Selection and Characterization of Somatic Cell Hybrids (Protoplast, Fusion)
Hauptmann, Randal Mark
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https://hdl.handle.net/2142/77657
Description
Title
Selection and Characterization of Somatic Cell Hybrids (Protoplast, Fusion)
Author(s)
Hauptmann, Randal Mark
Issue Date
1984
Department of Study
Plant Biology
Discipline
Botany
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Plant Physiology
Language
eng
Abstract
Fusion of two cell lines with different amino acid analog or herbicide resistances allows selection of heterokaryons from unfused and homologous fusion products through the use of resistance complementation.
Carrot and tobacco somatic cell hybrids were isolated using a carrot cell line resistant to S-(2-aminoethyl)-L-cysteine (AEC) and a tobacco cell line resistant to DL-5-methyltryptophan (5MT). Karyotypic analysis of the initial double resistant lines showed the presence of chromosomes from both species. With time, most lines lost the double resistance and this was correlated with the elimination of carrot chromosomes. In a few cases a translocation between a portion of a carrot chromosome and a tobacco chromosome was observed. In the hybrid lines the AEC uptake resistance was similar to that of the carrot parent as well as carrot-type levels of several free amino acids not characteristic of tobacco.
In the absence of continuous selection pressure further loss of carrot chromosomes was observed along with restoration of AEC sensitivity characteristic of the tobacco parent.
Cloned D. carota cell lines selected as double resistant to DL-5-methyltryptophan (5MT) and azetidine-2-carboxylate (A2C) were fused with a N-(phosphonomethyl) glycine (glyphosate) resistant cell line. Hybrids expressed resistances to 5MT in a semidominant fashion while A2C and glyphosate resistance was expressed as dominant, or semidominant, in a line specific manner.
Benomyl, which inhibits nuclear fusion in yeast, did not appreciably reduce nuclear fusion in plant cell hybrids, based on complementation frequencies of 5MT and A2C, or 5MT and S-2(aminoethyl)-L-cysteine (AEC) resistant cell lines fused with the glyphosate resistant line. No binucleate cells were observed in hybrid colonies and hybrids possessed additive parental chromosome numbers. Benomyl at concentrations up to 50 ppm did not exhibit toxic effects in suspension or protoplast cultures if dissolved by autoclaving or boiling.
The inhibitors iodoacetate, iodoacetamide, N-ethylmaleimide, diethylpyrocarbonate, AEC, A2C, 5MT, p-fluorophenylalanine, ethionine, and 5-fluorouracil were tested for use as universal hybrid selection agents by fusing glyphosate with 5MT and A2C resistant cell strains to serve as testable markers. Treatment of protoplasts with lethal concentrations prior to fusion did not result in metabolic complementation nor subsequent recovery of heterokaryons.
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