水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻(Oryz a sativa)受精过程中雌雄性细胞融合时的形态特征及时间进程, 确定合子期, 为花粉管通道转基因技术的实施提供理论依据。结果表明: 授粉后, 花粉随即萌发, 花粉管进入羽毛状柱头分支结构的细胞间隙, 继续生长于花柱至子房顶部的引导组织的细胞间隙中, 而后进入子房, 在子房壁与外珠被之间的缝隙中向珠孔方向生长, 花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞, 释放精子。精子释放前, 两极核移向卵细胞的合点端; 两精子释放于卵细胞与中央细胞的间隙后, 先后脱去细胞质, 然后分别移向卵核和极核, 移向卵核的精核快于移向极核的精核; 精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下: 授粉后, 花粉在柱头萌发; 花粉萌发至花粉管进入珠孔大约需要0.5小时; 授粉后0.5小时左右, 花粉管进入一个助细胞, 释放精子; 授粉后0.5-2.5小时, 精卵融合形成合子; 授粉后约10.0小时, 合子第1次分裂, 合子期为授粉后2.5-10.0小时; 授粉后1.0-3.0小时, 精核与两极核融合; 授粉后约5.0小时, 初生胚乳核分裂。     

水稻双受精过程的细胞形态学及时间进程的观察

应用常规石蜡切片和荧光显微镜观察水稻（Oryza sativa）受精过程中雌雄性细胞融合时的形态特征及时间进程,确定合子期,为花粉管通道转基因技术的实施提供理论依据。结果表明：授粉后,花粉随即萌发,花粉管进入羽毛状柱头分支结构的细胞间隙,继续生长于花柱至子房顶部的引导组织的细胞间隙中,而后进入子房,在子房壁与外珠被之间的缝隙中向珠孔方向生长,花粉与花粉管均具有明显的绿色荧光。花粉管经珠孔及珠心表皮细胞间隙进入一个助细胞,释放精子。精子释放前,两极核移向卵细胞的合点端：两精子释放于卵细胞与中央细胞的间隙后,先后脱去细胞质,然后分别移向卵核和极核,移向卵核的精核快于移向极核的精核：精核与两极核在向反足细胞团方向移动的过程中完成雌雄核融合。大量图片显示了雌雄性核融合的详细过程以及多精受精现象。水稻受精过程经历的时间表如下：授粉后,花粉在柱头萌发：花粉萌发至花粉管进入珠孔大约需要0．5小时：授粉后0．54,时左右,花粉管进入一个助细胞,释放精子：授粉后0．5—2．5小时,精卵融合形成合子：授粉后约10．0小时,合子第1次分裂,合子期为授粉后2．5-10．04,时：授粉后1．0-3．04,时,精核与两极核融合：授粉后约5．0小时,初生胚乳核分裂。’     

短柄五加开花后雌蕊的发育状态与受精作用的研究

短柄五加（ＥｌｅｕｔｈｅｒｏｃｏｃｕｓｂｒａｃｈｙｐｕｓＨａｒｍｓ．）开花当天,花药散粉,而雌配子体需经４～５ｄ才发育成熟。证实短柄五加为雄蕊先熟植物。开花第５天,成熟胚囊的比率为５７６９％,其余为退化和不育胚囊。开花第６天,胚囊开始受精。开花第１０天,受精胚囊占胚囊总数的５３５７％。柱头的可授期自开花后第４～５天开始,自花粉萌发至雌雄性核融合大约有２～３ｄ的间隔期。短柄五加受精过程与一般被子植物相同,其受精作用属于有丝分裂前配子融合类型。观察并统计了合子中雌性核仁的数目、存在状态,指出短柄五加合子中从雄性核仁出现到与雌雄性核仁融合为一个大核仁需经历３ｄ左右;如果以胚乳游离核数目为对照,大部分合子中雌雄性核仁的融合发生在３２～１２８个胚乳游离核时期。大多数合子是以雌雄性核仁融合为一个大核仁后进入合子分裂期;少数合子的雌雄性核仁不经融合也进入合子分裂期。观察到多精入胚囊、多精入卵以及成熟胚囊退化的现象。讨论了被子植物受精过程中有关受精终结的标志等问题。     

罗汉果双受精过程的细胞学观察

罗汉果（Ｓｉｒａｉｔｉａｇｒｏｓｖｅｎｏｒｉ（Ｓｗｉｎｇｌｅ）Ｃ．Ｊｅｍｅｙ）双受精过程属有丝分裂前配子融合类型,授粉后２４～４８ｈ,花粉管进入胚囊,穿过一个助细胞,放出两个精子。雌雄核融合和雄核与次生核融合同时发生在授粉后６２～７２,雄核与次生核融合速度快于配子融合,７２ｈ后即可见到初生胚乳核分裂。合子中的雌雄核仁在授粉后第５～６ｄ融合,授粉后８～９ｄ合成分裂形成二细胞胚。在双受精过程中,多次观察到有多条花粉管进入胚囊和多精入极核现象。原胚期有附加花粉管从珠孔进入。     

侧金盏花双受精进程研究

应用荧光显微镜和常规石蜡切片观察侧金盏花(Adonis amurensis)花粉管生长和受精作用的全过程。结果表明,侧金盏花为湿型柱头,授粉后1–2小时,花粉粒与柱头识别;授粉后2–4小时,花粉粒萌发;授粉后4–6小时,花粉管进入柱头。侧金盏花的受精模式为珠孔受精,授粉后10小时,精子被释放;授粉后30小时,精核与卵核融合;授粉后7天合子形成;授粉后15天合子进入分裂期,合子休眠期为8天。2个极核在受精前不融合,授粉后14–16小时,精核与1个极核融合;授粉后20–22小时,受精极核与另1个极核融合形成初生胚乳核。双受精作用属于有丝分裂前配子融合型。通过实验确定了侧金盏花受精过程的雌雄性细胞融合形态变化与相应经历的时间及其合子休眠期。研究结果丰富了侧金盏花胚胎学资料,对其今后的育种及转基因研究具有重要意义。     

星星草受精作用及其胚与胚乳早期发育的观察

利用常规石蜡切片法对星星草[Puccinellia tenuiflora(Griseb．)Scribn．et Merr．]受精过程及胚与胚乳的早期发育进行了观察,主要结论如下：(1)开花后2h,花粉管破坏1个助细胞,释放2个精子,精子呈逗点状。(2)开花后2～3h,2个精子分别移向卵细胞与极核。(3)开花后3～5h,精核分别贴附于卵细胞与极核的核膜上。(4)开花后5～10h,精核与卵核融合,雄性核仁出现,合子形成。(5)开花后5～6h,精核与极核融合,并出现雄性核仁,形成初生胚乳核,精核与极核的融合比与卵核融合要快。(6)开花后20h左右,合子分裂。(7)开花后8h,初生胚乳核。     

烟草中央细胞离体受精过程中雌雄核融合生活动态的记录

受精作用一直是植物生殖发育生物学研究的热点课题。近年发展尤为迅速。特别是诸如偏向受精等新概念的提出更进一步推动了对双受精作用的寻微探秘,日益显现出这一过程的精巧与复杂。但限于体内研究的局限性,对其中一些关键环节,如雌雄配子间的识别;配子融合过程中的相互作用;雄核在雌性细胞内迁移的动力学及雌雄核融合的时间进程与机制等仍知之甚少。离体受精操作及相关技术的建立［１～３］为探讨上述问题提供了新途径。我们在过去工作的基础上以烟草为材料进行了离体双受精研究,以视频增差显微观察系统首次记录到在生活状态下精核进…     

华贵栉孔扇贝♀×栉孔扇贝♂远缘杂交的受精细胞学观察

用HOECHST33258对华贵栉孔扇贝(Chlamys nobilis)♀×栉孔扇贝(Chlamys farreri)♂的受精卵进行染色,在荧光显微镜下观察其受精细胞学过程。观察表明:栉孔扇贝的精子能够使华贵栉孔扇贝卵子受精。精子入卵后呈一圆形亮点,体积稍有膨大成球形;授精后成熟卵母细胞释放出第一极体和第二极体后,精核解凝、稀疏、泡状化,形成雄性原核(male pronucleus);雌性原核(female pronucleus)在完成两次成熟分裂之后,染色质去浓缩,扩散膨大;雌雄原核相互靠拢,当雌雄原核膨胀到最大程度时,发生融合,形成合子。受精卵能够正常发育,完成第一次卵裂,卵裂时细胞核分离未观察到异常现象。与华贵栉孔扇贝和栉孔扇贝种内交配对照组相比,异源受精卵的细胞学过程明显滞后,同时其发生过程具明显的不同步现象。实验中还观察到少数的雌核发育现象。     

栉孔扇贝(♀)×虾夷扇贝( ♂ )受精细胞学观察

采用Bouin氏液固定、石蜡包埋、切片 ,用苏木精 伊红染色 ,在光学显微镜下观察栉孔扇贝 (♀ )与虾夷扇贝 (♂ )杂交的受精细胞学过程。尽管栉孔扇贝与虾夷扇贝同科不同属 ,但它们的杂交仍具有正常的受精细胞学程序。栉孔扇贝卵子处于第一次成熟分裂中期时接受虾夷扇贝的精子入卵 ,精卵混合后 6min精子入卵 ;8～ 1 0min精核略微膨胀 ;2 5～ 3 0min排出第一极体 ;1h左右 ,雌雄原核同时形成 ;1小时 3 0分钟左右 ,雌雄原核融合 ;2h左右开始卵裂。杂交过程中的精子入卵行为较本交迟缓 ,但杂交后代仍能正常发育     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

Live-cell imaging reveals the dynamics of two sperm cells during double fertilization in Arabidopsis thaliana

Flowering plants have evolved a unique reproductive process called double fertilization, whereby two dimorphic female gametes are fertilized by two immotile sperm cells conveyed by the pollen tube. The two sperm cells are arranged in tandem with a leading pollen tube nucleus to form the male germ unit and are placed under the same genetic controls. Genes controlling double fertilization have been identified, but whether each sperm cell is able to fertilize either female gamete is still unclear. The dynamics of individual sperm cells after their release in the female tissue remain largely unknown. In this study, we photolabeled individual isomorphic sperm cells before their release and analyzed their fate during double fertilization in Arabidopsis thaliana. We found that sperm delivery was composed of three steps. Sperm cells were projected together to the boundary between the two female gametes. After a long period of immobility, each sperm cell fused with either female gamete in no particular order, and no preference was observed for either female gamete. Our results suggest that the two sperm cells at the front and back of the male germ unit are functionally equivalent and suggest unexpected cell-cell communications required for sperm cells to coordinate double fertilization of the two female gametes.     

鲫鲤杂种一代(F1)自交二代(F2)的受精细胞学研究

荷包红鲫(♀)×湘江野鲤(♂)杂交产生的杂种一代(F     

Cytologic Observations on the Double Fertilization in Maize

1.The double fertilization is the type of the premitotic syngamy. 2. In 21 to 24 hours after pollination, most of the female nuclei fuse with the male nuclei. When the female nucleus fuses with the male nucleus, there are two situations in the appearance of the male nucleolus: one is that while the chromatin of the sperm nucleus relaxes gradually, a male nucleolus appears; the other is that after the chromatin of the sperm nucleus relaxes gradually, the male nucleus just appears in about 2 to 4 hours. 3. Generally, the fusion of the female nucleolus with the male nucleolus takes place before the division of the nygote. The nygote enters the stage of division when it has a jarge nucieojus; the zygote, in which the female nucleoius does not fuse with the male nucleolus, also can enter the stage of division. In 30 to 33 hours after pollination, the first division of the zygote occurs. The resting period of the zygote is about 9 hours 4. In the primary endosperm nucleus, the female nucleolus does not fuse with the male nucleolus. 24 to 26 hours after pollination, the primary endosperm nucleus begins the first division. The resting period of the primary endosperm nucleus is 2 to 4 hours. 5. Under the condition of artificial pollination, the fertilization of the fruit ears of maize proceeds sequencely, i.e. from the upper of the fruit ears to the lower the fertilization is fulfilled gradually.     

Cytological mechanisms of gynogenesis and sperm incorporation in unreduced diploid eggs of the clonal loach, Misgurnus anguillicaudatus (Teleostei: Cobitidae)

The loach Misgurnus anguillicaudatus comprises diploid clonal, triploid and diploid-triploid mosaic individuals in a wild population on Hokkaido island, Japan. When diploid eggs of clonal loaches are fertilized by haploid sperm of normal bisexual loaches, both diploid clonal and non-diploid aclonal individuals occur in the progeny. Flow cytometry and microsatellite analyses revealed that the occurrence of triploid, diploid-triploid and other progeny was essentially due to the genetic incorporation of sperm to diploid clonal genomes of unreduced eggs. In this study, we examined the influence of water temperature from fertilization to early embryogenesis on frequencies of diploid clonal and other progeny and observed that progeny of three out of four clonal females examined exhibited approximately constant rates of diploid clonal individuals (54.2-68.9%) at hatching stage. Thus, no drastic increase of non-diploid progeny was detected. However, the 28 degrees C group of the fourth clonal female gave significantly lower rate (28.1%) of diploid clonal progeny, suggesting that this temperature might be a critical or a borderline temperature inducing sperm incorporation. We also examined the cytological process by which diploid clonal and other aclonal progeny develop after fertilization. In some fertilized eggs, the sperm nucleus remained condensed throughout fertilization and early embryogenesis and never fused with the female pronucleus. This cytological observation concludes that clonal eggs develop by the mechanism of gynogenesis. However, some other eggs showed the cytological process of syngamy between the female pronucleus and an accidentally formed male nucleus, suggesting the formation of triploid progeny. The syngamy between an accidentally activated sperm nucleus with a male pronucleus-like structure and nucleus of a blastomere of gynogenetically developing clonal diploid embryo might produce a diploid-triploid mosaic individual.     

玉米双受精过程的细胞学观察

1.玉米双受精属于有丝分裂前配子融合的类型。2.授粉后21至24小时,大部分雌、雄性核发生融合。雌、雄性核融合时,雄性核仁的出现存在两种情况,其一是精核染色质逐渐分散的同时出现雄性核仁,其二是精核染色质逐渐分散后,约经2至4小时才出现雄性核仁。3.合子内的雌、雄性核仁若发生融合,一般在合子分裂前进行。合子以一个大核仁的形式进入分裂期;雌、雄性核仁不融合的合子同样可以进入分裂期。授粉后30至33小时,合子进行第一次分裂。合子静止期大约为9小时左右。4.初生胚乳核内的雌、雄性核仁不发生融合。授粉后24至26小时,初生胚乳核进行第一次分裂。初生胚乳核的静止期为2—4小时。5.人工授粉条件下,玉米果穗受精过程的进行有一定的顺序;即自果穗上部逐渐向下部完成受精作用。     

黑节草双受精过程及胚胎发育的研究

黑节草从传粉到受精约需１３０ｄ,精子在花粉管中形成,胚囊发育属蓼型胚囊,因反足细胞较早退化,故受精前胚囊多只由卵器和中央细胞组成。精卵核融合时,精核染色质进入卵核后凝集成颗粒状,并在原位与卵核的染色质融合,雌、雄性核仁一直维持至合子的第一次分裂期前。双受精作用正常,属于有丝分裂前配子融合类型,初生胚乳核发生２－３次分裂后逐渐退化消失,胚的发育局限于球形胚阶段。     

The Fusion of Male and Female Nuclei in Fertilization of Higher Plants

Studies on the fusion of male and female nuclei in fertilization of Helianthus an- nuus L., Triticum aestivan L., Gossypium hisutum L., Hosta caerulea Tratt., and Pinus tabulaeformis Carr. were made in the present work. The results are summarized as follows: 1. The essential process of the fusion of male and female nuclei during syngamy in four species of angiosperms studied may' be generalized as follows: (1) the male nucleus made contact with the female one, (2) followed by the fusion of nuclear membranes between the male and female nuclei. (3) then the despiralization of male spireme happened and male nucleolus made its appearance inside of the fertilized egg nucleus (4) the male chromatin dispersed and make its appearance indistinguishable from that of the female chromatin, (5) the male and female nucleoli fused together to form a larger nucleolus as a sign of completion of the fusion of the two nuclei. In the first mitotic division of the zygote there was only one common mitotic spindle. 2. The essential process of the fusion of egg and sperm nuclei during syngamy in a gymnosperm-Pinus tabulaeformis could also be outlined as follows: (1) the sperm nucleus made contact with the egg nucleue, (2) the fusion of nuclear membranes happened between the male and female nuclei, (3) the male and female ehromatins condensed to form two separate groups of chromatin threads together with the very apparent apperance of the male and female nucleoli at this stage, (4) the male and female chromosomes grouped respectively in their own spindles while both nucleoli disappeared, (5) then the two spindles fused together and all the chromosomes arranged to form a common equatorial plate, (6) finally two daugter nuclei resulted from the mitotic division. 3. Based on the facts that there were two different patterns of the fusion of male and female nuclei in fertilization discribed, all of these accounts are in general accord with the condition usually described that there are two types of fertilization, the pre- mitotic and postmitotie syngamy in higher plants. The type of angiosperm fertilization and the mechanism of promoting the zygote to divide after fertilization are discussed, and the nuclear fusion in sexual reproduction has been compared with that of somatic cell hybridization.     

番茄受精作用及其间隔期的研究

利用常规石蜡切片法研究了番茄受精作用的全过程,具体研究结果为:(1)授粉后2 h,花粉粒在柱头上萌发;约2~4 h,花粉管长入柱头,且末端膨大;约8 h后,生殖细胞进入分裂期;并于约两小时后,分裂为两个精细胞。(2)约14 h,花粉管进入子房腔;约18~24 h,花粉管进入胚囊,破坏一个助细胞,并在其珠孔端释放两个精子;随后被释放的精子移到卵细胞与次生核附近。(3)授粉后约30 h精核进入卵细胞;约34 h,精核与卵核融合,并在卵核内出现分散的雄性染色质,进而出现雄性核仁;44~50 h,雌、雄性核仁融合,形成合子;合子的休眠期为10 h左右。60 h之后,合子分裂形成二细胞原胚。(4)约26 h,另一个精子的精核与次生核核膜相贴伏,随后与之融合;约30~34 h,次生核内出现分散的雄性染色质,随之出现雄性核仁;约38~42 h,雌、雄性核仁融合,形成初生胚乳核。约44 h后,初生胚乳核进行有丝分裂,形成两个胚乳细胞。番茄胚乳发育属于细胞型。初生胚乳核无休眠期。(5)精子与次生核的融合比与卵核的融合快。(6)番茄的受精作用属于有丝分裂前配子融合类型。