Legume Res., 28 (4) : 235-243, 2005 SEEDCOAT SEM STUDIES OF SELECTED TAXA OF THE TRIBE PHASEOLEAE (FABACEAE) Baisakhi Bandyopadhyay, Subhas Chandra Santra and Mashahiro Kato [kparlment of Environmental Science, University of Kalyani, Nadia; West Bengal - 741 235, India ABSTRACT Morphological variability of seed coat surfaces of fifteen taxa of economically important legumes of the tribe Phaseoleae (cultivars of Glycine max, Erythrina variegata, Lab Jab purpureu5, Vigna unguiculata, CaJanus cajan) was studiedby scanningelectron microscopy (SEM). A distinctive pattern of surface features was shown by each taxon. Seeds were obovate to ovate In shape except In Erythrlna varlegata where they were oblanceolate, and the color varied from black and brown to derivatives of brown. Variations in spermoderm characters were observed at the varietal level also. INTRODUCTION The Phaseoleae combine in one tribe both the largest number of genera and the greatest economic importance within the Leguminosae. Apart from its many genera being the most highly cultivated legumes, a substantial portion of the remaining genera of this tribe contains members that are of considerable importance as food, fodder and ornament. External seed morphology is one of the important taxonomical characters. In the tribe Phaseoleae, the seed coat is generally smooth, but it may be patterned (e.g. Vigna). The hilum is the center of importance. Here, the funicle leaves behind a small aril, but sometimes the aril can be prominent (e.g. Cajaninae). Hilum length varies greatly. In almost all members of Phaseolinae and in some Erythrina sp. a dense mass of white tissue remains behind, thus forming a pithy pod which completely obscures the central portion of the hilum (Polhill and Raven, 1981). External seed morphology would perhaps be as important to taxonomy as flower morphology if equal attention were given to both. (Polhill and Raven, 1981). Scanning electron microscopy (SEM) has been employed to reveal the seed coat surface features of some Leguminosae. The major seed characterswere briefly summarized for the three subfamilies of Leguminosae and its 41 tribes by Gunn (1981). The recent works using SEM on the seed surface on Fabaceae were conducted out by different workers (Gonzalez-Andres and Oritz, 1995; Lopez et al., 2000). MATERIAL AND METHODS For SEM study, five varietiescif Glycine max (UPSM 19, Soya, JS2, PUSA and PK527), six varietiesof Vigna unguicujala [ST6B (White), ST-13, ST-IB (Black), ST-6A (Brown), ST-6B (Brown) and Hyd-White 1), two varieties of Cajanus cajan (B7 and 517); one vari-ety of Lablab purpureus (Sem Black) and Erythrina variegata were used. Varieties were selected for the study primarily based on common Indian legume cultivars. The material was fixed in FAA (formalin: acetic acid : 500A> ethyl alcohol = 5 : 5 : 90 v/v). For scanning electron microscopy, the material was dehydrated in an ethanol series, freeze dried and coated with platinum. Seed surface observations were made using a JSM 820S scanning electron microscope (20 kv). Measurements of length, breadth and dry weight were recorded separately for each species. RESULTS AND DISCUSSION Macromorphology and spermoderm Department of Biological Sciences, Graduate School of Science. University of Tokyo, Hongo 7-3-1, Tokyo 113-0033, Japan.
236 LEGUME RESEARCH. Table 1. Macro-morphological characters of some legume seeds Plant materials Shape Colour Mean dimension Weight (mg) Length±SE (mm) Breadth±SE (mm) Glycine max (L) Merr. UPSM19 Obevate Straw color 6.15±0.03 4.73±0.04 1.25±0.03 Soya Obovate YeIlowish brown 6.28±0.05 5.14±0.03 0.99±0.02 JS2 Obovate YeIlowish brown 7.21±0.17 5.92±0.02 0.98±0.09 PUSA Obovate Straw color 5.35±0.03 4.65±0.01 1.22+0.05 PK527 Obovate Straw color 6.28±0.04 4.25±0.02 2.12±0.41 Erythrlna varlegata L Oblanceolate Maroon 20.41±0.019 8.35±0.14 2.12±0.41 Lablab purpureus (L) Sweet Sem Black Vigna ungulculata (L) Walp. Ovate Black 10.12±0.04 6.25±0.04 1.5±0.05 ST-6B (White) Ovate Ughtbrown 4.75±0.04 2.39±0.21 0.21±0.01 ST-13 Ovate Ughtbrown 4.12±0.04 2.85±0.04 0.21±O.O2 ST-1B(B1ack) Ovate Black 4.53±0.02 2.24±0.21 0.21±0.04 ST-6A(Brown) Ovate Reddish 4.21±0.12 2.10±0.03 024±0.04 ST-6B (Brown) Ovate Brown 4.31±0.13 2.39±0.07 0.25±0.02 Hyderabad (White) Ovate Deep brown 4.31±0.17 2.12±0.03 0.25±0.01 Cajanus cajan (L) MiUsp B7 Obovate Brown 5.12±0.41 222±0.04 0.92+0.04 517 Obovate Brown 5.31±0.21 212±0.09 0.99±0.04 Table 2. Spermoderm features of some legume seeds Plant Materials Seed surface type Anticlinal wail Hilum Sinuous Straight Exposed Covered Glycine max (L) Merr. UPSM19 Rugose + - + Soya Punctieulatee + - + JS2 Rugose + + PUSA Captor Iidged + - + PK527 Rugose + - + Erythrlna varlegata L Ocellate + - + Lablab purpureus (L) Sweet SemBlack Maculo-reticulate + + VIgna unguiculata (L) Walp. ST-6B (White) Maculo-reticulate + - + ST 13 " + - + ST-1B(Black).. + + ST-6A(Brown).. + - + ST-6B (Brown).. + - + Hyderabad (White).. + + Cajanus cajan (L) MiUsp B7 CoIliculate + - + 517 Captor Iidged + - + features of seeds of different species and cultivars of legumes [fribe Phaseolae) were studied in detail. The macromorphological features are summarized in Table 1 and 2. In all the varieties, the seed coatwas impregnated with a wax deposition that varied in form and quality. The seeds of Glycine max cultivars were medium-sized, usually obovate in shape. In general, seed colors varied from brown to derivatives of brown. Spermoderm features of
different cultlvars of Glycine max as revealed through SEM are given In Tables 1 and 2, and in Figs. 11, 12, 23-30. The primary ornamentation was a rugose type In UPSM 19. The wax deposition was prominent. The irregular elevations were seen running In one direction. JS2 and PK 527 - the other cultivars of Glycine max had almost thesarne spermoderm features., But In 'SoYa', a punctlculate type of primary ornam~ntatlon was seen. Theseed surfacesweresmoothwith minutely punctate marks. IndistinCt spirally arranged tubercles were present. In 'Pusa', a captor-ridged primary ornamentation was seen with ridges Incomplete and mostly in half circles, each sultol,uldlng a central small ridge. Macromorphologlcal features of Erythrlna varlegata are listed In Tables 1 and 2 and shown In Ags. 9 and 10. The seeds were large, with a hard dry testa. Seed surface hairs were absent. The primary ornamentation of the seed surface was ocellate. So, It had an oblanceolate-shaped depression at the center, with a raised circular border. Smooth wax deposition was present at the apex. In Lablab purpureus (Sem Black cultivar) the seeds were black ih color, with hard testa, and were ovate In shape. Micropylar hairs were absent. Hilum was covered and the anticlinal wall sinuous. The primary ornamentation was macula-reticulate. Here, a network like struc:ture on the seed SUl'face could be seen, with minute blotches all over the surface (Ags. 5 and 6). Wax deposition was present over the whole surface of tile seed coat. In ~gna unguiculata, six cultivars were studied: ST-6B (White), ST-I3, ST-IB (Black), ST-6A (Brown), ST-6B (Brown) and Hyderabad (White) 1. The anticlinal wall was sinuous, with exposed hilum. The seeds were ovate-shaped, and surface hair was absent. The features showed a varietal constancy. Here. the color of the seed was brown or mixed brown. The Vol. 28, No.4, 2005 237 pattern of the primary ornamentation was almost the same In the varieties of Vigna unguiculata. A macula-reticulate type of ornamentation was seen. In the primary ornamentation, a network like structure. with minute blotches and scattered wax deposition all over the surface, was seen (Ags. 7, 8 and 13-22). In CaJanus cajan, the seeds were brown In color and medlurn-slzed; surface hair was absent. The hilum was exposed. sharply defined, large, conspicuous and sub-basal. placed at the broaderend on the ventral angle. In cultivar 517, the primary ornamentation was captor-ridged, with ridges incomplete and mostly in half circles, each surrounding a central smallridge with scattered wax deposition (Ags. 3 and 4). In B7, the primary ornamentation was colliculate. It WAS Closely spaced, with rounded broad elevations with smooth wax deposition. (Rgs. 1 and 2). The details of macro-morphological characters, ml_«;;ropylar hairs, hilum, and spermoderm features are summarized In Tables 1 and 2. A distinctive surface pattern was shown by each species of the tribe Phaseolae, and sometimes also by their cultivars. Seed Identification Is a meaningful part of seed testing, crop Improvement, archeology, paleobotany and most Importantly, taxonomy. However, only a meager pool of data about thesurface features ofthe legumes understudy Is available. The works of Nwanze etal. (1975) and Trivedi and Gupta (1986) have shown that seed surface patterns can also help In demarcat-ing varieties of legumes. Seeds are one of the distinctive features of spermatophytes; variation In seed size, shape. color and surface features are Important In identification of seeds. The seed surface patterns of ~gna (Fabaceae) varieties were described by Trivedi and Gupta (1986) who showed that seed surface patternsdiffered from species to species and sometimes In varieties
238 LEGUME RESEARCH Fig. 1, 2. Cajanus cajan-var. B7 Fig. 3, 4. Cajanus cajan-var. 517 'r- '.,~.. J. I,' ~, \... 'J') ~. t". '. "'") 1 "I "'.,.' 'I, f'.' I ~: '. "",\'..,,,'..\{ 'f~.. "l \' (, :( ",I", i \..,~ (' t~ ':>' 'II" '..1, '" ~ \ (I. "."',,,,\ I" '\'," I -',' 'l'f( ~ '~ 1.) "~'.\ 'f '10,'.,liA,'. '~ \~'"... ~ ~','" ~'".,:,-...,',:' ',.... \.1... '" f',"... 'j",,,;.,. I)...t,, "I I~ ")~ '" '-.,,. "1' ;,... -... ',"j'-, : I '. ',',.' -..'; ~,, --- 1129 17 11 1 >:2. :~1I)!0t'il IlDl6 f "... \ ~ '. _ \ " \ l;. Fig. 5, 6. LabJab purpureus var. Sem Black
Vol. 28, No.4, 2005 239 Fig. 7, 8. Vigna unguiculata var. Hyd-white 1 Fig. 9, 10. Erythrina variegata Fig. 11, 12. Glycine max var. Pusa
240 LEGUME RESEARCH Fig. 13, 14. Vigna unguiculata var. ST-13 Fig. 15, 16. Vigna unguiculata var. ST-IB (Black) Fig. 17, 18. Vigna unguiculata var. ST6A (Brown)
Vol. 28, No.4. 2005 241 Fig. 19, 20. Vi9na unguiculata var. ST 6B (White) Fig. 21, 22. Vigna unguiculata var. ST 6B (Brown) Fig. 23, 24. Glycine max var. Soya
242 LEGUME RESEARCH Fig. 25, 26. Glycine max var. JS2 Fig. 27, 28. Glycine max var. PK 527 Fig. 29, 30. Glycinemaxvar. UPSM19 Fig. 1-30. Scanning electron micrographs of seed coat patterns of the Legumes studied
Vol. 28, No.4, 2005 243 also. cultivars studied (ST-6B (white), ST-IB (black), Glycine max (L.) Merr. ofthe subtribe ST-6A (brown), ST-6B (brown) andst-13), ST Glycininae: the seeds were obovate and the IB (black) and ST-13 were more similar to primary ornamentation rugose in all the each other than to the other varieties. The varieties except in 'Soya' (in which a tubercles were the biggest in ST-6B (brown) puncticulate type of primary ornamentation and the smallest in ST-13 and ST-IB (black). was seen - the seed surface was smooth with These observations are supported by previous minute punctate spots) and in 'Pusa' (in which,works (Polhill and Raven, 1981; Marin et al., a captor ridged primary ornamentation was 1998). Cajanus cajan (L.) Millsp. of the subtribe seen, with ridges incomplete and mostly in half Cajaninae: in both the varieties the seedswere circles, each surrounding a central smallridge). obovate in shape and arils were prominent; So, the cultivars of Glycine max exhibited this observation tallies with that of Polhill and polymorphism. The only common feature of Raven (1981); In var. 517, the primary all the cultivars of Glycine maxwas the haziness ornamentation of the seed surface was captor of the cell boundaries. Erythrina variegate L. ridged, with, ridges in-complete and mostly in of subtribe Erythrininae: the seeds were half circles, each surrounding a small central oblanceate in shape. The primary ridge. In cultivar B7,. the primary ornamentation was ocellate type, with a ornamentation was colliculate with closely depression at the center and a raised circular spaced, rounded and broad elevations. The border. The hilum length varied greatly - an differences were prominent between the two observation that is supported by previous work varieties of Cajanuscajan. Wax deposition was (Polhill and Raven, 1981) Lablab purpureus smooth in B7, but not in cultivar 517. (L) Sweet, of subtribe Phaseolinae: the seeds Considering the above stated fact, it of the variety Sem Black were ovate in shape appears that macromorphological and and the primary ornamentation maculo- spermoderm features of each cultivated variety reticulate type. Here, a mesh-like structure was of legume showed the constancy in shape and found ontheseedsurface, with minute blotches spermoderm features. It is also interesting to all over the surface. Vigna unguiculata L. Walp. record the fact that colour and size of the of the subtribe Pha~eolinae: the seeds were cultivarsvaries considerably. However, there small and ovate in shape. The pattern of is exception in the genus Glycine max primary ornamentationwas macula-reticulate (Soyabean cultivars) where wide variation is with insignificant variations among the five surface features is noticed from SEM studies. REFERENCES Gonzalez-Andres, F. and Ortiz, J.M. (1995). Seed Sci. Technol., 23: 289-300. Gunn, C.R. (1981). Seed Topography in the Fabaceae. Seed Sci. Technol., 9. Lopez, J. et a/. (2000). Bot. J. Linn. Soc., 132(2): 97-120. Marin, P.O. et aj. (1998). Seed Sci. Technol., 26: 17-32. Nwanze, K.F. et a/. (1975). Environ. EntomoJ.. 4: 409-412. Polhill, R.M. and Raven, P.H. (1981). Advances in Legume Systematics. Royal Botanic Gardens, Kew. Trivedi, B.S. and Gupta, M. (1986). Phytomorphology, 36: 271-282.