Cytological Studies of Dominant GMS Sterile Line D3A in <i>Brassica napus</i> L.

Dai-xiang XU; Le-dong JIA; Rui WANG; Guo-qiang MA; Mou-zheng DUAN; Cun-min QU; Jia-na LI

doi:10.13718/j.cnki.xdzk.2020.01.003

2020 Volume 42 Issue 1

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Dai-xiang XU, Le-dong JIA, Rui WANG, et al. Cytological Studies of Dominant GMS Sterile Line D3A in Brassica napus L.[J]. Journal of Southwest University Natural Science Edition, 2020, 42(1): 16-21. doi: 10.13718/j.cnki.xdzk.2020.01.003

Citation:

Dai-xiang XU, Le-dong JIA, Rui WANG, et al. Cytological Studies of Dominant GMS Sterile Line D3A in Brassica napus L.[J]. Journal of Southwest University Natural Science Edition, 2020, 42(1): 16-21. doi: 10.13718/j.cnki.xdzk.2020.01.003

Cytological Studies of Dominant GMS Sterile Line D3A in Brassica napus L.

School of Agronomy and Biotechnology/Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China

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Corresponding author: Rui WANG ;
Received Date: 24/04/2018
Available Online: 20/01/2020
MSC: S565.4

Abstract

An experiment was made to detect the vital abortion periods and cytological characteristics of anther development in dominant GMS homozygous two-type line D3AB in Brassica napus L., so as to provide a theoretical basis for further study of the mechanism of male sterility. Stereoscopic examination showed that phenotypically, the flowers and floral development appeared normal in both D3A and D3B, but the development rate of the ovary stigmata of D3A was slightly greater than that of D3B. Meanwhile, the anthers gradually degenerated during the late development of flowers, which resulted in abnormal pollen release. The results of scanning electron microscopy analysis showed that the base anthers shrank and the epidermal cells were tightly arranged at the early development stages of anthers in D3A. However, with the development of flower buds, the pollen wall of D3A shrank obviously until loss of activity. Eventually the anthers of D3A could not normally crack and release pollen whereas the anthers in D3B normally cracked and released a large amount of mature pollen. Paraffin section analysis indicated that pollen mother cells were normally formed at the early flower stages in both D3A and D3B, but the structure of the pollen mother cells in D3A was loose, and a vacuole state began to appear in its tapetum. In the tetrad stage, highly vacuolized tapetum cells were detected in the pollen sacs of D3A, and no tetrad structures were noticed in it. The above-stated results indicated that the tapetum cells in the sterile line degraded prematurely and failed to be transformed to the secretory-type tapetum cells, and thus had no sufficient nutrients for their development at the tetrad stage and microspores were not formed to release pollen, thus leading to the sterility of D3A. In conclusion, the abortion of D3A may be the abnormal type of tapetum.
- dominant GMS,
- anther abortion,
- tapetum,
- cytological studies

References

[1]	GOLDBERG R B, BEALS T P, SANDERS P M. Anther Development:Basic Principles and Practical Applications[J]. The Plant Cell, 1993, 5(10):1217-1229. Google Scholar
[2]	MCCORMICK S. Control of Male Gametophyte Development[J]. The Plant Cell, 2004, 16(Suppl):S142-S153. Google Scholar
[3]	SORENSEN A, KRöBER S, UNTE U S, et al. The Arabidopsis ABORTED MICROSPORES (AMS) Gene Encodes a MYC Class Transcription Factor[J]. The Plant Journal, 2003, 33(2):413-423. doi: 10.1046/j.1365-313X.2003.01644.x CrossRef Google Scholar
[4]	YANG S L, XIE L F, MAO H Z, et al. TAPETUM DETERMINANT1 Is Required for Cell Specialization in the Arabidopsis Anther[J]. The Plant Cell, 2003, 15(12):2792-2804. doi: 10.1105/tpc.016618 CrossRef Google Scholar
[5]	ZHANG W, SUN Y L, CHEN C, et al. Regulation of Arabidopsis Tapetum Development and Function by DYSFUNCTIONAL TAPETUM1(DYT1) Encoding a Putative bHLH Transcription Factor[J]. Development, 2006, 133(16):3085-3095. doi: 10.1242/dev.02463 CrossRef Google Scholar
[6]	WILSON Z A, MORROLLl S M, DAWSON J, et al. The Arabidopsis MALE STERILITY1(MS1) Gene is a Transcriptional Regulator of Male Gametogenesis with Homology to the PHD-finger Family of Transcription Factors.[J]. Plant Journal, 2010, 28(1):27-39. Google Scholar
[7]	曾芳琴.油菜S45AB隐性核不育分子机理与应用研究[D].武汉: 华中农业大学, 2010.http://cdmd.cnki.com.cn/article/cdmd-10504-1011405122.htm Google Scholar
[8]	夏秀云.甘蓝型油菜雄性不育相关基因BnATA20功能验证[D].武汉: 华中农业大学, 2011.http://cdmd.cnki.com.cn/Article/CDMD-10504-1011404597.htm Google Scholar
[9]	杨莉芳, 刁现民.植物细胞核雄性不育基因研究进展[J].植物遗传资源学报, 2013, 14(6):1108-1117. Google Scholar
[10]	贾乐东.甘蓝型油菜隐性杂合两型系SLAB育性候选基因的筛选鉴定[D].重庆: 西南大学, 2017.http://cdmd.cnki.com.cn/Article/CDMD-10635-1017847254.htm Google Scholar
[11]	刘川.甘蓝型油菜隐性纯合两型系的形态学及转录组学分析[D].重庆: 西南大学, 2014.http://cdmd.cnki.com.cn/Article/CDMD-10635-1014264768.htm Google Scholar
[12]	傅寿仲, 伍贻美.甘蓝型油菜细胞质雄性不育"三系"及其杂种一代花器官形态变异规律[J].中国油料, 1980, 2(3):33-38. Google Scholar
[13]	CHOWDHURY J B, DAS K. Functional Male Sterility in Brown Sarson[J]. Indian J Genet Plant Breed, 1967, 27:143-147. Google Scholar
[14]	IWASAKI F, HOSODA T. Studies on Development of Floral Organs in Male Sterile Rape (Brassica Napus L.) with Morphological and Histochemical Methods[J]. Jap J Breed, 1970, 20(5):282-286. doi: 10.1270/jsbbs1951.20.282 CrossRef Google Scholar

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油菜作为重要的油料作物，不仅是食用油与饲料蛋白的主要来源，同时在工业上的应用也非常广泛，因此亟需提高油菜产量，以满足日益增长的需求.在农业生产中，杂种优势因能显著提高作物产量和改善作物品质被广泛应用，其中细胞核雄性不育因具有不育性稳定彻底、无胞质效应等优点，现已成为提高水稻、玉米、油菜等作物产量的重要途径.前人研究表明，致使雄性不育的过程极其繁杂，其中涉及到雄蕊形态建成、花粉囊中各组织分化等多形式、多过程的基因调控，具体调控机理尚未明确.

显性细胞核雄性不育可根据基因发生突变位点及数量不同，将其分为单基因控制的显性核不育、一对复等位基因控制的显性核不育和双基因控制的显性核不育3种不育类型.对D3AB进行遗传分析和验证，认为其是由双基因控制的显性核不育类型.在植物的生命过程中，花粉和花药的发育至关重要，期间涉及多种基因互作^[1-2].例如基因AMS^[3]以及水稻(Oryza Sativa)中的同源基因TDR都参与绒毡层及小孢子的发育过程，TPD1^[4]，DYT1^[5]及Ms1^[6]都参与绒毡层及小孢子的分化.目前，油菜中已发现影响绒毡层细胞发育的相关基因.曾芳琴^[7]报道的基因BnCYP704B1如果发生突变，会影响绒毡层细胞中脂类物质的分泌.夏秀云^[8]研究的基因BnATA02如果发生突变，会导致绒毡层细胞异常肥大.杨莉芳等^[9]综合前人的研究结果，将油菜不育类型分为花粉囊发育异常、减数分裂异常、胼胝质代谢异常、绒毡层发育异常、花粉壁发育异常及花药开裂异常6种类型.

本研究对显性核不育两型系材料D3AB通过体视镜观察花器官的形态特征，采用扫描电镜观察花药发育特点，最后通过石蜡切片观察，确定其发生败育的时期及特点.为深入研究D3A的不育机理奠定基础，同时为进一步推进油菜的杂种优势利用提供理论依据.

1. 材料与方法

1.1. 实验材料

实验材料是重庆市油菜工程技术研究中心选育的甘蓝型油菜显性细胞核雄性不育纯合两型系D3AB，不育材料D3A(MsMsrfrf)和可育材料D3B(MsMsRfrf)在花期呈现出明显的表型差异.研究材料D3AB种植于重庆市北碚区西南大学歇马试验基地，种植期间进行常规田间管理，在初花期分别取不育材料D3A和可育材料D3B的花蕾进行后续细胞学研究.

1.2. 实验方法

1.2.1. 体视镜样品的制备与观察

在初花期，分别取D3A和D3B不同发育时期的花蕾及盛开花朵置于冰盒中，进行分类整理后按顺序整齐固定摆放至载物台上进行观察拍照，然后依次将这些花蕾的萼片剥离进行观察拍照，最后再将其花瓣剥离至花蕊裸露并进行观察拍照，同时对盛开花朵进行观察拍照.

1.2.2. 扫描电镜样品的制备与观察

分别取D3A和D3B不同发育时期的花蕾材料进行雄蕊剥离，然后依次将这些雄蕊摆放于贴有专用导电胶的扫描电镜载物台上，并将其固定于扫描电镜样品舱中，进行观察拍照(分别对正、反面进行观察).

1.2.3. 石蜡切片的制备与观察

当油菜进入初花期后，在上午9：00-11：00分别取D3A和D3B不同时期的花蕾立即浸入FAA固定液(50%无水乙醇:冰醋酸:甲醛=90:5:5)中，抽真空30 min(若仍有花蕾漂浮，可重复抽真空操作)，进行48 h以上的固定处理，然后依次用百分浓度为50%，70%，90%，100%的乙醇进行梯度脱水，继续进行透明、浸蜡、包埋、切片、展片、脱蜡、复水等处理^[10]，并用1%甲苯胺蓝溶液染色2~3 min，最后用二甲苯-中性树胶(1:1)进行封片.将制作好的切片利用光学显微镜Nikon E600进行观察拍照.

3. 结语

关于油菜育性的研究，显性核不育主要包括3种不育类型：单基因控制的显性核不育、一对复等位基因控制的显性核不育及双基因控制的显性核不育.本研究中所采用的纯合两型系不育材料D3A和可育材料D3B是经过连续多代姊妹交获得，除了在花期可以观察到花器官的部分差异外，其他田间表型基本保持一致.本研究初步认为这一对材料是基于育性的近等基因系，对不育材料D3A和可育材料D3B的杂交F1代进行育性调查，其分离比例稳定在1:1，且可育株D3B自交后代不会出现育性分离，其遗传表现与单基因显性细胞核雄性不育机理相同^[11].

在花期出现花粉败育仅是细胞核雄性不育的表型体现，花粉囊发育的全过程及其分子机理研究是基础和关键.花粉和花药的发育在植物生命过程中至关重要，期间涉及多种基因互作^[1-2].傅寿仲等^[12]对宜3A进行研究认为，其在花粉母细胞时期前发育正常，在花粉母细胞减数分裂前期出现细胞染色体粘连；后期有的细胞出现染色体桥，在中期Ⅰ，Ⅱ及后期Ⅱ等阶段见到落后染色体. Chowdhury等^[13]对黄沙逊和褐沙逊油菜的核不育材料进行细胞学研究，认为在小孢子母细胞减数分裂时都正常，黄沙逊油菜雄性不育的产生在减数分裂后期，由于绒毡层在四分体后期退化，不能起到营养层的作用而导致雄性不育；褐沙逊油菜雄性不育的发生是在减数分裂后期，由于花粉壁细胞不正常地形成了坚实的厚壁引起花药未开裂. Iwasaki等^[14]用“Murasaki”核不育油菜为材料进行组织化学检查，结果表明到四分体时期为止，不育株与可育株的花器都还没有差异；四分体时期以后，不育株花药的绒毡层细胞比正常花药的厚而不易破碎，认为绒毡层细胞在花药成熟时期已经没有生理活力，花粉粒不能成熟而导致不育.

在本研究中，对甘蓝型油菜显性细胞核不育纯合两型系不育材料D3A和可育材料D3B不同时期的花蕾进行体视镜观察分析发现，D3A与D3B在外观形态上无明显差别，均能正常开花，且在雄蕊发育早期均表现为正常，后期D3A雄蕊退化萎缩，花粉囊不能正常开裂；不育材料D3A子房柱头发育的速度明显快于D3B.通过对不同时期的花药进行扫描电镜观察发现，D3A早期花药的基部开始皱缩，表皮细胞排列紧密.随着花蕾的发育，D3A花粉囊壁表皮细胞逐渐萎缩，最终塌陷使药室不能正常开裂释放花粉；D3B的花粉囊始终饱满，花药正常开裂，同时释放出大量成熟花粉粒.最后通过石蜡切片观察发现，D3A和D3B在花蕾发育早期都能形成花粉母细胞，但与D3B相比，D3A中的花粉母细胞结构松散，且绒毡层已开始呈现液泡化状态；进入四分体时期，不育材料D3A的花粉囊中观察到高度液泡化的绒毡层细胞，而且无四分体结构出现，表明绒毡层细胞已提前发生降解而未向分泌型转化，使四分体时期小孢子发育丧失了所必需的营养物质，因而不能形成小孢子并发育为成熟的花粉粒进行释放，最终导致不育.因此，本研究认为D3A的败育类型可能属于绒毡层发育异常败育类型.

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Cytological Studies of Dominant GMS Sterile Line D3A in Brassica napus L.