BRIEF REPORT |
Correspondence to: Adya P. Singh, Agricultural Plant Stress Center, Chonnam National University, Gwangju 500-757, Republic of Korea. E-mail: adyasingh@hotmail.com
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Summary |
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We developed a method involving air-drying of a rubber suspension after fixation in glutaraldehydetannic acid and postfixation in osmium tetroxide for SEM observation. For TEM immunolabeling the suspension was air-dried after osmium-only fixation. Whereas conventional methods failed to satisfactorily stabilize rubber particles, the methods described here proved successful in preserving their integrity.
(J Histochem Cytochem 51:11051108, 2003)
Key Words: rubber particles, air-drying, SEM, TEM immunolabeling
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Introduction |
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RUBBER is a natural renewable substance and a source of valuable products. Although over 2000 plants from various taxa produce rubber latex, the rubber tree (Hevea brasiliensis) is still the most important commercial source of rubber because of the high quantity and quality of rubber from its latex. However, because Hevea plantations are declining and because rubber from this plant can cause severe allergic reactions, there is a pressing need to find alternative sources of natural rubber and to understand the mechanism underlying rubber biosynthesis.
Electron microscopic examination of rubber particles (
We are presently characterizing rubber particles and the rubber biosynthetic process in a number of rubber-producing plants in search of alternative plant sources to obtain rubber of desirable properties (
Although the work in our laboratory involves characterization of rubber particles from several alternative plant sources, here we compare Hevea with only one other species, Ficus benghalensis. Rubber particles from these two sources were purified according to the procedure of
For SEM, the most effective preparation for retaining the natural form and size of rubber particles for both Ficus and Hevea involved air-drying after fixation in glutaraldehyde (containing tannic acid) and postfixation in osmium. The same fixation failed to retain these features of rubber particles when used in combination with critical point-drying (CPD). CPD caused considerable distortion in the shape of rubber particles from both Ficus and Hevea (illustrated only for Ficus; Fig 1) due to collapse of their surface. Although the extent of collapse was variable, with some particles suffering only slight to no distortion, most particles showed signs of severe collapse. In comparison, in the air-dried preparation particles from both Ficus (Fig 2) and Hevea (Fig 3) retained their natural shape and size. Ficus rubber particles were spherical, occurring predominantly as discrete individual particles in a range of sizes. Particle size measurements undertaken (
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The TEM immunolabeling method proved most effective in retaining the spherical shape of rubber particles and in specific labeling of the 24-kD protein. This involved fixation of rubber particle suspensions directly in osmium before preparing whole mounts and labeling. Fixation in paraformaldehydeglutaraldehyde, which is normally employed in immunocytochemistry, was largely ineffective in retaining the natural shape of rubber particles. The specificity of gold labeling was also variable. Most unsatisfactory was the method where labeling was carried out on unfixed material. Furthermore, only the rubber particles from Hevea were labeled for 24-kD protein. The particles from Ficus were not labeled in any of the preparation methods used and therefore are not illustrated. Illustrations have been selected to cover the range of preparation methods used. In unfixed preparations, rubber material appeared as highly irregular masses (Fig 4). The background labeling was high compared to the intensity of gold particles associated with the masses of rubber material (Fig 4). Fixation in paraformaldehydeglutaraldehyde led to some improvement in retention of particle shape and specificity of labeling (Fig 5). Particle shape varied from near-spherical to highly irregular. Labeling intensity was rather poor, although there was noticeable improvement in specificity over unfixed material. Dramatic improvements in retention of particle shape and in specificity and intensity of labeling were obtained when the material was directly fixed in osmium. Rubber particles of all sizes were spherical and labeled intensely with gold particles, closely associated with the surface of particles (Fig 6 and Fig 7). Background labeling was negligible.
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It was apparent that CPD, the method of choice for SEM with most biological specimens, was unsuitable for rubber particles because it greatly distorted their shape. Curiously, air-drying, rarely used in SEM because it causes severe shrinkage distortion of biological materials, was most satisfactory in stabilizing the shape and size of rubber particles when used in conjunction with prior fixation in glutaraldehydetannic acid and postfixation in osmium. There are indications that plant products, such as latex, are susceptible to extraction by dehydration fluids, such as acetone and ethanol (
Comparison provided here of particle shape and specificity of immunolabeling using various TEM preparation methods suggests that osmium-only fixation combined with air-drying proved to be the most suitable method. It is apparent from the highly irregular form of the rubber material that there was little or no stabilization of particle membranes in unfixed preparations. Consequent fusion and fragmentation of rubber particles may be the reason for the high background labeling in the unfixed material. Aldehyde fixation destabilizes latex particles, rendering them prone to fusion (
The methods recommended here for SEM and TEM immunolabeling with rubber particles are simple and rapid. Although they might be considered unconventional and unsuitable for most biological samples, they were most effective in our work because of their compatibility with the unique chemical and structural properties of rubber particles. These technical developments have made it possible to obtain valuable information on the architecture of rubber particles and have also helped clarify the role of 24-kD protein in rubber biosynthesis (
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Acknowledgments |
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Supported in part by a grant (No. R11-2001-003104-0) from Korea Science and Engineering Foundation (KOSEF) to the Agricultural Plant Stress Research Center, Chonnam National University. Adya Singh is grateful to KOSEF for the Brain Pool Scientist Award.
Received for publication February 4, 2003; accepted February 5, 2003.
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