甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究

doi:10.3724/SP.J.1006.2021.04207

摘要/Abstract

摘要：

明确甘蓝型油菜温敏核雄性不育系160S花器形态变化、花药败育的时期和细胞学特征, 初步探究败育的原因, 为深入研究不育系160S的内在分子调控机制提供理论基础, 也对其在油菜两系杂交育种中的实际应用具有指导意义。本研究在15℃和28℃条件下培养试验材料160S, 利用体式显微镜分别观察花发育形态特征; 采用醋酸洋红染色方法观察各时期小孢子发育形态; 通过石蜡切片和苏木精-伊红染色对可育植株(Male Fertile/160S-MF)和不育植株(Male Sterile/160S-MS)花药细胞学特征进行显微观察; TUNEL染色法检测花药发育各时期绒毡层细胞凋亡情况。160S-MF在15℃表现为可育, 雄蕊正常发育, 成熟的花药呈黄色, 形态饱满, 正常开裂, 表面一层有活性的花粉附着在上面; 28℃条件下, 160S-MS花朵的雌蕊、萼片与160S-MF花朵无差异, 但花瓣变小, 花丝变短, 雄蕊明显退化, 花药干瘪呈黄褐色, 无花粉粒附着在花药上, 表现出雄性完全不育。160S-MF的小孢子能正常发育为成熟有活力的花粉。而160S-MS由于雄蕊完全败育, 未观察到小孢子和花粉粒。160S-MS花药在造孢时期和花粉母细胞时期与160S-MF无明显差异, 但在减数分裂期, 160S-MS花药绒毡层形态和结构出现异常, 绒毡层细胞排列不整齐, 细胞空泡化, 伴随提前解体。同时花粉母细胞发育受阻, 无四分体结构形成, 最终在减数分裂期完成前形成空的花粉囊。TUNEL检测发现, 160S-MS花药绒毡层细胞在减数分裂期开始凋亡。本研究结果表明, 160S属花粉母细胞败育型不育系, 败育时期发生在减数分裂期, 绒毡层异常降解, 绒毡层未向腺质型转化, 不能提供花粉母细胞发育所需要的营养物质, 致使花粉母细胞发育受阻无法形成四分体结构, 从而导致小孢子无法形成, 花药形成空的花粉囊, 产生雄性不育。

关键词: 甘蓝型油菜, 温敏细胞核雄性不育, 花药败育, 细胞学研究

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

唐鑫, 李圆圆, 陆俊杏, 张涛. 甘蓝型油菜温敏细胞核雄性不育系160S花药败育的形态学特征和细胞学研究[J]. 作物学报, 2021, 47(5): 983-990.

TANG Xin, LI Yuan-Yuan, LU Jun-Xing, ZHANG Tao. Morphological characteristics and cytological study of anther abortion of temperature-sensitive nuclear male sterile line 160S in Brassica napus[J]. Acta Agronomica Sinica, 2021, 47(5): 983-990.

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花器特征 Flower trait	育性类型Types of male sterility
花器特征 Flower trait	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