甘薯细胞质传递的研究：精细胞的质体和线粒体及其DNA存在的状况

甘薯（ＩｐｏｍｏｅａｂａｔａｔａｓＬａｍ．）成熟花粉为二细胞型,在授粉后萌发之前生殖细胞分裂形成精细胞。仍在花粉粒中的两个精细胞大小和形状基本相似,细胞质中含丰富的质体和线粒体。细胞质ＤＮＡ特异荧光显示精细胞及产生它们的前细胞———生殖细胞中均含有丰富的类核。一对精细胞中类核的数量无明显的差异。精细胞中存在两种形态类核,大而荧光强的类核可能为质体类核,而小的荧光弱的类核为线粒体类核。双亲或父系质体遗传在被子植物中是少数,本研究结果为旋花科的除牵牛属和打碗花属外又提供了新的一属具这种遗传方式的细胞学证据。     

牵牛属生殖细胞和精细胞中细胞器DNA的研究

用ＤＡＰＩ荧光技术观察了大花牵牛（ Ｐｈａｒｂｉｔｉｓ ｌｉｍ ｂａｔａ Ｌｉｎｄｌ．）和圆叶牵牛（Ｐ． ｐｕｒｐｕｒｅａ（Ｌ．） Ｖｏｉｇｈｔ）生殖细胞的细胞质ＤＮＡ及其在精细胞形成过程中的动态。牵牛属这两个种的生殖细胞均为细长形,具有大量细胞器ＤＮＡ。刚形成的一对精细胞大多数一端钝,另一端呈尾状。较后期的精细胞多呈凸透镜状。生殖细胞分裂形成的一对精细胞多是同型的,也有的表现为异型。精细胞的核多偏于细胞的一端。在细胞质中有大量细胞器ＤＮＡ分布。精细胞中的细胞器ＤＮＡ 荧光点在大小及荧光强度上有所不同,可能代表线粒体和质体这两种不同的细胞器ＤＮＡ。大花牵牛与圆叶牵牛之间,无论是生殖细胞或精细胞在细胞形状和细胞质ＤＮＡ 分布状况上基本相似。一个明显的差异是,后者的细胞质ＤＮＡ 荧光点体积较小和荧光弱。研究表明,精细胞存在具有ＤＮＡ 的细胞器,为牵牛花细胞质具有双亲遗传或父系遗传的潜能提供了细胞学证据。本文还对研究的两个种的精细胞存在同型和异型的现象,以及精细胞在核质比率上的特点与质体双亲遗传的关系进行了讨论     

细胞质遗传并非都是母系遗传

细胞质遗传一般表现为具母系遗传的特征。随着ＤＮＡ分子标记技术的发展和应用,人们已发现在动物及植物中均存在有低频的线粒体ＤＮＡ单亲父系遗传及双亲遗传的现象,对质体ＤＮＡ遗传的研究表明,被子植物的质体ＤＮＡ大多表现为母系遗传。而裸子植物的质体ＤＮＡ则主要表现为父系遗传的方式,同时也出现存在其它的遗传规律。     

牵牛属质体DNA的父系遗传

利用限制性片段长度多态性（ＲＦＬＰ）技术研究了牵牛属（Ｐｈａｒｂｉｔｉｓ）植物质体ＤＮＡ 的遗传方式。结果表明,在牵牛（Ｐ． ｎｉｌ）×大花牵牛（Ｐ． ｌｉｍｂａｔａ）和大花牵牛×牵牛中,质体ＤＮＡ 为父系遗传。在大花牵牛×牵牛中,质体ＤＮＡ还有可能为双亲遗传。研究证明牵牛属为被子植物中具有质体父系遗传方式的第三个属。牵牛属质体父系遗传机制尚不清楚,作者认为母系质体及其ＤＮＡ 在受精后的释稀、排除和／或降解可能是质体父系遗传的基础     

牵牛属细胞质遗传的细胞学研究

牵牛属细胞质遗传的细胞学研究胡赞民，胡适宜（北京大学生命科学学院、北京１００８７）牵牛花质体遗传是属于单亲母系还是双亲遗传目前存在细胞学与遗传学证据的矛盾。为了弄清这一属（ｐｈａｒｂｉｔｉｓ）植物细胞质遗传的方式，本研究用此属的两个种，大花牵牛（ｐ．...     

王百合及兰州百合细胞质遗传的细胞学研究

描述了王百合（ＬｉｌｉｕｍｒｅｇａｌｅＷｉｌｓ．）及兰州百合（Ｌ．ｄａｖｉｄｉＤｕｃｈ．）质体和线粒体在生殖细胞及精细胞中的分布和细胞质中ＤＮＡ的状况。在刚形成的王百合的生殖细胞中含有少量的质体和大量的线粒体。当生殖细胞游离在营养细胞质中时,质体在生殖细胞中完全退化消失。ＤＡＰＩ荧光技术进一步证明,在成熟花粉、花粉管中的生殖细胞及其分裂形成的两个精细胞中无任何细胞器ＤＮＡ。兰州百合在小孢子分裂时质体严格地极性分布,造成了刚形成的生殖细胞中即无质体。兰州百合、麝香百合（Ｌ．ｌｏｎｇｉｆｌｏｒｕｍＴｈｕｎｂ．）及其杂种的ＲＦＬＰ分析,也证明兰州百合质体是单亲母系传递的。虽然在不同发育时期的生殖细胞及精细胞中可以观察到线粒体,但在雄配子体时期它们的ＤＮＡ已降解,因此雄性线粒体不能被传递至后代。研究结果提供了百合属的这两个种质体和线粒体具母系遗传方式的细胞学证据,并阐明父系细胞质不能作为遗传传递的机理。     

被子植物质体遗传的细胞学研究

植物细胞质遗传涉及细胞质中含DNA的两种细胞器——质体和线粒体从亲代至子代的传递。相对来说线粒体遗传的研究远不及质体的多,这可能是线粒体这种细胞器缺乏合适的表型突变体之故。高等植物质体遗传的研究历史可追溯到本世纪初在杂交试验中对叶色遗传的非孟德尔定律的发现,Baur在马蹄纹天竺葵(Pelargonium zonale)中从叶色突变体(白化体)的杂交遗传分析,发现了双亲质体遗传;而Correns在紫茉莉(Mirabilis jalapa)中则发现了单亲母本质体遗传(见Kuroiwa)。此后,对质体基因组突变性状遗传分析的研究,大量的资料说明了在被子植物中存在双亲质体遗传和单亲母系质体遗传两种类型,而后一种占大多数,仅少数是比较有规律的为双亲质体遗传或偶尔是双亲质体遗传。几十年来应用遗传分析的方法对被子植物质体遗传的研究,着重于揭示不同植物种质体的遗传是单亲母系或是双亲质体传递,以及探索杂种核基因对质体传递方式的影响。     

豌豆生殖细胞的发育—着重论述质体母系遗传的细胞学基础

超微结构的研究证明,豌豆（Ｐｉｓｕｍ ｓａｔｉｖｕｍ Ｌ．）生殖细胞自形成直至成熟花粉时期,始终存在少量质体和较多的线粒体。ＤＮＡ 荧光的观察表明,在发育早期的生殖细胞中不含细胞质ＤＮＡ 类核,但在成熟花粉的生殖细胞中有许多的类核。在花粉离体萌发过程中,随着花粉管的生长,生殖细胞中的类核逐渐降解。在花粉培养２４ ｈ 后,生殖细胞的类核全部消失。研究结果确定了豌豆质体母系遗传的细胞学基础,支持遗传分析及ＲＦＬＰ研究的结论,阐明了过去在细胞学上认为是双亲遗传的判断不正确的原因     

杜鹃精细胞的超微结构及DNA荧光观察——着重阐明质体双亲遗传的细胞学基础

迎红杜鹃 ( Rhododendron mucronulatum Turcz.)的成熟花粉为二细胞型 ,精细胞在花粉管中形成。花粉管中的两个精细胞及与营养核之间互相联结 ,形成雄性生殖单位。两个精细胞的细胞质中均含有丰富的细胞器 ,包括质体、线粒体、小泡及微管 ,内质网和高尔基体稀少。具正常结构的精细胞质体在切面上多呈环形或哑铃形 ,内膜不发达 ,基质电子密度高。线粒体为球形或棒状 ,基质电子密度较低。 DNA特异性荧光染色显示 ,生殖细胞及精细胞中均含有大量类核 ( nucleoid) ,两个精细胞中的类核数量无明显差异。结果证明了杜鹃精细胞中存在大量具 DNA的可遗传细胞器 ,为杜鹃属植物的双亲细胞质遗传方式提供了细胞学证据。     

菜豆花粉发育中质体和线粒体及其DNA存在的状况——着重阐明质体遗传的细胞学基础

应用电镜和DNA的DAPI荧光检测技术研究了菜豆(Phaseolus vulgaris L.)小孢子/花粉发育中质体和线粒体及其DNA存在的状况。观察表明:在小孢子分裂时质体全部分配到营养细胞中,初形成的生殖细胞已不含质体。线粒体和质体的DNA在花粉发育中也先后降解,生殖细胞从刚形成时发育至成熟花粉时期这两种细胞器DNA均不存在。研究结果为菜豆质体母系遗传提供了确切的细胞学证据。遗传分析的研究曾确定菜豆质体为双亲遗传,对与本研究结论不同的原因进行了讨论。     

Cytoplasmic Nucleoids in the Generative Cell,Sperm Cell and Egg Cell of Calystegia heder acea

Cytoplasmic nucleoids in the generative cell of mature pollens, sperm cells of pollens cultured in vitro and egg cell of mature embryo sac in Calystegia bederacea Wall. were studied by means of the DNA fluorochrome DAPI in conjunction with epitluorescence microscopy for in situ detection of cytoplasmic DNA in cells. Results showed that many cytoplasmic DNA nucleoids were present in the generative cell and speim cells. Two types of nucleoids were observed, one with big and strong fluorescent dots, and the other with small and weak fluorescence. Many dot-shaped and a few circle-shaped nucleoids were randomly distributed in the thin layered cytoplasm of the egg cell. It was suggested that different types of nucleoids might represent plastid DNA and mitochondrion DNA respectively. Results provided cytological data that Calystegia hederaeea had the potential of plastid DNA biparental inheritance, and the mode of which merits further study via molecular biological methods.     

Cytoplasmic Inheritance of Sweet Potato: with Respect to the Study of Plastids and Mitochondria and the Existence of Their DNA in Sperm Cells

The mature pollen of sweet potato ( Ipomoea batatas lam. ) was bicellular. After pollination generative cell divided into a pair of sperm cells before its germination. The pair of sperm cells remained in the hydrated pollen was similar in their shape and volume with enriched cytoplasmic plastids and mitochondria. The specific fluorescence of cytoplasm DNA indicated that the sperm cells and the generative cell contained numerous organelle nucleoids. The pair of sperm cells had no significant difference in their numbers of organelle nucleoids. Two kinds of organelle nucleoids existed in the pair of sperm cells. Tile ones as big and strong fluorescent dots appeared to be the plastid nucleoids and the others as tile small and weak fluorescent dots could be the mitochondrial nucleoid. Few of the angiosperms were of biparental or paternal plastid inheritance. The result of this study has provided the cytological evidence for another genus, Ipomoea, which is of biparental or paternal plastid inheritance besides Pharbitis and Calystegia in Convolvulaceae.     

Study on Organelle DNA Within the Generative Cell and Sperm Cells in Pharbitis

The organelle DNA in generative cell and its behavior during spermatogenesis in Pharbitis limbata and P. purpurea were observed by epifluorescence microscopy stained with 4',-6-diamidino-2-phenylindole (DAPI). In these two species, the generative cell is long and thin in which a great amount of cytoplasmic DNA is present. Most pairs of sperm cells are isomorphic, in which one end is obtuse and the other is elongate, but in a few pairs dimorphi sperms are present. The nucleus is located at one end of the cell. A lot of cytoplasmic DNA are distributed randomly throughout the cytoplasm. The size of organelle nucleoids and their fluorescence intensity are different in a sperm cell. The features of generative cell and sperm cell, and behavior of cytoplasmic DNA are similar in P. limbata and P. purpurea. The obvious differences between them are that the size and fluorescence intensity of organelle nucleoids in P. purpurea are respectively smaller and weaker than in P. limbata. The results showed that morning glory has potential of biparental or paternal cytoplasmic in heritance. Isomorphism and dimorphism of sperms, and the relationship between the ratio of nucleus and cytoplasm in sperm cell and the plastid biparental inheritance are discussed.     

Studies of Generative Cell Development in Pisum sativum--With Special Reference to Cytological Basis of Plastid Maternal Inheritance

It was proved by ultrastructural observations that few plastids and abundent mitochondria were ever present in the generative cell of Pisurn sativurn L. from its initiation to maturation. Fluorescence observations of DNA showed that many cytoplasmic DNA nucleoids were present in generative cell of mature pollen, but none in the early developing generative cell. During the germination of mature pollen in vitro, the cytoplasmic DNA nucleoids of the generative cell in the pollen tube degenerated gradually following the growth of the pollen tube and disappered completely 24 h after germination. The results provided a cytological basis for confirming the conclusion of plastid maternal inheritance in P. sativurn obtained from genetic study, and resolved the contradiction between results from cytological observation and genetic or RFLP analysis.     

Cytoplasmic DNA apportionment and plastid differentiation during male gametophyte development in Pelargonium zonale

In the male gametophyte of Pelargonium zonale, generative and sperm cells contain cytoplasmic DNA in high density compared to vegetative cells. Cytoplasmic DNA was examined using the DNA fluorochrome DAPI (4'6-diamidino-2-phenylindole) and observed with epifluorescence and electron microscopy. The microspore cell contains a prominent central vacuole before mitosis; mitochondria and plastids are randomly distributed throughout the cytoplasm. Following the first pollen grain mitosis, neither the vegetative cell nor the early generative cell display a distributional difference in cytoplasmic DNA, nor is there in organelle content at this stage. During the maturation of the male gametophyte, however, a significant discrepancy in plastid abundance develops. Plastids in the generative cell return to proplastids and do not contain large starch grains, while those in the vegetative cell develop starch grains and differentiate into large amyloplasts. Plastid nucleoids in generative and sperm cells in a mature male gametophyte are easily discriminated after DAPI staining due to their compactness, while those in vegetative cells stained only weakly. The utility of the hydrophilic, non-autofluorescent resin Technovit 7100 in observing DAPI fluorescence is also demonstrated.     

Heritable paternal cytoplasmic organelles in alfalfa sperm cells: ultrastructural reconstruction and quantitative cytology.

Sperm cells within pollen grains and pollen tubes of alfalfa (Medicago sativa L.) were observed at the ultrastructural level, and their plastid DNA was detected by DAPI (4,6-diamidino-2-phenylindole) staining. One sperm pair within the pollen grain and three sperm pairs within pollen tubes were reconstructed in three-dimensions from serial ultrathin sections. The two sperm cells are linked by cytoplasmic bridges in both pollen grains and tubes, and the vegetative nucleus is closely associated with the sperm cells within the pollen tube. The number of plastids and plastid nucleoids (DNA aggregates) in the sperm cell pair, collectively, is not significantly different from that in the generative cell; however, over 60% of the sperm cell plastids contain no DNA detectable with DAPI. The mean number of mitochondria in sperm cells is reduced from that in the generative cell (from 54 to 17), which suggests that paternal mitochondrial inheritance probably does not occur in the genotype investigated. Sperm cells of a pair may vary in their shape within the pollen grain and tube, but the number of plastids and mitochondria is not significantly different between the sperm cells. Therefore, heterospermy is not a factor determining cytoplasmic inheritance patterns in this species.     

Male gametophyte development inPlumbago zeylanica: cytoplasm localization and cell determination in the early generative cell

Summary The behavior of the generative cell during male gametophyte development inPlumbago zeylanica was examined by epifluorescence microscopy and electron microscopy with organelle nucleoid as a cytoplasm marker. When the thin sections stained with 4,6-diamidino-2-phenylindoIe (DAPI) were observed under an epifluorescence microscope, two types of fluorescence spots were detected in the cytoplasm of the pollen cells before the second mitosis. The spots emitting stronger fluorescence were confirmed as plastid nucleoids and those emitting dimmer fluorescence were mitochondrial nucleoids. Before the first mitosis, both plastid and mitochondrial nucleoids distributed randomly in the cytoplasm of the microspore. A small lenticular generative cell formed with attachment to the interior of the intine after the mitosis. Small vacuoles were found in the lenticular cell. In the cytoplasm of the lenticular cell, both plastid nucleoids and the small vacuoles were distributed randomly at the very beginning but began to migrate in opposite directions immediately. Plastid nucleoids aggregated to the side of the cell that faces the pollen center and the small vacuoles aggregated to the side of the cell that attaches to the inline. As the result, the lenticular generative cell appeared highly polarized in cytoplasm location soon after the first mitosis. In accordance with the definition of the cytoplasm polarization, the primary wall between the generative and the vegetative cells began to flex and the lenticular generative cell started to protrude towards the pollen center. When the generative cell peeled away from the inline, it was spherical in shape with the pole that aggregated plastids towards the vegetative nucleus. But the cell direction appeared to be transformed immediately. The pole that aggregated small vacuoles turned to the position towards the vegetative nucleus and the pole that aggregated plastid nucleoids turned to the position countering to the vegetative nucleus. A cellular protuberance formed at the edge of the pole that aggregated small vacuoles and elongated into a tapered end that got into contact with the vegetative nucleus. The polarization of the cytoplasm kept constant throughout the second mitosis. The small vacuoles that apportioned to the sperm cell which attached the vegetative nucleus (the leading sperm cell) disappeared during sperm cell maturation. Plastid nucleoids were apportioned to the other sperm cell (the trailing sperm cell) completely. Mitochondrial nucleoids became undetectable after the second mitosis.     

玉竹雄配子的发育——着重阐明质体在生殖细胞和营养细胞中的分布和变化

玉竹(Polygonatum simizui Kitag)小孢子在分裂前,质体极性分布导致分裂后形成的生殖细胞不含质体,而营养细胞包含了小孢子中全部的质体。生殖细胞发育至成熟花粉时期,及在花粉管中分裂形成的两个精细胞中始终不含质体。虽然生殖细胞和精细胞中都存在线粒体,但细胞质中无DNA类核。玉竹雄性质体的遗传为单亲母本型。在雄配子体发育过程中,营养细胞中的质体发生明显的变化。在早期的营养细胞质中,造粉质体增殖和活跃地合成淀粉。后期,脂体增加而造粉质体消失。接近成熟时花粉富含油滴。对百合科的不同属植物质体被排除的机理及花粉中贮藏的淀粉与脂体的转变进行了讨论。     

Changes of Plastids Mitochondria and Their DNA in the Egg Cell Before and After Fertilization in Pharbitis

The ultrastructure and cytoplasmic DNA in the egg cell and zygote of Pharbitis purpurea, (L.) Voyght and P. limbata Lindl. which were studied with electron microscopy and DNA epifiuorescence microscopy. The egg cell before fertilization was highly vacuolated with only a few cytoplasmic plastids and mitochondria. Plastids were spherical and/or rod- shaped containing 1 ～ 2 large starch grains. Most of the mitochondria were cup and/or circular. The cytoplasm in the zygote was much more abundant than that in the egg cell. The number of plastids and their electronic density were greatly increased, in most of which containing osmiophilic bodies. The mitochondria were rich and spherical-shaped in the zygote. Two types of cytoplasmic DNA nucleoids were detected in the egg cell, the more abundant one being big and circle-shaped and the other dot-shaped. Only dot-shaped nucleoids were present in the zygote. The content of nucleoids in the zygote was much less than that in the egg cell. Authors propose that some cytoplasmic DNA may degenerate after fertilization. The ultrastructural characteristics of the egg cell and the reduction of cytoplasmic DNA in the zygote may related to the mechanisms of plastid unipaternal inheritance in Pharbitis.