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Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 6 Number 7 (2017) pp. 2358-2363 Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2017.607.279 Seed Coat Permeability Studies in Wild and Cultivated Species of Soybean Subhash Chandra*, Raju R. Yadav, Shatakshi Poonia, Yashpal, Darasing R. Rathod, Ashish Kumar, S.K. Lal and A. Talukdar Division of Genetics, ICAR- Indian Agricultural Research Institute, New Delhi, India *Corresponding author ABSTRACT Keywords Glycine, Imbibation, Seed coat permeability, SCP, Seed longevity. Article Info Accepted: 23 June 2017 Available Online: 10 July 2017 Soybean [Glycine max (L.) Merr.] Seeds loose viability at a rapid pace under tropical and subtropical conditions. Poor seed longevity in cultivated soybean genotypes may associates with seed size, testa colour and seed coat permeability. Present study aims to understand kinetics of imbibation and evaluate the seed coat permeability in Glycine tomentella, Glycine soja and Glycine max. Out of all 125 genotypes of three species, 20 genotypes of wild annual and perennial species were small and hard seeded. All black seeded genotypes of three species including cultivated soybean were small in size, suggesting positive correlation of testa colour with seed size. There was strong positive correlation between 100 seed weight and seed coat permeability percentage based on 6 hours rapid imbibation test (0.83) and Seed coat permeability assessment in germination paper after 7 days (0.81). Kinetics of Imbibation suggest that G. max starts uptake water after some minutes, but seeds of G.tomentella imbibes water after 78 hours. Seed weight of G. soja genotype remained constant even after 4 days. Genotype of G. max (DS 9712) imbibes water up to 24 hours with steady linear curve, after that it becomes constant up to germination. Variability in seed coat permeability (44%) after 7 days slow imbibation in G. soja (DC 2008-1) suggests its use in introduction of optimum permeability behavior in G. max to slow down the extra leaching for improvement in seed longevity. Introduction Soybean is a major component in the diet of million people throughout the world. It plays vital role in nutritional security as it is a rich source of protein and oil. Although soybean is grown across the world, yet United States of America, Brazil, Argentina, China and India are the major producers. Taxonomically, the genus Glycine is divided into two subgenera: Glycine (with perennial species) and Soja (with annual species). Subgenus Soja includes the diploid species Glycine max (cultivated soybean) and Glycine soja (wild progenitor). Subgenus Glycine is currently consisting of 30 wild perennial species including Glycine tomentella, Glycine tabacina etc. (Singh et al., 2014). The typical cultivated soybean displays a bush-type growth habit with a stout primary stem and sparse branches, bearing large seeds with variable seed coat colors, whereas the wild type is a procumbent or climbing vine with a slender, many-branched stem bearing small, coarse black seeds. Soybean seeds are the important regenerative unit which cannot retain their viability indefinitely. Seed coat, the outer covering of every mature seed, is an important part of the 2358 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 seed and is associated with preservation of integrity of internal seed parts, protection of embryo against mechanical injuries and regulation of gaseous exchange between embryo and environment. It also offers tolerance to fungal activity, flooding, and protection to microbial attacks. The seed coat is also related to seed viability under delayedharvest field condition, seed longevity under humid environments and inhibition against rapid imbibation of water, which often deteriorates the germination (Kebede et al., 2014). Seeds with impermeable seed coat often results in adverse quality, and cost factors in processing seeds for vegetable oil and soy foods, and they affect the texture and consistency of products such as fermented soy food (Jang et al., 2015). Soybean seed reaches its maximum potential for germination and vigour at physiological maturity (Crookston et.al., 1978), which then gradually declines till harvest, followed by a more rapid decline thereafter. Loss of germination potential or viability is more acute in tropical and sub-tropical regions compared to temperate environments. However, hard seed coat (Tiwari et al., 1995), small seed size (Tiwari et al., 1989), black seed coat (Dassou et al., 1984) and tight attachment of the seed coat to the cotyledons (Kuchlan et al., 2010) are useful parameters for viability in tropical environment. Seeds of the wild type soybeans with hard seed coat exhibit better germination under tropical environment than the cultivated type. However, limited information is available on seed coat permeability and pattern of imbibitions in wild and cultivated germplasm of soybean. Therefore, the current study was conducted to evaluate seed coat permeability in the seeds of cultivated (G max) and two wild type soybean species (G soja and G tomentola) under ambient storage and to see their effect on seed viability. Materials and Methods Plant material The experimental material (125 genotypes) consisted of an accession of G tomentella, 19 accession of G soja and 105 genotypes of G max. The cultivated genotypes included high yielding popular cultivars, advanced breeding lines and exotic stocks (Table 1). The seeds, which were obtained from the Division of Genetics, IARI, New Delhi, varied in testa colour, seed size and seed weight. Seed coat color of all the 125 genotypes was recorded by visual comparison with the ‘color chart’ of Royal Horticultural Society, London under natural day light. Three replicates of 100 randomly selected seeds were taken for determination of seed weight. The mean value of the 3 replications was expressed in grams. Based on 100-seed weight, the genotypes were categorized as bold (>10 g), medium (8 10 g) and small (< 8 g) seeded type on basis of size diversity. For understanding the kinetics of imbibitions, the water uptake pattern was measured as per Hahlis and Smith (1997). Ten seeds of five genotypes viz., G. tomentella, DC 2008-1, PI 424079, DS9712 and JS 335 were soaked in 25 ml of distilled water at room temperature and their weight was recorded at every two hours interval up to 24 h. Three genotypes, DC 2008-1 (G. soja) and DS 9712 (G. max) and G. tomentella were used to understanding pattern of imbibation up to 96 hours with interval of 4 hours in another experiment. The rate of water uptake was depicted as percentage increase over the initial weight of the seeds with time. Seed coat permeability (SCP) was tested through Rapid and Slow imbibitions tests. For rapid imbibitions test, 2359 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 20 seeds of each genotype in 3 replications were soaked in 100 ml of distilled water for 6 hrs at room temperature. For slow imbibitions test, 50 seeds of each genotype in three replications were placed in water-moistened germination paper towels, which were then rolled up and placed vertically in plastic containers on a shelf in a germinator. The towels were maintained at near 100% humidity for 7 days. SCP (%) was recorded as percent of seeds imbibed water in respective tests (Kebede et al., 2014). As per Sun et al., (2015), the genotypes with SCP < 20% were categorized as hard-seeded and genotypes with SCP > 80% were grouped as soft-seeded. Data were analyzed for ANOVA, MSD, correlation coefficient using SAS software package version 9.4. All the replicated data were subjected to Tukey’s Studentized Range (HSD) Test. Results and Discussion possesses smaller seeds and vice versa. In this study, wide variation was observed among the genotypes for the seed weight. G. tomentella accession had the smallest seeds (100-seed wt. =0.5312g) and the G. max genotype Dsb19 had the largest seeds (100-seed wt. =13.52g). The 100-seed weight of the G. soja accessions ranged from 0.561g (DC 2008-1) to 2.072g (PI 406684) with an average of 1.26g, while that in G. max ranged from 5.06g (UPSL-34) to 13.52g (Dsb 19) and had an average weight of 8.43g (Table 2). Among the cultivated genotypes, yellow seeded genotypes had highest mean seed weight (9.27g/100 seeds), followed by brown (8.16g/ 100 seeds), yellow-green (7.46g/100seeds) and green (7.01g/100 seeds). Accordingly, all the 20 accessions of wild type soybean were categorized as small seeded, while 48, 42 and 20 genotypes of the cultivated type were classified as small, medium and large seeded, respectively. Seed color Dynamics of imbibition Seeds of all but one G. soja accessions were dull black; PI366120 had shiny black seeds. Seeds of the single G. tomentella accessions were also dull black. However, enough variability for color was observed in the seeds of cultivated type i.e. G max. Out of 105 G max genotypes, 69 had yellow seeds and 25 had black seeds. Besides, 6, 3 and 2 genotypes had yellow-green, brown and green seeds, respectively. During domestication, people preferred yellow colored seeds over black and hence most of the cultivated genotypes have yellow seeds. However, black colored genotypes like Kalitur are also popular locally. Imbibition pattern of seeds is primarily governed by permeability of the seed coat (Kuchlan et al., 2010). More permeable the seed coat more is the imbibitions and viceversa. It was observed that the cultivated genotypes viz., DS9712 and JS335 started imbibing quickly after soaking, and there was an increase in fresh weight of the seeds up to 24 hrs. However, no imbibitions were observed in the seeds of G. soja (DC 2008-1 and PI 424079) and G. tomentella accessions (Fig. 1). Therefore, the seeds of the G soja and G tomentella were classified as hardseeded while that of G max was rated as softseeded. Among the soft seeds, the rate of imbibitions was not uniform. It was more in JS335 (0.196 g/h) than DS9712 (0.032 g/h) in the first two hours, which was later reversed. Seed weight Weight of the seeds is an important trait. It influences consumers’ preferences and market price, as well. In general, wild type genotype It was further observed that seeds of G soja accession DC2008-1 did not imbibe even up 2360 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 to 96 hrs. It indicated that the seed coat of this accession is highly impermeable. Similarly, G. tomentella seeds started imbibing only after 78 hrs, of course at a very slow rate. On the other hand, seeds of DS9712 (G max) started imbibing minutes after soaking. There was a linear increase in rate of imbibitions up to 24 hrs after which it remained stagnant up to 72 hrs beyond which weight gain was observed due to germination. Table.1 Genotypes used in experiment for testing seed coat permeability S.No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 Genotype G. tomentella PI-464925-B PI-464925-A PI-561355 PI-464869-A PI-522183-B PI-464889-A PI-407294 PI-407292 PI-406684 PI-366120 PI-507805 PI-423991 PI-522229 PI-507830-B PI-326582-B PI-424051 B PI-424079 PI-424032 DC-2008-1 DS 9712 DS-76-1-2-1 G-2215 G-2132 G-2144 PK-1243 PK-1135 Bragg TGX1855-530 TGX1864-15F TAMS-38 UGM-34 S.No. 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 Genotype UPSL-117-B USSL-291 EC-456615 EC-457442 EC-457472 EC-471635 EC-457189 DS-9909 MAUS-164 PK-1347 TS-148-22 MACS-869 DS-22005 DS-9817 DS-9819 His-01 JS-94-67 KB-222 MACS-450 PK-1024 PK-1041 VLS-61 SL-679 SL-710 E-20 SL688 PKS-34 NRC-71 NRC-78 DS-2614 JS-20-05 DSb-15 S.No. 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 Genotype PS-1477 PS-1480 MACS-1336 SL-900 DSB-19 PS-1503 DSB-21 PS-1505 NRC-89 AMS-56 DS-2708 PS-1518 DS 61 DS 178 DS 76-37-2 DS 93-3 EC13969 EC343361A EC1021 EC36961 EC39873 EC97351 EC105790 G2265 G2344 G2608 JS 335 G2651 G3023 GUJ-SOY-1 IC-144409 IC-141446 S.No. 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 Genotype JAVA-16 J-231 JS-76-257 M253 MACS565 MAUS-2 NRC-21 PK-1225A UPSL-19 UPSL-29 UPSL-34 UPSL-736 EC-458342 EC 1023 DS MM 64 G 2603 G 2253 G 2601 MACS 694 EC 471919 EC 472134 G 395 UPSL 163 UPSL 332 DS 74 AMSS 34 NRC 7 PK 416 PK 472 (S.No. 1: G. tomentella; 2 to 20: G. soja; 21 to 125: G. max) Table.2 Descriptive statistics for 100 seed weight and Seed coat permeability among the three species of soybean Species G. max G. soja G. tomentella 100 seed Weight Range Mean 5.06-13.52 8.48 0.56- 2.07 1.26 0.53 0.53 SD 1.91 0.42 2361 SCP% (7 days basis) Range Mean SD 92.0-100 99.69 0.971 3.1 – 44.0 3.37 10.5 66.0 66.0 - Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 Fig.1 Pattern of water imbibation in wild and cultivated soybean Seed coat permeability Rapid permeability test: Permeability of the seed coat of all the 125 genotypes was checked after 6 hrs of soaking (rapid imbibitions) and 7 days of soaking (slow imbibitions). Those genotypes whose <20% seeds imbibed within 6 hrs were classified as hard seeded. Similarly, genotypes whose >80% seeds imbibed within 6 hrs were classified as soft-seeded. Accordingly, all the accessions of G. soja and G. tomentella were rated as hard-seeded as it did not imbibe any water (SCP=0%) within 6 hrs. Similarly, 81 genotypes of G. max out of 105 had 90-100% SCP and were classified as soft-seeded. Slow permeability test: Slow imbibitions test indicated 66% permeability in seeds of G. tomentella. Similarly, 14 out of 19 G. soja genotypes showed 0% permeability in the slow imbibitions test. G. soja accession DC2008-1 had 44% SCP and PI507830B, PI366120, PI464889A and PI424032 had 3%, 4%, 5% and 6% permeability, respectively. Therefore, all the accessions of G. soja were rated as hard-seeded. Genotypes of the cultivated soybean found to have soft seeds as all the genotypes had SCP ranging from 92- 100%. Mean value of SCP % in the slow permeability test were recorded as 3.37%, 66% and 99.69% in G. soja, G. tomentella and G. max, respectively. A very strong correlation (0.98) was observed between the result of SCP under rapid and slow permeability test. It indicated that rapid permeability test i.e. imbibitions of 6 hours can be effectively used for testing permeability of seeds. It will save time and energy. Similarly, seed weight found to have strong correlation with the seed coat permeability. So, large seeds would have high seed coat permeability. The morphological and physiological changes associated with domestication can be delimitated into adaptation syndromes resulting from natural or deliberate human selection (Harlan et al., 1992). Hard seed is an adaptive trait which ascertained in wild relatives of soybean and maintained longer viability in them. In the present study, all the accessions of G. soja except DC 2008-1 had permeability less than 7%. The seeds of DC2008-1 are highly viable (data not shown). Therefore, transferring partial permeable trait of DC 2008-1 to cultivated type might 2362 Int.J.Curr.Microbiol.App.Sci (2017) 6(7): 2358-2363 increase seed viability in soybean. Seed size found to have strong correlation with seed coat color and viability. Black genotypes of all the species including G.max had small seeds (<8 g) and in general were highly viable. Seed coat permeability was strongly associated with process of domestication (Liu et al., 2007). All genotypes of domesticated soybean are soft seeded. However, soft seeded genotypes are having short viability. Therefore, breeding efforts need to be oriented to increase viability of soybean seeds without compromising seed coat permeability. The information generated in this study would pave the way for developing soybean genotype with high viability and permeability. Acknowledgement The first author is thankful to the UGC and ICAR-IARI, New Delhi, India for the grant of Junior Research Fellowship and facilities during Ph.D. programme. References Crookston R. K. and Hill D. S. 1978 A visual indicator of the physiological maturity of soybean seed. Crop Sci., 18: 867-870 Dassou S & Kueneman E. A. 1984 Screening methodology for resistance to field weathering in soybean seed Crop Sci 24: 774-778 Hahalis, D.A. And Smith M.L. 1997 In Basic and Applied Aspects of Seed Bio. (Eds., R.H. Ellis, M. Black, A.J. Murdoch and T.D. Hang) pp 507-514. Kulwar. Acad. Publ. Boston Harlan JR. 1992. Crops and man, 2nd edn. Madison, WI: American Society of Agronomy, Inc. and Crop Science Society of America, Inc Jang S.J., Sato M., Sato K., Jitsuyama Y. Fujino K. and Mori H, et al., 2015 A Single-Nucleotide Polymorphism in an Endo-1,4-β-Glucanase Gene Controls Seed Coat Permeability in Soybean. 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Singh R.J. and Nelson R.L.2014 Methodology for creating alloplasmic soybean lines by using Glycine tomentella as a maternal parent Plant Breeding 133 624–631 Sun L., Miao Z., Cai C. and Zhang D et al., 2015 GmHs1-1, encoding a calcineurinlike protein, controls hard-seededness in soybean Nature Genetics 47:939-943 Tiwari S P & Bhatia V S 1995 Association of seed anatomical characters with seed longevity in soybean Seed Res 23: 3839 Tiwari S P & Joshi H J 1989 Correlation and path analysis for seed quality characters in soybean Oil Seed Res 16: 51-57 How to cite this article: Subhash Chandra, Raju R. Yadav, Shatakshi Poonia, Yashpal, Darasing R. Rathod, Ashish Kumar, S.K. Lal and Talukdar, A. 2017. Seed Coat Permeability Studies in Wild and Cultivated Species of Soybean. Int.J.Curr.Microbiol.App.Sci. 6(7): 2358-2363. doi: https://doi.org/10.20546/ijcmas.2017.607.279 2363