利用激光扫描共聚焦显微技术观察同源四倍体双胚苗水稻的胚胎学特征

利用激光扫描共聚焦显微技术对同源四倍体双胚苗水稻的受精过程、胚胎发育过程和双胚来源进行观察鉴定。研究结果表明,同源四倍体水稻和二倍体水稻在发生受精作用的时间上不存在很大的差异。双胚苗水稻的受精作用和胚胎发育表现出明显的特异性,其双胚苗性状有其胚胎学根源,双胚可能有4种来源,即来源于由双套胚囊形成的双胚、由多卵卵器中的卵细胞和类卵细胞分别通过受精作用形成双胚、由特异性的反足细胞通过异常发育后形成额外的幼胚、由胚乳细胞通过异常发育后形成胚乳型幼胚。通过对试验材料的受精频率及其结实率进行比较后发现,双胚苗水稻具有更弱的有性生殖能力,其遗传可塑性更强,这为离子束介导技术的研究找到了比较好的受体材料。     

利用激光扫描共聚焦显微术对同源四倍体水稻胚囊形成与发育的进一步观察

应用改进的整体染色透明激光扫描共聚焦显微术(WCLSM),对同源四倍体水稻PDER-2B-4x胚囊的形成与发育过程进行观察。发现其胚囊的形成发育过程与二倍体的一致,可以清楚地划分为8个发育时期,即孢原细胞形成期、大孢子母细胞形成期、大孢子母细胞减数分裂期、功能大孢子形成期、单核胚囊形成期、胚囊有丝分裂期、八核胚囊发育期和成熟胚囊期。除正常发育的过程外,大孢子发育的各个过程均出现一些异常现象,包括:细胞退化、核位置异常、核数目异常和细胞分化异常等。这些异常可能最终导致多种结构异常成熟胚囊的形成。     

四倍体大燕麦与六倍体裸燕麦的受精作用和胚胎发育

用石蜡切片法,对四倍体大燕麦(Avena magna L.)和六倍体裸燕麦(Avena nuda L.)杂交的受精作用和胚胎发育进行了观察。结果表明,六倍体裸燕麦花粉在四倍体大燕麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊。观察的168个四倍体大燕麦子房中,238%发生了双受精,产生了胚和胚乳;179%发生了单卵受精,只产生胚乳而无胚;总受精率为536%;成胚率为417%。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为四倍体大燕麦和六倍体裸燕麦杂交提供了细胞胚胎学证据。     

四倍体大燕麦与六倍体裸燕麦的受精作用和胚胎发育

用石蜡切片法,对四倍体大燕麦（Avena magna L.）和六倍体裸燕麦（Avena nuda L.）杂交的受精作用和胚胎发育进行了观察。结果表明,六倍体裸燕麦花偻在四倍体大燕麦柱头上萌发良好,花粉管可顺利长入花柱和胚囊,观察的168个四倍体大燕麦子房中,2.38%发生了双受精,产生了胚和胚乳;1.79%发生了单卵受精,只产生胚乳而无胚;总受精率为5.36%;成胚率为4.17%。由于胚乳的缺管或发育异常及败育,最终难以获得有生活力的种子,为四倍体大燕麦和六倍体裸燕麦杂交提供了细胞胚胎学证据。     

水稻双受精过程的共聚焦显微镜观察

(郑州大学离子束生物工程省重点实验室,郑州450052)摘要:首次利用核特异荧光染色与整体透明技术并利用激光扫描共聚焦显微镜(LSCM)对水稻双受精过程进行了观察。激光扫描共聚焦显微镜具有"组织与细胞CT"的功能,可以对整体组织进行扫描并构建三维结构。经荧光染色及透明处理后,在激光扫描共聚焦显微镜下经488nm激光激发,胚囊内各细胞的细胞核以及核仁发出明亮荧光,细胞核轮廓比较清晰,层次感强,并能清晰观察到胚囊内各细胞的结构特点和空间位置关系。不论是对开花前较小的成熟胚囊材料还是开花后较大的胚囊材料都取得了较好的观察效果。同传统的光学显微镜和荧光显微镜相比,其观察到的图像更清晰、更直观、更具立体感。     

四倍体双穗雀稗兼性无孢子生殖的研究

研究了四倍体双穗雀稗(Paspalum distichum L)无孢子生殖胚囊、胚胎发育以及假受精特点。当其大孢子母细胞发育至四分体阶段时,大多数情况下会发生四分体退化,同时有多个特化珠心细胞发育为1—3个无孢子生殖胚囊的现象。成熟无孢子生殖胚囊一般3核,包括1个卵细胞和2个极核。卵细胞在抽穗前就能自发分裂形成原胚团,而极核则在抽穗和传粉后参与假受精形成胚乳。当胚珠内存在多个无孢子生殖胚囊时,只是靠近珠孔端的1个无孢子生殖胚囊内的极核与精核结合,而其它的并不参与。种子成熟后出现很低频率的二胚苗。此外,还能观察到少量的有性生殖胚囊的发育以及有性生殖胚囊和无孢子生殖胚囊在同一胚珠中的发育现象,因此判断该类群为兼性无孢子生殖体。     

水稻(Oryza sativa L.)×狼尾草(Pennisetum sp.)胚胎发育的细胞学观察

1.狼尾草花粉可以在水稻柱头上萌发并能长入胚囊,但受精过程缓慢而且不正常,单受精现象经常出现。2.胚胎发育滞缓,长时间停留在球状胚阶段,很难进一步分化,而且不时败育,只在传粉后16—24天的一些胚囊中看到有简单分化的胚胎。3.胚乳发育异常,由游离核形成细胞的时间推迟到传粉后第八天。整个胚乳组织由形状及功能上均有很大差异的细胞团块组成。其中一部分细胞缺乏合成淀粉的能力,胚乳在发育过程中不时出现解体现象。4.在反足细胞附近的一些珠心细胞中出现多量淀粉积累,反映因杂交而出现的胚囊代谢上的某些变化。讨论了胚和胚乳发育困难的原因和得到杂种种子的可能性。     

黄花油点草胚胎发育研究

利用石蜡切片技术对百合科植物黄花油点草[Tricyrtis maculata(D.Don)Machride]双受精、胚及胚乳发育进行了研究,以明确其胚胎发育的特征,为百合科植物的系统研究提供生殖生物学资料。结果表明:(1)黄花油点草为珠孔受精;进入胚囊的2枚精子分别与卵细胞和中央细胞进行正常的双受精,其受精作用属有丝分裂前型。(2)受精后的初生胚乳核立即分裂,其发育方式为核型胚乳;早期的游离胚乳核沿胚囊的边缘分布,胚囊中央部位主要为胚乳细胞质,随着游离胚乳核数量的增加,胚乳核慢慢充满整个胚囊;当发育至球形胚早期阶段,在各胚乳核周围产生胚乳细胞壁,形成完整的胚乳细胞。(3)合子有较长的休眠时间,胚的发育方式为茄型;合子第一次有丝分裂为横裂,分裂后形成基细胞和顶细胞;基细胞经过3次横裂,形成一列胚柄细胞;顶细胞经过分裂形成胚体,依次形成球形胚、棒状胚和盾形胚。(4)种子成熟时胚无器官分化;成熟种子由种皮、胚和胚乳三部分组成。     

多胚水稻品系APⅣ不同类型胶囊的受精及其胚胎形成

通过GMA半薄切片技术对APⅣ不同类型水稻(Oryza sativa L.)胚囊的受精及其胚胎发育的研究表明,APⅣ中5-2-l型胚囊的3个卵细胞在少数情况下都可受精并发育形成3个胚;但多数情况只有 1个或2个卵细胞受精发育成1个胚或2个胚。6-2-0型和5-3-0型胚囊多个卵受精频率都很低。由此证明APIV多胚是来自如5-2-1型胚囊的多卵卵器胚囊多个卵细胞都受精的结果,其中3胚来自3个卵细胞受精发育,2胚来自2个卵细胞受精发育。双套结构胚囊受精最为复杂,多数情况是受精不正常,只有少数子房大、小胚囊中的卵细胞都能正常受精。大胚囊中的卵细胞受精发育可能是形成所谓中位胚(远离珠孔端胚)的主要原因。     

小麦x长穗偃麦草的受精和早期胚胎发育

用石蜡切片法,对小麦(Triticum aestivum)和长穗偃麦草(Elytrigia elongata)杂交的受精和早期胚胎发育进行了观察。结果表明,长穗偃麦草花粉在小麦柱头上萌发良好,花粉管可顺利长人花柱和胚囊。 观察的170个小麦子房中,17.65％发生了双受精,产生了胚和胚乳;9.41％发生了单卵受精,只产生胚而无胚乳;4.71％。发生了单极核受精,只产生胚乳而无胚;总受精率为31.77％;成胚率为27.06％。由于胚乳的缺乏或发育异常及败育,最终难以获得有生活力的种子。为小麦与长穗偃麦草远缘杂交提供了细胞胚胎学证据。     

SEED DEVELOPMENT IN CITRUS WITH SPECIAL REFERENCE TO 2X X 4X CROSSES

Comparative cytological and histological studies during embryogenesis of seeds from 2x X 2x and 2x x 4x crosses indicate that the ratio of ploidy level between embryo and endosperm is the most important factor affecting the course of seed development. The crosses produced seeds with the expected ploidy relationships between embryo, endosperm, and maternal tissue of 2:3:2 and 3:4:2 as well as the anomalous relationships 3:5:2, 4:6:2, and 6:10:2. All but 3:4:2 resulted in normal, germinable seeds. The ploidy level of the maternal tissue in relation to that of the embryo or endosperm did not appear to have any effect on seed development. About 92–99 % of seeds from 2x x 4x crosses containing triploid embryos with tetraploid endosperm aborted at different stages of embryogenesis. The abortion in all cases was preceded by abnormalities in the tetraploid endosperm. It is postulated that the unbalance of chromosome number between embryo and endosperm disturbs physiological relationships between these two tissues, leading first to the abortion of the endosperm and then of the embryo.     

五唇兰雌配子体发育和胚胎发生的研究

五唇兰的胚珠倒生型,具薄珠心,两层珠被。胚囊发育为双孢子葱型,成熟胚囊８核。从传粉到受精约５０ｄ,正常双受精。胚具５－６细胞的胚柄,种子成熟时胚柄及胚乳核消失,成熟种子只具单层细胞的种皮和一个未分化的珠珠形胚。     

Rice caryopsis development Ⅱ: Dynamic changes in the endosperm

The rice endosperm plays crucial roles in nourishing the embryo during embryogenesis and seed germination. Although previous studies have provided the general information about rice endosperm, a systematic investigation throughout the entire endosperm developmental process is still lacking. In this study, we examined in detail rice endosperm development on a daily basis throughout the 30‐day period of post‐fertilization development. We observed that coenocytic nuclear division occurred in the first 2 days after pollination (DAP), cellularization occurred between 3 and 5 DAP, differentiation of the aleurone and starchy endosperm occurred between 6 and 9 DAP, and accumulation of storage products occurred concurrently with the aleurone/starchy endosperm differentiation from 6 DAP onwards and was accomplished by 21 DAP. Changes in cytoplasmic membrane permeability, possibly caused by programmed cell death, were observed in the central region of the starchy endosperm at 8 DAP, and expanded to the whole starchy endosperm at 21 DAP when the aleurone is the only living component in the endosperm. Further, we observed that a distinct multi‐layered dorsal aleurone formed near the dorsal vascular bundle, while the single‐ or occasionally two‐cell layered aleurone was located in the lateral and ventral positions of endosperm. Our results provide in detail the dynamic changes in mitotic divisions, cellularization, cell differentiation, storage product accumulation, and programmed cell death that occur during rice endosperm development.     

腊梅的受精作用及胚胎发生

腊梅(Chimonanthus praecox)花两性,离心皮雌蕊着生在杯状花托上,柱头线形,干性。花粉经昆虫传播,落在柱头上1 d后萌发,第8d从珠孔进入,第14d左右完成双受精,为珠孔受精。胚乳为核型胚乳;初生胚乳核经短暂休眠进行核分裂,位于合点端的游离核首先形成细胞,并从合点向珠孔端细胞化,第37d胚乳充满整个囊腔。合子经过近2周的休眠后开始分裂,随着胚的发育,大部分胚乳降解,为胚的发育提供营养。合点端的胚乳细胞则侵入合点珠心组织,为胚进一步发育提供营养。其胚胎发生为柳叶菜型。     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

On the Fertilization of Oryza sativa L. × Sorghum vulgare Pets.

1.The pollen germination of Sorghum vulgate appeared normal on the stigma of the Oryza sativa, but the pollen tubes grew slowly in the style. Some of the pollen tubes may become enlarged in their tips or sometimes bursting, while others have continued to grow and entered the embryo sacs. 2. The growth rate of the pollen tubes varied widely. A few pollen tubes were observed in the embryo sacs of the materials 2 hours after pollination, but most of them entered the embryo sacs much later. 3. The zygote associated with a paucity of endosperm nuclei was observed in the materials 1 day after pollination. The double fertilization and 8–12-celled proembryo associated with a number of the free nuclei of the endosperm appeared with a rather high frequency (10.3%) in the materials 3 days after pollination. Some of them are normal in appearance and others may show more or less abnormalities. 4. No division figure was found except in one single case in which mitoses have occurred in both the proembryo and the endosperm. It is most likely that in such case the proembryo and the endosperm if left intact might develop further. 5. A 80-celled embryo was the biggest one which appeared in the materials 5 days after pollination. In general, no cells were ever formed in the endosperm, except in one instance among the 7 days materials the endosperm became cellular in micropylar end. In all other cases the endosperm either ceased to develop early or disorganized. The disorganized endosperm materials are considered to be utilized by the embryo. 6. In certain instances the free nuclei of the endosperm were not distributed at random. They were not equal in size and might fuse into giant nuclelei. 7. The most striking feature is that in the embryo sacs, in which double fertilization or proembryo and endosperm have occurred, a dark stained pollen tube was commonly present. This fact leads us to the conviction that in general only if a healthy pollen tube entered the embryo sac, double fertilization can take place and further development can proceed. 8. In certain cases the protoplasm of the embryo cells appeared scanty. It is apparently that the normal metabolism of the embryo was disturbed owing to the lack of nutrient, and the death of the embryo ensued. 9. No differentiated embryo was observed and no mature seeds were produced. The materials fixed 12 days after pollination showed a variety of abnormalities and collapses. The authors believe that the failure of seed production of rice X kaoliang was primarily due to the fact that the pollen tubes in the style grew too slowly to reach the embryo sacs in time. The consequence is that the double fertilization took place only in a late stage while the male and female gametes may have already become unhealthy. In addition, in this late stage the stored starch in the maternal tissues having gradually disappeared, the nutrient supply to the embryo sac was therefore limited and the young embryo and endosperm were finally in starvation.     

The Fertilization and the Development of Embryo and Endosperm in an Endangered Plant—Ranalisma rostratum Stapf (Alismataceae)

Ranalisma rostratum Stapf is a rare and endangered species． This paper deals with the fertilization and the development of embryo and endosperm in this plant．The embryogenesis is of Caryophyllad type and the development of endosperm belongs to Heobial type．Before fertilization,the two polar nuclei are located respectively at both ends of embryo sac. In most angiosperms with two polar nuclei,the polar nuclei may fuse eiher before fertilization to form a secondary nucleus or during fertilization called triple fusion． In Ranalisma rostratum Stapf, however, it is found that only in case when the micropylar polar nucleus is fertilized,it can move to the chalazal end and fuse with the chalazal polar nucleus．This phenomenon is very rare and the process must take more time to fulfil fertilization both polar nuclei． This feature of fusion of polar nuclei is therefore thought as a primitive character from the view of phylogeny．     

腊梅的受精作用及胚胎发生

腊梅 (Chimonanthuspraecox)花两性 ,离心皮雌蕊着生在杯状花托上 ,柱头线形 ,干性。花粉经昆虫传播 ,落在柱头上 1d后萌发 ,第 8d从珠孔进入 ,第 1 4d左右完成双受精 ,为珠孔受精。胚乳为核型胚乳 ;初生胚乳核经短暂休眠进行核分裂 ,位于合点端的游离核首先形成细胞 ,并从合点向珠孔端细胞化 ,第 37d胚乳充满整个囊腔。合子经过近 2周的休眠后开始分裂 ,随着胚的发育 ,大部分胚乳降解 ,为胚的发育提供营养。合点端的胚乳细胞则侵入合点珠心组织 ,为胚进一步发育提供营养。其胚胎发生为柳叶菜型。