Photos 5&6: Middle microspore stage. The fluorescence in tapetum is reduced and microspores increase in size. The double-layered exine and sporopollenin body wall of tapetum express extremely stong fluorescence. X320.
South China Agricultural University, Guangzhou 510642, China
The purpose of this study was to probe the relationships between tapetal
disintegration and pollen development, or the sporopollenin behavior during the
process of tapetal distintegration of a normal rice cultivar and its GMS
pollenless mutant.
The line I-15, GMS pollenless mutant used in this study was derived from the X\5\ generation of irradiated seeds of the American rice cultivar M-101 (Lu and Rutger 1984). The original cultivar served as the check.
The preparation of slides was done by the plastic thin sectioning method as described by Feder and O'Brien (1968). The sections were double stained on slides with PAS (periodic acid-Shiff's) and TBO (toluidine blue O). Tissue structures of the anthers were observed under microscope. Staining was done with the yellow fraction of pyronin B, specifically inducing the sporopollenin to fluorescence (Zhu 1983), and sporopollenin was detected under a fluorescence microscope. For viewing the sporopollenin behavior, two types of photographs were taken either of neighboring sections in some anthers, or of the same section in other anthers. That is, the section were first stained with the yellow fraction of pyronin B and photographed. And then, after being destained, they were stained with PAS and TBO again, and photographed at the same field.
The main results obtained are as follows: 1. In cultivar M-101, the process of tapetal disintegration might be divided into two phases: secreting sporopollenin at microspore developmental stage; and degrading non-sporopollenin substances at pollen developmental stage (Fig. 1). 2. In line I-15, GSM pollenless mutant, although a series of abnormalities occurred during the process of tapetal disintegration, both the timing of the start and the sequences of tapetal disintegration were similar to those of M- 101 (Fig.2).
Fig. 1. The behavior of sporopollenin during the process of tapetal
disintegration in cultivar M-101.
Photos 3&4: Early microspore stage. Tapetum begins to disintegrate and microspores form their primary exine. X320
Photos 5&6: Middle microspore stage. The fluorescence in tapetum
is reduced and microspores increase in size. The double-layered exine and
sporopollenin body wall of tapetum express extremely stong fluorescence. X320.
Photos 9 & 10: Vacuolate pollen stage. There is no sporopollenin but
still abundant non-sporopollenin substance in tapetum. X320.
Photos 11&12: Mature pollen stage. Tapetum completely disintegrates.
The pollen double-layered exine and sporopollenin body wall still exhibit
strong fluorescence. X160.
The odd number photographs were taken in the slides stained with PAS (periodic
acid-Schiff's) and TBO (toluidine bluo O) dyes. The even number photographs
were taken in the slides stained with the yellow fraction of pyronin B, which
was fluorescent.
Fig. 2. The behavior of sporopollenin during the process of tapetal
disintegration in the GMS pollenless mutant, line I-15.
Photos 13&14: Early microspore stage. In the same anther, the tapetum is still intact in two locules (C and D) and begins to disintegrate in other locules (A and B). X100.
Photos 15&16: Microspore exine thickens abnormally. X320.
Photos 19&20: The sporopollenin clumps form within the locule. X320.
Photos 23&24: Mature pollen stage. In four locules, the tapetum and
sporopollenin complexes have disintegrated almost completely. Sporopollenin is
in dissolved state. In locule D, the sporopollenin content is markedly less than
in other three locules. X160.
m: microspore, p: pollen, pw: primary wall, sb: sporopollenin body, sbw: sporopollenin body wall, sc: sporopollenin clump, sl: sporopollenin layer, t: tapetum.
References
Feder, N. and T.P. O'Brien, 1968. Plant microtechnique: some principles and new methods. Amer. J. Bot. 55(1): 123-142.
Lu, Y.G. and J.N. Rutger, 1984. Anther and pollen characteristics of induced genetic male sterile mutant in rice (Oryza sativa L.). Env. and Exp. Bot. 24(3):209-218.
Zhu, C., 1983. Using the yellow fraction of Pyronin B to induce fluorescence in sporopollenin fluorescence. Bulletin of Chinese Botany 1(1): 53-55. (in Chinese)
Sporopollenin-An alcohol found in the wall of spores, durable.