1 Introduction

The rare and endangered plant species Handeliodendron bodinieri (H. Lév.) Rehder (genus, Sapindaceae, Fig. 1) is native to China and only found distributed in karst areas (Fig. 2) including, Guangxi, Guizhou, and Yunnan (Cao et al. 2008; Wang et al. 2008). This woody, fuel-oil plant species is excellent for rock desertification control (Guo et al. 2019a; Guo et al. 2019b; Leng et al. 2020). However, due to its low seed germination rate, its vulnerability to insects, slow growth, shallow root system, and to the destruction of its habitat, it is currently on the verge of extinction and is therefore listed as a national, first-level protection species (He et al. 2012). Moreover, the high oil content of fat seeds is easily hydrolyzed and oxidized, thereby producing many toxic substances, such as malondialdehyde and free fatty acids, which pose a great threat to seed vigor and cause low seed germination-rate (Farhoosh et al. 2009), whereby, they represent serious limitations to the protection of the species. Seeds are the initial concentration of individual plants, and the basis for individual plant growth. Therefore, seed quality determines the strength of the individual to form a population.

Fig. 1. Introduction to Handeliodendron bodinieri, including H.bodinier (A); Leaf (B); Stamen (C); Pistil (D); Capsule (E); Seeds (F). A–E photo provided by Peng Jian.

Fig. 2. Distribution of Handeliodendron bodinieri, including Funing in Yunnan province; Xingyi, Anlong, Dushan, Pingtang, Libo, Sandu, Duyun in Guizhou province; Longlin, Tianlin, Leye, Lingyun, Tiandong, Longzhou, Tian’e, Fengshan, Nandan, Donglan, Huanjiang, Bama in Guangxi province.

One approach to solving the abovementioned limitations is to use optimized storage methods to preserve seed resources while improving seed germination rate, as well as seedling management and protection (Xie et al. 2020). Furthermore, among the various conservation methods designed for the long-term preservation of endangered species, seed storage has been proposed as one of the most convenient methods (Paunescu 2009; Engelmann 2011). Particularly, ultra-dry seed storage is a method for storing seeds at room or low temperatures, in which seed moisture content (MC) is reduced to an optimal level for safe long-term storage prior to sealing; as such technologies contribute to long-term seed storage without destruction of the integrity of cellular membrane structure and seed functions (Hoekstra et al. 2001), there is an increasing interest in ultra-dry storage technologies in the fields of agriculture and forestry (Yan 2017; Wawrzyniak et al. 2020) that may allow for the maintenance of seed viability, tissue and cell morphology integrity, and stability of the genetic material (Li et al. 2007; Li et al. 2010). Indeed, compared with traditional refrigeration methods, ultra-day seed storage is environmentally friendly, energy-saving, and simple (Ellis et al. 1995; Ellis 1998; Ellis et al. 1998). Therefore, a large number of seed species are currently stored using ultra-dry storage. Particularly, oily seeds such as Brassica napus L., Sesamum indicum L., Arachis hypogaea L., and Calocedrus macrolepis Kurz seeds are resistant to dry storage owing to their hydrophobic properties ( Ellis et al. 1986; Mira et al. 2019; Zhang et al. 2019). Further, ultra-dry storage has been proposed as an effective and simple technology to improve resource utilization efficiency (Pérez-García et al. 2007; Huo et al. 2015). Seed priming of ultra-dry seeds before germination can protect cell membrane integrity during water absorption, thereby improving seedling viability (Ellis et al. 1990; Van et al. 1994; Taylor et al. 1995; Jiang et al. 2020). Nonetheless, while ultra-dry storage has proved beneficial for seed storage, its effects upon seed germination and seedling growth thereafter, have rarely been studied.

The longevity of stored ultra-dry seeds is affected by MC, storage temperature, storage time, and other factors (Zheng et al. 1998). Ultra-dry stored seeds must not only germinate normally but seedlings must grow and develop normally. However, most studies have only focused on ultra-dry storage of seeds (Ellis et al. 1998; Wang et al. 2005), while there have been no reports on subsequent growth processes, such as seed germination and seedling growth.

Therefore, in this study, we aimed to develop a dry-storage method suitable for seed storage, sowing, and normal seedling growth of H. bodinieri. To this end, H. bodinieri seeds were dried to different MCs and then stored at room temperature or at 4 °C to find an optimum ultra-dry MC. Further, an optimal seed priming method was established to analyze the effect of ultra-dry storage treatment on seed sowing and seedling development. Finally, we measured various growth indicators including, plant height, stem diameter at ground level, and root length, as well as physiological indicators, such as antioxidant enzyme activity and malondialdehyde (MDA) content in seedlings grown from stored ultra-dry seeds and from seeds stored using the traditional method of wet-sand storage at 4 °C. We hypothesized that proper ultra-dry seed storage would allow adequate storage of H. bodinieri seeds and promote normal, healthy, seedling growth upon germination.

2 Materials and methods

2.1 Experimental materials

H. bodinieri seeds were collected in the karst area (24°32′30′′–25°06′10′′N, 106°08′10′′–106°50′35′′E) from the end of August to the beginning of September (one-third of the fruit cracked on the infructescence was used as criterion for maturity standard). Over 20 mother trees were selected for seed-harvesting at full fruiting period. Only healthy fruit branches were selected in each individual tree and within them, capsular fruits with a relatively uniform shape, size, appearance, and color were harvested for experimental use. The seed coat and aril were removed, and the seeds were placed in a dry environment at 25 °C. Mean 1000-seed weight and MC were 171.993 g and 9.62%, respectively, while mean seed length and width ranged from 8.70 to 11.30 and from 4.5 to 7.28 mm, respectively. Useless seeds accounted for 11.67% of all seeds harvested. Seedlings were grown in a container (15 × 10.6 × 13 cm) filled with loam soil collected from the Karst area (pH 7.25, total nitrogen: 5.95 g kg^–1, total phosphorus: 2.02 g kg^–1, total potassium: 0.28 g kg^–1, organic matter: 33.07 g kg^–1).

2.2 Experimental methods

2.2.1 Seed treatments

Using the silica gel drying method for ultra-dry treatment (Yan 2017), H. bodinieri seeds with different MCs (2.54% [mc1], 3.14% [mc2], 3.97% [mc3], and 4.77% [mc4]) were prepared. After the ultra-dry treatment, the seeds were packed in sealed double-layer aluminum foil bags and stored for 160 days either at room temperature (25 °C) or in a refrigerator at 4 °C. Seeds with an MC of 9.62% (mc5) without ultra-dry treatment were used as controls. As for storage in sand at 4 °C, seeds were surface-sterilized by soaking for 30 min in a 0.5% potassium permanganate solution, then rinsed with distilled water, and air-dried to reduce MC to 8.64%. The seeds were placed in three layers in a plastic container previously sterilized with alcohol and then topped with five layers of sand. Finally, the container was closed with a breathable lid and stored for 160 days in a refrigerator at 4 °C.

2.2.2Seed germination experiment

After storage for 160 days, the experimental seeds were soaked in distilled water for 24 h, disinfected by soaking for 15 min in a 0.5% potassium permanganate solution, and rinsed with sterile water. After removing the exopleura, seeds were placed in a petri dish and covered with filter paper. Four replicates, each consisting of 50 seeds, were evaluated for each storage method. Germination was evaluated in an incubator set to a constant temperature of 25 °C and a 12 h light/dark cycle. Water was replenished every day and seed germination was observed. Filter papers were replaced as needed. A seed was considered as germinated once the radicle length exceeded seed diameter. During the germination test, germination rate (GR), germination potential (GP), germination index (GI), and vitality index (VI) were calculated according to formulas reported by Ranal et al. (2006):

where, M₁ is the number of germinated seeds, M₂ is the normal number of germinated seeds within the days of germination potential, M is the number of seeds tested, S is the growth of seedlings, G_t is the number of germinated seeds on day t, and D_t is the corresponding number of germination days.

2.2.3 Priming treatment

Seeds with an MC of 4.77% were soaked for 2, 4, or 6 days in 5%, 10%, 15%, 20%, or 25% polyethylene glycol (PEG, molecular weight, 6000) solutions in Petri dishes. Thus, the test comprised 15 treatments, including all possible combinations of PEG concentration and storage times (Table 1). Simultaneously, saturation water vapor-priming was used as a control treatment, in which ultra-dried seeds were hydrated for 48 h in a sealed desiccator containing saturated CaCl₂ solutions (relative humidity (RH) of 35%), then for 48 h in a sealed desiccator containing saturated NaCl solutions (RH of 75%), and finally, for another 48 h in a sealed desiccator containing saturated water solutions (RH of 100%) at normal atmospheric temperature (25–30 °C) before germination assessment and subsequent experiments. Each treatment was evaluated using 50 seeds and four replicates. Germination was evaluated as described in 2.2.2.

2.2.4 Sowing experiment

The single experimental factor, i.e., storage treatment, was completely randomized. Each treatment was repeated three times using 20 seedlings in each repeat. Optimum MC for ultra-dry seed storage at 25 °C and 4 °C was used. Seeds stored in sand at 4 °C were used as controls. The sowing medium was saturated with water before planting.

2.2.5 Index measurements

Membrane permeability was measured according to the method reported by Blum et al. (1981). Soluble sugar (SS) content was determined using the anthrone method (Irigoyen et al. 1992). Soluble protein (SP) content was determined using Coomassie Brilliant Blue G-250 staining (Bradford 1976). MDA, a product of lipid peroxidation, was measured using the thiobarbituric acid assay described by Sudhakar et al. (2001). Superoxide dismutase (SOD) and peroxidase (POD) activities were measured using the methods reported by Dhindsa (1981).

The growth and physiological indicators were evaluated in one-year old seedlings. Seedling height and stem diameter were measured using a Vernier caliper, a band tape, and a ruler. The root index was measured with a digital scanner (Epson V750 Pro, Epson, Nagano-ken, Japan). Leaf area was measured with a portable leaf area meter (LI-3000C, LI-COR, Lincoln, NE, USA). Chlorophyll was measured using a chlorophyll meter (SPAD-502, Konica Minolta, Tokyo, Japan). Leaf net photosynthetic rate (Pn), stomatal conductance (G_s), and transpiration rate (Tr) in H. bodinieri seedling foliage was measured using a portable photosynthesis analyzer (LI-6400, LI-COR, Lincoln, NE, USA). Plant foliar total nitrogen (TN) was measured using a Vario Max CN analyzer (Elementar, Frankfurt, Germany). Plant foliar total K (TK) and total P (TP) were measured after digestion in hydrogen peroxide-sulfuric acid by the Kjeldahl method followed by standard colorimetric assays (O’Neill et al. 1970). SS and SP contents, SOD and POD activities, and MDA content were determined as described above for ultra-dry-stored seeds. Root activity was measured using the triphenyl tetrazolium chloride staining method described by Clemensson-Lindell (1994).

2.2.6 Evaluation of Handeliodendron bodinieri seedling quality after seed ultra-dry storage

The seedling growth-physiological index was used to evaluate seedling quality after the different ultra-dry storage treatments. Plant height, stem diameter at ground level, biomass, rhizome ratio, total root length, total root area, total root volume, leaf area, N, P, K, SOD, POD, MDA, SS, SP, root vitality, net photosynthesis, stomatal conductance, and transpiration were used as indicators of seedling quality. As the importance of each indicator was different, there was a substantial difference in dimension, and each indicator was a continuous variable. Therefore, the continuous nature of the membership function was used for standardization to eliminate the influence of dimension and determine the weight of each indicator in the comprehensive evaluation. Formula (5) was used to calculate indices positively correlated with the quality of H. bodinieri seedlings, and formula (6) was used to calculate indices negatively correlated with the quality of H. bodinieri seedlings.

where, F(X_i) is the membership value of each index, X_ij is the observed value of each index, and X_imax and X_imin are the maximum and minimum values of the i^th factor, respectively.

Principal component analysis was applied on the membership value to obtain the variance of the common factor of each index and then, the weight of each index was calculated as follows:

where, W_i is the weight of the i^th factor, and C_i is the common factor variance of the i^th factor.

The fuzzy set weighted synthesis method was used to calculate seedling quality index.

2.3 Statistical analysis

The effects of ultra-dry storage treatment of H. bodinieri seeds on seed storage, seed sprouting, plant growth, plant foliar N, plant foliar P, plant foliar K and plant physiology were evaluated by analysis of variance (ANOVA) in R (http://www.R-project.org/) (Ren et al. 2020a). The assumptions of normality of residuals and homogeneity of variances were assessed for all treatments, and data transformations were applied when appropriate to meet the assumptions (Ren et al. 2021). When main effects were significant, we used pairwise Duncan’s tests to determine significant differences among treatments. PCA biplots were generated using the package ggbiplot in R (Ren et al. 2020b).

3 Results

3.1 Effect of ultra-dry storage of seeds with different MCs

3.1.1 Effects of ultra-dry storage of seeds with different MCs on seed physiological indices

Physiological indices showed dynamic changes when seeds with different MCs were stored at room temperature or at 4 °C (Fig. 3). The MDA (p < 0.05) content and relative conductivity (p < 0.05) increased slowly as the time of storage increased, whereas SS (p < 0.05) and SP contents (p < 0.05), and SOD (p < 0.05) and POD (p < 0.05) activities first decreased and then increased. After 160 days of storage at room temperature, SS and SP contents, and SOD and POD activities of mc3 and mc4 seeds were relatively high, whereas the MDA content tended to decrease, which is best for seed storage. The relative conductivity of mc1 seeds was significantly higher than that of mc5 seeds. When stored at 4 °C, seeds with different MCs showed limited differences in the changes of various indices. Relative conductivity increased significantly as MC decreased, and soluble protein contents, SOD and POD activities, and MDA content varied at different time points. After 160 days, SS and SP contents, and POD activity decreased, whereas SOD activity and MDA content increased.

Fig. 3. Physiological analysis of Handeliodendron bodinieri seeds after ultra-dry treatment with different moisture content in 4 ℃ and room temperature. Storage at room temperature, the MDA (p < 0.05) content and relative conductivity (p < 0.05) increased slowly as the storage time increased, whereas soluble sugar (p < 0.05) and soluble protein (p < 0.05) contents and SOD (p < 0.05) and POD (p < 0.05) activities first decreased and then increased. stored at 4 °C, the relative conductivity (p < 0.05) increased obviously as the MC decreased, and the soluble sugar (p < 0.05) and soluble protein (p < 0.05) contents (p < 0.05), SOD (p < 0.05) and POD (p < 0.05) activities, and MDA (p < 0.05) content varied at different time points. View larger in new window/tab.

3.1.2 Effects of ultra-dry storage of seeds with different MCs on seed germination characteristics

After 160 days of storage at room temperature or at 4 °C (Table 2), ultra-dry-stored seeds with different MCs had significantly different GR, GP, GI, and VI (p < 0.05), indicating the MC had a significant effect on these parameters. GR, GP, GI, and VI of H. bodinieri seeds were higher at room temperature than at 4 °C. Further, GR, GP, GI, and VI of mc1 seeds were higher than those of mc5 (CK) seeds, and mc4 seeds performed the best. Meanwhile, GR, GP, GI, and VI of H. bodinieri seeds kept at 4 °C first increased and then decreased, with mc3 seeds performing best.

3.1.3 Comprehensive evaluation of the effects of ultra-dry storage of seeds with different MCs on germination and physiological indices

Growth and physiological index PCA factors of H. bodinieri seeds after ultra-dry storage treatment at room temperature or 4 °C are shown in Fig. 4. As can be seen for storage at room temperature, the first PCA axis (PC1) explained 45.1% of the total variation, while the second (PC2) explained 23.9% (Fig. 4A), for the total cumulative contribution rate of 69%, which indicated the relationship between MC and the various indicators. SOD activity, SS content, GR, GP, GI, and VI were mainly related to PC1, while POD activity was mainly related to PC2. Furthermore, mc1 (–0.26), mc2 (0.15), mc3 (0.32), and mc4 (0.60) treatments were associated with higher principal component scores than the control treatment (–0.80), indicating that ultra-dry storage at room temperature was conducive to adequate seed storage, with mc4 showing the best results.

Fig. 4. Physiological index PCA factor of Handeliodendron bodinieri seeds after ultra-dry treatment with room temperature(A) and 4 ℃(B). In plot A, factor 1 accounted for 45.1% of the variance, factor 2 accounted for 23.9% In plot B, factor 1 accounted for 49.1% of the variance, factor 2 accounted for 26.5%. SOD = superoxide dismutase, POD = peroxidase activity, MDA = malondialdehyde, SS = soluble sugar, SP = soluble protein, RC = relative conductivity, GR = germination rate, GP = germination potential, GI = germination index, VI = vitality index. View larger in new window/tab.

As for storage at 4 °C, PC1 explained 49.1% of the total variation and PC2 explained 26.5% (Fig. 4B). Thus, the total cumulative contribution rate was as high as 75.6%. SS and SP contents, GR, GP, GI, and VI were mainly related to PC1, while POD activity, MDA content, and relative conductivity were mainly related to PC2. The principal component scores for mc1 (–0.08) and mc4 (–0.71) were higher than those for the control (–0.15), whereas those for mc2 (0.02) and mc3 (0.92) were higher, indicating that there was little difference between ultra-dry low-temperature storage treatments, with mc3 showing the best results.

3.2 Effect of ultra-dry storage on seed sowing and seedling growth

3.2.1 Priming treatment of stored ultra-dry seeds

The ultra-dry stored H. bodinieri seeds showed significant differences in germination rate and germination index after priming (p < 0.05, Table 3). GR and GI values were higher after the 10 different PEG priming treatments, with 2-day and 4-day treatment times being higher than after seed priming with saturation water vapor. Differences among the 10 PEG treatments were significant (p < 0.05). GR and GI values after 2 days of PEG treatment were significantly higher than those after priming with saturation water vapor. GR and GI values decreased with increasing PEG-soaking time. As shown in Fig. 5, after two days of treatment, the highest PEG concentration had better results than the low concentration treatments, and at 20% PEG, GR and GI values were highest. After a 4-day priming treatment, low PEG concentrations yielded better results than high concentration treatments, with 10% PEG resulting in the highest GR and GI values. Furthermore, GR and GI values for the seeds after 6 days of treatment were lower than those of saturation water vapor-priming, likely because excessively long soaking times tended to suppress germination. Thus, PEG treatment should not be prolonged excessively.

Fig. 5. Germination rate (A) and germination index (B) change of polyethylene glycol treatments (x-axis) for Handeliodendron bodinieri seeds. The GR (p < 0.05) and GI (p < 0.05) were higher after the 10 different PEG priming treatments with treatment times of 2 days and 4 days were higher than after saturated water vapor priming (CK). View larger in new window/tab.

3.2.2 Effect of ultra-dry storage on the growth of Handeliodendron bodinieri seedlings

No clear effects of ultra-dry treatments were observed on plant height, stem diameter, leaf area, chlorophyll content, total root length, total root surface area, or TN (p > 0.05), whereas biomass, rhizome ratio, average root diameter, TK, and TP were significantly increased (p < 0.05). Specifically, the biomass (Fig. 6C), rhizome ratio (Fig. 6F), total root length (Fig. 6G), total root area (Fig. 6H), average root diameter (Fig. 6I), and TP (Fig. 6K) of seedlings grown from ultra-dry stored seeds were higher than those of sand-stored seeds, while those of ultra-dry seeds stored at room temperature were superior to those of ultra-dry seeds stored at 4 °C. Plant height (Fig. 6A) and stem diameter (Fig. 6B) of seedlings from seeds kept under room temperature ultra-dry storage were higher than those of seedlings grown from seeds kept in sand storage (4 °C) and superior to those of seedlings grown from seeds kept under low-temperature ultra-dry storage. TN (Fig. 6J), TK (Fig. 6K), and TP (Fig. 6L) of seedlings grown from low-temperature ultra-dry storage-treated seeds were higher than those of sand-stored seeds. In turn, leaf area (Fig. 6D) and chlorophyll content (Fig. 6E), were slightly lower in seedlings grown from ultra-dry-stored seeds than in those grown from seeds under sand storage, although in this case, the differences were not significant. Ultra-dry storage treatment of seedlings did not affect plant growth, and proper ultra-dry treatment even promoted plant height, root system development, and biomass formation, which was consistent with previous findings on the performance of Arachis hypogaea L. and Brassica napus L. (Hong et al. 2005).

Fig. 6. Effect of ultra-dry storage on plant growth of Handeliodendron bodinieri seedlings. The different letter indicate significant differences among different treatments, and the same letter indicates no significant difference (p < 0.05 Duncan’s). View larger in new window/tab.

As shown in Figs. 7A–C, SOD and POD activities, and MDA content of ultra-dry storage seedlings were consistent, but lower than those of seedlings obtained from sand-stored seeds. This indicated that ultra-dry treatment in a low-temperature environment alleviated the injury suffered by H. bodinieri seeds during storage. Seedling SS (Fig. 7D) and SP contents (Fig. 7E) were significantly improved under ultra-dry storage treatment compared to sand storage (p < 0.05). The SS content in seedlings from low-temperature ultra-dry storage seeds and the SP content in seedlings from room-temperature ultra-dry storage seeds were superior to those in seedlings grown from sand-stored seeds. As shown in Fig. 7F, ultra-dry treatment had no significant effect on root vitality (p < 0.05), nor did it affect seedling root condition. In terms of photosynthesis (Figs. 7G–I), ultra-dry treatment had no significant effect on Pn, Gs, or Tr (p < 0.05), and g_s and Tr of seedlings grown from low-temperature ultra-dry-stored seeds were greater to those of seedlings grown from sand-stored seeds, indicating that a proper low temperature during ultra-dry storage was conducive to normal photosynthesis of seedlings.

Fig. 7. Effect of ultra-dry storage on plant physiology of Handeliodendron bodinieri seedlings. The different letter indicate significant differences among different treatments, and the same letter indicates no significant difference (p < 0.05 Duncan’s). View larger in new window/tab.

3.2.3 Comprehensive evaluation of the effects of ultra-dry storage on the quality of Handeliodendron bodinieri seedlings

We used the common factor variance and weight of Handeliodendron bodinieri seedlings in ultra-dry storage (Table 4) to obtain a seedling quality index (Fig. 8). Quality evaluation scores for H. bodinieri seedlings from ultra-dry-stored seeds at 4 °C (0.59) and at room temperature (0.58) were significantly higher than that for seedlings grown from sand-stored seeds (0.37). The results revealed that the quality of H. bodinieri seedlings grown from ultra-dry-stored seeds was significantly higher than that of seedlings grown from sand-stored seeds, while there was no significant difference between quality of seedlings grown from seeds kept under room-temperature and low-temperature ultra-dry storage. These findings demonstrate that proper ultra-dry seed storage improves the quality of H. bodinieri seedlings.

Fig. 8. The seedling quality index of Handeliodendron bodinieri seedlings in ultra-dry storage. Seedling quality evaluation score of H. bodinieri seedlings from ultra-dry-stored seeds at 4 °C (0.59) and ultra-dry-stored seeds at room temperature (0.58) was significantly higher than that of seedling from sand-stored seeds (0.37).

4 Discussion

4.1 Ultra-dry storage tolerance of Handeliodendron bodinieri seeds

Seeds gradually age and lose their viability, as the seed lifespan is finite (Wawrzyniak et al. 2020). Storage-resistant seeds have a longer lifespan when stored ultra-dry; however, after a certain time, seeds will ultimately begin to age and deteriorate, which will negatively affect seed viability and seedling performance. Thus, to profit from the beneficial effects of ultra-dry storage on seed longevity and to maximize seed shelf life, it is important to find the optimum MC (Vertucci et al. 1993) at which to store ultra-dry seed. The effects of ultra-dry storage on seed vigor and early seedling establishment have to be thoroughly understood in order to produce high-quality seedling stands. Reports on seedling physiology after ultra-dry storage of seeds are scarce and confirm the possible impact on seed quality (Ellis et al. 1980; Ellis et al. 1998; Zhang et al. 2019). There are many factors that affect seed storage tolerance, including endogenous genetic factors, dormancy characteristics, maturity, firmness, MC, composition, and seed vitality. Additionally, external factors, such as storage temperature, moderation, physical factors, and chemical factors also affect seed storage tolerance (Ellis et al. 1980; Walters 1998; Holdsworth et al. 2008). Controlled storage conditions can delay seed deterioration and increase seed storage life. As a case in point, low-temperature storage and ultra-dry storage are effective methods for seed conservation (Harrington 1973; Wang et al. 2018). After H. bodinieri seeds were dried and dehydrated, their ultra-dry storage tolerance showed a strong relationship with seed contents of protective substances, cell membrane integrity, protective enzymes activity, and extent of lipid peroxidation. As seed MC decreases during storage, the cytoplasm becomes vitreous under the influence of sugars, whereby cell respiratory metabolism is inhibited and seed damage due to dehydration is reduced (Hendry 1993; Ballesteros et al. 2011). After ultra-dry treatment, the conformation of soluble proteins inside the seed changes, and interactions with other substances in the cell increases protein thermal stability, thereby preventing protein denaturation during dehydration and ensuring the integrity of cell membrane structure (Rajjou et al. 2008). Thus, during ultra-dry storage, favorable changes in soluble sugars within the seed and protein stability guarantee the desiccation tolerance of the seed. The changes in soluble sugar and protein contents during ultra-dry storage observed in our experiments suggested that the desiccation tolerance of H. bodinieri seeds was closely related to these contents (Figs. 3A, 3B). MDA is a well-established marker of oxidative stress (López-Fernández et al. 2018). Remarkably, MDA content of H. bodinieri seeds (mc1-mc4) was lower than that of the control seeds (mc5), whereas SOD and POD enzyme contents increased, which effectively maintained the corresponding enzyme activity and, consequently, membrane structure integrity.

Our experiments showed that ultra-dry seeds of H. bodinieri with an MC of 3.14% to 4.77% showed strong desiccation tolerance, indicating that this MC range is conducive to ultra-dry storage of H. bodinieri seeds.

4.2 Germination of ultra-dry stored seed requires seed priming

The effect of ultra-dry storage on seed vigor, tissue and cell morphology, and genetic material stability depends on the level of seed storage tolerance and the specific conditions of storage environment. However, even for storage-tolerant seeds, ultra-dry treatment may cause water to quickly enter the seed in response to a low water potential during seed germination (Ballesteros et al. 2011), which may cause damage to the cell membrane and result in solute leakage into the apoplast (Bewley et al. 2013). It is possible to reduce or eliminate such damage through proper osmotic adjustment and gradual priming before germination (Jisha et al. 2013), such as natural priming, PEG priming, or saturation water vapor priming, which can improve the quality parameters of aging seeds, such as the germination rate and seedling vigor; furthermore, priming may even restore the original germination rate (Bailly et al. 1998; Murthy et al. 2003).

In this study, natural priming was the most time-consuming priming treatment. On the other hand, compared with saturation water vapor priming (Fig. 5, CK), PEG priming significantly improved germination rate, seedling vigor, and seedling development of the ultra-dry-stored seeds. This may be attributable to the repairing effect of PEG on cells, enhancing antioxidant activity and reducing MDA accumulation (Butler et al. 2009). However, these findings were not a general occurrence, and PEG concentration greatly affected the effect of osmotic adjustment. If improperly used, PEG will not only exert an osmotic adjustment effect, but it may also negatively affect cell membrane repair (Draganic et al. 2012). For example, high concentrations of PEG inhibited the germination of Lycopersicon esculentum Mill., Avena sativa L., Castanea mollissima Blume, and Betula luminifera H.J.P.Winkl. seeds (Kester et al. 1997; Xia et al. 2016). This was consistent with the inhibitory effect of 25% PEG priming on the germination of H. bodinieri seeds, although such effect by PEG treatment after 6 days of storage was very weak (Fig. 5). Thus, priming of ultra-dry stored seeds can reduce seed damage and promote seed germination. Among the various priming methods evaluated to date, PEG priming reportedly has the best effect (Xia et al. 2016).

4.3 Ultra-dry treatment promoted seed storage feasibility and seedling growth

Importantly, ultra-dry treatment was beneficial for seed storage, and all physiological indices reached stable levels, such as observed herein for ultra-dry storage of H. bodinieri seeds (Figs. 3 and 4). Ultra-dry treatment improved germination capacity as well as seedling growth (Table 3 and Fig. 5). Seemingly, ultra-dry storage possibly provides a means to reduce or eliminate the damage caused by seed priming, which regulates the extent of cell water absorption and hydration status, thus stabilizing and synchronizing water absorption by seeds and resulting in successful seedling development (Fig. 5). Ultra-dry storage reportedly had positive effects on seedling growth, photosynthesis, and respiratory rate in oil-, starch-, and some protein-rich seeds, such as Arachis hypogaea L., Sesamum indicum L., Oryza sativa L., Stylosanthes guianensis (Aubl.) Sw., Jatropha curcas L., and C. mollissima seeds (Cui et al. 2014).

Ebone et al. (2019) proposed three stages in the deterioration of stored seeds: in phase I, suppression of the protective mechanism against oxidative damage occurs; in phase II: membrane damage results following lipid peroxidation; and in phase III, seed viability decreases to the point that germination is inhibited. Seeds generally enter phase II of deterioration only after experiencing the phase I, and viable seedlings with suppressed growth are characteristic of phase II. In this study, ultra-dry storage at room temperature or at 4 °C promoted H. bodinieri seedling biomass, root system development, and seedling P, consistently with the reported growth pattern of Xanthoceras sorbifolium Bunge, which belongs to the same family. However, the ability of the seed to maintain membrane functionality and resist lipid peroxidation depends on its tolerance to ultra-drying (Zhang et al. 2019). In Fagus sylvatica L. (Pukacka et al. 2007) and Populus nigra L. (Kalemba et al. 2015), germination ability and seedling quality were negatively correlated with seed lipid peroxidation.

During the ultra-drying process, seed MC rapidly decreases, resulting in a series of physiological changes within the seeds that may induce dehydration stress. This dehydration stress is maintained for a certain period of storage and stimulates an increase in protective enzyme activity and protective substances content; however, once the seed reaches a threshold level of dehydration, the content of protective substances decreases (Walters 2015). Nonetheless, cell membrane structure and function in the seed can be maintained through proper priming, thereby reducing seed damage, likely by removing harmful substances generated during germination and by repairing cell structure (Xia et al. 2016). All these processes require the participation of antioxidant enzymes, the contents of which increase during germination (Demir et al. 2007). Overall, our data indicated that ultra-dry stored H. bodinieri seeds did not enter phase II of deterioration.

Compared with ultra-dry stored H. bodinieri seeds, antioxidant enzyme activities and MDA content in conventionally stored seeds were not different. The increase in antioxidant enzymes after the sowing of ultra-dry stored seeds may occur to eliminate harmful substances, thus allowing for the observed promotion of normal, healthy seedling growth after ultra-dry storage (Figs. 6 and 7). Further, low-temperature storage was better than room temperature storage, which was more conducive to the accumulation of SS and SP in seedlings, thereby increasing photosynthesis rate and seedling absorption of N, P, and K. This phenomenon has also been reported in Brassicaceae Burnett and legumes (Zhu et al. 2007; Wawrzyniak et al. 2020).

5 Conclusions

This study showed that ultra-dry storage of H. bodinieri seeds prevented significant changes in seed viability or seedling vigor; furthermore, seed deterioration due to storage was minimized. Therefore, low-temperature ultra-dry storage was more conducive to seed germination and normal seedling growth thereafter, than room-temperature ultra-dry storage, such that the comprehensive index of seedling growth and the physiological activity were higher in the first case. Ultra-dry treatment was especially beneficial for the storage of H. bodinieri seeds with an MC of 3.14%–4.77%, and all physiological indices measured reached a stable level in these seeds. Ultra-dry storage of H. bodinier seeds may be used to reduce or eliminate seed damage caused by PEG seed-priming, thus promoting successful seedling emergence. As we confirmed desiccation tolerance of H. bodneri seeds, we believe that it is possible to classify the seeds of this species as of the orthodox storage type.

Author contributions

Conceptualization, Z.L. and J.P.; methodology, Z.L.; software, C.X.; validation, T.L.; formal analysis, Z.L.; investigation, S.G.; data curation, C.X.; writing and original draft preparation, C.X.; writing, X.X. and Z.L.; All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31560200).

Acknowledgments

This research were funded by the National Natural Science Foundation of China (31560200) and Guangxi University “Innovation and Entrepreneurship Training Program for College Students”. Xiaoxue Li, Meifang Jiang provided help for the experiment.

Conflicts of Interest

The authors declare that they have no conflict of interest.

References

Bailly C, Benamar A, Corbineau F, Côme D (1998) Free radical scavenging as affected by accelerated ageing and subsequent priming in sunflower seeds. Physiol Plant 104: 646–652. https://doi.org/10.1034/j.1399-3054.1998.1040418.x.

Ballesteros D, Walters C (2011) Detailed characterization of mechanical properties and molecular mobility within dry seed glasses: relevance to the physiology of dry biological systems. Plant J 68: 607–619. https://doi.org/10.1111/j.1365-313X.2011.04711.x.

Bewley JD, Bradford KJ, Hilhorst HMW, Nonogaki H (2013) Seeds: physiology of development, germination and dormancy 3rd edition. Seed Sci Res 23, article id 289. https://doi.org/10.1017/S0960258513000287.

Blum A, Ebercon A (1981) Cell membrane stability as a measure of drought and heat tolerance in wheat¹. Crop Sci 21: 43–47. https://doi.org/10.2135/cropsci1981.0011183X002100010013x.

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254. https://doi.org/10.1016/0003-2697(76)90527-3.

Butler LH, Hay FR, Ellis RH, Smith RD, Murray TB (2009) Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage. Ann Bot 103: 1261–1270. https://doi.org/10.1093/aob/mcp059.

Cao LM, Xia NH, Deng YF (2008) Embryology of Handeliodendron bodinieri (Sapindaceae) and its systematic value: development of male and female gametophytes. Plant Syst Evol 274: 17–23. https://doi.org/10.1007/s00606-008-0024-0.

Clemensson-Lindell A (1994) Triphenyltetrazolium chloride as an indicator of fine-root vitality and environmental stress in coniferous forest stands: applications and limitations. Plant Soil 159: 297–300. https://doi.org/10.1007/BF00009293.

Cui K, Wang H, Li K, Liao S, Li L, Zhang C (2014) Physiological and biochemical effects of ultra-dry storage on barbados nut seeds. Crop Sci 54: 1748–1755. https://doi.org/10.2135/cropsci2013.10.0680.

Demir I, Ozcoban M (2007) Dry and ultra-dry storage of pepper, aubergine, winter squash, summer squash, bean, cowpea, okra, onion, leek, cabbage, radish, lettuce and melon seeds at –20°C and 20°C over five years. Seed Sci Technol 35: 165–175. https://doi.org/10.15258/sst.2007.35.1.15.

Dhindsa R (1981) Leaf senescence: correlation with increased levels of membrane permeability and lipid peroxidation and increased levels of superoxide dismutase and catalase. J Exp Bot 32: 93–101. https://doi.org/10.1093/jxb/32.1.93.

Draganic I, Lekic S (2012) Seed priming with antioxidants improves sunflower seed germination and seedling growth under unfavorable germination conditions. Turk J Agric For 36: 421–428. https://doi.org/10.3906/tar-1110-16.

Ebone LA, Caverzan A, Chavarria G (2019) Physiologic alterations in orthodox seeds due to deterioration processes. Plant Physiol Biochem 145: 34–42. https://doi.org/10.1016/j.plaphy.2019.10.028.

Ellis RH (1998) Longevity of seeds stored hermetically at low moisture contents. Seed Sci Res 8: 9–10.

Ellis RH, Roberts EH (1980) Improved equations for the prediction of seed longevity. Ann Bot 45: 13–30. https://doi.org/10.1093/oxfordjournals.aob.a085797.

Ellis RH, Roberts EH (1998) How to store seeds to conserve biodiversity. Nature 395, article id 758. https://doi.org/10.1038/27361.

Ellis RH, Hong TD, Roberts EH (1986) Logarithmic relationship between moisture content and longevity in sesame seeds. Ann Bot 57: 499–503. https://doi.org/10.1093/oxfordjournals.aob.a087131.

Ellis RH, Hong TD, Roberts EH, Tao KL (1990) Low moisture content limits to relations between seed longevity and moisture. Ann Bot 5: 493–504. https://doi.org/10.1093/oxfordjournals.aob.a087961.

Ellis RH, Hong TD, Roberts EH (1995) Survival and vigour of lettuce (Lactuca sativa L.) and Sunflower (Helianthus annuus L.) seeds stored at low and very-low moisture contents. Ann Bot 76: 521–534. https://doi.org/10.1006/anbo.1995.1128.

Engelmann F (2011) Use of biotechnologies for the conservation of plant biodiversity. In Vitro Cell Dev-Pl 47: 5–16. https://doi.org/10.1007/s11627-010-9327-2.

Farhoosh R, Einafshar S, Sharayei P (2009) The effect of commercial refining steps on the rancidity measures of soybean and canola oils. Food Chem 115: 933–938. https://doi.org/10.1016/j.foodchem.2009.01.035.

Guo S, Li ZL, Xue JH (2019a) Establishment and optimization of comprehensive evaluation model for seed and seed oil traits of Handeliodendron bodinieri. Transactions of the CSAE 6: 314–322. https://doi.org/10.11975/j.issn.1002-6819.2019.06.038.

Guo S, Li ZL, Xue JH (2019b) Economic properties of seeds of Hahdeliodehdroh Bodihieri from different provenances. Scientia Silvae Sinicae 4: 84–96. https://doi.org/10.11707/j.1001-7488.20190409.

Harrington JF (1973) Biochemical basis of seed longevity. Seed Sci Technol 1: 453–461.

He R, Wang J, Huang HW (2012) Long-distance gene dispersal inferred from spatial genetic structure in Handeliodendron bodinieri, an endangered tree from karst forest in southwest China. Biochem Syst Ecol 44: 295–302. https://doi.org/10.1016/j.bse.2012.06.005.

Hendry GAF (1993) Oxygen, free radical processes and seed longevity. Seed Sci Res 3: 141–153. https://doi.org/10.1017/S0960258500001720.

Hoekstra FA, Golovina EA, Buitink J (2001) Mechanisms of plant desiccation tolerance. Trends Plant Sci 6: 431–438. https://doi.org/10.1016/S1360-1385(01)02052-0.

Holdsworth MJ, Bentsink L, Soppe WJJ (2008) Molecular networks regulating Arabidopsis seed maturation, after‐ripening, dormancy and germination. New Phytol 179: 33–54. https://doi.org/10.1111/j.1469-8137.2008.02437.x.

Hong TD, Ellis RH, Astley D, Pinnegar AE, Kraak HL (2005) Survival and vigour of ultra-dry seeds after ten years of hermetic storage. Seed Sci Technol 33: 449–460. https://doi.org/10.15258/sst.2005.33.2.17.

Huo P, Li J, Liu J, Zhang S, Shi S (2015) Cross adaptation of Medicago sativa seedlings germinated from ultra-dried seeds to saline and alkaline stresses. Int J Agric Biol 17: 860–868. https://doi.org/10.17957/IJAB/15.0015.

Irigoyen JJ, Emerich DW, Sanchez-Diaz M (1992) Water stress induced changes in concentrations of proline and total soluble sugars in nodulated alfalfa (Medicago sativa) plants. Physiol Plant 84: 55–60. https://doi.org/10.1111/j.1399-3054.1992.tb08764.x.

Jiang B, Wang LY, Xu CT, Yan M (2020) Hydropriming enhances the germination of aged ultra-dry wheat seeds. Seed Sci Technol 48: 57–63. https://doi.org/10.15258/sst.2020.48.1.08.

Jisha KC, Vijayakumari K, Puthur JT (2013) Seed priming for abiotic stress tolerance: an overview. Acta Physiol Plant 35: 1381–1396. https://doi.org/10.1007/s11738-012-1186-5.

Kalemba EM, Suszka J, Ratajczak E (2015) The role of oxidative stress in determining the level of viability of black poplar (Populus nigra) seeds stored at different temperatures. Funct Plant Biol 42: 630–642. https://doi.org/10.1071/FP14336.

Kester ST, Geneve RL, Houtz RL (1997) Priming and accelerated ageing affect L-isoaspartyl methyltransferase activity in tomato seed. HortSci 31: 632d-632. https://doi.org/10.21273/HORTSCI.31.4.632d.

Leng XH, Xue L, Wang J, Li S, Yang ZL, Ren H.D, Yao XH, Wu Z, Li JY (2020) Physiological responses of Handeliodendron bodinieri (Levl.) Rehd. to exogenous calcium supply under drought stress. Forests 11, article id 69. https://doi.org/10.3390/f11010069.

Li Y, Feng H-Y, Chen T, Yang X-M, An L-Z (2007) Physiological responses of Limonium aureum seeds to ultra-drying. J Integr Plant Biol 49: 569–575. https://doi.org/10.1111/j.1744-7909.2007.00452.x.

Li Y, Qu J, Zhang W, An L, Xu P, Li Y (2010) Impact of ultra-dry storage on vigor capacity and antioxidant enzyme activities in seed of Ammopiptanthus mongolica. Bot Stud 51: 465–472.

López-Fernández MP, Moyano L, Correa MD, Vasile F, Maldonado S (2018) Deterioration of willow seeds during storage. Sci Rep 8, article id 17207. https://doi.org/10.1038/s41598-018-35476-3.

Mira S, Nadarajan J, Liu U, Gonzalez-Benito ME, Pritchard HW (2019) Lipid thermal fingerprints of long-term stored seeds of brassicaceae. Plants 8, article id414. https://doi.org/10.3390/plants8100414.

Murthy UMN, Kumar PP, Sun WQ (2003) Mechanisms of seed ageing under different storage conditions for Vigna radiata (L.) Wilczek: lipid peroxidation, sugar hydrolysis, Maillard reactions and their relationship to glass state transition. J Exp Bot 54: 1057–1067. https://doi.org/10.1093/jxb/erg092.

O’Neill JV, Webb RA (1970) Simultaneous determination of nitrogen, phosphorus and potassium in plant material by automatic methods. J Sci Food Agric 21: 217–219. https://doi.org/10.1002/jsfa.2740210501.

Paunescu A (2009) Biotechnology for endangered plant conservation: a critical overview. Rom Biotech Lett 14: 4095–4103.

Pérez-García F, González-Benito ME, Gómez-Campo C (2007) High viability recorded in ultra-dry seeds of 37 species of Brassicaceae after almost 40 years of storage. Seed Sci Technol 35: 143–153. https://doi.org/10.15258/sst.2007.35.1.13.

Pukacka S, Ratajczak E (2007) Age-related biochemical changes during storage of beech (Fagus sylvatica L.) seeds. Seed Sci Res 17: 45–53. https://doi.org/10.1017/S0960258507629432.

Rajjou L, Debeaujon I (2008) Seed longevity: survival and maintenance of high germination ability of dry seeds. C R Biol 331: 796–805. https://doi.org/10.1016/j.crvi.2008.07.021.

Ranal MA, de Santana DG (2006) How and why to measure the germination process? Rev Bras Bot 29: 1–11. https://doi.org/10.1590/s0100-84042006000100002.

Ren H, Huang B, Fernández-García V, Miesel J, Yan L, Lv C (2020a) Biochar and rhizobacteria amendments improve several soil properties and bacterial diversity. Microorganisms 8, article id 502. https://doi.org/10.3390/microorganisms8040502.

Ren H, Qin X, Huang B, Fernández-García V, Lv C (2020b) Responses of soil enzyme activities and plant growth in a eucalyptus seedling plantation amended with bacterial fertilizers. Arch Microbiol 202: 1381–1396. https://doi.org/10.1007/s00203-020-01849-4.

Ren H, Warnock DD, Tiemann LK, Quigley K, Miesel JR (2021) Evaluating foliar characteristics as early indicators of plant response to biochar amendments. For Ecol Manage 489, article id 119047. https://doi.org/10.1016/j.foreco.2021.119047.

Sudhakar C, Lakshmi A, Giridarakumar S (2001) Changes in the antioxidant enzyme efficacy in two high yielding genotypes of mulberry (Morus alba L.) under NaCl salinity. Plant Sci 161: 613–619. https://doi.org/10.1016/S0168-9452(01)00450-2.

Taylor AG, Lee SS, Beresniewicz MM, Paine DH (1995) Amino acid leakage from aged vegetable seeds. Seed Sci Technol 1: 113–122.

Van Bilsen D, Hoekstra FA, Crowe LM, Crowe JH (1994) Altered phase behavior in membranes of aging dry pollen may cause imbibitional leakage. Plant Physiol 104: 1193–1199. https://doi.org/10.1104/pp.104.4.1193.

Vertucci CW, Roos EE (1993) Theoretical basis of protocols for seed storage II. The influence of temperature on optimal moisture levels. Seed Sci Res 3: 201–213. https://doi.org/10.1017/S0960258500001793.

Walters C (1998) Understanding the mechanisms and kinetics of seed aging. Seed Sci Res 8: 223–244. https://doi.org/10.1017/S096025850000413X.

Walters C (2015) Orthodoxy, recalcitrance and in-between: describing variation in seed storage characteristics using threshold responses to water loss. Planta 242: 397–406. https://doi.org/10.1007/s00425-015-2312-6.

Wang J, Gao P, Kang M (2008) Isolation and characterization of polymorphic microsatellite markers for the endangered tree Handeliodendron bodinieri (Sapindaceae). Conserv Genet 9: 727–729. https://doi.org/10.1007/s10592-007-9363-6.

Wang WQ, He A, Peng SB, Huang JL, Cui KH, Nie LX (2018) The effect of storage condition and duration on the deterioration of primed rice seeds. Front Plant Sci 9, article id 172. https://doi.org/10.3389/fpls.2018.00172.

Wang XF, Jing XM, Lin J (2005) Starch mobilization in ultradried seed of maize (Zea mays L.) during germination. J Integr Plant Biol 47: 443–451. https://doi.org/10.1111/j.1744-7909.2005.00088.x.

Wawrzyniak MK, Michalak M, Chmielarz P (2020) Effect of different conditions of storage on seed viability and seedling growth of six European wild fruit woody plants. Ann For Sci 77, article id 58. https://doi.org/10.1007/s13595-020-00963-z.

Xi CL, Xiao X, Gu S, Jian MF, Li ZL (2020) Research trends of the rare and endangered plant - Handeliodendron bodinieri. Mol Plant Breed 18: 1725–1730. https://doi.org/10.13271/j.mpb.018.001725.

Xia FS, Chen LL, Yan HF, Sun Y, Li ML, Mao PS (2016) Antioxidant and ultrastructural responses to priming with PEG in aged, ultra-dry oat seed. Seed Sci Technol 44: 556–568. https://doi.org/10.15258/sst.2016.44.3.12.

Yan M (2017) The preliminary study on the optimum moisture content of ultra-dry storage and its related chemicals in seeds from six crop species. Plant Genet Resour 15: 506–514. https://doi.org/10.1017/S1479262116000216.

Zhang JP, Wang HY, Liao SX, Cui K (2019) Appropriate ultra-low seed moisture content stabilizes the seed longevity of Calocedrus macrolepis, associated with changes in endogenous hormones, antioxidant enzymes, soluble sugars and unsaturated fatty acids. New Forest 50: 455–468. https://doi.org/10.1007/s11056-018-9670-4.

Zheng GH, Jing XM, Tao KL (1998) Ultradry storage cuts cost of gene bank. Nature 393: 223–224. https://doi.org/10.1038/30383.

Zhu C, Chen J (2007) Changes in soluble sugar and antioxidant enzymes in peanut seeds during ultra dry storage and after accelerated aging. Seed Sci Technol 35: 387–401. https://doi.org/10.15258/sst.2007.35.2.14.

Total of 66 references.