Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía)

plants Article Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía) Óscar Martínez 1, Verónica Arjones 1, María Victoria González 2and Manuel Rey 1,*   Citation: Martínez, Ó.; Arjones, V.; González, M.V.; Rey, M. Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía). Plants 2021,10, 1164. https://doi.org/10.3390/ plants10061164 Academic Editors: Itziar A. Montalbán and Paloma Moncaleán Received: 15 May 2021 Accepted: 4 June 2021 Published: 8 June 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Departamento de Biología Vegetal y Ciencia del Suelo, Universidad de Vigo, Campus Universitario, 36310 Vigo, Spain; [email protected] (Ó.M.); arjonesgonzalezver[email protected] (V.A.) 2Departamento de Biología Funcional, Universidad de Santiago de Compostela, Campus Sur, 15872 Santiago de Compostela, Spain; [email protected] *Correspondence: mr[email protected]; Tel.: +34-986812596 Abstract: The low induction rates of somatic embryogenesis are one of the main limitations in its routine application in the grapevine (Vitis vinifera L.). The use of an induction medium containing histone deacetylase inhibitors (trichostatin A and, mainly, sodium butyrate) resulted in an improvement of the embryogenic responses in grapevine (cv. Mencía) cotyledonary and recently germinated somatic embryos. The relative expression of several grapevine genes related to embryogenic competence or encoding histone deacetylase enzymes was studied in cotyledonary somatic embryos that were cultured in the presence of 0.5 mM sodium butyrate. The results showed a significant overexpression of the BBM and VvSERK2 genes after 24 h of culture, whereas the VvWOX2 gene was underexpressed less in treated versus untreated explants. The results suggest that the inhibitor may trigger a molecular response related to an increase in embryogenic competence and changes in the expression of associated genes. The treatment with sodium butyrate also produced significant variations in the expression of several histone deacetylase enzyme-encoding genes. These results may enhance the possibility of obtaining somatic embryos, reducing the seasonal constraints associated with the use of floral explants in grapevines. Keywords: somatic embryogenesis; Vitis vinifera; gene expression; sodium butyrate; trichostatin A; BABY BOOM;SERK2; histone deacetylase-encoding genes 1. Introduction Somatic embryogenesis is the physiological process by which somatic cells, under appropriate induction conditions, undergo a redifferentiation process and acquire embryogenic cell competences. Subsequently, these cells undergo a series of morphological and biochemical changes that culminate in the formation of somatic embryos and the generation of new plants [ 1 ]. Somatic embryogenesis is a powerful biotechnological tool that is particularly useful in species such as the grapevine. In this species, genetic improvement by traditional methods is inefficient due to factors such as severe inbreeding depression, a long-life cycle with late sexual maturity (which makes breeding tedious and time consuming), extreme heterozygosity, sex incompatibility (which makes the transfer of traits among Vitis species impossible due to their different numbers of chromosomes), and the difficult development of new cultivars due to consumer preferences for wines from specific cultivars [2]. The application of somatic embryogenesis to grapevines began in the late 1970s [ 2 , 3 ], and since then, the number of protocols available for different cultivars has increased exponentially [ 3 ]. Thus, the efficiency of the induction of somatic embryogenesis depends Plants 2021,10, 1164. https://doi.org/10.3390/plants10061164 https://www.mdpi.com/journal/plants Plants 2021,10, 1164 2 of 19 mainly on the interaction of the genotype, the culture medium, the explant used, and the degree of development of the explant itself [4–6]. The most successful explants for inducing somatic embryogenesis in grapevines are reproductive structures such as anthers [ 6 ], ovaries [ 6 , 7 ], stigmas [ 8 ], stamen filaments [ 9 , 10 ] or whole flowers [ 11 ]. In contrast, the acquisition of embryogenic competence using vegetative structures such as leaves, petioles, and nodal explants has been achieved on a few occasions and with low induction rates [ 2 , 3 , 12 ]. The practical implication of this phenomenon is that the establishment of new embryogenic crops is mostly restricted to the grapevine’s flowering period only, when the reproductive structures are available at the appropriate stage of development (just one week per year). For this reason, the establishment of a methodology to obtain somatic embryos from other grapevine tissues represents a key step in optimizing the use of somatic embryogenesis in this species [13]. However, although some of the genes involved in the acquisition of embryogenic competence have been identified, the exact mechanism that regulates the whole process is not clear, preventing the application of this technique on a routine basis. One of the alternatives to increase the embryogenic capacity of recalcitrant tissues is the modification of the expression of genes related to embryogenic competence, such as BABY BOOM (BBM), Somatic Embryogenesis Receptor Kinase (SERK), Leafy Cotyledon (LEC), or Wuschel (WUS)-related homeobox (WOX). The ectopic expression of the SERK1 and the BBM genes has been shown to increase the efficiency of the initiation of somatic embryogenesis in Arabidopsis [ 14 , 15 ], while the overexpression of the WUS gene in different tissues of this species (roots, petioles, stems, and leaves) induces the formation of somatic embryos [ 16 ]. Furthermore, the pattern of expression of the LEC1 and LEC2 genes has been observed to be similar to that of WUS during the somatic embryogenesis of Arabidopsis [ 17 ], suggesting that the LEC genes are also involved in this process. In grapevines, Schellenbaum et al. [ 18 ] characterized three SERK genes and analyzed the putative existence of LEC genes; they found a single sequence (VvL1L) showing the characteristic domains of the Leafy cotyledon1-Like (L1L) proteins of Arabidopsis. The VvAP2- 16 gene, belonging to the apetala2 (AP2) family, has been identified as a putative BBM gene in grapevines [ 19 ]. A total of 12 WOX genes have been characterized in grapevines, which were named VvWUS and VvWOXn, according to their similarity with the sequences already described in Arabidopsis [20]. Recently, it was discovered that reversible changes in histone acetylation play an essential role in the regulation of gene expression during plant regeneration (recently reviewed by the authors in [ 21 , 22 ]). In general terms, the acetylation of histone lysine residues produces a relaxation of the chromatin structure, and this phenomenon is associated with increased gene activity [ 23 , 24 ]. In contrast, the elimination of these acetyl groups leads to a compaction of chromatin, often related to repression and gene silencing [ 25 ]. The balance between histone acetylation and deacetylation is controlled by the activity of histone acetyltransferases (HATs) and histone deacetyltransferases (HDACs), respectively [ 26 ]. The number of coding genes for these enzymes is high, and there are several gene families for each group of enzymes. Thus, two categories of HATs have been characterized according to their cellular distribution: type A, which is responsible for acetylation at the nuclear level, and type B, which catalyzes the acetylation of histone H4 in the cytoplasm. In the case of HDACs, the HD2 family (which is exclusive to plants), the sirtuin family (SIR2), and the RPD3/HDA1 superfamily have been described [27]. In grapevines, a total of 7 HAT enzyme-encoding genes and 13 HDAC-encoding genes have been identified [ 27 ]. Of these 13 HDAC genes, one belongs to the HD2 family and has been named HDT in grapevines; two belong to the sirtuin family and have been named SRT; and the remaining 10 are part of the RPD3/HDA1 superfamily and have been named HDA. Treatment with HDAC inhibitors has been shown to increase histone acetylation [ 28 ] and influence many physiological processes [ 29 ]. Furthermore, these inhibitors have been shown to partially arrest the progression of germination, thus maintaining the embryogenic Plants 2021,10, 1164 3 of 19 potential of Pinus and spruce embryos [ 30 ] and increasing the embryogenic response in Brassica gametes [ 31 ]. Trichostatin A (TSA) and sodium butyrate (NaB) are the most widely used histone deacetylase inhibitors in plants [30–38]. In this work, we studied the effect of the inhibitors of histone deacetylases, NaB and TSA, on the embryogenic response of different grapevine explants derived from embryogenic cultures of the cv. Mencía [ 10 , 39 ]. We have also tried to better understand the role that HDAC inhibitors play in the embryogenic response in grapevine explants. With that goal, we analyzed the expression of several embryogenesis-related and HDAC- encoding grapevine genes. Since it has been suggested that the gene response to HDAC inhibitors occurs during the first hours of their application [ 32 , 40 , 41 ], we studied the expression of those genes in cotyledonary somatic embryos that were cultured for 24 h and 48 h in the presence of NaB. 2. Results 2.1. Effect of Histone Deacetylase Inhibitors on the Embryogenic Potential of Different Grapevine Explants Four different explants (cotyledonary somatic embryos, recently germinated somatic embryos, and shoot apices and leaves from both in vitro -grown plants) obtained from cultures of somatic embryo aggregates of Vitis vinifera L. cv. Mencía, were cultured in an induction medium [ 39 ] and supplemented with different concentrations of NaB (0.5, 2 or 5 mM) or TSA (0.5, 2, or 5 µ M). In all of these explants, three types of responses were observed. The first was the maintenance of their original morphology with progressive necrosis (Figure 1A). The second response was the formation of a non-embryogenic callus that was yellowish in color with some whitish areas and a watery appearance (Figure 1B). The third response was the formation of proembryos on the surface of the explants (Figure 1C), except in leaves. The formation of proembryos on the shoot apices was occasionally observed only in the presence of 0.5 mM NaB and 0.5 µ M TSA (data not recorded), but these proembryos were not able to proliferate further and finally became necrotic and died. The proembryos originating from cotyledonary or recently germinated somatic embryos proliferated rapidly through secondary embryogenesis, leading to the formation of small groups of somatic embryo aggregates (Figure 1D). These aggregates developed normally using previously described procedures [ 10 ] and allowed the regeneration of plantlets without phenotypic alterations (Figure 1E). Histological analysis revealed that the non-embryogenic callus had an irregular structure, with cells of different sizes and shapes (Figure 2A), low proliferative activity, as revealed by their low-density cytoplasm (Figure 2C), and a low number of nuclei (Figure 2E). In contrast, the somatic embryo aggregates presented a more uniform structure, with a higher number of cells of high proliferative activity, as revealed by their smaller size (Figure 2B), dense cytoplasm (Figure 2D), and bright nuclei mainly at the periphery of those structures (Figure 2F), where the formation of proembryos could be observed (Figure 2D). The effect of NaB and TSA on the embryogenic response in cotyledonary somatic embryos (Figure 3) and recently germinated (Figure 4) somatic embryos was recorded after four and eight weeks of culture. The highest percentages of embryogenic response were obtained using cotyledonary somatic embryos in which somatic embryos were newly formed on their surface after 4 weeks of culture in all media tested, except in the presence of 5 mM NaB (Figure 3A), in which no response was observed. Of all the HDAC inhibitor treatments, the best results were obtained with 0.5 mM NaB (15%), although this was not significantly different from the results obtained with the control medium. In all cases, the results obtained in the presence of TSA were very low. The embryogenic response generally increased in cotyledonary somatic embryos after eight weeks of culture (Figure 3B) in the presence of HDAC inhibitors. The highest variation between the fourth and eighth weeks of culture was observed in the presence of 2 mM NaB, in which the embryogenic response increased from 2.4% to 10.3%. The best results were obtained again for the cotyledonary somatic embryos that were cultured in the presence of 0.5 mM NaB, with the percentage (30% on average) being significantly higher Plants 2021,10, 1164 4 of 19 than that of the control and all other inhibitor treatments. Additionally, no response was observed in the presence of 5 mM NaB. Plants 2021, 10, x FOR PEER REVIEW 4 of 21 Figure 1. Responses observed in grapevine cv. Mencía cotyledonary somatic embryos. (A) Cotyledonary somatic embryo with no response and signs of necrosis after eight weeks of culture in an induction medium with 5 mM NaB. (B) Nonembryogenic callus originating from cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors for eight weeks. (C) Proembryos formed on the surface of cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. (D) Somatic embryo aggregate formed by secondary embryogenesis of proembryos formed from cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. (E) Grapevine microplant regenerated from somatic embryos induced in cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB. Bars: 1 mm (A,B); 0.5 mm (C,D); 1 cm (E). Histological analysis revealed that the non-embryogenic callus had an irregular structure, with cells of different sizes and shapes (Figure 2A), low proliferative activity, as revealed by their low-density cytoplasm (Figure 2C), and a low number of nuclei (Figure 2E). In contrast, the somatic embryo aggregates presented a more uniform structure, with a higher number of cells of high proliferative activity, as revealed by their smaller size (Figure 2B), dense cytoplasm (Figure 2D), and bright nuclei mainly at the periphery of those structures (Figure 2F), where the formation of proembryos could be observed (Figure 2D). Figure 1. Responses observed in grapevine cv. Mencía cotyledonary somatic embryos. ( A ) Cotyledonary somatic embryo with no response and signs of necrosis after eight weeks of culture in an induction medium with 5 mM NaB. ( B ) Nonembryogenic callus originating from cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors for eight weeks. ( C ) Proembryos formed on the surface of cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. ( D ) Somatic embryo aggregate formed by secondary embryogenesis of proembryos formed from cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. ( E ) Grapevine microplant regenerated from somatic embryos induced in cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB. Bars: 1 mm (A,B); 0.5 mm (C,D); 1 cm (E). In the recently germinated somatic embryos, the embryogenic response after four weeks of culture (Figure 4A) was generally lower than that described above in the cotyledonary somatic embryos (Figure 3A). In this culture period, the recently germinated somatic embryos showed no embryogenic response in the control medium without inhibitors, and the best results (5.6%) were obtained in the presence of the highest NaB concentration (5 mM). In addition, an embryogenic response was also obtained with this kind of explant in the presence of 2 mM NaB, 0.5 µ M TSA, and 5 µ M TSA. After eight weeks of culture, the embryogenic response generally increased in the recently germinated somatic embryos (Figure 4B). The highest increase in the embryogenic response was observed in the presence of 2 mM NaB or 0.5 mM NaB. In addition, the response in the control medium without inhibitors was remarkable, although the embryos had no response after four weeks of culture (Figure 4A). No statistically significant differences between the treatments were observed in the response of the recently germinated somatic embryos. Plants 2021,10, 1164 5 of 19 Plants 2021, 10, x FOR PEER REVIEW 5 of 21 Figure 2. Histological analysis of non-embryogenic calli (A,C,E) and somatic embryo aggregates (B,D,F) formed from grapevine cv. Mencía cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors or supplemented with 0.5 mM NaB. Sections were stained with calcofluor (A,B), toluidine blue (C,D), or DAPI (E,F). Bars: 200 μm. The effect of NaB and TSA on the embryogenic response in cotyledonary somatic embryos (Figure 3) and recently germinated (Figure 4) somatic embryos was recorded after four and eight weeks of culture. The highest percentages of embryogenic response were obtained using cotyledonary somatic embryos in which somatic embryos were newly formed on their surface after 4 weeks of culture in all media tested, except in the presence of 5 mM NaB (Figure 3A), in which no response was observed. Of all the HDAC inhibitor treatments, the best results were obtained with 0.5 mM NaB (15%), although this was not significantly different from the results obtained with the control medium. In all cases, the results obtained in the presence of TSA were very low. Figure 2. Histological analysis of non-embryogenic calli ( A , C , E ) and somatic embryo aggregates ( B , D , F ) formed from grapevine cv. Mencía cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors or supplemented with 0.5 mM NaB. Sections were stained with calcofluor (A,B), toluidine blue (C,D), or DAPI (E,F). Bars: 200 µm. Necrosis in the cotyledonary somatic embryos increased with time spent in the culture and due to the use of HDAC inhibitors (Table 1). In general, necrosis was more frequent in the media supplemented with NaB than with TSA and was significantly higher in the cotyledonary somatic embryos that were cultured in the presence of 2 mM NaB, both after four and eight weeks of culture. However, the use of 0.5 mM NaB, which showed the best embryogenic response in the cotyledonary somatic embryos (Figure 3), produced the lowest necrosis percentages of all the NaB treatments. The recently germinated somatic embryos showed a lower necrosis response than cotyledonary embryos (Table 1), although the percentage of necrotic explants also increased from four to eight weeks of culture. The recently germinated somatic embryos that were cultured in the presence of 0.5 or 2 mM NaB, which showed the highest embryogenic response (Figure 4B), also showed the lowest necrosis rate after eight weeks of culture (Table 1). The most significant and highest necrosis rate in the recently germinated somatic embryos was observed after eight weeks of culture in the presence of 5 mM NaB; this necrosis rate was similar to that observed in the cotyledonary somatic embryos during the same culture period in the same medium. Plants 2021,10, 1164 6 of 19 Plants 2021, 10, x FOR PEER REVIEW 6 of 21 Figure 3. Effect of HDAC inhibitors (NaB and TSA) on embryogenic responses in grapevine cv. Mencía cotyledonary somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after (A) 4 and (B) 8 weeks of culture is shown. Different letters indicate statistically significant differences (p < 0.05). Figure 3. Effect of HDAC inhibitors (NaB and TSA) on embryogenic responses in grapevine cv. Mencía cotyledonary somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after ( A ) 4 and ( B ) 8 weeks of culture is shown. Different letters indicate statistically significant differences (p< 0.05). Plants 2021, 10, x FOR PEER REVIEW 7 of 21 Figure 4. Effect of HDAC inhibitors (NaB and TSA) on the embryogenic response in grapevine cv. Mencía recently germinated somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after (A) 4 and (B) 8 weeks of culture is shown. There were no statistically significant differences between any of the treatments (p < 0.05). The embryogenic response generally increased in cotyledonary somatic embryos after eight weeks of culture (Figure 3B) in the presence of HDAC inhibitors. The highest variation between the fourth and eighth weeks of culture was observed in the presence of 2 mM NaB, in which the embryogenic response increased from 2.4% to 10.3%. The best results were obtained again for the cotyledonary somatic embryos that were cultured in the presence of 0.5 mM NaB, with the percentage (30% on average) being significantly higher than that of the control and all other inhibitor treatments. Additionally, no response was observed in the presence of 5 mM NaB. In the recently germinated somatic embryos, the embryogenic response after four weeks of culture (Figure 4A) was generally lower than that described above in the cotyledonary somatic embryos (Figure 3A). In this culture period, the recently germinated somatic embryos showed no embryogenic response in the control medium without inhibitors, and the best results (5.6%) were obtained in the presence of the highest NaB concentration (5 mM). In addition, an embryogenic response was also obtained with this kind of explant in the presence of 2 mM NaB, 0.5 μM TSA, and 5 μM TSA. After eight weeks of culture, the embryogenic response generally increased in the recently germinated somatic embryos (Figure 4B). The highest increase in the embryogenic response was observed in the presence of 2 mM NaB or 0.5 mM NaB. In addition, the response in the control medium without inhibitors was remarkable, although the embryos had no response after four weeks of culture (Figure 4A). No statistically significant differences between the treatments were observed in the response of the recently germinated somatic embryos. Necrosis in the cotyledonary somatic embryos increased with time spent in the culture and due to the use of HDAC inhibitors (Table 1). In general, necrosis was more frequent in the media supplemented with NaB than with TSA and was significantly higher in the cotyledonary somatic embryos that were cultured in the presence of 2 mM NaB, both after four and eight weeks of culture. However, the use of 0.5 mM NaB, which Figure 4. Effect of HDAC inhibitors (NaB and TSA) on the embryogenic response in grapevine cv. Mencía recently germinated somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after ( A ) 4 and ( B ) 8 weeks of culture is shown. There were no statistically significant differences between any of the treatments (p< 0.05). Plants 2021,10, 1164 7 of 19 Table 1. Percentage of necrosis in grapevine (cv. Mencía) cotyledonary and recently germinated somatic embryos that were cultured in an induction medium supplemented with different concentrations of HDAC enzyme inhibitors (NaB and TSA) or without them (control). Means and standard error are shown. Different letters indicate statistically significant differences (p< 0.05). Inhibitor Treatments Cotyledonary Somatic Embryos Recently Germinated Somatic Embryos Necrosis (%) after 4 Weeks Necrosis (%) after 8 Weeks Necrosis (%) after 4 Weeks Necrosis (%) after 8 Weeks Control 7.50 ±3.66 a 16.07 ±8.38 a 0.00 ±0.00 a 27.50 ±11.06 a 0.5 mM NaB 26.00 ±9.09 ab 40.00 ±9.89 ab 0.00 ±0.00 a 0.00 ±0.00 a 2 mM NaB 72.72 ±8.07 c 83.83 ±6.05 c 0.00 ±0.00 a 5.00 ±3.33 a 5 mM NaB 38.89 ±5.79 b 55.08 ±1.89 b 16.67 ±9.32 a 58.33 ±11.79 b 0.5 µM TSA 2.00 ±1.33 a 29.00 ±6.79 ab 7.50 ±5.34 a 20.00 ±7.26 a 2µM TSA 7.00 ±2.60 a 29.00 ±6.74 ab 7.50 ±5.34 a 17.50 ±9.17 a 5µM TSA 25.00 ±7.03 ab 56.00 ±7.48 b 17.50 ±3.82 a 25.00 ±6.45 a 2.2. Effect of the Histone Deacetylase Inhibitor NaB on the Expression of Somatic Embryogenesis- Related Genes and HDAC-Encoding Genes in Grapevine Cotyledonary Somatic Embryos Due to the positive effect of the induction medium with 0.5 mM NaB on the recovery of the embryogenic responses of the cotyledonary somatic embryos (Figure 3), we studied the effect of this treatment on the expression of the genes related to embryogenic competence (BBM,VvSERK1,VvSERK2,VvL1L,VvWUS, and VvWOX2) in these explants. In addition, the expression of several genes encoding grapevine histone deacetylase enzymes (HDT1, SRT1,SRT2,HDA1,HDA2,HDA3,HDA4,HDA6,HDA7-9, HDA8,HDA10, and HDA11) was also studied to investigate whether the inhibition of histone deacetylase enzymes had any effect. After 24 h of culture, the BBM and VvSERK2 genes, related to embryogenic competence, were significantly overexpressed in the cotyledonary somatic embryos that were cultured in the presence of 0.5 mM NaB (Figure 5A). Although the expression of the BBM gene was not significantly different in the cotyledonary somatic embryos treated with NaB compared to those that were untreated, the expression of VvSERK2 and VvWOX2 was different. These two genes were significantly less underexpressed in the cotyledonary somatic embryos that were cultured in the presence of 0.5 mM NaB (Figure 5A). On the other hand, the VvSERK1 and VvWUS genes were significantly underexpressed in the cotyledonary somatic embryos after 24 h of culture in the induction medium with or without 0.5 mM NaB. After 48 h of culture, BBM was the only gene to be significantly overexpressed in the cotyledonary somatic embryos, with a higher relative expression level after 24 h of culture. The VvSERK1 and VvWOX2 genes remained significantly underexpressed. VvWOX2 was less underexpressed in the presence of 0.5 mM NaB (Figure 5B). On the other hand, the VvSERK2 gene was not overexpressed after 48 h in the presence of NaB, whereas the VvWUS gene was not underexpressed. No differences between the treatments with or without NaB were observed for any gene. The results obtained for the HDAC-encoding genes showed that after 24 h of culture of the cotyledonary somatic embryos in NaB and NaB-free media (Figure 6A), the HDT1 and HDA2 genes were significantly overexpressed in those embryos that were cultured in the presence of 0.5 mM NaB. In addition, these genes were differentially and significantly expressed with respect to the embryos that were cultured in the induction medium without NaB, in which their expression did not change during the first 24 h of culture. On the other hand, the SRT1,SRT2, and HDA4 genes were significantly underexpressed in the cotyledonary somatic embryos that were cultured for 24 h in the control medium without NaB (Figure 6A). Their expression was significantly different from that observed in the cotyledonary somatic embryos that were cultured in the presence of 0.5 mM NaB, in which their expression did not change during the first 24 h of culture. During this culture period, the HDA3 and HDA8 genes were significantly underexpressed in the cotyledonary somatic embryos that were cultured with or without 0.5 mM NaB, whereas Plants 2021,10, 1164 8 of 19 the HDA11 gene was significantly underexpressed in the embryos that were cultured only in the presence of 0.5 mM NaB. After 48 h of culture, the number of genes whose expression was different between the treatments with or without NaB (Figure 6B) was less than that after 24 h of culture (Figure 6A). Of the genes whose expression was significantly different in the NaB treatment with respect to the control, only the SRT1,HDA2, and HDA4 genes remained the same. In addition, the HDA8 gene expression was significantly different between the treatments with or without NaB after 48 h of culture (Figure 6B). This is because this gene was not underexpressed in the embryos that were cultured in the presence of 0.5 mM NaB. The HDA1 gene was the only gene whose expression significantly changed in the cotyledonary somatic embryos after 48 h of culture in the presence of 0.5 mM NaB (Figure 6B), in which it was overexpressed. The expression did not change in the embryos that were cultured without NaB. Plants 2021, 10, x FOR PEER REVIEW 10 of 21 . Figure 5. Relative expression of genes related to somatic embryogenesis in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for 24 (A) and 48 (B) h. The mean values of two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p < 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while red squares framing genes indicate statistically significant differences (p < 0.05) between the NaB and no NaB treatments. Figure 5. Relative expression of genes related to somatic embryogenesis in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for 24 ( A ) and 48 ( B ) h. The mean values of two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p< 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while red squares framing genes indicate statistically significant differences (p< 0.05) between the NaB and no NaB treatments. Plants 2021,10, 1164 9 of 19 Plants 2021, 10, x FOR PEER REVIEW 11 of 21 Figure 6. Relative expression of HDAC-encoding genes in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for (A) 24 and (B) 48 h. The mean values of the two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p < 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while the red squares framing genes indicate statistically significant differences (p < 0.05) between the NaB and no NaB treatments. 3. Discussion Since the development of the first somatic embryogenesis protocols in grapevines [3], almost all have been based on the use of reproductive structures as explants. Less commonly, somatic embryos have also been obtained from tissues derived from vegetative structures, such as leaves and petioles [5,42–46], tendrils [47], or stem nodal explants [12,48]. The best induction results from vegetative tissues have been obtained from nodal segments [12,48], with efficiencies of up to 10–20% using an induction medium containing unusually high concentrations of auxins and cytokinins [12]. In this work, the highest rate of embryogenic response after 8 weeks of culture was obtained with cotyledonary somatic embryos and recently germinated somatic embryos. Although apices and leaves did not show any stable embryogenic response, the formation of proembryos was observed in shoot apices that were cultured in an induction medium supplemented with 0.5 mM NaB or 0.5 μM TSA, yet the proembryos did not proliferate and finally became necrotic. However, their formation provides the first evidence that HDAC inhibitors could be useful in the induction of embryogenesis in this type of explant. These results confirm the importance of the degree of tissue differentiation in terms of embryogenic competence. Although it is often assumed that plant cells are totipotent, this assertion has only been possible to demonstrate in certain cells, mostly belonging to juvenile or poorly differentiated tissues [49]. Thus, the initiation of the embryogenic pathway is restricted to a small number of cells that have the potential to activate genes related to somatic embryogenesis [1]. In grapevines, this phenomenon results in the rates of the Figure 6. Relative expression of HDAC-encoding genes in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for ( A ) 24 and ( B ) 48 h. The mean values of the two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p< 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while the red squares framing genes indicate statistically significant differences (p< 0.05) between the NaB and no NaB treatments. 3. Discussion Since the development of the first somatic embryogenesis protocols in grapevines [ 3 ], almost all have been based on the use of reproductive structures as explants. Less commonly, somatic embryos have also been obtained from tissues derived from vegetative structures, such as leaves and petioles [ 5 , 42 – 46 ], tendrils [ 47 ], or stem nodal explants [ 12 , 48 ]. The best induction results from vegetative tissues have been obtained from nodal segments [ 12 , 48 ], with efficiencies of up to 10–20% using an induction medium containing unusually high concentrations of auxins and cytokinins [12]. In this work, the highest rate of embryogenic response after 8 weeks of culture was obtained with cotyledonary somatic embryos and recently germinated somatic embryos. Although apices and leaves did not show any stable embryogenic response, the formation of proembryos was observed in shoot apices that were cultured in an induction medium supplemented with 0.5 mM NaB or 0.5 µ M TSA, yet the proembryos did not proliferate and finally became necrotic. However, their formation provides the first evidence that HDAC inhibitors could be useful in the induction of embryogenesis in this type of explant. These results confirm the importance of the degree of tissue differentiation in terms of embryogenic competence. 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