In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants.

Abstract

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.

Detection of changes in the nuclear phase and evaluation of male germ units by flow cytometry during in vitro pollen tube growth in Alstroemeria aurea

“…The in vitro fertilization (IVF) technique, in which isolated male and female gametes are directly fused under artificial conditions, has been developed for higher plants (Kranz and Lörz 1993;Kranz et al 1998;Uchiumi et al 2007). IVF is considered a useful approach for overcoming cross incompatibility and additionally has potential as an experimental tool for the analysis of fertilization processes or early embryogenesis (Sauter et al 1998;Scholten et 5 al.…”

Endosperm Development after Fusion of Isolated, Single Maize Sperm and Central Cells in Vitro

“…Fusion of a sperm with a central cell was performed mainly electrically, as described previously by Kranz et al (1991aKranz et al ( , 1991b and Kranz and Lörz (1993), using the technique of Koop and Schweiger (1985b), or chemically in a calcium-containing medium (Faure et al, 1994;Kranz and Lörz, 1994).…”

“…These dishes were located in a 3.5-cm-diameter plastic dish filled with 1.5 mL of a maize feeder suspension (Kranz et al, 1991a). For culture, a modified Murashige and Skoog medium (Murashige and Skoog, 1962) was used as described by Kranz and Lörz (1993). In some experiments, to prevent bursting of floating cells on the surface of the medium, unfertilized and fertilized central cells were transferred onto the membrane of a Millicell-CM dish that had been filled with 100 L of a semisolidified mannitol solution (600 mosmol/kg H 2 O; 0.5 and 0.75%, respectively, ultralow gelling temperature agarose, type IX; Sigma).…”

Isolation of protoplasts from viable sperm cells of common buckwheat (Fagopyrum esculentum Moench.)

“…Several research groups have developed different techniques for isolating male gametoplasts from a variety of plant species at different microspore and pollen stages (Tanaka 1988;Zhou 1988;Russell 1991). In maize, fusion products of egg cells and sperm cells have been obtained and cultured in vitro (Kranz and Lórz 1993;Kranz et al 1995). Thus, isolation and fusion of gametoplasts might prove to be a valuable tool in future tissue culture techniques utilized for the improvement of this species.…”

“…Cereal grain sperm cells are natural protoplasts (Cass 1973) in that they lose their cell walls during pollen maturation. If isolation, purification and handling of these sperm cells can be optimized, angiosperm male gametes may represent unique cells for protoplast fusion (Kranz et al 1993;Dumas et al 1984), particularly in the case of cereal grains.…”