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Die Aufnahme doppelt-markierter DNS in isolierte Protoplasten von Petunia hybrida

1973; Elsevier BV; Volume: 69; Issue: 3 Linguagem: Inglês

10.1016/s0044-328x(73)80069-8

0044-328X

Franz Hoffmann,

Carcinogens and Genotoxicity Assessment

Isolated protoplasts of higher plants possibly render a system, which in a particular way makes approachable transformation experiments in intact higher plants. In this connection the incorporation of DNA is of predominant interest. The present paper deals about DNA-incor-poration. Protoplasts of the mesophyll of leaves of Petunia hybrida (white flowering pure line 34d10) were taken as the DNA-receptors, the DNA-donor was the red flowering cyanidin type. The DNA fed was double labeled with 3H and 14C of an isotopic ratio of 10:1. After a 12 h feeding period the protoplasts were analysed for DNA. In order to do this the nuclei were isolated and the nuclear and cytoplasmic DNA were collected by CsCl equilibrium density gradient centrifugation in separate runs. The determination of the isotopic labeling showed the isotopic ratio of the DNA bands well matched with the isotopic ratio of the DNA fed (fig. 6). Under the experimental conditions 0,04% of the radioactivity fed were found in the band of the nuclear DNA, and 0,007% in the band of cytoplasmic DNA. Together with autoradiographic data given previously (Hoffmann und Hess, 1973) the present results indicate an at least partially undegraded incorporation of the fed DNA into the cytoplasm and the nuclei of the protoplasts. Furthermore, the present paper deals about the applicability of DEAE-dextran as a stimulating agent for an incorporation of DNA in the present experiments. The damage caused in protoplasts by the polycation as a function of the DEAE-dextran concentration (tab. 1) and the influence of different salts on this effect (tab. 2) was studied. On the basis of uv-spec-troscopical data (fig. 4) and the results concerning DNase activity in the presence of the DEAE-dextran/DNA complex (fig. 5) the application of DEAE-dextran is critically discussed and rejected for use in experiments as under discussion. An improved method is given for the isolation of highly purified nuclei with a high yield from small amounts of material (fig. 3). Isolierte Protoplasten von Petunia hybrida werden mit 3H/14C-markierter DNS eines anderen Petunien-Genotyps gefüttert. Nach Isolierung der Kern- und cytoplasmatischen DNS wird in diesen Fraktionen ein Isotopenverhältnis gefunden, das annähernd dem der Donor-DNS entspricht. Die Verwendbarkeit von DEAE-Dextran als DNS-Aufnahmestimulans wird untersucht und für Versuche unserer Zielsetzung abgelehnt. Zur Gewinnung von Zellkernen wird für spezielle Probleme ein überlegenes Verfahren angeboten.