Impaginato 239 Adv. Hort. Sci., 2022 36(3): 239­245 DOI: 10.36253/ahsc­12192 Contrasting aspects of the physical and physiological dormancy by seeds from four peach rootstocks R.D. Menegatti 1, M.A. Lima 1, A.G. Souza 2 (*), O.J. Smiderle 3, V.J. Bianchi 1 1 Federal University of Pelotas, Capão do Leão, Brazil. 2 Centro University of Maringá, Maringá, Brazil. 3 Brazilian Agricultural Research Corporation Embrapa Roraima, Boa Vista, Brazil. Key words: Pre­germination treatment, Prunus persica, seed propagation. Abstract: Considering that cultivars of peach rootstocks selected to be propa­ gated by seeds show variation in the degree of physiological dormancy and peculiarities regarding the limitation imposed by the endocarp, it is essential to define the cold stratification period and the ideal temperature to be used dur­ ing the pre­germination treatment. Isolated for each cultivar, knowledge of these variables which will provide viability in the production of peach root­ stocks via seeds, in the presence of the endocarp, at a larger scale. Two germi­ nation tests were carried out, in a completely randomized experimental design and 4 x 3 factorial scheme, with four replications, each one consisting of 25 endocarps. The first experiment was performed with four cultivars (Aldrighi, Capdebosq, Okinawa Roxo and Tsukuba 1) and three stratification tempera­ tures (1°C, 4°C and 7°C) for a period of 90 days. For the second experiment, the same cultivars were used, two temperatures (7°C for ‘Aldrighi’ and ‘Capdebosq’ and 4°C for ‘Okinawa Roxo’ and ‘Tsukuba’) and three stratification periods (40, 80 and 120 days), followed by sowing in substrate under greenhouse conditions for a period of 45 days, with subsequent endocarp breaking and re­sowing. The pre­germination treatment at 7°C for 90 days is sufficient to obtain high germi­ nation percentage of ‘Aldrighi’ and ‘Capdeboscq’ seeds in the presence of the endocarp. Under the stratification conditions tested, the seeds of ‘Okinawa Roxo’ and ‘Tsukuba 1’ require rupture of the endocarp to reactivate the germi­ native embryonic process. 1. Introduction The production of peach rootstocks [Prunus persica (L.) Batsch] is tra­ ditionally done by seed, being the simplest and the most inexpensive method when compared to clonal propagation (Martins et al., 2014; Fischer et al., 2016). Despite the fact that stone fruit production is very important in Brazil, the country still presents relatively low orchard aver­ age productivity, mainly in Rio Grande do Sul state (Fischer et al., 2016). Factors associated with this low productivity include pest incidence, dis­ (*) Corresponding author: alineufla@hotmail.com Citation: MENEGATTI R.D., LIMA M.A., SOUZA A.G., SMI­ DERLE O.J., BIANCHI V.J., 2022 ­ Contrasting aspects of the physical and physiological dor‐ mancy by seeds from four peach rootstocks. ­ Adv. Hort. Sci., 36(3): 239­245. Copyright: © 2022 Menegatti R.D., Lima M.A., Souza A.G., Smiderle O.J., Bianchi V.J. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/ index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the origi­ nal author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 18 October 2021 Accepted for publication 29 June 2022 AHS Advances in Horticultural Science Short note https://doi.org/10.36253/ahsc-12192 http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2022 36(3): 239­245 240 eases, climate and seedling quality, mainly in the form of rootstock production; in general, they are obtained from seeds discarded by the canning indus­ try and, therefore, with no genetic identity (Souza et al., 2018). Currently, at Federal University of Pelotas and Embrapa Temperate Climate, a wide range of root­ stock cultivars for peach seedling production are available to growers (Souza et al., 2018; Menegatti et al., 2019 a; Souza et al., 2019 a; Menegatti et al., 2020). However, peach seeds of different genotypes exhibit considerable divergence in germination rate as a result of the variability in the degree of physical and physiological dormancy of the seeds, constituting a limitation for commercial seedling production of the specie (Souza et al., 2017; Menegatti et al., 2019 b). Souza et al. (2017) and Souza et al. (2019 b) sug­ gest that the combination of physical and physiologi­ cal dormancy presented by seeds of temperate cli­ mate species, such as peach, may be one of the main factors responsible for the variation in the germina­ tion percentage of rootstocks seeds. This variation requires the exposure of seeds to different combina­ tions of time and low temperatures to overcome dor­ mancy and, consequently, to the maximizing and homogeneity of germination. According to Souza et al. (2016) the overcoming of this dormancy can be achieved through techniques that promote the rup­ ture of the endocarp, popularly known as the stone. Endocarp removal, suggested as a method for obtain­ ing high germination percentages in peach seeds (Souza et al., 2017), is an operation that results in an additional cost for the nurseryman, mainly due to the high cost of the procedure, which must be performed manually and individually in order not to compromise the physical integrity of seed structures (Wagner Júnior et al., 2013). Considering that cultivars of peach rootstocks selected to be propagated by seeds show variations in the degree of physiological dormancy and peculiar­ ities regarding the limitation imposed by the endo­ carp, it is essential to define the cold stratification period and the ideal temperature to be used during the pre­germination treatment, isolated for each cul­ tivar (cv.), which will provide viability in the produc­ tion of peach rootstocks via seeds, in the presence of the endocarp, at a large scale. Therefore, this study aimed to evaluate contrasting aspects of physical and physiological dormancy shown by seeds of different peach rootstock cultivars. 2. Materials and Methods The present study was carried out with plant material obtained from ripe fruits harvested from clonal matrix plants of peach cvs. Aldrighi, Capdeboscq, Okinawa Roxo and Tsukuba 1, from Federal University of Pelotas Peach rootstock Germplasm Collection. Two different experiments were performed, both referring to germination tests. In the first one, the endocarps were packed in tulle bags and placed in plastic boxes (40 x 27 x 10 cm) and covered with fine vermiculite, previously and periodi­ cally moistened with distilled water and fungicide solution (Benlate 500 ­ 2 g L­1). Then the plastic boxes were placed in Biochemical Oxygen Demand (BOD), in different temperatures, where they remained for 90 days in cold weather for stratification, with four cvs. (Aldrighi, Capdebosq, Okinawa Roxo and Tsukuba 1) and three stratification temperatures (1°C, 4°C and 7°C), with four repetitions, each one consisting of 25 endocarps. At the end of the germination test (at 90 days), the percentage of seed germination was determined, with germinated seeds considered as those that pre­ sented radicle protrusion of at least two millimeters (MAPA, 2009). With these data, it was possible to determine the ideal temperature to be used during the pre­germinative treatment of the seeds of each peach rootstock cv. in the presence of the endocarp. From these results, the second experiment was performed in which the endocarps were conditioned and kept under similar conditions to the previous experiment but were subjected to different stratifica­ tion periods using the temperatures predetermined in experiment I, specifically 7°C for cvs. Aldrighi and Capdebosq and 4°C for cvs. Okinawa Roxo and Tsukuba 1, and three stratification periods (40, 80 and 120 days), with four repetitions, each one con­ sisting of 25 endocarps. At 40, 80 and 120 days, the germination percentage was determined according to MAPA (2009), and the endocarps in which the seeds did not germinate during the cold stratification process were sown, at 1.0 cm depth, in polystyrene trays of 72 cells (114 cm3 per cell) containing as sub­ strate composed of 25% orchard soil + 25% vermi­ culite + 25% medium sand + 25% commercial. The trays were kept under controlled greenhouse condi­ tions, with irrigation as needed, for a period of 45 days. After this period, the percentage of seedlings Menegatti et al. ‐ Contrasts of physical and physiological domancy of peach rootstock seeds 241 emerged under greenhouse conditions was deter­ mined (MAPA, 2009), and the endocarps in which the seeds did not germinate were removed from the trays and broken to extract the seeds using a manual lathe. The seeds were then re­sown in the polystyrene trays, under the same conditions men­ tioned above, and kept under greenhouse conditions to evaluate seedling emergence. The percentage of seedling emergence was daily monitored, and the experiment was finished at 15 days after the direct re­seeding during which, in three consecutive evaluations, no new seedlings emerged in any of the treatments. All analyses was performed with the program SigmaPlot version 10.0 (Systat, 2006). 3. Results and Discussion The seeds of cvs. Aldrighi and Capdeboscq pre­ sented superior results regarding to germination per­ centage in the presence of endocarp when stratified at 7°C for 90 days (Table 1), suggesting that such con­ ditions are adequate to overcoming seed dormancy and appropriate for inducing germination. A temper­ ature of 7°C employed in cold stratification, for a period of 90 days the cvs. Aldrighi and Capdeboscq provided average values of germination percentage of 82 and 80% respectively, results considered satis­ factory according to MAPA (2009). Souza et al. (2017) evaluated the stratification of the seeds of these same cultivars, under identical conditions, but for a period of 60 days, registered lower values, which were: 56.5 and 21%, respectively, indicating that the period of 60 days is not sufficient to overcome the dormancy process and, consequently, to obtain satis­ factory germination results. These results also suggest that the 30­day increase, from the 60 days suggested by Souza et al. (2017), to 90 days proposed in the present study, for the pre­germinative treatment of seeds from these cultivars. in the presence of endocarp, might have promoted significant changes in the hormonal bal­ ance that controls the process of physiological dor­ mancy, thereby making the seed metabolism more active and the embryo able to resume development. Additionally, it was observed that when the stratifi­ cation temperature was reduced to 4°C and 1°C, the germination percentages decreased significantly for ‘Capdeboscq’ and ‘Aldrighi’, being about 54% and 15%, respectively (Table 1). The seeds of cvs. Okinawa Roxo and Tsukuba 1 showed low average germination percentages at the end of the stratification period, regardless of the temperature tested. The best results were obtained in the treatment in which the endocarps remained in stratification at 4°C for 90 days, which were: 10% and 11%, respectively (Table 1). These results are superi­ or to those obtained by Souza et al. (2017), who reported zero germination rate for these two cvs., when the seeds in the presence of the endocarp were stratified at 7°C, for a period of 60 days. Although these results suggest that the prolonga­ tion of seed stratification period in the presence of the endocarp and the reduction in temperature from 7°C to 4°C, allow expressive increases in the germina­ tion percentage of seeds of these cultivars, these val­ ues are still considered unsatisfactory according to MAPA (2009). Based on the best results obtained in the first experiment, seeds from the four rootstocks were subjected to stratification temperatures that were considered most appropriate, with reductions and extensions of the stratification period. Thus, ‘Aldrighi’ and ‘Capdeboscq’ endocarps were placed to germinate at 7°C and ‘Tsukuba 1’ and ‘Okinawa Roxo’ were stratified at 4°C. In figure 1 A and B, the effects of the prolongation of the cold stratification period (in days) on the seed germination percentage in the presence of the endo­ carp are shown under the ideal temperatures of 7°C for ‘Aldrighi’ and ‘Capdeboscq’, and 4°C for ‘Okinawa Roxo’ and ‘Tsukuba 1’, that were previously described as ideal for pre­germinative treatment. The prolongation of the cold stratification period induced an increase in the germination percentage, with the maximum obtained at 120 days independent of cv. evaluated, however, the most significant increase in germination rate was registered for ‘Aldrighi’ (Fig. 1). These results corroborate with the research done by Wagner Júnior et al. (2013), in which they observed superiority in the effect of cold stratification on seeds Table 1 ­ Mean germination values (%) of four peach rootstocks, in accordance of the temperature used in the pre­ger­ mination cold stratification treatment for 90 days Temperature (°C) Germination (%) Aldrighi Capdeboscq Okinawa Tsukuba 1 1°C 15 12.5 2 4 4°C 54 52.5 10 11 7°C 82 80 2 0 Adv. Hort. Sci., 2022 36(3): 239­245 242 of some Prunus cvs. according to the number of hours of accumulated cold increases. It is important to highlight that at 120 days of stratification at 7°C, ‘Aldrighi’ and ‘Capdeboscq’ pre­ sented an average germination percentage of 82% (Fig. 1A), similar to that obtained before 90 days of cold stratification at this same temperature (Table 1). These results indicate, with greater reliability, the use of pre­germinative stratification treatment at 7°C for 90 days to overcome seed dormancy of these culti­ vars, in the presence of the endocarp, and may have greater impact on the large­scale production of root­ stocks. of ‘Capdeboscq’ as a rootstock in the production of peach seedlings in southern Brazil (Souza et al., 2018) for a long time. However, the use of this genotype is not currently recommended due to attributes such as the high susceptibility to different species of the genus Meloidogyne (Almeida et al., 2015, Souza et al., 2019 c), a parasitic nematode, and the adherence of the fruit pulp in the endocarp. Removal of the pulp necessitates the cleaning of the endocarps, facilitat­ ing the potential development of pathogens that may compromise the phytosanitary quality of the posthar­ vest seeds. These characteristics are also exhibited by ‘Aldrighi’, discouraging the use of these particular cultivars, and promoting of the use of cutlivars with opposite characteristics. The increase in germination percentage with an extension of the stratification period was also observed in ‘Okinawa Roxo’ and ‘Tsukuba 1’ seeds kept at 4°C (Fig. 1B). However, at 120 days of stratifi­ cation, satisfactory germination rates were not reached (MAPA, 2009), indicating that the increase in the number of cold hours beyond this period is not the main factor determining the low germination rate of these cultivars. Thus, it is suggested that physical limitations imposed by the presence of the endocarp may con­ tribute decisively to the low seed germination rate of these cvs., as already evidenced by Wagner Júnior et al. (2013) and Fischer et al. (2016), which suggests that the endocarp is a highly lignified structure, that confers high resistance to water absorption. This fac­ tor could compromise seed germination due to the difficulty of the embryo to overcome such a barrier during initial stages of the germination process when imbibition of water is critical. For these same four cultivars, Souza et al. (2017) obtained germination rate of 100% when seeds were stratified without endocarp at 7°C for 25 days. On the other hand, when the seeds were stratified at 7°C for 60 days, without endocarp removal, there were 0 and 0,7% of germination rates for ‘Tsukuba 1’ and ‘Okinawa Roxo’, respectively, suggesting that in addi­ tion to the presence of the endocarp, the early matu­ ration of the fruits of these cvs., in relation to the other genotypes, directly influence into the physio­ logical quality of the seeds, especially in relation to the embryo maturation. According to Pérez­Jiménez et al. (2012) when an embryo is not fully developed, it requires maintenance under specific conditions for a period of time to induce full development in order to ensure the establishment of a new seedling. Fig. 1 ­ Mean values of seed germination (%) in the presence of the endocarp, of the peach rootstocks cultivars, in Biochemical Oxygen Demand (BOD), in accordance of the period used in the pre­germination treatment. Besides ensuring germination efficiency, the use of this pre­germination treatment to overcome seed dormancy of these cutlivars excludes mechanical pro­ cedures such as endocarps rupture, a method com­ monly indicated for many of the seeds of the Prunus genus of commercial interest in which the endocarp acts as a physical limitation to the initiation of the germination process (Souza et al., 2019 c). The superior seed germination rate and the lower degree of physical dormancy has promoted the use Menegatti et al. ‐ Contrasts of physical and physiological domancy of peach rootstock seeds 243 The differences in germination percentage of seeds previously treated with cold stratification by different temperatures and periods, followed by sow­ ing in the presence of the endocarp in trays contain­ ing commercial substrate, and kept for 45 days under greenhouse conditions are shown in figures 2 and 3. Seeds of ‘Aldrighi’ stratified previously at 7°C during the periods of 40 and 80 days (Fig. 2), and the seeds of ‘Capdeboscq’ stratified for 40 days (Fig. 2B), fol­ lowed by sowing in the greenhouse did not demon­ strate a continuity in seed germination. For these treatments, a higher percentage of germination was expected during maintenance in the greenhouse given that during the period they were kept in BOD the values were considered low. Different behaviour was exhibited by seeds submitted to stratification treatment at 7°C for 120 days, for these two cultivars (Fig. 2A and B), since they had already shown high germination percentage in BOD, not showing signifi­ cant increases in the percentage of germination in the greenhouse. On the other hand, the seeds of ‘Okinawa Roxo’ and ‘Tsukuba 1’ did not show high germination per­ centage either under BOD conditions or when sub­ mitted to different stratification periods at 4°C fol­ lowed by a period under greenhouse conditions (Fig. 3). This suggests once more the necessity of ruptur­ ing of the endocarps for the continuity of the germi­ native process by the embryo. Souza et al. (2019 c) and Menegatti et al. (2019 a) point out that in some peach rootstock cvs. the physical barrier imposed by the endocarp may be more determinant in germina­ tion than the low temperature and the cold stratifica­ tion period. The results show that physiological seed dormancy is always present and it needs to be over­ come with cold stratification so that the germination occurs, but besides physiological dormancy, the pres­ ence of the endocarp may also limit or even prevent Fig. 2 ­ Mean values of increase in percentage (%) of germinated seeds after pre­germination stratification treatment in accordance with the period, (A) 'Aldrighi' and (B) 'Capdeboscq', at 7°C, followed by sowing of the seeds in the presence of the endocarp in trays containing com­ mercial substrate and kept for 45 days in greenhouse (GR). Dotted lines on darker bars indicate the limit of the average germination percentage obtained during stratifi­ cation in Biochemical Oxygen Demand (BOD), and the additional germination obtained after 45 days in greenhouse. Fig. 3 ­ Mean values of percentage increase (%) of germinated seeds after pre­germination stratification treatment in accordance of the period, (A) 'Okinawa Roxo' and (B) 'Tsukuba 1', at 4°C, followed by seed sowing in the pre­ sence of the endocarp, in trays containing commercial substrate and kept for 45 days in greenhouse (GR). Dotted lines on darker bars indicate the limit of the ave­ rage germination percentage obtained during stratifica­ tion in Biochemical Oxygen Demand (BOD), and the addi­ tional germination obtained after 45 days in greenhouse. 244 Adv. Hort. Sci., 2022 36(3): 239­245 seed germination of some cultivars resulting in non­ uniform seedlings, as demonstrated by Souza et al. (2019 c), Souza et al. (2017), and the present study. Among non­germinated seeds of ‘Aldrighi’ and ‘Capdeboscq’ after pre­germination treatment, fol­ lowed by sowing and maintenance in trays for 45 days in the greenhouse, when the endocarp was rup­ tured, a high percentage of nonviable seeds was found (mostly rotten), This percentage was highest with the cold stratification treatment for 40 days (Table 2). Moreover, the results showed that the increase of the cold stratification period induced a reduction in the number of dead seeds of these same cultivars. These results show that the presence of the endocarp, involving the seeds of these cultivars., doesn’t totally restrict the entry of water and cold to overcome dormancy, but probably the accumulation of 40 and 80 days of cold was not enough to promote effective changes in hormonal balance. This response was not observed in the great majority of ‘Okinawa Roxo’ and ‘Tsukuba 1’ seeds (Table 2), which when extracted from the endocarp were intact, with no turgidity, i.e., no evidence of water entering, which allowed the re­seeding and maintenance in the greenhouse again, for a period of 15 days. After this period, it was possible to observe a significant increase in the germination rate directly proportional with the extension of the stratification period employed, reaching a maximum value of 60 and 58%, respectively, after the stratification period of 120 days. The results obtained in this study reinforce the results published by Souza et al. (2017), which com­ pared the seed germination rate of ‘Tsukuba 1’ after cold stratification in the presence and absence of the endocarp and found that the germination rate was zero in the presence of the endocarp. In contrast, the authors obtained approximately 95% germination when the physical barrier (endocarp) was eliminated. Similar response was also obtained by Souza et al. (2017), having found that seeds without endocarp of ‘Okinawa Roxo’ and ‘Tsukuba 1’ achieved germina­ tion percentages greater than 90%. In ‘Okinawa Roxo’ and ‘Tsukuba 1’, the presence of the endocarp, besides preventing the entry and absorption of water by the embryo, evidenced by the absence of turgidity in the seeds present inside the endocarp even after stratification for 120 days, may also have prevented the leaching of the inhibitor substances present in the seeds. This factor would make the germination process in the pres­ ence of the endocarp more demanding regarding the stratification period. This behavior occurs espe­ cially in cultivars with greater resistance to rupture of the endocarp, as is the case with these two previ­ ously mentioned cultivars (Souza et al., 2017) and ‘Okinawa Roxo’ according to work by Fischer et al. (2016). 4. Conclusions Pre­germinative treatment at 7°C for 90 days is efficient to obtain high germination percentages of seeds of ‘Aldrighi’ and ‘Capdeboscq’ in the presence of the endocarp. Total removal of the endocarp from the seeds of cultivars ‘Okinawa Roxo’ and ‘Tsukuba 1’ enables a more efficient overcoming of physical and physiological dormancy and allows for a greater opti­ mization germination rate these cultivars that could benefit large­scale peach rootstock production. Pre­germination treatment at 4°C for 80 days is not efficient to obtain high germination percentages of seeds of ‘Okinawa Roxo’ and ‘Tsukuba 1’ in the Table 2 ­ Mean values of the percentage of non­germinated seeds after pre­germination stratification treatment, 'Aldrighi' and 'Capdeboscq', at 7°C (B) 'Okinawa Roxo' and 'Tsukuba 1' at 4°C, for different periods, and kept for 45 days in greenhouse, per­ centage of dead seeds after this period, percentage of seeds germinated after the whole process followed by endocarp rup­ ture and re­sowing and percentage of lost seeds %S= percentage of seeds; GR= greenhouse; BOD= Biochemical oxygen demand. days in BOD at 7°C + 45 days in GR days in BOD at 4°C + 45 days in GR Aldrighi Capdeboscq Okinawa Roxo Tsukuba 1 40 80 120 40 80 120 40 80 120 40 80 120 % S Non­germinated 100 46 17 82 16 13 100 96 75 96 89 79 % S dead 94 12 11 78 12 8 0 2 1 0 3 2 % S germinated after rupture­endocarp 0 17 2 0 2 3 32 44 60 20 40 58 % S lost 6 17 4 4 2 2 68 5 14 76 46 19 Menegatti et al. ‐ Contrasts of physical and physiological domancy of peach rootstock seeds 245 presence of the endocarp. 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