Successful transfer of the chloramphenicol transferase gene to Black Mexican Sweet (BMS) protoplasts by polyethylene glycol

Polyethylene glycol (PEG) technique has great promise for the transfer of genes into cereals and other graminaceous monocots, which are not readily transformed by Agrobacterium tumefaciens. This method is a desirable alternative to microinjection and electroporation since it is quick, inexpensive, reliable, and relatively nontoxic to transformed cell populations. In our laboratory, protoplast viability after PEG treatment is 57 to 77%.

The method developed by Krens for PEG transfer of genes to tobacco (Krens et al., Nature 296:72, 1982) has subsequently been modified by several laboratory groups (I. Potrykus, Plant Cell and Tissue Culture Vol. III, 289-302, 1987). One experimental variation of Krens' method, suitable for use with P.S. Chourey's BMS cell line, was recently sent to us by S. Howell, University of California, San Diego. We have made further adaptations of this technique so that we can now obtain excellent gene transfer to protoplasts of BMS-M, a fast-growing subline of BMS derived by C.E. Green and kindly given to us by D.A. Somers, University of Minnesota.

For PEG gene transfer experiments we use 5 x 106 protoplasts (S.R. Ludwig et al., Theor. Appl. Genet. 71:344, 1985) resuspended in 0.75ml of 0.2M mannitol and 80mM CaCl2 (pH 5.6). Twenty to 50µg plasmid DNA and 50µg sonicated calf thymus DNA are added to the cells and mixed thoroughly. Cells and DNA are then combined with 0.75ml 50% PEG 8000 solution, made in Krens' F medium, pH 7.2. The PEG solution is added slowly, a drop at a time, and the mixture is then incubated at 28 C for 30min without shaking. At the end of incubation the PEG-cell mixture is diluted slowly from 25% PEG to 2% PEG with F medium, in the manner of Krens. Cells are then collected by gentle centrifugation and resuspended in growth medium with 8% (439mM) mannitol.

To test our methods we have transformed BMS-M protoplasts with p CaMv 11 CN. In this vector, kindly provided by M. Fromm (Genes and Development, in press), the chloramphenicol transferase (CAT) gene is regulated by the cauliflower mosaic virus (CaMV) 35s promoter and the enhancer activity of an Adh intron fragment (I1). Transferred protoplasts were incubated for 40h before assay for CAT activity (Fromm, Proc. Natl. Acad. Sci. USA 82:5824, 1987).

Fig. 1 shows the results of one experiment. In PEG-treated cells transformed with 50µg p CaMV I1 CN DNA, the conversion of chloramphenicol to 1-and 3-acetate chloramphenicol was 37%. This represents a 520-fold increase over background. We obtain similar results when only 20µg DNA is used in the transformation.

Figure 1. Expression of CAT genes transferred into BMS-M protoplasts by PEG treatment. Lanes: 1, 5 x 106 PEG-treated protoplasts with 50µg pCaMVI1 CN DNA; 2, 5 x 106 PEG-treated protoplasts with no added DNA; 3, 1000 units of chloramphenicol transferase from E. coli plus 0.1 uCi 14C chloramphenicol. a, 1,3-diacetyl chloramphenicol. b, 3-acetylchloramphenicol. c, 1-acetylchloramphenicol. d, 14C chloramphenicol.

M. Antonelli, M. Moreno and J. Stadler

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