Determination of suitable electrofusion parameters for the somatic hybridization of mint protoplasts with the Eppendorf Multiporator®
Ellen Nissing & Andreas Müller1, Natascha Weiß2
1) Phytowelt GreenTechnologies GmbH, Nettetal, Germany 2) Eppendorf AG, Hamburg, Germany
Somatic hybridization has opened up new possibilities in the area of plant breeding. Here, electrofusion represents
a very effective method for the fusion of the protoplasts. The combination of the Eppendorf Multiporator
with various fusion chambers enables optimal adjustment to the respective experimental conditions. This method
provides fusions using little material for establishing a system as well as mass fusions in the context of breeding
approaches. For the purposes of the application, the electrical parameters for the alignment and the fusion of the
protoplasts are first optimized in order to achieve efficient fusion and a high degree of cell vitality. The established
protocol can then be used in a reliable and user-friendly fashion in routine applications.
Cell fusion provides the basis for the creation of cells with
new properties. This technique can thus be used in a wide
range of applications, e.g. basic research in natural sciences,
medical tasks, biotechnological applications and
in plant breeding. In addition to the fusion of mammalian
cells, the creation of plant hybrids through the fusion of
protoplasts is of great importance; a process known as
somatic hybridization (SH).
In plant breeding, this method offers advantages in comparison
to classic approaches. Genetic diversity can thus
be increased in a short time, and it is possible to create
plant lines that can not or can only very inefficiently result
from natural cross-breeding. Neither the identification and
isolation of specific genes nor knowledge concerning the
regulation of the relevant properties is thereby necessary.
In addition, SH overcomes the transformation limitation of
being able to introduce only a few genetic factors into a
given genetic background, and enables the multiplication of
the chromosome number or of the ploidy for the purpose
of increasing the yield (heterosis effect).
In one of the first experiments in this area, the work group
around Power was able to fuse corn with oat protoplasts
in 1970 . Two years later, the first report concerning
interspecific hybridization through protoplast fusion of two
different tobacco types was published . The potential
of SH is also demonstrated by the cultivation of optimized
potatoes and citrus fruits [3, 4]. Another example is found
in the decontamination of soil (heavy metals) in industrial
usage through the transfer of zinc tolerance .
Established methods are the chemically induced fusion
of cells with polyethylene glycol (PEG)  and electrofusion
. As already often described, electrofusion has the
advantage that it can be carried out quickly and easily,
causes less damage to the protoplasts and is very efficient
[8-10]. This technique consists of three phases: In the first
so-called alignment, a dipole in the cells is induced through
a weak, inhomogeneous, alternating field.
The membranes are brought into close contact with one
another due to the mutual attraction. The subsequent,
very brief electrical pulse with high field strength creates
an electrical breakdown of the cell membrane, thus initiating
the fusion. During the last phase (post-alignment), the
cell membranes remain in contact until stable membrane
bridges have formed between the fusing cells. The fusion
product is then rounded off.
Using the example of mint, the following will describe the
determination of electrical parameters for the fusion of
protoplasts. The objective was to create a new type of mint
more suitable for the production of menthol than previously
used types. In order to achieve this, menthol-rich types are
combined with types with a high biomass .
Materials and Methods
The following mint types were used for electrofusion:
Mentha piperita var. Piperita multimentha, Mentha piperita
var. Piperita mitcham and Mentha suaveolens Ehrh. in vitro
growing plant material was used, as the yield and regeneration
rate for greenhouse material are too low .
Electrofusion device and accessories
The Eppendorf Multiporator is an electroporation device
into which a module for electrofusion can be integrated.
Various fusion chambers can be connected to the machine
using special inserts (Fig. 2 A). The Micro fusion chamber
from Eppendorf (electrode distance of 0.2 mm) is used for
optimizing the parameters under direct microscopic control
(Fig. 2 B). Larger chambers, such as the Eppendorf Helix
fusion chamber with an electrode gap of 0.2 mm and a filling
volume of 250 µl (Fig. 2 A), and the plate fusion chamber
from Phytowelt (filling volume of 5 ml, electrode gap of
1 mm), are used for routine applications.
The isolation of the protoplasts was done according to a
modified method from Sato et al. . For electrofusion,
the cell density was set in 0.7 M sorbitol to 5.0 – 9.0 x 105 protoplasts/ml and the suspensions of the fusion partner
were pipetted into the chambers in the same ratio. The
electrodes of the Micro fusion chamber were thereby completely
covered in solution, while 250 µl was used in the
Helix fusion chamber and 2 ml in the Phytowelt chamber.
The respective parameters set on the Multiporator are
listed in the result section.
The fusion was followed by the determination of the vitality
on the basis of morphological changes such as uneven
distribution of the chloroplasts or deformation of the protoplasts.
In order to detect the fusion, the cell nuclei of
fusing protoplasts were stained with DAPI (4.6-Diamidino-
2-Phenylindol) (see also Fig. 4B).
In order to enable targeted selection of somatic hybrids,
mint types with varying cell culture properties were
selected: One type, which can be regenerated well from
protoplasts, was combined with a fusion partner with poor
capacity for regeneration. The type with good regeneration
properties was treated with iodine acetamide (IOA), which
results in an inability of the protoplasts to divide. Hybrids
in which the genetic information of both partners are mixed
thus have a selection advantage in comparison to fusion
products consisting of only one type or non-fused protoplasts.
The electrical fusion parameters of voltage, pulse duration
and number of pulses were optimized for the „alignment
phases“ and the fusion pulse. The results are presented as
a representation of the Multiporator display:
It should be noted that the strength of the electrical field
(the decisive parameter for the electrofusion) is derived
from the set voltage and the distance between the electrodes.
When using electrofusion chambers with a varying
electrode gap, the set voltage must be adjusted in accordance
with the following formula in order to result in the
same field strength (field strength [V/cm] = voltage [V] /
electrode gap [cm]).
Determining the parameters for the alignment
The optimization of the parameters for the alignment phases
is carried out by applying alternating voltages within
a range from 1.5 - 3 V in 0.5 V steps to the Micro fusion
chamber. After 2.5 V the protoplasts stretched extensively
and burst. As shown in Fig. 3, as well as by Sato et al. ,
the setting of 1.5 V was quite suitable for the alignment.
This voltage could be directly adopted for the Helix fusion
chamber. A voltage of 7.5 V was set for the Phytowelt
fusion chamber with an electrode gap of 1 mm.
The alignment duration was tested in a range from 30 - 60 s
in 10 s steps. An alternating field of 40 s proved to be sufficient.
The same parameters were also used for the postalignment
Optimization of the pulse length and number of pulses
The minimum field strength necessary to achieve membrane
breakthrough can be derived from the size of the cell
on the basis of the given formula.
The cell diameter (d) of the protoplasts varies greatly,
meaning that a mean value of 25 µm was used. This results
in a critical field strength of 533.33 V/cm for a simple
membrane breakthrough. Because the fusion of two cells
is involved, the value is doubled to 1,066.7 V/cm. In arithmetical
terms this means a voltage of 21.3 V for a 0.2 mm
chamber is set on the Multiporator. On this basis, a voltage
of 25 V was set to determine the pulse duration (τ) and the
number of pulses (n) in the Micro fusion chamber. The following
parameter combinations were analyzed:
All three conditions resulted in no negative effect on
the vitality of the protoplasts. The fusions were counted
between the electrodes of the Micro fusion chamber
microscopically, the criteria being that the protoplasts be
extremely large, lie very close to one another or be shaped
like an eight. Combination no. 2 resulted in 9 fusions, while
6 – 7 fusions per preparation were obtained for the other
two preparations. All other experiments were carried out
with a pulse duration of 25 µsec and 2 pulses.
Optimization of the pulse voltage
In order to determine the pulse voltage, eight fusions were
carried out with successively greater field strengths in a
range from 750 – 2,500 V/cm. The vitality and fusion rate
of the protoplasts were then determined. „Two cell fusions“
of the cell nuclei stained with DAPI were counted without
taking multiple fusions into account (Fig. 4). The results are
shown in Figure 5. Only the Phytowelt chamber was used
for further experiments, as larger sample volumes were to
The vitality of the protoplasts decreased with an increasing
fusion rate. A vitality of 82 % or 81 % could be obtained
with the voltages 150 and 170 V, while the fusion rate was
still adequate at 25 % or 24 %.
Fusion of the protoplasts of two mint types for the creation
of somatic hybrids
The electrofusions were carried out with the following
parameters: 170 V, 25 µs, 2 x and 7.5 V, 40 s for alignment
and post-alignment. Two different approaches were thereby
realized; fusions without or with active selection of the
1) Fusion without active selection
In order to obtain an optimal fusion without external influences,
e.g. by selection measures, the protoplasts from
M. piperita var. Piperita mitcham (rich in menthol) and M.
suaveolens Ehrh. (high biomass) were used directly for the
fusion. The density of the protoplast mix was set to 6.3 x
105 protoplasts/ml. Calli originating both from parent cells
and from fusion products were anticipated as a result. The
fusion rate was 21 % with a protoplast vitality of 97 %. A
total of approx. 6,600 calli were regenerated. These are
currently being evaluated
2) Fusion with active selection
Protoplasts of M. piperita var. Piperita mitcham (rich in
menthol) and M. suaveolens Ehrh. (high biomass) were
used for targeted electrofusion with selection. Following the
IOA treatment of the protoplasts of M. piperita var. Piperita
multimentha, a mixture with a density of 5.5 x 105 protoplasts/
ml was fused. There was a selection advantage for
the regeneration of hybrids due to the IOA treatment and
the poor regeneration of M. suaveolens Ehrh. The fusion
rate and the vitality were no longer determined following
the experiment, as it was already shown in advance that
the vitality of the protoplasts is not negatively effected, and
that the fusion rate fluctuates only mildly when the same
parameters are chosen. Approx. 640 calli could be regenerated.
Using the example of mint, it was shown that the
Multiporator, when used with optimized electrical parameters,
is very suitable for the somatic hybridization of various
types. Fusion rates of 16 - 27 % were achieved in the
project described, whereby the vitality of the protoplasts,
which reached up to 97 %, was not significantly affected.
Previously published results with a fusion rate of 6 % 
could thus be considerably improved. The basis of the success
was formed by the easy operation of the Multiporator,
which enabled efficient and error-free work, and the considerable
flexibility of the device, as it can be used for
applications ranging from optimization to commercial mass
use, depending upon the fusion chamber used. This means
that the Multiporator is an efficient and target-oriented tool
for cell fusion, which also satisfies the quality and efficiency
requirements of companies specializing in cell fusion,
such as Phytowelt GreenTechnologies GmbH.
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Information on the Multiporator system is available from Eppendorf AG (www.eppendorf.com). Phytowelt GreenTechnologies GmbH (www.phytowelt.com) can be contacted with questions concerning the plate fusion chamber or tissue culture technology.
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