Direct gene transfer via polyethylene glycol

Abstract

SUMMARY: Polyethylene glycol can be used to induce DNA uptake into plant protoplasts. Procedures for isolation, culture and transformation of N. tabacum protoplasts are described and can be adapted for other dicot and monocot species. Criteria for proof of transformation are discussed.

Structure, expression, chromosomal location and product of the gene encoding ADH1 inPetunia

“…[18], and aliquots of 1.5 × 107 protoplasts were placed into centrifuge tubes. To each tube, 100/~g of herring sperm DNA was added.…”

“…An equal volume of a solution containing 40~o PEG 8000, 0.4 M mannitol and 0.1 M Ca(NO3)2"4H20, pH 8.0, was immediately added, and the solution mixed gently. After a ten-minute period, 5 volumes of W5 [18] were slowly added, and the protoplasts pelleted as above. The pellet was resuspended in K3 [18] containing 0.4 M glucose and 0.5 M mannitol at a density of 5 x 105/ml.…”

“…After a ten-minute period, 5 volumes of W5 [18] were slowly added, and the protoplasts pelleted as above. The pellet was resuspended in K3 [18] containing 0.4 M glucose and 0.5 M mannitol at a density of 5 x 105/ml. After a 48-hour incubation at 26 °C in the dark, the protoplasts were removed to a centrifuge tube, one volume of W5 was slowly added and mixed, and the protoplasts were pelleted.…”

Plant genetic engineering and genetically modified crop breeding: history and current status

“…The generation and use of plant protoplasts (cells from which the walls have been stripped by treatment with enzymes) was pioneered at Nottingham University by Cocking in 1961 [58] . Protoplasts proved to be very suitable for the introduction of DNA or viruses and a further important development was the introduction of polyethylene glycol to greatly enhance the uptake of exogenous DNA by protoplasts [59] . In addition to adding DNA fragments, it proved possible to transfer chromosomes between cells of different species by protoplast fusion, forming cell hybrids known as cybrids.…”