Enzymatic isolation of viable nucelli at the megaspore mother cell stage and in developing embryo sacs in Nicotiana tabacum

Summary Nucelli and developing embryo sacs were enzymatically isolated from ovules of Nicotiana tabacum. Megaspore mother cells, tetrads, uninucleate, binucleate, four-nucleate, eight-nucleate and mature embryo sacs were obtained. The isolated embryo sacs were intact and living, and maintained their original shape and organization. Cytoplasmic streaming was clearly observed. Prolonged incubation of ovules or reincubation of isolated embryo sacs in the maceration mixture resulted in the liberation of the gametophytic cells as individual, living protoplasts.     

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     

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.     

Isolation and Identification of Viable Embryo Sacs in Several Angiosperm Species

Since the enzymatic technique for isolating embryo sac (ES) has been established on fixed materials of several angiosperms as well as on fresh ovules of Antirrhinum majus in our lab, further works on isolation of viable ESs were carried on. Fresh ovules were macerated in a solution of enzymes, sucrose with or without potassium dextran sulphate by a microshaker at 28–30℃ for several hours. The enzymes included pectinase, cellulase, snailase and pectolyase Y-23, the combination and concentration of which varied with the plant species and the developmental stages of ESs. To date the mature ESs of Helianthus annuus, A. majus and Nicotiana tabacum and the ESs after fertilization with proembryo and endosperm cells in the two former species were well isolated. Nomarski interference contrast and Hoechst 33253 fluorescence microscopical observations revealed that the ESs retained their cell structure and were rich of ergastic substances. Fluorochromasia induced by fluorescein diacetate further proved that they were really viable.     

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.     

Production of seedlings from ovules excised at the zygote stage in Lilium spp.

The present study was undertaken to establish a culture system for ovules excised at the zygote stage in Lilium spp. Ovules of Lilium × `Connecticut King' and L. × `Enchantment' were excised together with placental tissue 3, 5, and 10 days after pollination (DAP) and cultured on B5 medium and half-strength B5 medium containing sucrose at different concentrations. In vitro embryo development in ovules cultured at 3 DAP was influenced by the basal media and the sucrose concentration. The half-strength B5 medium with 9% sucrose was the best condition, but only a few ovules isolated from placental tissue developed into seedlings. Application of embryo culture, in which embryos were excised from ovules after 14 weeks of ovule-with-plancetal-tissue culture, greatly improved the production of seedlings. The present study indicates that a two-step culture procedure, ovule-with-placental-tissue culture and embryo culture, make it possible to produce seedlings from ovules just after fertilization.     

Isolation of protoplasts from female gametophytes of Torenia fournieri

Protoplasts from the cells of mature embryo sacs (ES-protoplasts) of Torenia fournieri were obtained during incubation of ovules in an enzyme solution. Four protoplasts which arose from each embryo sac were connected together after isolation, or aggregates of the egg cell protoplast and two synergide protoplasts dissociated from the protoplast of the central cell. The ES-protoplasts stayed viable for 2 weeks in culture, but they did not regenerate cell walls.Abbreviations ES embryo sac - FAA fixative (formalin : acetic acid : alcohol = 1 : 1 : 18) - FDA fluorescein diacetate - PAS periodic acid Schiff reaction - 2,4-D 2,4-dichlorophenoxyacetic acid     

A study of morphology,cytology and sterility in interspecific hybrids and amphidiploids of Nicotiana knightiana X N. umbratica

Summary Interspecific hybrids and amphidiploids of Nicotiana knightiana Goodspeed (n= 12)x N. umbratica Burbidge (n = 23) resembled either parent in some characters and were intermediate in other characters. The F₁ hybrids (2n = 35) showed mostly univalents during meiosis, while the amphidiploids (2n = 70) formed bivalents almost regularly. The former were completely sterile and the latter fully male fertile but predominantly female sterile. This female sterility was due to disintegration of the embryo sacs leading to collapsed ovules. The few fertile ovules, however, showed normal development of embryo sac and embryo. The occurrence of fertile and sterile ovules was believed to be due to segregation of the genes governing sterility.     

Callose in the walls of mature embryo sac ofTorenia fournieri

Summary During the course of a fluorescence microscopic investigation on the extra-ovular micropylar portion of the embryo sacs ofTorenia fournieri Lind. (Scrophulariaceae) a callosic wall was found which surrounded it almost completely until the time of anthesis. In addition, the walls of young synergids and the filiform apparatus also showed callosic fluorescence. Treatments with PAS reaction revealed a PAS-positive substance filling up the locular cavity. Our attempts to induce fluorochromasia by employing fluorescein diacetate failed, indicating the low permeability of the callosic wall around the embryo sac. It is assumed that the callose wall around the embryo sac isolates the latter from the contents of the locular cavity whereas the callose in the synergid walls may represent an intermediate stage in the maturation of these walls; the filiform apparatus is mainly composed of callose.     

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.     

Embryological Studies on Narcissus tazetta var. chinensis

Chinese narcissus (Narcissus tazetta var. chinensis Roem) blooms but has no seeds. Embryological studies on the species were conducted to discover the causes of its sterility. Its anther wall is composed of four layers of cells, and its tapetum is of the secretory type. The cytokinesis of microspore mother cells is of the successive type, and the tetrad is tetrahedral. During meiosis of microspore mother cells, some chromosomes lagged, and several micronuclei were found in tetrads. Only 27.7% of the pollen grains contained full cytoplasm, and 1.3% of them germinated in culture medium. No pollen grain, however, could germinate on the stigma. The ovary is trilocular with axile placenta, and the ovules are bitegmic, tenuinucellate, and anatropous. Its embryo sac is of the polygonum type. Most embryo sacs degenerated, and only about 4.5% of the ovules contained a normal embryo sac with an egg cell, two synergids, three antipodal, and a central cell containing two polar nuclei. One reason for the sterility of Chinese narcissus is the abnormality of microsporogenesis and megasporogenesis, in which only a few functional pollen grains and embryo sacs are produced. The other reason is that the pollen grains cannot germinate on the stigma. This paper was translated from Journal of Xiamen University (Natural Science), 2005, 44(1) (in Chinese)     

Embryo sac isolation inArabidopsis thaliana: A simple and efficient technique for structure analysis and mutant selection

Arabidopsis thaliana has been widely used as a model plant in gene function analysis. However, its tiny flower and curved embryo sac make it difficult to study gene expression during megagametogenesis, fertilization, and early embryogenesis, especially in the screening of mutants from those developmental processes. The techniques currently available are sectioning and whole-mount clearing of ovules; however, sectioning is time consuming and laborious for quantitative analysis, and whole-mount clearing, makes clear cytological observation impossible. Reported here is a simple and efficient method based on enzymatic isolation of embryo sacs that enables both quantitative analysis and elaborate cytological observation for gene expression investigation and mutant screening.     

The development of onion (<Emphasis Type="Italic">Allium cepa</Emphasis> L.) Embryo sacs <Emphasis Type="Italic">in vitro</Emphasis> and gynogenesis induction in relation to flower size

Summary The development of embryo sacs (ES) in vitro and induction of gynogenesis were studied in onion flower bud culture. Explants were divided into three groups according to their size at inoculation: (a) small flower buds (2.3–3.0 mm in diameter); (b) medium flower buds (3.1–3.7 mm); and (c) large flower buds (3.8–4.4 mm). For histological study, excised ovaries were fixed at inoculation and then at 3-d intervals until day 12, and after 2 and 3 wk of culture. Some explants were cultured until embryo emergence, i.e., 3–5 mo. In total, 2592 ovules were examined histologically. At inoculation, 83% of ovules in small flower buds contained a megaspore mother cell; in 17% of ovules, two-nucleate ES occurred. In medium flower buds two-nucleate, four-nucleate, and mature ES were present at frequencies of 15%, 46%, and 40%, respectively. In large flower buds, only mature ES occurred. In vitro conditions did not disturb meiosis and megagametophyte development in non-degenerated ovules. Regardless of the developmental stage at inoculation, only mature ES occurred on day 12. Gynogenic embryos were found after 2 wk of culture, indicating that embryos developed in mature ES exclusively. Embryos were detected in 5.4% of histological studied ovules; however, the number of embryos after 3–5 mo. was higher (12.4%). The parthenogenetic origin of the embryos is discussed. In addition, ES containing endosperm only (6.5%) and both endosperm and embryo (0.4%) were observed.     

Nuclear DNA amount during sporogenesis and gametogenesis in sexual and aposporous buffelgrass

Nuclear DNA amounts (C values) were measured in Feulgen-stained sections of anthers and ovules of sexual plant B-2s (genotype aaaa) and aposporous cultivar Higgins (genotype AAaa) of buffelgrass (Pennisetum ciliare). The mass of the unreplicated nuclear genome of a gamete equals 1C DNA. In both lines, pollen mother cell nuclei were 4C before leptotene; anther wall, dyad, 1-nucleate pollen, and generative cell nuclei were 2C; microspore tetrad, enlarging microspore, and sperm nuclei were 1C. The tapetum persisted as uninucleate cells with 4C DNA. Archespores (2-4C) of both lines initiated meiosis to form megaspore tetrad nuclei with 1-2C DNA. In B-2s, chalazal megaspores (2-4C) formed reduced 8-nucleate Polygonum type embryo sacs, and sacs at 2- and 4-nucleate stages showed distributions with peaks near C1 and C2, corresponding to G1 and G2 cell cycle phases; this is characteristic of active mitosis. Nuclei of 8-nucleate sacs and of eggs and polars were 1C, indicating chromosomes were not duplicated before fertilization. Antipodal nuclei had levels from 1 to 36C, possibly due to polyteny or endopolyploidy. In Higgins, aposporous initials and 2-nucleate embryo sacs showed bimodal distributions of 2n nuclei with peaks at 2C and 4C DNA. Nuclei of newly formed 4-nucleate Panicum type aposporous sacs and of polars were 2C; aposporous eggs stained too faintly for reliable measurement.Names of products are included for the benefit of the reader and do not imply endorsement or preferential treatment by USDA     

Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr.

Protoplasts of the ectomycorrhizal ascomycete Cenococcum geophilum were isolated from mycelium grown in liquid medium. The method was optimized with regard to culture conditions, preincubation, lytic enzyme system, pH value of the incubation medium, osmotic buffer and incubation temperature for C. geophilum strains SIV and 1448. The yields were 1-3·10⁸ and 7·10⁶ protoplasts per gram fresh weight for C. geophilum SIV and C. geophilum 1448, respectively. Protoplasts from C. geophilum SIV exhibited plasma membrane integrity close to 100% (fluorescein diacetate staining). At least 50% of the protoplasts contained a nucleus (staining with acridine orange). The regeneration of protoplasts from C. geophilum is described for the first time. The regeneration frequency was up to 13%, and, dependent on the conditions of culture (liquid medium, agarose, agar), four types of regeneration patterns could be distinguished Regenerated protoplasts of C. geophilum were capable of forming mycorrhizas with spruce (Picea abies) seedlings.     

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.     

An embryological study ofPlatanthera bifolia (Orchidaceae)

Flowers ofPlatanthera bifolia were hand-pollinated and fixed in FPA₅₀ after 2, 5, 7, 14, and 21 days. Ovules, made transparent in Herr's clearing fluid, were investigated using confocal scanning laser microscopy. Pollination initiates the megasporogenesis. Two days after pollination dyads are frequent. Three days later most embryo sacs contain two nuclei. Seven days after pollination the embryo sacs are 4–8-nucleate and some are organized, and a week later all embryo sacs are organized and fertilization takes place. The embryo sac development follows thePolygonum type. Twenty-one days after pollination the egg nuclei have been fertilized and the embryo sacs contain 2- to many-celled embryos. A suspensor is formed during early stages of embryo development but degenerates later. Fertilization of the central nucleus does not lead to endosperm development.     

Different patterns of callose wall formation during megasporogenesis in two species ofOenothera (Onagraceae)

The homozygousOenothera hookeri Torr. etGray shows the typical pattern ofOnagraceae with ± callose on the external walls of megaspore mother cells and tetrads. Megasporogenesis is heteropolar, and the micropylar megaspore is the mother-cell of the 4-celled embryo sac. The complex-heterozygousOenothera biennis L. during megasporogenesis generally has callose not on the external cell walls but only on the transversal walls of the tetrad. In 95% of the ovules both the external chalazal and the micropylar megaspores develop to embryo sac mother-cells. Megasporogenesis is homopolar, and competition between two developing embryo sacs for nutrition in the ovule occurs. The embryo sac with the stronger genotype wins the race against the other one. Polarity phenomena during ontogeny of the female gametophyte are related to nutritional supply and hormonal induction from the ovule. The introduction of a developmental-physiological point of view into the discussion about the evolution of the embryo sac inOnagraceae is therefore justified.Stipendiatin der Alexander von Humboldt-Stiftung 1974/76.     

Protoplast fusion-derived Ogura male sterile cauliflower with cold tolerance

Cauliflower (Brassica oleracea L. ssp. botrytis) protoplasts with Ogura male sterile and fertile B. oleracea cytoplasms were fused, producing plants with an array of organellar types. Plants with Ogura mitochondria were male sterile; those with B. oleracea chloroplasts were cold tolerant. In some fusions, unfused parental protoplasts were eliminated by double inactivation with iodoacetate and gamma-irradiation; in others, fused protoplasts were physically isolated by micromanipulation or by cell sorting. Double inactivation fusions produced the most plants, including many which were male sterile, female fertile, cold tolerant and diploid.Abbreviations IA iodoacetate - FDA fluorescein diacetate - CMS cytoplasmic male sterility - mtDNA mitochondrial DNA     

Factors influencing protoplast isolation from Coffea arabica cells

Cultured plant cells such as Coffea arabica L. cells, accumulate low concentration of secondary metabolites. One way to obtain high-producing plant cell cultures is to prepare single cell clones by using protoplast systems. Identification of limiting factors should facilitate the development of an isolation procedure that can generate adequate yields of intact and viable protoplasts Coffea arabica L. suspension cells. The most suitable conditions for protoplasting were as follows: 6 g of fresh tissue were plasmolysed in 100 ml of K ₃ salts (Nagy & Maliga 1976) containing 0.5 M sucrose for 1 h at 24°C. Then, 1 g of preplasmolysed cells were incubated in 10 ml of cellulase R10 (1%), macerozyme R10 (0.8%) and driselase (0.5%) in preplasmolysis medium. The protoplasts were collected and purified after 15 h of lytic reaction in the dark, at 28°C. More than 75% and 95% of the cells were converted into protoplasts when 5 and 8 day-old suspensions respectively were used for the release step. A number of viable protoplasts ranging from 3.5×10⁶ to 4.6×10⁶ P g^-1 fresh weight was obtained corresponding to an increase by a factor 10 to 15 of the protoplast yield obtained by Acuna & De Pena (1991).Abbreviations BAP 6-benzylamino purine - BSA Bovine Serum Albumin - 2,4-d 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - MES 2-(N-morpholino)ethanesulfonic acid - NAA naphthalene acetic acid - PI propidium iodide - PCV Packed Cell Volume - fw fresh weight