Plant Physiol.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Plant Physiology 59:301-308 (1977)
© 1977 American Society of Plant Biologists

This Article
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Web of Science (23)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Uchimiya, H.
Right arrow Articles by Murashige, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Uchimiya, H.
Right arrow Articles by Murashige, T.
Agricola
Right arrow Articles by Uchimiya, H.
Right arrow Articles by Murashige, T.
Articles

Quantitative Analysis of the Fate of Exogenous DNA in Nicotiana Protoplasts 1

Hirofumi Uchimiya2 and Toshio Murashige

a Department of Plant Sciences, University of California, Riverside, California 92502

After a 5-hour incubation of protoplasts of Nicotiana tabacum L. `Xanthi' with 3H-DNA (7.26 µg/ml) from N. tabacum L. `Xanthi nc' 3.5% of the initial radioactivity was found in acid-insoluble substances of the protoplasts. The addition of DEAE-dextran and poly-L-lysine to the incubation medium nearly doubled radioactivity adsorption. The absorption was inhibited by 2,4-dinitrophenol, KCN, and low temperature (0 C); this inhibition could not be reversed by exogenous ATP. About 500 tobacco plants established from protoplasts of a normally tobacco-mosaic virus-susceptible cultivar that had been allowed to absorb DNA prepared from a resistant cultivar did not show transfer of the virus-resistant gene.

A detailed analysis was performed of the disposition of exogenous DNA in plant protoplasts, by employing Escherichia coli3H-DNA and Nicotiana glutinosa protoplasts. In 5 to 20 hours, about 10% of the 3H-DNA entered the protoplasts. Competition experiments between the 3H-DNA and unlabeled DNA or thymidine showed that the entry occurred as undegraded 3H-DNA. Examination of intraprotoplast fractions revealed that 60 to 80% of the absorbed radioactivity resided in the "soluble" fraction of the cytoplasm and 20% in the nuclear fraction. The mitochondrion fraction also contained measurable radioactivity. Sizing on sucrose density gradients showed that the bulk of the absorbed E. coli DNA had been depolymerized. Of the incorporated radioactivity, 15% was accountable as DNA, exogenous as well as resynthesized, and 15% as RNA, protein, and other cell constituents. DNA/DNA hybridization test indicated that 17.6% of the re-extractable 3H-DNA retained homology with the E. coli DNA; this was equivalent to 2.6% of the absorbed radioactivity. Resynthesized receptor protoplast DNA was represented by a fraction at least 1.7% of the total absorbed radioactivity. The amount of bacterial DNA remaining in protoplasts suggests that each protoplast retained 2.3 x 10–15g donor DNA, or approximately half of the E. coli genome.

2 Present address: Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024.

1 This research was supported in part by an Elvenia J. Slosson Fellow-ship in Ornamental Horticulture awarded to T. M.






HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
ASPB Publications PLANT PHYSIOLOGY® THE PLANT CELL
Copyright © 1977 by the American Society of Plant Biologists