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.     

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 and preservation of the living embryo sac ofCrinum asiaticum L. var.japonicum baker

The enzymatic maceration method was used to isolate an intact embryo sac ofCrinum asiaticum and its component cells. Best results were obtained when using enzyme solutions that contained pectinase hemicellulase, cellulase and pectolyase. Aseptic ovules were incubated in the enzyme solution for 1.5 hr at 25 C. This allowed the isolation of embryo sacs to yield up to 20% of the amount present. An isolated embryo sac usually consists of an egg cell, synergids, antipodals and a central cell. Some embryo sacs can be digested as gametophytic protoplast. The size, shape and position of the isolated embryo sac seemingly possessed similarities with those of the fixed embryo sac in the ovary. An isolated embryo sac can be in a living state when the result of the fluorochromatic reaction (FCR) and protoplasmic streaming is positive. When cultured in proper media, 68% of the isolated gametophytic protoplasts were observed to have sustained their positive FCR for more than 1 month.     

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.     

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     

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.     

Development of Ovules and Embryo sacs in Ostrya virginiana (Betulaceae) and Its Systematic Significance

The development of ovules and embryo sacs in Ostrya virginiana was studied for the first time. Most ovaries had two ovules which were anatropous, unitegmic and crassinucellate. The ovule usually possessed several archesporial ceils which divided periclinally into the upper parietal cell and the lower sporogenous cell. The sporogenous cell functioned directly as megaspore mother cell. The tetrad of megaspores was linear in arrangement, and every megaspore might be functional. One ovule often contained 2- 6 embryo sacs and the embryo sac belongs to Polygonum type. It can be concluded from the present data that all ovules among the genera of the Betulaceae are unitegrnic. There are more groups with the phenomenon of multiple embryo sacs in anemophic plants such as Betulaceae, Casuarinaceae, Graminae, Jnglandaceae, Myricaceae, Simaroubaceae, Ulmaceae, than in entomophilous plants. Multiple embryo sacs also occur among some parasitic plants and saprophytes, e.g. Orobanchaceae, Cassytha in Lauraceae, Cuscuta in Convolvulaceae and Utricularia in Lentibulariaceae. It may be inferred that the characteristic of multiple embryo sacs be an evolutionary adaptation of those plants with lower pollination rate to increase the rate of fertilization. Finally, a comparison of embryological characters among the genera of the Betulaceae shows that the family is of a number of common embryological characters, such as multicellular archesporium, multiple embryo sacs in one ovule, and a long interval between pollination and fertilization. The diversity and systematic significance of several embryological characters among the “higher” hamamelid families are also discussed. It is still hard to explain the phy-logenetic relationships among those families clearly only with.     

Embryo Sacs of Hevea brasiliensis and Carica papaya Isolated with Enzyme-Squash Technique

The enzyme-squash technique had been employed to study the development of megaspores and embryo sacs from tropical plants of Hevea brasiliensis Muel-Arg. and Carica papaya L. Ovules fixed with FAA or FPA were enzymatieally macerated in 3% pectinase cellulase solution or in 3–4% snailase solution for 4–6 h, at 28–30℃ to isolate the embryo sacs after being squashed. The whole structural images of megaspores and embryo sacs at different developmental stages were observed and identified without staining under Olympus BH-2 phase contrast microscope or light microscope after ron-aceto-carmine staining.     

Isolation of individual egg cells and zygotes in Alstroemeria followed by manual selection with a microcapillary-connected micropump

AIMS: To develop a procedure for isolating living egg cells and zygotes from Alstroemeria ovules. SCOPE: An attempt was made to isolate egg cells and zygotes from the ovules of Alstroemeria aurea. The ovules were histologically observed using a clearing procedure which revealed the localization and sizes of the embryo sacs and egg apparatus within the ovules. For the isolation of egg cells, ovules were cut into sections with a surgical blade and treated with an enzyme solution. Subsequently, these ovule sections were dissected using a glass needle under an inverted microscope. Egg cells successfully isolated by this procedure were collected using microcapillaries connected to a micropump. For zygote isolation, ovules were excised from ovaries 24 h after self-pollination. By treating excised ovules with an enzyme solution and subsequently dissecting them using a glass needle, zygotes were successfully isolated from the ovules and collected with a microcapillary. The isolated zygotes were associated with pollen tubes and one of the synergids. Egg cells and zygotes were viable for up to 2 h following isolation, as determined by fluorescein diacetate staining. CONCLUSIONS: The procedures for isolating egg cells and zygotes in Alstroemeria were established, and each egg cell and zygote was captured with a microcapillary.     

OVULE ABORTION IN QUERCUS (FAGACEAE)

This study deals with the occurrence and relative abundance of four different types of abortive ovules in three species of Quercus. It was found that, contrary to previous literature, fertilization does not always occur in the abortive ovules. The most common type of abortive ovule is the one in which a normal embryo sac develops, yet fertilization does not occur. The absence of embryo sacs and the occurrence of empty embryo sacs account for abortion in other ovules. All types of abortive ovules can occur in the same ovary. It is proposed that all of the ovules that develop a normal embryo sac are potential seeds, but the first one to be fertilized suppresses the normal development of the others.     

Micromanipulation of male and female gametes ofNicotiana tabacum: I. Isolation of gametes

The isolation of male and female gametes is a precondition for the micromanipulation of flowering plant gametes. To reflect their condition at fertilization, isolated gametes need to be physiologically mature and vigorous. Sperm cells are isolated from pollen tubes grown on cut styles using the in vivo/in vitro technique. Embryo sacs are isolated 2 days after anthesis using brief treatments of minimal concentrations of cell-wall-digesting enzymes on ovules of emasculated flowers. Egg cells are then mechanically separated from the embryo sac, allowing unambiguous identification of cells. Two days is usually the minimum required for the pollen tube to penetrate the ovule and effect fertilization in vivo.     

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。     

蓝猪耳卵细胞和合子的分离

蓝猪耳(Torenia fournieri)胚囊部分裸露出胚珠,在光学显微镜下能清楚观察到卵细胞和助细胞的形态结构.用解剖和酶解-解剖两种方法都能分离出生活卵细胞.用前种方法机械分离出的卵细胞数量较少(5%),但避免了酶对配子识别研究的干扰.在后种方法中加入0.1%纤维素酶和0.1%果胶酶既能使分离更加容易操作,又对卵细胞没有致命伤害,能在短时间内分离出较多的卵细胞(18%).用酶解-解剖方法也可分离出授粉14 h后的合子细胞.     

Ovule Development and Determination of Seed Number Per Pod in Oilseed Rape (Brassica napus L.)

Seed number per pod at maturity over the terminal raceme ofsingle plants of oilseed rape is closely correlated to the percentageof ovules with complete embryo sacs (ovule fertility) at floweropening. Approximately one-third of the ovules did not containan embryo sac and sterility, due to the absence of embryo sac,accounted for most of the difference between the numbers ofovules and seeds. Within the terminal raceme, both a decreasedproportion of fertile ovules and a lower number of ovules perovary in apical flowers contributed to the lower number of seedsper pod in the mature apical pods compared to the basal ones.A study of ovule development before flower opening showed thatdifferences in the differentiation of the embryo sacs arosebefore the buds were 40 mm long and probably involved the stagesof meiosis II and/or differentiation of the chalazal megaspore. Key words: Oilseed rape, ovule development, seed number per pod     

Female Gametophyte Development in Maize: Microtubular Organization and Embryo Sac Polarity

The developmental stages of the maize embryo sac were correlated with the corresponding silk lengths of ear florets in the female inflorescence. The development of embryo sacs in the ovules of spikes occurs in a gradient pattern with the initiation of the embryo sac beginning at the base of the ear and progressing to the top. At the beginning of meiosis, the presence of conspicuous cortical microtubules coincides with the extensive elongation of the megasporocyte. The spindles at metaphase I and II align along the long axis of the megasporocyte leading to the linear alignment of the dyad and tetrad of megaspores. During megagametogenesis, micropylar and chalazal nuclei of the embryo sac undergo synchronized divisions and migration at the second and third mitosis. Radiate perinuclear microtubules are present during the interphase of the second and third mitosis, and inter-sister nuclear microtubules occur at the late four-nucleate embryo sac. The configuration and orientation of the spindles, phragmoplasts, and pairs of nuclei result in precise positioning of the nuclei. The fusion of the polar nuclei and the formation of a microtubule organizing center-like structure in the filiform apparatus occur right after the first division of the antipodal cells. The different patterns of organization of microtubules in the cells of the mature embryo sac reflect their structural adaptations for their future function.     

Isolation of egg cells and zygotes of Torenia fournieri L. and determination of their surface charge

Egg cells of Torenia fournieri were isolated from embryo sacs 1 day after anthesis using enzymatic digestion or mechanical dissection. About 5% of the egg cells and zygotes (2-3 from 50 ovules) could be mechanically dissected within 2 h. When 0.1% cellulase and 0.1% pectinase were added to the mannitol isolation solution, about 18% of the egg cells (8-10 from 50 ovules) could be isolated within 2 h. The egg cells isolated by mechanical dissection could be used for in vitro fertilization studies without any of the potentially deleterious effects of the enzymes on the plasma membrane of egg cell. The egg cells isolated using enzymatic digestion could be used in the study of the molecular biology of female gamete because more egg cells could be isolated with this technique. Using enzymatic digestion, over 10 zygotes from 50 ovules (over 20%) were isolated from the pollinated ovules. Coupled with our successful isolation of mature sperm cells, the isolation of egg cells of T. fournieri will make in vitro fertilization possible in a dicotyledon plant.     

Confocal microscopy of whole ovules for analysis of reproductive development: the elongate1 mutant affects meiosis II

Analysis of female meiosis (megasporogenesis) and embryo sac development (megagametogenesis) in angiosperms is technically challenging because the cells are enclosed within the nucellus and ovule tissues of the female flower. This is in contrast to male sporogenesis and gametogenesis where development can readily be observed through the easily dissectable developing anthers. Observation of embryo sac development is a particular problem in crassinucellate ovules such as those of maize. To overcome the problems in observing reproductive development, we developed a simple Feulgen staining procedure optimized for use with confocal microscopy to observe reproductive progression in the crassinucellate ovules of maize. The procedure greatly facilitates the observation of nuclei and cell structures of all stages of megasporogenesis and embryo sac development. The high resolution obtained using the technique enabled us to readily visualize chromosomes from individual cells within ovule tissue samples of maize. A propidium iodide staining technique was also used and compared with the Feulgen-based technique. Static cytometry of relative DNA content of individual nuclei was possible using Imaris software on both Feulgen and propidium iodide-stained samples. The techniques also proved successful for the observation of Arabidopsis and Hieracium aurantiacum female gametophyte and seed development, demonstrating the general applicability of the techniques. Using both staining methods, we analysed the maize meiotic mutant elongate1, which produces functional diploid instead of haploid embryo sacs. The precise defect in meiosis from which diploid embryo sacs arise in elongate1 has not previously been reported. We used confocal microscopy followed by static cytometry using Imaris software to show that the defect by which diploid embryo sacs arise in the maize mutant elongate1 is the absence of meiosis II with one of the dyad cells directly initiating megagametogenesis.     

酶解压片分离沙田柚胚襄与胚胎发育研究

用２．５％纤维素酶与２．５％果胶酶混合液在３０℃下对已固定的沙田柚（Ｃｉｔｒｕｓｇｒａｎｄｉｓｖａｒ．ｓｈａｔｉｎｙｕＨｏｒｔ．）胚乳酶解６—１０小时,分离了开花前、授粉至受精间隔期、合子休眠期、胚和胚乳发育各期的胶囊．用Ｏｌｙｍｐａｓ相差显微装置进行观察与摄影,在胚囊中显示了沙田柚胚胎发育各主要阶段的结构特征。分析了酶解压片技术在柑桔属植物胚胎发育研究的前景     

Enzymatic isolation of living embryo sacs of Petunia

Summary A 20%–25% yield of isolated and living embryo sacs of Petunia hybrida L. was obtained using an enzymatic maceration mixture containing 3% driselase (soluble fraction only), 0.1% MES buffer, pH 5.5, and 8% mannitol. For each maceration ± 450 ovules were incubated in 1 ml enzyme solution for 2 h at 30° C in a shaking waterbath (150 rpm). Subsequently, the enzyme solution was replaced by Brewbaker and Kwack's medium, pH 6.5, supplemented with 10% mannitol (BKM). Gentle agitation of the suspension resulted in the liberation of embryo sacs, which were then collected with a micropipette using a dissecting microscope and transferred to fresh BKM. The embryo sacs isolated are intact and living, and have maintained their original shape and organization When stored in BKM at room temperature the isolated embryo sacs remain alive for 8 h. Storage at 4° C results in a prolongation of viability of up to 80 h. Prolonged incubation of ovules or reincubation of isolated embryo sacs in the maceration mixture results in the liberation of the gametophytic cells as individual, living protoplasts.     

Inorganic salts modify embryo sac development in sexual and aposporous Cenchrus ciliaris

Summary Sexual and aposporously apomictic plants of buffelgrass (Cenchrus ciliaris L.) form megaspore tetrads. In sexual plants the chalazal megaspore develops into a single Polygonum type embryo sac. In aposporous plants the megaspores degenerate, and one or more un-reduced nucellar cells form Panicum type embryo sacs. Apospory is conditioned by gene A; the dominant allele of gene B is epistatic to A and preserves sexual reproduction. We recently observed that heavy application of (NH₄)₂SO₄ to the soil induced multiple embryo sacs in a sexual line. Therefore we tested the effect of salt stress on embryo sac formation in sexual and aposporous genotypes. One molar solutions of CaCl₂, NaCl, (NH₄)₂SO₄, NH₄Cl, NaNO₃, or Na₂SO₄ were applied to the soil of greenhouse plants every day or two starting at the archespore stage. Some of the pistils in salt-treated plants of sexual genotypes AaBb, aaBb, and aabb showed features not seen in untreated controls: (1) multiple Polygonum type embryo sacs in 1%–7% of pistils depending upon the salt; (2) embryo sacs without antipodals (0%–7%); (3) embryo sacs protruding through the micropyle (1%–16%). Some pistils of salt-treated obligately aposporous lines, but not controls, developed Polygonum type embryo sacs (4%–13%) and protruding embryo sacs (0%–6%). There was no ion specificity for induction of abnormal features. We postulate that salt stress suppresses the developmental priority of nucellar embryo sacs over megaspores in aposporous lines and of the chalazal megaspore over other megaspores in all lines. This may permit megaspores of aposporous plants to form reduced Polygonum type gametophytes, and permit more than one megaspore to form reduced embryo sacs in all lines. Protrusion of sacs and failure of antipodal formation in reduced embryo sacs may be the consequence of uncoordinated expansion of the embryo sacs and surrounding tissue.Joint contribution of the Department of Biology, The Pennsylvania State University, and USDA-ARS, U.S. Regional Pasture Research Laboratory. Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA