Effect of Culture Temperatures on the Initial Growth Performance of Seedlings Germinated from Cryostored Seeds of a Tropical Tree Parkia nitida Miq. (Fabaceae, Mimosoideae)

Abstract

Seedling growth is one of the most important stages for the establishment of natural and artificial regeneration. For the first time, the initial growth and biomass allocation of seedlings germinated from cryostored seeds of Parkia nitida were analyzed. P. nitida is an economically and ecologically important timber tree species distributed in Central and South America. Cryostored seeds germinated quickly after priming by scarifying a part of the seedcoat with emery paper, reaching a germination percentage of 94%. Thirteen weeks after germination, the seedlings grew to a height of 16.5 to 60.0 cm. The results of our study, under different day/night alternating culture temperatures, showed that culture temperature had a direct correlation with seedling growth, total biomass allocation, and biomass partitioning. The greatest growth (height, diameter, and number of node sections) and greatest biomass allocation (leaf, stem, and root weight) were recorded under alternating temperatures of 30/25 °C, and these decreased with decreasing culture temperatures to 25/20 °C and 20/15 °C. Shoot:Root (S:R) ratios also decreased with decreasing culture temperatures, but a statistically significant difference (p < 0.05) was only observed between 20/15 °C and 30/25 °C. However, significant differences were not observed in Photosynthetic:Non-photosynthetic organ ratios among the different alternating culture temperatures. This study provides fundamental information for the production of good-quality seedlings of the fast-growing tropical trees of the legume family.

1. Introduction

Parkia (Fabaceae, Mimosoideae) is a pantropical genus of 30 or more species, about half of which occur in the neotropics, from Honduras to southeastern Brazil [1]. The genus is the taxonomically most diverse in the rainforest of the Amazon basin [2,3]. Parkia nitida Miq. (syn. Parkia oppositifolia Spruce ex Benth.), one of the most common Parkia species in neotropics, is a medium- to large-sized tree up to 40 m high, widely distributed in Central and South America from southern Panama to the central region of Amazonia [1,4,5]. This species is a timber tree of economic and ecological importance due to its wood and the possibility of rapid growth in degraded areas [6]. Additionally, the pod gums of this species represent one of the most important food sources in the diet of monkeys, parrots, deer, peccaries, and rodents [4,7]. Moreira and Franco [8] reported that the pods produce 13.5% extracted gum based on dry weight, and can be a substitute for arabic gum. Moreover, numerous reports demonstrate that plants from the genus Parkia possess medicinal values to treat several ailments, such as diabetes, diarrhea, wounds, hypertension, cough, chronic piles, conjunctivitis, and measles [9].

P. nitida is a fast-growing species with great potential for reforestation activities, especially for plantations in degraded areas [10,11]. However, one of the greatest difficulties in the use of tropical tree species in agroforestry systems, afforestation, and the restoration and recovery of degraded areas is to obtain good quality seedlings on a commercial scale [12]. Seed germination and seedling growth are two important stages for the establishment of natural and artificial regeneration. Additionally, seed storage is an important aspect to consider in afforestation plans, especially for species that do not produce seeds regularly. Among storage methods, cryopreservation is now one of the most powerful tools available in germplasm collections for the long-term storage of valuable genetic material [13]. It has been shown that it is possibles to maintain a high germination rate for an extended period of time, and it has extreme importance as a permanent method for genetic resource conservation. The successful storage of seeds from several important Amazonian trees, including P. nitida, was reported using the cryopreservation method [14]. On the other hand, the seed dormancy mechanism, which accompanies the impermeability of their covers to water, is one of the simplest but most effective means of preventing or delaying germination [15]. This dormancy mechanism is commonly exhibited by legume species with hard-coated seeds [16]. The seeds of P. nitida have hard-coated seeds, which prevents germination. Chemical or mechanical scarification is reported as an effective method to break dormancy in this species, which consequently accelerates and improves the germination percentage [15,17].

The location of the nursery where these stored seeds will be grown commercially, i.e., the growing environment feasible for seedling growth, is also an important issue. Basic knowledge, such a suitable temperature for P. nitida, will contribute not only to the feasibility of ex situ seedling production, but also to understanding the temperature adaptability and predicting the future distribution of this species. However, there is no information on the initial patterns of growth and biomass allocation in P. nitida seedlings under different temperature conditions. The analysis of biomass allocation patterns is crucial for the evaluation of the performance of plants experiencing different environmental conditions. Biomass allocation ratios are often used in the literature to standardize biological data, and one of the most used ratios is the Shoot:Root (S:R) ratio, which summarizes the trade-off between above- and below-ground investments [18]. The hormetic response of the S:R ratio can have an important role in forestry for producing seedlings with desired characteristics to achieve maximum health/productivity and resilience under plantation conditions [19]. Biomass partitioning is one of the keys to a plant’s ability to compensate for limited environmental resources and thus survive and succeed in competition [20]. Understanding how plant biomass is distributed between roots, stems, and leaves is central to many questions in life-history evolution, ecology, and ecosystem studies [21]. Notwithstanding the above, the growth and biomass partitioning mechanism is a very complex process influenced by many factors and variables, making it difficult to understand and sometimes be misinterpreted.

Here, we describe for the first time the initial growth and biomass allocation in Parkia nitida seedlings grown under different culture temperatures in controlled environment chambers. Growth parameters (height, basal diameter, number of nodal sections, dry basis weight of leaves, stems, and roots) and biomass allocation ratios (S:R ratio and Photosynthetic:Non-photosynthetic organ ratio) were evaluated to compare the effects of different culture temperatures on initial growth and biomass partitioning.

The main objective of this study was to know the initial growth patterns and biomass allocations under different temperature conditions, focused on determining the optimal biomass partition that allows obtaining vigorous seedlings with good performance in the field.

2. Material and Method

2.1. Plant Materials

The pods containing seeds were collected in July 1987 at the Alexander von Humboldt National Forest, Pucallpa, Peru, within the experimental area belonging to the National Institute for Agricultural Innovation (INIA-Pucallpa). The ripe pods were collected directly from the mother tree and subsequently dried in the shade at room temperature until they opened naturally. The average seed sizes were 17.2 × 9.0 × 6.2 mm (length × width × thickness) and the average seed weight and moisture content were 717.9 mg and 14.0%, respectively. Moisture content of seeds was determined by measuring the change in weight after drying the sample in an electric oven (Hirayama MFG. Corp., Tokyo, Japan) at 103 °C for 17 h. The seeds were stored for about one year at 5 °C before the cryostorage test.

2.2. Cryostorage of Seeds

Cryostorage and seed germination tests were performed in August 1988. The 5 mL Nunc cryotubes (Thermo Scientific, MA, USA), containing five seeds in each, were fixed in aluminium canes and stored in a BioCane^TM 20 cryo tank (Thermo Scientific, Waltham, MA, USA) with liquid nitrogen (–196 °C) for 1 day. After storage in liquid nitrogen, the samples were removed from the tank and warmed by rapidly transferring cryotubes in a water bath at 40 °C for 2 to 3 min. After warming, the cryotubes containing samples were kept at room temperature before preparing them for germination tests.

2.3. Priming Treatment and Seed Germination

Before sowing, cryostored and control unstored seeds were primed by scarifying a part of the seedcoat with emery paper as shown in Figure 1A. After the priming, seeds were disinfected with 70% Ethanol for 3 min and then with 5% (w/v available chlorine) sodium hypochlorite solution (Wako Pure Chemical, Osaka, Japan) for 45 min. After surface-sterilization, seeds were rinsed five times with sterile distilled water for 5 min each time inside a laminar flow cabinet. Subsequently, the samples were aseptically cultured in glass plates containing 1/4 MS medium [22] (MS medium with basal salts reduced to the quart concentration from the standard), supplemented with 20 g L⁻¹ sucrose and solidified with 6 g L⁻¹ agar (Wako Pure Chemical, Osaka, Japan). The pH was adjusted to 5.8 prior to autoclaving the medium for 15 min at 121 °C. Germination tests were conducted at 25 °C under photon flux density of 45–65 µmol m⁻² s⁻¹ for 16 h daily. Each treatment had 5 replicates (Petri dish) with each replicate containing 25 seeds. Control seedlings from unstored seeds were not used for subsequent growth analysis.

2.4. Growth of Seedlings under Different Culture Temperatures

Approximately 2 to 3 weeks after germination, seedlings were carefully transferred to Wagner’s pots ICW-2 (ICM Co., Ltd., Tsukuba, Japan) containing vermiculite and irrigated daily with a nutrient solution modified from Nagao [23], containing in mg L⁻¹: NH₄NO₃ 143.0, NaH₂PO₄·2H₂O 55.1, KCl 47.1, CaCl₂·2H₂O 52.5, MgSO₄·7H₂O 61.0, Fe-III EDTA 25.0, Cu EDTA 0.1, Mn EDTA 0.1, Zn EDTA 0.1, H₃BO₃ 1.5, KI 0.01, CoCl₂·6H₂O 0.005, and MoO₃ 0.005. Every day in the morning, at the same time, the nutrient solution was supplied by automatic pumping to the culture tank until it flooded to approximately two-thirds of the height of the Wagner pot containing the seedlings. The nutrient solution level was maintained for 1 h and then the nutrient solution was automatically returned to the storage tank. The nutrient solution was changed once every two weeks. The seedlings were cultured inside growth chambers at 15/20 °C; 20/25 °C; and 25/30 °C alternating temperatures, 12 h day/12 h night, respectively. The day culture condition period was under photon flux density of 365–400 µmol m⁻² s⁻¹ for 12 h daily, whereas the night culture condition period was in darkness. The relative humidity condition for all chambers in the day/night culture conditions was set to 75%. Each treatment contained ten seedlings.

After 13 weeks of culture, the growth of seedlings was evaluated considering the following parameters: height, basal diameter, number of nodal sections, dry basis weight of leaves, stems, and roots, as well as the S:R ratio and Photosynthetic:Non-photosynthetic organ ratio to compare the effects of different culture temperatures on the initial growth and biomass allocation. The S:R ratio was calculated based on the dry matter weight of the aerial part (shoot dry weight, which includes the leaves and stem dry weight) and the underground part (roots’ dry weight). The dry weight of plant materials was determined after 72 h at 80 °C. The Photosynthetic:Non-photosynthetic organ ratio (leaf vs. stem and root) was also calculated as described by Monsi and Saeki [24].

2.5. Data Analysis

Differences in seed germination in two weeks after sowing were tested using generalized linear models (GLMs). Seed germination per Petri dish was the response variable and with or without cryostorage was the explanatory variable. The response variable was assumed to follow a binomial distribution. To investigate whether factors other than temperature (i.e., seedling size) affected biomass allocation, we first checked the correlation between each growth and biomass allocation parameter variable. The R package GGally [25] was used for pairwise comparison of Pearson’s correlation coefficients and drawing. For comparison with other parameters for which a normal distribution is assumed, the allocation values were log-transformed during the comparison according to Poorter and Sack [18]. The initial growth and allocation pattern of seedlings under different temperature conditions was evaluated using GLMs and multiple comparisons of Tukey’s HSD test (BH adjusted). GLMs were constructed with each growth measurement and allocation value as response variables and growth conditions as explanatory variables. For the same reason described as above, the error distribution of the response variable was assumed to follow a gamma distribution with log link function for the allocation patterns (S:R ratio and Photosynthetic:Non-photosynthetic organ ratio). The number of nodes, which is the count data, was assumed to follow a Poisson distribution, and the rest followed a Gaussian distribution (i.e., linear regression models). Subsequent multiple comparisons between temperature conditions were performed using the R package multicomp [26]. These analyses were performed under R version 4.3.1 [27].

3. Result

3.1. Seed Germination

The cryostored seeds germinated almost simultaneously about three to five days after sowing under aseptically conditions (Figure 1B). Two weeks after sowing, the germination rate reached an average percentage of about 94% and 92% for cryostored and unstored (control) seeds, respectively. No statistical difference was found between them (Pr(>|z|) = 0.625, GLM analysis). Additionally, no differences concerning the morphological aspect of seedlings were observed.

3.2. Initial Growth and Biomass Partitioning

Significant correlations were found among the respective coefficients (Figure 2). Significant but weak correlations were observed between S:R ratios and growth parameters (r = 0.401–0.631), except for root dry weight (r = 0.244). Conversely, almost all the comparisons between Photosynthetic:Non-photosynthetic organ ratios and growth parameters were not significant (r = 0.104–0.398).

GLM analyses showed that statistically significant differences (p < 0.05) were observed in terms of height, diameter, leaf, stem, and root weights among the three different culture temperatures, whereas regarding the number of node sections, a significant difference was only observed between the 30/25 °C and 20/15 °C culture temperatures (Figure 3). The most significant growth (height: 40.0–69.0 cm, diameter: 8.7–10.5 mm, and number of node sections: 7–10) and the greatest biomass allocation (leaf: 8.9–17.1 g, stem: 3.6–7.4 g, and root weight: 2.5–4.2 g) was recorded under alternating temperatures of 30/25 °C, which decreased with decreasing culture temperatures, recording the worst growth (height: 16.5–26.0 cm, diameter: 4.0–6.7 mm, and number of node sections: 3–6) and lowest biomass allocation (leaf: 0.6–4.6 g, stem: 0.6–1.8 g, and root weight: 0.3–1.6 g) at 20/15 °C. (Figure 1C,D and Figure 3).

S:R ratios also decreased with decreasing culture temperatures (ranges from 3.42 to 7.03, 3.80 to 6.55, and 2.61 to 6.48 for 30/25 °C, 25/20 °C, and 20/15 °C, respectively), but only a statistically significant difference (p = 0.005) was observed between the alternating temperatures of 20/15 °C and 30/25 °C (Figure 4A). On the other hand, statistically significant differences were not observed (p > 0.05) in Photosynthetic:Non-photosynthetic organ ratios among the different culture temperatures. Ranges from 1.10 to 1.94, 1.17 to 1.91, and 0.74 to 1.78 were achieved for 30/25 °C, 25/20 °C, and 20/15 °C, respectively (Figure 4B).

4. Discussion

4.1. Seed Germination

Due to the high germination rate of the cryostored seeds being the result of a storage period of only one day, this does not guarantee that P. nitida seeds can be preserved for a long time at low storage temperatures. However, because there were no differences in germination percentages compared with the control, this result suggests that the seeds of this species are suitable for storage in liquid nitrogen. Therefore, more research to clarify this potential is very important for future afforestation programs and the germplasm conservation of Parkia species. On the other hand, the rapid germination and the high percentage of conversion into seedlings confirm the effectiveness of the priming applied to the seeds. In contrast, without scarification treatment, most seeds are not able to germinate [15]. The mechanical scarification of seed coats with emery paper (Figure 1A) was reported as the best priming method for breaking dormancy in P. nitida and Schizolobium amazonicum seeds [15]. Similarly, Cruz, et al. [17] reported that treatments using H₂SO₄ and mechanical scarification of seeds were the best treatments for breaking dormancy in P. nitida seeds. Seed dormancy is a frequent phenomenon in tropical species, causing slow and non-uniform germination. To overcome dormancy in legume species with a hard seed coat, treatments such as scarification on abrasive surfaces and hot water are efficient [28]. The hydropriming of seeds also increases germination rates and reduces the germination period [6]. In addition, aseptic germination in our study confirms the effectiveness of the seed surface-sterilization method for P. nitida, as reported by Maruyama and Hosoi [14].

4.2. Initial Growth and Biomass Partitioning

When growing P. nitida seedlings under different temperature conditions, the highest tree height and biomass were achieved in the 30/25 °C treatment, which is closest to the native climate (23.0 to 29.9 °C: average annual minimum and maximum temperatures in Pucallpa, Peru, 1991–2021, Climate-data.org: https://climate-data.org/ (accessed on 8 July 2024). The growth environment differed from the native climate; that is, as temperatures decreased in the present study, seedling size and biomass decreased. When producing good-growing seedlings, it is necessary to nurse them in a suitable growing environment that would be consistent with that of the natural distributions. On the other hand, it will be necessary as a future topic to analyze whether cryostorage affects the acquisition of resistance to cold temperatures. Additionally, careful consideration must be given to subsequent biomass allocations.

The seedling size, biomass in the shoot (stem and leaf) and root, and the S:R ratio decreased proportionally with decreasing temperatures. In addition, the allocation of each organ and the S:R ratio were also dependent on the seedling size. Pooter et al. [29] reported that low temperatures decrease the fraction of stems and leaves and increase the root mass fraction. Similarly, the S:R ratio average (about 2.3) of two larch species was minimal in comparison with that (5.1) of Scots pine stands, which grow on the continuous and non- or discontinuous permafrost regions in Siberia, respectively [30]. In our study, however, although the total biomass allocation, biomass partitioning, and S:R ratio decreased with decreasing culture temperatures, the decrease in biomass was proportional both in the above- and below-ground part, without a clear investment of biomass allocation to the roots. This result suggests that, due to the plant growth rate and, consequently, seedling size being positively related to the temperature of the culture, the proportional decrease in S:R ratio with decreasing temperature was a seedling size effect rather than a direct response of biomass partitioning to environmental conditions. Moreover, although reduced water uptake rates are often a likely cause of increased biomass allocation to roots, the optimal S:R ratio generally depends on the availability and ratio of light and nutrients [31]. Species in areas where water availability is high typically show small biomass allocations to roots and large biomass allocations to stems and leaves. Inversely, plants in low-rainfall habitats might benefit from a larger root system, as they will compete better for water [32,33,34]. Likewise, models of optimal biomass allocation in plants predict decreasing root allocation with increasing nutrient availability [34]. These reports were consistent with the relatively small biomass allocation to roots and large biomass allocation to stems and leaves achieved in P. nitida growing under favourable conditions (high availability of water, nutrients, and light).

On the other hand, the data recorded in terms of Photosynthetic:Non-photosynthetic organ ratios among the different culture temperatures (p > 0.05) (Figure 4B) were consistent with those reported for Acer platanoides, in which, despite the changes in the distribution of biomass due to plant size, the proportion of biomass in the foliage (proportion of photosynthetic organs) remained constant; while for Quercus robur, the proportion of foliage biomass decreased with increasing plant size [35]. The photosynthetic organ ratio in the biomass allocation patterns is considered an important parameter because photosynthesis strongly influences relative growth rates in a wide range of plant species and growth environments [36]. In experiments on temperate rainforest tree seedlings, Lusk, et al. [37] reported that at low nutrient availability, no clear differences in S:R ratio were apparent between poor-site and fertile-site species, whereas at high nutrient availability, the S:R ratio was markedly higher in fertile sites, resulting from differences in biomass allocation to stems (not leaves). In Pinus species, it was reported that S:R ratio is correlated with DBH, height, and age, but the relationship among them is hardly constant [38]. Drew and Ledig [39] showed that the ratio of S:R in loblolly pine seedlings remains constant for at least the first two years of development. Conversely, Cannell and Willet [40] showed that the S:R ratio varied seasonally in Sitka spruce and lodgepole pine, a fact they attributed to the periodicity of shoot development, which is rigidly controlled by photoperiod and temperature. Compared with herbaceous plants, woody plants allocate a more significant proportion of their biomass to non-photosynthetic organs, particularly wood. However, a great difference is observed within the species of woody flora. In addition, seedlings of woody species also varied significantly in their biomass allocation to leaves [41].

Much data in the literature prove that the relationship between the various organs is dependent on the developmental stage of the plant [42,43,44], water regime [33,45,46], nutrient availability [37,47,48,49,50,51,52], environment temperature [29,53,54,55], and availability of light [24,56,57,58,59]. Moreover, it is well established that biomass allocation patterns are not constant during plant ontogeny, which implies changes in the distribution of biomass according to the growth rate [35,43]. Thus, in most of the species that changed biomass allocation in response to the nutrient treatment, these changes were primarily a consequence of plant size [60,61], or in several cases, ontogenetical shifts in biomass allocation may have been wrongly interpreted as plastic responses to the environment [29,62]. Additionally, biomass allocation patterns may vary from species to species.

In summary, despite the fact that the relationships between metabolic production, growth, and patterns of biomass partitioning are fundamental to ecological and evolutionary theory, many aspects regarding how metabolic and biomass production are used to maintain and build the plant body remain unclear [21,29,62,63,64]. Furthermore, considering that phenotypic plasticity is one of the major means by which plants can cope with environmental factor variability, the extent to which this may influence environmental condition changes still remains largely unknown because the results are sometimes controversial [65,66]. Regardless, the size and ratio of above-ground to below-ground biomass may often have an important effect on seedlings’ vigour and growth on planting [67,68]. Although the optimal S:R ratio for planting Parkia seedlings has not been determined, the fact that this study was able to show that growth and S:R ratio can be controlled by temperature will be an important finding for future Parkia afforestation.

Despite more research being needed to improve our results, this is the first reported study on Parkia nitida to our knowledge. Therefore, we believe that this research can be helpful as a basis for future studies on producing good quality seedlings for afforestation, as well as for understanding and perhaps predicting the response of Parkia species to fluctuations in environmental factors, including climate change. Future research directions focused on determining the optimal biomass partitioning to obtain vigorous seedlings with good performance in the field will be a priority to put the research results into practice in Parkia species as well as in other fast-growing tropical forest trees.

5. Concluding Remarks

In conclusion, our experiments under controlled environmental conditions showed that culture temperature had a direct correlation with seedling growth, total biomass allocation, and biomass partitioning in Parkia nitida seedlings. This finding suggests that the biomass distribution, both in the above-ground and underground part of the plant, can be controlled by the culture temperature. On the basis of this knowledge, future research directions focused on determining the optimal biomass distribution to obtain vigorous seedlings with good performance in the field will be a priority in order to put the research results into practice in Parkia species.