Method for the generation of a monoclonal plant cell line
The present invention relates to the field of plant
biotechnology. In particular, the present invention relates to
the generation of a native (wild-type) or transgenic
monoclonal plant cell line from a heterogeneous population of
plant cells through flow cytometric sorting. A s will be
apparent for a skilled person, the invention also comprises to
use the monoclonal plant cell line for the regeneration of
whole fertile plants.
During the past decades, enormous efforts have been dedicated
to the establishment and culturing of plant-based systems for
the accumulation and harvesting of native or heterologous
proteins and secondary metabolites. The literature provides a
vast quantity of evidential material that proves the utility
of plant-based systems to produce a large variety of desired
substances that are either secreted into the culture medium or
isolated from the producing cells, tissues, organelles or even
whole plants or parts thereof. Likewise, a broad range of
transformation protocols exist that ensure the establishment
of either stably or transiently transformed plant material.
However, there is still a need for a reliable, relatively
cost-efficient and rapid technology to obtain high yields of a
desired product from plant cells.
It has been repeatedly reported that transformation of a
population of plant cells such as a plant suspension culture
frequently results in transgenic cultures that exhibit cells
with highly heterogeneous (mixed) and inconsistent expression
levels of the target protein related to the mixture of
epigenetically different cells within the primary
heterogeneous cell population. Within recombinant cell lines
the heterogeneity in transgene expression demonstrates a
serious problem in terms of production rates.
A main problem is that high-producing clones are often rare
events within a transformation assay and it is very time
consuming to establish a homogeneous high-producing cell line.
A still ongoing technical challenge, therefore, is the elite
transgenic event production and recovery from a freshly
transformed or already transgenic plant culture.
For flow cytometric sorting such as e.g. FACS application,
single spherical cells must be obtained from the usually
aggregated plant cell population or culture by enzymatic
digestion of the cells to liberate protoplasts. For most plant
species, however, the regeneration of single protoplasts is
hampered by the necessity to be maintained at certain
population densities.
A reliable and reproducible procedure for the regeneration of
a single transgenic cell/protoplast or for the regeneration of
a whole fertile plant therefrom (especially after flow
cytometric sorting) has hitherto not been described.
The present invention is thus primarily concerned with the
provision of a plant-based system to produce high levels of
desired native or recombinant products that makes use of a
non-transformed or transgenic monoclonal plant cell line
generated from a heterogeneous (mixed) population of plant
cells such as a suspension culture and overcomes the problems
of the prior art, in particular with respect to the rapid
separation and subsequent regeneration of single (transgenic)
protoplasts until the formation of a microcolony that can be
used to establish a monoclonal plant cell line that,
preferably, is capable of producing and accumulating high
quantities of the desired product. It is clear for a skilled
person that the present invention likewise enables to provide
whole fertile plants regenerated from the established
monoclonal plant cell line.
Contrary to many currently used and developed systems that are
based on the use of intact plants or at least intact and
differentiated plant tissue, the use of suspension cells has
the advantage that homogeneous material can be reproducibly
produced under controlled, aseptic and contained conditions.
There are currently two principal strategies to produce
recombinant proteins in plants, namely (i) the generation of
stable transgenic plants or suspension cell lines or (ii) the
transient expression of heterologous gene(s) after infecting
the plant expression hosts (plant, tissue or cells) with a
bacteria (e.g. Agrobacterium) , a virus (e.g. Tobacco mosaic
virus, Potato virus X/Y, Cowpea mosaic virus and many others),
or a combination of both (e.g. magnif ection) to enable the
host to express the heterologous genetic information (DNA or
RNA) . In the alternative and as known in the art, the genetic
information can also be introduced into the plant expression
host by established mechanical means such as e.g.
elect roporation or laser perforation.
Although the invention is preferably concerned with the use of
stably transformed plant cell material, systems for the
transient expression having the advantage of speed (gene-toproduct,
time-to-market, emergency response) as well as the
possibility to achieve accumulation levels that are much
higher than those that can typically be obtained in stably
transformed transgenic plants or parts thereof such as cells
may also be involved in the method according to the present
invention .
According to the invention, there is provided a method for the
generation of a native (wild-type, non-transformed) or
transgenic monoclonal plant cell line from a heterogeneous
population of plant cells. The method comprises to firstly
provide said population of plant cells such as e.g. plant
suspension cells forming the source plant cell material that
is subjected to the further steps comprised by the method
according to the invention. Usually, this plant cell material
can easily be derived from e.g. a heterogeneous plant
suspension culture which, preferably, has been cultivated
under controlled and/or aseptic conditions. The source cells
can be (stably/transiently) transformed transgenic cells or
wild-type (native, non-transformed) cells able to produce and
accumulate a desired product.
Since the method according to the invention uses flow
cytometric sorting such as e.g. FACS technology to separate or
isolate single, i.e. individualized protoplasts, these have to
be prepared from a population of plant cells as provided above
using materials and methods known in the art. According to a
preferred embodiment, these protoplasts are transformed and
capable of (i) producing a fluorescent marker protein or
polypeptide, (ii) producing a desired product, and/or (iii)
surviving in presence of a selection agent. The preferred
sorting criteria for flow cytometric sorting are cell
granularity as a marker for e.g. qualitative characteristics
such as apoptosis, and cell size. The preferred sorting
criteria for FACS can be selected from the group comprising
the genetic background (e.g. ploidy, aneuploidy) , mutants
transgenics, gene exchange products, and fluorescence (e.g.
autof luorescence (chloroplast s, metabolites), fluorescent
proteins or enzyme-mediated fluorescence). It is to be
understood that the use of a selection agent is not necessary.
Thus, the protoplast does not necessarily have to be
transformed with a nucleic acid sequence conferring an
appropriate resistance.
After the separation or isolation of single (transformed)
protoplasts by flow cytometric sorting such as e.g. FACS, each
single transformed protoplast is regenerated until the
formation of a microcolony (microcallus ) by co-cultivation in
the presence of feeder cell material. The plant source origin
is not limited but restricted to those lines, varieties and
species whose protoplasts have the potential to regenerate
until the formation of a microcolony or microcallus. The
present invention is thus applicable to all plant varieties
and species for which a regeneration protocol has been
established or will be provided in the future. In view of the
aspect according to the invention concerning the further
regeneration of the monoclonal microcolony or plant cell line
into whole fertile plants, it is to be understood that this
aspect can be carried out with all plant varieties and species
for which a regeneration protocol has been established or will
be provided in the future.
Subsequently, the microcolony is separated or removed from the
feeder cell material and cultivated until the formation of a
monoclonal plant cell line.
According to a preferred embodiment, the next step comprised
by the method according to the invention therefore relates to
the generation of a monoclonal callus tissue by (i)
transferring the microcolony or microcallus to solid
cultivation medium and (ii) cultivating the microcolony or
microcallus in the presence of at least one selection agent
until the formation of a transgenic callus tissue from which a
transgenic monoclonal plant cell line can be established by
transferring the callus tissue to liquid cultivation medium.
A s will be appreciated by the skilled person, the microcolony
can also be removed or separated from the feeder cell material
by mechanical means such as e.g. by clone picking. In this
case, no selection agent is needed and the cells comprised by
the microcolony do not need to display resistance against any
selection agent.
According to a preferred embodiment, the cells comprised by
the heterogeneous population of plant cells are native (e.g.
wild-type) or non-t ransgenic cells that, before being
subjected to flow cytometric sorting, are stably or
transiently transformed with at least one expression vector
comprising at least one heterologous nucleic acid sequence
operably linked to a functional promoter, wherein said at
least one heterologous nucleic acid sequence codes for a
desired product. According to a further embodiment, the at
least one expression vector comprises at least two
heterologous nucleic acid sequences operably linked to (a)
functional promoter (s), wherein said at least two heterologous
nucleic acid sequences code for a fluorescent marker protein
or polypeptide and for a resistance against a selection agent
or for a desired product. If desired, the cells may
additionally comprise a heterologous nucleic acid sequence
that codes for a desired product to be accumulated in the
transgenic monoclonal plant cell line as provided according to
the invention.
The term "heterologous" as used herein indicates that the
gene/sequence of nucleotides in question have been introduced
into plant cells by using genetic engineering, i.e. by human
intervention. A heterologous sequence of nucleotides may
comprise the coding sequence for a fusion protein comprised of
a fusion partner that may be formed, for example, in part by a
plant protein that may be fused to a non-plant protein which
may be termed a hybrid plant :non-plant fusion protein for the
purposes of the present invention. Alternatively, a fusion
protein may be one which is formed of fusion partners that are
of non-plant origin. A heterologous gene may augment the
expression of a protein of interest from an endogenous
equivalent gene, i.e. one which normally performs the same or
a similar function, or the inserted sequence may be additional
to the endogenous gene or other sequence. Nucleic acid
heterologous to a cell may be non-naturally occurring in the
cultivated cell type, variety or species. Thus, the
heterologous nucleic acid may comprise a coding sequence of,
or derived from, a particular type of organism, such as a
plant or mammalian species, e.g. of human, ovine, bovine,
equine, or porcine species, placed within the context of a
cultivated cell such as a BY2 cell derived from tobacco. A
further possibility is for a nucleic acid sequence to be
placed within a cultivated target cell in which it or a
homologue is found naturally, but wherein the nucleic acid
sequence is linked and/or adjacent to nucleic acid which does
not occur naturally within the cell, or cells of that type or
species or variety of plant, such as operably linked to one or
more regulatory sequences, such as a promoter sequence, for
control of expression. Furthermore, synthetic (artificial)
nucleic acid sequences can be used as well.
"Vector" is defined to include, inter alia, any plasmid,
cosmid, phage, or viral vector in double or single stranded
linear or circular form which may or may not be self
transmissible or mobilizable, and which can transform a
prokaryotic or eukaryotic host and exists extrachromosomally
(e.g. autonomous replicating plasmid with an origin of
replication) . Specifically included are shuttle vectors by
which is meant a DNA vehicle capable, naturally or by design,
of replication in two different host organisms, which may be
selected from actinomycetes and related species, bacteria and
eucaryotic (e.g. higher plant, mosses, mammalian, yeast or
fungal) cells.
"Expression vector" refers to a vector in which a nucleic acid
is under the control of, and operably linked to, an
appropriate promoter or other regulatory elements for
transcription in a host cell such as a microbial or plant
cell. The vector may be a bi- functional expression vector
which functions in multiple hosts. In the case of genomic or
subgenomic DNA, this may contain its own promoter or other
regulatory elements and in the case of cDNA this may be under
the control of an appropriate promoter or other regulatory
elements for expression in the host cell.
A "promoter" is a sequence of nucleotides from which
transcription may be initiated of DNA operably linked
downstream (i.e. in the 3 ' direction on the sense strand of
double-stranded DNA) .
"Operably linked" means joined as part of the same nucleic
acid molecule, suitably positioned and oriented for
transcription to be initiated from the promoter.
The term "inducible" as applied to a promoter is well
understood by those skilled in the art. In essence, expression
under the control of an inducible promoter is "switched on" or
increased in response to an applied stimulus. The nature of
the stimulus varies between promoters. Some inducible
promoters cause little or undetectable levels of expression
(or no expression) in the absence of the appropriate stimulus.
Other inducible promoters cause detectable constitutive
expression in the absence of the stimulus. Whatever the level
of expression is in the absence of the stimulus, expression
from any inducible promoter is increased in the presence of
the correct stimulus.
The invention also embraces use of a variant of any of these
sequences. A variant protein shares homology with, or is
identical to, all or part of the sequences discussed above.
For the expression of recombinant proteins, a suspension of
recombinant Agrobacteria or viruses (vectors) containing the
genetic information for the proteins of interest is applied to
the plant suspension cells mentioned above in a manner known
in the art. The vector infects the plant cells and transmits
the genetic information. Preferably, the plant cell material
to be transformed is provided in high density with only small
amounts of media being present so that the vector suspension
can be applied just by dropping or spraying. This preferred
embodiment of transformation has several practical advantages
with respect to handling, automation, containment, up-scaling
and waste production and removal. In the alternative, known
techniques such as particle bombardment, elect roporation and
the like can be used as known in the art.
Suitable promoters include the Cauliflower Mosaic Virus 35S
(CaMV 35S) . The promoter may be selected to include one or
more sequence motifs or elements conferring developmental
and/or tissue-specific regulatory control of expression.
A s already mentioned, the at least one selectable genetic
marker, that may be desired to be produced, may be included in
the construct or be provided in a second construct, such as
those that confer selectable phenotypes such as resistance to
antibiotics or herbicides (including but not limiting e.g.
kanamycin, hygromycin, phosphinotricin, chlorsulf uron,
methotrexate, gentamycin, spectinomycin, imidazolinones and
glyphosate) .
Alternatively, the plant suspension cells used for the
preparation of protoplasts can also be provided from an
already transgenic heterogeneous plant suspension culture
comprising transgenic cells.
The (transgenic) monoclonal plant cell line established
according to the invention can be treated or cultivated in the
presence of precursors, inducers, hormones, stabilizers (e.g.
compatible solutes), inhibitors, RNAi/siRNA molecules,
signaling compounds, enzymes (e.g. pectinase), and/or
elicitors in addition to or instead of the vector suspension,
for the production of recombinant proteins or metabolites.
According to a preferred embodiment, the desired product is
selected from the group consisting of heterologous proteins or
polypeptides (e.g. blood products, cytokines, growth hormones,
therapeutic/diagnostic/industrial enzymes, vaccines, full-size
antibodies or various antibody derivates), secondary
metabolites (e .g . Phenylpropanoids, Alkaloids, Terpenoids,
Quinones or Steroids), and markers for the diagnosis or
analysis of gaseous, solid or fluidic (chemical) compounds and
substances .
Genes of interest include those encoding proteins which are
themselves natural medicaments such as pharmaceuticals or
veterinary products. Furthermore, genes of interest also
include any other recombinant protein such as e.g. technical
enzymes, toxines, or recombinant proteins conferring for new
agronomic input and output traits.
Heterologous nucleic acids may encode, inter alia, genes of
bacterial, fungal, plant or non-plant origin such as fusion
proteins as alluded to hereinabove or animal origin. Poly
peptides produced may be utilized for producing polypeptides
which can be purified therefrom for use elsewhere. Proteins
that can be produced in a process of the invention include
heterodimers , such as FSH, immunoglobulins, fusion antibodies
and single chain antibodies. Furthermore, the above genes may
be altered to produce proteins with altered characteristics
such as a modified glycane structure. However, the invention
does also allow to use synthetic genes such as artificial
sequences that, as such, do not exist in nature.
Such proteins include, but are not limited to retinoblastoma
protein, p53, angiostatin, and leptin. Likewise, the methods
of the invention can be used to produce mammalian regulatory
proteins. Other sequences of interest include proteins,
hormones, such as follicle stimulating hormone, growth
factors, cytokines, serum albumin, hemoglobin, collagen,
thaumatin, thaumatin-like proteins, epidermal growth factors
such as VEGF, etc.
As will be appreciated by the skilled artisan, the invention
enables to produce a large variety of proteins and
polypeptides including (recombinant) proteins of
pharmaceutical relevance (such as e.g. vaccines, antibodies,
therapeutical enzymes, allergens and hypoallergens ,
antimicrobial peptides, structural proteins such as elastin
and collagen for use as biocompatible coating materials,
virus-like particles, protein bodies etc.), (recombinant)
proteins of nutritional value (food and feed additives),
(recombinant) proteins for diagnostic applications (such as
e.g. enzymes, antibodies and engineered antibodies, other
enzyme or fluorescent fusion proteins, antigens to be used as
positive controls, binding ligands for protein arrays),
(recombinant) proteins of technical relevance (such as e.g.
binding ligands for affinity sorbents, high value enzymes,
biocatalysts ), and recombinant proteins improving agronomic
input or output traits.
Generally speaking, heterologous nucleic acids may be
expressed by any appropriate process used in the art or they
may be transcribed or expressed as follows:
(i) transient expression of 'naked DNA e.g. comprising a
promoter operably linked to the heterologous sequence of
interest ,
(ii) expression from an expression vector, such as a
replicating vector. Generally speaking, those skilled in the
art are well able to construct vectors and design protocols
for transient recombinant gene expression. Suitable vectors
can be chosen or constructed, containing appropriate
regulatory sequences, including promoter sequences, terminator
fragments, polyadenylat ion sequences, enhancer sequences,
marker genes and other sequences as appropriate. For further
details see, for example, Molecular Cloning: a Laboratory
Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor
Laboratory Press or Current Protocols in Molecular Biology,
Second Edition, Ausubel et al. eds ., John Wiley & Sons, 1992.
(iii) expression from a non-integrating vector.
It will be understood that these categories are not mutually
exclusive, for instance because a non-integrating vector may
also be an expression vector etc.
A s will be appreciated by the skilled artisan, the at least
two heterologous nucleic acid sequences coding for a
fluorescent marker protein or polypeptide or for an enzyme
producing a fluorescent molecule and for the heterologous
protein of interest (desired product) may be provided either
(i) in polycist ronic configuration comprised by a single
expression cassette on the same vector, (ii) in a tandem
configuration with at least two different expression cassettes
on the same vector, or (iii) in at least two different
expression cassettes on different vectors, wherein the tandem
configuration is preferred.
According to a further aspect, the invention thus also
provides a method for the production of at least one desired
product preferably selected from the group consisting of
heterologous proteins or polypeptides, secondary metabolites,
and markers. The method comprises to use the (transgenic)
monoclonal plant cell line as established according to the
invention in order to produce and accumulate the at least one
desired product which is subsequently obtained or isolated
from the producing cells or from the cultivation medium.
Thus, in one aspect of the invention, there is disclosed use
of a preferably stably transformed monoclonal plant cell line
additionally capable of generating mRNA encoding a desired
product such as a heterologous target protein generated by
transcription from an introduced nucleic acid construct
including the target nucleotide sequence operably linked to a
promoter .
The "introduced nucleic acid" will thus include the
heterologous nucleic acid sequence as a DNA sequence provided
in the form of a construct that is capable of giving rise to
the production and accumulation of the desired product.
Thus in a preferred aspect of the invention, there is
disclosed a method of achieving stable expression of a
heterologous nucleotide sequence in a monoclonal plant cell
line, which method comprises the step of stably introducing
into a target cell at least a first nucleic acid sequence
comprising a heterologous nucleotide sequence coding for the
desired product.
In one embodiment there is provided a method of generating at
least an extracellular heterologous protein, which method
comprises the steps of:
(i) stably introducing into a target cell comprised by the
starting population of plant cells a first nucleic acid
comprising the nucleotide sequence coding for the heterologous
protein or desired product;
(ii) preparing protoplasts from plant suspension cells
provided from said plant suspension culture, wherein the
protoplasts are additionally transformed and capable of (i)
producing a fluorescent marker protein or polypeptide and (ii)
surviving in presence of a selection agent;
(iii) separating single transformed protoplasts by subjecting
the preparation of protoplasts to FACS;
(iv) regenerating a separated single transformed protoplast
until the formation of a microcolony or microcallus by cocultivation
in the presence of feeder cell material;
(v) generating a monoclonal callus tissue by (i) transferring
the microcolony or microcallus to solid cultivation medium and
(ii) cultivating the microcolony or microcallus in the
presence of at least one selection agent until the formation
of a transgenic callus tissue;
(vi) establishing a transgenic monoclonal plant cell line by
transferring the callus tissue to liquid cultivation medium;
and
(vii) causing or permitting expression from the nucleic acid
of the heterologous protein or desired product by providing
appropriate cultivation conditions, and
(viii) harvesting the accumulated heterologous protein or
desired product from the producing cells.
The isolation may be by entirely conventional means, and may
or may not entail partial or complete purification.
Naturally, the man skilled in the art will recognize that more
than one gene may be used in the, or each, construct. Multiple
vectors (each including one or more nucleotide sequences
encoding heterologous protein of choice) may be introduced
into the target cells as described herein or elsewhere. This
may be useful for producing e.g. multiple subunits e.g. of an
enzyme.
The fluorescent marker protein or polypeptide can be any
protein detectable by fluorescence such as GUS, fluorescent
proteins such as GFP or DsRed, luciferase etc. Preferably, the
reporter is a non-invasive marker such as DsRed or GFP.
According to a further aspect, the invention provides a method
for the identification of higher expressing insertion loci by
cell sorting comprising the steps of transforming cells (e.g.
as described in Example IB below) e.g. with a construct
containing fluorescent protein 1 , and identifying and
separating single high fluorescent protein 1 producing cells
by FACS including the regeneration to microcolony and
suspension culture and the gene exchange e.g. with fluorescent
protein 2 , and identifying and separating rare gene exchange
products.
The invention will now be further described with reference to
the following non-limiting Figures and Examples. Other
embodiments of the invention will occur to those skilled in
the art in the light of these.
Figure 1 is a schematic drawing illustrating the structure of
an expression cassette used for the preparation of the
transgenic MTED BY-2 line as described herein. In particular,
the figure illustrates the T-DNA of the pTRAkc ::MTED plant
expression vector used for the transformation of BY-2
suspension cells.
LB and RB : left and right border of the T-DNA; Pnos and pAnos :
promoter and terminator of the nopaline synthase gene; nptll:
coding sequence of the neomycine phosphotransferase gene; SAR :
scaffold attachment region; P35SS and pA35S: promoter with
duplicated enhancer and terminator of the Cauliflower mosaic
virus (CaMV) 35S gene; CHS: 5'-UTR of the chalcone synthase
from Petroselinum crispum; SP: signal peptide; HC and LC :
coding sequence of the heavy and light chain of the M12
antibody; TL: 5'-UTR of the tobacco etch virus (TEV) ; TP:
transit peptide; DsRed: coding sequence for the red
fluorescent protein from Discosoma spec.
Examples
Example 1
Rapid generation of elite-producing monoclonal cell lines
after a transformation event
A . Tobacco cell culture
The wild type suspension culture of Nicotiana tabacum cv.
Bright Yellow 2 (BY-2) was maintained in darkness under
sterile conditions as 50 ml aliquots in 100 ml glass
Erlenmeyer flasks at 26°C, with a constant orbital agitation
of 180 rpm. The cultivation medium comprised basal MSMO medium
(pH 5.8) supplemented with sucrose (3%, w/v) and 1 mg/1 2,4-
dichlorophenoxyacetic acid. Subculture was done at day 7
intervals by transfer of 5% (v/v) of the cells into 50 m l
fresh medium.
For protoplast preparation the suspension cell culture was
subcultured by transfer of 2% (v/v) into 50 m l fresh medium.
B .Accelerated generation of transgenic events for subsequent
sorting
BY-2 wild type suspension cells were cultivated as described
in section A . In parallel transgenic Agrobacterium tumefaciens
harbouring a construct comprising several expression cassettes
on the same vector (see Fig. 1 ) were cultivated in YEB medium
containing the appropriate antibiotic (0 .5% Nutrient Broth,
0 .1% Yeast Extract, 0.5% Peptone, 0.5% Sucrose, 2 M MgS0 4, pH
7.4) on an orbital shaker at 160 rpm and 27 °C to an OD oonm of
1 . Three days after subcul tivation 3 ml BY-2 wild type cells,
200 nM acetosyringone and 150 m ΐ agrobacteria (OD oonm = 1 )
were co-cultivated in Petri dishes in the dark. After 3 days
of co-cultivation at room temperature the BY-2 cells were
resuspended in 10 ml of BY-2 medium supplemented with 200 mg/1
cefotaxime. The cells were transferred to a 50 ml sterile tube
and washed twice by centrif ugation (850 g , 5 min) in order to
remove agrobacteria . After resuspension of the cell pellet
cultivation of the transformed BY-2 cells takes place in 100
m l shake flasks using 20-50 m l BY-2 medium supplemented with
cefotaxime and a suitable selective agent (180 rpm, 26°C) .
After regeneration of a proper suspension (packed cell volume
approximately 50-60 % ) the cells can be subcultured for
protoplast preparation (see section C ) . This method requires
14 to 2 1 days to establish a transgenic suspension culture
that can be used for subsequent protoplast generation (C) and
flow cytometric sorting (D) .
C . Protoplast preparation and cell wall regeneration
Actively growing cell cultures were used 3 days after
subculture for sedimentation of cells by centrif ugation at 850
g for 5 min in sterile conical plastic centrifuge tubes. The
supernatant was removed and cells were resuspended in 10 m l of
PNT digestion solution (3.6 g/1 Kao Michayluk basal salts
(Duchefa) , 0.4 M sucrose, 0.5 mg/1 NAA, 1 mg/1 BAP) comprising
1% (w/v) cellulase and 0.3% (w/v) macerozyme. The cell-enzyme
suspension was placed into 6 cm Petri dishes sealed with an
adhesive tape. Digestion was carried out overnight (16-18 h )
at 26°C in the dark with gentle agitation. Protoplasts were
filtered through a 100-pm nylon mesh and subsequently floated
to the surface during centrif ugation (104 g for 8 min) . The
pellet and the medium interface were removed and protoplasts
washed twice with PNT solution. Protoplasts were resuspended
in 5 solution (154 m NaCl, 125 CaCl 2, 5 mM KC1, 5 m
glucose, pH 5.6) and sedimented by centrif ugation at 76 g for
2 min. Protoplasts were cultured in the modified regeneration
medium 8p2c (see table 1 below; optimized from 8p medium)
after gentle resuspension. Usually the described procedure
resulted in 7xl0 5 protoplasts per ml with an average percentage
of 74% viable protoplasts.
Protoplasts were regenerated for 3 days at 26°C in the dark to
initiate cell wall regeneration. The resulting protoplasts
were sieved again through a 100-pm nylon mesh and then
transferred to a sterile sample introduction tube for FAC
sorting. Single protoplasts were sorted into each well of a
96-well microtiter plate containing non-t ransgenic wild type
feeder protoplasts or cells (see section D ) .
BY-2 wild type protoplasts, which were used as feeder
protoplasts were adjusted to approximately 2 x 103 cells/ml
8p2c medium using a Fuchs-Rosenthal counting chamber. Fifty
microlitres of these protoplasts were transferred to each well
of a 96-well microtiter plate so that approximately 100 wild
type feeder protoplasts were transferred into each well.
Table 1 : Composition of the 8p2c medium (pH 5.6)
- Kao und Michayluk basal salt mixture (Duchefa)
- Kao und Michayluk vitamine solution (Sigma)
0.02 mg/l p-Aminobenzoic acid
2 mg/l L-Ascorbic acid
0.01 mg/l Biotin
1 mg/l D-Calcium pantothenate
1 mg/l Choline chloride
0.4 mg/l Folic acid
100 mg/l Myo-inositol
1 mg/l Nicotin amide
1 mg/l Pyridoxine HC1
0.2 mg/l Riboflavin
1 mg/l Thiamine HC1
0.01 mg/l Vitamin A
0.02 mg/l Vitamin B12
0.01 mg/l Vitamin D
- Organic acids (pH 5.5 with NH OH)
20 mg/1 Sodium pyruvate
40 mg/1 Malic acid
40 mg/1 Citric acid
40 mg/1 Fumaric acid
- Sugar and sugar alcohols
0.25 g/1 Sucrose
250 mg/1 Mannose
68.4 g/1 Glucose
250 mg/1 Rhamnose
250 mg/1 Fructose
250 mg/1 Cellobiose
250 mg/1 Ribose
250 mg/1 Sorbitol
250 mg/1 Xylose
250 mg/1 Mannitol
- Hormones
0.2 mg/1 2.4-D
0.5 mg/1 Zeatin
1.0 mg/1 NAA
- 2% (v/v) Coconut water
- 500 mg/1 Casamino acid
D . Flow cytometric analysis and sorting
The FACS Vantage (DIVA option, BD Bioscience) instrument with
a 488 n / 635 nm Argon ion laser was used for sorting of
transgenic plant protoplasts. The sheath fluid, a phosphate
buffered saline (PBS pH 7.4), was sterilized by autoclaving
and by passage through a 0.22 m filter. Prior to sorting the
sample tubes were cleared of residual ethanol by passage of
sterile sheath fluid. The cytometer system/sorting settings
were aligned using commercial standard autof luorescent
calibration particles. The flow sorter was operated at 488 nm
with a laser output of 175 W . Prior to sorting the electronic
sort windows were positioned based upon signals collected for
forward light scatter, side light scatter and fluorescence of
a protoplast culture sample in order to define the strongly
fluorescent population. The signals were displayed as dot
plots using the DIVA software (BD Bioscience) . Sort regions
were defined by creating gates first around the population of
viable protoplasts and second, based on the first gate, around
the population of strongly fluorescent protoplasts. Sorting
was performed through a 200- m h flow tip with a system sheath
pressure of 4-6 psi, a drop frequency of approx. 7 kHz and a
sample flow rate of approx. 1.000 events/sec.
Using the described sorting parameters a plating efficiency of
20% (i.e. 20% of the wells contained intact and viable single
sorted protoplasts) was achieved.
E . Regeneration of single sorted protoplasts by co-cultivation
with nurse/feeder protoplasts
Prior to the sorting of highly fluorescent single protoplasts
each well of a 96-well microtiter plate was filled with 50 m ΐ
of sterile 8p2c regeneration medium containing approx. 100 N .
tabacum cv . BY-2 wild type protoplasts as feeder cells. The
single sorted transgenic protoplasts were analysed by inverse
fluorescence microscopy at different time points to verify
single cell deposition after the sorting process and also to
monitor the proliferation and microcolony formation (14-20
days after sorting) of the transgenic protoplasts. The
cultivation of sorted protoplasts in 96-well plates took place
at 26°C to 27°C in the dark, the plates were closed with a
sterile lid and sealed with adhesive tape.
Transgenic microcolonies were then transferred to solid
regeneration medium (0.8 % (w/v) agar), containing an
antibiotic selection marker (e.g. kanamycin) . Therefore, the
microcallus tissue including the feeder cells present in the
wells were gently resuspended by pipetting and transferred
using a pipette with a wide tip end. Subsequently, the wells
and also the transferred microcalli on the solid regeneration
medium were analysed by inverse fluorescence microscopy to
verify the successful transfer of the transgenic and
fluorescent microcolonies . Upon the transfer transgenic
microcalli were grown for 14-20 days and transferred to fresh
plate containing solid regeneration medium including the
selection marker. Callus tissue with a size of approximately 2
cm in diameter was used to establish suspension cultures by
transfer of the cell material to 5 ml cultivation medium
(described in section A ) in 50 ml plastic tissue culture
flasks. These flasks were cultured as described in section A
until the cell suspension was grown up to a packed cell volume
of about 50-60% for transfer to 100 ml glass Erlenmeyer
flasks. The cultivation of the transgenic monoclonal
suspension culture was performed as described in section A .
The described feeder cell strategy permits the regeneration of
about 50% of the initially sorted intact and viable single
protoplasts (i.e. ca . 10% of the single protoplasts sorted
into the wells of a 96-well microtiterplate developed to
microcalli ).
F . Verification of the successful elimination of feeder cell
survival during regeneration of sorted single protoplasts
A procedure has been developed that enables the reliable
regeneration of single FACS selected protoplasts to monoclonal
suspension cultures. Since single protoplasts have to be
regenerated after sorting, feeder cells are required to
support regeneration and proliferation of sorted single
protoplasts. Because the feeder protoplasts are temporarily
co-cultivated with sorted fluorescent target protoplasts it is
mandatory to exclude a survival of feeder protoplasts during
the regeneration of monoclonal cultures.
The potential contamination of single sorted transgenic and
fluorescent BY-2 cells with feeder protoplasts has been
investigated. Transgenic cells have been transformed with a
construct containing a GFP-KDEL expression cassette and an
AHAS selection marker (conferring Imazethapyr resistance) .
Single BY-2 protoplasts transformed with this construct and
producing GFP were sorted into 96-well plates containing
protoplasts of a transgenic cell line containing a DsRed
expression cassette and a nptll selection marker (conferring
kanamycin resistance) . In a second experiment, single BY-2
protoplasts transformed with the DsRed expression cassette and
a nptll selection marker were sorted into 96-well plates
containing protoplasts of the transgenic cell line producing
GFP. After regeneration the resultant GFP and DsRed
fluorescent cultures were analyzed with respect to their
resistance towards imazethapyr or kanamycin and their
fluorescence (green versus red) . Callus tissue from both
approaches was plated on selection medium containing either
1.5 mM imazethapyr or 100 mg/L kanamycin. Cell growth was
evaluated visually after 14 days of incubation. All of the
tested calli (20 in total) grew exclusively on medium plates
containing their specific selective agent. In brief, GFP/AHAS
transformed calli grew on imazethapyr but not on kanamycin
containing plates whereas DsRed/kanamycin transformed calli
grew only on kanamycin plates. This observation clearly
demonstrated that regenerated transgenic cell lines were not
contaminated with the respective feeder cell line. A potential
contamination with feeder cells was additionally assessed by
flow cytometric analysis. The regenerated GFP and DsRed
suspension cultures were analyzed with respect to their
optical properties elicited by the fluorescent proteins GFP or
DsRed, respectively. This observation clearly demonstrated
that regenerated transgenic cell lines were not contaminated
with the respective feeder cell line. All tested BY-2 cultures
showed exclusively the expected fluorescence pattern. Cultures
established after sorting of GFP transformed cells show only
green fluorescence while cultures producing DsRed were
detected exclusively in the red fluorescence channel. In the
case of a contamination with feeder cells a fluorescence
signal in both channels would have been expected. The result
of the cytometric analysis confirmed the efficient removal of
feeder cells on selection plates as demonstrated before by the
resistance test.
G . Analysis of monoclonal transgenic suspension cultures
The monoclonal suspension cultures were first analysed
regarding their percentage of highly fluorescent cells.
Therefore, protoplasts were prepared as described in section
C . For the flow cytometric determination of the portion of
fluorescent protoplasts the FACS Calibur Instrument (BD
Bioscience) was used. Based on BY-2 wild type protoplasts the
setting parameters (e.g. amplification of light and
fluorescence scatter multipliers) were adjusted and the
samples were measured. After gating the viable population, the
distribution of this population in the fluorescence channel
was used to set a threshold, which excluded all background
signals caused by the wild type autof luorescence . According to
this threshold the percentage of fluorescent protoplasts
within the improved protoplast cultures derived from a single
protoplast was calculated. Flow cytometric analyses of the
monoclonal suspension cultures producing the recombinant DsRed
protein displayed homogeneously distributed cells of similar
and strong fluorescence intensities (narrow fluorescent
peaks) . The calculation of the DsRed fluorescent cell portions
resulted in percentages ranging between 78-88% strongly
fluorescent cells.
The accumulation levels for the recombinant protein can be
determined by different procedures (e.g. enzyme linked
immunosorbent assay (ELISA) ). Therefore, the cells were
centrifuged (850 g , 5 in ), resuspended in 3 Vol. extraction
buffer (PBS pH 6 , 5 2-mercaptoethanol, 5 EDTA, 10
ascorbic acid) and disrupted by sonication. The extract was
separated from cell debris by another centrif ugation step (20
min., 16 000 g ) and used for analysis. Immunological analysis
of the M12 antibody accumulation in 5 dpi cell extracts of
suspension cultures transformed with pTRAkc:MTED revealed up
to 118 ± 20 g/g fresh weight (1.5 fold higher than using the
conventional generation method i.e. callus generation and
screening) .
Example 2
Generation of monoclonal cell lines from a heterogeneous
transgenic suspension culture
A . Tobacco ce l culture
The transgenic Nicotiana tabacum cv. Bright Yellow 2 (BY-2)
suspension culture MTED#18 producing the ER-retarded human
full-size IgGl antibody M12 and the plastid targeted
fluorescent protein DsRed was maintained in the dark under
sterile conditions as 50 ml aliquots in 100 m l glass
Erlenmeyer flasks at 26°C, with a constant orbital agitation
of 180 rpm. BY-2 wild type cells were cultivated under the
same conditions as control. The cultivation medium comprised
basal MSMO medium at pH 5.8 supplemented with sucrose (3%,
w/v) and 1 mg/l 2,4-dichlorophenoxyacet ic acid. Subculture was
done at day 7 intervals by transfer of 5% (v/v) of the cells
into 50 ml fresh medium.
The transgenic suspension culture has been generated by
gro acteri -mediated transformation of N . tabacum cv. BY-2
cells followed by an antibiotic based selection and subsequent
separation of transformed callus tissue. The callus tissues
were screened according to their antibody production by
immunological assays (Dotblot and ELISA) and the best
candidate was used for suspension culture establishment (=
cell line MTED# 18). The specific M l2 antibody production of
the parental MTED#18 culture was 13 q /q fresh cell weight (10
mg/L) . Flow cytometric analysis revealed that the transgenic
culture consists of two subpopulat ions with only 24 % of the
viable population producing the fluorescent marker protein
DsRed .
For protoplast preparation the suspension cell culture was
subcultured by transfer of 2% (v/v) into 50 ml fresh medium.
B . Protoplast preparation and cell wall regeneration
See example 1 , section C .
Usually the described procedure resulted in 5xl0 5 protoplasts
per ml with an average percentage of 62.2 of viable transgenic
protoplasts.
C . Flow cytometric analysis and sorting
The instrument settings and pre-arrangement s were done as
described in example 1 , section D .
Single strongly fluorescent plant protoplasts (1-2% of all
sorted protoplasts) were sorted into cell deposition device
(i.e. microtiter plates) in single cell mode. One protoplast
per well, consistent to the second gated criteria, was sorted
into 96-well plates filled with 50 mΐ of sterile 8p2c
regeneration medium containing approx. 100 wild type
protoplasts as feeders. The 96-well plates were closed using a
sterile lid and sealed by an adhesive tape. The actual number
of recovered protoplasts was determined by inverse
fluorescence microscopy. The flow cytometric sorting of
protoplasts in single cell mode resulted in a plating
efficiency of approximately 20% wells containing one intact
and viable protoplast per well.
D . Regeneration of sorted protoplasts at low densities
The regeneration of single sorted protoplasts was performed as
described in example 1 , section E .
The flow cytometric sorting of highly fluorescent protoplasts
in the single cell mode resulted in approximately 20% of the
wells containing only one sorted protoplast. 50% of these
single protoplasts started proliferation and could be used to
establish suspension cultures.
E . Analysis of monoclonal transgenic suspension cultures
To determine the percentage of fluorescent protoplasts in
improved suspension cultures derived from a single protoplast
a flow cytometric analysis was performed as described before
(experiment 1 , section D ) . The M12 antibody accumulation
levels were determined by an enzyme linked immunosorbent assay
(ELISA) . Therefore, the suspension cells were centrifuged (850
g , 5 min), resuspended in 3 Vol. extraction buffer (PBS pH 6 ,
5 m 2-mercaptoethanol, 5 m EDTA, 10 m ascorbic acid) and
disrupted by sonication. The extract was separated from cell
debris by a centrif ugation step (20 min, 16000 g ) and used for
analysis. Due to the chosen set up (Fc capture and LC
detection) only fully assembled antibodies were detected.
After one FACS round the monoclonal suspension cultures showed
significantly improved accumulation levels for both
recombinant proteins: 3.7-fold enriched percentage of DsRed
fluorescent cells (90%) and 11-fold increase of the M12
antibody (145 yg/g fresh weight or 9.3 fold on mg/L level (93
mg/L) ) when compared to the parental suspension culture.
F . Repetition of suspension culture improvement
In order to further increase and stabilize recombinant protein
productivity of transgenic monoclonal suspension cultures
steps B-E can be repeated. The same conditions for protoplast
generation sorting and regeneration as described before were
applied .
The second sorting round resulted in a further increase of
antibody accumulation: 182 g/g fresh weight or 113 mg/L,
which is a 14-fold and 11.3-fold increase compared to the
parental culture.
A third round of sorting of the best producing 2nd generation
monoclonals resulted in 3rd generation monoclonal cultures
producing similar accumulation levels indicating that the
maximum level was achieved.
G . Stability of elite producing monoclonal cultures in terms
of target protein productivity
The stability of FACS derived monoclonal cell lines was
investigated exemplary for 3 monoclonal lines. Monoclonal cell
lines were subcultured in a 7 day cycle (refer to example 1 ,
section A ) while both recombinant target proteins were
measured at 2 month intervals always on day 5 after
subculture. Over a period of 12 month it has been demonstrated
for the 1st generation of monoclonal cultures that these
cultures still produce high and stable amounts of the M12
antibody per gram fresh weight. Only slight variations of
antibody levels in the bi-monthly sampling intervals (caused
by cell cultivation variations) were observed.
Analysis of the 2nd generation monoclonals verified cell line
stability by showing similar or slightly increased
accumulation levels of both recombinant proteins M12 antibody
and DsRed compared to the 1st generation monoclonal culture
they were derived from. Over all the analyzed monoclonal
cultures of the 2nd generation appear more stable in terms of
target protein production (less variations in bi-monthly
sampling intervals) compared to 1st generation monoclonal
cultures. During a period of 12 month two of the three
analyzed monoclonal cultures were found to be highly stable
regarding their percentage of DsRed fluorescent cells in the
total population as well as M12 antibody accumulation.

Claims
Method for the generation of a monoclonal plant cell line
from a heterologous population of plant cells, comprising
the following steps:
(a) provision of a heterologous population of plant cells;
(b) preparation of protoplasts from said heterologous
population of plant cells;
(c) separation of single protoplasts by subjecting the
preparation of protoplasts to flow cytometric sorting;
(d) regeneration of a separated single transformed
protoplast until the formation of a microcolony by cocultivation
in the presence of feeder cell material;
(e) removal of the microcolony from the feeder cell
material and cultivation of the microcolony until the
formation of a monoclonal plant cell line.
Method according to claim 1 , further comprising the
regeneration of the monoclonal plant cell line as obtained
in step (e) into whole fertile plants.
Method according to claim 1 or 2 , wherein the protoplasts
as prepared in step (b) are transformed and capable of (i)
producing a fluorescent marker protein or polypeptide, (ii)
producing a desired product, and/or (iii) surviving in
presence of a selection agent.
Method according to claim 3 , wherein the desired product is
selected from the group consisting of heterologous proteins
or polypeptides, secondary metabolites, and markers for
diagnostic or analytic purposes.
5 . Method according to any one of the preceding claims,
wherein the separation of single protoplasts in step (c) is
effected by subjecting the protoplasts to FACS.
6 . Monoclonal plant cell line, obtainable or obtained by
carrying out the method as defined in any one of claims 1
to 5 .
7 . Whole fertile plant, obtainable or obtained by carrying out
the method as defined in any one of claims 2 to .