eCDC48\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Cell division cycle protein 48 homolog\u003c/p\u003e\n\u003cp\u003eDAPs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Differentially accumulated proteins\u003c/p\u003e\n\u003cp\u003eDTT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Dithiothreitol\u003c/p\u003e\n\u003cp\u003eFDR \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;False discovery rate\u003c/p\u003e\n\u003cp\u003eIBA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Indole-3-butyric acid\u003c/p\u003e\n\u003cp\u003eMS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Murashige and Skoog\u003c/p\u003e\n\u003cp\u003emtCS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Citrate synthase mitochondrial\u003c/p\u003e\n\u003cp\u003ePA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Polyamines\u003c/p\u003e\n\u003cp\u003ePGRs \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Plant growth regulators\u003c/p\u003e\n\u003cp\u003ePut \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Putrescine\u003c/p\u003e\n\u003cp\u003eTCA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Trichloroacetic acid\u003c/p\u003e\n\u003cp\u003eSpd \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Spermidine\u003c/p\u003e\n\u003cp\u003eSpm \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Spermine\u003c/p\u003e\n\u003cp\u003eWPM \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Woody Plant Medium\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e The authors thank Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) and the Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ) for funding. This study was also financed in part by the Coordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior - Brazil (CAPES) - Finance Code 001. LSP, TRO, VPMA and KRS are thankful for the scholarship funding provided by FAPERJ.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e LPS and CSC conceived the study, designed the experiments and wrote the manuscript. LSP was responsible for the in vitro culture of shoots and ex vitro rooting experiments and performed the statistical analyses. LSP and VPMA were responsible for PA analyses. LSP, TRO and VS were responsible for the proteomic analyses. All the authors read and approved the final manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This research was supported by the Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico (CNPq) (444453/2014-8; 309303/2019-2) and the Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Amparo \u0026agrave; Pesquisa do Estado do Rio de Janeiro (FAPERJ) (E26/202.969/2016; E26/202.533/2019). This study was also financed in part by the\u003c/p\u003e\n\u003cp\u003eCoordena\u0026ccedil;\u0026atilde;o de Aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior\u0026mdash;Brazil (CAPES)\u0026mdash;Finance Code 001. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031999. All identified proteins are available in the supplementary material.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e PXD031999.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial interests\u003c/strong\u003e The authors declare they have no financial interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest/Competing interests\u003c/strong\u003e The authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAhanger MA, Aziz U, Alsahli A, Alyemeni MN, Ahmad P (2020) Combined kinetin and spermidine treatments ameliorate growth and photosynthetic inhibition in \u003cem\u003eVigna angularis\u003c/em\u003e by up-regulating antioxidant and nitrogen metabolism under cadmium stress. Biomolecules 10:147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom10010147\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAhmad N, Javed SB, Khan MI, Anis M (2013) Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of \u003cem\u003eVitex trifolia\u003c/em\u003e: an important medicinal plant. Acta Physiol Plant 35:2493\u0026ndash;2500. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11738-013-1285-y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAhmad P, Abdel Latef AA, Abd_Allah EF, Hashem A, Sarwat M, Anjum NA, Gucel S (2016) Calcium and potassium supplementation enhanced growth, osmolyte secondary metabolite production, and enzymatic antioxidant machinery in cadmium-exposed chickpea (\u003cem\u003eCicer arietinum\u003c/em\u003e L.). Front Plant Sci 7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2016.00513\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAmarante L, Lima JD, Sodek L (2006) Growth and stress conditions cause similar changes in xylem amino acids for different legume species. Environ Exp Bot 58:123\u0026ndash;129. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envexpbot.2005.07.002\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eArag\u0026atilde;o VPM, Navarro BV, Silva AT, Silveira V, Santa-Catarina C (2017a) Micropropagation of \u003cem\u003eCariniana legalis\u003c/em\u003e (Martius) O. Kuntze, an endangered hardwood tree from the Brazilian Atlantic Forest. Plant Cell Culture \u0026amp; Micropropagation 13:41\u0026ndash;50\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eArag\u0026atilde;o VPM, Reis RS, Silveira V, Santa-Catarina C (2017b) Putrescine promotes changes in the endogenous polyamine levels and proteomic profiles to regulate organogenesis in \u003cem\u003eCedrela fissilis\u003c/em\u003e Vellozo (Meliaceae). Plant Cell Tiss Org Cult 130:495\u0026ndash;505. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-017-1239-y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eArag\u0026atilde;o VPM, Ribeiro YRS, Reis RS, Macedo AF, Floh EIS, Silveira V, Santa-Catarina C (2016) In vitro organogenesis of \u003cem\u003eCedrela fissilis\u003c/em\u003e Vell. (Meliaceae): the involvement of endogenous polyamines and carbohydrates on shoot development. Plant Cell Tiss Org Cult 124:611\u0026ndash;620. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-015-0919-8\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBarakat A, Szick-Miranda K, Chang I-F, Guyot R, Blanc G, Cooke R, Delseny M, Bailey-Serres J (2001) The organization of cytoplasmic ribosomal protein genes in the \u003cem\u003eArabidopsis\u003c/em\u003e genome. Plant Physiol 127:398\u0026ndash;415. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.010265\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBaron K, Stasolla C (2008) The role of polyamines during in vivo and in vitro development. In Vitro Cell Dev Biol Plant 44:384\u0026ndash;395. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-008-9176-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eBunn E, Turner SR, Dixon KW (2011) Biotechnology for saving rare and threatened flora in a biodiversity hotspot. In Vitro Cell Dev Biol Plant 47:188\u0026ndash;200. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-011-9340-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eC\u0026aacute;novas FM, Avila C, Cant\u0026oacute;n FR, Ca\u0026ntilde;as RA, de la Torre F (2007) Ammonium assimilation and amino acid metabolism in conifers. J Exp Bot 58:2307\u0026ndash;2318. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erm051\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChen D, Ren Y, Deng Y, Zhao J (2010) Auxin polar transport is essential for the development of zygote and embryo in \u003cem\u003eNicotiana tabacum\u003c/em\u003e L. and correlated with ABP1 and PM H\u003csup\u003e+\u003c/sup\u003e-ATPase activities. J Exp Bot 61:1853\u0026ndash;1867. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/erq056\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eCousins AB, Pracharoenwattana I, Zhou W, Smith SM, Badger MR (2008) Peroxisomal malate dehydrogenase is not essential for photorespiration in \u003cem\u003eArabidopsis\u003c/em\u003e but its absence causes an increase in the stoichiometry of photorespiratory CO\u003csub\u003e2\u003c/sub\u003e release. Plant Physiol 148:786\u0026ndash;795. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.108.122622\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDa Costa C, De Almeida M, Ruedell C, Schwambach J, Maraschin F, Fett-Neto A (2013) When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings. Front Plant Sci 4. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2013.00133\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDamerval C, De Vienne D, Zivy M, Thiellement H (1986) Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat‐seedling proteins. Electrophoresis 7:52\u0026ndash;54\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ede la Torre F, Ca\u0026ntilde;as RA, Pascual MB, Avila C, C\u0026aacute;novas FM (2014) Plastidic aspartate aminotransferases and the biosynthesis of essential amino acids in plants. J Exp Bot 65:5527\u0026ndash;5534. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/eru240\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDias PC, Oliveira LS, Xavier A, Wendling I (2012) Estaquia e miniestaquia de esp\u0026eacute;cies florestais lenhosas do Brasil. Pesquisa Florestal Brasileira 32:453. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.4336/2012.pfb.32.72.453\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDistler U, Kuharev J, Navarro P, Levin Y, Schild H, Tenzer S (2014) Drift time-specific collision energies enable deep-coverage data-independent acquisition proteomics. Nat Meth 11:167\u0026ndash;170. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nmeth.2767\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eDutra NT, Silveira V, Azevedo IG, Gomes-Neto LR, Facanha AR, Steiner N, Guerra MP, Floh EIS, Santa-Catarina C (2013) Polyamines affect the cellular growth and structure of pro-embryogenic masses in \u003cem\u003eAraucaria angustifolia\u003c/em\u003e embryogenic cultures through the modulation of proton pump activities and endogenous levels of polyamines. Physiol Plant 148:121\u0026ndash;132. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-3054.2012.01695.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFermino-Junior PCP, Scherwinski-Pereira JE (2012) Germina\u0026ccedil;\u0026atilde;o e propaga\u0026ccedil;\u0026atilde;o in vitro de cerejeira (\u003cem\u003eAmburana acreana\u003c/em\u003e (Ducke) A.C.Smith - Fabaceae). Cienc Florest 22:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5902/198050985074\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eFoan CC, Othman RY (2006) In vitro direct shoot organogenesis and regeneration of plantlets from leaf explants of Sentang (\u003cem\u003eAzadirachta excelsa\u003c/em\u003e). Biotechnology 5:337\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3923/biotech.2006.337.340\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGhosh S, Pal A (2013) Proteomic analysis of cotyledonary explants during shoot organogenesis in \u003cem\u003eVigna radiata\u003c/em\u003e. Plant Cell Tiss Org Cult 115:55\u0026ndash;68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-013-0340-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGiri CC, Shyamkumar B, Anjaneyulu C (2004) Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees 18:115\u0026ndash;135. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00468-003-0287-6\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eGupta AK, Harish, Rai MK, Phulwaria M, Agarwal T, Shekhawat NS (2014) In vitro propagation, encapsulation, and genetic fidelity analysis of \u003cem\u003eTerminalia arjuna\u003c/em\u003e: a cardioprotective medicinal tree. Appl Biochem Biotechnol 173:1481\u0026ndash;1494. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12010-014-0920-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHazubska-Przybył T (2019) Propagation of juniper species by plant tissue culture: A mini-review. Forests 10:1028. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/f10111028\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHeringer AS, Santa-Catarina C, Silveira V (2018) Insights from proteomic studies into plant somatic embryogenesis. Proteomics 18:e1700265. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pmic.201700265\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eHussain SA, Ahmad N, Anis M (2018) Synergetic effect of TDZ and BA on minimizing the post-exposure effects on axillary shoot proliferation and assessment of genetic fidelity in \u003cem\u003eRauvolfia tetraphylla\u003c/em\u003e (L.). Rend Lincei Scienze Fis e Naturali 29:109\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12210-018-0667-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eIUCN (2021) The IUCN Red List of Threatened Species. Version 2021-3. International Union for Conservation of Nature. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.iucnredlist.org\u003c/span\u003e\u003c/span\u003e. Accessed 31 Jan 2021\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eJing H, Strader LC (2019) Interplay of auxin and cytokinin in lateral root development. Int J Mol Sci 20:486\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKodym A, Leeb CJ (2019) Back to the roots: protocol for the photoautotrophic micropropagation of medicinal Cannabis. Plant Cell Tiss Org Cult 138:399\u0026ndash;402. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-019-01635-1\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKoyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D (2000) Overexpression of mitochondrial citrate synthase in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e improved growth on a phosphorus-limited soil. Plant Cell Physiol 41:1030\u0026ndash;1037. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/pcp/pcd029\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKoyama H, Takita E, Kawamura A, Hara T, Shibata D (1999) Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium. Plant Cell Physiol 40:482\u0026ndash;488. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/oxfordjournals.pcp.a029568\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eKusano T, Berberich T, Tateda C, Takahashi Y (2008) Polyamines: essential factors for growth and survival. Planta 228:367\u0026ndash;381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-008-0772-7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLapaille M, Thiry M, Perez E, Gonz\u0026aacute;lez-Halphen D, Remacle C, Cardol P (2010) Loss of mitochondrial ATP synthase subunit beta (Atp2) alters mitochondrial and chloroplastic function and morphology in \u003cem\u003eChlamydomonas\u003c/em\u003e. Biochim et Biophys Acta (BBA) - Bioenergetics 1797:1533\u0026ndash;1539. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbabio.2010.04.013\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLerin J, Aragao VPM, Reis RS, Silveira V, Santa-Catarina C (2019) Proteomic profile and polyamine contents are modulated by light source to promote in vitro shoot development in \u003cem\u003eCariniana legalis\u003c/em\u003e (Martius) O.Kuntze (Lecythidaceae). Plant Cell Tiss Org Cult 137:329\u0026ndash;342. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-019-01574-x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLerouxel O, Mouille G, And\u0026egrave;me-Onzighi C, Bruyant M-P, S\u0026eacute;veno M, Loutelier-Bourhis C, Driouich A, H\u0026ouml;fte H, Lerouge P (2005) Mutants in DEFECTIVE GLYCOSYLATION, an \u003cem\u003eArabidopsis\u003c/em\u003e homolog of an oligosaccharyltransferase complex subunit, show protein underglycosylation and defects in cell differentiation and growth. Plant J 42:455\u0026ndash;468. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1365-313X.2005.02392.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLiu L, Li C, Song S, Teo ZWN, Shen L, Wang Y, Jackson D, Yu H (2018) FTIP-Dependent STM Trafficking Regulates Shoot Meristem Development in \u003cem\u003eArabidopsis\u003c/em\u003e. Cell Rep 23:1879\u0026ndash;1890. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.celrep.2018.04.033\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eLloyd G, McCown B (1981) Commercially-feasible micropropagation of mountain laurel, \u003cem\u003eKalmia latifolia\u003c/em\u003e, by use of shoot-tip culture. Combined Proceedings, International Plant Propagators\u0026apos; Society 30:421\u0026ndash;427\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMartinez V, Ingwers M, Smith J, Glushka J, Yang T, Bar-Peled M (2012) Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi \u003cem\u003eMagnaporthe grisea\u003c/em\u003e and \u003cem\u003eBotryotinia fuckeliana\u003c/em\u003e. J Biol Chem 287:879\u0026ndash;892. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M111.287367\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMcAllister CH, Wolansky M, Good AG (2016) The impact on nitrogen-efficient phenotypes when aspartate aminotransferase is expressed tissue-specifically in \u003cem\u003eBrassica napus\u003c/em\u003e. New Negatives in Plant Science 3\u0026ndash;4:1\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.neps.2016.03.001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMeng Y, Xing L, Li K, Wei Y, Wang H, Mao J, Dong F, Ma D, Zhang Z, Han M, Zhao C, Tahir MM, Zhang D (2019) Genome-wide identification, characterization and expression analysis of novel long non-coding RNAs that mediate IBA-induced adventitious root formation in apple rootstocks. Plant Growth Regul 87:287\u0026ndash;302. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10725-018-0470-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMitrović A, Jano\u0026scaron;ević D, Budimir S, Pristov JB (2012) Changes in antioxidative enzymes activities during \u003cem\u003eTacitus bellus\u003c/em\u003e direct shoot organogenesis. Biol Plant 56:357\u0026ndash;361\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMujib A (2005) In vitro regeneration of sandal (\u003cem\u003eSantalum album\u003c/em\u003e L.) from leaves. Turk J Bot 29:63\u0026ndash;67\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMulkidjanian AY, Galperin MY, Koonin EV (2009) Co-evolution of primordial membranes and membrane proteins. Trends Biochem Sci 34:206\u0026ndash;215. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tibs.2009.01.005\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eMurashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473\u0026ndash;497. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-3054.1962.tb08052.x\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eNanjo Y, Skultety L, Uvackova L, Klubicova K, Hajduch M, Komatsu S (2012) Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings. J Proteome Res 11:372\u0026ndash;385. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/pr200701y\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eOliveira TR, Arag\u0026atilde;o VPM, Moharana KC, Fedosejevs E, do Amaral FP, Sousa KR, Thelen JJ, Ven\u0026acirc;ncio TM, Silveira V, Santa-Catarina C (2020) Light spectra affect the in vitro shoot development of \u003cem\u003eCedrela fissilis\u003c/em\u003e Vell. (Meliaceae) by changing the protein profile and polyamine contents. Biochimica et Biophysica Acta (BBA) -. Proteins and Proteomics 1868:140529. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbapap.2020.140529\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePacurar DI, Perrone I, Bellini C (2014) Auxin is a central player in the hormone cross-talks that control adventitious rooting. Physiol Plant 151:83\u0026ndash;96. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/ppl.12171\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePal AK, Acharya K, Ahuja PS (2012) Endogenous auxin level is a critical determinant for in vitro adventitious shoot regeneration in potato (\u003cem\u003eSolanum tuberosum\u003c/em\u003e L.). J Plant Biochem Biotechnol 21:205\u0026ndash;212. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s13562-011-0092-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eParimalan R, Giridhar P, Ravishankar G (2011) Enhanced shoot organogenesis in \u003cem\u003eBixa orellana\u003c/em\u003e L. in the presence of putrescine and silver nitrate. Plant Cell Tiss Org Cult 105:285\u0026ndash;290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-010-9865-7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePassamani LZ, Bertolazi AA, Ramos AC, Santa-Catarina C, Thelen JJ, Silveira V (2018) Embryogenic competence acquisition in sugarcane callus is associated with differential H\u003csup\u003e+\u003c/sup\u003e pump abundance and activity. J Proteome Res 17:2767\u0026ndash;2779. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.jproteome.8b00213\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePatel AK, Phulwaria M, Rai MK, Gupta AK, Shekhawat S, Shekhawat NS (2014) In vitro propagation and ex vitro rooting of \u003cem\u003eCaralluma edulis\u003c/em\u003e (Edgew.) Benth. \u0026amp; Hook. f.: an endemic and endangered edible plant species of the Thar Desert. Sci Hortic 165:175\u0026ndash;180. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2013.10.039\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePerez-Riverol Y, Bai J, Bandla C, Garc\u0026iacute;a-Seisdedos D, Hewapathirana S, Kamatchinathan S, Kundu DJ, Prakash A, Frericks-Zipper A, Eisenacher M, Walzer M, Wang S, Brazma A, Vizca\u0026iacute;no JA (2022) The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res 50:D543\u0026ndash;d552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/nar/gkab1038\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePerveen S, Anis M, Aref IM (2013) In vitro plant regeneration of \u003cem\u003eAlbizia lebbeck\u003c/em\u003e (L.) from seed explants. For Syst 22:241\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.5424/fs/2013222-03261\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePhillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. In Vitro Cell Dev Biol Plant 55:242\u0026ndash;257. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-019-09983-5\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePhulwaria M, Shekhawat NS, Rathore JS, Singh RP (2013) An efficient in vitro regeneration and ex vitro rooting of \u003cem\u003eCeropegia bulbosa\u003c/em\u003e Roxb. - A threatened and pharmaceutical important plant of Indian Thar Desert. Ind Crops Prod 42:25\u0026ndash;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.indcrop.2012.05.013\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePieruzzi FP, Dias LLC, Balbuena TS, Santa-Catarina C, dos Santos ALW, Floh EIS (2011) Polyamines, IAA and ABA during germination in two recalcitrant seeds: \u003cem\u003eAraucaria angustifolia\u003c/em\u003e (Gymnosperm) and \u003cem\u003eOcotea odorifera\u003c/em\u003e (Angiosperm). Ann Bot 108:337\u0026ndash;345. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aob/mcr133\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePijut PM, Beasley RR, Lawson SS, Palla KJ, Stevens ME, Wang Y (2012) In vitro propagation of tropical hardwood tree species - a review (2001\u0026ndash;2011). Propag Ornam Plants 12:25\u0026ndash;51\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003ePijut PM, Woeste KE, Vengadesan G, Michler CH (2007) Technological advances in temperate hardwood tree improvement including breeding and molecular marker applications. In Vitro Cell Dev Biol Plant 43:283\u0026ndash;303. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-007-9026-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eR Core Team (2014) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRanaweera KK, Gunasekara MTK, Eeswara JP (2013) Ex vitro rooting: A low cost micropropagation technique for Tea (\u003cem\u003eCamellia sinensis\u003c/em\u003e (L.) O. Kuntz) hybrids. Sci Hortic 155:8\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.scienta.2013.03.001\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRancour DM, Dickey CE, Park S, Bednarek SY (2002) Characterization of AtCDC48. Evidence for multiple membrane fusion mechanisms at the plane of cell division in plants. Plant Physiol 130:1241\u0026ndash;1253. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.011742\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eReis RS, de Moura Vale E, Heringer AS, Santa-Catarina C, Silveira V (2016) Putrescine induces somatic embryo development and proteomic changes in embryogenic callus of sugarcane. J Proteom 130:170\u0026ndash;179. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jprot.2015.09.029\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eReis RS, Vale EM, Sousa KR, Santa-Catarina C, Silveira V (2021) Pretreatment free of 2,4-dichlorophenoxyacetic acid improves the differentiation of sugarcane somatic embryos by affecting the hormonal balance and the accumulation of reserves. Plant Cell Tiss Org Cult 145:101\u0026ndash;115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-020-01995-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eResendis-Antonio O, Hern\u0026aacute;ndez M, Salazar E, Contreras S, Batallar GM, Mora Y, Encarnaci\u0026oacute;n S (2011) Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling. BMC Syst Biol 5:120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1752-0509-5-120\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eRůžička K, \u0026Scaron;im\u0026aacute;\u0026scaron;kov\u0026aacute; M, Duclercq J, Petr\u0026aacute;\u0026scaron;ek J, Zaž\u0026iacute;malov\u0026aacute; E, Simon S, Friml J, Montagu MCEV, Benkov\u0026aacute; E (2009) Cytokinin regulates root meristem activity via modulation of the polar auxin transport. Proc Natl Acad Sci U S A 106:4284\u0026ndash;4289. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.0900060106\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSahai A, Shahzad A (2013) High frequency in vitro regeneration system for conservation of \u003cem\u003eColeus forskohlii\u003c/em\u003e: a threatened medicinal herb. Acta Physiol Plant 35:473\u0026ndash;481. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11738-012-1090-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSahu J, Khan S, Sahu RK, Roy A (2014) Micropropagation of \u003cem\u003eDalbergia sissoo\u003c/em\u003e Roxb. through tissue culture technique. Pak J Biol Sci 17:597\u0026ndash;600. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3923/pjbs.2014.597.600\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSanta-Catarina C, Maciel SdC, Pedrotti EL (2001) Germina\u0026ccedil;\u0026atilde;o in vitro e embriog\u0026ecirc;nese som\u0026aacute;tica a partir de embri\u0026otilde;es imaturos de canela sassafr\u0026aacute;s (\u003cem\u003eOcotea odorifera\u003c/em\u003e Mez). Rev Bras Bot 24:501\u0026ndash;510. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0100-84042001000500004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSanta-Catarina C, Silveira V, Balbuena TS, Viana AM, Estelita MEM, Handro W, Floh EIS (2006) IAA, ABA, polyamines and free amino acids associated with zygotic embryo development of \u003cem\u003eOcotea catharinensis\u003c/em\u003e. Plant Growth Regul 49:237\u0026ndash;247. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10725-006-9129-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eSanta-Catarina C, Silveira V, Scherer GFE, Floh EIS (2007) Polyamine and nitric oxide levels relate with morphogenetic evolution in somatic embryogenesis of \u003cem\u003eOcotea catharinensis\u003c/em\u003e. Plant Cell Tissue and Organ Culture 90:93\u0026ndash;101. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-007-9259-7\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eScholl J, Dengler L, Bader L, Forchhammer K (2020) Phosphoenolpyruvate carboxylase from the cyanobacterium \u003cem\u003eSynechocystis\u003c/em\u003e sp. PCC 6803 is under global metabolic control by PII signaling. Mol Microbiol 114:292\u0026ndash;307. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/mmi.14512\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eShukla S, Shukla SK, Mishra SK (2008) In vitro plant regeneration from seedling explants of \u003cem\u003eStereospermum personatum\u003c/em\u003e D.C.: a medicinal tree. Trees 23:409. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00468-008-0290-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eStein VC, Ferreira TR, Rossato M, Macedo BF, da Silva FF, Paiva R, Paiva LV (2017) Establishment and in vitro multiplication of \u003cem\u003eCalophyllum brasiliensis\u003c/em\u003e. Acta Hortic 1155:149\u0026ndash;156. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.17660/ActaHortic.2017.1155.20\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eStevens ME, Pijut PM (2018) Rapid in vitro shoot multiplication of the recalcitrant species \u003cem\u003eJuglans nigra\u003c/em\u003e L. Vitro Cell Dev Biol Plant 54:309\u0026ndash;317. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11627-018-9892-3\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eStuepp CA, Wendling I, Xavier A, Zuffellato-Ribas KC (2018) Vegetative propagation and application of clonal forestry in Brazilian native tree species. Pesqu Agropecu Bras 53:985\u0026ndash;1002. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1590/S0100-204X2018000900002\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTaylor DJ, Devkota B, Huang AD, Topf M, Narayanan E, Sali A, Harvey SC, Frank J (2009) Comprehensive molecular structure of the eukaryotic ribosome. Structure 17:1591\u0026ndash;1604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.str.2009.09.015\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTiburcio AF, Altabella T, Bitri\u0026aacute;n M, Alc\u0026aacute;zar R (2014) The roles of polyamines during the lifespan of plants: from development to stress. Planta 240:1\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-014-2055-9\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTomaz T, Bagard M, Pracharoenwattana I, Lind\u0026eacute;n P, Lee CP, Carroll AJ, Str\u0026ouml;her E, Smith SM, Gardestr\u0026ouml;m P, Millar AH (2010) Mitochondrial malate dehydrogenase lowers leaf respiration and alters photorespiration and plant growth in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Physiol 154:1143\u0026ndash;1157. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.110.161612\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eTsugeki R, Terada S (2015) The \u003cem\u003eArabidopsis\u003c/em\u003e ortholog of the DEAH-box ATPase Prp16 influences auxin-mediated development. Plant Signal Behav 10:e1074369. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15592324.2015.1074369\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWatt G, Leoff C, Harper AD, Bar-Peled M (2004) A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in \u003cem\u003eArabidopsis\u003c/em\u003e. Plant Physiol 134:1337\u0026ndash;1346. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.103.037192\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWhite JT, Cato T, Deramchi N, Gabunilas J, Roy KR, Wang C, Chanfreau GF, Clarke SG (2019) Protein methylation and translation: Role of lysine modification on the function of yeast elongation factor 1A. Biochemistry 58:4997\u0026ndash;5010. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acs.biochem.9b00818\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWillick IR, Plaxton WC, Lolle SJ, Macfie SM (2019) Transcriptional and post-translational upregulation of phosphoenolpyruvate carboxylase in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (L. Heynh) under cadmium stress. Environ Exp Bot 164:29\u0026ndash;39. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.envexpbot.2019.04.018\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eWybouw B, De Rybel B (2019) Cytokinin - A Developing Story. Trends Plant Sci 24:177\u0026ndash;185. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tplants.2018.10.012\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eXiao R, Li L, Ma Y (2019) A label-free proteomic approach differentiates between conventional and organic rice. J Food Compos Anal 80:51\u0026ndash;61. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jfca.2019.04.004\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eYan H, Liang C, Yang L, Li Y (2009) In vitro and ex vitro rooting of \u003cem\u003eSiratia grosvenorii\u003c/em\u003e, a traditional medicinal plant. Acta Physiol Plant 32:115. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11738-009-0386-0\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZeng Q, Han Z, Kang X (2019) Adventitious shoot regeneration from leaf, petiole and root explants in triploid (\u003cem\u003ePopulus alba\u003c/em\u003e \u0026times; \u003cem\u003eP. glandulosa\u003c/em\u003e)\u0026times; \u003cem\u003eP. tomentosa\u003c/em\u003e. Plant Cell Tiss Org Cult 138:121\u0026ndash;130. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-019-01608-4\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhang T, Feng P, Li Y, Yu P, Yu G, Sang X, Ling Y, Zeng X, Li Y, Huang J, Zhang T, Zhao F, Wang N, Zhang C, Yang Z, Wu R, He G (2018) VIRESCENT-ALBINO LEAF 1 regulates leaf colour development and cell division in rice. J Exp Bot 69:4791\u0026ndash;4804. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/jxb/ery250\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eZhou H, Li M, Zhao X, Fan X, Guo A (2010) Plant regeneration from in vitro leaves of the peach rootstock \u0026lsquo;Nemaguard\u0026rsquo; (\u003cem\u003ePrunus persica\u003c/em\u003e \u0026times; \u003cem\u003eP. davidiana\u003c/em\u003e). Plant Cell Tiss Org Cult 101:79\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11240-010-9666-z\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Adventitious rooting, Auxin, Cytokinin, In vitro propagation, Plant proteomics, Polyamines","lastPublishedDoi":"10.21203/rs.3.rs-1419531/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1419531/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eDalbergia nigra\u003c/em\u003e is an endangered species from the Brazilian Atlantic Rainforest, and \u003cem\u003ein vitro\u003c/em\u003e propagation can be applied for the conservation of this species. The aim of this study was to establish \u003cem\u003ein vitro\u003c/em\u003e propagation and ex vitro rooting in D. \u003cem\u003enigra\u003c/em\u003e and evaluate the alterations in polyamines (PAs) and protein profiles during shoot development\u003cem\u003e.\u003c/em\u003e The effect of MS and WPM culture media on \u003cem\u003ein vitro\u003c/em\u003e germination was tested (%). For shoot induction, explants of apical and cotyledonary nodal segments from 45-day-old seedlings were inoculated in WPM culture medium supplemented with benzyladenine (BA; 0, 2.5 and 5 μM). Shoots obtained \u003cem\u003ein vitro\u003c/em\u003e without and with 2.5 μM BA were rooted \u003cem\u003eex vitro\u003c/em\u003e with different concentrations (0, 100 and 500 μM) of indole-3-butyric acid (IBA). The best growth of seedlings was obtained in WPM culture medium. Treatment with 2.5 μM BA significantly increased the length of shoots by increasing free putrescine contents and the accumulation of proteins associated with shoot elongation, such as aspartate aminotransferase, elongation factor, calreticulin-3, and cell division cycle protein 48. Ex vitro rooting was obtained in all treatments of IBA, and the use of auxin was not necessary. The BA used for shoot multiplication significantly affected rooting, reducing the induction and number of roots but increasing the length of roots. This study showed the relevance of cytokinin, PAs and proteomic profiles on \u003cem\u003ein vitro\u003c/em\u003e shoot development, as well as the auxin and cytokinin balance on \u003cem\u003eex vitro\u003c/em\u003e rooting in \u003cem\u003eD. nigra.\u003c/em\u003e\u003c/p\u003e","manuscriptTitle":"Benzyladenine Effects On Polyamine Contents And Proteomic Profiles During In Vitro Shoot Development And On Ex Vitro Rooting In Dalbergia Nigra (Vell.) Allemão Ex Benth. (Fabaceae)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-03-16 15:05:53","doi":"10.21203/rs.3.rs-1419531/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2022-03-15T04:47:35+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-03-14T14:58:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-03-08T18:22:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2022-03-04T09:40:31+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"18f10473-ad87-4fda-9ede-64f29fac2c11","owner":[],"postedDate":"March 16th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-05-23T08:21:54+00:00","versionOfRecord":[],"versionCreatedAt":"2022-03-16 15:05:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1419531","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1419531","identity":"rs-1419531","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}