Isolation of Viable Embryo Sacs and Their Protoplasts of Nicotiana tabacum

Isolation of fixed and fresh embryo sacs has been reported. However,the isolation of protoplasts of embryo sac elements is reported here for the first time.The protoplasts of egg cell, synergids, central cell and antipodal cells have been isolated with the retaining of their viability. Though this is a preliminary work, it indicatesthe potentiality of isolation of naked female gametes of angiosperms, which may beused in genetic manipulation and plant biotechnology. Nicotiana tabacum was grown in the greenhouse of the Department of Biology,Peking University. From opened and unpollinated flowers, the ovaries were removedand sterilized with 70% alcohol. The ovules were dissected out from those ovaries andfollowed by incubation (4–8 hrs. 28℃) in anenzyme solution containing 2% driselase, 0.65 M mannitol and 0.25% potassium dextran sulfate. Ovules from 3 4 ovariescould be incubated with 1 ml of enzyme solution in a 3 cm petri dish. All these manipulations and the following procedures were carried out under sterile conditions. Afterincubation, ovules were washed 3 times with a washing solution of 0.65 M mannitol.The isolated embryo, sacs and their protoplasts were obtained by gently squashing digested ovules in a small volume of washing solution on a slide. When the fresh ovules were incubated 3–3.5 hrs in the enzyme solution, the embryosacs may be successfully isolated in an intact manner, either for mature or immatureembryo sacs. The isolated embryo sac looked plump, viable and very distinct in itsstructure. If the isolated embryo sacs were incubated in 0.01% fluorescein diacetate(FDA) used as a test for the viability of the embryo sac, and observed under fluorescein microscope, the cytoplasm of all embryo sac elements, including egg cell, synergids,central cell and antipodal cells, showed strong fluorescence. It is proved that these iso-lated embryo sacs are still viable. When the incubation of ovules was prolonged as to 8 hrs in certain cases, theboundary wall of the embryo sac may be partially digested and the protoplasts of embryo sac elements came out from micropylar or chalazal end after squashing. The difference of the protoplasts derived from different embryo sac elements could be recognized by their relative size and other characteristics. The egg protoplast is smallerthan that of the synergid. However, the protoplasts of antipodal cells were. obviouslysmaller than that of egg. But the central cell protoplast was the largest among theseprotoplasts and possessed two polar nuclei and a very large central vacuole. All theseisolated protoplasts of embryo sac elements were also proved viable with FDA method. The importance of isolated protoplasts of embryo sac elements is discussed withrespect to genetic manipulations.     

Observations on enzymatically isolated,living and fixed embryo sacs in several angiosperm species

A technique has been developed for isolating embryo sacs (ESs) by enzymatic maceration. Ovules were macerated in a mixture of pectinase, cellulase and, in some cases, snailase and pectolyase Y-23. The ovular tissues were removed and the ESs were isolated in toto. Embryo sacs were isolated from both fixed and fresh ovules of Antirrhinum majus L., Helianthus annuus L. and Nicotiana tabacum L. Fluorochromasia by fluorescein diacetate showed that the ESs isolated from fresh ovules were viable. The method has promise for various histochemical and cell-physiological studies and quite possibly also for in-vitro culture of ESs.Abbreviations ES embryo sac - FDA fluorescein diacetate - FPA formalin-propionic acid 50% alcohol (5:5:10, by vol.) - H33258 Hoechst 33258     

Isolation of Embryo Sac with the Enzyme-Squash Technique

The enzyme-squash technique is especially suited for studying the development of megaspores and embryo sacs in angiosperms. Ovulles are fixed in FPA, FAA or Carnoy's for 2–24 hrs. After passing through a series of alcohol and distilled water, they are treated in an aqueous solution of 2% Driselase. for 3-6 hrs. at 28℃. Ovules are then transferred with a drop of laeto-phenol-glycerin fluid to a slide, covered with a cover glass. By the aid of gently tapping and pressing the cover glass, as that of ordinary squash method, cells of ovules are separated and the megaspores or embryo sacs are isolated, and then examined with phase contrast optics With this technique we have successfully isolated the megaspores and embryo sacs in different developmental stages from fixed ovules of several plant species, including Atropa belladonha, Vanilla fragrans, Belamcanda chinensis, Platycodon grandifIorus and Oenothera odorata. The rosults of the experiments indicate that this technique for isolation of embryo sac has several advantages it is suitable both in tenuinucellate and crassinucellate ovules the manipulation of this technique is rather simple and the special instruments are net required the difficulties of the separation of the embryo sac from its nucellar epidermis or tissues of the chalazal end can be overcome by this method. The initial results of isolation of vital embryo sacs from living ovules has been gotten with this technique.     

The possibility of obtaining seeds following placental pollinationin vitro

The paper aims at verifying the possibility of obtaining seeds following placental pollination in more than 20 species of dicotyledons and monocotyledons. Excised placentae with ovules were cultivated in solid nutrient media in Petri dishes. Pollen was transferred from anthers onto placentae, its germinating capacity being enhanced by a drop of a 5 per cent sucrose in boric acid in certain cases. Fertilization of ovules resulting in the formation of viable seeds was obtained withNicotiana tabacum, Melandrium album andAgrostemma githago. Although withPaeonia officinalis, Viola tricolor, Antirrhinum majus, Galanthus nivalis, Narcissus pseudonarcissus, Clivia miniata andTulipa gesneriana normal seed set occurred, the seeds did not germinate even after being stimulated with GA₃. The results indicate that the findings may be used for further embryological studies. The species are listed in which no seeds were obtained.     

金鱼草珠被绒毡层壁囊的分离与鉴定

由金鱼(Antirrhinum majus L.)草开花后的胚珠中分离出抗乙酰解、抗酶解、对强氧化剂有一定抗性但不抗二乙醇胺的细胞壁囊状结构。切片原位观察表明,它是珠被绒毡层的内壁,能被苏丹 IV 染色与金胺 O 荧光染色。它全面地包围胚囊内正在发育的胚和胚乳,仅在合点与珠孔两端开口,而在此二处分别有胚乳吸器与胚柄与外相通。这一结构的功能可能是保证营养物质定向导入胚囊以及防止珠被绒毡层分泌的水解酶侵蚀胚囊等。     

Observations on Megasporogenesis and Megagametophyte Development in Paulownia sp and Sesamum indicum by Enzymatic Maceration Technique

A technique has been recently developed in our lab to isolate embryo sacs by means of enzymatic maceration of ovules (Zhou and Yang, 1982). In the pre sent paper this method was adopted in observing the whole processes of mtgasporgcnsis and megagametophyte development in Pauiownia sp. and Sesamum indicum. FPA fixed ovules were macerated in peetinase-eellulase solution with a microshaker, eleared in lactophenol, and then observed under a microscope with Nomarski interference contrast or phase contrast equipments. In both species, various developmental stages, from megasporocyte till mature embryo sac, were successfully identificd and described (Plate Ⅰ and Ⅱ). As a kind of microtechnique, enzymatic method shows some merits as its rapidness in specimen preparation and convenience for obtaining whole structural image Several technical points are discussed hereof.     

Identification of genes expressed in the Arabidopsis female gametophyte

The angiosperm female gametophyte typically consists of one egg cell, two synergid cells, one central cell, and three antipodal cells. Each of these four cell types has unique structural features and performs unique functions that are essential for the reproductive process. The gene regulatory networks conferring these four phenotypic states are largely uncharacterized. As a first step towards dissecting the gene regulatory networks of the female gametophyte, we have identified a large collection of genes expressed in specific cells of the Arabidopsis thaliana female gametophyte. We identified these genes using a differential expression screen based on reduced expression in determinant infertile1 (dif1) ovules, which lack female gametophytes. We hybridized ovule RNA probes with Affymetrix ATH1 genome arrays and validated the identified genes using real-time RT-PCR. These assays identified 71 genes exhibiting reduced expression in dif1 ovules. We further validated 45 of these genes using promoter::GFP fusions and 43 were expressed in the female gametophyte. In the context of the ovule, 11 genes were expressed exclusively in the antipodal cells, 11 genes were expressed exclusively or predominantly in the central cell, 17 genes were expressed exclusively or predominantly in the synergid cells, one gene was expressed exclusively in the egg cell, and three genes were expressed strongly in multiple cells of the female gametophyte. These genes provide insights into the molecular processes functioning in the female gametophyte and can be used as starting points to dissect the gene regulatory networks functioning during differentiation of the four female gametophyte cell types.     

The Ultracytochemical Localization of ATPase Activity in the Ovules of Antirrhinum majus L.

The ultracytochemical localization of ATPase activity was carried out by the method of lead precipitation in the ovules of Antirrhinum majus L. No ATPase activity is observed in the egg apparatus, but some in the polar nuclei, cytoplasm and plasma membrane of the central cell. Between the embryo sac wall and the cuticle surrounding it, there is a gap where some filamentand vesicle-like structures were demonstrated by conventional staining method, and much of ATPase activity is found there. At the chalaza of the ovule, a lot of ATPase particles are found irt the nuclei, plasma membranes and the thick and loose wall of the hypostase cells. The particles of ATPase in the hypostase and those in the gap surrounding embryo sac are continuously distributed through the intervals of the cuticle at the chalazal end of the embryo sac. Some of ATPase particles are found on the plasma membranes and plasmadesmata of integument ceils, noticeably much more in the nucleoplasm of the integumentary tapetum. According to the ATPase distribution pattern in the ovules, we suggest that the function of the integumentary tapetum and hypostase is secretion, and that the gap surrounding the embryo sac may be an apoplastic ehannal for nutrient flow into the embryo sac.     

Development of the male and female gametophyte in Capsicum annuum L. var. California Wonder

The study of reproductive organs and their developmental stages during sporogenesis and gametogenesis is an important step for basic and applied sciences. In this order and to develop knowledge about Capsicum annuum L. (Solanaceae), flower buds in different developmental stages were investigated by light and scanning electron microscopy. The bell pepper bisexual flowers have homoantherous stamens with tetrasporangiate anthers. The pattern of anther wall formation is basic type, while previously described as dicotyledonous type. Microspore mother cells meiosis has simultaneous cytokinesis and tetrads are tetrahedral. Two-celled mature pollen grains are tricolporate with scabrate ornamentation. The gynoecium is bicarpellate and epigynous, with hemianatropous, unitegmic and tenuinucellate ovules. After megaspore mother cell meiosis, cytokinesis is simultaneous. The chalazal megaspore of the linear tetrad functions as the functional megaspore. Embryo sac development is of monosporic Polygonum type and consists of seven cells. We give the first reproductive calendar for the species that allows prognostication of gametogenesis on the basis of sporophytic parameters related to reproductive organ development and floral bud form and size. Some of these developmental characteristics should be useful for comparative studies and investigation of phylogenetic relationships within Solanaceae family.     

酶法分离胚囊的荧光显微观察

用荧光显微技术对酶法分离的胚囊进行了三方面的研究:(1)利用与DNA结合的荧光染料Hoechst 33258对金鱼草、蚕豆、芝麻、烟草、龙葵等植物的分离胚囊??染色,可以观察胚囊中各组成细胞的核以及合子和胚乳的核。(2)用金胺O和樱草黄两种荧光染料染色,证明胚囊壁中含有耐果胶酶与纤维素酶作用的成分,可能是角质,因而虽经上述酶处理仍能保持胚囊的整体性。(3)用脱色苯胺蓝染色观察了被果胶酶分离的泡桐大孢子发生的各期材料,对这一过程中胼胝质的动态作了描述。以上各项实验表明:酶法分离技术与荧光显微技术相结合,是研究胚囊细胞生物学的一种简便有效的方法。     

雌、雄配子体间及雌配子体成员细胞间的通讯

被子植物的双受精是一个复杂而精密的调控过程.成功的受精依赖于配子体的正确发育以及雌配子体与雄配子体间的相互识别.研究表明,雌配子体自身成员细胞间存在广泛的胞间通讯.这种通讯不仅影响不同细胞的发育进程,也决定细胞的发育命运,从而保证雌配子体的正常发育.此外,雌配子体与雄配子体间存在胞间通讯,这种胞间通讯是雌配子体与雄配子体间相互识别的分子基础,精确调控了雄配子体准确进入珠孔、在雌配子体内适时停止伸长、尖端破裂并在特定位置释放精细胞等过程.本文概述了这些方面的最新进展,梳理胞间通讯的途径与信号,并展望了未来雌、雄配子体间及雌配子体成员细胞间通讯的研究方向与可能的应用前景.     

Isolation of embryo sacs from Dianthus ovules by enzymatic treatments and microdissection

 Mature ovules of Dianthus (Caryophyllaceae) were histologically observed by clearing and serial sectioning to characterize the cells of the embryo sac. The results show that the mature embryo sac was located deep inside the hemitropous ovule due to thick nucellar tissue at the micropylar region. For the isolation of the embryo sacs, ovules were collected from ovaries of flowers 1 day after anthesis, and treated with an enzyme solution for digesting cell walls on a gyratory shaker. After 12 h of enzyme treatment, these ovules were dissected using a glass needle under an inverted microscope to release the embryo sacs. The embryo sacs, characterized by their specific size, were successfully released by these successive treatments. The viability of the embryo sacs was more than 80% as assessed with fluorescein diacetate staining. Fluorescent staining with 4,6-diamidino-2-phenylindole revealed the nuclei of the egg apparatus in the isolated embryo sacs. The procedure for isolating embryo sacs established in this study will offer a new approach to further in vitro studies on fertilization in Dianthus. Received: 20 January 1999 / Revision received: 12 July 1999 / Accepted: 17 August 1999     

Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.)

Summary Isolation of viable egg cells of perennial ryegrass (Lolium perenne L.) has been accomplished. After an enzyme incubation, ovules disintegrated into loose cells upon mechanical manipulation. The egg cells could be identified between the bulk of sporophytic cells derived from the macerated ovules. The morphology of the isolated egg cell corresponds to the morphology of the egg cell in situ and is comparable to the morphology of egg cells of other monocotyledons and angiosperms. Two hours after isolation the egg cells were still viable. The protocol proved reproducible and the yield was determined at 10%.     

荔枝胚胎败育与酚类抑制物质的关系

在荔枝 (LitchichinensisSonn .)胚胎败育发生期 ,以系统溶剂法从正常或败育胚珠中初步提取酚类抑制物质 ,通过TLC分离与纯化 ,用GC MS联用仪进一步分离鉴定 ,并以标准品核对。试验首次从荔枝胚珠中分离鉴定出酚类抑制物质对羟基苯甲酸 (p_HBA)。生物活性测定表明 ,p_HBA是一种很强的生长抑制物质。在败育胚珠中其含量及IAA氧化酶活性均显著高于正常胚珠 ,IAA水平则明显低于正常胚珠 (P <0 .0 1)。因此认为 ,p_HBA参与了荔枝胚胎发育的调节 ,高含量的p_HBA是通过促进IAA侧链的氧化并影响促进和抑制生长的物质之间的平衡而导致荔枝胚胎的败育     

A New Method for Embryo Sac Isolation and In Situ Fusion of Egg and Synergid Protoplasts in Nicotiana tabacurn

A new method combining enzymatic maceration with osmotic shock was developed for isolation of living embryo sac and its protoplasts in Nicotiana tabacum L. The principle of this method was that the ovules submitted to enzymatic treatment and osmotic shock could release embryo sacs along with some internal ovular cells through either the funicle cut end or the micropyle. Factors affecting embryo sac isolation were investigated, including concentration of mannitol as a shock osmoticum and in enzymesolution ,duration of enzymatic maceration,and duration of osmotic shock. As a result a procedure was established: Ovules at mature embryo sac stage were macerated for 2. S h in 1 %–1.5% cellulase R-10 and 0. 5% macerozyme R-10 (or 1% Pectinase,Serva) dissolved in 13% mannitol solution using microshaker,followed by osmotic shock for 15–30 min with enzyme free 8% mannitol solution and gentle agitation using a pipette. Using a capillary,50–70 embryo sacs could be collected manually in one hour. The embryo sacs thus isolated could be kept viable from which protoplasts of egg cell and other componcnt cells could be further isolated. An additional interesting phenomenon was that osmotic shock often caused in situ fusion the protoplasts of egg cell and synergids. The rate of fusion ranging 9%—71.9% could be controlled by modification of the procedure. This phenomenon merits further attention both from basic and practical point of view. The present method gives the advantages of faciliting isolation and promoting good harvest of viable embryo sacs/female protoplasts within a relative short time.     

Megagametogenesis in Halophila johnsonii, a threatened seagrass with no known seeds,and the seed-producing Halophila decipiens (Hydrocharitaceae)

Megagametogenesis, the development of a megaspore into an embryo sac, has been identified in the seagrass Halophila johnsonii, a threatened species with no known sexual reproduction or seeds. Megagametogenesis in H. johnsonii was compared with megagametophyte development in Halophila decipiens, a related species known to readily produce viable seeds. In both species, ovules were structurally similar, megaspore mother cells were seen in premeiotic ovules, and linear tetrads and megagametophytes with two to eight nuclei were present in postmeiotic ovules. However, H. decipiens postmeiotic ovules had a chalazal pouch that was absent in the postmeiotic ovules of H. johnsonii. Late-stage H. decipiens ovules also contained embryos, indicating that they had been fertilized, whereas all late-stage H. johnsonii ovules were degrading and showed no signs of fertilization. These observations suggest that meiosis does occur in H. johnsonii megasporocytes, leading to the formation of viable megagametophytes and egg cells that could be fertilized if pollination occurred. Thus, the lack of seed set is due to a lack of pollination rather than any loss of capacity to produce seeds in this species.     

The immunolocalization of plasma membrane H+-ATPase in the transfer cell region of Brassica napus (Brassicaceae) ovules

Unfertilized mature ovules of Brassica L. contain an abundance of starch in the integument cells from the micropyle to a plane approximately at the level of the central cell polar nuclei. Inside the embryo sac central cell, in the coinciding region, there are transfer cell-like wall projections with plasma membranes appressed to their inner surfaces. H⁺-ATPase is present along the inner surfaces of the wall projections as indicated by reactivity with antibody raised against plasma membrane H⁺-ATPase. A number of mitochondria are in close association with wall projections in the region of the egg apparatus. Antibody raised against corn plasma membrane H⁺-ATPase cross reacts with a protein of the same size in extracts of Brassica napus indicating that the two species contain a similar plasma membrane H⁺-ATPase.     

Improved ultrastructural preservation ofPetunia andBrassica ovules and embryo sacs by high pressure freezing and freeze substitution

Summary In order to improve the ultrastructural preservation of the female gametophyte ofPetunia x hybrida andBrassica napus we tested several cryofixation techniques and compared the results with those of conventional chemical fixation methods. Ovules fixed with glutaraldehyde and osmium tetroxide in the presence or absence of potassium ferrocyanide showed poor cell morphological and ultrastructural preservation. In ovules cryo-fixed by plunging into liquid propane, the cell morphology was well preserved. However, at the ultrastructural level structure-distorting ice crystals were detected in all tissues. Due to the large size of the ovules, cryofixation by plunging in liquid propane is not adequate for ultrastructural studies. In contrast,P. x hybrida andB. napus ovules cryo-fixed by high pressure freezing showed improved cell morphological as well as ultrastructural preservation of the embryo sac and the surrounding integumentary tissues. The contrast of the cellular membranes after freeze substitution with 2% osmium tetroxide and 0.1% uranyl acetate in dry acetone was high. At the ultrastructural level, the most prominent improvements were: straight plasma membranes which were appressed to the cell walls; turgid appearing organelles with smooth surface contours; minimal extraction of cytoplasmic and extracellular substances. In contrast to the chemically fixed ovules, in high pressure frozen ovules numerous microtubules and multivesicular bodies could be distinguished.     

Variation in the costs and benefits of mutualism: the interaction between yuccas and yucca moths

Summary Yucca moths are both obligate pollinators and obligate seed predators of yuccas. I measured the costs and net benefits per fruit arising for eight species of yuccas from their interaction with the yucca moth Tegeticula yuccasella. Yucca moths decrease the production of viable seeds as a result of oviposition by adults and feeding by larvae. Oviposition through the ovary wall caused 2.3–28.6% of ovules per locule to fail to develop, leaving fruit with constrictions, and overall, 0.6–6.6% of ovules per fruit were lost to oviposition by yucca moths. Individual yucca moth larvae ate 18.0–43.6% of the ovules in a locule. However, because of the number of larvae per fruit and the proportion of viable seeds, yucca moth larvae consumed only 0.0–13.6% of potentially viable ovules per fruit. Given both oviposition and feeding effects, yucca moths decreased viable seed production by 0.6–19.5%. The ratio of costs to (gross) benefits varied from 0% to 30%, indicating that up to 30% of the benefits available to yuccas are subsequently lost to yucca moths. The costs are both lower and more variable than in a similar pollinator-seed predator mutualism involving figs and fig wasps.There were differences between species of yuccas in the costs of associating with yucca moths. Yuccas with baccate fruit experienced lower costs than species with capsular fruit. There were also differences in costs between populations within species and high variation in costs between fruit within populations. High variability was the result of no yucca moth larvae being present in over 50% of the fruit in some populations, while other fruit produced up to 24 larvae. I present hypotheses explaining both the absence and high numbers of larvae per fruit.