Note: Descriptions are shown in the official language in which they were submitted.
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13414~ 1
5-14872/1-3/ZFM
Transformation of hereditary material of plants
The present invention relates to a novel process for transforming
hereditary material of plar.~ts and to the plant products obtained by
said process.
Plants having novel and/or improved properties can be produced by
introducing new genetic information into plant material.
In view of the rapid rise in world population and the concomitant
increase in the need for food and raw materials, increasing the
yield of useful plants as well as the increased extraction of plant
storage substances, and in particular advances in the field of
nutrition and medicine, are among the most urgent tasks of biologic-
al research. In this connection, the following essential aspects
may be mentioned by way of example: strengthening the resistance of
useful plants to unfavourable soil or climatic conditions as well as
to disease and pests; increasing resistance to plant protective
agents such as insecticides, herbicides, fungicides and bacteri-
cides; and a useful change in the nutrient content or of the harvest
yield of plants. Such desirable effects could be produced generally
by induction or increased formation of protective substances,
valuable proteins or toxins. A corresponding influence on the
hereditary material of plants can be brought about, for example, by
inserting a specific foreign gene into plant cells without utilising
conventional breeding methods.
The transfer of novel DNA sequences into plant cells using
genetically manipulated plant infecting bacteria has been described
in the literature in a number of publications, for example Nature,
Vol. 303, 209-213 (1983); Nature, Vol. 304, 184-187 (1983); Scien-
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~3414~1
tific American 248(6), 50-59 (1983); EMBO-Journal 2(6), 987-995
(1983); Science 222, 476-482 (1983); Science 223, 247-248 (1984); or
Proc. Natl. Acad. Sci. USA 80, 4803-4807 (1983). In these publi-
cations, the natural properties of these bacteria for infecting
plants were utilised to insert new genetic material into plant
cells. So far such insertion has been made using preferably
Agrobacterium tumefaciena itself or the Ti plasmid thereof, and also
cauliflower mosaic virus.
In contrail-stinction thereto, the novel process of this invention
makes possible the direct transfer of a gene without the use of
biological vectors. Pathogens have been used as vectors in the known
processes. As the process of this invention is performed without
pathogens, the limitations imposed by the host specificity of
pathogens also do not apply. The development of the plants on which
the novel process of transformation is carried out is not impaired
by said process.
In addition to the process for transforming hereditary material of
plants, the present invention also relates to the products obtain-
able by said process, in particular protoplasts and plant material
derived therefrom, for example cells and tissues, in particular
complete plants that have been regenerated from said protoplasts and
the genetically identical progeny thereof.
Within the scope of the present invention, the following definitions
aPPlY:
gene: structural gene with flanking expression
signals
structural gene: protein-coding DNA sequence
expression signals: promoter signal and termination signal
plant expression
signal: expression signal that functions in plants
promoter signal: signal that initiates transcription
termination signal: signal that terminates transcription
1341471
21489-6725
enhancer signal: signal that promotes transcription
replication signal: signal that makes possible DNA
replication
integration signal: DNA sequence that promotes the
integration of the gene into genomic DNA
hybrid gene: gene constructed from heterologous DNA
sequences, i.e. DNA sequences of
different origin that may be natural as
well as synthetic DNA sequences
carrier DNA: a neutral (i.e. not participating in the
function of the gene) DNA sequence
flanking the gene
isolated gene: DNA sequence coding for a single protein
and separated from the original DNA
NPT II gene: neomycin 3'-phosphotransferase gene, type
II, of transposon Tn 5 [Rothstein, S.J.
and W.S. Retznikoff, Cell 23, 191-199
(1981)]
genomic DNA: DNA of the plant genome (total or part
thereof).
The following drawings illustrate embodiments of the
invention.
Fig. 1 shows the construction of plasmids pABDI and
pABDII comprising the NPT II gene under the control of the 5' and
3' expression signals of gene VI of the cauliflower mosaic virus
[CaMV] as described in Example 1 a).
Fig. 2 shows the construction of plasmid pCaMV6Km
comprising within a defective CaMV genome the kanamycin-resistant
3a 1 3 4 1 4 7 1
gene NPT II in substitution of the CaMV gene VI, as described
in Example 2a).
The present invention is concerned with a novel
process for the transformation of hereditary material of
plants, which process comprises transferring a gene direct
into plant cells without the aid of natural systems for
infecting plants. Such transformation is accordingly a
vector-free transformation. In this vector-free
transformation, the foreign gene for insertion is under the
control of plant expression signals. The vector-free
t ransformat ion of plant genes is preferably carried out by
introducing a foreign gene for insertion into plant cells
together with plant protoplasts acting as recipients (receptor
protoplasts) into a suitable solution and leaving them until
the gene has been taken up by the protoplasts.
The invention provides a process for stable
integration of foreign DNA under control of plant expression
signals, said foreign DNA being unaccompanied by T-DNA border
regions of a Ti-plasmid, into hereditary material of a plant
protoplast, which process comprises
(a) contacting said foreign DNA, which is under the control
of plant expression signals and unaccompanied by the T-DNA
border regions of the Ti-plasmid, with a plant protoplast in a
medium and under conditions that renders the plant protoplast
permeable to DNA molecules for a period of time sufficient for
the DNA to be taken up by the plant protoplast, wherein the
21489-6725
3b 1 3 4 1 4 7 1
direct gene transfer is carried out by a technique selected
from the group consisting of:
a) polyethylene glycol treatment;
b) electroporation and polyethylene glycol treatment;
c) electroporation and heat shock treatment;
d) heat shock, electroporation and polyethylene glycol
t reatment ;
e) heat shock treatment and polyethylene glycol
t reatment ;
(b) expressing and replicating the gene being stably
incorporated into the plant genome.
The invention also provides a process for the
production of a transgenic plant, which comprises
(a) contacting a foreign DNA, which is under the control of
plant expression signals and unaccompanied by T-DNA border
regions of a Ti-plasmid, with a plant protoplast in a medium
and under conditions that renders the plant protoplast
permeable to DNA molecules for a period of time sufficient for
the DNA to be taken up by the plant protoplast, wherein the
direct gene transfer is carried out by a technique selected
from the group consisting of=
1) polyethylene glycol treatment;
2) electroporation and polyethylene glycol treatment;
3) electroporation and heat shock treatment;
4) heat shock, electroporation and polyethylene glycol
treatment;
21489-6725
30041-7
3 c '"
,~ ~. b:
5) heat shock treatment and polyethylene glycol
treatment;
(b) expressing and replicating the gene being stably
incorporated into the plant genome,
(c) regenerating transgenic plants from the previously
transformed plant cells or protoplasts.
The invention also provides a transgenic plant
cell comprising stably integrated into the plant genome a
DNA which is wholly or partially heterologous with respect
to the recipient cell and has been recombined such that it
essentially consists of a structural gene and flanking plant
active expression signals, which are heterologous to said
structural gene, with the proviso that the integrated DNA
does not contain part from plant pathogens causing the
natural plant infectious properties of said pathogen.
The invention also provides use in a genetic cross
for obtaining progeny plants of a plant comprising a
transgenic plant cell comprising stably integrated into the
plant genome a DNA, which is wholly or partially
heterologous with respect to the recipient cell and has been
recombined such that it essentially consists of a structural
gene and flanking plant active expression signals, which are
heterologous to said structural gene, with the proviso that
the integrated DNA does not contain parts from plant
pathogens causing the natural plant infectious properties of
said pathogen.
As protoplasts it is preferred to use those of a
single plant species or of a systematic unit which is a
suborder of a species.
F
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1341471
The foreign gene and the protoplasts are conveniently left in the
solution for a pert.nd of time ranging from several seconds to
several hours, preferably from 10 to 60 minutes, most preferably for
30 minutes.
The process of this invention is susceptible of broad application.
Thus it is possible to transfer any structural genes of plant
origin, for example the zein gene [Weinand, U., et al., Mol. Gen.
Genet. 182, 440-444 (1981)), of animal origin, for example the TPA
gene [tissue -type plasminogen activator gene; Pennica, D., et al.,
Nature 301, 214-221 (1983)], of microbial origin, for example the
NPT II gene, or also of synthetic origin, for example the insulin
gene [Stepien, P., et al., Gene 24, 289-291 (1983)], into hereditary
material of plants, provided that the structural genes are flanked
by expression signals which are expressed in plants and which
expression signals may be of plant, animal, microbial or synthetic
origin.
The transferred genes, consisting of structural gene and flanking
expression signals, may be naturally occurring genes as well ae
hybrid genes. In the process of this invention, it is preferred to
use those genes whose expression signals are of animal or, in
particular, of plant or synthetic origin. Exemplary of such genes
are:
a) complete genes of plants consisting of the structural gene with
its natural expression signals;
b) completely synthetic genes consisting of a structural gene of
synthetic origin, flanked by expression signals of synthetic origin;
c) structural genes of plant origin, flanked by plant expression
signals, with the structures and expression signals originating from
various plant species;
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d) structural genes of plant origin, flanked by expresafon signals
of synthetic origin;
e) structural genes of animal, microbial or synthetic origin,
flanked by expression signals of plant origin; or
f) structural genes of animal or microbial origin, flanked by
expression signals of synthetic origin.
Most preferred are structural genes of bacterial origin, flanked by
expression signals of plant origin, in particular those originating
from plant viruses. Particularly suitable expression signals for use
in the process of this invention are the expression signals of gene
VI of cauliflower mosaic virus.
The hybrid genes are prepared by microbiological techniques which
are known per se, retaining the reading frame of the coding for the
proteins to be produced by the plant cell. Such techniques are known
and are described e.g. in the following publications: "Molecular
Cloning", Maniatis, T., Fritsch, E.F. and J. Sambrook, Cold Spring
Harbor Laboratory, 1982, and "Recombinant DNA Techniques",
Rodriguez, R.L. and R.C. Tait, Addison-Wesley Publishing Comp.,
London, Amsterdam, Don Mills. Ontario, Sydney, Tokyo, 1983.
To integrate the foreign gene into the genomic DNA of the plant
cell, it is advantageous if the gene, consisting of structural gene
and plant expression signals, is flanked by neutral DNA sequences
carrier DNA). The carrier DNA may consist of two linear DNA strands,
so that the construction to be inserted into the plant cell is a
linear DNA molecule. The DNA sequence prepared for the gene trans-
formation can, however, also have an annular structure (plasmid
structure). Such plasmids consist of a DNA strand into which the
foreign gene containing the expression signals is integrated. The
carrier DNA can be of synthetic origin or can be obtained from
naturally occurring DNA sequences by treatment with suitable
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1341471
restriction enzymes. Thus, for example, naturally occurring plasmids
which have been opened with a selective restriction enzyme are
suitable for use as carrier DNA.
Exemplary of such a plasmid is the readily obtainable pUC8 plasmid
(described by Messing, J. and J. Vieira, Gene 19, 269-276, 1982).
Fragments of naturally occurring plasmida can also be used as
carrier DNA. For example, the deletion mutant for gene VI of
cauliflower mosaic virus can be used as carrier DNA.
The prohability of the genetic transformation of a plant cell can be
enhanced by different factors. Accordingly, as is known from
experiments with yeast, the number of successful stable gene
transformations increases
1) with the increasing number of copies of the new genes per cell,
2) when a replication signal is combined with the new gene, and
3) when an integration signal is combined with the new gene.
The process of this invention is therefore susceptible of especially
advantageous application when the transferred gene is combined with
a replication signal which is effective in plant cells or with an
integration signal which is effective in plant cells, or which is
combined with both signals.
The expression of a gene in a plant cell depends on the trans-
cription frequency of the gene in a messenger RNA sequence. It is
therefore advantageous if the new gene is combined with an enhancer
signal that promotes this transcription. Methods meriting particular
attention are those for transferring a gene which is combined with
replication, integration and enhancer signals that are effective in
plants.
It is further of great technical advantage if the transferred gene
has a selective marker function, i.e. if the transformed plant cells
can be separated from the non-transformed plant cells under specific
conditions of selection. A marker function of this kind permits the
'- i3414~1
process to be carried out efficiently in that only those plant cells
need to be regenerated to calls or complete plants by microbiolog-
ical techniques, the hereditary material of which plants contains a
gene capable of expression that permits marker-specific methods of
selection.
Whereas protoplasts, cell culture cells, cells in plant tissues,
pollen, pollen tubes, egg-cells, embryo-sacs or zygotes amd embryos
in different stages of development are representative examples of
plant cells which are suitable starting materials for a transfor-
mation, protoplasts are preferred on account of the possibility of
using them direct without further pretreatments.
Isolated plant protoplasts, cells or tissues can be obtained by
methods which are known per se or by methods analogous to known
ones.
Isolated plant protoplasta which are also suitable starting mater~~
isle for obtaining isolated cells and tissues can be obtained from
any parts of the plant, for example from leaves, embryos, stems,
blossoms, roots or pollen. It is preferred to use leaf protoplasts.
The isolated protoplasts can also be obtained from cell cultures.
Methods of isolating protoplasts are described e.g. in Gamborg, O.L.
and Wetter, L.R., Plant Tissue Culture Methods, 1975, 11-21.
The transfer of the new genes into plant cells is effected direct
without using a natural system for infecting plants such as a plant
bacterium, a plant virus, or transfer by insects or phytopathogenic
fungi. This is achieved by treating the plant cells which it is
desired to transform direct with the gene to be transferred by
introducing the foreign gene and plant protoplasta into a suitable
solution and leaving them therein until the foreign gene has been
taken up by the protoplasts. The transformation frequency can be
increased by combining this step With techniques which are employed
in microbiological research for gene transfer, for example by
treatment with poly-L-ornithine or poly-L-lysine, liposome fusion,
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1~414~1
DNA protein complexing, altering the charge at the protoplast
membrane, fusion with microbial protoplasts, or calcium phosphate
co-precipitation and, in particular, by treatment with polyethylene
glycol, heat shock and electroporation, as well as a combination of
these last three mentioned techniques.
Suitable solutions into which the foreign gene and the receptor
protoplasts are introduced are preferably the osmotically stabilised
culture media employed for protoplast cultures.
Numerous culture media are already available which differ in their
individual components or groups of components. However, the composi-
tion of all media is in accordance with the following principle:
they contain a group of inorganic ions in the concentration range
from about 10 mg/~, to several hundred mg/~, (so-called macroelementa
such as nitrate, phosphate, sulfate, potassium, magnesium, iron), a
further group of inorganic ions in maximum concentrations of several
mg/~, (so-called microelements such as cobalt, zinc, copper,
manganese), then a number of vitamins (for example inositol, folic
acid, thiamine), a source of energy a.nd carbon, for example
saccharose or glucose, and also growth regulators in the form of
natural or synthetic phytohormones of the auxin and cytokinin
classes in a concentration range from 0.01 to 10 mg/~. The culture
media are additionally stabilised osmotically with sugar alcohols
(for example mannitol) or sugar (for example glucose) or salt ions
(for example CaCl2), and are adjusted to a pH in the range from 5.6
to 6.5.
A more detailed description of conventional culture media will be
round, for example, in Koblitz, H., Methodische Aspekte der Zell-
und Gewebezuchtung bei Gramineen unter besonderer Berucksichtigung
der Getreide, Kulturpflanze XXII, 1974, 93-15J.
A particularly suitable technique of gene transformation is
"polyethylene glycol treatment", where the term "polyethylene
glycol" within the scope of this invention denotes not only the
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13414~1-
substance polyethylene glycol itself, but will also be understood as
generic term for all substances that likewise modify the protoplast
membrane and are employed e.g. in the field of cell fusion. The term
thus also comprises other polyhydric alcohols of longer chain
length, for example polypropylene glycol (425 to 4000 g/mole),
polyvinyl alcohol or polyhydric alcohols whose hydroxyl groups are
partially or completely etherified, as well as the detergents which
are commonly employed in agriculture and tolerated by plants, and
which are described e.g. in the following publications:
"Mc Cutcheon's Detergents and Emulsifiers Annual"
MC Publishing Corp., Ridgewood New Jersy, 1981;
Stache, H., "Tensid-Taschenbuch",
Carl Hanser Verlag, Munich/Vienna, 1981
If polyethylene glycol itself is used (as in Examples 1 to 3, 5 and
7), then it is preferred to use a polyethylene glycol having a
molecular weight in the range from 1000 to 10,000 g/mole, preferably
from 3000 to 8000 g/mole.
Of the above mentioned substances, it is preferred to use poly-
ethylene glycol itself.
A substantial and reproducible transformation frequency of 10 5 is
achieved by means of the techniques described above. However, this
frequency can be greatly improved on by the appropriate techniques
described in more detail hereinafter.
In the polyethylene glycol treatment, the procedure can be for
example such that either a suspension of the protoplasts is added to
a culture medium and then the gene, which is normally employed as
plasmid, is added in a mixture of polyethylene glycol and culture
medium, or, advantageously, protoplasts and gene (plasmid) are first
added to the culture medium and then polyethylene glycol is added.
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13414~1 .
In the process of this invention, electroporation and heat shock
treatment have also proved particularly advantageous techniques.
In electroporation (Neumann, E. et al., The EMBO Journal 7, 841-845
(1982)], protoplasts are transferred to an osmoticum, for example a
mannitol/magnesium solution and the protoplast suspension is
introduced into the electroporator chamber between two electrodes.
By discharging a condenser over the suspension, the protoplasts are
subjected to an electrical impulse of high voltage and brief
duration, thereby effecting polarisation of the protoplast membrane
and opening of the pores in the membrane.
In the heat treatment, protoplasts are suspended in an osmoticum,
for example a solution of mannitol/calcium chloride, and the
suspension is heated in small containers, for example centrifuge
tubes, preferably in a water bath. The duration of heating will
depend on the chosen temperature. In general, the values are in the
range of 40°C for 1 hour and 80°C for 1 second. Optimum results
are
obtained at a temperature of 45°C over 5 minutes. The suspension is
subsequently cooled to room temperature o. ~.~~wer.
It has also been found that the transformation frequency can be
increased by inactivating the extracellular nucleases. Such an
inactivation can be effected by using divalent cationa that are
tolerated by plants, for example magnesium or calcium, and also
preferably by carrying out the transformation a.~: a high pH value,
with the optimum pH range being from 9 to 10.5.
Surprisingly, the selective use of these different methods results
in the enormous increase in transformation frequency that has long
been an objective in the field of genetic engineering.
The lower the transformation frequency in gene transformation, the
more difficult and time-consuming it is to find the few cloned cells
resulting from the transformed cells from among the enormous number
of non-transformed clones. Where the transformation frequency is
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low, the use of conventional screening techniques is almost or
completely impossible, unless the gene employed is one with selec-
tive marker function (e.g. resistance to a specific substance). Low
transformation frequency thus requires a very substantial investment
in time and effort when using genes without marker function.
In transformations using genes without marker function it is only
possible to employ conventional screening techniques for finding
cloned cells by selection efficiently and successfully if the
transformation frequency is in the order of percentages (about
10 2). As will be shown below, the desired transformation frequency
can now be achieved by means of the process of this invention.
Surprisingly, the specific use of different techniques in the
process of this invention results in an enormous increase in the
previously attained transformation frequency up to 1 to 2
Combining foreign gene and receptor protoplasta before employing the
other techniques such as polyethylene glycol treatment, electro-
poration and heat shock treatment brings about an improvement in
transformation frequency of the order of about a power of ten as
compared with a procedure in which the sequence of the steps
employed is different.
Electroporation effects a 5- to 10-fold, and heat shock treatment a
10-fold or greater, improvement in transformation frequency.
A combination of two or three of the following techniques has proved
advantageous: polyethylene glycol treatment, heat shock treatment
and electroporation, with particularly good results being obtained
by employing these techniques after the foreign gene and protoplasta
have been introduced into a solution. The preferred technique is
heat shock treatment before the polyethylene glycol treatment and
the optional subsequent electroporation. In general, the additional
electroporation effects a further increase in transformation
-12- 1341471
frequency; but in some cases the results obtained by heat shock and
polyethylene glycol treatment are no longer essentially improved by
additional electroporation.
Just as the techniques can be combined with one another, so it is
also possible to combine the use of divalent cations which are
tolerated by plants and/or carrying out the transformation at pH 9
to 10.5 both with individual techniques, as well as combined
techniques, preferably with polyethylene glycol treatment, heat
shock treatment and electroporation. The numerous combination
possibilities permit the process of this invention to be adapted
very well to the respective conditions.
The combination of heat shock treatment, polyethylene glycol
treatment and, optionally, electroporation subsequent to the already
existing combination of foreign gene and receptor protoplasts
results in a transformation frequency of 10 2 to 10 3.
Accordingly, the process of this invention permits a high transfor-
mation frequency to be achieved without utilising biological vectors
for the transformation, for example cauliflower mosaic virus or
Agrobacterium.
An advantageous method comprises for example transferring proto-
plasts to a mannitol solution and mixing the protoplast suspension
so obtained with the aqueous solution of the gene. The protoplasts
are then incubated in this mixture for 5 minutes at 45°C and
subsequently cooled to 0°C over 10 seconds. Then polyethylene glycol
(mol. wt. 3000 to 8000) is added until the concentration is in the
range from 1 to 25 %, preferably about 8 %. After cautious thorough
mixing, treatment is carried out in an electroporator. The proto-
plast suspension is then diluted with culture medium and the
protoplasts are taken into culture.
-13- 13414 71
The process of this invention is suitable for the transformation of
all plants, especially those of the systematic groups Angiospermae
and Gymnospermae.
Among the Gymnoapermae, the plants of the Coniferae class are of
particular interest.
Among the Angiospermae, plants of particular interest are, in
addition to deciduous trees and shrubs, plants of the following
families: Solanacese, Cruciferae, Compositae, Liliaceae, Vitaceae,
Chenopodiaceae, Rutaceae, Bromeliaceae, Rubiacese, Theaceae,
Musacese or Gramineae and of the order Leguminosae, in particular of
the family Papilionacese. Preferred plants are representatives of
the Solanaceae, Cruciferae and Graminese families.
To be particularly mentioned are plants of the species Nicotiana,
Petunia, Hyoscyamus, Brasaica and Lolium, as for example,
Nicotiana tabacum, Nicotiana plumbagenifolia, Petunia hybrids,
Hyoscyamus muticus, Brasaica napus, Brasaica raps and Lolium
multiflorum.
In the field of transformation of plant cells, interest focuses in
particular on the high yield cultivated plants such as maize, rice,
wheat, barley, rye, oats and millet.
All plants which can be produced by regeneration from protoplasts
can also be transformed utilising the process of this invention. So
far it has not been possible to manipulate genetically representa-
tives of the Graminese family (grasses), which also comprises
cereals. It has now been shown that graminaceous cells, including
cereal cells, can be transformed genetically by the above described
method of direct gene transformation. In the same way, transforma-
tion of cultivated plants of the genus Solanum, Nicotiana, Brassica,
Beta, Pisum, Phaseolus, Glycine, Heliauthus, Allium, wheat, barley,
oat, Setaria, rape, rice, Cydonia, Pyrus, Malus, Rubus, Fragaria,
-14- 1341471
Prunus, Arachis, Secale, Panicum, Saccharum, Coffea, Camellia, Musa,
Ananas, Hitis or Citrus is possible and desirable, even if the total
yields and crop areas are smaller worldwide.
The proof of transformed genes can be adduced in a manner known per
se, for example by crossing analyses and molecular biological
assays, including in particular the Southern blot analysis and
enzyme activity tests.
The Southern blot analysis can be carried out for example as
follows: the DNA isolated from the transformed cells or protoplasts
is electrophoresed in 1 % agarose gel after treatment with restric-
tion enzymes and transferred to a nitrocellulose membrane [Southern,
E.M., J. Mol. Biol. 98, 503-517 (1975)), and hybridised with the DNA
whose existence it is desired to establish and which was nick-trans-
lated [Right', W.J., Dieckmann, M., Rhodes, C. and P. Berg, J. Mol.
Biol. 113, 23%- 51, (1977)) (DNA specific activity 5 x 108 to
10 x 108 c.p.m./ug). The filters are washed 3 times for 1 hour with
an aqueous solution of 0.03 M sodium citrate and 0.3 M sodium
chloride at 65°C. The hybridised DNA is visualised by darkening an
X-ray film for 24 to 48 hours.
Testing for enzyme activity - explained in more detail in the assay
for aminoglycoside phosphotransferase (enzyme for kanamycin-specific
phosphorylation) - can be carried out for example as follows: Callus
or leaf pieces (100 to 200 mg) are homogenised in 20 p,~, of extrac-
tion buffer in an Eppendorf centrifuge tube. The buffer is modified
from that used by Herrera-Estrella, L., DeBlock, M., Messens,
E., Hernalsteens, J.-P., Van Montagu, M. and J. Schell, EMBO J. 2,
987-995 (1983) omitting bovine serum albumin and adding 0.1 M
sucrose. The extracts are centrifuged for 5 minutes at 12000 g and
bromophenol blue is added to the supernatant to a final concen-
tration of 0.004 %. The proteins in 35 uJ~ of supernatant are
separated by electrophoresis in a 10 % non-denaturing polyacrylamide
gel. The gel is covered with a layer of agarose gel containing
kanamycin and r-32P labelled ATP, incubated, and the phosphorylated
~ls" 1341471
reaction products are transferred to Whatman*p81 phosphocelluloae
paper. The paper is washed 6 times with deionised water at 90°C and
then autoradiographed.
The following Examples illustrate the present invention in more
detail but without limiting the scope thereof. They describe the
construction of a hybrid gene and the insertion thereof in carrier
DNA sequences of cyclic character, the transfer of said hybrid gene
into plant cells, selection of the transformed plant cells and
regeneration of completo plants from the trannformod plant cells as
well as the genetic crossing and molecular biological analysis
thereof.
In the Examples, the process of the invention is illustrated as
follows:
1) by transformation of tobacco plants by transfer of the NPT II
gene by joining promoter and termination signals of the CaMV gene VI
to the NPT II gene, inserting said gene into the pUC8 plaemid and
transferring the resultant chiroaeric plasmid into isolated tobacco
protoplasts by polyethylene glycol treatment;
2) by transformation of plants of the genus Brassica by transfer
of the NPT II gene by joining promoter and termination signals of
the CaMV gene VI to the NPT II gene, inserting this construction
instead of the CaMV gene VI into the CaMV genome, and transferring
the resultant chimaeric plasmid into isolated Brassica protoplasts
by polyethylene glycol treatment, and
3) by transformation of plants of the genus Lolium by transfer of
the NPT II gene by joining promoter and termination signals of the
CaMV gene YI to the NPT II gene, inserting said gene into the pUC8
plasmid, and transferring the resultant chimaeric plasmid into
isolated Lolium protoplasts by polyethylene glycol treatment.
*Trade-mark
21489-6725
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1341471
Further, the advantageous effect on the transformation by heat shock
treatment and electroporation as well as the combined method of heat
shock treatment, polyethylene glycol treatment and electroporation
after combining protoplasts and NPT II gene will be exemplified.
Example 1: Transformation of cells of Nicotiana tabacum c.v. Petit
Havana SRI by transfer of the NPT IIgene
a) Construction of the pABDI plasmid
The freely available plasmids pKm 21 and pKm 244 [Beck, E. et al.,
Gene 19, 327-336 (1982)] are cut with the PstI restriction endonucle-
ase. The fragments of the plasmids which are used for recombination
are purified by electrophoresis in 0.8 % agarose gel. The plasmid
pKm 21244 resulting from the combination of the fragments contains a
combination of the 5'- and 3'-Bal 31 deletions of the NPT II gene,
as described by Beck et al in Gene 19, 327-336 (1982). Joining the
promoter signal of cauliflower mosaic virus to the HindIII fragment
of the plasmid pKm 21244 is effected by constructing the linker
plasmid pJPAX. The coupling plasmid pJPAX is obtained from the
plasmids pUC8 and pUC9 [Messing, J. and J. Vieira, Gene 119, 269-276
(1982)]. 10 base pairs of the linker sequence of the plasmid pUC9
are deleted by restriction at the HindIII and SalI sites and the
resultant cohesive ends are filled in by treatment with the poly-
merase I Klenow fragment [Jacobson, H. et al., Eur. J. Biochem. 45,
623, (1974)] and ligating the polynucleotide chain, thus restoring
the HindIII site. An 8 base pair synthetic XhoI linker is inserted
at the Smal site of this deleted linker sequence. Recombination of
the appropriate XorI and HindIII fragments of the plasmid pUC8 and
of the modified plasmid pUC9 yields the plasmid pJPAX with a
partially asymmetric linker sequence containing the following
sequence of restriction sites: EcoRI, SMaI, BamHI, SalI, PstI,
HindIII, BamHI, XhoI and EcoRI. Joining of the 5' expression signals
of the CaMV gene VI and the HindIII fragment of the NPT II gene is
carried out on the plasmid pJPAX by inserting the promoter region of
the CaMV VI gene between the PstI and HindIII sites. The plasmid so
-17- 141471
obtained is restricted at its single HindIII site and the HindIII
fragment of the plasmid pKm 21244 is inserted into this restriction
site in both orientations, yielding the plasmids pJPAX CaKmø and
pJPAX CaKm . To provide an EcoRV site near the 3'-terminal region
of the NPT II hybrid gene, a BamHI fragment of the plasmid pJPAX
CaKmø is inserted into the BamHI site of the plasmid pBR 327
[Soberon, X. et al., Gene 9, 287-305 (1980)], yielding the plasmid
pBR 327 CaKm. The EcoRV fragment of the plasmid pBR 327 CaKm, which
contains the new DNA construction, is used to replace the EcoRV
region of the CaMV gene VI, which is cloned at the SalI site in the
plasmid pUC8, thereby placing the protein-coding DNA sequence of
the NPT II gene under the control of the 5' and 3' expression
signals of the cauliflower mosaic gene VI. The plasmids so obtained
are designated pABDI and pABDII respectively (q.v. Fig. 1).
b) Transformation of protoplasts of Nicotiana tabacum c.v. Petit
Havana SRI by transfer of the NPT gene as part of the plasmfd
pABDI by PEG treatment
Tobacco protoplasta at a population density of 2~106 ger ml are
suspended in 1 ml of K3 medium [q.v. Z. Pflanzenphysiologie 78,
453-455 (1976); Mutation Research 81 (1981) 165-175], containing
0.1 mg/~, of 2,4-dichlorophenoxyacetic acid, 1.0 mg/~, of 1-naphthyl-
acetic acid and 0.2 mg/~ of 6-benzylaminopurine, which protoplasta
have been obtained beforehand from an enzyme suspension by flotation
on 0.6 molar sucrose at pH 5.8 and subsequent sedimentation (100 g
for 5 minutes) in 0,17 M calcium chloride at pH 5.8. To this suspen-
sion are added, in succession, 0.5 ml of 40 % polyethylene glycol
(PEG) with a molecular weight of 6000 in modified (adjusted again to
pH 5.8 after autoclaving) F-medium [Nature 296, 72-74 (1982)] and
65 u~, of an aqueous solution containing 15 ug of the plasmid pABDI
and 50 ug of calf thymus DNA. This mixture is cultured for 30
minutes at 26°C with occasional agitation and subsequent stepwise
dilution with F medium. The protoplasts are isolated by centrifuging
(5 minutes at 100 g) and resuspended in 30 ml of fresh K3 medium.
Further incubation is carried out in 10 ml portions in Petri dishes
of 10 cm diameter at 24°C and in the dark. The population density
-18- 1341471
is 6.3~10'' protoplasts per ml. After 3 days the culture medium in
each dish is diluted with 0.3 parts by volume of fresh K3 medium and
incubated for a further 4 days at 24°C and 3000 lux. After a total
of 7 days, the clones developed from the protoplasts are embedded
in a culture medium solidified with 1 l of agarose and containing
50 mg/~, of kanamycin, and cultured at 24°C in the dark by the bead
type culture method [Plant Cell Reports, 2, 244-247 (1983)]. The
culture medium is replaced every 5 days by fresh nutrient solution
of the same kind.
c) Regeneration of kanamycin-resistant tobacco plants
After 3 to 4 weeks of continued culturing in kanamycin-containing
culture medium, the resistant calli of 2 to 3 mm diameter are
transferred to agar-solidified LS culture medium [Physiol. Plant 18,
100-127 (1965)), containing 0.05 mg/~ of 2,4-dichlorophenoxyacetic
acid, 2 mg/~, of 1-naphthylacetic acid, 0.1 mg/~ of 6-benzylamino-
purine, 0.1 mg/x of kinetin and 75 mg/~, of kanamycin. Kanamycin-
resistant Nicotiana tabacum Petit Havana SRI plants are obtained by
inducing shoots on LS medium containing 150 mg/~, of kanamycin and
0.2 mg/~ of 6-benzylaminopurine, and subsequent rooting on T medium
[Science 163, 85-87 (1969)].
d) Detection of the NPT II gene in hereditary material of plants
Samples of 0.5 g of callus of the transformed cell cultures or leaf
tissue of the plants regenerated therefrom are homogenised at 0°C in
15 % saccharose solution containing 50 mmoll~ of 1-ethylenediamine
N,N,N',N'-tetraacetic acid (EDTA), 0.25 mol/~, of sodium chloride and
50 mmol/~ of a,a,a-tris(hydroxymethyl)methylamine hydrochloride
(TRIS-HC1) at pH 8. Centrifugation of the homogenate for 5 minutes
at 1000 g gives a crude nuclear pellet which is resuspended at pH
8.0 in 15 % saccharose solution containing 50 mmol/~, of EDTA and
50 mmol/~ of TRIS-HC1. Sodium dodecyl sulfate is added to a final
concentration of 0.2 % and heated for 10 minutes to 70°C. After
cooling to 20°-25°C, potasasium acetate is added to the mixture
to a
concentration of 0.5 mol/,~. This mixture is incubated for 1 hour at
0°C. The precipitate is centrifuged for 15 minutes at 4°C in a
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~341471 -
microcentrifuge. The DNA is precipitated from the supernatant with
2.5 volumes of ethanol at 20°-25°C. The isolated DNA is
dissolved in
a solution of 10 mmol of TRIS-HCl containing 10 ug/ml of ribonucle-
ase A. After incubation for 10 minutes at 37°C, proteinase K is
added to a concentration of 250 250 ug/ml and incubation is conti-
nued for 1 hour at 37°C. The proteinase K is removed by phenol and
chloroform/ isoamyl alcohol extractions. The DNA is precipitated
from the aqueous phase by addition of 0.6 part by volume of a
0.6 molar solution of sodium acetate in isopropanol and dissolved
in 50 u~ of a solution containing 10 mmol/~ of TRIS-HC1 and 5 mmol/~
of EDTA at pH 7.5. This preparation yields DNA sequences which
contain substantially more than 50,000 base pairs. Restriction of
this DNA with EcoRV endonuclease, hybridisation of the fragments
with radioactively labelled HindIII fragments of the NPT II gene
and comparison with the plasmid pABDI show, in Southern blot
analysis, the presence of the NPT II gene in the cell nucleus DNA of
the transformed Nicotiana tabacum cells.
e) Evidence of the transfer of the transformed gene to sexual
offspring and of its heredity as normal plantgene
Extensive genetic crossing analyses and detailed molecular biolo-
gical studies (for example Southern blot analysis of the DNA of the
plant genome; investigation of the enzyme activity of the amino-
glycoside phoaphotransferase, i.e. the enzyme for the kanamycin-
specific phosphorylation) with the genetically transformed plants
(first generation and progeny) have yielded the following results:
1. the baceterial gene is stably integrated into the plant genome;
2. it is normally unchanged and regularly transferred to crossed
progeny;
3. its heredity corresponds to that of a natural, simple dominant
plant gene;
4, the molecular analysis by DNA hybridisation and enzyme test
confirms the results of the genetic crossing analysis;
2° 1341471
5. the genetically transformed plants retain their normal, natural
phenotype during the treatment, i.e. no undesirable modifications
are observed.
These results show that the process of this invention for the direct
transfer of a gene into protoplasts affords the best mode of
specifically transforming plant material genetically. The genetic
transformation is stable and unwanted modifications in the genotype
of the plant do not occur. Parallel results are also obtained when
carrying out the transformation described in the foregoing Example
with Nicotiana plumbagenifolia, Petunia hybrids, Hyoscyamus muticus
and Brassica napus.
Example 2: Transformation of cells of Brassica raps c.v.
Just Right by transfer of the NPT II gene
a) Construction of the plasmid pCaMV6Km
The plasmid pBR 327 CaKm+ described in Example la is digested with
restriction endonuclease EcoRV and the EcoRV restriction fragment
containing the kanamycin-resistant gene (NPT II) is used to replace
the EcoRV fragment of the plasmid pCa20-Bal I, which fragment
contains the gene VI of cauliflower mosaic virus, yielding the
plasmid pCaMV6Km (Fig. 2). The plasmid Ca20-Bal I is a chimaeric
CaMV plasmid which is derived from the natural deletion mutant
CM4-184. The entire region II is missing from this plasmid, except
for the first 5 codons and the translation stop signal TGA. An XhoI
coupling component was inserted immediately before the stop codon
in region II.
b) Transformation of protoplasta of Brassica rape c.v. Just Right
by transfer of the NPT gene as part of the plasmid pCaMV6Km by
PEG treatment
Brassica raps protoplasts are washed with a suitable osmoticum and
suspended in a population density of 5~106 per ml in a culture
medium prepared according to Protoplasts 83, Proceedings Experientia
Supplementum, Birkhauser Verlag, Basel, Vol. 45 (1983), 44-45. 40 %
21 1341471
polyethylene glycol (PEG) with a molecular weight of 6000, dissolved
in modified F medium (pH 5.8) (q.v. Example lb), is mixed with the
protoplast suspension to a final concentration of 13 % PEG. To this
mixture is added immediately a solution of 10 yrg of plasmid pCaMV6Km
digested with endonuclease SalI, and 50 ug of calf thymus DNA in
60 ug of water. With occasional agitation, the mixture is incubated
for 30 minutes at 20°-25°C. Then 3 x 2 ml of modified F medium
(6 ml
in all) and 2 x 2 ml of culture medium (4 ml in all) are added at
5 minute intervals. The protoplast suspension is transferred to
10 cm Petri dishes and made up to a total volume of 20 ml with
additional culture medium. These protoplast suspensions are
incubated in the dark for 45 minutes at 26°C. The protoplasts are
isolated by sedimentation for 5 minutes at 100 g, taken up in an
initially liquid and then solidifying agarose gel culture medium and
cultured by the bead type culture method (Plant Cell Reports 2,
244-247 (1983)]. After 4 days, in the development stage of the first
cell division, kanamycin is added to the. cultures in a concentration
of 50 mg/~. The liquid culture medium surrounding the agaroae
segments is replaced every 4 days by fresh kanamycin-containing
nutrient solution. After 4 weeks the kanamycin-resistant clones are
ibolated and then further cultured by providing them weekly with
kanamycin-containing nutrient solution (50 mg/~).
c) Detection of the NPT II gene in the hereditary material of
the plants
The presence of the NPT II gene in the cell nucleus of the transform-
ed Brassica raps cells can be detected by isolation of the cell
nucleus DNA and restriction thereof and hybridisation of the DNA
fragments as described in Example 1 d).
Example 3: Transformation of protoplasts of graminaceous plants of
the species Lolium multiflorum
Protoplasts of Lolium multiflorum (Italian ryegrass) are taken up
at a concentration of 2~106 per ml in 1 ml of 0.4 molar mannitol at
pH 5.8. To this suspension are added, in succession, 0.5 ml of 40 %
polyethylene glycol (PEG) with a molecular weight of 6000 in
1341471
- 22 _ 21489-6725
modified (pH 5.8) F medium [Nature 296, 72-74 (1982)], and 65 u~, of
an aqueous solution containing 15 ug of the plasmid pABDI and 50 ug
of calf thymus DNA. This mixture is incubated for 30 minutes at 26°C
with occasional agitation and subsequently diluted with F medium, as
described in Nature 296 (1982), 72-74. The protoplasts are isolated
by centrifugation (5 minutes at 100 g) and taken up in 4 ml of CC
culture medium [Potrykus, Harms, Lorz, Callus formation from cell
culture protoplasts of corn (Zea Mays L.), Theor. Appl. Genet. 54,
209-214 (1979)] and incubated in the dark at 24°C. After 14 days the
developing cell cultures are transferred to the same culture medium,
but with the the antibiotic G-418 (commercially available; GIBCO
EUROPE P:oduct Catalogue, Catalogue No. 0661811). G-418 is toxic to
Lolium cells at a concentration of 25 mg/,t and permits solely the
further development of cells which have taken up the bacterial gene
for kanamycin resistance. G-418 is a kanamycin analog with
substantially better activity in~graminaceous cells than kanamycin
itself. Resistant cell colonies are transferred to agar medium (the
same medium as above, 25 ml/~, G-418, without osmoticum) and, after
reaching a size of several grams fresh weight per cell colony,
analysed for the presence of the bacterial gene and for the biolo-
gical activity of the gene. The former analysis is made by hybridi-
sation of a radioactively labelled DNA sample of the gene with DNA
which has been isolated from the cell culture; while the latter is
made by detecting the enzyme activity by phosphorylation of
kanamycin with radioactive ATP. Both molecular analyses yielded
unequivocal pzoof of the genetic transformation of the cell colonies
which had been selected on G-418. The assays constitute the first
proof of the genetic transformation of protoplasts of graminaceous
plants and furthermore prove that, in principle, protoplasts of
grasses can be genetically manipulated by the described process, The
possibility of genetically manipulating cultivated grasses, for
example cererals, is thus also afforded.
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1341471
Example 4: Transformation of cell culture cells of Nicotiana
tabacum by transferring the NPT gene by means of
electroporation
Protoplasts are produced by sedimentation from 50 ml of a log phase
suspension culture of the nitrate reductase deficiency variant of
Nicotiana tabacum, cell strain nia-115 [Miiller, A.J. and R. Grafe,
Mol. Gen. Genet. 161, 67-76 (1978)j, and resuspended in 20 ml of
enzyme solution [2 % Cellulase Onozuka R-10, 1 % Mazerozym R-10 and
0.5 x Driselase*(available from Chemische Fabrik Schweizerhalle,
Basel) in a wash solution (0.3 M mannitol, 0.04 M calcium chloride
and 0.5 9~ 2-(N-morpholino)ethanesulfonic acid), adjusted to pH 5.6
with KOHj and incubated for 3 hours on a gyratory shaker at 24°C.
The protoplasts are then separated from undigested tissue by
filtering them through a 100 um mesh sieve. An equal volume of 0.6 M
sucrose is added and the suspension is centrifuged for 10 minutes
at 100 g. The protoplasts floating on the surface are collected and
washed 3 times by sedimentation in the wash solution.
Transformation is carried out by electroporation. The chamber of a
DialogR "Porator" (available from Dialog GmbH, Harffstr. 34, 4000
Dusseldorf, West Germany) is sterilised by washing with 70 % ethanol
and then 100 9~ ethanol and dried by a current of sterile air from a
ventilator with laminary air flow). The protoplasts are suspended at
a concentration of 1 x 106/ml in 0.4 M mannitol solution, ad~uated
with magnesium chloride to a resistance of I.4 kOhm and pABDI DNA is
added in a concentration of 10 ~rg/ml. 0.38 ml samples of this
protoplast suspension are subjected 3 times at 10 second intervals
to a charge of 1000 volts or to a charge of 2000 volts. The proto-
plasts are then cultured in a concentration of 1 x 105/ml in 3 ml of
AA-CH medium [AA medium of Glimelius, K. et al., Physiol. Plant. 44,
273-277 (1978)j, modified by increasing the inositol concentration
to 100 mg/~, and the saccharoae concentration to 34 g/~, as well as
by adding 0.05 ml/~, of 2-(3-methyl-2-butenyl)adenine, and which is
solidified by a 0.6 ~ content of agarose (Sea Plaque, FMC Corp.,
Marine Colloids Division, P.O. Box 308, Rockland, Maine 04841, USA).
~'--Ti,uc~e ~ m a r/~
2~ X341471
After 1 week, the agarose layer containing the protoplasts is
transferred to 30 ml of liquid AA-CH medium which contains 50 mg/~,
of kanamycin. After 3 weeks, during which time half the liquid
medium is replaced weekly by fresh medium of the same composition,
the transformed cell colonies can be observed visually. Four weeks
after being transferred to the medium containing kanamycin, these
cell colonies are transferred to AA medium (Glimelius, K. et al.,
Physiol. Plant. 44, 273-277 (1978); 0.8 % agar), which contains
50 mg/~. of kanamycin, for further culturing and investigation.
Confirmation of the successful transformation is by DNA hybridi-
sation and testing for the enzyme activity of aminoglycoaide
phosphotransferase.
Analogous assays with protoplasts of Brassica rape and Lolium
multiflorum also result in successful transformations.
Example 5: Transformation of cells of Nicotiana tabacum by
transfer of the NPT II gene by electroporation
The preparation of the electroporator is as described in Example 4
and of the protoplasts as in Example 1.
For transformation, protoplasts of Nicotiana tabacum are resuspended
in a concentration of 1.6 x 106/ml in mannitol solution (0.4 M,
buffered with 0.5 9~ w/v of 2-(N-morpholino)ethanesulfonic acid; pH
pH 5.6). The resistance of the protoplast suspension is measured in
the porator chamber (0.38 ml) and adjusted to 1 to 1.2 kOhm with
magnesium chloride solution (0.3 M). 0.5 ml samples are put into
capped plastic tubes (5 ml volume) to each of which are added
initially 40 p~ of water containing 8 ug of pABDI (linearised with
SmaI) and 20 ug of calf thymus DNA, and then 0.25 ml of polyethylene
glycol solution (24 ~o w/v in 0.4 M mannitol). Nine minutes after
addition of the DNA, 0.38 ml portions are put into the pulse chamber
and 10 minutes after the addition of DNA, the protoplast suspensions
present in the chamber are subjected to 3 impulses (1000-2000
volts) at 10 second intervals. The treated portions are put into
Petri dishes of 6 cm diameter and kept for 10 minutes at 20°C.
Then
.. . ~ - ~3414~1
- 25 - 21489-6725
3 ml of K3 medium containing 0.7 9~ w/v of Sea Plaque agarose are
added to each Petri dish and the contents of the dish are thoroughly
mixed. After solidification of the contents of each dish, the
cultures are kept for 1 day at 24°C in the dark and then for 6 days
in light. The protoplast-containing agarose is then cut into
quarters and introduced into liquid medium. The protoplasts are then
cultured by the bead type culturing method. Callus tissues which are
obtained by selection of the transformed material with kanamycin
and plants regenerated therefrom contain the NPT II enzyme (amino-
glycoside phosphotransferase) as product of the NPT II gene.
Electroparation induces a 5- to 10-fold increase in the frequency of
transformation compared with the method without electroporation.
Analogous assays with Brassica raps c.v. Just Right and Lolium
multiflorum also bring about an increase in the frequency of
transformation of the same order~of magnitude.
Example 6: Transformation of cells of Nicotiana tabacum by
transfer of the NPT II gene b~ means of heat shock
Protoplasta isolated from leaves or cell cultures of Nicotiana
tabacum are isolated as described in Examples 1 and 4 and trans-
ferred to an osmotic medium as described in the preceding Examples.
The protoplast suspensions are kept for 5 minutes at 45°C, cooled
with ice for 10 seconds and then the plasmid pABDI is added as
described in Examples 1 and 4. The heat shock treatment increases
the transformation frequency by a factor of 10 or higher compared
with a transformation carried out without this treatment.
Analogous assaya with the protoplasts and plasmids described in
Examples 2 and 3 also bring about an increase in the frequency of
transformation of the same order of magnitude.
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1341471
Example 7: Transformation of different plant cells by transfer of
the NPT II gene by combining.protoplasta and gene as
first step and subsequent combined treatment
Protoplasts of the plants:
Nicotiana tabacum c.v. Petit Havana SRI (A),
Brassica rapa c.v. Just Right (B) and
Lolium multiflorum (C)
are isolated and transferred to an osmotic medium as described in
Example 5. The protoplast suspensions of A) and C) are mixed with
the plasmid pABDI (Example la), and those of B) with the plasmid
pCaMV6Km (Example 2a) as described in Examples 1 to 3, but without
simultaneous treatment with polyethylene glycol. The protoplast
suspensions are then subjected to a heat shock treatment as des-
cribed in Example 6, then to a polyethylene glycol treatment as
described in Examples 1 to 3; and finally subjected to electron
poration as described in Example 5..The transformation frequency in
this proced;~re is in the range from 10 3 to 10 2, but may be from 1
to 2 % depending on the conditions. (The transformation frequency in
Examples 1 to 3 is in the order of about 10 5). Results in the range
from 10 3 to 10 2 are also obtained if, after combining protoplasts
and plasmids, the subsequent steps of heat shock, polyethylene
glycol treatment and electroporation are employed in different
sequence.
