Storage potential of Eugenia uniflora Lam. seeds incubated in different osmotic solutions and temperatures

ABSTRACT:

The ex situ conservation of recalcitrant seeds has been one of the biggest challenges in seed technology. Different strategies have emerged in recent years and among these strategies, osmotic conditioning at controlled temperatures has shown excellent results. Therefore, the objective of this research was to store recalcitrant seeds of E. uniflora through osmotic conditioning in order to reduce the metabolism of these seeds and extend their storability. The seeds were stored at temperatures of 10 and 25 °C in PEG solutions at 0.0 (water), -0.5, -1.0, -1.5, and -2.0 MPa, as well as without moistening, for 3, 6, 12, and 18 months. There was a reduction in metabolism in the treatments with PEG, and seed viability was maintained after 18 months at 25 °C and -1.5 MPa.

Index terms: conservation; osmoconditioning; recalcitrant seeds

RESUMO:

A conservação ex situ de sementes recalcitrantes tem sido um dos maiores desafios da tecnologia de sementes. Diferentes estratégias surgiram nos últimos anos e dentre essas estratégias, o condicionamento osmótico em temperaturas controladas tem demonstrado excelentes resultados. Portanto, o objetivo desta pesquisa foi armazenar sementes recalcitrantes de E. uniflora por meio de condicionamento osmótico, a fim de reduzir o metabolismo dessas sementes e ampliar sua capacidade de armazenamento. As sementes foram armazenadas nas temperaturas de 10 e 25 °C em soluções de PEG a 0,0 (água), -0,5, -1,0, -1,5 e -2,0 MPa e sem umedecimento, durante 3, 6, 12 e 18 meses. Os resultados demonstraram redução do metabolismo nos tratamentos com PEG, e a viabilidade das sementes foi mantida após 18 meses a 25 °C e -1,5 MPa.

Termos para indexação: conservação; osmocondicionamento; sementes recalcitrantes

INTRODUCTION

The inclusion of seeds sensitive to desiccation, i.e., recalcitrant seeds, in germplasm banks is one of the main challenges in seed conservation. Such seeds lose viability when dehydrated or kept under refrigerated conditions (Roberts, 1973; Umarani et al., 2015; Wyse et al., 2018). However, sensitivity to desiccation and to low temperatures can vary among species, and up to now, there is no efficient storage method that can include the recalcitrant seeds of all species (Barbedo, 2018; Breman et al., 2021). It is estimated that around 8% of all flora is composed of species that have desiccation-sensitive seeds, and this may rise to 18.5% in tropical and subtropical regions (Wyse and Dickie, 2017). Therefore, developing technologies that allow recalcitrant seeds to be conserved in germplasm banks is an urgent need, especially due to concerns over loss of plant diversity.

Among the attempts to conserve these seeds, water control through osmoconditioning has shown promising potential by regulating the intense metabolism of recalcitrant seeds of some species. Polyethylene glycol 6000 (PEG) has been widely used for that purpose, above all to store seeds of this type (Andréo et al., 2006; Pereira et al., 2020; Pelissari et al., 2022; Cécel and Barbedo, 2023).

Seed metabolism can be indirectly analyzed by respiration rate, allowing more accurate diagnosis of the condition of the seeds under storage conditions (Lamarca and Barbedo, 2012). The amount of oxygen (O₂) consumed by the seeds, as well as the amount of carbon dioxide (CO₂) released, allow the intensity of seed metabolism to be quantified and the degree of efficiency of the seed and environment conditions to be identified for extended storage. The respiratory quotient (RQ), obtained by the quotient between the CO₂ released and the O₂ consumed, then provides information regarding the type of respiration, whether aerobic or anaerobic; and the difference between the O₂ consumed and the CO₂ released indicates whether other oxidation processes are occurring, such as those resulting from the action of reactive O₂ species (Kader and Saltveit, 2002; Bragante et al., 2018).

Eugenia uniflora L. (pitanga), native to the Atlantic Forest, Cerrado, and Pampa biomes and not endemic to Brazil, is of great ecological importance; it produces fruit with pharmacological and food potential and has potential for use as an urban tree (Bourscheid et al., 2011; Moura et al., 2018; Bittencourt et al., 2021; Mazine et al., 2024). The seeds of this species are sensitive to desiccation and prove to have interesting traits for the study of recalcitrant seed conservation in general. The aim of this study was to analyze the changes in respiratory rates of E. uniflora seeds resulting from incubation under controlled hydration, as well as the effects of this incubation on the storability of these seeds.

MATERIAL AND METHODS

Obtaining plant material - Ripe fruit from E. uniflora was collected from ten mother plants in the state park Parque Estadual das Fontes do Ipiranga, São Paulo, Brazil (23°38’30.7” S, 46°37’14.2” W), between 26 Oct. 2021 and 13 Nov. 2021. Seeds were manually extracted from the fruit under running water using trays and sieves. Excess water was removed from the seeds using filter paper and the seeds were stored in transparent polyethylene bags at 8 °C in a BOD incubation chamber until the experiments began, not exceeding seven days.

Physical and physiological determinations - The moisture content (expressed as a percentage, wet basis) and the dry matter content (in g.seed^-1) were determined using the air-circulation laboratory oven method for 17h at 103 °C (Brasil, 2009). The water potential (expressed in MPa) was measured by a WP4 potentiometer (Decagon Devices, Inc.; Pullman, WA, USA), based on dew point temperature (Delgado and Barbedo, 2012). Both measurements were performed with two replications of three seeds each.

Germination was evaluated by placing the seeds on Germitest® (germination testing) paper previously moistened with tap water, with two sheets as a base and one over the seeds. These papers were rolled up and placed in transparent polyethylene plastic bags with perforations (ten 0.5-mm holes) and placed in a climate-controlled room (25 °C) under continuous light. Weekly evaluations were made up to 60 days, recording the number of germinated seeds (root emergence of 0.5 cm) and those that produced normal seedlings, i.e., with roots and shoots that developed without defect (Delgado and Barbedo, 2007). Four replications of nine seeds each were used.

Preparation of the osmotic incubation solutions - The osmotic solutions were prepared by dissolving different amounts of solid PEG crystals in distilled water. A magnetic stirrer was used to homogenize the solution in the preparation process. The amounts of PEG were determined from the table proposed by Villela et al. (1991), preparing solutions of -0.5, -1.0, -1.5, and -2.0 MPa.

Analysis of the respiratory rates of seeds incubated in the different osmotic solutions - Seed samples were weighed and distributed into 3 replications of 20 seeds, placed on two sheets of Germitest® paper, and covered by a third sheet. All the seeds were moistened until saturation with PEG solutions at the water potentials of -0.5, -1.0, -1.5, and -2.0 MPa, as well as with distilled water (0.0 MPa). The sheets with the seeds were rolled and placed in 600-mL, transparent, hermetic glass containers (respirometers - Lamarca and Barbedo, 2012; Bonjovani and Barbedo, 2019), with lids sealed using Parafilm® (plastic wrap), and incubated in a BOD chamber at 10 °C and at 25 °C in darkness. Free seeds were also incubated in the respirometers, i.e., seeds without moistening in paper rolls (control).

The beginning of the experiment was considered to be when the containers were sealed (Lamarca and Barbedo, 2012; Bonjovani and Barbedo, 2019). The seed respiration rate was analyzed after 12 hours of incubation, calculated according to consumption of O₂ and release of CO₂ (Kader and Saltveit, 2002). To obtain the O₂ consumption and CO₂ release rates, the gases were collected from inside the containers with a hypodermic needle inserted into a rubber septum in the respirometer lid, as described in Manoel et al. (2024), and measured with the Model 6600 gas analyzer (Illinois Instruments, Inc., Johnsburg, IL, USA). The values obtained as percentage of volume were converted to partial gas pressure and then to µmols by the Clapeyron equation, PV = nRT. The number of mols of each gas was then divided by the seed dry matter of the respirometers and the incubation time. Furthermore, the Respiratory Quotient (RQ) and the oxidation rate were calculated, as previously described. Three replications of fifteen seeds each were used.

Seed storage in the osmotic solutions - Seed samples were distributed at equal distance on two sheets of Germitest® paper (28 × 19 cm, sterilized), for four replications of ten seeds, and covered by a third sheet. All were previously moistened with 30 mL of distilled water (0.0 MPa) or with 30 mL of the previously described PEG solutions. Then the sheets were rolled up and placed in polyethylene bags, which were closed with twist ties and perforated (10 holes) to allow gas exchanges. They were then stored at 2 temperatures (10 and 25 °C) in the dark. The control consisted of seeds placed in perforated plastic bags, without moistening. All the treatments were stored for 3, 6, 12, and 18 months, and at the end of each period, the seeds were evaluated for germination and moisture content, as previously described. At 6 and 18 months of storage, the seeds were also evaluated regarding their water potential, according to the methodology already described.

Analysis of variance (ANOVA) was used on the data, and the means were compared with each other by Tukey’s test (5%). When necessary, the data were transformed using √(x+0.5) to provide for normality (Santana and Ranal, 2004).

RESULTS AND DISCUSSION

The recently collected E. uniflora seeds had 95% germination, 92% normal seedlings, 52.4% moisture content, 0.17 g.seed^-1 dry matter, and water potential of -2.56 MPa.

Incubation of the seeds at the different temperatures and osmotic potentials resulted in different respiratory rates (Figure 1). At 25 °C, nearer the natural condition of these seeds, incubation in water or at lower osmotic potentials (-0.5 to -2.0 MPa) resulted in a lower CO₂ release than in the seeds incubated without any moistening (control). That may indicate a higher respiratory rate of the control seeds. Nevertheless, the O₂ consumption data did not show differences among the treatments, indicating that the difference in CO₂ release was probably due to oxidative processes not resulting from respiration. The RQ data and the oxidation rates (Figure 1) support this possibility, since an RQ near 1.0 indicates respiration using carbohydrates as a probable substrate, whereas RQs lower than 0.6 suggest the presence of other oxidative reactions (Lamarca and Barbedo, 2012).

Figure 1
O₂ consumption, CO₂ release, respiratory quotient (RQ), and oxidative rates of E. uniflora seeds incubated for 12 h at the temperatures of 10 °C (blue) and 25 °C (orange), in the following treatments: control (C), water (0.0), and PEG at the osmotic potentials of -0.5, -1.0, -1.5, and -2.0 MPa. Columns with the same letter do not differ from each other (Tukey’s test, 5%).

The seeds incubated at the lower temperature, 10 °C, had less release of CO₂, especially those of the control (Figure 1), suggesting lower respiratory rates than in the seeds incubated at 25 °C. This lower respiratory intensity could result in greater storage potential. However, the values of O₂ consumption, of RQ, and of oxidative rates show that the reduction in temperature intensified the oxidative processes, and may speed deterioration of the seeds (Chandra et al., 2021). Interestingly, however, at the osmotic potentials of -1.0, -1.5, and -2.0 MPa, the seeds had higher oxidative rates, which may indicate some stress condition from low temperature.

In general, the seeds had lower respiratory rates at 10 °C than at 25 °C, which was also seen for seeds of other species within the family, such as Eugenia pyriformis Cambess., E. brasiliensis Lam., and Myrcianthes pungens (O. Berg) D. Legrand (Lamarca et al., 2020; Guardia et al., 2020; Cécel and Barbedo, 2021; 2023). However, the reduction in temperature brought about an increase in oxidative rates, as also observed for recalcitrant seeds of Inga vera subsp. affinis (DC.) T.D. Pennington (Bonjovani and Barbedo, 2014). If, on the one hand, reduction in respiratory rates could increase storability, on the other hand, the increase in oxidative rates could have an opposite effect. In fact, there is the recommendation of storing seeds in germplasm banks under low O₂ concentration conditions in the environment (Groot et al., 2015), which could reduce these oxidation rates, as observed in seeds of Pinus densiflora (Gerna et al., 2022), of lettuce, and of onion (Schwember and Bradford, 2011).

Storage of seeds under these different conditions showed that, already in the first period (3 months), germination occurred within the container itself (Figure 2). The number of seeds that germinated during storage varied depending on the temperature and the type of incubation. In storage at 25 °C and at the potentials of 0.0, -0.5, and -1.0 MPa, more than 90% of the seeds had already germinated (Figure 3), which would be expected given the high respiratory rates (Figure 1). The reduction in water availability to -1.5 MPa decreased these germination values to around 50%, and an even greater reduction in this availability, to -2.0 MPa, practically impeded germination in the container. Nevertheless, the seeds remained viable. In reducing the storage temperature to 10 °C, there was also generally reduction in seed germination during storage, except for the seeds incubated in water (0.0 MPa) and in PEG solution at -0.5 MPa (Figure 3), which also exhibited higher respiratory rates than the seeds incubated at lower osmotic potentials (Figure 1). The seeds of the control treatment hardly germinated during storage, probably because of reduction in their moisture content (Table 1) to levels below that tolerated by these seeds (Delgado and Barbedo, 2007). It is noteworthy that while incubation without solutions (or water) resulted in rapid reduction in seed moisture content, incubation in solutions with different water potentials resulted in seeds with different levels of hydration, generally lower as the water potentials decreased (Table 1).

Figure 2
Germination and formation of normal and abnormal seedlings from Eugenia uniflora seeds. A: germinated seed, with emergence of primary root; B: normal seedling with developed shoots and roots; C: abnormal seedling formed during storage; D: abnormal seedling formed during storage but with resumption of shoot development during the germination test; E and F: seeds removed after 12 months of storage in the control, soaked in water (0.0 MPa), or in PEG solutions with different osmotic potentials (-0.5, -1.0, -1.5, and -2.0 MPa) at 10 °C (E) or 25 °C (F).

Figure 3
Specific germination (%) of E. uniflora seeds after each storage period of 3 (A and B), 6 (C and D), 12 (E and F), and 18 (G and H) months at 10 °C (blue) and 25 °C (orange) in the following treatments: control (C, without moistening), water (0.0), and PEG at the osmotic potentials of -0.5, -1.0, -1.5, and -2.0 MPa.

Table 1
Moisture content and water potential (Ψw) in E. uniflora seeds after removal from BOD chambers at the periods of 3, 6, 12, and 18 months in the following treatments: control (without moistening), water (0.0), and PEG at the potentials of -0.5, -1.0, -1.5, and -2.0 MPa, at 10 and 25 °C.

Storage temperature	Type of incubation	Moisture content (%)				Water potential (MPa)
		Period (months)				Period (months)
		3	6	12	18	6	18
10 °C	Control	26	13	12	18	-70.5	-40.1
	0.0	57	55	64	69	-1.2	-1.8
	-0.5	56	53	58	45	-2.1	-3.3
	-1.0	49	51	56	56	-3.4	-3.7
	-1.5	56	55	52	50	-4.2	-4.4
	-2.0	52	51	47	48	-4.6	-5.6
25 °C	Control	19	20	15	18	-44.9	-52.0
	0.0	65	71	58	58	-1.1	-2.0
	-0.5	60	65	51	37	-2.6	-20.8
	-1.0	58	63	55	32	-5.1	-22.6
	-1.5	63	56	31	31	-5.3	-23.5
	-2.0	58	47	44	31	-5.9	-26.2

All the seeds, whether or not they germinated in the container itself, were placed to germinate in the germination test. The total number that germinated was thus considered as all the seeds that continued the germination that had begun in storage with those that began germination in the germination test. The seeds that germinated during storage but that did not continue their development during the germination test were recorded as not germinated. Likewise, those that had germinated during storage and had already died and that were placed under the germination test were also recorded as not germinated.

Conservation of seed viability during storage was clearly better at the lower temperature, 10 °C, than at 25 °C (Figure 4), as would be expected for recalcitrant seeds (Barbedo, 2018) and also from the lower respiratory rates (Figure 1). Even at the lower temperature, the seeds lost viability already at 6 months when not kept in a moist substrate (control). When kept in a substrate with total water availability, they began to lose vigor after 12 months of storage, as can be seen by their ability to produce normal seedlings (Figure 4). The seeds kept in PEG solutions did not show this same pattern of deterioration (Figure 4); they were likely highly affected by the lower respiratory rates, especially at the potentials below -0.5 MPa (Figure 1).

Figure 4
Germination percentages (lighter-colored bars) and development of normal seedlings (darker bars) in the germination test of E. uniflora seeds stored for 3 (A and B), 6 (C and D), 12 (E and F), and 18 (G and H) months at 10 °C (blue) and 25 °C (orange) in the following treatments: control (C, without moistening), water (0.0), and PEG at the osmotic potentials of -0.5, -1.0, -1.5, and -2.0 MPa. Columns with the same letter do not differ from each other (Tukey’s test, 5%).

Seeds stored at the higher temperature deteriorated more rapidly, and not only did the control seeds quickly lose vigor, but also the seeds kept in the water substrate. They did not produce normal seedlings after the first 3 months of storage, whereas seeds kept in a PEG solution at -0.5 MPa deteriorated after 6 months of storage (Figure 4). After 18 months of storage, only the seeds kept in a PEG solution at -1.5 MPa maintained viability and the ability to produce normal seedlings.

Analyzing the germination and normal seedling production values (Figure 4) together with the respiration and oxidative rates (Figure 1), it is clear that both the reduction in temperature and in water availability result in two important consequences: reduction in respiratory rates, which would be favorable to storage, and increase in oxidative rates, which would negatively affect storage. Perhaps for that reason, some treatments stood out, such as storage at -0.5 MPa when the temperature was low, and storage at -1.5 MPa when the temperature was higher. In both cases, there may be a point at which greater reduction in respiratory rates coincided with a smaller increase in oxidative processes.

Eugenia spp. are well adapted to 25 °C (Gomes et al., 2016). At low temperatures, the mobility of water molecules and metabolic processes are reduced (Nadarajan et al., 2023). That may explain the decrease in germination during storage at 10 °C, as was also observed by Inocente and Barbedo (2019) and Cécel and Barbedo (2021). The high moisture content and accumulation of reserves in seeds at an advanced maturity stage result in germination, and removal of water from the seeds leads to an increase in solutes and, consequently, reduction in metabolism (Bewley et al., 2013, Marcos-Filho, 2015). However, it is noteworthy that when water availability declined using the PEG treatment at -1.5 MPa, even at 25 °C, it was possible to maintain practically 50% of the seeds viable, and many were able to produce normal seedlings, even after 18 months of storage (Figure 4H). This 18-month time is quite significant for recalcitrant seeds stored at a high temperature, since Eugenia spp. seeds more quickly become inviable at ambient temperatures (Silva et al., 2019). Moreover, finer adjustment in the PEG concentration, among other factors, will likely allow an even longer storage period. It is important to mention that the seeds germinating during storage did not totally compromise the viability of the treatments with PEG, for the resumption of germination by shoot production resulted in normal seedlings, as already shown in seeds of E. involucrata DC. (Inocente and Barbedo, 2021) and in E. brasiliensis Lam. (Cécel and Barbedo, 2023). Therefore, the results of the present study indicate the considerable potential of use of osmotic solutions to increase the storability of recalcitrant seeds. The results also show the importance of oxidative processes in the loss of viability of seeds during storage.

CONCLUSIONS

E. uniflora seeds can be stored for up to 18 months at 25 °C when kept at -1.5 MPa.

ACKNOWLEDGMENTS

Our thanks to the Instituto de Pesquisas Ambientais de São Paulo - IPA and to its Seed Research Laboratory; to the Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP for funding (Process: 17/50341-0); and to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES for the Master’s degree scholarship granted to the first author.

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Publication Dates

• Publication in this collection
  23 Sept 2024
• Date of issue
  2024

History

• Received
  18 Feb 2024
• Accepted
  23 July 2024