Intact Ovules Research Articles

Innovative sensory methods to access acceptability of mixed polymer semisoft ovules for microbicide applications.

Vaginal microbicides are a promising means to prevent the transmission of HIV, empowering women by putting protection under their control. We have been using gel technology to develop microbicides in the intermediate texture space to overcome shortcomings of current solid and liquid forms. We recently formulated semisoft ovules from mixed polymer combinations of carrageenan and Carbopol 940P to overcome some of the flaws with our previous generation of formulations based solely on carrageenan. To determine the user acceptability of the reformulated gels, women first evaluated intact semisoft ovules before evaluating ovules that had been subjected to mechanical crushing to simulate samples that represent post-use discharge. Women then evaluated combinations of intact and discharge samples to understand how ovule textures correlated with texture of the resulting discharge samples. Carbopol concentration directly and inversely correlated with willingness to try for discharge samples and intact samples, respectively. When evaluating intact samples, women focused on the ease of inserting the product and preferred firmer samples; conversely, when evaluating discharge samples, softer samples that resulted in a smooth paste were preferred. Significant differences between samples were lost when evaluating pairs as women made varying trade-offs between their preference for ease of inserting intact ovules and acceptability of discharge appearance. Evaluating samples that represent different stages of the use cycle reveals a more holistic measure of product acceptability. Studying sensory acceptability in parallel with biophysical performance enables an iterative design process that considers what women prefer in terms of insertion as well as possibility of leakage.

Simulating in ovulo osmotic potentials and O2 tensions normalize growth and pigmentation of immature cotton embryos

Oxygen tensions and osmotic potentials are important physiological factors of plant growth and development. To optimize these variables for cotton (Gossypium hirsutum L.) embryo culture, we quantified dissolved O2 (dO2) tensions, osmotic potentials, and pH at several locations in cotton ovules during embryony. Clark O2 microelectrodes were micromanipulated into intact ovules at an angle lateral to the developing embryo, and dO2 tensions were determined in integuments, nucelli and embryos. Ovular osmotic potentials and pH were determined from extracted ovule sap using vapor pressure osmometers and pH microelectrodes. Dissolved O2 tensions near or in embryos decreased from 104 mmol m−3 at 5 days post-anthesis (DPA) to 83 mmol m−3 at 18 DPA. Osmotic potentials of ovule sap decreased from −0.70 megapascals (MPa) at 2 DPA to −1.12 MPa at 8 DPA but then increased to −0.84 MPa by 17 DPA. Ovule sap pH at 5–17 DPA varied inconsistently and ranged from 5.4 to 6.5. Based on these results, a factorial experiment with two osmotic and three O2 treatments was designed. Immature embryos of cotton cultivar HS-26 were randomly assigned to the treatment combinations and cultured for 33 days. Oxygen treatments did not affect embryo growth, and there were no differences among treatments with regard to percentage of embryos that progressed to a more advanced stage of embryo development. However, cotyledons of embryos grown without osmotic adjustment were abnormally large, and embryos exposed to these treatments were abnormally brown. Browning was less severe for embryos exposed to low O2 tensions. Growth and pigmentation were most normal for embryos simultaneously exposed to O2 tensions and osmotic potentials that best simulated the observed in ovulo conditions.

Reproductive Behavior of Induced Allotetraploid ×Chitalpa and In Vitro Embryo Culture of Polyploid Progeny

×Chitalpa tashkentensis Elias & Wisura is a sterile intergeneric hybrid [Catalpa bignonioides Walt. × Chilopsis linearis (Cav.) Sweet]. To restore fertility in ×Chitalpa the following were evaluated: 1) oryzalin as a polyploidization agent, 2) fertility of induced polyploids, and 3) in vitro culture methods for embryo rescue of interploid crosses. Meristems of ×Chitalpa `Pink Dawn' were submerged in an aqueous solution of 150 μm oryzalin for 0, 6, 12, or 24 hours and ploidy analyzed via flow cytometry. As treatment duration increased, recovery of diploids decreased as mixoploids and shoot mortality increased. Two tetraploid shoots occurred in the 24-hour treatment. Four tetraploids and two cytochimeras were stabilized in total. Tetraploids flowered sparsely; however, cytochimeras flowered profusely and these were used to study fertility at the tetraploid level. Diploid ×Chitalpa `Pink Dawn' pollen was essentially nonviable, but cytochimera pollen stained and germinated equal to or greater than pollen of C. bignonioides and C. linearis `Bubba'. Cytochimera ×Chitalpa were selfed yielding tetraploid seedlings, crossed with C. bignonioides to yield triploids, but failed in reciprocal crosses with C. linearis `Bubba' and `Burgundy Lace'. To increase recovery of triploids, germination of triploid and tetraploid embryos was investigated, as either intact ovules or excised embryos, on Schenk and Hildebrandt (SH) basal salts supplemented with sucrose at 20, 40, and 80 g·L-1, presence or absence of 2% coconut-water, and gibberellic acid (GA3) at 0, 1, 2, or 4 μm, and harvested weekly beginning 2 weeks after pollination (WAP). Germination of triploids (cytochimera ×Chitalpa × diploid C. bignonioides) and tetraploids (selfed cytochimera ×Chitalpa) were greatest with excised embryos at 7 WAP on SH supplemented with sucrose at 20 g·L-1 and ≥1 μm GA3. Germination of triploids (diploid C. linearis × cytochimera ×Chitalpa) was <5% at 4, 5, or 6 WAP on the same medium as above. Oryzalin effectively induced polyploidy and restored fertility in ×Chitalpa `Pink Dawn'. Successful crosses between hybrid and parental taxa of different ploidy levels, coupled with embryo culture will facilitate a ×Chitalpa breeding program. Chemical names used: 4(dipropylamino)-3,5-dinitrobenzenesulfonamide (oryzalin).

Maturation of Arabidopsis Seeds Is Dependent on Glutathione Biosynthesis within the Embryo

Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.

Regeneration of the barley zygote in ovule culture

An ovule culture technique has been established for barley that allows the regeneration of plants from zygotes. An average of 1.3 plantlets per ovule could be regenerated from more than 60% of the cultured ovules and about 75% of the regenerated plantlets developed into normal, fertile plants. The same regeneration frequencies were obtained in intact ovules and in ovules where the two integuments had been removed from the micropylar region. Unfertilized ovules and ovules where the fertilized eggs had been destroyed by a microinjection needle did not give rise to embryo-like structures. Plants could be regenerated from the zygote at the same frequency at developmental stages from immediately after fertilization until the formation of bicellular embryos. This tissue culture system appeared to be largely independent of genotype since similar regeneration frequencies were obtained in two different barley cultivars, Igri and Alexis, that in anther and microspore culture behave differently. The same technique has also been applied successfully in the wheat cultivar Walter.

Somatic Embryogenesis and Plant Development in Eight Cultivars of Pecan

The influence of stage of fruit development and plant growth regulators on somatic embryogenesis and the relation of cultivar response on somatic embryogenesis and subsequent plant development have been investigated in eight cultivars of pecan [Carya illinoensis (Wangenh.) C. Koch]. Explants from the micropylar region of the ovule were more embryogenic when removed from fruits in the liquid endosperm stage than were intact ovules from less-mature fruits or from cotyledonary segments of more-mature fruits. Explants conditioned on medium containing auxin alone or auxin + cytokinin produced more somatic embryos than medium containing cytokinin alone. Under the conditions of this study, frequency of embryogenesis, as well as the germination of somatic embryos leading to plant development, indicated appreciable variation among cultivars. Plant development was greatest by far from somatic embryos of `Schley' than other cultivars studied.

Megasporogenesis in normal and a synaptic-mutant (sy-2) of Solanum commersonii Dun.

Nomarski differential interference contrast microscopy was used to study megasporogenesis in intact ovules of Solanum commersonii Dun., following staining with Mayer's hemalum and clearing with methyl salicylate or cedarwood oil. Previous studies in Solanum have observed the Polygonum type of megasporogenesis, in which a linear tetrad of megaspores is formed. The three micropylar megaspores then degenerate, leaving the chalazal megaspore to divide mitotically to form the egg sac. Contrary to expectation, only 30% of the observed sporads within the same ovary were tetrads. Triads, including some with one deteriorating cell at the micropylar end, were the predominant form. Many dyads, which are an intermediate stage in megasporogenesis, were found with one cell prematurely deteriorating. These observations can be explained if the micropylar daughter cell of the dyad stage, which formed after telophase I, began deteriorating before the second meiotic division, such that it never underwent the second division. The chalazal daughter cell would still undergo the second meiotic division, followed by death of the new micropylar megaspore.Key words: Solanum, potato, megasporogenesis, S. commersonii, ovule, clearing.

A rapid methyl salicylate clearing technique for routine phase‐contrast observations on female meiosis in Solanum

SUMMARYA rapid clearing technique, involving methyl salicylate clearing and phase‐contrast microscopy, that enables large‐scale observations on meiosis and embryo sac development in intact ovules of potato, was developed. With high resolution phase‐contrast optics the details observed in cleared ovules were as well defined as in microtome sections and ‘abnormalities' associated with desynapsis and 2n‐megaspore formation were readily detected.In potato this ovule clearing technique offers certain advantages over earlier clearing and staining‐clearing techniques and permits bulk preparation of ethanol (70%) stored ovaries shortly (1·5–2 h) before observation.

Turgor‐sensitive transport in developing seeds of legumes: the role of the stage of development and the use of excised vs. attached seed coats

Abstract After removal of the embryo from developing seeds of Vicia faba L. and Pisum sativum L., the ‘empty’ ovules were filled with a substitute medium (pH 5.5) and the effect of the osmolality of this solution on assimilate transport was exandned. In pulse‐labelling experiments with a mixture of [3H]sucrose and [14C]α‐andnoisobutyric acid (AIB), a solute concentration of 400 mol m−3 (100 mol m3− sucrose + 300 mol m−3 mannitol) was too low to maintain sugar and andno acid transport into empty ovules of V. faba in a very early stage of development (embryo dry weight < 100 mg) on the same level as transport into intact ovules within the same fruit. A 550‐mol m−3 solution could maintain the normal rate of transport. In experiments with seeds in a more advanced stage of development (embryo dry weight > 250 mg), transport of labelled sucrose and AIB into empty ovules filled with a 400‐mol m−3 solution was practically equal to transport into intact ovules within the same fruit. Experiments without isotopes, on sugar and andno acid release from the seed coat, confirmed the important role of the osmotic environment. A very low osmolality of the solution (e.g. 50 mol m−3 mannitol) enhanced net efflux of assimilates from excised seed coats and cotyledons, by inhibiting resorption from the apoplast.

Effect of the Osmotic Environment on the Balance between Uptake and Release of Sucrose and Amino Acids by the Seed Coat and Cotyledons of Developing Seeds ofPisum sativum

Effect of the Osmotic Environment on the Balance between Uptake and Release of Sucrose and Amino Acids by the Seed Coat and Cotyledons of Developing Seeds of<i>Pisum sativum</i>

Overcoming cross-incompatibility between Nicotiana tabacum L. and N. rustica L. by test-tube pollination and ovule culture.

Interspecific hybrids between Nicotiana tabacum (=) and N.rustica can not be obtained by conventional crossing because of cross-incompatibilit.y. Two techniques, test-tube pollination and ovule culture, were combined to overcome cross-incompatible combination. Whole placentae with intact ovules of N.tabacum were pollinated in vitro by dusting pollen grains of N.rustica. Developed ovules were excised from the placentae and transferred to ovule culture. Finally, 12 hybrid plants were obtained by the combination of these two techniques. The hybrid plants were intermediate between their parents in their morphological appearance and had 48 chromosomes which were the additive sum of the haploid chromosomes of the parents. They failed to produce fertile pollen and were completely self-sterile. A fertile chromosome-doubled hybrid was obtained by colchicine treatment. From the results, it is considered that the combination of two techniques, test-tube pollination and ovule culture, is useful for overcoming cross-incompatibility between distantly related species.

Turgor‐sensitive sucrose and amino acid transport into developing seeds of Pisum sativum. Effect of a high sucrose or mannitol concentration in experiments with empty ovules

Sugar and amino acid transport into empty ovules of Pisum sativum L. cv. Marzia was examined. In fruits containing 4–6 developing seeds, the embryo was removed from four ovules. After this surgical treatment, each empty seed coat was filled with a solution (pH 5.5) containing a low (0, 50 or 200 mM), medium (350, 400 or 500 mM) or high (0.7 or 1 M) concentration of sucrose and/or mannitol. In pulse‐labelling experiments with sucrose and α‐aminoisobutyric acid (AIB), transport of sucrose and AIB into an empty ovule filled with a solution containing a high sucrose concentration was the same as transport into an ovule filled with a mannitol solution of similar osmolarity, demonstrating that a high sucrose concentration in the seed coat apoplast affects phloem transport of sucrose and AIB into the seed coat only by the osmotic effect. The osmolarity of a given solution filling the seed coat cavity appeared to be important for phloem transport of sucrose and AIB into empty ovules.In our experiments, 350 mM appeared to be the optimal concentration for sucrose and AIB transport into the cavity within an empty ovule, giving results comparable with transport into intact ovules. A lower osmolarity of the solution induced less transport. Very high sucrose or mannitol concentrations caused a strong inhibition of sucrose and AIB unloading from the seed coat, so that transport into the empty ovules was inhibited. A low (strongly negative) but not too low osmotic potential of the solution in the seed coat apoplast seems necessary to maintain a normal rate of phloem transport into developing seeds. Apparently, the “sink strength” of developing seeds is turgor‐sensitive.

A NEW CLEARING‐SQUASH TECHNIQUE FOR THE STUDY OF OVULE DEVELOPMENT IN ANGIOSPERMS

The simple, efficient method described here for the study of ovule and megagametophyte development in angiosperms provides for the extension of investigation beyond the limits imposed by the traditional but arduous section technique. Excised pistils previously fixed in FPA50 and stored in 70 % ethanol are placed in a clearing fluid composed of lactic acid (85 %), chloral hydrate, phenol, clove oil, and xylene (2:2:2:2:1, by weight). After 24 hr, ovules dissected from the ovularies are transferred with some of the fluid to a slide, covered so that the cover glass is supported laterally by two permanently affixed covers, and examined with phase contrast optics. The unique action of the clearing fluid permits the study of cellular structure with the phase oil objective focused at any focal plane within the ovule. Downward focusing thus reveals a series of optical sections in the sagittal, frontal, or transverse plane depending on the orientation of the ovule. Orientation can be altered by a slight shifting of the cover glass on the lateral support mounts. The ovules become quite fragile in the clearing fluid. Pressure applied to the cover glass gradually breaks the ovule apart without disrupting the structural integrity of individual cells. This squash procedure provides for extending observations to cytological features of megasporocytes, megaspores, and megagametophytes previously identified in intact ovules. The new method is applied here to the study of ovule development in two unrelated species, Cassia abbreviata Oliver var. granitica Bak. f. (Leguminosae) and Ludwigia uruguayensis (Camb.) Hara. (Onagraceae). For best results, the ovules of Ludwigia must be pretreated in lactic acid (85 %) for 24 hr prior to application of the clearing fluid. Other methods for pretreatment likely will be required as the technique is applied to a wider range of flowering plant species.

Untersuchung der ersten Entwicklungsphasen von Embryo und Endosperm der Art Jasione montana L. an lebendem Material

Growth of the zygote and the first phases of the endosperm development of Jasione montana L. in isolated intact ovules was studied. The zygote begins to grow simultaneously with the first division of the primary endosperm nucleus. It forms a long outgrowth into the embryo sac. A distinct oil droplet occurs in the basic part of the zygote, which disappears after the development of the embryo is advanced.