JOURNAL OF ELECTRON MICROSCOPY TECHNIQUE 18:207-208 (1991)
Rapid Communication Methods of Onion Seed Preparation for Scanning Electron Microscope Studies of the Seed Coat
Yasseen Mohamed-Yasseen', Birute P. Jakstys2 and Walter E. Splittstoesseg 'Dept. Horticulture, 201 Vegetable Crops; 2Center for Electron Microscopy, 84A Bevier Hall; U. Illinois, Urbana, IL 61801
Scanning electron microscopy (SEM) has been used to study seed structure (Chuang and Heckard, 1972). Seeds with shrunken, cracked, or ruptured seed coats are usually short-lived (Bass, 1980). Seed shrinkage is an indication of a weak seed coat, resulting in cracks and damage which ultimately leads to loss of seed vigor or even death (Abdul-Baki and Anderson, 1970). This paper compares five different methods of seed preparation to observe shrinkage and cracks in onion seed coats. Sweet Spanish onion (Allium cepa L.) seeds were prepared for SEM as follows: seeds were 1) untreated sputter coated with gold/palladium and observed in a JEOL U3 SEM; 2) cryofixed (frozen in propane pre-cooled by liquid nitrogen), coated with gold/palladium at -SOT and observed at -8pC in an Amray 1000A SEM); 3) prepared using conventional techniques. The seeds were first cleaned in 0.1M potassium phosphate buffer p H 7.2; fixed in 4% glutaraldehyde and postfixed in 1% osmium tetroxide in the same buffer for 2 hr at 24OC; dehydrated in an ethanol series; critical point dried with CO,; sputter coated with gold/palladium and immediately observed with the SEM; 4) prepared as in 3, but without postfixation in osmium tetroxide; and 5) fixed in 100% ethanol at 3 2 T for 48 hr; critical point dried and sputter coated with gold/palladium. No detectable differences were seen between seeds prepared by the conventional method (Fig. l), seeds prepared and observed under low temperature (Fig. 2), without preparation (Fig. 3), seeds not postfixed with osmium tetroxide and critical point dried (Fig. 4), seeds metal coated only, and those fixed in 100% ethanol. No one method of onion seed preparation for SEM seemed to be better than another and the methods of preparation appeared not to have been responsible for shrinkage or crack formation. Observation of untreated seeds at the dissecting microscope level suggested that shrinkage and perhaps cracks in the seed coat were already present. The existence of fungal growth in seed cracks (Fig. 4) supports the suggestion that the cracks in the seed coat were already present prior to seed preparation for SEM. At low magnifications, no differences in seed coat appearance were observed between methods of seed preparation. However, it was easier to study fine details of seeds prepared by the conventional method (Fig. 1) than seeds observed without preparation (Fig. 3). Inspection of seed lots by low magnification SEM would enable seed companies to determine quickly and efficiently the amount of seed coat shrinkage and cracking and, thus, enable the companies to predict onion seed quality. 01991 WILEY-LISS, INC.
Received January 19, 1991; accepted in revised form March 1, 1991
208
Y. MOHAMED-YASSEEN ET AL
Fig. 1. SEM micrograph showing onion seed coat shrinkage. Specimen was prepared by method 3. (X20).
Fig. 2. SEM micrograph showing onion seed prepared by method 2. (X30). Fig. 3. SEM micrograph showing fungi in the hilum area. Prepared by method 1. (X390). Fig. 4. SEM micrograph the seed coat showing fungal growth in seed cracks and hilurn area. Prepared by method 4. (X100). References Abdul-Baki, A.A. and J.D. Anderson (1990). Crop Sci. 10:31-34. Bass, L.N. (1980). Hort Rev. 1:117-141. Chuang, T.L. and L.R. Heckard (1972). Amer. J. Bot. 59:258-265.
Rapid Communication Methods of Onion Seed Preparation for Scanning Electron Microscope Studies of the Seed Coat
Yasseen Mohamed-Yasseen', Birute P. Jakstys2 and Walter E. Splittstoesseg 'Dept. Horticulture, 201 Vegetable Crops; 2Center for Electron Microscopy, 84A Bevier Hall; U. Illinois, Urbana, IL 61801
Scanning electron microscopy (SEM) has been used to study seed structure (Chuang and Heckard, 1972). Seeds with shrunken, cracked, or ruptured seed coats are usually short-lived (Bass, 1980). Seed shrinkage is an indication of a weak seed coat, resulting in cracks and damage which ultimately leads to loss of seed vigor or even death (Abdul-Baki and Anderson, 1970). This paper compares five different methods of seed preparation to observe shrinkage and cracks in onion seed coats. Sweet Spanish onion (Allium cepa L.) seeds were prepared for SEM as follows: seeds were 1) untreated sputter coated with gold/palladium and observed in a JEOL U3 SEM; 2) cryofixed (frozen in propane pre-cooled by liquid nitrogen), coated with gold/palladium at -SOT and observed at -8pC in an Amray 1000A SEM); 3) prepared using conventional techniques. The seeds were first cleaned in 0.1M potassium phosphate buffer p H 7.2; fixed in 4% glutaraldehyde and postfixed in 1% osmium tetroxide in the same buffer for 2 hr at 24OC; dehydrated in an ethanol series; critical point dried with CO,; sputter coated with gold/palladium and immediately observed with the SEM; 4) prepared as in 3, but without postfixation in osmium tetroxide; and 5) fixed in 100% ethanol at 3 2 T for 48 hr; critical point dried and sputter coated with gold/palladium. No detectable differences were seen between seeds prepared by the conventional method (Fig. l), seeds prepared and observed under low temperature (Fig. 2), without preparation (Fig. 3), seeds not postfixed with osmium tetroxide and critical point dried (Fig. 4), seeds metal coated only, and those fixed in 100% ethanol. No one method of onion seed preparation for SEM seemed to be better than another and the methods of preparation appeared not to have been responsible for shrinkage or crack formation. Observation of untreated seeds at the dissecting microscope level suggested that shrinkage and perhaps cracks in the seed coat were already present. The existence of fungal growth in seed cracks (Fig. 4) supports the suggestion that the cracks in the seed coat were already present prior to seed preparation for SEM. At low magnifications, no differences in seed coat appearance were observed between methods of seed preparation. However, it was easier to study fine details of seeds prepared by the conventional method (Fig. 1) than seeds observed without preparation (Fig. 3). Inspection of seed lots by low magnification SEM would enable seed companies to determine quickly and efficiently the amount of seed coat shrinkage and cracking and, thus, enable the companies to predict onion seed quality. 01991 WILEY-LISS, INC.
Received January 19, 1991; accepted in revised form March 1, 1991
208
Y. MOHAMED-YASSEEN ET AL
Fig. 1. SEM micrograph showing onion seed coat shrinkage. Specimen was prepared by method 3. (X20).
Fig. 2. SEM micrograph showing onion seed prepared by method 2. (X30). Fig. 3. SEM micrograph showing fungi in the hilum area. Prepared by method 1. (X390). Fig. 4. SEM micrograph the seed coat showing fungal growth in seed cracks and hilurn area. Prepared by method 4. (X100). References Abdul-Baki, A.A. and J.D. Anderson (1990). Crop Sci. 10:31-34. Bass, L.N. (1980). Hort Rev. 1:117-141. Chuang, T.L. and L.R. Heckard (1972). Amer. J. Bot. 59:258-265.
