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Preparation of Nucleic Acids and a Genetic Transformation Attempt in Maize 1
Author(s) -
Coe E. H.,
Sarkar K. R.
Publication year - 1966
Publication title -
crop science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.76
H-Index - 147
eISSN - 1435-0653
pISSN - 0011-183X
DOI - 10.2135/cropsci1966.0011183x000600050012x
Subject(s) - nucleic acid , biology , dna , sodium , chloroform , nucleoprotein , biochemistry , saline , chromatography , botany , chemistry , organic chemistry , endocrinology
Nucleoproteins and nucleic acids of apparently high quality can be prepared from maize seedlings by simple modifications of saline extraction and chloroform deproteinization procedures used to isolate transforming principles in bacteria. Seedlings are disintegrated by brief blending (2 minutes) with saline‐EDTA‐sodium lauryl sulfate; the mash is strained, brought to 1M with sodium perchlorate, and emulsified with chloroform; nucleoproteins are precipitated in crude form from the aqueous fraction with ethanol. Analyses indicate that protein content can be greatly reduced by additional deproteinizations, and that ribonucleic acid can be reduced relative to deoxyribonucleic acid by winding the highly fibrous precipitates. Crude precipitates dissolved in saline‐citrate, isolated from genetic material dominant for three plant characters and four seed characters, were injected into the apical meristem region of recessive recipient seedlings, repeating one to three times. Spermine, a stabilizing agent for deoxyribonucleic acid, was included in some of the treatments; part of the treated seedlings were irradiated with 1,000R of X rays immediately following injection. No phenotypic effects indicative of transformation in the treated plants were observed; five exceptional seeds (in 26,000 produced from intercrosses among treated plants) were derived from outcross contamination. Competence, nucleases, penetration, susceptibility of loci, numbers treated, and degradation are indicated as problems that may need to be considered and solved in order to achieve transformation in higher plants.