New Proposal to Increase Soybean Seed Vigor: Collection Based on Pod Position

Abstract

The seed lots were evaluated based on their viability and vigor, which vary according to their origin and the locations where the seeds were produced. However, differences in vigor can be observed within a single seed lot, resulting from the deposition of photoassimilates. In this context, the hypothesis of this study is that distinct locations on the plant may produce seeds with different physiological quality. Therefore, the objective of this work was to evaluate how pod position influences the vigor of seeds from different soybean genotypes. Field experiments were conducted during the 2021/22 and 2022/23 crop seasons in Brazil. The experimental design was a randomized complete block with four replications and 32 soybean populations from the UFMS/CPCS Breeding Program. During the R4, R5, R6, and R7 reproductive stages of soybean, at the time of pod formation, the plants in each block were tagged with string to delimit the uppermost point at which pods had formed. Tagging was carried out as each stage change was verified, at approximately eight-day intervals. When analyzing how the pod position of the plant influences seed physiological variables, we found that position P1 was responsible for the best results for the variables evaluated, with the exception of genotypes G18 and G28. This result indicates that pods from the first position produce seeds with greater germination capacity and a higher ability to generate normal seedlings. However, the genotypes are still under development and, therefore, do not yet exhibit stability. Nevertheless, the results obtained highlight the relationship between the pod position of the plant and seed physiological variables. The position of the pods on the soybean plant influences the physiological quality of the seeds. In general, the P1 position, when the plants are in the R4 reproductive stage, with fully developed pods measuring 2 cm on one of the four upper nodes of the stem, is responsible for the best results in seed physiological quality tests for most of the soybean genotypes evaluated. These results indicate that pod position should be considered in breeding and seed production programs, since genotypes with greater physiological stability in the upper positions may be preferential in selection strategies. In the future, studies in different environments and evaluation of biochemical traits may confirm these patterns and contribute to the development of cultivars with higher seed quality and physiological uniformity.

1. Introduction

Soybean (Glycine max (L.) Merril) is an important legume and one of the most highly valued oilseeds worldwide due to its composition, which is responsible for providing plant-based protein [1]. Soybean yield depends on meeting the crop’s requirements throughout its growth and development stages, thereby preventing quantitative and qualitative losses in the harvested seeds [2]. The reproductive phases, which include seed composition, pod formation, and seed filling, are critical periods in soybean cultivation because they are directly associated with the plant’s photosynthetic efficiency. During these stages, reductions in photosynthetic performance compromise the ownership and transport of photoassimilates, which can affect both yield and physiological quality of the seeds. Thus, variations in the light microenvironment and the position of the pods on the plant can influence the availability of assimilates during filling, resulting in differences in seed vigor and predictability [3,4]. These limitations negatively affect cell division and seed development [5].

Seed production is influenced by the number of flowers produced by the mother plant and the proportion of flowers that develop into pods, both of which represent critical periods for the formation of high-quality seeds [6]. Therefore, soybean seed quality results from its genetic, physiological, physical, and sanitary attributes [7]. Physiological potential is estimated through viability and vigor, metrics that determine the seed’s ability to germinate rapidly and uniformly [8]. The use of low-vigor seeds in agricultural production generates abnormal seedlings or ungerminated seeds, resulting in wasted labor and financial resources. Therefore, it is necessary to develop vigor screening technology to avoid low vigor or seed death, thus improving agricultural productivity [9].

Germination is the term used to assess seed viability through the emergence of the primary root and the formation of a normal seedling, characterized by well-developed root and shoot systems [10]. However, conducting germination tests in the laboratory under optimal conditions for the seeds often leads to discrepancies when these seeds are sown in the field [11]. For this reason, vigor tests are employed, subjecting seeds to adverse conditions and evaluating their capacity, speed, and uniformity of germination [12].

Vigor assessment is performed through specific tests and comparison among seed lots, as lots with higher vigor percentages consist of seeds that germinate uniformly. This results in rapid stand establishment, thereby contributing to crop quality and yield [13]. Therefore, differences observed among seed lots are expected; however, variation in seed vigor within the same lot raises questions regarding its causes, since, theoretically, the lot should be homogeneous. The lack of standardization within the same batches for evaluating vigor is a problem for estimating physiological vigor, particularly for samples that exhibit similar germination rates [14]. One reason for this variation is the position of the pod that gave rise to the seed.

The light intensity received by different parts of the soybean plant varies, especially due to shading of the lower leaves, which results in reduced stomatal conductance and lower photosynthetic performance [15]. In contrast, leaves in the upper parts of the plant, even at early developmental stages, receive greater quantity and quality of sunlight, resulting in improved stomatal conductance and photosynthetic performance [16]. A reduction in the photosynthetic rate implies less synthesis and redistribution of photoassimilates to the reproductive organs, which can compromise seed filling and maturation [17]. Thus, plant positions with lower photosynthetic efficiency tend to produce seeds with less accumulated reserves, resulting in reduced predictive and physiological vigor [18].

Based on this, the hypothesis of this study is that different regions of the plant can produce seeds with distinct physiological quality. Understanding these patterns is relevant not only for harvest management and obtaining seed lots with greater vigor, but also for genetic improvement programs, which can use this information to select genotypes with a greater capacity to produce high-quality seeds in different plant positions. The originality of this work lies in the integrated approach between pod position and seed physiological vigor in multiple soybean genotypes, considering the precise definition of canopy positions and their practical application in seed selection and production programs. Therefore, the objective of this study was to evaluate how pod position influences seed vigor in different soybean genotypes.

2. Materials and Methods

2.1. Field Experiments

Field experiments were conducted during the 2021/22 and 2022/23 crop seasons at the experimental area of the Federal University of Mato Grosso do Sul, Chapadão do Sul Campus, MS, Brazil. The site is located at 18°41′33″ S and 52°40′45″ W, at an altitude of 810 m.

The soil is classified as a Dystrophic Red Latosol with a clayey texture, and its characteristics in the 0–0.2 m layer are as follows: pH (H₂O) = 6.2; exchangeable Al, Ca + Mg, K, sum of bases, and CEC with values of 0.0, 4.31, 0.2, 2.3, and 5.1 cmolc dm⁻³, respectively; P = 41.3 mg dm⁻³; organic matter = 19.74 g dm⁻³; and V and m = 45% and 0.0%, according to soil analysis. The regional climate is classified as Tropical Savanna (AW) according to the Köppen–Geiger classification. Precipitation and average temperature during the 2021/22 and 2022/23 seasons are presented in Figure 1.

The experimental design was a randomized complete block with four replications and 32 soybean genotypes from the UFMS/CPCS Breeding Program, all with indeterminate growth habit. Experimental plots consisted of five rows, each 1.5 m long, spaced 0.45 m apart, with a population of 15 plants per meter.

Manual sowing was performed at the beginning of the rainy season in both years, with the area prepared using conventional tillage involving plowing and harrowing. Seeds were treated with fungicide (Pyraclostrobin + Thiophanate-methyl), insecticide (Fipronil), and inoculant (Bradyrhizobium spp.) at a rate of 200 mL of each product per 100 kg as recommended by the manufacturer, used in the same region as was chosen in [19]. Crop management practices for pest, pathogen, and weed control were carried out as needed for the crop.

2.2. Pod Tagging on Soybean Plants

At the reproductive stage of soybean, during pod formation, the plants in each block were marked using string to delimit the uppermost point of pod development.

Table 1. Pod tagging on soybean plants.

Position	Reproductive Stage (R)	Description	Representative Figure
P1	R4	Fully developed pods, 2 cm in length, located at one of the four uppermost nodes of the stem.
P2	R5	Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development.
P3	R6	Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development.
P4	R7	Beginning of physiological maturity of the seeds, with at least one mature pod present on the main stem.

Source: Prepared by the authors, adapted from Oliveira Junior [20].

presents the reproductive stage and a description of each tag position.

Tagging was performed as each developmental stage was identified, at approximately eight-day intervals. All soybean plants were tagged to obtain a greater quantity of seeds for physiological testing. Each tagging was carried out in a specific experimental block: plants at position P1 were tagged in block 1, those at position P2 in block 2, position P3 in block 3, and position P4 in block 4. Thus, each block received a single tagging, and subsequently, each plot within the block was subdivided into four replications for the physiological seed quality tests.

2.3. Harvest and Seed Separation

At harvest, when the plants reached the R8 stage, pods from each plant were collected up to the previously tagged point. Thus, pods at position P1 were harvested only up to the corresponding tag, and the same procedure was followed for pods at positions P2, P3, and P4. Untagged plants were discarded. Pods were manually opened, and seeds were identified according to their genotype and stored in a room at 16 °C to preserve seed quality until testing.

2.4. Seed Analysis

For physiological quality assessment, seeds were first subjected to moisture content determination. Once adequate moisture levels were confirmed, the following tests were conducted: germination (GERM), first germination count (FGC), and tetrazolium vigor (TZVG) and viability (TZVB) tests.

Moisture content was determined using the oven method: two subsamples of approximately 4.0 g of seeds were placed in containers, weighed, and then kept in a forced-air oven for 24 h at 105 ± 3 °C. The methodology followed the guidelines proposed by Ref. [21], and results were expressed as a percentage on a wet basis.

For the GERM test, four subsamples of 50 seeds were placed on Germitest paper moistened with distilled water at a volume equivalent to 2.5 times the weight of the paper, and then kept in a germinator for eight days at 25 °C [21]. Results were expressed as the percentage of normal seedlings, i.e., those with well-developed shoots and roots, after eight days. FGC test was performed concurrently with the germination test, by counting the number of normal seedlings five days after the test was set up, with results also expressed as a percentage.

The tetrazolium test involved pre-soaking four subsamples of 50 seeds on Germitest paper moistened to 2.5 times its weight, kept in a germinator at 25 °C for 16 h. After pre-soaking, seeds were placed in cups with a 2,3,5-triphenyltetrazolium chloride solution and incubated in a B.O.D. chamber at 40 °C in the dark for 3 h [21]. After incubation, seeds were washed with distilled water, sectioned, and individually evaluated for vigor (TZVG) and viability (TZVB), with results expressed as percentages. The methodology follows the recommendations of [22].

2.5. Statistical Analysis

Given the assumptions of normality and homogeneity of variances, which yielded satisfactory results, the analysis of variance was performed. Data were subjected to analysis of variance (ANOVA) using the F-test. The effects of pod position and genotype were considered fixed, while the effect of growing season was considered random. When fixed effects and their interactions were significant, means were grouped using the Scott–Knott test [23]. In all cases, a 5% significance level was adopted.

A Pearson correlation and a principal component analysis (PCA) plot was also constructed to analyze the correlations among the studied variables. Statistical analyses were performed using R version 4.1.0 [24] software.

3. Results

Seed moisture content ranged from 11.5 to 12.0%. The individual effects of growing season, pod position, and genotype were significant for first germination count (FGC), germination (GERM), tetrazolium vigor (TZVG), and tetrazolium viability (TZVB) (

Table 2. Summary of the analysis of variance for first germination count (FGC), germination (GERM), tetrazolium vigor (TZVG), and tetrazolium viability (TZVB) in 32 soybean genotypes evaluated according to pod position on the plant during two crop seasons in Chapadão do Sul, MS, Brazil.

SV	DF	FGC	GERM	TZVG	TZVB
Crop Season (S)	1	154,907.24 *	6033.97 *	44,249.45 *	4289.10 *
Position (P)	3	37,454.86 *	5350.66 *	15,385.28 *	1320.64 *
Genotypes (G)	31	532.09 *	171.59 *	452.09 *	161.81 *
P × G	93	316.98 *	116.58 *	329.64 *	141.15 *
P × S	3	20,701.37 *	1797.50 *	5928.25 *	1021.24 *
G × S	31	137.84 *	66.55 *	256.22 *	50.74 *
P × S × G	92	149.27 *	58.89 *	175.35 *	56.42 *
Residual	769	27.67	29.21	47.69	34.24
CV (%)	-	6.82	5.97	9.74	6.42

* Significant by the F-test at 5% probability; SV: sources of variation; DF: degrees of freedom; CV: coefficient of variation.

). There were also significant two-way and three-way interactions among these factors for all evaluated variables. The interaction among factors demonstrates that pod position on the plant, in combination with genotype, influences the physiological quality variables of seeds. Therefore, further analyses of the fixed effects of pod position × genotype are presented in

Table 3. Unfolding the interaction between genotypes and pod positions on the plant for the seed physiological variable first germination count percentage (FGC), during two crop seasons in Chapadão do Sul, MS, Brazil.

Genotype	P1	P2	P3	P4
G1	98.5 aB	79.5 bB	76.0 bA	59.5 cB
G2	97.5 aA	76.0 bCc	77.5 bA	70.5 bA
G3	100.0 aA	71.5 bC	77.5 bA	64.5 bB
G4	99.5 aA	96.5 aA	76.5 bA	67.5 bA
G5	98.0 aA	87.5 bA	77.0 cA	66.5 cA
G6	94.5 aA	80.5 bB	74.0 bA	87.5 aA
G7	100.0 aA	81.5 bB	77.5 bA	71.5 bA
G8	99.0 aA	77.5 bB	74.0 bA	72.5 bA
G9	96.0 aA	75.5 bC	79.5 bA	70.0 bA
G10	97.5 aA	75.5 bC	79.5 bA	63.5 cB
G11	100.0 aA	73.5 bC	79.5 bA	63.5 cB
G12	99.0 aA	72.0 bC	77.5 bA	60.0 cB
G13	100.0 aA	67.0 cC	76.5 bA	60.5 cB
G14	99.0 aA	62.5 cC	75.0 bA	56.5 cB
G15	97.5 aA	66.0 bC	57.5 bB	58.5 bB
G16	99.5 aA	71.0 bC	74.5 bB	61.5 cB
G17	91.5 aA	74.0 bC	64.0 bB	63.5 bB
G18	72.0 aB	72.5 aC	60.0 bB	62.5 bB
G19	97.0 aA	70.5 bC	71.5 bA	60.0 cB
G20	96.0 aA	67.5 cC	81.0 bA	64.5 cB
G21	94.5 aA	73.5 bC	70.5 bA	65.0 bB
G22	97.5 aA	74.5 bC	70.5 bA	63.5 cB
G23	93.5 aA	70.5 bC	73.5 bA	59.0 cB
G24	95.5 aA	65.5 bC	62.5 bB	58.5 bB
G25	98.0 aA	77.5 bB	74.5 bA	67.5 bA
G26	88.0 aA	78.5 aB	71.5 bA	67.0 bA
G27	92.5 aA	71.0 bC	73.5 bA	71.5 bA
G28	60.5 bC	73.0 aC	81.5 aA	68.5 bA
G29	97.0 aA	77.5 bB	77.5 bA	62.5 cB
G30	89.0 aA	80.5 aB	72.5 bA	69.5 bA
G31	82.0 aB	81.0 aB	76.5 bA	65.5 bB
G32	93.5 aA	73.5 bC	77.0 bA	73.5 bA

Equal lowercase letters in rows and uppercase letters in columns indicate no significant difference by the Scott–Knott test at 5% probability. P1-(R4): Fully developed pods, 2 cm in length, located at one of the four uppermost nodes of the stem; P2-(R5): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development. P3-(R6): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development; P4-(R7): Beginning of physiological maturity of the seeds, with at least one mature pod present on the main stem.

,

Table 4. Unfolding the interaction between genotype and pod position on the plant for the seed physiological variable germination percentage (GERM), during two crop seasons in Chapadão do Sul, MS, Brazil.

Genotype	P1	P2	P3	P4
G1	98.5 aA	95.0 aA	91.5 bA	85.0 bB
G2	98.0 aA	91.0 bA	92.5 bA	87.5 bA
G3	100.0 aA	87.5 bB	92.5 bA	86.5 bA
G4	99.5 aA	96.5 aA	92.0 bA	87.5 bA
G5	98.5 aA	95.0 aA	92.0 aA	85.5 bB
G6	96.0 aA	95.5 aA	88.5 bA	86.5 bA
G7	100.0 aA	96.5 aA	92.5 aA	82.5 bB
G8	99.5 aA	92.5 bA	91.5 bA	93.5 bA
G9	97.0 aA	90.5 bA	94.5 aA	89.5 bA
G10	98.0 aA	90.5 aA	94.5 aA	77.5 bB
G11	100.0 aA	89.5 bB	94.5 aA	84.5 bB
G12	99.0 aA	91.5 bA	92.0 bA	84.5 cB
G13	100.0 aA	88.5 bB	91.5 bA	81.0 cB
G14	99.0 aA	82.5 cC	90.5 bA	83.5 cB
G15	99.0 aA	81.0 bC	86.5 bB	84.5 bB
G16	99.5 aA	86.0 bB	89.5 bA	84.0 bB
G17	94.5 aA	89.0 aB	82.5 bC	82.0 bB
G18	86.0 aB	87.5 aB	78.0 bC	84.5 aB
G19	98.0 aA	85.5 bB	86.5 bB	82.5 bB
G20	96.0 aA	82.5 bC	96.0 aA	82.5 bB
G21	96.5 aA	88.5 bB	90.0 bA	81.5 cB
G22	98.5 aA	89.5 bB	87.5 bB	82.5 cB
G23	95.0 aA	85.5 bB	91.5 aA	87.5 bA
G24	97.0 aA	80.5 bC	84.2 bB	82.5 bB
G25	99.0 aA	92.5 bA	92.0 bA	88.5 bA
G26	91.0 aA	93.5 aA	88.0 aB	86.5 aA
G27	94.5 aA	86.0 bB	87.5 bB	93.5 aA
G28	83.5 bB	88.5 bB	95.5 aA	86.0 bA
G29	98.0 aA	92.5 bA	91.0 bA	79.5 cB
G30	93.0 aA	95.5 aA	89.5 bA	86.5 bA
G31	90.0 aA	96.0 aA	91.0 aA	91.5 aA
G32	95.0 aA	88.5 aB	91.5 aA	90.5 aA

Equal lowercase letters in rows and uppercase letters in columns indicate no significant difference by the Scott–Knott test at 5% probability. P1-(R4): Fully developed pods, 2 cm in length, located at one of the four uppermost nodes of the stem; P2-(R5): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development. P3-(R6): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development; P4-(R7): Beginning of physiological maturity of the seeds, with at least one mature pod present on the main stem.

,

Table 5. Unfolding the interaction between genotype and pod position on the plant for the seed physiological variable tetrazolium vigor percentage (TZVG) during two crop seasons in Chapadão do Sul, MS, Brazil.

Genotype	P1	P2	P3	P4
G1	93.0 aA	71.5 bA	67.0 bA	58.5 cB
G2	88.5 aA	61.0 bB	69.5 bA	60.5 bB
G3	85.5 aA	70.5 bA	70.0 bA	58.0 cB
G4	85.0 aA	87.5 aA	67.4 bA	72.5 bB
G5	85.0 aA	78.5 aA	68.0 bA	67.0 bB
G6	89.0 aA	75.5 bA	73.5 bA	72.0 bA
G7	83.0 aA	77.0 aA	63.0 bB	70.5 bA
G8	83.0 aA	72.5 bA	63.0 bB	67.0 bA
G9	82.0 aA	68.0 bB	67.0 bA	72.0 bA
G10	75.0 aB	65.5 aB	65.0 aA	70.5 aA
G11	86.0 aA	68.5 bB	66.5 bA	68.0 bA
G12	83.5 aA	65.5 bB	68.0 bA	67.0 bA
G13	89.5 aA	67.0 bB	66.0 bA	68.0 bA
G14	84.0 aA	73.5 bA	68.5 bA	68.5 bA
G15	92.0 aA	60.5 bB	68.5 bA	61.0 bB
G16	87.0 aA	70.5 bA	71.0 bA	70.5 bA
G17	92.5 aA	58.0 bB	57.0 bB	64.5 bB
G18	54.0 cC	71.0 aA	57.5 bB	69.5 aA
G19	73.0 aB	63.0 aB	61.5 aB	66.0 aA
G20	85.5 aA	68.0 bB	64.0 bB	59.0 bB
G21	89.0 aA	70.5 bA	66.5 bA	57.5 cB
G22	90.5 aA	71.5 bA	68.5 bA	71.0 bA
G23	82.0 aA	74.0 bA	70.5 bA	69.0 bA
G24	76.5 aB	64.5 bB	67.5 bA	65.0 bA
G25	71.0 aA	64.0 bB	58.5 bB	70.5 aA
G26	76.0 aB	67.0 bB	57.5 bB	62.5 bB
G27	87.0 aA	67.5 bB	68.0 bA	72.0 bA
G28	54.0 bC	61.0 bB	75.5 aA	57.5 bB
G29	85.0 aA	67.0 bB	68.0 bA	65.5 bA
G30	85.0 aA	64.0 bB	67.0 bA	64.0 bB
G31	85.0 aA	74.5 bA	66.0 bA	75.5 bA
G32	84.5 aA	62.5 bB	64.0 bB	72.0 bA

Equal lowercase letters in rows and uppercase letters in columns indicate no significant difference by the Scott–Knott test at 5% probability. P1-(R4): Fully developed pods, 2 cm in length, located at one of the four uppermost nodes of the stem; P2-(R5): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development. P3-(R6): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development; P4-(R7): Beginning of physiological maturity of the seeds, with at least one mature pod present on the main stem.

and

Table 6. Unfolding the interaction between genotype and pod position on the plant for the seed physiological variable tetrazolium viability percentage (TZVB) during two crop seasons in Chapadão do Sul, MS, Brazil.

Genotype	P1	P2	P3	P4
G1	98.0 aA	91.0 bB	89.5 bA	81.5 cA
G2	97.5 aA	84.5 bB	88.5 bA	83.5 bA
G3	95.0 aA	86.5 bB	86.5 bA	85.0 bA
G4	96.0 aA	96.0 aA	91.5 aA	89.5 aA
G5	97.5 aA	90.5 aB	94.0 aA	89.5 aA
G6	99.0 aA	94.5 bA	91.5 bA	90.0 bA
G7	94.0 aA	92.5 aA	96.5 aA	88.5 aA
G8	96.0 aA	93.0 aA	94.0 aA	93.0 aA
G9	99.0 aA	86.0 bB	96.5 aA	92.0 bA
G10	98.0 aA	85.5 bB	93.5 aA	91.5 aA
G11	98.0 aA	90.5 bB	97.0 aA	86.5 bA
G12	91.5 aA	90.0 aB	89.5 aA	87.5 aA
G13	91.5 aA	87.5 aB	91.5 aA	89.0 aA
G14	99.0 aA	89.0 bB	93.0 bA	88.5 bA
G15	96.5 aA	89.5 bB	91.5 bA	87.5 bA
G16	98.5 aA	92.5 bA	93.0 bA	90.5 bA
G17	95.5 aA	88.0 aB	93.0 aA	93.5 aA
G18	70.0 bB	88.5 aB	93.0 aA	91.0 aA
G19	98.0 aA	84.5 bB	93.0 aA	90.0 bA
G20	95.0 aA	89.5 aB	87.5 aA	88.0 aA
G21	91.5 aA	91.0 aB	93.5 aA	88.0 aA
G22	93.0 aA	90.0 aB	87.5 aA	89.5 aA
G23	97.0 aA	90.0 bB	90.0 bA	90.5 bA
G24	96.5 aA	88.0 bB	89.0 bA	89.0 bA
G25	96.0 aA	93.0 aA	89.5 aA	93.5 aA
G26	98.0 aA	89.0 bB	91.0 bA	87.5 bA
G27	94.5 aA	91.5 aA	89.0 aA	90.5 aA
G28	67.5 bB	90.0 aB	91.0 aA	87.5 aA
G29	95.0 aA	90.5 aB	90.5 aA	88.5 aA
G30	96.0 aA	96.5 aA	88.5 bA	86.5 bA
G31	94.0 aA	95.0 aA	90.5 aA	89.0 aA
G32	95.5 aA	93.5 aA	88.5 bA	88.5 bA

Equal lowercase letters in rows and uppercase letters in columns indicate no significant difference by the Scott–Knott test at 5% probability. P1-(R4): Fully developed pods, 2 cm in length, located at one of the four uppermost nodes of the stem; P2-(R5): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development. P3-(R6): Pods at grain-filling stages, perceptible to touch and exhibiting varying degrees of grain development; P4-(R7): Beginning of physiological maturity of the seeds, with at least one mature pod present on the main stem.

.

Regarding unfolding of the FGC variable, it was observed that most genotypes showed higher FGC percentages at position P1, with the exception of genotypes G4, G6, G18, G26, G28, G30, and G31 (Table 3). Genotypes G4, G18, G26, G30, and G31 exhibited the highest PCG percentages at positions P1 and P2. Genotype G6 showed the highest FGC percentages at positions P1 and P4, while genotype G28 had the highest FGC percentages at positions P2 and P3.

Regarding pod positions on the plant, except G1, G28, and G31, there was no difference in FGC percentage among genotypes at position P1. At position P2, genotypes G4 and G5 showed the highest FGC percentages, differing from the others. Position P3 exhibited the lowest PCG percentages for genotypes G15, G16, G17, and G18. At position P4, genotypes G2, G4, G5, G6, G7, G8, G9, G25, G26, G27, G28, G30, and G31 had the highest FGC percentages.

Regarding unfolding of the GERM variable, genotypes G2, G3, G8, G12, G13, G14, G15, G16, G19, G20, G21, G22, G23, G24, G25, and G29 showed the highest GERM percentages at position P1 (Table 4). Genotypes G1, G4, G6, G17, G18, and G30 presented higher GERM percentages at positions P1 and P2.

Genotypes G11, G13, G16, G20, G21, and G23 showed higher GERM percentages at positions P1 and P3. Genotype G26 exhibited the highest GERM percentage at position P2. Genotype G27 had the highest GERM percentages at positions P1 and P4. Genotype G31 showed the highest GERM percentages at positions P2, P3, and P4, while genotype G32 had the highest GERM percentages at positions P1, P3, and P4.

There was no statistical difference among genotypes for GERM percentage at position P1, except for genotypes G18 and G28. At position P2, genotypes G1, G2, G4, G5, G6, G7, G8, G9, G10, G12, G25, G26, G29, G30, and G31 presented the highest GERM percentages. At position P3, only genotypes G15, G17, G18, G19, G22, G24, G26, and G27 did not show high GERM percentages for this position. At position P4, genotypes G2, G3, G4, G6, G8, G9, G23, G25, G26, G27, G28, G30, G31, and G32 had the highest GERM percentages.

Regarding the unfolding of TZVG variable, it was observed that, except the genotypes G4, G5, G7, G10, G18, G19, G25, and G28, the remaining genotypes showed the highest TZVG percentages at position P1 (Table 5). Genotypes G4, G5, and G7 had the highest TZVG percentages at positions P1 and P2. Genotypes G10 and G19 showed no statistical difference among pod positions for the TZVG variable. Genotype G18 showed the highest TZVG percentages at positions P2 and P4, and genotype G25 had the highest TZVG percentages at positions P1 and P4.

Regarding pod positions on the plant, genotypes G10, G18, G19, G24, G26, and G28 were the only ones that did not show high TZVG percentages at position P1. At position P2, genotypes G1, G3, G4, G5, G6, G7, G8, G14, G16, G18, G21, G22, G23, and G31 presented the highest TZVG values. At position P3, genotypes G7, G8, G17, G18, G19, G20, G25, G26, and G32 had the lowest TZVG percentages, differing from the others. At position P4, genotypes G1, G2, G3, G4, G5, G15, G17, G20, G21, G26, G28, and G30 differed from the other genotypes by presenting the lowest TZVG values.

Regarding unfolding of the TZVB variable, genotypes G1, G2, G6, G9, G14, G15, G16, G23, G24, and G26 showed the highest TZVB percentages at position P1 (Table 6). Genotypes G4, G5, G7, G8, G12, G13, G17, G20, G21, G22, G25, G27, G29, and G31 did not show any difference in TZVB among the studied positions.

The genotypes showed variation in the percentage of TZVB according to the pod’s position on the plant. In general, G10 and G11 stood out in the upper positions (P1, P3, and P4), while G18 and G28 showed the highest values in the intermediate and lower positions (P2, P3, and P4), differing from the others with 70% and 67% TZVB, respectively. Genotypes G30 and G32 showed the highest values in positions P1 and P2. In position P2, most genotypes (G4, G6, G7, G8, G16, G25, G27, G30, G31, and G32) exhibited superior performance, and in positions P3 and P4 there was no significant difference between the genotypes. Correlation analyses indicated a positive association between PCG and GERM at positions P1, P2, and P3 (r = 0.8–0.9), and between PCG and TZVB at positions P1 and P2 (r = 0.5–0.8). GERM also correlated with TZVG and TZVB, especially at P1, while TZVG and TZVB showed a moderate correlation at positions P1 and P2 (Figure 2).

According to the principal component analysis, the physiological variables of the seeds had stronger associations with P1 (Figure 3). This result indicates that the pods in the first position produce seeds with a higher germination capacity, corroborating previously presented results.

When analyzing how pod position on the plant influences seed physiological variables, we found that position P1 was responsible for the best results for the variables evaluated, with the exception of genotypes G18 and G28. This result indicates that pods from the first position produce seeds with greater germination capacity and a higher ability to generate normal seedlings. However, the genotypes are still under development and, therefore, do not yet exhibit stability. Nevertheless, the results obtained highlight the relationship between pod position on the plant and seed physiological variables.

4. Discussion

Viability and vigor reach their maximum potential when the seed attains physiological maturity, at which point it ceases to receive photoassimilates from the mother plant and is ready to be harvested and perform its functions [25]. During pod and seed development, a large proportion of the photoassimilates produced are allocated to their formation and filling, with yield losses being directly related to reductions in photosynthetic performance [5]. Soybean production is limited by several abiotic factors, such as temperature, precipitation, and light availability [26]. Among these factors, light availability is directly related to photosynthetic capacity, such that the locations where seeds are found influence photosynthetic uptake, and when photosynthetic performance is low, poor synthesis and translocation of assimilates occurs for soybean seeds [27].

Soybean genotypes exhibit higher percentages of first germination count (PCG) when at position P1, even when sharing the best results with other positions. Leaves play a fundamental role in the production and transport of photoassimilates to the seeds [28]. Photosynthetically active radiation (PAR) intercepted and leaf area index (LAI) are parameters used to estimate crop growth, encompassing the period of node and axillary bud development, during which flowers are formed, giving rise to pods and seeds [29]. Photosynthetic activity is dependent on light availability; under shaded conditions, a decline in photosynthetic rate and leaf area may occur [16]. Thus, the pods located at position P1, because they formed earlier, possibly had a higher priority in directing the photoassimilates produced by the mother plant. Even under shade, the early development of these pods ensured an adequate supply of assimilates during seed filling, resulting in greater accumulation of reserves and, consequently, greater vigor. Therefore, to enhance the study results, analyses of secondary metabolites and antioxidant enzymes can aid in understanding these responses, and would be interesting to develop in future work.

Regarding FGC, it was observed that positions P1 and P3 provided better results for the genotypes studied, indicating that pods formed at these positions produce more vigorous seeds compared to other positions. When leaves develop in shady conditions, they produce a greater number of light-harvesting complexes (LHCs), which are part of the photosystems and help to better utilize available light. This adaptation leads to an increase in chlorophyll b content, responsible for expanding the range of light that the plant can absorb, increasing the photosynthetic apparatus [30,31]. This result may be related to the fact that pods at position P3 receive greater amounts of direct light, while leaves at position P1, due to shading, are thinner than those exposed to normal light.

Additionally, pods at position P1 benefit from being the first to develop and, as initial sinks, receive a greater amount of photoassimilates, while seeds at position P3 exhibit vigor due to the photosynthetic contribution of leaves at that position [32]. Soybean plants may exhibit determinate growth habit, characterized by larger leaves throughout the plant, or indeterminate growth habit, with smaller leaves in the upper canopy and larger leaves in the middle and lower canopy [33]. These characteristic influences light interception, photosynthesis, and the availability of photoassimilates [34].

Soybean seeds are composed of approximately 40% protein, 21% oil, and 39% other compounds such as carbohydrates, which can undergo changes and impact seed quality, as protein synthesis depends on nitrogen mobilization following leaf senescence, and carbohydrate and lipid synthesis relies on the integrity of the photosynthetic system [35], this content may vary depending on the cultivar [36]. Seed reserve accumulation occurs in three phases: an initial phase of rapid cell division and tissue differentiation, an effective seed filling phase, and a maturation phase [33]. However, the initial and final phases differ only in terms of the chemical fractions involved, with the deposition of carbohydrates, proteins, and lipids [37,38]. As mentioned earlier, adaptation regarding light seeking aims to optimize the use of light in shaded environments, ensuring the maintenance of photosynthetic activity, albeit at a lower intensity. Since photosynthesis is the main source of assimilates used during seed filling, alterations in the photosynthetic efficiency of leaves can modify the pattern of reserve deposition (carbohydrates, proteins, and lipids) in the different stages of seed development [39]. Thus, variations in light distribution within the canopy, associated with the position of the pods on the plant, can result in differences in the physiological quality of the seeds produced [40].

Based on these findings, it is evident that the position of the pod on the plant influences the physiological quality of the developing seeds, particularly in relation to vigor. Future research on this topic could focus on quantifying the biochemical components present in the seeds and elucidating their relationship with the deposition of photoassimilates by the mother plant. Additionally, identifying the main chemical elements associated with seed vigor and viability would contribute to a deeper understanding of these physiological differences. Among the study’s limitations, the experiment was carried out at a single site, which may restrict the extrapolation of the results to other environmental conditions. Furthermore, the lack of complementary biochemical analyses limits the interpretation of the mechanisms, highlighting the need for future studies to investigate amino acid profiles and secondary metabolites. The analysis of secondary metabolites would add important information to the study because these compounds are closely linked to physiological and biochemical mechanisms that determine the quality of sensations, especially in terms of vigor, tolerance, and stress tolerance [25,41].

5. Conclusions

The position of the pods on the soybean plant influences the physiological quality of the seeds. In general, the P1 position, when the plants are in the R4 reproductive stage, with fully developed pods measuring 2 cm on one of the four upper nodes of the stem, is responsible for the best results in seed physiological quality tests for most of the soybean genotypes evaluated. These results indicate that pod position should be considered in breeding and seed production programs, since genotypes with greater physiological stability in the upper positions may be preferential in selection strategies. In the future, studies in different environments and evaluation of biochemical traits may confirm these patterns and contribute to the development of cultivars with higher seed quality and physiological uniformity.