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.