Journal of Experimental Botany, Vol. 54, No. 383, pp. 699±706, February 2003
DOI: 10.1093/jxb/erg079
RESEARCH PAPER
Genes encoding two essential DNA replication activation
proteins, Cdc6 and Mcm3, exhibit very different patterns
of expression in the tobacco BY-2 cell cycle
Gerardas Dambrauskas1,4, Stephen J. Aves1, John A. Bryant1,3, Dennis Francis2 and Hilary J. Rogers2
1
2
School of Biological Sciences, Washington Singer Laboratories, University of Exeter, Exeter EX4 4QG, UK
School of Biosciences, Cardiff University, PO Box 915, Cardiff CF10 3TL, UK
Received 4 July 2002; Accepted 14 October 2002
Abstract
Very little is known about the expression patterns in
plants of genes that encode proteins involved in the
initiation of DNA replication. Partial cDNA sequences
that encode Cdc6 and Mcm3 in tobacco have been
isolated. The sequences were used as probes in
northern blots which suggested that, in the cell cycle
of synchronized tobacco BY-2 cells, expression of
CDC6 is con®ned to late G1 and S-phase whereas the
expression of MCM3 is not con®ned to any particular
cell cycle phase. These data were con®rmed and
extended by real-time PCR measurements of mRNA
abundance through the cell cycle. CDC6 exhibits a
very clear peak of expression in S-phase whereas
MCM3, expressed at a much lower level than CDC6,
is not cell-cycle-regulated. These patterns of cell
cycle gene expression resemble those found in the
®ssion yeast Schizosaccharomyces pombe rather
than those in budding yeast or mammalian cells.
Key words: BY-2, Cdc6, cell cycle, DNA replication, Mcm3,
tobacco.
Introduction
The initiation of DNA replication at eukaryotic origins of
replication is a complex multi-step process (reviewed in
Bryant et al., 2001). In summary, origins marked by the
origin±recognition complex are recognized and bound by
the `loading factors' Cdt1 and Cdc6. This permits the
binding of the MCM (mini-chromosome maintenance)
complexes, leading to the expulsion of the loading factors.
3
4
The helicase activity of the MCM complexes initiates the
unwinding of the double helix leading to the formation of a
replication bubble and the establishment of two replication
forks. Binding of the primase-polymerase-a loading
factor, Cdc45, then occurs, followed by loading on to the
template of the primase-polymerase-a complex itself.
This multi-step process involves proteins that are
required transiently at each origin, such as Cdt1, Cdc6
and Cdc45, and proteins required throughout replication of
the complete replicon, such as the MCM proteins (Labib
and Dif¯ey, 2001) and primase-polymerase-a (required
not only for the initiation of the leading strand but also for
the initiation of each Okazaki fragment). In common with
most other proteins involved in plant DNA replication,
very little is known about expression patterns of the genes
encoding these important proteins during the cell cycle.
Sabelli et al. (1996) suggested, on the basis of the
distribution of mRNA as detected by in situ hybridization,
that the expression of MCM3 in maize roots was restricted
to only a part of the cell cycle, but it was not possible to
ascertain which phase that was. For CDC6, however, there
are clear data from Arabidopsis thaliana cell cultures
showing that expression is limited to late G1 and S-phase
(de Jager et al., 2001; Castellano et al., 2001).
In other eukaryotes, two general patterns of gene
expression have been observed. In budding yeast,
Saccharomyces cerevisiae, a very high proportion of the
genes involved in DNA replication are expressed only in
S-phase (or in late G1 plus S-phase) under the control of the
MCB (MluI cell cycle box) elements in their promoters
(Johnston and Lowndes, 1992; Koch and Nasmyth, 1994).
A very similar pattern occurs in Metazoa, although some of
the genes, such as those encoding replicative enzymes, are
To whom correspondence should be addressed. Fax: +44 (0)1392 264668. E-mail: [email protected]
Present address: Department of Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
Journal of Experimental Botany, Vol. 54, No. 383 ã Society for Experimental Biology 2003; all rights reserved
700 Dambrauskas et al.
expressed at signi®cant levels outside S-phase, but are
further up-regulated in S-phase (Yamaguchi et al., 1996;
Zhao and Chang, 1997; Huikeshoven and Cotterill, 1999).
In ®ssion yeast, Schizosaccharomyces pombe, by contrast,
the expression of only a few DNA replication genes is
con®ned to S-phase. These include cdt1, cdc18 (homologous to CDC6 of S. cerevisiae), cdc22 (large subunit of
ribonucleotide reductase) and cig2 (a B-type cyclin)
(Gordon and Fantes, 1986; Fernandez Sarabia et al.,
1993; Kelly et al.,1993; Connolly and Beach, 1994; ObaraIshihara and Okayama, 1994; Hofmann and Beach, 1994).
Other DNA replication genes that have been investigated
in S. pombe, including those encoding the initiating DNA
polymerase and the MCM proteins, fail to show a peak of
expression at any particular cell cycle phase.
Using an easily synchronizable cell line should help to
resolve the timing of expression of regulatory genes of
S-phase in the plant cell cycle. Therefore, the expression of
genes encoding replication initiation proteins in tobacco
(Nicotiana tabacum) BY-2 cells is studied here. For this
study, two genes, CDC6 and MCM3 that show different
expression patterns in S. pombe but similar patterns in
S. cerevisiae and in Metazoa, were selected initially.
Expression of these genes in synchronized cultures, using
two different techniques for mRNA assay, has been
rigorously investigated. The results clearly show that the
expression of tobacco CDC6 is con®ned to late G1 and
S-phase, whereas the expression of MCM3 occurs throughout the cell cycle.
Materials and methods
Maintenance and synchrony of tobacco BY-2 cells
The tobacco BY-2 cell line (derived from Nicotiana tabacum cv.
Bright Yellow-2) was maintained as described by Nagata et al.
(1992). The cells were synchronized by a 24 h incubation of
stationary phase cells (7 d since previous subculture) in a medium
containing 5 mg ml±1 aphidicolin (Herbert et al., 2001). The cells
were released from the aphidicolin block by thorough washing in
BY-2 medium and then transferred into 100 ml fresh medium.
The mitotic index was determined by staining nuclei with 0.1 mg
ml±1 bis-benzamide tri-hydrochloride, Hoechst 33258, solution
(Sigma) in the presence of 2% Triton X-100 and examining them
under UV light (365 nm) with a Nikon EFD-3 microscope.
Preparation of probes for northern hybridization
N. tabacum complementary DNA (cDNA) was made as described
below (see Reverse transcription) and probes were generated from
the cDNA by PCR. Primers for MCM3 were based on the pea (Pisum
sativum) homologue (Accession no. AJ012750). Primers for CDC6
were based on the A. thaliana homologue (Accession no.
AJ293020). Primers for GAPdH were based on the tobacco cytosolic
glyceraldehyde-3-phosphate dehydrogenase sequence (Accession
no. M14419). The primers are listed in Table 1. Thirty cycles of PCR
were performed from cDNA at 95 °C for 2 min, 55±65 °C
(depending on the primers used) for 30 s and 72 °C for 1 min. The
products were cloned into pGEMâ-T Easy Vector (Promega) and
sequenced. The probe (196 bp) for A. thaliana histone H4 was a gift
from Dr Nicole Chaubet-Gigot (Strasbourg).
Table 1. Primers used for ampli®cation of sequences from
cDNA
Target
Sequence (5¢ to 3¢)
MCM3
MCM3
CDC6
CDC6
GAPdH
GAPdH
CTG CGT CTG GTC AGC TCC CAA GAA
CTG TCA GCA AGA ACC ATT GCC CCA
GGG TGT CCA GGA ACT GGA AGT
GCG CTC CTG CAG ACA CAT AGA GCC T
GCT GCT GAC TTG AAG GGT GGT GCC
CCC CAT TCA TTG TCA TAC CAC GAC
RNA extraction and Northern blots
Total RNA was isolated from BY-2 cells using TRI ReagentÔ
(Sigma) following the manufacturer's protocol. RNA samples
(30 mg) were separated on 1% agarose formaldehyde gel and
transferred onto Nylon 66-Blotting membrane (Fluka). The probes
were labelled with 32P using Ready-To-Go DNA Labelling Beads
(Amersham Biosciences) and hybridized in Northern MaxÔ
hybridization solution (Ambion) overnight at 42 °C. The membrane
was washed with 13 SSPE, 0.1% SDS for 10 min at 42 °C followed
by two washes with 0.53 SSPE 0.1% SDS for 10 min at room
temperature. The membrane was exposed to X-ray ®lm (Fuji) with
an intensifying screen.
Reverse transcription
Complementary DNA was synthesized in a 50 ml reaction volume. A
sample of total RNA (10 mg) was incubated at 65 °C for 5 min with
500 ng of oligo-dT primer (Sigma), then cooled for 10 min to 22 °C
to allow oligo-dT to anneal to the poly(A) tail of mRNA. 5 ml of 103
®rst strand buffer (Stratagene), 40 units of RNase Inhibitor (Sigma),
dNTPs to 200 mM, and 80 units of M-MuLV reverse transcriptase
(Stratagene) were added to the reaction mix which was incubated at
37 °C for 1 h. To inactivate reverse transcriptase the reaction was
incubated at 90 °C for 5 min. The cDNA was then ready for real-time
PCR.
Primers used for real-time PCR
The primers were based on the N. tabacum MCM3, CDC6, H4, and
GAPdH sequences previously characterized (see above). The
speci®city of the ampli®ed products was veri®ed by gel electrophoresis and sequencing prior to use in real-time PCR reactions. The
primers for real-time PCR are shown in Table 2.
Real-time quantitative PCR
Quantitative PCR was performed with the real-time PCR system,
LightCyclerÔ (Roche Diagnostics, Mannheim, Germany), using
SYBRâ Green I double strand DNA binding dye. The ampli®cation
of a target with the primers listed in Table 2 was carried out in a total
volume of 5 ml containing 1 pmol of each primer, 5 mM MgCl2, 1 ml
LightCyclerÔ DNA Master SYBRâ Green I (containing 1.25 units
of Taq polymerase, 103 Taq buffer (500 mM KCl, 100 mM TrisHCl, pH 8.3), dNTPs each at 2 mM, 103 SYBRâ Green I; Roche
Diagnostics) and 100 ng of cDNA prepared as described above. The
ampli®cation protocol comprised four phases: (i) denaturation of the
template DNA, (ii) ampli®cation of the target DNA, (iii) melting
curve analysis for product identi®cation, and (iv) cooling the rotor
and the thermal chamber. The initial template denaturation was at
95 °C for 30 s, followed by 40 three-step cycles of template
denaturation at 95 °C with a 0 s hold, primer annealing at 63 °C for
7 s and extension at 72 °C for 17 s. Fluorescence data were collected
after each extension step. Melting curve analysis was performed by
heating the template at 95 °C with a 0 s hold, then cooling to 65 °C
with a 15 s hold and ®nally raising to 95 °C with a 0.1 °C s±1
Cdc6 and Mcm3 in the tobacco BY-2 cell cycle 701
Table 2. N. tabacum primers used for the real-time PCR LightCyclerÔ system
Target
Amplicon length
Sequence (5¢®3¢)
Tm (°C)
GAPdH
GAPdH
MCM3
MCM3
Histone H4
Histone H4
CDC6
CDC6
452
452
550
550
320
320
320
320
GCA CTA CCA ACT GCC TTG CAC CT
GGC AAT TCC AGC CTT GGC ATC G
CTG CGT CTG GTC AGC TCC CAA GAA C
CTG TCA GCA AGA ACC ATT GCC CCA GC
GGC ACA GGA AGG TTC TGA GGG ATA ACA
TAA CCG CCG AAA CCG TAG AGA GTC C
GAG AAG CAG CAG CCA GCA GGC ACG
GCA CAT AGT TCC AAT GCT TGA GGC
64.2
64.0
67.9
68.0
66.5
66.3
69.6
62.7
temperature transition rate while continuously monitoring the
¯uorescence. All other phases were performed with a 20 °C s±1
transition rate. Fluorescence was analysed using LightCyclerÔ
Analysis Software. The crossing point for each reaction was
determined using the Second Derivative Maximum algorithm and
manual baseline adjustment. Concentrations of standards were
determined by measuring the A260.
Reporter gene construction and plant cell transformation
The cauli¯ower mosaic virus 35S promoter (CaMV35S) was
removed from the uidA reporter gene plasmid pCAMBIA1305.2
(CAMBIA, Canberra, Australia) by digestion with EcoRI and NcoI.
The A. thaliana MCM3 promoter was ampli®ed using standard PCR
procedures. The forward primer introduced an EcoR1 restriction site
and the reverse primer introduced an Nco1 restriction site. Forward
primer: 5¢-CCTTTTGAATTCTCT GCCCTATCG-3¢. Reverse primer: 5¢-TTACCATGGTTCTTAACAAAATCTGGGTTC-3¢. The
ampli®ed promoter was subcloned into the pGEMâ-T Easy vector
(Promega), checked by sequencing, then excised by digestion with
EcoRI and NcoI and cloned into the pCAMBIA1305.2 vector
upstream of the uidA (gus) reporter gene to create an
MCM3promoter-uidA fusion. The plasmid was introduced into
Agrobacterium tumefaciens LBA 4404 and used to transform
tobacco BY-2 cells, essentially as described by An (1987).
Hygromycin-resistant calli were pooled and reintroduced into
suspension culture. The transgenic cell suspension was maintained
by subculturing 5 ml of stationary-phase cells into 95 ml of fresh
medium supplemented with timentin (ticarcillin/potassium clavulanate) (250 mg ml±1) and hygromycin (100 mg ml±1). After two rounds
of subculture, timentin was omitted from the medium.
Results
CDC6 and MCM3 in tobacco
In order to generate homologous probes for northern
blotting, speci®c fragments of the tobacco CDC6
(Accession no. AJ298071) and MCM3 (Accession no.
AY077639) cDNAs were ampli®ed by PCR, cloned and
sequenced. Alignments with homologous predicted protein
sequences from other plants (Fig. 1) show a high degree of
identity between species for Mcm3: over the regions
analysed Nicotiana tabacum Mcm3 is 81% identical with
Mcm3 of Arabidopsis thaliana, 79% with Pisum sativum,
and 81% with Zea mays. From the limited data available
Cdc6 appears to be less conserved, showing only a 66%
identity between the corresponding proteins in N. tabacum
and A. thaliana (Fig. 1).
Expression of CDC6 and MCM3 in the tobacco cell
cycle
To determine the cell cycle expression patterns of tobacco
CDC6 and MCM3, RNA samples from tobacco BY-2 cells
synchronized by aphidicolin block and release were
analysed. Mitotic indices determined at 1 h time intervals
after release from the block indicated a high degree of
synchrony (Fig. 2a) and this was con®rmed by probing
northern blots with the S-phase marker histone H4
(Fig. 2b). The length of the S-phase observed in these
experiments was c. 3.5 h, similar to that reported for BY-2
cells in other recent publications (Sorrell et al., 1999;
Herbert et al., 2001). An obvious phase-related expression
pattern of CDC6 was observed, with the peak levels of
mRNA occurring in S-phase, after which expression
declined so as to be undetectable by northern hybridization
by late G2/early M-phase (Fig. 2b). By contrast, the
expression of MCM3 did not show any cell cycle-related
pattern: there were ¯uctuations in the amounts of mRNA
detected by northern hybridization (Figs 2b, 3), but these
did not show any clear correlation with cell cycle phase
and indeed varied between different experiments. These
variations were ascribed to dif®culties in detecting scarce
mRNAs (see below) using northern hybridization,
although a high loading of total RNA (30 mg) was used.
Similar patterns of expression for CDC6 and MCM3 were
observed in cells that had been subjected to a double-block
regime (aphidicolin and propyzamide) (data not shown).
Real-time PCR analysis of CDC6 and MCM3 mRNA in
the tobacco cell cycle
In order to con®rm and extend the results obtained by
northern hybridization a further series of experiments was
carried out in which estimates of relative mRNA
abundances were made by quantitative RT-PCR using
the LightCyclerÔ system. The concentration of glyceraldehyde 3-phosphate dehydrogenase (GAPdH) mRNA in
each sample was determined to correct for differences in
the amount of RNA (northern analysis did not reveal any
¯uctuations of GAPdH expression during the cell cycle:
Fig. 2b). The expression of histone H4 was again used as a
marker for S-phase (Fig. 4). Some histone H4 expression
was detected at all time points but the obvious peak of
702 Dambrauskas et al.
Fig. 1. (a) Alignment of tobacco Mcm3 predicted protein sequence (Nt, accession no. AAL76090) with the corresponding sequences from pea
(Ps,: CAA10166), Arabidopsis (At: CAA03887), and maize (Zm: AAD48087). (b) Alignment of tobacco Cdc6 predicted protein sequence (Nt
Accession no: CAC18552) with Arabidopsis Cdc6b (At: CAC83650).
expression during the ®rst 2±3 h after release from the
aphidicolin block clearly indicated that the bulk of cells
were in S-phase during that period. Comparison of the
expression patterns of CDC6 and MCM3 again revealed
clear differences: MCM3 did not show a phase-related
peak of expression whereas the expression of CDC6 was
strongly correlated with S-phase (Fig. 4). These data
clearly con®rm the conclusions from the Northern
hybridization data and also show that, during S-phase,
the CDC6 message is three to four times more abundant
than the MCM3 message.
Reporter gene expression driven by the MCM3
promoter
Synchronized cells that had been transformed with the
uid-A (gus) gene under the control of the Arabidopsis
thaliana MCM3 promoter provided further con®rmation of
the expression pattern of MCM3. Figure 5 shows an RTPCR analysis of gus mRNA through the cell cycle: there is
no phase-related peak of expression, in strong contrast to
the expression pattern of the resident histone H4 gene.
Discussion
The cloning and characterization of speci®c fragments of
CDC6 and MCM3 from tobacco add to the very small
number of plants from which these sequences have been
isolated and extends previous work from this group on
MCM3 in pea (Accession no. AJ012750; Bryant et al.,
2001).
These results are the ®rst to show expression patterns in
the tobacco cell cycle of genes directly involved in DNA
replication and clearly indicate that the two genes studied
here exhibit very different patterns of expression. The
expression of CDC6, encoding one of the MCM loading
factors, in BY-2 cells is similar to that of RNR2
(ribonucleotide reductase small subunit) in being strongly
correlated with DNA replication (Philipps et al., 1995;
Cdc6 and Mcm3 in the tobacco BY-2 cell cycle 703
Fig. 2. Expression within the tobacco BY-2 cell cycle of MCM3,
CDC6 and histone H4. (a) Time-course of tobacco BY-2 mitotic index
after release from the aphidicolin block. (b) Detection by northern
hybridization of mRNAs encoding Cdc6, Mcm3, histone H4, and
GAPdH throughout the cell cycle after release from the aphidicolin
block. Cell cycle phases were determined as previously described
(Herbert et al., 2001).
Chaboute et al., 1998). In this respect, it resembles the
expression pattern of CDC6 homologues in nearly all other
eukaryotes in which it has been investigated, including
S. pombe (Kelly et al., 1993) and Metazoa (Hateboer et al.,
1998; Yan et al., 1998). These results are also similar to
those very recently obtained with cultured cells of
Arabidopsis thaliana (de Jager et al., 2001; Castellano
et al., 2001). Indeed, the patterns of expression of both
CDC6 and histone H4 after release from the aphidicolin
block in A. thaliana resemble very closely those reported
here for tobacco, albeit that the data presented here for
tobacco provide a more detailed analysis than that carried
out with A. thaliana.
Ramos et al. (2001), however, report that CDC6 is
apparently strongly expressed in cultured A. thaliana roots
treated with oryzalin (a mitotic metaphase inhibitor), but is
not expressed in roots treated with hydroxyurea which
were presumed to be held in S-phase. However, allocation
to cell cycle phase may have been problematic because it is
noted that histone H4 was expressed as strongly in roots
held in M-phase as in control roots that had not been
blocked at any cell cycle phase. The likelihood is therefore,
based on results from tobacco (this paper) and A. thaliana
cell cultures (de Jager et al., 2001; Castellano et al., 2001),
that CDC6 expression occurs in late G1 and in S-phase.
In animals, the expression or up-regulation of genes in
late G1 and in S-phase is dependent on transcription
factors of the E2F family (Zhao and Chang, 1997;
Fig. 3. Relative expression within the tobacco BY-2 cell cycle of
MCM3, CDC6 and histone H4 analysed by northern hybridization.
Data were normalized against the expression of GAPdH, a
housekeeping gene.
Hateboer et al., 1998; Leone et al., 1998; Yan et al., 1998).
Thus E2F transcription factors are the functional equivalents of the DSC1 transcription factors in S. cerevisiae that
recognize the MCB elements. Regulation by E2F transcription factors has now been shown for a limited number
of plant genes, including a CDC6 of A. thaliana (de Jager
et al., 2001; Castellano et al., 2001) and the tobacco genes
704 Dambrauskas et al.
Fig. 5. RT-PCR detection of gus expression driven by the Arabidopsis
thaliana MCM3 promoter in aphidicolin synchronized tobacco BY-2
cells. Histone H4 expression was used to indicate S-phase. GAPdH is
presented as the loading control.
Fig. 4. Relative expression within the tobacco BY-2 cell cycle of
MCM3, CDC6 and histone H4 analysed by real-time PCR. Data were
normalized against the expression of GAPdH, a housekeeping gene.
Error bars represent the range of values between two replicates within
an individual experiment.
encoding the small subunit of ribonucleotide reductase
(Chaboute et al., 1998) and PCNA (Egelkrout et al., 2001;
Kosugi and Ohashi, 2002).
The expression pattern of MCM3, in showing no clear
phase-related peaks, resembles that of mcm3 in S. pombe,
but contrasts with that in S. cerevisiae and in mammals
where there is a peak of expression in S-phase (see
Introduction). It is thus interesting that the promoter of the
MCM3 gene in A. thaliana contains two E2F transcription
factor recognition sites (see accession no. AJ000058) and
that this promoter drives the expression of a reporter gene
in tobacco cells in a similar cell cycle phase-independent
manner to that observed with the resident MCM3 gene.
Thus possession of E2F recognition sites within a promoter
is not enough to confer a late G1-S expression pattern.
However, it is noted that, in a similar series of
experiments recently reported by Stevens et al. (2002),
the A.thaliana MCM3 promoter did confer on the gus gene
a clear pattern of expression in G1-S of the tobacco BY-2
cell cycle. These authors further concluded that the two
E2F binding sites were both involved in regulating this
pattern of expression. The reason for this difference
between the results of Stevens et al. and those presented
here is not clear. However, in their paper no analysis of
expression of the resident MCM3 gene in BY-2 cells was
presented, whereas the conclusions reached by the present
authors are based on extensive and repeated analysis of the
expression of the tobacco MCM3 gene.
In relation to research on MCM3 in other plants, Sabelli
et al. (1999) were unable to detect Zea mays MCM3
mRNA by northern hybridization whereas in this laboratory a 2.7 kb message has been detected in tobacco (this
paper) and a 2.9 kb message has been detected in pea
(Moore, 1999; Bryant et al., 2001). Sabelli et al. (1999)
ascribe their failure to an inability to incorporate suf®cient
radiolabel into the probe, but it is just as probable that the
problem resides in the low abundance of the mRNA (see
Results). However, Sabelli et al. (1996) had earlier
Cdc6 and Mcm3 in the tobacco BY-2 cell cycle 705
reported that in situ hybridization of MCM3 mRNA
showed an uneven distribution in Z. mays root meristems,
consistent with expression being con®ned to cells in
particular, but unidenti®ed phases of the cell cycle. The
data obtained with cultured tobacco cells presented here
clearly do not support this suggestion, although the
possibility of differences between dicots and monocots is
acknowledged.
Thus far, the limited data available for plants suggest
greater similarity to S. pombe in which few genes other
than cdt1 and cdc18 (=CDC6) show G1-S phase-speci®c
cell cycle expression (see Introduction), than to
S. cerevisiae and to Metazoa in which many DNA
replication genes show S-phase-related peaks of expression. The signi®cance of these expression patterns for plant
development is not clear. However, it can be stated that
regulation of the activity of Mcm3 (and, most probably,
other MCM proteins) must rely on post-transcriptional
mechanisms. The need for further study of the regulation
of the expression of these genes, especially in whole
plants, is clear. Such research is now in progress.
Acknowledgements
GD gratefully acknowledges ®nancial support from the Lithuanian
government, from the UK Overseas Research Scholarship scheme
and from the University of Exeter (Stratton Scholarship). We also
thank Dr Nicole Chaubet-Gigot for providing the histone H4 probe,
CAMBIA (Canberra, Australia) for the provision of vector
pCAMBIA1305.2, Professor Janet Hemingway and Dr Caroline
Wickham for use of the LightCyclerÔ systems in their laboratories,
and Dr David Sorrell for many useful discussions on working with
tobacco BY-2 cells.
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