In vitro fertilization of single,isolated gametes of maize mediated by electrofusion

Summary Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released from pollen grains after rupture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cellulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs of gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.     

Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

From in vitro fertilization to early embryogenesis in maize

Summary The development of in vitro fertilization methods in plants, the characterization of developmental mutants, and the adaptation of molecular biology techniques to construct cDNA libraries from minute samples, all represent important recent technical break-throughs. They allow the study of fertilization and early embryogenesis at a molecular level and considerable improvement in the under-standing of higher plant reproduction can be predicted over the next few years. Important biological questions, such as polyspermy, gamete fusion physiology, asymmetrical cell division, embryo axis formation, can now be addressed experimentally in maize, which appears as a major study model in this area.Abbreviations PCR polymerase chain reaction - RFLP restriction fragment length polymorphism     

In vitro pollination as a model for studying fertilization in maize (Zea mays L.)

Summary In vitro pollination was conducted using excised segments of maize female spikelets to determine the effects of age and silk length on fertilization efficiency and developmental pattern. Ovary development after 15 days resulted in: (1) normal kernels, (2) abnormal kernels and (3) enlarged ovaries; the percentages of each class varied with age. Evidence of double fertilization was observed in both normal and abnormal kernels. In vitro fertilization was traced using silk excision and autoradiography with ³²P-radiolabelled pollen and occurred between 4 and 7 h after the pollination of 4.5-cm-long silks. This study supports the validity of the in vitro pollination method for studying fertilization and emphasizes the importance of using a developmentally sensitive index (silk length) for establishing female developmental stage.     

Egg activation in flowering plants

Compared to animals and algae, egg activation in flowering plants is still poorly understood because of the inaccessibility and complexity of the fertilization process which is double and internal. However, the development of in vitro fertilization (IVF) systems in maize and a few other plants, despite some limitations, offers new possibilities for the study of early post- fusional events and signals leading to egg activation under defined conditions. This review reports recent data on calcium events induced by gamete fusion during maize IVF and presents perspectives on the role of calcium in egg activation and in early development. Received: 2 December 2000 / Accepted: 7 June 2001     

In vitro fertilization as a tool for investigating sexual reproduction of angiosperms

In vitro fertilization (IVF) of isolated male and female gametes of flowering plants was first accomplished in the last decade. Successful isolation of male and female gametes, and culturing of in vitro zygotes to form new plants, is a prelude to the use of IVF for research into the cellular and molecular control of fertilization in higher plants and its application as a tool in biotechnology. Genes unique to male and female gametes and zygotes of higher plants, although currently incompletely characterized, are expected to permit direct molecular dissection of fertilization. By applying IVF and microculture to zygotes and endosperm obtained by both in vivo and in vitro methods, newly activated fusion products may be observed and manipulated in media where they are directly accessible to the techniques of molecular cell biology. IVF and zygote culture may also offer potential for creating new hybrid plants by fusing isolated gametes from different species to produce unique zygotes and ultimately plants that would be impossible to obtain using typical crossing techniques. Transformation and regeneration frequencies using IVF may also be high enough to avoid the necessity of adding controversial antibiotic and herbicide resistant genes to screen transformed products. This review describes advances using IVF in plant sexual reproduction and discusses its potential in the genetic improvement of flowering plants.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

Micromanipulation of male and female gametes ofNicotiana tabacum: II. Preliminary attempts for in vitro fertilization and egg cell culture

This research is part of an attempt to establish an in vitro fertilization system in tobacco to aid in understanding mechanisms of fertilization. Fusions of isolated male and female gametes were induced in a polyethylene glycol solution. Fusion appears similar to that in maize. One nuclear division of both an unfertilized egg cell and a synergid was induced in KM8p medium with 1 mg/l 2,4-dichlorophenoxyacetic acid in a microchamber culture; one cellular division of the egg cell was also induced in the same medium in solid-drop culture. The osmolality of suspension culture feeder cells was critical for the development of these cells. These results indicate that in vitro fertilization is possible in tobacco, which would be the first such system in dicots.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - PEG Polyethylene glycol     

Quantitative,three-dimensional ultrastructure of isolated corn (Zea mays) sperm cells

Summary Five isolatedZea mays sperm cells, taken from the same population as used for a previous morphometric study, were serially ultrathin sectioned and computer-reconstructed to yield three-dimensional images as well as quantitative data. All cells were found to be essentially spherical and to contain a full complement of cell constituents except plastids and microtubules. The nuclei of three cells were highly curved into a C or V shape while the other two nuclei were not curved, but were more spherical to disc shaped. The three curved nuclei were heterochromatic in appearance, the other two were more euchromatic. Mitochondria were closely associated with the nuclei, were predominately in the form of large, variously shaped complexes, and ranged in number from 7 to approximately 74 per cell. Dictyosomes tended not to be close to the nucleus and ranged in number from 6 to 23 per cell. The endoplasmic reticulum was similarly not typically associated with the nucleus, and varied from extensive sheet-like areas to small membranous whorls. In addition to confirming the findings of previous studies on isolated corn sperm cells and providing new three-dimensional and distribution data, the results of the present work underscore the existence of a high degree of morphological variability amongZea mays sperm cells of a population.Abbreviations ER endoplasmic reticulum - SD standard deviation     

Production of normal,germinable and viable pollen from in vitro-cultured maize tassels

Summary Immature tassel meristems (1.0–1.5 cm long) of Zea mays L. inbred, Oh43, and single cross hybrid, Se60, cultured on a nutrient liquid medium underwent extensive development through to maturity and produced normal, mature, trinucleate pollen grains. The grains germinated on nutrient agar and on receptive silks and also produced viable kernels. No differences were observed between in vitro-produced pollen and in vivo pollen (pollen from greenhouse-grown plants) in characteristics such as pollen size, in vitro and in situ germination, and pollen tube growth in vitro. The kernels produced with in vitro pollen grew into mature plants (in vitro plants) which were similar to in vivo plants (plants produced with in vivo pollen), with no significant differences for all the morphological characteristics measured, and no phenotypic and cytological abnormalities. Gel electrophoresis of polypeptides revealed no major differences between in vitro and in vivo seedlings. This demonstration of fertilization and production of normal, uniform plants with pollen from cultured tassels has significant potential in basic and applied research studies.     

In vitro fertilization: analysis of early post-fertilization development using cytological and molecular techniques

Methods have been developed which enable us to obtain in vitro fusion of pairs of sperm and egg cells, and sperm and central cells of angiosperms. Cultured products of such cell fusions develop progressively into zygotes, embryos and fertile plants, and endosperm, respectively. In vitro fusion of isolated gametes allows precisely timed examination of the earliest developmental processes following fertilization. When cultured, in vitro produced zygotes and primary endosperm cells organize themselves independently, and without any requirement for supporting tissues. This technology thus constitutes a unique model system for studies of early stages of zygotic embryogenesis and endosperm development. Following the adaptation of molecular techniques for use with only a few cells, it has proved possible to investigate developmental processes in these systems. This review describes the successful combination of molecular techniques with in vitro fertilization methods, and highlights results obtained with small numbers of reproductive cells isolated by microdissection.     

Results of a diallel trial and a breeding experiment for in vitro aptitude in maize

Summary Some characteristics of in vitro culture of somatic tissues of maize were analysed by a diallel trial. Eight genetically different pure strains, chosen for their aptitudes, were used. The results show that there is considerable genetical variation for the characteristics of in vitro culture and that it should be possible to breed for aptitude to in vitro culture. The linear regression of hybrids on mid-parent reveals a significant heritability for such aptitude. Through selection we have improved plant regeneration after a long period of callus growth.     

In vitro double fertilization in Nicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy

In vitro double fertilization in tobacco was carried out with attention to fusion behavior and gamete interaction. Structural and cytological events indicating possible reaction to the fusion of sperm-egg and especially sperm-central cell were recorded by video-enhanced microscopy. Generative cells were fused with the egg cell or central cell as a control system to better understand gamete interaction. As early as adherence of the male cell, the female cell showed response by means of cytoplasm strand formation. After gamete fusion, cytoplasm activation in the egg cell was observed as long distance movement of organelles. In fertilized central cells, however, fusion did not result in notable cytological change within 30 min. Male nuclear movement recorded in the female cell illustrated two different patterns of movement which showed similarity to organelle movement. The dynamics of male and female nuclear fusion after in vitro fertilization was also recorded in the central cell. It revealed that the fusion process requires only a few seconds and is similar to that of gamete fusion in vitro. This may offer a new clue for understanding how female and male nuclei attract, adhere and finally fuse each other. Received: 13 October 1999 / Revision accepted: 6 December 1999     

Factors affecting in vitro maturation of isolated maize microspores

Summary An in vitro method to simulate pollen development was developed in maize (Zea mays L.). Microspores at the late uninucleate to early binucleate stage were isolated and cultured under various conditions. Cell viability, starch content and the formation of the three nuclei as found in normal mature pollen were monitored during the course of the culture. Media composition was modified in order to promote starch accumulation and frequency of mitosis, while maintaining the viability of the microspores. Under the best conditions, up to 12% of the microspores matured in vitro into trinucleate, starch-filled viable pollen grains which were unable to germinate or produce seeds. At different stages during in vitro maturation, proteins patterns were analyzed and compared with their in vivo equivalent and the patterns were only partially similar.     

Analysis of pollen-tube growth in cultured maize silks

Summary In vitro pollen-tube growth in maize was studied using an in vitro pollination system. In the cut-silk method, ovaries with silks were placed on medium in vitro, whereafter the silk was cut and the upper part of the silk was pollinated. Pollen tubes were not able to bridge the space between the two silk parts. Even when silk parts were tightly connected, pollen tubes still were not able to pass the cut ends and reach the lower silk part. Pollen-tube growth rates and the direction of tube growth were not influenced by the presence or absence of an ovary. Prepollination did not have any influence on pollen-tube growth rate. Measurements of pollen-tube growth rate also showed that there was no population effect, i.e. growth rate was not stimulated by pollination with an excess of pollen grains. We found that the direction in which maize pollen grew was determined only by the positioning of the silk hairs.     

In vitro culture of immature tassels of an inbred field variety of Zea mays,cv. Oh43

The medium and conditions which permit in vitro culture of immature tassels of Zea mays cv. Oh43 are reported. Final fresh weight, total number of spikelets and the number of normal spikelets were enhanced by optimal concentrations of sucrose (0.3 M), kinetin (10^–7 M) and casein hydrolysate (30 mg l^–1) when added to the Murashige and Skoog salts, White's vitamins and glycine and inositol. A single cultured tassel produces up to 200 normal spikelets which contain anthers bearing germinable pollen.     

Electrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process,fusion of sperm cells with synergids and central cells,and cell reconstitution in maize

Summary Fusion products were created by the electrofusion of single sperm cells with single synergids and central cells. The synergid was also fused with the sperm cell, occasionally in the presence of adhering second synergids, egg cells, and central cells. Single egg cells were fused with single sperm cells in the presence of adhering synergids and the central cell. Cytoplasmic organelles were transmitted through the fertilization process by electrofusion using cytoplasts of maize mesophyll cells. Cell reconstitution was achieved by fusion of one or two sperm cells with single enucleated protoplasts, thus creating a haploid or a diploid cell.     

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

Summary. In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively. Correspondence and reprints: Agricultural Research Institute, Hungarian Academy of Sciences, Brunszvik utca 2, 2462 Martonvásár, Hungary.     

Effects of ultrashort gamete co-incubation time on porcine in vitro fertilization

A reduction in co-incubation time has been suggested as an alternative method to reduce polyspermic fertilization. The aim of this study was to evaluate the effect of short periods of gamete co-incubation during pig in vitro fertilization. A total of 2833 in vitro matured oocytes were inseminated with thawed spermatozoa and coincubated for 0.25, 1, 2, 3, 7, 10 min and 6 h. The oocytes from the 0.25–10 min groups were washed three times in modified Tris-buffered medium (mTBM) medium to remove spermatozoa not bound to the zona and transferred to the same medium (containing no spermatozoa) until 6 h of co-incubation time were completed. After 6 h, presumptive zygotes from each group were cultured in NCSU-23 medium for 12–15 h to assess fertilization parameters. After each period of co-incubation, 45–50 oocytes from each group were stained with Hoechst-33342 and the number of spermatozoa bound to the zona was counted. Although the number of zona bound spermatozoa increased (p < 0.05) with the co-incubation time, no increase was observed in penetration rates among groups from 2 min to 6 h of co-incubation time (ranging from 53.5 ± 2.8 to 61.3 ± 2.6%). Similarly, the efficiency of fertilization reached a maximum for the 2 min of co-incubation group with values ranging between 32.3 ± 2.4 and 41.9 ± 2.5%. The reduction of co-incubation time did not affect the monospermy rate (range: 71.3 ± 3.4–80.2 ± 3.8%) and the mean number of spermatozoa/oocyte (range: 1.2 ± 0.4–1.4 ± 0.5). These results show that, under our in vitro conditions, high penetration rate can be obtained with co-incubation times as short as 2 min, although monospermy could not be improved using this strategy.     

In vitro double fertilization in Nicotiana tabacum (L.): polygamy compared with selected single pair somatic protoplast and chloroplast fusions

In vitro polygamy was studied mainly by using isolated sperm and central cells of tobacco in order to elucidate the mechanism that might be involved in preventing in vivo polygamy. In 17.5% 4000 M.W. polyethylene glycol, only when two sperm cells were made close enough to each other and adhered to a female cell simultaneously was polygamy possible. If one sperm cell fused with the egg or central cell, within 30 min another sperm cell could not fuse with the same egg or central cell. Similar phenomena were found in selected single somatic cell fusion. When more than two protoplasts adhered to each other simultaneously, fusion was always successful; after two protoplasts fused, within 30 min the fusion products could not fuse with another protoplast under the same conditions. This comparative study revealed this characteristic to be shared by both sexual and somatic cell fusion. However, after cytoplasm reorganization was complete in the fusion product, it was possible for the fusion product to fuse with the third protoplast. This indicates that the obstruction to additional fusion was present only during a certain period after the preceding fusion under certain condition. The possible reason for the effect is discussed. Received: 7 March 2000 / Revision accepted: 15 June 2000