Die Aufnahme bakterieller DNA in quellende und keimende Pollen von Petunia hybrida und Nicotiana glauca

Abstract

Summary The uptake of bacterial DNA into swelling and germinating pollen grains of Petunia hybrida and Nicotiana glauca was investigated: 1. Autoradiography. A germination medium for pollen material from N. glauca was worked out (85 % germination). Pollen of N. glauca was incubated in this medium containing 14C-labelled DNA from Rhizobium leguminosarum (existing methods for the isolation of DNA from Rhizobium were improved), treated with DNase and used for autoradiography. The results (figs. 1–5) are consistent with a DNA uptake but can not prove it. 2. CsCl gradient centrifugation. a) A method for the isolation of native DNA from pollen grains was worked out. It includes DNase treatments for the removal of superficially bound DNA and treatments with Triton X-100 for the removal of DNase insensitive complexes eventually formed between the exogenous DNA and constituents of the pollen surfaces. Furthermore, Triton X-100 weakens the exine and thus enables the pollen to be released from the exine (figs. 5–7). b) Unripe pollen grains of P. hybrida were incubated with 3H-labelled DNA from a strain of E. coli carrying R-factors against kanamycin. By CsCl gradient centrifugation of the DNA isolated from the pollen grains not only the pollen DNA but also the bacterial DNA — the main genome as well as the episomal DNA — could be detected (fig. 8). Ripe pollen grains of N. glauca were germinated in germination medium containing 3H-labelled DNA from Rhizobium leguminosarum . DNA was isolated from the pollen material and fractionated by CsCl gradient centrifugation. The bacterial as well as the plant DNA were present in the gradient (fig. 9). All the experiments were carried out under semisterile conditions. To prevent bacterial growth the media contained a mixture of cloxacillin and ampicillin. Furthermore, the reisolation of the episomal DNA in the petunia experiments excludes any intensive bacterial degradation and reutilization of the labelled exogenous DNA respectively DNA building units. The results presented here could be relevant in two lines of investigations: 1. Unripe pollen grains could be incubated with exogenous DNA and than grown up to haploid, eventually genetically altered plants following the methods worked out by Nitsch (1974). 2. Ripe pollen grains could be germinated in exogenous DNA. Using this pollen material for fertilizations perhaps a DNA transfer from pollen to egg cell could be mediated. From the resulting seeds eventually genetically altered plants could be grown up (Hess, 1974). Pollen incubated with exogenous DNA could be used for normal fertilization and seed formation (Hess, in preparation; Hess et al., in preparation).