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.

Plant imprinted genes show parent-of-origin expression in seed endosperm, but little is known about the nature of parental imprints in gametes before fertilization. We show here that single differentially methylated regions (DMRs) correlate with allele-specific expression of two maternally expressed genes in the seed and that one DMR is differentially methylated between gametes. Thus, plants seem to have developed similar strategies as mammals to epigenetically mark imprinted genes.

The phylogeny based on the homeodomain (HD) amino acid sequence of the WOX (WUSCHEL-related homeobox gene family) was established in the 3 major radiations of the Poaceae family: Pooideae (Brachypodium distachyon), Bambusoideae (Oryza sativa), and Panicoideae (Zea mays). The genomes of all 3 grasses contain an ancient duplication in the WOX3 branch, and the cellular expression patterns in maize and rice indicate subfunctionalization of paralogues during leaf development, which may relate to the architecture of the grass leaf and the encircling of the stem. The use of maize WOX gene family members as molecular markers in maize embryo development for the first time allowed us to visualize cellular decisions in the maize proembryo, including specification of the shoot/root axis at an oblique angle to the apical-basal polarity of the zygote. All molecular marker data are compatible with the conclusion that the embryonic shoot/root axis comprises a discrete domain from early proembryo stages onward. Novel cell fates of the shoot and the root are acquired within this distinct morphogenic axis domain, which elongates and thus separates the shoot apical meristem and root apical meristem (RAM) anlagen in the maize embryo.

Early events, such as formation of the cell wall, first nuclear division and first unequal division of the zygote, were examined following in vitro fusion of single egg and sperm protoplasts of maize (Zea mays L.). The time course of these events was determined. The formation of cell wall components was observed 30 sec following egg—sperm fusion and proceeded continuously thereafter. Within 15 h after fusion most of the organelles became more densely grouped around the nucleus of the zygote. In the in vitro produced zygote the location of the cell organelles and of the dividing nucleus showed polarity. Two nucleoli were first observed 18 h after gamete fusion. The zygotic nucleus remained undivided for about 40 h. The first cell division was observed 40–60 h, generally 42–46 h, after egg—sperm fusion. The non‐fused egg cell could be triggered to sporophytic development in vitro by pulses of high amounts of 2,4‐D. Without such a treatment, cultured egg cells of different maize lines did not divide. Although nuclear fusion seemed to occur, fusion products of two egg cells also did not divide. Cell wall formation was incomplete and non‐uniform, showing a polarity of cultured egg cells and fusion products of two egg protoplasts. Cell division was also induced after fusion of maize egg with sperms of genetically remote species, such as Coix, Sorghum, Hordeum or Triticum. These gametic heterologous fusion products developed to microcalli. Moreover, cell division occurred in fusion products of an egg and a diploid somatic cell‐suspension protoplast from maize.

We demonstrate here the possibility of regenerating phenotypically normal, fertile maize plants via in vitro fertilization of isolated, single sperm and egg cells mediated by electrofusion. The technique leads to the highly efficient formation of polar zygotes, globular structures, proembryos, and transition-phase embryos and to the formation of plants from individually cultured fusion products. Regeneration of plants occurs via embryogenesis and occasionally by polyembryony and organogenesis. Flowering plants can be obtained within 100 days of gamete fusion. Regenerated plants were studied by karyological and morphological analyses, and the segregation of kernel color was determined. The hybrid nature of the plants was confirmed.

SummaryAll four members of a gene family, which are highly expressed in the cells of the female gametophyte (ZmES1±4: Zea mays embryo sac), were isolated from a cDNA library of maize egg cells. High expression of ZmES genes in the synergids around the micropylar region was detected in thin sections of maize ovaries. Single-cell RT±PCR analyses with the various cells of the female gametophyte con®rmed the expression in synergids and also showed expression in the egg cell and central cell, and low expression in the antipodals. The expression of the whole gene family is suppressed after fertilization of the embryo sac, and expression in two-cell or later embryo stages or other tissues of maize could not be detected. In order to investigate ZmES mRNA gradients in the highly polarized and vacuolized cells of the maize embryo sac, a whole-mount in situ protocol with isolated single cells was developed: as for total RNA, ZmES transcripts are uniformly distributed in the cytoplasm of egg cell, synergids and central cell. ZmES genes encode small, cysteine-rich proteins with an N-terminal signal peptide, probably for translocation into the embryo sac cell wall. The four ZmES proteins display high sequence identity with each other, and the proposed tertiary structure of the mature peptides is similar to that of plant and animal defensins. The function of ZmES1±4 during the fertilization process is discussed.

A PCR-based genomic scan has been undertaken to estimate the extent and ratio of maternally versus paternally methylated DNA regions in endosperm, embryo, and leaf of Zea mays (maize). Analysis of several inbred lines and their reciprocal crosses identified a large number of conserved, differentially methylated DNA regions (DMRs) that were specific to the endosperm. DMRs were hypomethylated at specific methylation-sensitive restriction sites upon maternal transmission, whereas upon paternal transmission, the methylation levels were similar to those observed in embryo and leaf. Maternal hypomethylation was extensive and offers a likely explanation for the 13% reduction in methyl-cytosine content of the endosperm compared with leaf tissue. DMRs showed identity to expressed genic regions, were observed early after fertilization, and maintained at a later stage of endosperm development. The implications of extensive maternal hypomethylation with respect to endosperm development and epigenetic reprogramming will be discussed.

These authors contributed equally to this work. SummaryK channels control K homeostasis and the membrane potential in the sieve element/companion cell complexes. K channels from Arabidopsis phloem cells expressing green¯uorescent protein (GFP) under the control of the AtSUC2 promoter were analysed using the patch-clamp technique and quantitative RT-PCR. Single green¯uorescent protoplasts were selected after being isolated enzymatically from vascular strands of rosette leaves. Companion cell protoplasts, which could be recognized by their nucleus, vacuole and chloroplasts, and by their expression of the phloem-speci®c marker genes SUC2 and AHA3, formed the basis for a cell-speci®c cDNA library and expressed sequence tag (EST) collection. Although we used primers for all members of the Shaker K channel family, we identi®ed only AKT2, KAT1 and KCO6 transcripts. In addition, we also detected transcripts for AtPP2CA, a protein phosphatase, that interacts with AKT2/3. In line with the presence of the K channel transcripts, patch-clamp experiments identi®ed distinct K channel types. Time-dependent inward rectifying K currents were activated upon hyperpolarization and were characterized by a pronounced Ca 2 -sensitivity and inhibition by protons. Whole-cell inward currents were carried by single K -selective channels with a unitary conductance of approximately 4 pS. Outward rectifying K channels (approximately 19 pS), with sigmoidal activation kinetics, were elicited upon depolarization. These two dominant phloem K channel types provide a versatile mechanism to mediate K ¯uxes required for phloem action and potassium cycling.