Mitochendrial DNA was isolated from cotton (Pima S-5) with sufficient purity for endonuclease restriction analysis. Development of the protocol was aided by the use of a monitoring technique that allowed assessment of mtDNA purity and integrity during the isolation steps. The technique involved deproteinization of a small aliquot of the extract with CsCl in a microfuge tube with centrifugation followed by agarose get electrophoresis.

The tissue source was 4-day-old etiolated seedlings homogenized in a Waring blender at low speed (60 see) with 1:4 w/v extracting medium. Inclusion of 1.2M NaCl as osmoticum in the extracting buffer greatly improved subsequent digestion of contaminating nuclear DNA with DNAase. Lysis of the mitochondria was accomplished with sarcosyl and protease K. The mtDNA was banded in CsCl by centrifugation at 40,000 rpm for 40 hr. The product was restricted by Hind III, Bam HI, Eco RI, Pst I and Bst Ell. The last two enzymes gave the most uniform distribution of fragment sizes. The sum of the fragments indicate that the mitochondrial genome of cotton is exceptionally large compared to most other plants that have been examined. Cotton rivals muskmelon in this respect.