甘蓝型油菜显性细胞核雄性不育系雌性不孕的细胞学研究
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摘要
雌雄配子体的正常发育、结合和完成双授精过程是被子植物世代交替过程中的标志事件。在雄配子体发育的过程中,如果出现外在或者内在条件的变化使发育过程中断,将造成雄性不育。同样情况发生在雌配子发育过程中亦将导致雌性不孕。雌配子体的正常发育对种子的形成尤为重要,一旦发生雌性不孕将不能通过类似雄性不育利用外来正常花粉进行弥补。因此,在生产中必须避免雌性不孕的发生,否则将严重影响作物的产量。在对甘蓝型油菜显性细胞核雄性不育的转育过程中,发现不育株中雌性结实率都存在不同程度的下降,其中FM195AB-1和FM195AB-2表现严重雌性不孕。通过对以上材料的柱头形态观察、花粉管萌发实验、石蜡切片观察花药与胚囊结构、压片法观察花药减数分裂过程等手段,对FM195AB不育株雌性不孕产生的原因进行了较深入的研究,获得的结果如下：
1.结实率调查表明,在不同遗传背景不育系的不育株中,雌性不孕程度有所差异,可育株结实完全正常。原始不育系Rs1046AB中不育株的每角果平均种子为5.5粒,而FM195AB中的绝大多数角果不能结实,极少数只有1-2粒,二者结实率差异极显著。而杂合型不育系不育株中每角果粒数显著高于纯合型不育系。
2.雌性不孕不受环境条件影响,雌性不孕只发生在雄性不育株上,表明雌性不孕极可能是显性核不育基因在雌性和雄性发育途径中的不同表现,或者控制雄性不育和雌性不孕性状的基因紧密连锁。纯合型不育系和杂合型不育系之间雌性不孕程度存在差异暗示着该性状与基因的剂量效应有关。
3.扫描电镜观察发现FM195AB可育株和不育株的柱头形态无明显差异。采用正常可育花粉人工授粉四小时后,花粉在可育株和不育株的柱头上均可萌发,说明不育株雌蕊的柱头不是导致雌性不孕的原因。在FM195AB不育株和可育株的花柱和子房中,花粉管起初都能正常延伸。但在花粉管进入胚珠的前期,可育株和不育株中存在明显差异：可育株中花粉管形态正常,通过激光激发是不规则的线状,上面有箭头状的亮点,花粉管能大量的延伸至胚珠,多条花粉管包裹胚珠；而在不育株中,花粉管形态异常,花粉管卷曲严重,虽能延伸至胚珠附近,但是花粉管不能包裹胚珠。
4.对比观察FM195AB中不育株和可育株的胚囊发育过程发现在不育株中,不孕胚囊存在几种类型,比例各不相同。
5.对FM195AB不育株花药和胚囊的对比观察显示,花药败育发生在花粉母细胞的减数分裂时期,没有观察到四分体的形成,而在同时期的雌蕊胚珠中未发现功能大孢子的形成,且在随后的胚珠发育中,未发现正常的单核胚囊,从而推测不育株的胚珠中减数分裂异常。对花药减数分裂进行观察发现,败育发生在减数分裂的间期。综合上述观察结果表明：不育株内花药和胚珠的败育时期相同,均发生在减数分裂阶段。但在胚珠中,少数胚囊可以逃逸。
根据上述实验结果,推测FM195AB不育株中胚珠减数分裂异常是导致雌性不孕产生的根本原因。胚珠发育异常一方面不能形成正常功能的胚囊(及雌配子体),另一方面不能在双受精过程中引导花粉管的正常延伸和进入珠孔,从而导致雌性不孕。由于花药和胚珠中雌雄配子体发育异常的相似性,推测雌性不孕和雄性不育均是显性核不育基因Ms造成的结果,暗示着ms在雌性配子体减数分裂过程中均发挥着重要作用.
Male-female gametophyte development, fusion and double fertilization are important marks of metagenesis in flowering plants. If some unusual happen in internal and external condition during the continuous development process of male gametophyte, male gametophyte would be sterile, and all the same in the female gametophyte. The female gametophyte is an essential structure for seeds formation in flowering plants. Female sterility can't be made up by healthy pollens, so need to avoid in agricultural production. In the study of sterility in dominant genic male-sterile Brassica napus, we found different degrees of phenomenon of female sterility expressed in male-sterile line Rs1046AB and derivative male-sterile lines FM195AB-1 and FM195AB-2. To overcome this negative trait, study the causes of female sterility of male-sterile line, the main results as follows:
1. Different levels of female sterility in numbers of male-sterile lines, the average seed numbers is 5.5 per pod in original male-sterile line Rs1046A, the least is 1-2, the seed rates of both male-sterile lines has significant differences. The number of seed per pod of heterozygous male-sterile lines is significantly higher than homozygous.
2. Female-sterile occurs only in sterile plants of dominant genic male-sterile lines, it isn't impacted by environmental, this phenomenon shows female-sterile and male sterile are linked closely, it's like that the dominant male gene expresses two different effects in development of female and male. Occurrence of female-sterile may be associated with the dosage effect of sterility genes.
3. Stigmas of Brassica napus Rs1046AB and FM195AB have no obvious differences. Pollen grains are germinating soon after they locates in the stigma, pollen grains in the stigmas of Brassica napus Rs1046B are germinating normally 4h after artificial pollination, it indicated that abnormale pistil stigmas is not the cause of female-sterile. Pollen tubes elongate in stylar and gather nearby the ovules successfully in FM195AB, there is significant difference before pollen tubes enter the ovule in FM195AB:the morphology of pollen tubes is normal in fertile plants, it's just like irregular lines with arrow-shaped bright spot, lots of pollen tubes gather nearby the ovules, and enwrap ovules; but in sterile plants, the morphology of pollen tubes is abnormal, coiled serious, pollen tubes gather nearby the ovules successfully, but not enwrap.
4. Analyze female gametophyte development of FM195AB before fertilization, we found five major types of abnormalities with different proportion in the mature embryo sac in FM195A.
5. Comparing the development stage of embryo sac with the anther, we found the meiosis of the microsporocyte is abnormal, nor form tetrad. In the same period of the pistil, there is no formation of the functional megaspore, and in subsequent ovule development, there is no mononuclear embryo, thus speculate that meiosis is abnormal in ovules of sterile plants. In anther, exception occurs in the period of meiotic interphase. The above observation showed:abortion of anthers and ovules happened in the same period, both in meiosis, but in ovules, a few sac developed normally.
Based on the above results, we speculate that the meiotic abnormalities leads to female-sterile. Abnormal ovule development could not form the normal functional sac, on the other hand could not guide the pollen tube extension and enter into the ovule. As the similarity of the abnormal development in anthers and ovules, suggesting dominant male sterile gene Ms causes the male and female sterility, it implied ms plays an important role in meiosis of the female gametophyte.
1.结实率调查表明,在不同遗传背景不育系的不育株中,雌性不孕程度有所差异,可育株结实完全正常。原始不育系Rs1046AB中不育株的每角果平均种子为5.5粒,而FM195AB中的绝大多数角果不能结实,极少数只有1-2粒,二者结实率差异极显著。而杂合型不育系不育株中每角果粒数显著高于纯合型不育系。
2.雌性不孕不受环境条件影响,雌性不孕只发生在雄性不育株上,表明雌性不孕极可能是显性核不育基因在雌性和雄性发育途径中的不同表现,或者控制雄性不育和雌性不孕性状的基因紧密连锁。纯合型不育系和杂合型不育系之间雌性不孕程度存在差异暗示着该性状与基因的剂量效应有关。
3.扫描电镜观察发现FM195AB可育株和不育株的柱头形态无明显差异。采用正常可育花粉人工授粉四小时后,花粉在可育株和不育株的柱头上均可萌发,说明不育株雌蕊的柱头不是导致雌性不孕的原因。在FM195AB不育株和可育株的花柱和子房中,花粉管起初都能正常延伸。但在花粉管进入胚珠的前期,可育株和不育株中存在明显差异：可育株中花粉管形态正常,通过激光激发是不规则的线状,上面有箭头状的亮点,花粉管能大量的延伸至胚珠,多条花粉管包裹胚珠；而在不育株中,花粉管形态异常,花粉管卷曲严重,虽能延伸至胚珠附近,但是花粉管不能包裹胚珠。
4.对比观察FM195AB中不育株和可育株的胚囊发育过程发现在不育株中,不孕胚囊存在几种类型,比例各不相同。
5.对FM195AB不育株花药和胚囊的对比观察显示,花药败育发生在花粉母细胞的减数分裂时期,没有观察到四分体的形成,而在同时期的雌蕊胚珠中未发现功能大孢子的形成,且在随后的胚珠发育中,未发现正常的单核胚囊,从而推测不育株的胚珠中减数分裂异常。对花药减数分裂进行观察发现,败育发生在减数分裂的间期。综合上述观察结果表明：不育株内花药和胚珠的败育时期相同,均发生在减数分裂阶段。但在胚珠中,少数胚囊可以逃逸。
根据上述实验结果,推测FM195AB不育株中胚珠减数分裂异常是导致雌性不孕产生的根本原因。胚珠发育异常一方面不能形成正常功能的胚囊(及雌配子体),另一方面不能在双受精过程中引导花粉管的正常延伸和进入珠孔,从而导致雌性不孕。由于花药和胚珠中雌雄配子体发育异常的相似性,推测雌性不孕和雄性不育均是显性核不育基因Ms造成的结果,暗示着ms在雌性配子体减数分裂过程中均发挥着重要作用.
Male-female gametophyte development, fusion and double fertilization are important marks of metagenesis in flowering plants. If some unusual happen in internal and external condition during the continuous development process of male gametophyte, male gametophyte would be sterile, and all the same in the female gametophyte. The female gametophyte is an essential structure for seeds formation in flowering plants. Female sterility can't be made up by healthy pollens, so need to avoid in agricultural production. In the study of sterility in dominant genic male-sterile Brassica napus, we found different degrees of phenomenon of female sterility expressed in male-sterile line Rs1046AB and derivative male-sterile lines FM195AB-1 and FM195AB-2. To overcome this negative trait, study the causes of female sterility of male-sterile line, the main results as follows:
1. Different levels of female sterility in numbers of male-sterile lines, the average seed numbers is 5.5 per pod in original male-sterile line Rs1046A, the least is 1-2, the seed rates of both male-sterile lines has significant differences. The number of seed per pod of heterozygous male-sterile lines is significantly higher than homozygous.
2. Female-sterile occurs only in sterile plants of dominant genic male-sterile lines, it isn't impacted by environmental, this phenomenon shows female-sterile and male sterile are linked closely, it's like that the dominant male gene expresses two different effects in development of female and male. Occurrence of female-sterile may be associated with the dosage effect of sterility genes.
3. Stigmas of Brassica napus Rs1046AB and FM195AB have no obvious differences. Pollen grains are germinating soon after they locates in the stigma, pollen grains in the stigmas of Brassica napus Rs1046B are germinating normally 4h after artificial pollination, it indicated that abnormale pistil stigmas is not the cause of female-sterile. Pollen tubes elongate in stylar and gather nearby the ovules successfully in FM195AB, there is significant difference before pollen tubes enter the ovule in FM195AB:the morphology of pollen tubes is normal in fertile plants, it's just like irregular lines with arrow-shaped bright spot, lots of pollen tubes gather nearby the ovules, and enwrap ovules; but in sterile plants, the morphology of pollen tubes is abnormal, coiled serious, pollen tubes gather nearby the ovules successfully, but not enwrap.
4. Analyze female gametophyte development of FM195AB before fertilization, we found five major types of abnormalities with different proportion in the mature embryo sac in FM195A.
5. Comparing the development stage of embryo sac with the anther, we found the meiosis of the microsporocyte is abnormal, nor form tetrad. In the same period of the pistil, there is no formation of the functional megaspore, and in subsequent ovule development, there is no mononuclear embryo, thus speculate that meiosis is abnormal in ovules of sterile plants. In anther, exception occurs in the period of meiotic interphase. The above observation showed:abortion of anthers and ovules happened in the same period, both in meiosis, but in ovules, a few sac developed normally.
Based on the above results, we speculate that the meiotic abnormalities leads to female-sterile. Abnormal ovule development could not form the normal functional sac, on the other hand could not guide the pollen tube extension and enter into the ovule. As the similarity of the abnormal development in anthers and ovules, suggesting dominant male sterile gene Ms causes the male and female sterility, it implied ms plays an important role in meiosis of the female gametophyte.
引文
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