Effect of Seed Coat Characteristics on Seed Quality in Soybean [Glycine max (L) Merrill] Genotypes with Contrasting Seed Longevity Traits

Soybean [Glycine max (L) Merrill] is a native of eastern Asia and belongs to botanical family Fabaceae. It contains 20 percent oil and 38-42 percent high quality protein possessing high level of essential amino acids like lysine (5%), minerals (4%), phospholipids (2%) and vitamins A and D (Hymowitz and Harlan, 1983). At physiological maturity, the soybean seed reaches its maximum potential for germination and vigour. This potential is short lived and reduces substantially. A number of seed characters, which are under genetic control such as seed size, per cent hard seededness, permeability and oil content are reported to be associated with seed quality in soybean.
       
The seed coat is the seed’s primary defense against adverse environmental conditions. It is the main modulator of interactions between the internal structures of the seed and the external environment. A hard seed coat protects the seed not only from mechanical stress but also from microorganism invasion and also from temperature and humidity fluctuations during storage. Phenolic compounds in the seed coat contribute to seed hardness and inhibition of microorganism growth. Lignins and phenolic polymers  found in the cell wall are believed to contribute to the compressive strength, resistance to degradation by microbial  attack and  water  permeability to the polysaccharide  protein  matrix of the cell wall (Francisco et al., 2008). The seed coat is one of the main determinants of seed germination, vigour and longevity potentials. However, there are no inter-varietal reports on seed coat characterization. Hence, seed coat characterization of soybean genotypes showing contrasting longevity patterns needs to be addressed to develop soybean varieties with better seed longevity.  Therefore, the present study was undertaken to study the Effect of seed coat characteristics on seed quality in soybean [Glycine max (L) Merrill] genotypes with contrasting seed longevity traits.

The present study was carried out both in the experimental farm and laboratory of Division of Seed Science and Technology, IARI, New Delhi during 2015-16 and 2016-2017 using 24 soybean genotypes having contrasting seed longevity traits, comprising 12 good (black-seeded) storers and 12 poor (yellow-orange seeded)  as the experimental material, which was procured from Soybean Section, Division of Genetics, ICAR-IARI, New Delhi. About 200 g of untreated freshly harvested soybean seeds from each genotype were packed in muslin cloth bags and stored in the laboratory of Division of Seed Science and Technology under ambient storage environment (average 25±2°C and 65±5% RH). Various parameters were recorded to categorize the soybean genotypes using established methodologies and protocols, as under.
 
Seed coat colour and seed hilum colour
 
Both the parameters were recorded by using Royal Horticultural Society (RHS) colour chart during the harvest stage under natural day light.
 
Seed coat percentage
 
Seed coats from 5 g seeds in two replications were removed carefully with the help of forceps after applying a gentle stroke to seeds placed in-between the muslin cloth. Seed coat and cotyledons were collected and weighed separately. The total weight of seed coat and cotyledon was considered as the seed weight. The proportion of seed coat was calculated as follows:  
Mechanical strength of the seed coat
 
Mechanical strength of the seed coat was measured as the first break point on the graph of seed cracking using the texture analyzer (Stable Micro System) following the method of Kuchlan (2006). The seed was placed on the stationary plate of the instrument. The plane perpendicular to the plane of the hilum was parallel to the surface of the plate. The force was applied by gradually increasing the force, which was plotted along the Y-axis. The duration of force applied (until the seed is cracked) was plotted along the X-axis. There was a gradual increase in the graph with the increase of force and at a certain point of time there was a sudden drop on the graph which indicated the first break, on the seed coat surface. Further increase in the force along the graph and next drop on the graph was recorded due to breakage and subsequently for the pieces of seed.
 
Seed coat thickness and seed coat histology
 
Various steps as below were undertaken to study these parameters:
 
Dehydration of samples and block preparation
 
Dehydration of samples was done using TBA series.
 
Seed samples
 
Fixed in FAA→Wash in 30%/50%/70% Ethanol →  55%TBA (2hr)→ 75%TBA (overnight)→ 85% TBA(1hr)→   95% TBA(1hr) →ax (blocks).

After that paraffin blocks were prepared. These blocks were subjected to microtome sectioning and drying.
 
Staining techniques
  50% Alcohol (5min) → Safaranin (45 min) → 50% Alcohol (5min) → 70% Alcohol (5 min) → 95% Alcohol (5min)  Fast Green (a dip only) →95% Alcohol (5 min) → Xylene:  Alcohol (1:1) (5 min) →Pure Xylene → DPX Mount.
       
Thickness of the seed coat was measured using phase contrast microscope (Leica software) which is connected to a computer. Finally, using phase contrast microscope, the thickness of parenchyma cells (epidermis), thickness of palisade parenchyma (hypodermis) and length of anticlinal cells were measured (μm). The stained seed coat sections were viewed using phase contrast microscope which is connected to a computer. Photographs were recorded using the above set up.
 
Lignin content
 
Lignin content of the seed coat was determined colorimetrically. The sample was extracted in NaOH solution and the aliquot was adjusted to pH 7.0 and 12.3. The amount of lignin was calculated by the difference between A 245 (pH 7.0) and A 350 (pH 12.3), i.e. E 350/sample. Moisten 100 mg of oven dried experimental material in a mortar with water. Add 2 ml of NaoH to residue and extract 70-80°C for 12-16hr. Cool, add 0.45 ml of 2N HCl and adjust the pH to 7 or 8 with NaoH. Make up the volume to 3 ml with water. Centrifuged at 2000 g for 5 min. Collect the supernatant. To 0.8 ml of extract, add 0.8 ml of 0.1 M sodium phosphate buffer, pH 7.0. To another aliquot of 0.8 ml extract, add 0.8 ml of 0.1N NaoH pH 12.3. Measure the absorbance at 245 and 350. Derive the lignin concentration from the difference between A245 and A350 on pH 7.0 and 12.3, samples diluted with buffer and NaoH, respectively. Express the amount of lignin as E 350/sample.
 
Statistical analysis
 
The analysis of variance was done using single factor analysis using the “F” test. Critical difference (CD) was also used to test the difference between any two means. The mean data of both fresh and stored seeds was jointly analyzed using paired t-test two samples for means to compare whether there was significant difference between the values of fresh and stored seeds.

Seed coat colour and seed hilum colour
 
Black seeded genotypes performed better as compared to yellow seeded ones. Further, the seed coat colour remained unchanged with advanced seed storage of eight months (Table 1). The black-seeded genotypes with white hilum maintained seed quality higher than IMSCS (70%) than yellow-seeded genotypes with brown to grey hilum after eight months of laboratory ambient storage. These results were in accordance with earlier work of Kumar (2005) and Sooganna (2015) in soybean. Further, black seed coat is also associated with resistance to field weathering (Mugnisjah et al., 1987) and better resistance against pathogen (Starzinger and West, 1982) and hence, is classified as a better storer. Better physiological quality of genotypes with black coats as compared to genotypes with yellow coats was also reported by Bahry et al., 2017 in soybean.
 


Seed coat percentage
 
With respect to good storers, the average seed coat percentage for fresh seed and after eight months of storage, was recorded to be 9.4146% and 9.4143%, respectively. Genotype G-2614 showed maximum seed coat percentage (10.01%) followed by G-2251 (9.83%), whereas M-11913 recorded least seed coat percentage. However, with respect to poor storers, the average seed coat percentage for fresh seed and after eight months of storage, was recorded to be 7.6059% and 7.6051%, respectively. Genotype 761(137) recorded maximum seed coat percentage (8.244%) followed by genotypes 250 (129) which recorded 8.046% and 241(128) which recorded 7.17%. Further, the black seeded soybean genotypes recorded higher seed coat percentage than yellowish orange genotypes. However, no significant difference was observed between fresh and stored seeds, irrespective of quality of seed. The study recorded black seeded genotypes viz. G-2614 and G-2251 with higher seed coat percentage as better performers (Table 1).
 
Mechanical strength of seed coat
 
There were significant variations in mechanical strength of seed coat among good and poor storers in the freshly harvested seeds (Table 1). Hard seed coat has been observed to be very beneficial in maintaining the seed viability and vigour. With respect to the mechanical strength of seed coat for freshly harvested seeds, significant variations were recorded among good and poor storers. However, no clear association was reported between the mechanical strength of the seed and their seed quality, after eight months of ambient laboratory stored soybean seeds (Sooganna, 2015). On the contrary, Zahid (2013) reported a significant reduction in soybean seed coat hardness from 36 months stored seeds. This can be attributed to the fact that the black-seeded genotypes are land races which are rich in anthocyanin, an antioxidant and have less oil; which accounts for better storability of black-seeded genotypes with greater mechanical strength.
 
Seed coat histology
 
The stained seed coat sections were viewed and photographed using phase contrast microscope. Soybean seed coat consisted of an epidermis of one cell layer (cutinized palisade cells), a hypodermis of single layer of large cells with thick anticlinal walls having intercellular spaces (Fig 1). The pictorial representations revealed that the hour-glass cells, were more in number, uniform in shape and distribution along the cross section of the seed coat in good storer varieties, viz. AMSS-34, G-2265 as compared to the yellow-seeded  poor storer varieties 218(123), 241(128) (Fig 2).
 




Seed coat thickness
 
Thickness of the seed coat was measured using phase contrast microscope (Leica software) which was connected to a computer. The thickness of parenchyma + palisade parenchyma and length of anticlinal cells was measured and is presented in Table 2. Seed coat thickness in soybean genotypes ranged between 193 to 200 ìm. Small seeded soybean seeds have thicker seed coat tissues which helps in better maintenance of viability in contrast to the bold seeds where seed viability is not better maintained.
 


Lignin content
 
The higher the lignin content in the seed coat, the better is the expected resistance to mechanical damage. In the present study, significant difference was recorded for lignin content between good and poor storers; however, no significant change was observed in lignin content after storage for eight months (Table 3). In soybean, seed coat lignin also plays an important role in physical and physiological seed traits (Alvarez et al., 1997). The soybean seed coat is awfully thin and squat in lignin content and provides little protection to the weak radicle that lies in a susceptible location straight beneath the seed coat. The steadiness of seed coat lignin yet in storage, can be explained by the insolubility and intricacy of the lignin polymer.

Genotypic variations were recorded in seed coat characteristics and soybean seed quality. Black-seeded genotypes with white hilum were found to be better performers. Seed coat and hilum colour remained unchanged with seed storage. In the present study, no clear association could be established between the mechanical strength of the seed and their seed quality after eight months of laboratory ambient stored seeds. Transverse sections of the seed coat of soybean seeds depicted three observable cell layers: an outer epidermis, palisade cell layer and an hourglass cell. The hour-glass cells were more in number, uniform in shape and distribution along the cross section of the seed coat in good storers (black-seeded) than those to poor storers (yellow-seeded) genotypes. Black-seeded genotypes with white hilum, thick seed coat and higher seed coat (%) with more and uniform hours glass cells attributed to better seed quality in soybean.

None.