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Acta Agronomica Sinica ›› 2021, Vol. 47 ›› Issue (5): 983-990.doi: 10.3724/SP.J.1006.2021.04207

• RESEARCH NOTES • Previous Articles    

Morphological characteristics and cytological study of anther abortion of temperature-sensitive nuclear male sterile line 160S in Brassica napus

TANG Xin(), LI Yuan-Yuan, LU Jun-Xing, ZHANG Tao*()   

  1. College of Life Sciences, Chongqing Normal University, Chongqing 401331, China
  • Received:2020-09-09 Accepted:2020-11-13 Online:2021-05-12 Published:2020-12-15
  • Contact: ZHANG Tao E-mail:1047910732@qq.com;zht2188@126.com
  • Supported by:
    Natural Science Foundation of Chongqing(cstc2012jjA80010);Science and Technology Bureau Foundation Project Chongqing(cstc2017jcyjAX0108);Scientific and Technological Research Program of Chongqing Municipal Education Commission(KJQN201900533)

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Abstract:

The main purpose of this study was to explore the flower morphologies, anther abortion times and cytological characteristics of the thermo sensitive genic male sterile line 160S in Brassica napus. The results will provide a theoretical basis for further study on the molecular regulation mechanism and guide significance for its practical application in two-line hybrid breeding of rape. The 160S were grown at 15℃ and 28℃. The flower morphology, microspore, anther structure and tapetum at different stages of fertile plants (Male Fertile/160S-MF) and sterile plants (Male Sterile/160S-MS) were observed by stereomicroscope with carmine acetate staining, paraffin section, hematoxylin eosin staining and TUNEL staining. The anther was yellow, plump and dehiscent with high pollen viability at 15℃. At 28℃, there was no differences in pistil and potato chips between 160S-MS and 160S-MF flowers, but the petals and the filaments of 160S-MS were smaller and shorter. The stamens of 160S-MS degenerated obviously and its anthers were shriveled in yellowish brown without pollen grains, indicating completely male sterility. But interestingly, the 160S-MF developed normally with higher pollen viability. However, microspore and pollen grains were not observed in 160S-MS due to complete abortion of stamens. Paraffin section results showed that there was no significant difference between 160S-MS and 160S-MF in sporulation and pollen mother cell stages. The morphology and structure of tapetum of 160S-MS appeared some abnormal appearances, such as irregularly cell arrangement, vacuolization, and premature disintegration at meiosis stage. Meanwhile, the development of pollen mother cells were hindered and no tetrads structure were formed leading to the empty pollen sac before the completion of meiosis. TUNEL assay showed that the tapetum of 160-MS began to apoptosis at meiosis stage. In summary, these results showed that 160S belongs to pollen mother cell abortion type male sterile line, whose abortion stage occured during meiosis stage and the tapetum cells degraded prematurely and failed to be transformed to the glandular type, so that the nutrients needed for pollen mother cell development were not provided. The tetrad structures and microspores can not be formed, which leaded to the formation of empty anther sac and resulted in male sterility.

Key words: Brassica napus, thermo-sensitive genic male sterility, anther abortion, cytological study

Fig. 1

Flower morphology of 160S-MF and 160S-MS A: the flower of 160S-MF; B: the flower stamens of 160S-MF; C: the petal and sepal of 160S-MF; D: the flower of 160S-MS; E: the flower stamens of 160S-MS; F: the petal and sepal of 160S-MS."

Table 1

Comparison of the flower traits between 160S-MF and 160S-MS (mm)"

花器特征
Flower trait		 育性类型Types of male sterility
160S-MF 160S-MS
花冠径Diameter of corolla 18.97±1.68 15.58±0.78**
花瓣长Length of petal 13.98±1.16 12.30±0.57**
花瓣宽Width of petal 7.48±0.78 5.88±0.37**
雌蕊长Length of pistil 9.24±1.13 9.80±0.72
四强雄蕊长Length of four stronger stamens 9.38±0.45 6.11±1.09**
二弱雄蕊长Length of two weaker stamens 6.99±0.81 4.89±1.20**
四强花药长Length of four stronger stamens’ anther 2.25±0.15 1.80±0.08**
花丝长Length of four long filaments 7.44±0.46 4.69±0.89**
花柄长Length of stalk 29.13±1.38 16.50±1.27**
萼片长Length of sepal 7.10±0.82 6.61±0.51

Fig. 2

Microspores morphology of 160S-MF and 160S-MS A: terads of 160S-MF; B: early microspores of 160S-MF; C: uninucleate microspores of 160S-MF; D: binucleate microspores of 160S-MF; E: pollen grains of 160S-MF; F: pollen grains of 160S-MS. The arrow points to the nucleus of the cells."

Fig. 3

Microscopical structure of anthers at different developmental stages in 160S-MF A, B: sporogenous cell stage; C: pollen mother cell stage; D: meiotic stage; E: tetrad stage; F: late uninucleate stage; G: binucleate stage; H, I: mature pollen; Epi: epidermal; Mid: middle layer; En: endothecium; T: tapetum; Sp: sporogenous cell; PMC: pollen mother cell; Te: tetrad; M: microspore; Po: pollen."

Fig. 4

Microscopical structure of anthers at different developmental stages in 160S-MS A, B: sporogenous cell stage; C: pollen mother cell stage; D, E: meiotic stage; F: locule of aborted anther; Epi: epidermal; Mid: middle layer; En: endothecium; T: tapetum; Sp: sporogenous cell; PMC: pollen mother cell."

Fig. 5

TUNEL detection of anther programmed cell dead at different developmental stages in 160S-MF and 160S-MS A: 160S-MF at pollen mother cell stage; B: 160S-MS at pollen mother cell stage; C: 160S-MF at meiotic stage; D and E:160S-MS at meiotic stage; F: 160S-MF at tetrad stage; G:160S-MF at binucleate stage; T: tapetum; PMC: pollen mother cell; Te: tetrad; M: microspore. The fluoresce of nuclei stained by propidium iodide is red, while green fluorescence is TUNEL-positive nuclei staining (arrow)."

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