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Title:Effect of Liposome Encapsulation of Cauliflower Mosaic Virus and Its Dna on Plant Infectivity and Culture of Turnip (Brassica Rapa L.) Protoplasts, Their Uptake and Expression of Exogenous Dna
Author(s):Ulrich, Thomas Henry
Department / Program:Agronomy
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Biology, Plant Physiology
Abstract:An infectivity test was developed for assessing the effect of liposome encapsulation of cauliflower mosaic virus (CaMV) and its DNA. When grown at temperatures below 24 C, inoculated half-leaves of turnip (Brassica rapa L. cv. Just Right) produced distinct lesions. Decolorizing and staining leaves for starch increased 2 to 3 times the number of observable lesions. This starch lesion assay for CaMV provided infectivity-dilution curves with linear response regions and allowed accurate estimates of unknown virus concentrations.
Encapsulation of CaMV with liposomes composed of egg-lecithin, dicetylphosphate, and lysolecithin in a mol % ratio of 70:24:5, respectively, reduced the relative infectivity of CaMV. Unencapsulated CaMV labeled with tritium remained associated with liposomes when both were centrifuged together in sucrose density gradients. The infectivity of CaMV extracted with detergent from liposomes was not altered. Therefore, the adsorption of CaMV to liposomes effectively reduced the infectious pool of virus available for infection, suggesting that CaMV has a strong affinity at least for the artificial membrane of these liposomes. In contrast, a mixture of CaMV-DNA and liposome encapsulated CaMV-DNA was more infectious than the same quantity of CaMV-DNA alone. However, DNase treatment of this mixture or DNase treatment followed by purification of liposome encapsulated CaMV-DNA by gel filtration reduced the relative infectivity of CaMV-DNA substantially.
Protoplasts from mesophyll cells of turnip plants were isolated from peeled leaves treated with a mixture of cellulase and pectinase. Up to 36% of the protoplasts formed cell clumps in liquid media that was replenished weekly. Cell divisions began in 4 days and callus cells projected from aggregates in 14 days. Calli transferred from liquid to several solid media grew rapidly and one medium induced root formation. One liquid medium induced greening of calli, but no shoots were formed.
The expression of CaMV-DNA as newly synthesized virus particles was used to demonstrate the uptake of exogenous DNA by turnip protoplasts. Protoplasts were treated with purified CaMV-DNA and cultured in medium with {('32)P}. The production of CaMV in DNA treated protoplasts was detected as the result of the base resistant, acid precipitable property of {('32)P}-DNA of CaMV which co-sedimented with unlabeled CaMV in sucrose density gradients. Results taken together indicated that {('32)P}-CaMV was produced in turnip protoplasts treated with CaMV-DNA. Virus production was most efficient in protoplasts washed with only mannitol before DNA treatment and cultured 4 days in medium with normal (10('-3) M) phosphate levels. This CaMV-DNA infection system should allow efficient optimization of DNA uptake and expression in plant protoplasts.
Issue Date:1980
Description:91 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1980.
Other Identifier(s):(UMI)AAI8026606
Date Available in IDEALS:2014-12-14
Date Deposited:1980

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