Abstract

Protoplasts were isolated from tobacco and barley leaves in sucrose or mannitol using commercially available cellulases and macerozymes. Barley growth and protoplast isolation and purification conditions were optimized so that protoplasts were obtained in high yields free of unwanted debris and organelles. A technique for processing barley and tobacco protoplasts for examination by scanning electron microscopy was developed in which protoplasts seem to have maintained their structural integrity. Barley and tobacco protoplasts took up 3H-B. subtilis DNA, 125I-B. subtilis DNA or 125I-M. luteus DNA as a linear function of time (0-6 hr) and DNA concentration (0-200 μg/ml). Up to 16 pg of exogenous DNA was taken up per protoplast of which approximately one half became nuclear associated. Protoplasts were viable after the uptake as shown by standard staining and culturing techniques. Approximately 20% of the DNA taken up after typical 4 hr uptake reactions was of average gene size (5-10 x 105 daltons), and therefore of potential significance to host gene expression.

Degree

PhD

College and Department

Physical and Mathematical Sciences; Chemistry and Biochemistry

Rights

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