There is ample scope of rectification of different categories or states of soybean plant and seed morphological characteristics and inclusion of new characters for classification of soybean varieties and future testing of new varieties.
Our findings indicated that several varieties exhibited discrepancies in trait identification as per DUS guidelines. Moreover, our analysis suggested opportunities to expand the DUS test guidelines (2009) by incorporating additional traits and exploring a broader range of existing plant or seed characteristics.
Seedling hypocotyl pigmentation
Anthocyanin pigmentation in the hypocotyl region of soybean seedlings is a critical morphological trait for differentiating various cultivars. The existing guidelines for Distinctness, Uniformity and Stability (DUS) testing classify soybean hypocotyl anthocyanin pigmentation into two categories: present and absent. Our research identified significant variations in the intensity of this pigmentation among the surveyed soybean varieties. In the analyzed collection of notified varieties, the intensity of hypocotyl anthocyanin pigmentation ranged from very high to very low. Based on these observations, soybean varieties can be classified into four distinct categories: (i) High, (ii) Medium, (iii) Low and (iv) Absent (Plate 1).
In contrast, under the previous classification, 48.3% of varieties were considered absent, with 51.7% classified as present. The varieties exhibiting high pigmentation included NRC 130, NRC 131, MAUS 158, JS 335 and Punjab Soya 1. Conversely, varieties such as JS 93-05 and NRC 7 demonstrated medium pigmentation, while JS 20-69, NRC 138, PK 416, RKS 18, SL 295 and SL 525 had low pigmentation. Varieties characterized by the total absence of pigmentation were JS 20-116, JS 20-98, JS 20-29 and JS 20-34, among others (Table 1). The coloration of hypocotyls is correlated with flower color; specifically, the presence of hypocotyl pigmentation is associated with purple flowers, while its absence correlates with white flowers.
Palmer and Payne (1979) reported that flower color is determined by the dominant/recessive alleles of the W1 locus, which pleiotropically regulates hypocotyl color. Our observations suggest that even minimal anthocyanin presence in the seedling hypocotyl relates to the occurrence of white flowers. The hypocotyl anthocyanin color is governed by the T and W1 loci. The W1 gene encodes a flavonoid 32, 52 -hydroxylase (F32 52 H), which is crucial for the synthesis of flavonoids exhibiting the 32 ,42 ,52 B-ring hydroxylation pattern (
Buzzell et al., 1987;
Zabala and Vodkin, 2007). Furthermore,
Murai et al., (2016) highlighted that soybean plants with the dominant W1 allele exhibit purple hypocotyls regardless of the T locus, while recessive w1 and t alleles lead to green hypocotyls and recessive w1 paired with dominant T results in light anthocyanin or bronze hypocotyls. In our study, it was apparent that varieties demonstrating low anthocyanin pigmentation in hypocotyls were also closely linked to the presence of white flowers.
This investigation underscores the importance of hypocotyl pigmentation as a morphological marker in soybean breeding, seed production and classification, contributing to the advanced understanding of genetic traits associated with flower color and overall plant characterization.
Leaf shape
In accordance with established testing guidelines, three leaf shape categories have been recognized: (a) Round ovate, (b) Pointed ovate and (c) Lanceolate. Notably, the distinction between pointed ovate and round ovate leaf shapes is minimal, with the primary differentiating factor being the leaf tip morphology. The width-to-length ratios for these two categories often overlap across different varieties, making differentiation challenging. Certain varieties, such as JS 90-41, exhibit characteristics that de
viate significantly from the pointed ovate classification, displaying neither a perfect pointed ovate nor lanceolate form; these are more accurately described as triangular. Therefore, it is advisable to expand the classification of leaf shapes to include: (i) Lanceolate, (ii) Triangular, (iii) Pointed Ovate and (iv) Round ovate (Plate 2) (Table 1). According to
Bernand and Weiss (1973), the leaf shape in question is governed by a single gene, where the homozygous dominant state (LnLn) results in broader leaves, while the recessive homozygous state (lnln) produces narrower leaves. The
UPOV (2022) has also provided a similar classification for this trait.
Leaf size
The ratio of leaf length to leaf width L:W has been found to be a significant factor in classifying soybean leaf shapes. This ratio ranges from 1.11 to 2.71 and can be divided into two main categories. The first category, with an L: W ratio ranging from 1.11 to 1.87, encompasses pointed ovate and round ovate leaf types. The second category, with ratios between 2.50 and 2.71, includes only lanceolate leaf types. Notably, the L: W ratio for lanceolate leaves was significantly higher (LSD 0.1903) than that of the other groups. Higher L: W ratios indicate a more pointed leaf shape, with ratios of 1.87 (JS 90-41), 1.84 (JS 2) and 1.83 (JS 20-34) reflecting a transition towards triangular shapes (Fig 1). According to
Sawada (1991), a Leaf Shape Index (LSI) value exceeding 2.6 suggests narrow leaves, while values below this threshold indicate broad leaves Plate 3.
In the current DUS Test guidelines, leaf size has not been included as a criterion, despite being a prominent morphological characteristic for differentiating plant varieties. Leaf size has been assessed in terms of length, width and area. Significant variation in leaf length was observed among varieties, ranging from 7.7 cm in Davis to 16.2 cm in PS 1347 (Plate 3). The leaf width varied from 4.67 cm in PS 1024 to 11.17 cm in Swarn Vasundhara. Lanceolate varieties such as PS 1024, JS 93-05, RVS 18, PS 1347 and JS 95-60 exhibited relatively narrow leaves, measuring between 4.67 cm to 5.67 cm. In contrast, pointed and round ovate leaves ranged from 4.67 cm to 11.17 cm. Notably, certain varieties such as PS 1024 (4.67 cm), Davis (5.17 cm), Gaurav (5.33 cm) and JS 80-21 (5.67 cm) displayed widths smaller than that of the lanceolate type JS 95-60 (5.67 cm), indicating a narrower leaf shape within their respective Similar categories had been adopted by
UPOV (2022).
Furthermore, significant differences in leaf area were noted among the various soybean varieties, with areas ranging from 30.9 cm² to 116.8 cm² (LSD at 10.96). Generally, varieties with larger leaf areas tended to belong to the pointed or round ovate categories, in comparison to some lanceolate varieties, such as PS 1347, RVS 18, JS 95-60, JS 93-05 and PS 1024, which had leaf areas of 59.0 cm², 58.0 cm², 53.4 cm², 43.3 cm² and 42.2 cm², respectively. Among the lanceolate types, PS 1347, RVS 18 and JS 95-60 exhibited significantly larger areas than JS 93-05 and PS 1024 (Fig 2). For pointed ovate and round ovate leaf types, the distribution of leaf area among varieties was quite broad.
Additionally, smaller seed size varieties such as Davis, Gaurav, SL 96 and JS 20-34 had leaf areas ranging from 30 cm² to 50 cm² Varieties with medium leaf sizes, measuring between 51 cm² and 70 cm², included Lee, JS 2, MACS 450 and JS 97-52. Lastly, those with larger leaf sizes (greater than 70 cm²) included RKS 24, MAUS 158, NRC 86, PS 1029, Karune, NRC 130 and Swarn Vasundhara (Plate 3) and (Fig 2).
Leaf blistering
Leaf blistering refers to the degree of roughness on the surface of leaves. This characteristic can be categorized into two distinct types: smooth and rough. Previously, leaf blistering was not included in DUS (Distinctness, Uniformity and Stability) testing guidelines (
Anonymous, 2009) due to a lack of sufficient reference varieties. However, with the recent introduction of new varieties exhibiting this trait, there is an opportunity to add this characteristic to the test guidelines for improved variety classification.
Example varieties that display leaf blistering include NRC 130, NRC 131 and Karune. In alignment with UPOV (Union for the Protection of New Varieties of Plants) guidelines, which classify this characteristic into five categories (i) absent, (ii) very weak, (iii) weak, (iv) medium, (v) strong and (vi) very strong (
UPOV, 2022) the incorporation of leaf blistering into DUS testing protocols could be beneficial. Incorporating these available reference varieties, the characteristic of leaf blistering could be effectively integrated into the DUS testing guidelines of the PPV and FRA (Protection of Plant Varieties and Farmers’ Rights Authority), under the following categories, Leaf Surface: (i) Smooth and (ii) Rough (Plate 4) (Table 1).
Inflorescence pattern in soybean varieties
The study of inflorescence patterns across various released and notified soybean varieties has revealed notable variation in this characteristic. The inflorescence typically presents as an axillary raceme on both the main stem and its branches. In certain varieties, flowers cluster in groups of four to five at the nodes of the main stem and branches, exhibiting a sessile arrangement. Conversely, other varieties feature flowers on elongated peduncles, where the length of the peduncle is greater compared to the shorter peduncles observed in sessile arrangements.
Schaik and Probst (1958) distinguished between pedunculate inflorescences-characterized by long peduncles with sessile flowers and found that the pedunculate form (Se) is dominant over the sessile form (se). In light of these findings, a new characteristic for DUS (Distinctness, Uniformity and Stability) testing is proposed, leading to the categorization of soybean inflorescences into two distinct types: (i) Pedunculate Inflorescence and (ii) Sessile Inflorescence (Plate 5). Examples of soybean varieties with Pedunculate Inflorescence include Ankur, JS 93-05, JS 76-205, JS 95-60, MAUS 158, PS 1024, JS 20-34 and MACS 1520. Conversely, varieties exhibiting Sessile Inflorescence include Hardee, Pusa 97-12, MACS NRC 1667, NRC 142 and NRC 150. Notably, some sessile varieties, such as Type 49, JS 2, KHSb 2, Pusa 24, JS 335, SL 295 and MACS 1188, display very small peduncles (Table 1). Currently, this characteristic has yet to be addressed by UPOV (International union for the protection of new varieties of plants).
Pod pubescence
Pod pubescence refers to the presence of hairs on soybean plants and their pods. This trait is influenced by specific genetic loci-Pd1, Ps and Pl-located on linkage groups D1a+Q, H and K on chromosome 9 (
Cregan et al., 1999). According to
Bernard and Singh (1969), a sparse or semi-sparse allele is dominant at the Ps locus, yielding various types of pubescence: Glabrous (P1), curly pubescence (Pc), dense pubescence (Pd), sparse pubescence (Ps) and puberulent (p2). The presence or absence of hairs on the plants and pods serves as a distinct identification marker for soybean varieties. In accordance with DUS (Distinctness, Uniformity, Stability) guidelines, pubescence is classified simply as either present or absent. However, our research has revealed that some varieties classified as pubescence absent display a low intensity of pubescence, resulting in potential confusion. Notably, certain plant types exhibit nearly non-existent pubescence. One such variety, Karune, was recently released and is characterized by its minimal pubescence on both plants and pods.To enhance clarity in classification, there is need to revise the categories of pubescence into three distinct states: (i) presence of pubescence, (ii) puberulent (sparsely present) and (iii) absence of pubescence (Plate 6) (Table 1).
Seed color
According to the DUS guidelines, soybean seed colors are classified into four categories: yellow, greenish-yellow, green and black. When yellow and black-seeded lines are crossed, they segregate into yellow, brown and black seeded progenies. Notably, while brown-seeded lines are not represented among currently released and notified varieties, sources are available within germplasm collections.
The color of the soybean seed coat is a polygenic trait influenced by multiple loci, specifically I, R, T, W1 and O (
Senda et.al., 2002). The homozygous recessive genotype results in a self-colored seed coat. The R and T loci determine specific seed coat colour such as black (iRT), brown (irT), or buff (irt) by regulating anthocyanin and proanthocyanin pigments. In the DUS Test guidelines, seed coat colors are currently categorized into four groups: 1. Yellow 2. Greenish-yellow 3. Green 4. Black. To enhance this classification, it is recommended to introduce an additional category for brown, leading to a total of five categories for soybean seed coat color
i.e. 1. Yellow 2. Greenish-yellow 3. Green 4. Brown 5. Black (Plate 7) (Table 1).
Pod color
The DUS testing guidelines set forth by the PPV andFRA recognize three pod color categories: Yellow, brown and black. However, this study reveals that the brown coloration observed across various years and soybean varieties could be refined into a more detailed classification. We propose the introduction of five distinct categories: (i) Yellow, (ii) Light brown, (iii) brown, (iv) Dark brown and (v) Black (Plate 8) (Table 1). This fine categorization would enhance the differentiation of soybean varieties. Additionally, it is noteworthy that the UPOV guidelines encompass a total of seven categories based on available variability (
UPOV, 2022).
Seed hilum color in soybean
According to the DUS test guidelines, the seed hilum color is classified into five distinct categories: yellow, grey, brown, black and variegated. Within the brown category, there exists a variation in the intensity of the hue among different soybean varieties, ranging from dark brown to light brown (Table 1). The color of the hilum in soybeans is controlled by multiple genes, notably the I locus, which comprises four alleles (I, ii, ik and i) responsible for flavonoid pigmentation. The presence of the I allele leads to a complete inhibition of pigmentation. Other genes, including T, R and O, also influence hilum color, particularly in varieties exhibiting the recessive i allele. For genotypes ii and ik, additional interactions with genes such as TR, TRO, Tro, tRW1, tRw1 and tr can result in various colors, including black, brown, red brown, imperfect black, buff and yellow. Notably, when the I allele is present, the TR and tRW1 genes will yield a grey hilum, while the other genes will produce a yellow hilum (
Specht and Williams, 1978;
Palmer and Stanley, 1979). To enhance the classification of soybean varieties based on hilum color, it is recommended to subdivide the brown category into dark brown and light brown. Consequently, the following refined categories for hilum color are proposed: (i) yellow, (ii) grey, (iii) light brown, (iv) dark brown, (v) black and (vi) variegated (Plate 9) (Table 1). In alignment with UPOV guidelines, the brown hilum color is typically categorized into three groups: light brown, red brown and dark brown.
