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.

Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (somatic embryogenesis receptor-like kinase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

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.

Transposition of the maize autonomous controlling element Activator (Ac) and a nonautonomous derivative, Dissociation (Ds), was investigated in tobacco cells. Tobacco protoplasts were transformed with Ti-plasmid vectors that contained Ac or Ds flanked by short maize wx gene sequences. The structures of the elements and surrounding wx and T-DNA sequences were investigated in nine Ac and five Ds tobacco transformants by digestion with restriction enzymes, Southern blotting, and hybridization using specific probes. In four of the nine Ac transformed lines, Ac had excised from its original position in the T-DNA and inserted at new sites in the tobacco genome. Ds did not excise from its original T-DNA position in any of the transformants examined. Two Ac fragments and cellular flanking sequences were cloned from a line of tobacco in which Ac had transposed. Fragments, comprised of sequences flanking the newly integrated Ac elements, were used as hybridization probes to normal tobacco DNA and to the tobacco DNA from which they were isolated. The Ac copies were integrated into repetitive tobacco DNA sequences. Two tobacco fragments containing empty Wx donor sites were cloned from the DNA of the same Ac transformant and sequenced. Both sequences are among the types of excision products observed to result from Ac-catalyzed excision events in maize. Our results indicate that the maize controlling elementAc is capable of self-catalyzed transposition in tobacco. '-1 kb-

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.