Plant Molecular Biology Manual AI: 1-16, 1994.

© 1994 Kluwer Academic Publishers. Printed in Belgium.

PEG-mediated direct gene transfer and electroporation

ROLAND BILANG, ANDREAS KLOTI, MARTIN SCHROTT and

INGO POTRYKUS
Institute of Plant Sciences. Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092
Zurich, Switzerland

Introduction

a) Transformation of protoplasts

For many years of genetic manipulation in plants, direct uptake of naked DNA
by plant protoplasts has been the sole alternative to Agrobacterium tumefaciens-
mediated gene transfer. The first experiments demonstrating direct gene trans-
fer included the delivery of isolated plasmid DNA to protoplasts of petunia and
tobacco in the presence of poly-L-ornithine or polyethylene glycol (PEG)
[1-4]. During the following years, protoplast transformation mediated by PEG
[5] or electroporation [6] was substantially simplified and their efficiency in
model systems was increased by several orders of magnitude (reviewed by
Paszkowski et al. [7]).
The production of transgenic plants via direct gene transfer to protoplasts
depends on protoplast-to-plant regeneration and on efficient selection systems
for transgenic clones. Early gene transfer experiments focused on protoplasts
of Solanaceae species that are easily regenerable, and on the use of the bacterial
gene for neomycin phosphotransferase (npt II), conferring antibiotic resistance
to transformed clones (Table 1). During the past few years, protoplast-to-plant
regeneration was achieved for many other plant species. Transgenic plants of
the model plant Arabidopsis thaliana, of important crops such as J aponica and
Indica rice varieties, maize, and forage grasses have been obtained (Table 1).
Natural resistance of many monocotyledonous species to the antibiotic kana-
mycin [8,9] made the development of other selection systems necessary. In
addition to the npt II gene, the genes for hygromycin-phosphotransferase (hpt)
[ 10] and phosphinotricin-acetyltransferase (pat) [11] have proven useful for
the selection of stably transformed colonies in mono- and dicotyledonous
species (Table 1). Other selectable markers in use are streptomycin-phospho-
transferase [12], a mutant acetolactate synthase from Arabidopsis thaliana
conferring resistance to sulfonylurea herbicides [13], and a mutant dihydro-
folate reductase, conferring resistance to methotrexate [9].
PEG- and electroporation-mediated gene transfer is simple and efficient:
dozens of protoplast samples can be treated in a single experiment, and
thousands of individual transgenic plants can be obtained in model systems

PMAN-AI/I
Table I. Stable transformation of plants via DNA-mediated direct gene transfer to protoplasts

Year Plant species Trans- Type of Selectable marker Reference

formation transgenics gene 2/
technique' Selecting agent

1984 Nicotiana tabacum C Fertile plants npt II/kanamycin [4]

1985 Lotium multiflorum C Callus npt II/G-418 [8]
1985 Triticum monococcum C Callus npt II/kanamycin [44]
1986 Brassica campestris C Callus npt II/kanamycin [45]
1987 Petunia hybrida C Plants npt II/kanamycin [46]
1987 Brassica napus E Callus npt II/kanamycin, [47]
paromomycin
1988 Panicum maximum E Callus dlifr/methotrexate [9]
1988 Oryza sativa (Japonica) E Fertile plants hpt/hygromycin [48]
1988 Dactytis glomerata C,E Plants hpt/hygromycin [49]
1989 Solanum tuberosum E Fertile Plants npt II/paromomycin [50]
hpt/hygromycin
als/chlorosulfuron
1989 Arabidopsis thaliana C Fertile plants hpt/hygromycin [51, [52]
1990 Oryza sativa (Indica) C Fertile plants hpt/hygromycin [53]
1992 Festuca arundinacea C Fertile plants hpt/hygromycin [54]
pat/phosphinotricin
1993 Zea mays C Fertile plants nptll/kanamycin [55]
pat/phosphinotricin

'Transformation of protoplasts was performed by (E) electroporation or by (C) chemical

methods, i.e. treatment with PEG. 2npt II, neomycin phosphotransferase gene; hpt, hygromycin
phosphotransferase gene (both from Escherichia coli); pat, phosphinotricin acetyltransferase gene
(Streptomyces ssp.); dhfr, dihydrofolate reductase gene (Mus musculus); als, mutant acetolactate
synthase (Arabidopsis thaliana).

with tobacco. Manipulation of nucleic acids prior to transformation is possible,

and there are no host-range limitations. These advantages allowed the develop-
ment of a number of transient and integrative gene expression assays, which
are important tools for the investigation of the regulatory mechanisms of gene
expression.
Among the most important parameters that affect the efficiency of PEG-
mediated gene transfer to Nicotiana protoplasts are the concentration of
magnesium or calcium ions in the incubation mixture, the presence of inert
carrier DNA, and the molecular weight and concentration of PEG [5]. The
physical configuration of nucleic acids has an impact on gene transfer efficiency:
linearized double-stranded plasmid DNA molecules are more efficiently ex-
pressed and integrated into the genome than are supercoiled forms [e.g. 5, 14].
After delivery to protoplasts, single-stranded DNA molecules were efficiently
used as templates for in vivo duplex formation followed by genomic integration
[15, 16]. mRNA molecules transferred to electroporated protoplasts of dicoty-
ledonous and monocotyledonous species were efficiently translated [17].
Multiple copy integration of the foreign DNA and rearrangements of the

PMAN-Al/2