Seed Quality and Seedling Production of Sequoia sempervirens, Sequoiadendron giganteum, and Pseudotsuga menziesii

Abstract

Given the ecological significance and potential for afforestation and carbon sequestration of these species, this study contributes to optimizing nursery practices for successful regeneration and conservation efforts. Thus, this research assessed the physical and physiological seed quality and seedling production of Sequoia sempervirens, Sequoiadendron giganteum, and Pseudotsuga menziesii. For seed characteristics the following were tested: (I) Tetrazolium at concentrations: 0.0%, 0.1%, 0.5%, 1.0%; (II) moisture content and thousand-seed weight; (III) in-lab germination; and (IV) the classification of seeds’ viability through the use of a seed blower. Meanwhile, seedling production was tested: (I) five compositions of substrates and (II) doses of a controlled-release fertilizer (14-14-14): 0.0, 2.0, 4.0, 6.0, 8.0 g L⁻¹, for S. giganteum. The seed evaluations revealed no significant effect of tetrazolium concentrations on determining their viability. The water content results classify all species as orthodox. All species’ seeds were classified as small according to the weight of a thousand seeds. A maximum of 41% germination was observed for both S. sempervirens and S. giganteum, this value was 56% for P. menziesii, attributed to non-viability and emptiness. The seed blower increased germination by more than 20% for S. giganteum and almost 40% for P. menziesii. Seedling production was affected by the substrates, and a dosage of 4.0 g L⁻¹ of the controlled-release fertilizer is recommended for S. giganteum.

1. Introduction

Introducing new species into the Brazilian forestry sector is crucial for diversifying the timber market. Currently, most planted forests in Brazil consist of the genus Pinus and Eucalyptus, covering approximately 10 million hectares [1]. In this context, the potential of Sequoia species (Sequoia sempervirens) and Giant Sequoia (Sequoiadendron giganteum) as valuable alternatives for expanding the forestry market is noteworthy. Cultivating these species can enhance the diversity of timber products, create new economic opportunities, and bolster the sustainability of the Brazilian forestry sector.

Sequoia sempervirens (D. Don) Endl., known simply as “Sequoia”, is a hexaploid plant species from the Cupressaceae family [2], native to the west coast of California in the United States. Sequoiadendron giganteum (Lindl.) Buchholz, also a member of the Cupressaceae family discovered in the mid-nineteenth century, is popularly known also as “Sequoia” or “Giant Sequoia” and “Sierra Redwood”. Due to their advantageous characteristics of rapid growth and wood quality under favorable conditions, their introduction into the Brazilian forestry sector might be an interesting alternative to the Pinus genus.

These trees’ impressive resilience and longevity have attracted the interest of scientists and businesses. While the species are largely protected within their natural ranges, they flourish and are prized for their decorative qualities and potential for timber production outside these areas [3]. Furthermore, alongside Sequoias, Pseudotsuga menziesii (Douglas-fir) can also be considered a viable alternative species.

Pseudotsuga menziesii (Mirb.) Franco, a conifer from the Pinaceae family native to the western coast of North America, is renowned as of the most commercially valuable lumber species globally [4]. To successfully cultivate these trees, it is crucial to have a comprehensive understanding of aspects related to seed production, both sexual and asexual, as well as the behavior of the seeds and seedlings. This knowledge is essential for the development of effective cultivation techniques.

Hence, the assessment of the initial seed quality from different species to effectively study propagation and seedling growth is fundamental [5], being evaluated based on physiological and physical characteristics. To assess the physiological quality of seeds, it is crucial to verify their viability, typically achieved through germination and tetrazolium tests. The germination test, commonly used to assess physiological quality, takes around 21 days for S. sempervirens and P. menziesii and 28 days for S. giganteum [6].

However, the tetrazolium test, also used for the same purpose, is more efficient in terms of completion time and results, making it a suitable option for seed quality analysis, especially for recalcitrant and slow-germinating seeds. However, there is no defined methodology for this test for S. sempervirens and S. giganteum. The identification of physical and physiological seed quality impacts germination and seed requirements [7,8,9]. Nevertheless, even though the factors related to seed germination are crucial for determining their quality, seedling production may involve many others.

The production of forest seedlings in nurseries is vital for ecological restoration, silviculture, and biodiversity conservation, but it is a complex process influenced by various factors. These conditions include the choice of appropriate substrates that directly influence root development, nutrient absorption, and seedling quality [10]. Substrate fertilization is crucial as it significantly accelerates development and reduces production costs [11].

In today’s market, there is a wide variety of fertilizers available with differences in chemical composition, physical form, and solubility rate [12,13]. Controlled-release fertilizers have emerged as an advantageous alternative for seedling production. These fertilizers are incorporated into the substrate during its preparation, eliminating the need for subsequent fertilization during seedling cultivation, resulting in higher-quality seedlings [14,15].

Thus, the objective of this study was to develop a methodology for conducting the tetrazolium test on Sequoia sempervirens, Sequoiadendron giganteum, and Pseudotsuga menziesii seeds. In addition, the objective was to evaluate the physical and physiological quality of these seeds before and after storage and to evaluate the effectiveness of commercial substrates in the production of seedlings for these species. Finally, this study analyzed the impact of different doses of controlled-release fertilizer on the production of Sequoiadendron giganteum seedlings.

2. Materials and Methods

2.1. Seminal Material

The seeds of Sequoia sempervirens were obtained from the United Kingdom, Sequoiadendron giganteum from Romania, and Pseudotsuga menziesii from Portugal. These seeds were collected between July and August 2020 and transported to the Forest Seed Laboratory at the University of Santa Catarina State in Lages, SC, Brazil. Upon arrival, the seeds were divided into two lots for experimentation, labeled as Lot A1 and Lot A2. Both lots were stored in waterproof packaging, below 7 °C, and relative humidity around 10%–20%, for a period of 3 months in the collection region and for another 3 months in the university’s laboratory. After this preliminary storage period, the first portion (Lot A1) was evaluated, while the second portion (Lot A2) remained stored for nearly two years until being evaluated.

2.2. Tetrazolium Test Methodology Definition

To establish the methodology of the tetrazolium test for Sequoia sempervirens and Sequoiadendron giganteum as well as to develop an alternative approach for Pseudotsuga menziesii, the protocol recommended in the Seed Analyses Rules (RAS) [6] was adopted. In this sense, different concentrations were tested. For each species, three tetrazolium treatments were applied (0.1%, 0.5%, and 1%).

The procedure started with the immersion of the seeds in water for a period of 18 h. This step aimed to activate their metabolism and facilitate the absorption of the tetrazolium solution. Subsequently, the seeds were longitudinally sectioned (along the embryo) and placed in plastic containers. Afterward, the seeds were submerged in solutions of 2,3,5-triphenyl tetrazolium chloride concentrations, corresponding to the treatments of 0.1%, 0.5%, and 1.0%, respectively. It is worth noting that the 1.0% concentration was recommended as standard by RAS for Pseudotsuga sp.

After their immersion into the abovementioned solutions, the seeds were kept in a dark environment at 30 °C for 6 h. The RAS recommends 12 h of incubation, but preliminary tests carried out to optimize the methodology indicated that a specific incubation period of 6 h is sufficient for staining the seed tissues. This procedure allowed the assessment of seed viability based on the color reactions. After the coloring period, the seeds underwent a washing process with running water, followed by the evaluation of the uniformity and intensity of the tissue coloration, categorizing the seeds as viable or non-viable.

The classification of embryo viability included four distinct subclasses. Subclass 1 is for viable embryos, which have characteristics such as a color ranging from bright pink to light red, and tissues with a normal and firm appearance. Non-viable embryos are represented by subclasses 2, 3, and 4. Subclass 2 includes embryos with more than 80% discolored tissues, while subclass 4 refers to all tissues completely discolored (Figure 1).

Subclass 3 was specifically defined for P. menziesii due to the typical coloration observed in its non-viable embryos (Figure 2).

The number of deteriorated, empty, and preyed seeds was counted. Seeds with a blackish-red coloration and/or flaccid consistency were categorized as deteriorated seeds (Figure 3).

A germination test was conducted as a control treatment, compared with the results from the tetrazolium test. The test consisted of 100 seeds arranged on blotting paper in four germination boxes (transparent plastic containers, 11 × 11 cm) at 25 °C under constant light, in Biochemical Oxygen Demand (BOD)-type germination chambers. The procedure followed the guidelines established by RAS [6].

These seeds were previously moistened with distilled water in an amount corresponding to 2.5 times the dry weight of the paper. The final evaluation of the germination percentage was planned for the 21st day after sowing; however, it was replaced by a final evaluation at the 30th day due to the slow germination characteristic exhibited by the seeds. Both the germination and tetrazolium tests were conducted in a completely randomized design with four treatments and four repetitions, each consisting of 25 seeds, totaling 100 seeds per treatment, applied to both portions of Lot A1 and Lot A2.

2.3. Physical and Physiological Quality

The seeds underwent assessments for both physical (moisture content and thousand-seed weight) and physiological (germination and tetrazolium) qualities. To determine the water content (WC), 4.0 g of seeds were distributed in two replicates of 2.0 g each, following the guidelines of RAS [6]. The samples were first placed in aluminum foil and then taken to an oven at 105 ± 2 °C for a drying period of 24 h, and finally, the seeds were transferred to a desiccator containing silica gel for 15 min followed by weighing on an analytical balance of 0.0001 g accuracy. The results were calculated as a percentage based on the wet weight of the seed, applying the equation:

$$\mathrm{W}\mathrm{C}\left(\%\right)={\displaystyle \frac{\mathrm{w}\mathrm{e}\mathrm{t} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{d}\mathrm{r}\mathrm{y} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}}{\mathrm{w}\mathrm{e}\mathrm{t} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}-\mathrm{T}\mathrm{a}\mathrm{r}\mathrm{e}}}$$

(1)

For the thousand-seed weight, eight replicates of 100 seeds each were used, and the weight in grams was measured, with the sum calculated [6].

A germination test was also conducted to assess seed viability. The test used 100 seeds placed in germination boxes (clear plastic boxes, 11 × 11 cm) on blotting paper moistened with distilled water (2.5 times the dry weight of the paper) at 25 °C under constant light, in a BOD-type germination chamber.

The analysis of germination percentage was supplemented with an assessment of seed fate 30 days after sowing. Seed conditions included: germinated, hard, deteriorated, and empty. Seeds were categorized as germinated if they showed a radicular protrusion of at least 1 mm and normal seedlings, while seeds were considered deteriorated exhibited a blackish-red color filling and/or a flaccid texture (Figure 3).

Lastly, a tetrazolium test was performed on seeds that passed the germination test but did not germinate, nor did they present fungal or bacterial contamination and were not flaccid, thus, being categorized as “hard seeds”. The reasons for this behavior may be physiological or physical dormancy or substances that inhibit germination. However, it is only after the tetrazolium test that it is possible to know whether these seeds were potentially viable. For the test, the seeds were longitudinally sectioned, meaning they were cut along the embryo, and then placed in plastic containers. These containers were immersed in a solution containing 2,3,5-triphenyl tetrazolium chloride at a concentration of 0.1%. The seeds were kept in the dark at a temperature of 30 °C for 12 h to allow for the color reaction.

After the coloring period, the seeds were rinsed with running water and subjected to the evaluation of the colorations that appeared in the tissues. The analysis considered the uniformity and intensity of the colorations exhibited by the various seed components. Based on this analysis, the seeds were classified into two categories: viable and non-viable. To determine viability, the criteria mentioned and described in the previous section were adopted.

The germination test was conducted in a completely randomized design, using four replicates, each consisting of 25 seeds, totaling 100 seeds.

2.4. Seed Processing Using a Seed Blower

Only the seeds of Lot A1 were subjected to the beneficiation process using a blower. A South Dakota model seed blower (DeLeo, Porto Alegre/RS) was used to separate the seeds. This equipment regulates the airflow velocity by dividing the original sample into light and heavy fractions. A valve numbered from 0 to 25 cm is used to manually adjust the airflow velocity, in this experiment, the equipment was calibrated to a 2 cm opening with a runtime lasting 5 min. After this beneficiation step, the heavy fraction of seeds underwent a germination test as previously described.

After conducting a germination test on the heavy fraction of blown seeds that had not previously germinated, also known as “hard seeds,” a tetrazolium test was performed to determine embryo viability. The viability criteria established four subclasses to assess embryo viability, with subclasses 2, 3, and 4 indicating non-viable seeds, and subclass 1 indicating viable seeds (refer to Figure 1 and Figure 2). The germination test was conducted using a completely randomized design with four replicates, each containing 25 seeds, totaling 100 seeds. Data evaluation involved comparing the percentage of germinated seeds with and without the blower beneficiation.

2.5. Nursery Seedling Production

The research was conducted at the Forest Nursery of the University of Santa Catarina State, located in Lages, Santa Catarina, Brazil. For the production of seedlings, seeds without being separated by lots were directly sown into seedbeds containing a commercial substrate consisting of peat, vermiculite, organic residue, and limestone. Additionally, a controlled-release fertilizer (CRF) with a 14-14-14 (NPK) formulation at a rate of 5 g L⁻¹ was homogenously applied to the substrate, with a release period of up to 4 months. The seedbeds were covered with a 2 cm layer of expanded vermiculite and protected by shade cloth with a light interception capacity of 70%.

Once the seedlings reached approximately 5 cm in height, they were transplanted into plastic tubes with a capacity of 280 cm³. Experiments with different commercial substrates and doses of controlled-release fertilizer were performed using these seedlings (

Table 1. Experiments with seedling growth were conducted for the species using different substrates and doses of controlled-release fertilizer (CRF).

Experiments	Species	CRF	Substrates
Commercial substrates	S. sempervirens, S. giganteum and P. menziesii	6 g L−1	SI-peat, vermiculite, class A organic residue (toasted rice husk), and limestone; SII-peat and carbonized rice husk; SIII-pine bark, vermiculite, and ashes; SIV-pine bark, ashes, vermiculite, peat, sawdust, and bio-stabilized materials; SV-a mixture of commercial substrate (60%), composed of peat and carbonized rice husk, supplemented with N (0.04%), P2O5 (0.04%), K2O (0.05%), and calcitic limestone (1.5%), with crushed pine cones (40%)
CRF doses	S. giganteum	0 g L−1 2 g L−1 4 g L−1 6 g L−1 8 g L−1	pine bark, ashes, vermiculite, peat, sawdust, and bio-stabilized materials

).

A fifth commercial substrate (substrate V) was tested for S. giganteum due to the greater availability of seedlings. After the treatments were applied, the seedlings remained in the greenhouse for approximately 180 days, with irrigation through micro-sprinklers three times a day for 5 min each.

The physical-chemical characterization of the substrates (Figure 4) was determined in the Substrate Laboratory of the Department of Horticulture and Silviculture at the Federal University of Rio Grande do Sul, following the Normative Instruction No. 17 of the Ministry of Agriculture, Livestock and Supply [16] and Fermino [17].

The hydrogen potential (pH), determined in water, at a dilution of 1:5 (v/v), expressed the values 6.05, 5.98, 4.49, 5.35, and 5.55 H₂O for the substrates I, II, III, IV, and V, respectively. Electrical conductivity was obtained in a 1:5 (v/v) solution, and the results for substrates I, II, III, IV, and V were 0.16, 1.86, 0.16, 1.25, and 0.33 mScm⁻¹, respectively.

All experiments were conducted using a completely randomized design. Ten replicates of four plants were used in the experiment to evaluate different substrates for Sequoia sempervirens and Pseudotsuga menziesii. For Sequoiadendron giganteum, ten replicates of eight plants were used. In the experiment analyzing the effects of different doses of CRF, ten replicates were employed, each consisting of seven plants.

For both experiments, the survival rate (%) was assessed at 60 days, while the morphological characteristics such as height (cm) and stem diameter at the soil surface (mm) were evaluated 180 days after transplantation. The ratio between height (h) and stem diameter at the soil surface (SDSS) ratio (h/SDSS) was calculated.

2.6. Statistical Analysis

The data were subjected to a test to check the assumption of normality at 5% significance (Shapiro–Wilk) and homogeneity of variances also at 5% significance (Bartlett). With these assumptions, an analysis of variance was conducted at 5%, and if significance was observed, the averages were compared using a t-test or a Scott-Knott test at a 5% probability level. All statistics were performed using the statistical software System for Analysis of Variance-SISVAR [18] and R 4.4.1 [19].

3. Results

3.1. Tetrazolium Test, Physical and Physiological Quality, Seed Processing

No significant interactions or relevant differences were observed for S. giganteum, and S. sempervirens regarding tetrazolium concentrations and seed lot categories, except for P. menziesii, whereas a considerable difference for non-viable seeds when evaluating the factors individually was noted (Figure 5).

The data analysis of seed viability indicates that a large proportion of the seeds are non-viable, deteriorated, or empty, exceeding 50%. Overall, S. giganteum has a higher percentage of empty seeds, while S. sempervirens has a higher percentage of non-viable seeds. However, both conditions have a significant number of deteriorated seeds.

For the results of P. menziesii, it can be observed that there is a high percentage of non-viable seeds in the Lot A1. The highest percentage of this condition was observed at 0.1% for the tetrazolium concentrations.

Additionally, it was found that some seeds were contaminated with larvae of insects belonging to the Corticaria genus, Latridiidae family, and Coleoptera order (27% of seeds from Lot A1 and 20% from Lot A2). This was evident from the observation that only the larva was found inside the seed upon opening, indicating that it had consumed all of the seed’s contents, leaving only the seed coat intact. (Figure 6).

The results of water content and the weight of one thousand seeds presented similarities between the two lots of each species (

Table 2. Water content (WC) and weight of one thousand seeds (WTS) of the seeds of Sequoiadendron giganteum, Sequoia sempervirens, and Pseudotsuga menziesii.

Species	WC (%)		Sig.	WTS (g)		Sig.
	Lot A1	Lot A2		Lot A1	Lot A1
S. giganteum	11.0	10.0	ns	4.920	4.484	ns
S. sempervirens	8.0	9.4	ns	3.865	4.044	ns
P. menziesii	9.0	9.7	ns	12.370	11.111	ns

Sig. = significance; ns = not significant by the t-test at a 5% probability.

).

In terms of seed viability, differences were observed between the lot categories. For P. menziesii, the germination of Lot A1 seeds was below 50%, whereas the stored seeds (Lot A2) exhibited germination rates above this threshold (

Table 3. Seed viability of Sequoiadendron giganteum, Sequoia sempervirens, and Pseudotsuga menziesii classified as germinated, hard, deteriorated, and empty in both portions of the lot.

Species	Classification	Lot A1	Sig.	Lot A2	Sig.
S. giganteum	Germinated	34	A	41	A
	Hard	11 (42% viable TZ)	A	6 (32% viable TZ)	A
	Deteriorated	0	A	43	B
	Empty	55	B	10	A
S. sempervirens	Germinated	41	A	18	B
	Hard	17 (19% viable TZ)	A	16 (35% viable TZ)	A
	Deteriorated	42	A	66	B
	Empty	0	-	0	-
P. menziesii	Germinated	15	B	56	A
	Hard	34 (21% viable TZ)	B	16 (38% viable TZ)	A
	Deteriorated	50	B	24	A
	Empty	1	A	4	A

Uppercase letters indicate a significant effect in the row by the Scott-Knott test at a 5% probability. Sig. = significance; TZ = tetrazolium test performed on the hard seeds after the germination test period.

).

In general, it was observed that the germination of seeds in both categories might be influenced by external factors since the stored seeds (Lot A2) exhibited a 41% higher germination rate compared to the seeds from Lot A1.

Due to the high percentage of observed empty and deteriorated seeds, seed processing was carried out using a seed blower to enhance seed quality by eliminating non-viable seeds. (

Table 4. Viability of seeds from Lot A1 with and without using seed blower processing.

Species	Classification	Blown	Sig.	Not blown	Sig.
S. giganteum	Germinated	56	A	34	B
	Hard	2 (0% viable TZ)		11 (91% viable TZ)
	Deteriorated	42	B	55	A
S. sempervirens	Germinated	43	A	41	A
	Hard	15 (59% viable TZ)		17 (93% viable TZ)
	Deteriorated	42	A	42	A
P. menziesii	Germinated	63	A	15	B
	Hard	6 (50% viable TZ)		34 (41% viable TZ)
	Deteriorated	31	B	51	A

Uppercase letters indicate a significant effect in the row by the Scott-Knott test at a 5% probability. Sig. = significance.; TZ = tetrazolium test.

).

Based on the data, the seed blower processing was significantly beneficial for S. giganteum and P. menziesii, as it resulted in an increase in the percentage of germinated seeds and a decrease in deteriorated seeds. There was no significant effect on S. sempervirens.

3.2. Nursery Seedling Production

In the nursery, the seedlings presented a similar germination rate to the laboratory results, around 30% to 40% of germination. Of the germinated seeds, a significant portion showed a reliable survival response in the first 60 days, with significant differences being observed, except for S. sempervirens, which had no significant effect regarding the substrates. The height, stem diameter at the soil surface, and the relationship between both variables were significant for the substrates (

Table 5. Survival (%), height (cm), stem diameter at the soil surface (SDSS) (mm), and the relationship between these variables (h/SDSS) of the nursery-stage seedlings of Sequoiadendron giganteum, Sequoia sempervirens, and Pseudotsuga menziesii, evaluated 180 days after the installation of the experiments.

Species	Treatments	Survival (%)	Height (cm)	SDSS (mm)	h/sdss
S. giganteum	Substrate I	99.0 a	19.5 a	3.7 a	5.4 a
	Substrate II	43.0 b	7.6 b	1.7 b	4.5 b
	Substrate III	95.2 a	13.1 a	3.6 a	3.7 b
	Substrate IV	96.4 a	15.2 a	3.4 a	4.4 b
	Substrate V	98.0 a	11.6 a	2.4 b	5.2 a
	0 g L−1	93.0 a	3.7 c	1.0 c	3.4 b
	2 g L−1	99.0 a	13.4 b	3.4 b	3.9 a
	4 g L−1	94.0 a	15.4 a	4.4 a	3.5 b
	6 g L−1	86.0 b	13.2 b	3.4 b	3.9 a
	8 g L−1	98.4 a	17.3 a	4.4 a	4.0 a
P. menziesii	Substrate I	75.0 a	-	-	-
	Substrate II	48.0 b	-	-	-
	Substrate III	83.5 a	-	-	-
	Substrate IV	62.5 b	-	-	-
S. sempervirens	Substrate I	95.0 a	26.9 b	3.7 a	7.3 b
	Substrate II	93.0 a	23.5 b	3.0 b	7.9 b
	Substrate III	98.0 a	28.8 b	3.2 b	9.0 a
	Substrate IV	100.0 a	34.8 a	3.6 a	9.6 a

Lowercase letters indicate a significant effect in the column by the Scott-Knott test at a 5% probability. CRF = controlled-release fertilizer; “-“—there are no results because of the loss of all plants before evaluation by a plague.

).

The data for the height and SDSS (stem diameter at the soil surface) of S. giganteum and S. sempervirens seedlings in different commercial substrates show variations in growth conditions, indicating a significant effect on the substrates. In the case of S. giganteum, substrate II had significantly lower average height and average SDSS, while the others were not significantly different for these variables. For S. sempervirens, substrate IV reached the highest average height, while the largest SDSS was obtained with substrate I, while substrate II had the lowest height and SDSS.

For P. menziesii, substrate III resulted in the highest average for the survival variable. Meanwhile, substrate II had the lowest average, similar to the results obtained for the Sequoias.

It was possible to observe a significant influence of the CRF treatments on the evaluated morphological variables for S. giganteum. For height, the best performance was obtained with the highest applied dose; in contrast, the lowest average height was found when no fertilization was used. Similarly, for the SDSS variable, the dose of 0 g L⁻¹ resulted in the lowest average, while the highest average was achieved with 4 g L⁻¹ and 8 g L⁻¹.

4. Discussion

4.1. Tetrazolium Test, Physical and Physiological Quality, Seed Processing

Redwood seeds (S. sempervirens and S. giganteum) do not exhibit dormancy [2,20]. Hence, the high percentage of deteriorated and non-viable seeds observed in both batches is caused by other external and internal factors (Figure 5). One explanation for these results is inadequate pollination, resulting in the formation of empty or incomplete seeds. This can be attributed to factors such as lack of pollinators, unfavorable weather conditions, or problems with flower structure, gamete viability, and embryo maturation, which can cause immature embryos and low viability [21,22,23]. Other factors such as poor nutrition, diseases, pathogens, genetics, and hybridization can produce empty and/or malformed seeds [24,25].

Gius [23] studied redwood embryonic development in depth to understand seed inviability, identifying critical stages and factors that influence this process. In addition to the factors abovementioned, tannins, a phenolic compound present in the form of esters or heterosides that are common in conifers, can inhibit germination, as they can suppress the activity of essential enzymes, such as those involved in the mobilization of reserves and embryo growth [26]. This makes it difficult to identify non-viable seeds, as redwood seeds appear healthy, but are often filled with tannins, resulting in low viability. Seeds determined as inviable by the tetrazolium test were, for the most part, visually normal, with intact embryos. However, internal defects in the seeds, such as wrinkled, emptiness, or missing embryos, could not be detected by external visual methods, reflected in the inviability of the seeds [27].

Douglas-fir (P. menziesii) seeds on the other hand have dormancy that cold stratification can break down [28]. The present study did not perform cold stratification to observe seed germination behavior. However, pretreatment of immersion in water for 24 h was used, which may have influenced the germination results. According to Stein and Owston [28], most seed lots benefit from stratification, but some do not require stratification, while some are even harmed by it. Several pretreatments instead of stratification can stimulate germination or reduce damage caused by pathogens. Furthermore, Lot A2 was stored at low temperatures; it might have inadvertently experienced some stratification, potentially explaining the higher viable seed rates observed in this lot. Further investigation into stratification would be warranted for this species as a follow up to this research.

Seed deterioration is a complex problem affecting several species’ conservation and reproduction, including Redwoods and Douglas-fir.

Based on the tetrazolium results, opting for the concentration of 0.1% allows for cost-effective use of the solution, optimizing the material used in the test since there is not a wide variation in the viability data. According to Marcos Filho, Cícero & Silva [29], the tetrazolium test can encompass various solution concentrations, depending on the species under analysis, seed preparation process, and tegument permeability. In the context of forest species seeds, these concentrations vary between 0.05% and 1.0%. Santos, Vieira & Panobianco [30] concluded that a 0.2% concentration of tetrazolium for 4 h was the ideal methodology for evaluating the viability of Pinus taeda seeds. However, it can be noted that lower concentrations are also recommended. This is justified by reducing the reagent cost optimizing the detection of chromatic changes and identifying different lesion categories [8].

The same proportions of tetrazolium concentrations found to be most suitable for evaluating viability, as identified in this study, were also observed for Araucaria angustifolia. According to Oliveira et al. [31], the use of 0.1% or 0.5% concentrations applied for one hour at 25 °C emerged as an effective alternative methodology compared to the conventional tetrazolium test for this species in the RAS [6].

Regarding the cut made to the seeds, the RAS recommends one longitudinal cut before staining, and after a period of 18 h, expose the embryo and remove the integument prior to the assessment [6]. An additional period is required for the latter, making this procedure impractical for routine laboratory analysis. In the alternative procedure proposed, a longitudinal cut is made adjacent to the embryo to expose it without the need to remove the integument, and only the half containing the embryo is stained, while the other one is discarded. The advantages of this procedure are the ease and speed of the proposed cut, whereas its only inconvenience is the greater difficulty of washing seeds after staining, leading to the separation of the embryo from the endosperm cavity. However, this issue can be effectively addressed by carefully maintaining the flaccid tissue surrounding the embryo during double washing and gently removing it before assessment.

The low water content observed (Table 2) in the analysis of physical and physiological quality resembles the typical characteristic of orthodox seeds, which can withstand drying and cooling, allowing them to be stored for extended periods without losing their viability [32,33,34]. On the other hand, S. giganteum and S. sempervirens seeds can also be classified as intermediate, which tolerate dehydration between 7% and up to 10% of moisture and cannot withstand low temperatures for prolonged periods [35]. For P. menziesii, the data align with what has been reported by Stein & Ownston [28], who stated that the storage of seeds from this species should be at −18 °C or near a moisture content ranging from 5% to 9%, in well-sealed plastic bags. If stored under these conditions, viability should be preserved for many years.

Through the weight of one thousand seeds (Table 2), they are classified as small, weighing less than 200 g, according to the criteria defined by the RAS [6]. The weight of one thousand seeds can be used, for example, to estimate the number of seeds needed for sowing in a specific area, which helps optimize the use of seeds, reduce losses, and improve establishment [36].

When observing the germination test results, it can be affirmed that the seeds from Lot A1 have a lower viable rate than the stored seeds, except for S. sempervirens, which did not present differences (Table 3). The storage process may have provided more favorable conditions for preserving the viability of these seeds, resulting in a higher germination rate. These results emphasize the importance of proper seed production and storage practices to ensure the best quality and viability. Control and proper maintenance of environmental conditions during storage are essential to preserve the viability and germination capacity of seeds over time.

Thus, the differences found and mentioned earlier between the lots of the same species are likely related to factors linked to seed storage. As mentioned by Toledo & Marcos Filho [37], storage conditions can affect seed viability, as storage does not improve the physiological quality of the lot but preserves it when performed properly. It is noteworthy that potentially viable seeds were still observed after the completion of the germination test, as evidenced by the tetrazolium test (Table 3). This fact may suggest the need for a longer period to complete the test or the possibility of seed dormancy. Nevertheless, according to Olson et al. [2], low germination in Sequoia seeds is generally attributed to a high percentage of non-viable seeds (greater than 75%, according to the authors), and not dormancy. This corroborates with the information obtained in the present study, whereas the rates of non-viable seeds (deteriorated and empty) exceeded 50%.

When examining the data for germinated seeds of S. sempervirens and P. menziesii, discrepancies can be observed between the lot categories, with differences of 23% and 41%, respectively (Table 3). However, it is important to consider that this divergence is possibly not solely associated with storage but also with the presence of fungi during the germination test period.

Hansen and Muelder [27] determined 11% of fungi damage in the S. sempervirens seeds, and only a small fraction, 32%, were deemed free of infections and capable of germination. Fungal infections can severely damage the embryo, reducing or eliminating its viability. A study conducted by Fonseca et al. [38] on the quality of Pinus elliottii seeds revealed a high fungal infestation that had a direct impact on germination results. In seeds with a lower level of fungal contamination, the germination percentage reached 91%, while in seeds with contamination levels exceeding 70%, germination was negatively affected.

It is important to mention that a detailed survey of the frequency and identification of the fungi present was not conducted. Therefore, it is recommended that future studies focus on investigating the incidence of these microorganisms and their relationship with seed germination in these species. A more in-depth analysis in this regard will contribute to a better understanding of the results of the present study.

The seed blower proved to be an efficient method for removing empty seeds, contributing to an improvement in seed physiological quality (Table 4), as demonstrated in previous studies on various forest species [32,39,40,41]. Krugman and Boe [32] and Faria et al. [39] observed the removal of underdeveloped, empty, and resin-filled seeds using seed blowers, while Shibata et al. [40] and Feitosa et al. [41] highlighted the positive impact of this equipment on germination rates. Furthermore, seed blowers have been shown to effectively remove seeds contaminated with fungi, improving overall seed quality and germination success. However, seed processing was not conducted on the stored seeds in this study due to the low prevalence of empty seeds, as indicated by the germination test (Table 3), making it unnecessary.

4.2. Nursery Seedling Production

The optimization of height growth in forest seedlings should be balanced with other quality characteristics to ensure successful establishment in different environments, such as the stem diameter of the seedlings. According to Gomes & Paiva [42], stem diameter can be used to qualify seedlings suitable for planting, indicating that higher values are related to greater survival and growth in the field.

Based on the results obtained for the evaluated morphological variables, it can be stated that there is an influence of the different substrates tested, and these results are directly related to the physical and chemical characteristics of the substrates and their compositions (Table 5). Thus, Sequoias do not respond well to high density substrates, low easy-available water content (EAW), and high electrical conductivity (CE), as substrate II has the highest wet density (804.05 kg m⁻³) and dry density (349.58 kg m⁻³), the lowest EAW (11.35%), and high CE (1.86 ms cm⁻¹) (Figure 4). These characteristics can result in growth limitations and difficulties in removing the seedlings for shipment, as well as damage to plant growth in the absence of frequent irrigations, leading to water deficiencies, osmotic stress due to the high concentration of soluble salts in the substrate solution, and even plant death [43,44].

Based on the composition, substrates I and IV show a positive effect on the development of the seedlings. This is due to their compositions, which include components such as peat, vermiculite, and organic and inorganic residues. As stated by Oliveira et al. [45], the formulation of substrates containing both organic and inorganic nutrient sources, in appropriate concentrations, has a direct influence on both biomass production and the vigor of forest species seedlings.

The morphological variables height and stem diameter of P. menziesii seedlings could not be measured due to an unexpected attack by insects, which caused total defoliation of the seedlings, resulting in the death of multiple individuals. The deterioration in the quality of seedlings due to insect damage has the potential to negatively affect both tree survival and growth. To mitigate these impacts, several management strategies can be adopted. The use of insecticides is an option, but the more sustainable approach involves the integration of control tactics. This can include the selection of resistant genotypes, the adoption of appropriate cultural practices, such as soil moisture management, and the implementation of biological control through the introduction of natural predators [46].

The index resulting from the “h/diameter” ratio, also called the robustness quotient, is one of the most important morphological parameters for estimating seedling growth because it provides information on the slender index of the seedling [47]. From the data obtained for S. giganteum and S. sempervirens, it can be observed that the index ranged from 3.7 to 5.4 and 7.3 to 9.6, respectively (Table 5). This indicates that S. sempervirens may be more fragile in response to handling and climatic conditions such as wind, frost, and drought, resulting in additional costs for replanting [48]. However, despite presenting high index values, the results are still below 10, consistent with the h/diameter reference proposed by Hunt [49] and Carneiro [47].

For the height variable and stem diameter (Table 5), the 4.0 g L⁻¹ may indicate a better response of CRF, values above this dose indicate spending on fertilization. The non-use of fertilization resulted in poor-quality seedlings, with seedlings measuring less than 4 cm in height and 1 mm in diameter. The implementation of controlled-release fertilizers in forest seedlings, especially conifers, has been widely investigated to assess their impact on initial development, with an emphasis on the height variable. The gradual and controlled release of nutrients provided by these fertilizers can offer significant advantages in terms of vertical seedling growth, a crucial factor for healthy plant establishment.

Several studies focused on conifers, such as the work of Stape, Binkley & Ryan [50] with Pinus elliottii, highlighted that the application of controlled-release fertilizers resulted in substantial increases in seedling height. Similar results were found by Salcido-Ruiz [51], who investigated different types of controlled-release fertilizers in Pinus engelmannii seedlings. In this study, seedlings treated with these fertilizers showed greater diameter growth compared to unfertilized seedlings.

The evaluation of controlled-release fertilizers also showed positive effects on the height variable of native conifer species, as demonstrated by Rossa et al. [52] in Araucaria angustifolia seedlings. The proper dosage of these fertilizers resulted in a significant increase in seedling height, emphasizing the importance of this type of fertilization for vertical plant development. The convergence of these results reinforces the importance of controlled-release fertilizers as an effective strategy to promote height growth in conifer seedlings. The application of these fertilizers appears to have a direct and positive impact on this aspect, contributing to vigorous seedling establishment in different forest contexts.

It is important to emphasize that the Sequoiadendron giganteum seedlings used in the fertilization experiment had a 35% mortality rate, probably caused by the excessive heat observed one month before the evaluation of these variables. This observation is important to understand the unexpected behavior of the average below the others, identified at the 6 g L⁻¹ dosage.

Planting these species in places with conditions similar to their place of origin can help in the conservation of genetic characteristics, allowing the study of the species’ interactions with the new environment, and assisting in research on ecology, physiology, and genetics. Therefore, it is a useful ex situ conservation tool [53]. However, the combination of ex situ and in situ conservation techniques is the most appropriate approach to preserve biodiversity.

5. Conclusions

The proposed methodology for the tetrazolium test in Sequoiadendron giganteum and Sequoia sempervirens, alongside the alternative method for Pseudotsuga menziesii, involving seed immersion in water for 18 h followed by immersion in a 0.1% tetrazolium solution at 30 °C for 6 h, is an effective procedure to assess seed viability.

The seed moisture content ranged from 8.0% to 11.0%, suggesting classification as orthodox seeds, and the weight of one thousand seeds indicated lightweight and small seeds (from 3.865 g to 12.370 g). The lots were very similar in physical quality for all species, but there were some differences in physiological quality. The stored lot had a higher quantity of viable seeds for S. giganteum and P. menziesii, with no differences for S. sempervirens. The main cause of the low germination rates was the high percentage of non-viable seeds.

The most suitable substrates for seedling production were SI (peat, vermiculite, organic residue class A [roasted rice husk], and limestone) for Sequoiadendron giganteum, SIV (pine bark, ash, vermiculite, peat, sawdust, and bio-stabilized material) for Sequoia sempervirens, and SIII (pine bark, vermiculite, and ash) for Pseudotsuga menziesii. Sequoiadendron giganteum’s dose of 4 g L⁻¹ of controlled-release fertilizer was more favorable for seedling development.