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Polyamines influence morphogenesis and

caffeine biosynthesis in in vitro cultures of
Coffea canephora P. ex Fr.

Article in Acta Physiologiae Plantarum · March 2007

DOI: 10.1007/s11738-007-0110-x

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Acta Physiol Plant (2008) 30:217–223
DOI 10.1007/s11738-007-0110-x

ORIGINAL PAPER

Polyamines influence morphogenesis and caffeine biosynthesis

in in vitro cultures of Coffea canephora P. ex Fr.
Vinod Kumar Æ P. Giridhar Æ A. Chandrashekar Æ
G. A. Ravishankar

Received: 2 June 2006 / Revised: 29 August 2007 / Accepted: 26 September 2007 / Published online: 15 November 2007

Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2007

Abstract The influence of polyamines, polyamine Abbreviations

inhibitors and ethylene inhibitors were tested in Coffea NEC Non-embryogenic callus
canephora for in vitro morphogenetic response and caf- BA 6-Benzylaminopurine
feine biosynthesis. Coffea canephora produced non- IAA Indole acetic acid
embryogenic and embryogenic calli. Somatic embryos AgNO3 Silver nitrate
were produced only from the embryogenic callus. Endo- PA Polyamine
genous polyamine pools were estimated in these tissues. Put Putrescence
Somatic embryos were subjected to secondary embryo- SAM S-Adenosyl methionine
genesis under the influence of putrescine, silver nitrate and DFMA-a DL-Difluromethyl arginine
specific inhibitors of polyamine biosynthesis. Estimation of DFMO-a DL-Difluromethyl ornithine
endogenous total polyamines revealed that embryogenic
callus contained 11-fold more spermine and 3.3-fold higher
spermidine when compared to non-embryogenic callus.
Incorporation of polyamines resulted in 58% explant Introduction
response for embryogenesis when compared to control with
42% response. Incorporation of silver nitrate resulted in Plant embryogenesis represents the most definitive stages
65% response for embryogenesis. Incorporation of poly- of the plant life cycle, with the overall architectural pattern
amine biosynthetic pathway inhibitors DFMO and DFMA of the mature organism established during a relatively short
resulted in 83% reduction in embryogenic response with interval in many plant species (Thomas 1993). Endogenous
concomitant increase in caffeine levels by two-fold as and exogenously administered hormones play a crucial role
compared to control. These results have clearly demon- in somatic embryogenesis. Polyamine pools and ethylene
strated that polyamines play a crucial role in pathway are interlinked and known to redirect the cell
embryogenesis and caffeine biosynthesis. towards embryogenesis when present in optimum concen-
trations (Feirer et al. 1984). Secondary embryogenesis
Keywords Caffeine
Coffea canephora
Polyamines
process requires a fine balance in the reprogramming of
S-adenosyl-L-methionine
Somatic embryogenesis cells towards differentiation and maturation.
Polyamines (PAs) viz. putrescine, spermidine and
spermine are known to play important role in various cel-
Communicated by J. Kepczynski.
lular processes (reviewed by Bais and Ravishankar 2002).
Chemically, they are non-protein, straight chain, aliphatic
V. Kumar
P. Giridhar
A. Chandrashekar
amines. Polyamines are known to be involved in DNA
G. A. Ravishankar (&) replication, cell division, protein synthesis, responses to
Plant Cell Biotechnology Department,
abiotic stress, rhizogenesis, flower development and in
Central Food Technological Research Institute,
Mysore 575 020, India vitro flower induction (reviewed by Bais and Ravishankar
e-mail: [email protected] 2002). Polyamines are reported to inhibit ethylene

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218 Acta Physiol Plant (2008) 30:217–223

biosynthesis in plants (Apelbaum et al. 1981). Evidence Materials and methods

from several studies have indicated that polyamines play a
crucial role in somatic embryogenesis and regeneration of Explant source and media
several plant species (reviewed by Bais and Ravishankar
2002; Kumar and Rajam 2004). Seeds of Coffea canephora selection 274 was obtained
Putrescine is synthesized from ornithine as well as from from Central Coffee Research Institute (CCRI) Balehon-
arginine through ornithine decarboxylase and arginine nur, India. The seeds were germinated in vitro after surface
decarboxylase respectively. Putrescine is used as a pre- sterilization with 0.2% mercuric chloride for 10 min fol-
cursor together with the aminopropyl moiety derived from lowed by immersion in a solution containing 1% sodium
S-adenosyl-L-methionine (SAM) after decarboxylation, to hypochlorite for 15 min followed by three rinses in sterile
synthesize spermidine and spermine (Evans and Malmberg distilled water. Seedlings with fully opened cotyledonary
1989). Apart from this, silver nitrate is known to regulate leaves were obtained after 3 months of culture. Cotyle-
morphogenesis through various means. It is known to be a donary leaf explants (20 mm2) were cut from the leaf blade
potent inhibitor of ethylene action (Beyer 1976) and known with a scalpel excluding the mid-vein and margins and
to increase polyamine pools. suspended in 50 mg l–1 cystine HCl solution to prevent
Coffee is an important plantation crop. Commercial browning. Two successive media were used for the
coffee production relies mainly on two species: Coffea induction of callus. The hormone regimes were selected on
arabica and C. canephora. Somatic embryogenesis is a the basis of previous work reported by Van Boxtel and
highly useful method for large-scale propagation of Coffea Berthouly (1996). The callus induction medium contained
sp. Somatic embryos and secondary somatic embryos are 2.26 lM 2,4-D, 2.45 lM IBA and 9.94 lM 2-iP or
widely considered to be of single cell origin, hence is 4.53 lM 2,4-D and 9.94 lM 2-iP. The callus multiplica-
advantageous for transformation studies. We have devel- tion medium contained 4.53 lM 2,4-D and 17.76 lM BA
oped embryogenesis and regeneration systems (Giridhar or 4.53 lM 2,4-D and 4.44 lM BA in a medium com-
et al. 2003, 2004a, 2004b, 2004c), which is highly useful in prising Murashige and Skoog medium (Murashige and
the clonal propagation of coffee. Coffee leaves and seeds Skoog 1962), B5 vitamins (Gamborg et al. 1968) supple-
contain purine alkaloids in high amounts. In vitro cultures mented with 40 mg l–1 cystine HCl. Somatic embryo
of Coffea sp. contain purine alkaloids such as caffeine, induction was obtained in a medium comprising 2.85 lM
theobromine and theophylline (Frischknecht and Baumann IAA and 1.11 lM BA. Embryo maturation medium com-
1985). Caffeine is the major alkaloid in coffee. Caffeine prised half strength MS medium, devoid of growth
biosynthesis comprises sequential methylations at N-7-, regulators. Mature torpedo shaped embryos were used for
N-3- and N-1- of the xanthine ring catalyzed by S-adeno- secondary embryogenesis. Medium used for secondary
syl-L-methionine (SAM) dependent methyltransferases embryogenesis comprised half strength MS salts, B5 vita-
(Koshiishi et al. 2001). mins, 2.85 lM IAA, 1.11 lM BA alone or along with
Briefly, the caffeine biosynthesis pathway in this either of 40 lM AgNO3, 50 mM putrescine and 1 mM
plant is xanthosine ? 7-methylxanthosine ? 7-methyl- DFMA + 1 mM DFMO. Control medium comprised 0.5
xanthine ? 3,7-dimethylxanthine (theobromine) ? 1,3,7- 2.85 IAA and 1.11 lM BA. Media pH was adjusted to 5.8
trimethylxanthine (Caffeine). All the methylation reactions and autoclaved at 121C, 1.2 kg/cm2 pressure for 15 min.
are catalyzed by N-methyl transferases, and S-adenosyl For studies on caffeine levels under polyamine treat-
methionine is the methyl donor for the methylation steps ments, callus cultures were used. Embryogenic callus lines
(Mazzafera et al. 1994). Recently we have isolated the were derived according to the procedure mentioned above.
promoter for N-methyl transferase associated with caffeine The callus was grown for 60 days in a medium comprising
biosynthesis (Satyanarayana et al. 2005) and this also may MS salts and vitamins, 4.53 lM 2,4-D, 17.76 lM BAP and
play a crucial role in regulating caffeine biosynthesis in a either of putrescine 50 mM, spermidine 50 mM, spermine
tissue specific and genotype specific manner. Although the 50 mM, DFMA 1 mM, DFMO 1 mM, DFMA + DFMO
metabolic steps are known in the biosynthetic pathway, the 1 mM each, AgNO3 50 lM, DFMA 1 mM + DFMO and
regulation of the caffeine biosynthesis in coffee tissues is 1 mM + AgNO3 50 lM.
not fully understood.
Polyamines, ethylene and caffeine biosynthetic path-
ways are S-adenosyl-L-methionine dependent. The Experimental conditions
objective of this work is to study the influence of poly-
amines, polyamine inhibitors and ethylene inhibitors on The leaf explants were cultured with their adaxial side in
somatic embryogenesis and caffeine accumulation using in contact with the medium. Five torpedo shaped somatic
vitro cultures of Coffea canephora. embryo explants in ten replicates were inoculated per

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Acta Physiol Plant (2008) 30:217–223 219

treatment for secondary embryogenesis response. Approx- Ambion, USA). All the plastic wares were treated with
imately, five 50 mg callus clumps each were inoculated in 0.1% DEPEC and the working area, electrophoresis tank
individual Petri plates for embryogenic response. The Petri and other required materials were treated with RNase Zap
plates were closed with parafilm. Cultures were incubated (Ambion, USA). The control and tissues subjected to
at 25 ± 2C in dark for 45 days for each treatment. polyamine and polyamine inhibitor treatment were har-
vested, frozen in liquid nitrogen and RNA was extracted
immediately. Quality and concentration of RNA were
Chemicals checked on denaturing agarose gel. All the RNA samples
were subjected to DNase (Ambion, USA) treatment to
Putrescine, spermine, spermidine and S-adenosyl-L-methi- avoid possible artifact amplifications from contaminant
onine (SAM) were obtained from sigma (USA). DFMO genomic DNA.
and DFMA were procured from Marrion Merrel Research N-methyltransferase gene specific primers were
foundation (Cincinatti, Ohio, USA). AgNO3 was obtained designed across the intron. First-strand cDNAs were syn-
from (Qualigens, India). These were added to the media thesized from 2 lg of total RNA in 20 ll final volume,
after filter sterilization using a 0.22 lM filter (Sortorius) to using MuLV reverse transcriptase (Ambion USA) and
obtain the desired concentration range. All the chemicals oligo-dT(18) primer (Sigma USA) following the manu-
were of analytical grade and the solvents of high perfor- facturer’s instructions. To quantify template quantities, the
mance liquid chromatography grade (Qualigens, India). RT-PCR reaction was stopped in the early exponential
phase (30th cycle) to maintain initial differences in target
transcript quantities. PCR was performed as explained
Extraction and estimation of endogenous polyamines above using the primer sequences NMTF-50 CGA GGA
GTC CAT GCA TTT TT 30 and NMTR-50 CCT CCT CAA
The extraction of endogenous polyamines was carried out CCA TGC ACT TT 30 . A volume of 10 ll from each PCR
by acid hydrolysis of perchloric acid. PAs were analyzed reaction was fractionated on a 1.5% (w/v) agarose gel in
by benzoylation and estimated using the method adopted Tris–acetate EDTA buffer and stained with 0.5% (w/v)
by Flores and Galston (1982). Averages of triplicates were ethedium bromide. The gels were photographed with a
expressed in lg polyamine per g FW of the tissue. Digital Imaging System (HeroLab, GMBH, Germany). The
intensity of the ethidium-bromide-stained DNA was ana-
lyzed using intensity histograms.
Estimation of caffeine

Caffeine extraction from the samples was carried out using Statistical analysis
80% ethyl alcohol. Estimation of caffeine was carried out
by high performance liquid chromatography on a Bond- A total of 10 explants were used for each treatment and the
apak C18 column (5 l · 250 m) with 50 mM sodium experiments were repeated thrice and the mean ± SE val-
acetate (pH 5.0), methanol and tetrahydrofuran in the ratio ues of the results are presented. Only optimal hormonal and
91:8:1. The parameters were controlled by a Shimadzu LC growth regulators concentration data were provided in the
10-AS liquid chromatograph equipped with a dual pump results.
and a UV spectrophotometric detector (Model SPD-10 A)
set at 270 nm. The recorder Shimadzu C-R7A chromatopac
was set at a chart speed of 2.5 cm/min. The injection Results
volume was 10 ll, and injected with a Rheodyne 7125
injector. Peak identification was achieved by comparing Induction of in vitro callus cell lines
with the retention time of standards (SIGMA, USA) and
confirmed by spiking the standards with individual sam- The seeds were germinated in 60 days of culture in a
ples. HPLC analysis was carried out for each treatment in germination medium. However, the first cotyledonary
triplicate. leaves opened only after 3 months. The germination per-
centage ranged from 40 to 50%. Callus initiation from leaf
explants started at the end of 1 month. Maximum response
Analysis of N-methyl transferase transcript levels for callus induction, i.e., 68% was obtained in a medium
comprising 2,4-D, IBA and 2 iP (Table 1). On subcultur-
The total RNA was extracted from an equal amount of ing, the callus in the same medium did not possess
coffee callus using a total RNA extraction kit (RNeasy kit, embryogenic potential (data not shown). This callus was

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220 Acta Physiol Plant (2008) 30:217–223

Table 1 Callus initiation in Coffea canephora after 45 days of embryos that matured and turned to torpedo stage were
culture cultured on half strength MS medium with B5 vitamins for
Media: half MS salts and B5 vitamins Percentage explants 45–60 days. These torpedo embryos were able to undergo
showing callus initiation repeated cycles of secondary embryogenesis when cultured
2,4 D lM IBA lM 2-iP lM
on the medium comprising 2.85 lM IAA and 1.11 lM
2.26 2.45 9.94 68 ± 2.6 BAP alone or with putrescine/AgNO3 (Fig. 3). Incorpora-
4.53 0 9.94 36 ± 2.2 tion of polyamines resulted in 58% explant response for
0 0 0 – embryogenesis when compared to control with 42%
response. These embryos were creamish green, bold and
showed vigorous development (Fig. 1c). Incorporation of
grown in a callus multiplication medium for 45 days. A silver nitrate resulted in 65% response for embryogenesis.
medium comprising 4.53 lM 2,4-D and 17.76 lM BA When polyamine biosynthetic pathway was inhibited by
resulted in rapid multiplication of the callus. Two kinds of DFMO and DFMA, 83% reduction in embryogenic
callus were obtained in the callus multiplication medium response was observed when compared to control (Fig. 3).
i.e., embryogenic callus (EC) and non-embryogenic callus Most of the explants turned brown and the embryos formed
(NEC). The non-embryogenic callus was black in color in this medium did not mature to give plantlets (Fig. 1d)
(Fig. 1a) and this cell line was unable to undergo
embryogenesis in subsequent experiments. Embryogenic
callus was creamish in color and friable (Fig. 1b). At the Influence of polyamines on caffeine content
end of 45 days of culture, the explants showed 52%
embryogenic callus and 28% non-embryogenic callus There was no significant difference in caffeine levels in
(Table 2). polyamine treated cultures. However, treatment with
DFMA and DFMO resulted in two-fold increase in the
caffeine levels (Fig. 4). Incorporation of silver nitrate in
Endogenous polyamine pool the culture medium decreased the caffeine to 0.48% DW
when compared to the control with 0.6% DW caffeine.
The total polyamine estimation data revealed difference in Incorporation of silver nitrate and polyamine synthesis
the endogenous spermine levels (Fig. 2). The embryogenic inhibitors resulted in 0.76% DW caffeine content (Fig. 4).
callus contained 11-fold more spermine and 3.3-fold higher
spermidine when compared to NEC. There was no signif-
icant difference in the putrescine levels in embryogenic and Analysis of N-methyl transferase transcript levels
non-embryogenic callus (Table 3)
There was no difference in the transcripts of N-methyl
transferase under polyamine or polyamine inhibitor treat-
Somatic embryogenesis and secondary embryogenesis ment as evident from RT-PCR analysis (Fig. 5). Therefore,
the increase in caffeine accumulation in callus cultured on
Somatic embryogenesis was obtained in a medium com- polyamine synthesis inhibitors may be due to more avail-
prising 2.85 lM IAA and 1.11 lM BAP. These globular ability of S-adenosyl-L-methionine for caffeine biosynthesis

Fig. 1 Response of Coffea canephora in vitro cultures for embryo- c Somatic embryogenesis under the influence of 40 lM putrescine.
genesis under the influence of putrescine and polyamine synthesis d Inhibition of embryogenic response under the influence of
inhibitors. a Non-embryogenic callus. b Embryogenic callus. polyamine biosynthetic pathway inhibitors DFMA and DFMO

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Acta Physiol Plant (2008) 30:217–223 221

Table 2 Multiplication of first generation callus in Coffea canephora after 45 days of culture
Media: half MS salts and B5 vitamins Percentage explants Percentage explants
producing embryogenic producing non-embryogenic
2,4 D lM BAP lM callus callus

4.53 17.76 52 28 ± 1.4

4.53 4.44 31 40 ± 2.8
0 0 – 15 ± 1.1

1400 80
Put Spd Spm

% Explant response for

70
1200
60

embryogenesis
1000 50
Micrograms / g FW

40
800 30
20
600
10
0
400
Control Putrescine AgNO3 40µM DFMA+DFMO
50mM 1mM each
200
Treatment

0
Non embryogenic Embryogenic Globular somatic Torpado somatic
Fig. 3 Influence of polyamine (putrescine), ethylene action inhibitor
callus callus embryo embryo (silver nitrate) and polyamine biosynthetic pathway inhibitors
(DFMA + DFMO) on somatic embryogenesis response in Coffea
Fig. 2 Endogenous polyamine levels in selected stages of somatic canephora
embryogenesis and callus lines of Coffea canephora
responses (reviewed by Bais and Ravishankar 2002).
and may not due to the upregulation of N-methyltransferase Polyamines are reported to promote shoot multiplication
genes involved in caffeine biosynthesis. (Chi et al. 1994; Bais et al. 2001) and in vitro flowering in
Cichorium intybus (Bais et al. 2000). In our studies,
exogenous administration of putrescine resulted in increase
Discussion in embryogenesis, suggesting the promotive role of poly-
amines in embryogenesis in Coffea canephora. In an earlier
The accumulation of spermine in embryogenesis may be study, Beatriz et al. (1994) studied the effect of exogenous
essential for the shift from callus to embryogenesis. A fine administration of polyamines in the concentration range of
balance of different polyamines may be required for 0, 50 and 100 lM. The results indicated that there is sig-
embryogenesis. This is evident from our experimental nificant difference in embryogenesis. However
results that treatment with polyamine biosynthesis inhibi- incorporation of 50 lM spermidine with high dose of
tors resulted in drastic reduction in embryogenesis putrescine resulted in the promotion of embryogenesis in
response. Similar kind of response was observed in dif- C. canephora genotype N-123 (Beatriz et al. 1994). In
ferent plant systems with regard to morphogenetic preliminary experiments we have found that low concen-
tration of polyamines (50–100 lM) do not elicit any
Table 3 Embryogenesis response of Coffea canephora callus cul-
morphogenetic response and this was supported by an
tured in the following medium in 45 days earlier work (Beatriz et al. 1994). We found that higher
concentrations of polyamines are required for embryo-
Media: half MS salts and B5 vitamins Percentage explant s
producing globular genesis in Coffea canephora, and the promotive effect of
IAA lM BAP lM embryos polyamines were confirmed by incorporation of polyamine
inhibitors in the culture medium.
2.85 1.11 85 ± 2.8
Polyamines are known to elicit morphogenetic respon-
0 8.88 10a ± 0.85
ses when incorporated in high concentration (Bais et al.
0 0 –
2001). Our observations indicate that incorporation of
No embryos were formed in black non-embryogenic callus 50 mM putrescine promotes embryogenesis and 1 mM
a
Embryogenic callus multiplication was observed each polyamine synthesis inhibitors significantly inhibits

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222 Acta Physiol Plant (2008) 30:217–223

1.4
Caffeine content (%DW)
Ethylene synthesis
1.2
Inhibits ethylene action
1
0.8 Caffeine synthesis S adenosyl-L-methionine Silver ions
0.6
0.4 Promote
0.2 Polyamine synthesis
SAM synthase
0
1 2 3 4 5 6 7 8 C
Treatments Embryogenesis
Organogenesis
Fig. 4 Influence of putrescine, silver nitrate and polyamine biosyn- In vitro multiple shoots
Methionine In vitro rooting,
thetic pathway inhibitors on caffeine content in Coffea canephora flowering
callus cultures. Bars: 1, Put 50 mM; 2, Spm 50 mM; 3, Spd 50 mM;
4, DFMA 1 mM; 5, DFMO 1 mM; 6, DFMA + DFMO 1 mM each;
Fig. 6 Interplay of polyamine and caffeine biosynthesis pathway and
7, AgNO3 50 lM; 8, DFMA 1 mM + DFMO 1 mM + AgNO3
its modulation by S-adenosyl methionine
50 lM

synthesis inhibitors. Miyazaki and Yang (1987) reported the

influence of putrescine and AgNO3 on the competitive uti-
lization of S-adenosyl-L-methionine. Utilization of SAM by
putrescine for its conversion to spermidine would possibly
result in a lower availability of SAM for ethylene biosyn-
thesis (Bais et al. 2001). Reports on somatic embryogenesis
in carrot (Roustan et al. 1990) indicate that the potent eth-
ylene action inhibitor AgNO3 helps in increasing ADC
activity, which in turn increases the levels of endogenous
Fig. 5 N-methyl transferase transcript levels by RT-PCR. Lanes: M polyamines in carrot embryogenic cultures. There is strong
100 bp marker, template from 1, Genomic DNA; 2, cDNA from
evidence that there is competition for SAM between the
control Coffea callus; 3, cDNA from Coffea callus cultured on 50 mM
putrescine; 4, cDNA from Coffea callus cultured on DFMA + DFMO biosynthetic pathways of polyamines and ethylene (Minocha
1 mM each, AgNO3 50 lM; 5, cDNA from Coffea callus cultured on et al. 1990). The formation of caffeine is closely associated
DFMA 1 mM + DFMO 1 mM + AgNO3 50 lM with SAM because the three methylation steps in the caffeine
embryogenesis response. These results are in agreement biosynthetic pathway use SAM as the methyl donor
with earlier reports (Feirer et al. 1984; Bais et al. 2001) (reviewed by Ashihara and Suzuki 2004). In our view, the
where polyamines play a crucial role in embryogenesis and increase in caffeine levels in embryos cultured on polyamine
plant morphogenesis. synthesis inhibitors may be due to more availability of S-
Silver ion is a potent inhibitor of ethylene action (Beyer adenosyl- L -methionine for caffeine biosynthesis. These
1976) and has been found to enhance plant regeneration in results also indicate that SAM may be playing a crucial role
different systems (Bais et al. 2000; Reddy et al. 2001; Hyde in regulating caffeine biosynthesis in coffee beans and down
and Phillips 1996). Since polyamines were reported to regulation of SAM may lead to low caffeine coffee plants.
promote embryogenesis (Feirer et al. 1984), the promotive The possible pathways are depicted in Fig. 6.
effect of ethylene inhibitors such as AgNO3 on regenera- In the investigation reported here, we were able to
tion was thought to be due to enhanced polyamines establish a correlation between endogenously and exoge-
synthesis. Pua et al. (1996) clearly described the synergistic nously administered polyamines with embryogenesis and
effect of AgNO3 and putrescine on shoot regeneration in caffeine biosynthesis.
Chinese radish. Ethylene is known to be a potent inhibitor
Acknowledgments This work was supported by a research grant
of embryogenesis and morphogenesis (Bais et al. 2001). from Department of Biotechnology, Government of India. VK is
Our earlier reports suggested that ethylene inhibitors thankful to CSIR for the award of research fellowship. The institu-
increase somatic embryogenesis and secondary embryo- tional support of Dr. V. Prakash, Director, CFTRI, is gratefully
acknowledged.
genesis in Coffea sp. (Giridhar et al. 2004b). Polyamines
also known to inhibit ethylene biosynthesis (Apelbaum
et al. 1981) and this may be one of the reasons for
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Acta Physiol Plant (2008) 30:217–223 223

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