Effects of Auxin (Indole-3-butyric Acid) on Adventitious Root Formation in Peach-Based Prunus Rootstocks

  Citation: Justamante, M.S.; Mhimdi, M.; Molina-Pérez, M.; Albacete, A.; Moreno, M.Á.; Mataix, I.; Pérez-Pérez, J.M. Effects of Auxin (Indole-3-butyric Acid) on Adventitious Root Formation in Peach-Based Prunus Rootstocks. Plants 2022,11, 913. https://doi.org/ 10.3390/plants11070913 Academic Editor: Giovanna Frugis Received: 9 March 2022 Accepted: 28 March 2022 Published: 29 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 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/). plants Article Effects of Auxin (Indole-3-butyric Acid) on Adventitious Root Formation in Peach-Based Prunus Rootstocks María Salud Justamante 1,†, Mariem Mhimdi 1,†, Marta Molina-Pérez 1, Alfonso Albacete 2,‡ , MaríaÁngeles Moreno 3, Inés Mataix 4and JoséManuel Pérez-Pérez 1,* 1 Instituto de Bioingeniería, Universidad Miguel Hernández, 03202 Elche, Spain; [email protected] (M.S.J.); [email protected] (M.M.); [email protected] (M.M.-P.) 2Departmento de Nutrición Vegetal, CEBAS-CSIC, 30100 Murcia, Spain; [email protected] 3Department of Pomology, Estación Experimental de Aula Dei-CSIC, 50059 Zaragoza, Spain; [email protected] 4Invisa Biotecnología Vegetal S.L., 30410 Caravaca de la Cruz, Spain; [email protected] *Correspondence: jmper[email protected]; Tel.: +34-966-658-958 † These authors contributed equally to this work. ‡ Present address: Departamento de Producción Vegetal y Agrotecnología, Instituto Murciano de Investigación y Desarrollo Agrario y Medioambiental (IMIDA), 30150 Alberca Las Torres, Spain. Abstract: Several Prunus species are among the most important cultivated stone fruits in the Mediterranean region, and there is an urgent need to obtain rootstocks with specific adaptations to challenging environmental conditions. The development of adventitious roots (ARs) is an evolutionary mechanism of high relevance for stress tolerance, which has led to the development of environmentally resilient plants. As a first step towards understanding the genetic determinants involved in AR formation in Prunus sp., we evaluated the rooting of hardwood cuttings from five Prunus rootstocks (Adafuel, Adarcias, Cadaman, Garnem, and GF 677) grown in hydroponics. We found that auxin-induced callus and rooting responses were strongly genotype-dependent. To investigate the molecular mechanisms involved in these differential responses, we performed a time-series study of AR formation in two rootstocks with contrasting rooting performance, Garnem and GF 677, by culturing in vitro microcuttings with and without auxin treatment (0.9 mg/L of indole-3-butyric acid [IBA]). Despite showing a similar histological structure, Garnem and GF677 rootstocks displayed dynamic changes in endogenous hormone homeostasis involving metabolites such as indole-3-acetic acid (IAA) conjugated to aspartic acid (IAA-Asp), and these changes could explain the differences observed during rooting. Keywords: vegetative propagation; Prunus rootstocks; hormone profiling; auxin homeostasis 1. Introduction The root system of plant species is of great importance for plant anchorage and the absorption of water and nutrients from the soil [ 1 ]. Adventitious rooting is a multifactorial response that leads to the formation of new roots from aerial organs and, if necessary, to the establishment of a complete and autonomous plant when the main root system is absent [ 2 ]. Wound-induced adventitious root (AR) formation is crucial for the clonal propagation of forest and horticultural species. Deprivation of the original root system interrupts the supply of water, nutrients, and plant hormones, such as cytokinins formed in the roots, which in turn leads to the accumulation of other downwardly transported metabolites, such as auxin, at the basal region of the stem near the wound [ 3 ]. In response to excision, a new developmental program is initiated at the base of the stem near the wound, ultimately leading to the generation of a new root system. The Prunus genus belongs to the Rosaceae family and includes approximately 200 species, some of which are commercially important, such as the cherry tree (Prunus cerasus L.), the Plants 2022,11, 913. https://doi.org/10.3390/plants11070913 https://www.mdpi.com/journal/plants Plants 2022,11, 913 2 of 18 peach tree (Prunus persica (L.) Batsch), or the almond tree (Prunus dulcis (Mill.) DA Webb, syn. Prunus amygdalus Batsch) [ 4 ]. The economic importance of this genus of stone fruit trees lies in the various uses of its species as a source of food and other resources such as wood or oil, in addition to its use as ornamental plants [4]. The commercial production of many species of the genus Prunus involves the use of appropriate rootstocks belonging to the same or other species of the same genus. The rootstocks constitute the main support of the shoots of the varieties of interest, being responsible for the absorption of water and nutrients, and also for providing resistance to soil pathogens, as well as tolerance to stressful environmental conditions [ 5 ]. In Spain, the economic importance of some Prunus species is unquestionable, placing this country as the first European producer of peach and nectarine with more than one million tons [ 6 , 7 ]. However, the cultivation of these plant species entails a series of shortcomings and problems that must be addressed to ensure the sustainability and economic profitability of the crops [ 6 ]. The presence of limestone soils in the Mediterranean region implies abiotic stresses that limit the cultivation of most stone fruit species. In these soils, iron chlorosis and root asphyxia predominate, both associated with tree mortality and nutrient deficiency [ 8 – 10 ]. In addition, the increasing salinization of soils and the scarce availability of water are other relevant problems that also acquire special relevance in the Mediterranean region [ 8 ]. Likewise, the climate change experienced in the last two decades had serious effects, highlighting an increase in summer temperatures and a shorter duration of the winter period, which results in the advancement of the flowering date and the consequent increase in the risk against spring frost [ 6 ]. Thus, the search for varieties with lower winter cold requirements and with flowering appropriate to the climatic zone of their cultivation are priority objectives in rootstock improvement programs in most European countries [8,11]. GF 677 is known to be the most widely used peach–almond hybrid in the Mediterranean region and shows greater adaptability to limestone and low nutrient soils, which are common in this region [ 10 , 11 ]. On the contrary, despite the high vigor of the Cadaman rootstock, it is known as a hard-to-root genotype [ 12 ]. Garnem is widely used as a rootstock due to its high adaptability in both irrigated and non-irrigated soils [ 13 ], although waterlogging conditions should be avoided [ 10 , 14 ]. Furthermore, previous results show that Garnem is easily propagated both as hardwood cuttings and in vitro grown microshoot cuttings [ 9 ]. In addition, other rootstocks such as Adafuel and Adarcias display high to moderate rooting capacity, respectively, as previously described [ 15 ], and are suitable for peach production in low nutrient calcareous soils [14,16]. Only two complete reference genomes are described in Prunus sp., the peach genome and the Japanese apricot genome [ 17 ]. In this sense, the use of new high-throughput sequencing methods will allow us to improve our understanding of the molecular networks that regulate the biology of this species, information that can be used in breeding programs [ 17 ]. In addition, the development of new, more effective auxins, as well as the identification of possible rooting cofactors (auxin synergists), are also critical areas of research, which will be key in the vegetative propagation of elite genotypes and the expansion of stone fruit species cultivation [18]. In this work, we studied the rooting performance of five Prunus rootstocks, in which the differences in rooting capacity under substrate conditions were confirmed. Since the study of rooting capacity under field conditions is difficult to carry out, we established an experimental design based on hydroponic culture, which allowed us to quantify the differences in regeneration among the studied genotypes. Based on the results obtained from this study, two genotypes with contrasting rooting behavior were selected for detailed in vitro phenotypic and hormonal analyses. 2. Results 2.1. Rooting Response in Five Prunus Rootstocks during Hydroponic Culture This project was initiated to evaluate the rooting performance of several Prunus rootstocks used for the mass production of grafted stone fruit trees: four peach–almond Plants 2022,11, 913 3 of 18 hybrids, Adafuel, Adarcias, Garnem, and GF 677, and one peach–Chinese wild peach hybrid, Cadaman (Table 1). The selected rootstocks represent a wide range of genetic backgrounds and showed, in previous studies, large differences in some traits related to rooting [ 11 , 16 , 19 ]. GF 677 and Cadaman were chosen due to their great use in Spain, since both rootstocks represented 71% of the total number of rootstocks used, with 50% and 21%, respectively [11]. Table 1. Prunus rootstocks used in this work. Rootstock Name Species Origin Adafuel P. dulcis x P. persica CSIC, Spain Adarcias P. dulcis x P. persica CSIC, Spain Cadaman P. persica x P. davidiana IFGO, Hungary and INRA, France Garnem P. dulcis x P. persica CITA, Spain GF 677 P. dulcis x P. persica INRA, France CSIC, Consejo Superior de Investigaciones Científicas. INRA, Institut National de la Recherche Agronomique. CITA: Centro de Investigación y Tecnología Agroalimentaria de Aragón. By conducting a small-scale trial, we confirmed that the rooting response of hardwood stem cuttings after auxin treatment and grown for 82 days in a commercial substrate under controlled environmental conditions was highly genotype-dependent (Figure S1a). For a detailed characterization of the contrasting rooting responses observed in hardwood cuttings from selected genotypes, we devised a hydroponic culture system. This system allowed us to periodically evaluate multiple parameters associated with effective rooting (Tables S1 and S2), such as the percentage of callus response or the number of ARs, which were measured at 32, 50, and 90 days after planting (dap). AR performance was also estimated by establishing different categories according to the number of ARs formed at 90 dap (Figure S1b), and a visual callus-stage scale was also established (Figure S1c). 2.1.1. Callus Formation and Rooting Efficiency Adafuel and Garnem were the first genotypes to initiate a regenerative response in the basal region of the stem, with 83.5% and 46.3% callus formation at 32 dap, respectively (Figure 1a). The earliest formation of ARs was observed in Garnem, with 6.0% of cuttings rooted at 32 dap (Figure 1a). At 90 dap, Adafuel reached the highest regenerative response, followed by Adarcias, Garnem, and GF 677 (Figure 1b). Adafuel and Garnem presented the highest percentage of rooted cuttings, with 70.0% and 67.4%, respectively. In contrast, Cadaman had the lowest regenerative response in terms of the callus and AR formation of the studied genotypes, and the percentage of rooted cuttings only reached 3.4% at 90 dap (Figure 1a,b). In addition to the regenerative response (which was highest in Adafuel), the effective rooting of hardwood cuttings was dependent on the development of many functional ARs. Garnem and GF 677 cuttings were the first genotypes to develop 10 or more ARs at 50 dap (Figure 1c). At the end of the experiment (Figure 1d), the rooted Garnem cuttings had the highest number of ARs (8.8 ± 1.2), followed by GF 677 (7.0 ± 1.8) and Adafuel (6.1 ± 0.5). Therefore, the adventitious rooting of hardwood cuttings was effectively evaluated under hydroponic conditions (Figures 1e and S1b), as both the regenerative responses and rooting capacity of the cuttings confirmed the contrasting characteristics of the studied genotypes under commercial conditions. 2.1.2. Correlations between Regenerative Responses We noticed that, in all cases, callus formation in the basal region of the cuttings preceded AR initiation. We measured the callus area at the end of the experiment from stored images, and the highest values were found in Adafuel (0.82 ± 0.05 cm 2 ), followed by Garnem (0.56 ± 0.06 cm 2 ), Adarcias (0.41 ± 0.03 cm 2 ), and Cadaman (0.21 ± 0.07 cm 2 ). GF 677 showed the lowest average callus area (0.19 ± 0.02 cm 2 ). For the rapid determination of callus growth, we established a visual classification of callus stages (Figure S1c). Callus Plants 2022,11, 913 4 of 18 stage was positively correlated with the measured callus area (p-value = 0.000, Pearson) (Figure S1d), confirming that the visual assignment of callus stage is a reliable parameter to qualitatively assess callus growth. Furthermore, we found no significant association (p-value > 0.05; Pearson) between the callus area, AR number, and maximum AR length (Figure S1d), suggesting the independent regulation of these regenerative responses. Plants 2022, 11, x FOR PEER REVIEW 4 of 18 Figure 1. Rooting performance of hardwood cuttings of five Prunus rootstocks during hydroponic growth. (a,b) The regenerative response was estimated by the percentage of cuttings with callus and adventitious roots (ARs) at (a) 32 days after planting (dap) and (b) 90 dap. The number of samples varies from 59 to 72. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. (c,d) Rooting capacity at (c) 50 and (d) 90 dap according to the indicated categories. Letters indicate significant differences (p-value < 0.01) for the average number of ARs between the genotypes studied. The number of samples varies from 7 to 56. (e) Representative images of the genotypes studied after 90 days of growth in hydroponic culture. Scale bar: 7.5 cm. 2.1.2. Correlations between Regenerative Responses We noticed that, in all cases, callus formation in the basal region of the cuttings preceded AR initiation. We measured the callus area at the end of the experiment from stored images, and the highest values were found in Adafuel (0.82 ± 0.05 cm2), followed by Garnem (0.56 ± 0.06 cm2), Adarcias (0.41 ± 0.03 cm2), and Cadaman (0.21 ± 0.07 cm2). GF 677 showed the lowest average callus area (0.19 ± 0.02 cm2). For the rapid determination of callus growth, we established a visual classification of callus stages (Figure S1c). Callus stage was positively correlated with the measured callus area (p-value = 0.000, Pearson) (Figure S1d), confirming that the visual assignment of callus stage is a reliable parameter to qualitatively assess callus growth. Furthermore, we found no significant association (pvalue > 0.05; Pearson) between the callus area, AR number, and maximum AR length (Figure S1d), suggesting the independent regulation of these regenerative responses. Due to the low rooting performance of Adarcias and Cadaman, these two rootstocks were discarded for further studies. Of the other three rootstocks, Garnem and GF 677 were Figure 1. Rooting performance of hardwood cuttings of five Prunus rootstocks during hydroponic growth. ( a , b ) The regenerative response was estimated by the percentage of cuttings with callus and adventitious roots (ARs) at ( a ) 32 days after planting (dap) and ( b ) 90 dap. The number of samples varies from 59 to 72. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. (c,d) Rooting capacity at ( c ) 50 and ( d ) 90 dap according to the indicated categories. Letters indicate significant differences (p-value < 0.01) for the average number of ARs between the genotypes studied. The number of samples varies from 7 to 56. ( e ) Representative images of the genotypes studied after 90 days of growth in hydroponic culture. Scale bar: 7.5 cm. Due to the low rooting performance of Adarcias and Cadaman, these two rootstocks were discarded for further studies. Of the other three rootstocks, Garnem and GF 677 were selected to be studied during in vitro culture since both showed significant differences in the percentage of AR response (p-value < 0.05; Chi-square) as well as in the AR number (p-value < 0.01). Plants 2022,11, 913 5 of 18 2.2. Regenerative Response of Garnem and GF 677 during In Vitro Rooting We performed two in vitro experiments (E1 and E2) with Garnem and GF 677 microcuttings provided by a private company (Figure S2a, see Section 4). They remained in the preincubation medium for 20 and 11 days, respectively. The imaging of explants at different time points (Figure S2b,c) allowed us to quantify different regeneration parameters. We measured the AR response and rooting capacity of the starting material (Figure S3a) and found that in both cases, Garnem showed higher rooting performance than GF 677 (Figure S3b,c). Furthermore, the rooting performance of the starting material was positively correlated with the time spent in the preincubation medium (Figure S3b,c). 2.2.1. Histology To understand the cellular events leading to the contrasting AR responses observed within the studied genotypes, we analyzed cross-sections of the basal region of the microcuttings. Some structural elements could be identified in these sections: pith, xylem, cambium, phloem, cortex, and epidermis (Figure 2a,d). The growth of the stem microcuttings in the preincubation medium caused the stems to initiate secondary growth. In fact, we observed regular organization in the cells of the epidermis, cortex, and pith, while the cambium cells were highly vacuolated and with defined nuclei. No striking anatomical differences were found between the two studied genotypes. Plants 2022, 11, x FOR PEER REVIEW 5 of 18 selected to be studied during in vitro culture since both showed significant differences in the percentage of AR response (p-value < 0.05; Chi-square) as well as in the AR number (p-value < 0.01). 2.2. Regenerative Response of Garnem and GF 677 during in vitro Rooting We performed two in vitro experiments (E1 and E2) with Garnem and GF 677 microcuttings provided by a private company (Figure S2a, see Section 4). They remained in the preincubation medium for 20 and 11 days, respectively. The imaging of explants at different time points (Figure S2b,c) allowed us to quantify different regeneration parameters. We measured the AR response and rooting capacity of the starting material (Figure S3a) and found that in both cases, Garnem showed higher rooting performance than GF 677 (Figure S3b,c). Furthermore, the rooting performance of the starting material was positively correlated with the time spent in the preincubation medium (Figure S3b,c). 2.2.1. Histology To understand the cellular events leading to the contrasting AR responses observed within the studied genotypes, we analyzed cross-sections of the basal region of the microcuttings. Some structural elements could be identified in these sections: pith, xylem, cambium, phloem, cortex, and epidermis (Figure 2a,d). The growth of the stem microcuttings in the preincubation medium caused the stems to initiate secondary growth. In fact, we observed regular organization in the cells of the epidermis, cortex, and pith, while the cambium cells were highly vacuolated and with defined nuclei. No striking anatomical differences were found between the two studied genotypes. Figure 2. Histological comparison between Garnem and GF 677 microcuttings. No structural differences were observed between Garnem (a,b) and GF 677 (c,d) tissue microcuttings before AR formation. Scale bar 200 µM (a,c) and 100 µM (b,d). Cx, cortex; Ph, phloem; Ca, cambium; Xy, xylem; P, pith; and Ep, epidermis. Figure 2. Histological comparison between Garnem and GF 677 microcuttings. No structural differences were observed between Garnem ( a , b ) and GF 677 ( c , d ) tissue microcuttings before AR formation. Scale bar 200 µ M ( a , c ) and 100 µ M ( b , d ). Cx, cortex; Ph, phloem; Ca, cambium; Xy, xylem; P, pith; and Ep, epidermis. 2.2.2. Auxin Treatment Improves Callus Formation and Enhances AR Number After we pooled all the data from E1 and E2, we performed a multivariate ANOVA to determine which factor(s) contributed the most to the observed variation (Table S3). No significant differences (p-value > 0.05) were found in the AR number at 15 days after excision (dae) with respect to the experiment (Figure S3d), container, or spatial position within the container (Table S3). These results indicate that, despite the differences in starting material, the timing of wounding activates the regeneration response similarly, so this point was defined as 0 dae. Plants 2022,11, 913 6 of 18 The regenerative response was periodically analyzed based on the percentage of callus and AR formation after the excision of the basal region of the microcuttings (Figure 3). Without exogenous auxin, both rootstocks were able to develop calluses at the base of the stem all through the wound, but callus emergence in Garnem arose earlier than in GF 677 (Figure 3a). Treatment with 0.9 mg/L of indole-3-butyric acid (IBA) had a significant effect in anticipating callus formation in Garnem and GF 677 (Figure 3a). A similar trend was observed for AR formation (Figure 3b) but with some delays in callus formation. In all cases, callus formation preceded AR formation, and the IBA-treatment anticipated AR formation in Garnem and GF 677. For each genotype and treatment, we estimated the time when half of the explants showed a regenerative response (i.e., producing callus or ARs), which we dubbed as the RR50 (Table 2). Table 2. RR50 values (expressed in dae) obtained for callus and AR emergence from E1 and E2. Genotype (Treatment) Callus Emergence AR Emergence Garnem (mock) 9.7 15.7 Garnem (IBA) 6.3 8.9 GF 677 (mock) >30 >30 GF 677 (IBA) 12.7 16.3 We found significant differences (p-value < 0.05; Chi-square) in callus and AR formation in terms of genotype and treatment (Figure 3c,d). At 9 dae, the highest percentage of callus formation was found in Garnem treated with IBA (81.3%), where about 60% of their explants developed ARs (Figure 3c). In both genotypes, the IBA-supplemented medium produced a significant increase (p-value < 0.05; Chi-square) in callus and AR formation, although GF 677 showed lower values for callus and AR formation than Garnem ( Figure S3a–e ). Interestingly, both IBA-treated GF 677 explants and non-treated Garnem explants displayed non-significant differences (p-value = 0.07; Chi-square) in callus and AR formation (Figure 3d), confirming that the addition of exogenous auxin can improve the poor rooting response of GF 677 microcuttings. IBA-supplemented medium also significantly (p-value = 0.0000; Chi-square) improved the regenerative response in Garnem, as almost all explants developed ARs at the end of the experiment (Figure 3d). The results corresponding to the rooting capacity at 30 dae are shown in Figure 3e. IBA-treated Garnem microcuttings developed eight or more roots per explant in more than half of them (65.7%). In this way, GF 677 treated with IBA reached a similar AR capacity than non-treated Garnem, while the number of ARs per microcutting increased significantly (p-value < 0.01) in Garnem treated with IBA (Figure 3e). Although the number of ARs in IBA-treated microcuttings increased relative to those grown in mock medium, the length of the ARs in the IBA-supplemented medium was much shorter (Figure 3f). 2.2.3. Improved Rooting Performance on Prunus Rootstocks by an IBA Pulse As it is well known that prolonged incubation with auxin can cause excessive callus formation, which could limit effective rooting [ 20 ], we tested a 24 h incubation treatment with 0.9 mg/L IBA in a third experiment (E3), hereinafter referred to as the IBA pulse (IBAp). We found that IBAp-treated microcuttings showed premature callus formation compared to microcuttings grown on medium without IBA (i.e., mock condition) (Video S1). In GF 677 in mock condition, callus formation began around 8 dae, while IBAp treatment induced earlier callus formation in GF 677, at a rate similar to untreated Garnem (Figure 4a). The IBAp treatment of Garnem also improved callus formation (Figure 4a), but to a similar degree as continuous IBA treatment (Figure 3a). Interestingly, IBAp treatment markedly decreased the initiation of AR formation in both genotypes (Figure 4b). In fact, AR formation in IBAp-treated microcuttings was observed at 7 dae in 38.1% and 16.7% of Garnem and GF 677, respectively (Video S2). Unlike the treatment with continuous IBA (Figure 3), the treatment with IBAp produced AR initiation in GF 677 even earlier than Garnem in mock conditions and, subsequently, the percentage of AR formation at 9 dae was significantly Plants 2022,11, 913 7 of 18 higher (p-value = 0.0000; Chi-square) in GF 677 IBAp-treated than in untreated Garnem (Figure 4c). However, at 20 dae there were no significant differences (p-value = 0.088; Chi-square) between Garnem in mock conditions and IBAp-treated GF 677 regarding the regenerative response of the microcuttings (Figure 4d). Therefore, IBAp treatment accelerated AR initiation by 6.0 days in Garmen and 11.9 days in GF 677, arising in both cases from previously established calluses (Table 3and Video S2). Plants 2022, 11, x FOR PEER REVIEW 7 of 18 Figure 3. Rooting performance of Garnem and GF 677 microcuttings grown in vitro. (a,b) Appearance of (a) callus and (b) ARs over time in microcuttings treated with and without auxin (0.9 mg/L IBA). Letters indicate significant differences (p-value < 0.05) at 30 days after excision (dae). (c,d) Regenerative response estimated by the percentage of cuttings with callus and ARs at (c) 9 days after excision (dae) and (d) 20 dae. The number of samples varies from 36 to 100. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. (e) Rooting capacity measured at 30 dae. Letters indicate significant differences (p-value < 0.01) for the average number of ARs. (f) Representative images of Garnem and GF 677 microcuttings at the end of the experiment. Scale bar: 2.5 cm. Figure 3. Rooting performance of Garnem and GF 677 microcuttings grown in vitro . ( a , b ) Appearance of ( a ) callus and ( b ) ARs over time in microcuttings treated with and without auxin (0.9 mg/L IBA). Letters indicate significant differences (p-value < 0.05) at 30 days after excision (dae). ( c , d ) Regenerative response estimated by the percentage of cuttings with callus and ARs at ( c ) 9 days after excision (dae) and ( d ) 20 dae. The number of samples varies from 36 to 100. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. ( e ) Rooting capacity measured at 30 dae. Letters indicate significant differences (p-value < 0.01) for the average number of ARs. ( f ) Representative images of Garnem and GF 677 microcuttings at the end of the experiment. Scale bar: 2.5 cm. Plants 2022,11, 913 8 of 18 Plants 2022, 11, x FOR PEER REVIEW 9 of 18 Taken together, these results indicate that a short pulse of IBA is sufficient to trigger AR initiation in Prunus microcuttings, shortening the time required under in vitro conditions. Figure 4. Rooting performance of Garnem and GF 677 microcuttings after a 24 h pulse of IBA. (a,b) Appearance of (a) callus and (b) ARs over time in microcuttings treated with and without a 24 h pulse of IBA 0.9 mg/L (IBAp). Letters indicate significant differences (p-value < 0.05) between categories. (c,d) Regenerative response estimated by the percentage of cuttings with callus and ARs at (c) 9 days after excision (dae) and (d) 20 dae. The number of samples varies from 11 to 48. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. (e) Rooting capacity measured at 20 dae. Letters indicate significant differences (p-value < 0.01) for the average number of ARs. The number of samples in the rooting capacity graph varies from 11 to 12. (f) Representative images of Garnem and GF 677 microcuttings at the end of the experiment. Scale bar: 2.5 cm. Figure 4. Rooting performance of Garnem and GF 677 microcuttings after a 24 h pulse of IBA. ( a , b ) Appearance of ( a ) callus and ( b ) ARs over time in microcuttings treated with and without a 24 h pulse of IBA 0.9 mg/L (IBAp). Letters indicate significant differences (p-value < 0.05) between categories. ( c , d ) Regenerative response estimated by the percentage of cuttings with callus and ARs at ( c ) 9 days after excision (dae) and ( d ) 20 dae. The number of samples varies from 11 to 48. Letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in callus regenerative response, and white letters indicate significant differences (p-value < 0.05; Chi-square) between genotypes in AR regenerative response. ( e ) Rooting capacity measured at 20 dae. Letters indicate significant differences (p-value < 0.01) for the average number of ARs. The number of samples in the rooting capacity graph varies from 11 to 12. ( f ) Representative images of Garnem and GF 677 microcuttings at the end of the experiment. Scale bar: 2.5 cm. Plants 2022,11, 913 9 of 18 Table 3. RR50 values (expressed in dae) obtained for callus and AR emergence from E3. Genotype (Treatment) Callus Emergence AR Emergence Garnem (mock) 7.6 13.5 Garnem (IBAp) 6.7 7.5 GF 677 (mock) >20 >20 GF 677 (IBAp) 11.9 8.1 Rooting capacity at the end of the experiment (20 dae) was significantly improved (p-value < 0.01) by IBAp in both genotypes (Figure 4e). About 50–60% of GF 677 and Garnem microcuttings produced more than five ARs on IBAp, and around 20% of Garmen microcuttings on IBAp had more than eight ARs (Figure 4e). Unlike what was found in continuous IBA incubation where ARs were short (Figure 3f), IBAp-treated ARs were able to elongate to a similar degree as untreated microcuttings (Figure 4f). We then compared the rooting performance of the different treatments at 20 dae by studying the number and the maximum length of the ARs (Figure S4a,b). These results confirmed that IBAp had a positive effect on effective rooting in both genotypes. On the one hand, GF 677 IBAp displayed a significant increase (p-value < 0.01) both in the number of ARs (3.6 ± 0.6) and in the maximum AR length (66.7 ± 3.7 mm) compared to those found in mock condition (0.8 ± 0.4 ARs and 24.2 ± 7.3 mm) (Figure S4a,b). On the other hand, although Garnem produced more ARs in IBA (8.1 ± 0.45 ARs) than in IBAp ( 5.7 ±0.7 ARs ), their ARs were longer in IBAp conditions (62.5 ± 5.4 mm) (Figure S4b). In fact, IBAp treatment produced higher shoot growth of both genotypes compared to untreated or continuous IBA conditions (Figure S4b,c), confirming the better performance of the AR system. We wondered if IBAp could trigger AR formation after a short incubation under in vitro conditions. In a further trial, microcuttings were maintained for 96 h under in vitro conditions after IBAp treatment and then transferred to substrate conditions for about three weeks. We found significant differences (p-value < 0.05, Chi-square) in plant survival and shoot growth due to IBAp treatment (Figure S5a,b). At the end of the experiment, the number of ARs was significantly higher (p-value < 0.05) in Garnem and GF 677 microcuttings that were treated with IBAp, compared to the untreated ones (Figure S5b). Furthermore, the AR system was highly developed in IBAp-treated microcuttings (Figure S5c,d). Taken together, these results indicate that a short pulse of IBA is sufficient to trigger AR initiation in Prunus microcuttings, shortening the time required under in vitro conditions. 2.2.4. Hormone Profiling of Garnem and GF 677 Microcuttings during IBA-Induced Rooting Next, we measured the endogenous levels of 17 hormone metabolites in the basal region of microcuttings at 0, 4, and 8 dae (see Section 4). For most of them, we found non-significant differences (p-value > 0.05) in their endogenous levels regarding time and replicates (Table S4). Indole-3-acetic acid (IAA) levels were similar in Garnem and GF 677 at excision time (0 dae) and significantly increased (p-value = 0.000) with IBA treatment in both genotypes and at similar levels (Figure 5a). In addition, the levels of IAA conjugated with isoleucine (IAA-Ile) and with aspartic acid (IAA-Asp) were slightly increased in GF 677 with respect to those found in Garnem at 0 dae (Figures 5b and S6a). In all cases, IBA treatments significantly (p-value < 0.005) increased the endogenous levels of inactive IAA derivatives, such as IAA-Ile or IAA-Asp, as well as those of methyl-IAA (MeIAA) (Figures 5b and S6a,b). Several bioactive cytokinins were measured: trans-zeatin (tZ), cis-zeatin (cZ), dihydrozeatin (DHZ), and N6-( ∆ 2-Isopentenyl)adenine (iP) (Table S4). The levels of tZ, cZ, and DHZ in the basal region of the microcuttings were not significantly different (p-value > 0.05) between Garnem and GF 677 at the time of excision (0 dae), which were otherwise significantly (p-value < 0.01) increased by IBA treatment, either by continuous or pulsed IBA incubation (Figures 5c and S6c,d). These results indicate that exogenous IBA mainly induced the accumulation of tZ and cZ in the basal region of the microcuttings. On the other hand, iP levels were low and mostly unchanged in Plants 2022,11, 913 16 of 18 5. Conclusions The application of exogenous auxin led to an improvement in the rooting performance of the genotypes studied. While continuous IBA exposure promoted callus formation, a 24 h IBA pulse (IBAp) promoted highly efficient AR formation in Prunus microcuttings. IBAp treatment was easily applied, and the effect was proven to be reproducible in our experiments; therefore, it is highly recommended for commercial in vitro propagation strategies. An integrative approach based on selective transcriptome profiling will allow us to identify the key regulatory pathways involved in AR performance in Prunus species, which could then be used to breed rootstocks with enhanced rooting ability to increase production under challenging environmental conditions. Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/plants11070913/s1, Figure S1: Regenerative response of studied Prunus rootstocks and correlation among different regeneration parameters, Figure S2: Adventitious rooting evaluation in Prunus at in vitro culture, Figure S3: AR formation in Garnem and GF 677 microcuttings, Figure S4: AR formation in Prunus microcuttings with different treatments, Figure S5: Ex vitro acclimation of Prunus microcuttings after IBAp treatment, Figure S6: Hormonal profiling of Garnem and GF 677 microcuttings during IBA-induced rooting (II), Table S1: Regenerative parameters studied in this work, Table S2: Raw data of hydroponic assay, Table S3: Raw data of in vitro assays, Table S4: Raw data of hormone profiling, Video S1: E1 and E2 regenerative response at different time points, Video S2: E3 regenerative response at different time points. Author Contributions: Conceptualization, J.M.P.-P.; methodology, J.M.P.-P. and M.S.J.; validation, M.S.J. and M.M.; formal analysis, M.S.J., M.M., M.M.-P. and A.A.; investigation, M.S.J., M.M., A.A. and M.M.-P.; resources, J.M.P.-P., I.M. and M.Á.M.; data curation, M.S.J. and M.M.; writing—original draft preparation, J.M.P.-P., M.S.J. and M.M.; writing—review and editing, J.M.P.-P., M.Á.M. and A.A.; supervision J.M.P.-P.; project administration, J.M.P.-P.; funding acquisition, J.M.P.-P. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. M.M. is a research fellow of the Generalitat Valenciana (GRISOLIAP/2019/098). Data Availability Statement: Data supporting reported results can be found in the Supplementary Materials. Acknowledgments: We would like to thank María Gomáriz Clemente (SATDI-UMH) for her technical support. Conflicts of Interest: The authors declare no conflict of interest. References 1. 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