Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts
The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in licorice G. glabra L. suspension protoplasts by electroporation after introduction of the chimeric plasmid pDNt23-CaMV35S-nos-cat (pDNt23-root-specific and CaMV 35S promoters, nopaline synthase nos-terminator, and selectable...
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| Date: | 2004 |
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Інститут молекулярної біології і генетики НАН України
2004
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| Cite this: | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts / P.G. Kovalenko // Біополімери і клітина. — 2004. — Т. 20, № 5. — С. 421-428. — Бібліогр.: 28 назв. — англ. |
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| author | Kovalenko, P.G. |
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| citation_txt | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts / P.G. Kovalenko // Біополімери і клітина. — 2004. — Т. 20, № 5. — С. 421-428. — Бібліогр.: 28 назв. — англ. |
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| description | The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in licorice G. glabra L. suspension protoplasts by electroporation after introduction of the chimeric plasmid pDNt23-CaMV35S-nos-cat (pDNt23-root-specific and CaMV 35S promoters, nopaline synthase nos-terminator, and selectable NPT-II gene). Maximum of CAT activity in the licorice protoplasts was observed in 50 h after electroporation. Together with recent advances in the cell isolation and electroporation procedures, this system allows to study expression of the root-specific promoter introduced into the licorice cells. The level of total flavonoids production in 4 weeks culture was tested in the electroporated cell lines. Additionally, these electroporated cells were treated by elicitors (yeast extract and fungal bioextract). The total flavonoids production increased by 2–5 times in comparison with the non-electroporated licorice cells.
Суспензійні протопласти солодцю Glycyrrhiza glabra L. трансформували химерною плазмідою pDNt23-CaMV35S-nos методом електропорації, для чого було підібрано оптимальні параметри цього процесу. Експресію підтверджували за допомогою репортерного гена хлорамфеніколацетилтрансферази (CAT). Максимальна його активність у трансформованих клітинах солодки спостерігалася через 50 год. Отримані трансформовані клітинні лінії продовжували культивувати протягом місяця для визначення сумарної фракції флавоноїдів. Трансформовані клітинні агрегати солодцю додатково обробляли біотичними еліситорами. Вміст флавоноїдів в оброблених клітинних агрегатах у 2–5 разів перевищував контроль
Суспензионные протопласты солодки G. glabra L. трансформировали методом электропорации химерной плазмидой pDNt23-CaMV35S-nos, для чего были подобраны оптимальные параметры этого процесса. Экспрессию подтверждали с помощью репортерного гена хлорамфениколацетилтрансферазы (CAT). Максимальная его активность в трансформированных клетках солодки наблюдалась спустя 50 ч. Полученные трансфорированные клеточные линии продолжали культивировать в течение месяца для определения суммарной фракции флавоноидов. Трансформированные клеточные агрегаты солодки до полнительно обрабатывали биотическими элиситорами. Содержание флавоноидов в обработанных агрегатах в 2–5 раз превышало контроль.
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ISSN 0233-7657. Біополімери і клітина. 2004. Т. 20. № 5
МОЛЕКУЛЯРНА І КЛІТИННА БІОТЕХНОЛОГІЇ
Transient gene expression and total flavonoids
production in the electroporated licorice
Glycyrrhiza glabra L. suspension protoplasts
P. G. Kovalenko
Institute of Molecular Biology and Genetics National Academy of Sciences of Ukraine
Zabolotnoho str. 150, Kyiv, 03143 , Ukraine
E-mail:omg@imbg.org.ua
The bacterial chloramphenicol acety(transferase (CAT) gene was expressed in licorice G. glabra L.
suspension protoplasts by electroporation after introduction of the chimeric plasmid pDNt23-CaMV35S-
nos-cat (pDNt23-root-specific and CaMV 35S promoters, nopaline synthase nos-terminator, and selectable
NPT-II gene). Maximum of CAT activity in the licorice protoplasts was observed in SO h after
electroporation Together with recent advances in the cell isolation and electroporation procedures, this
system allows to study expression of the root-specific promoter introduced into the licorice cells. The level
of total flavonoids production in 4 weeks culture was tested in the electroporated cell lines. Additionally,
these electroporated cells were treated by elicitors (yeast extract and fungal bioextract). The total flavonoids
production increased by 2—5 times in comparison with the non-electroporated licorice cells.
Introduction. At present, in biotechnology great atten
tion is paid to plant tissue cultures, because the plant
cells are able to synthesize specific compounds, espe
cially various important secondary metabolites which
are useful as medicines and food additives. Although
lots of attempts have been made on plant cell suspen
sion culture as a source of the important metabolites.
Dedifferentiated plant cells are heterogeneous in the
genetic composition and the contents of economically
important metabolites are often lower than in donor
plants.
The suspension cells can grow more rapidly than
original field plants, which makes it possible to
increase the expectable productivity of in vitro culture.
The productivity can be also enhanced by optimiza
tion of cultivation conditions, cell selection and gene
tic transformation. Recent advances in recombinant
DNA technology allow to generate new transgenic
plant species that is of high importance for the
pharmaceutical industry and new drug design. It is
necessary to have a system of genetic transformation
that is simple and effective.
Electroporation is widely used to produce stable
© P G KOVALENKO, 2 0 0 4
genetic transformation in both prokaryotic and euka-
ryotic organisms. It is used extensively as an efficient
method for studing the transient gene expression.
Suspension protoplasts electroporation allows to
introduce foreign DNA into a great variety of the
plant species. To establish a genetic transformation
system via electroporation, it is necessary to have an
cell competent for the electroporation process, and an
in vitro culture system, which permits a high fre
quency of regeneration. In plants, the major limitation
of recovering stable transformants by protoplasts
electroporation is the requirement for an efficient
protoplast-to-plant regeneration scheme. The effects
of an electric field on cell walls are still unknown
more fully. Biological membranes are composed of
phospholipids, amphipatic molecules having a hydro-
philic head group attached to a hydrophobic tail.
They can be polarized in electric fields. Electric
pulses raise the transmembrane potential, promoting
transient pore formation due to the increased dipole
moment of the hydrophilic lipid heads, allowing
charged macromolecules to migrate through the pores
and eventually reach the nucleus where they can
promote genetic transformation.
The roots of European licorice (G. glabra L.,
421
mailto:omg@imbg.org.ua
KOVALENKO P. G.
Leguminosae, tribe Astragalaceae) and other members
of this pharmacologically important genus contain
many flavonoids. Flavonoids represent a large group
of phenolic secondary metabolites widespread in
plants and involved in different plant functions. Some
flavonoids like liquiritin, isoliquiritin, liquiritigen, lu-
curaside and others are known to possess anti
allergic, anti-inflammatory, anti-viral and anti-carci
nogenic activities [1—4] . However, very often the
biosynthetic potential of the callus mass and sus
pension culture can be decreased in comparison with
the original plant tissue [5] .
As reported in our previous papers [9, 15] , the
plant DNA fragments with transcription promoted
activity were selected from the pool of random tobacco
nuclear DNA fragments, and one of them (pDNt23)
was shown to have the root-specific expression in
transgenic plants. The chimeric plasmid pDNt23-
35SCaMV-CAT-nos-3 (pDNt23-root specific promo
ter, nos-nopaline synthase terminator and had the
selectable gene as neomycin phosphotransferase II-
NPT-II) was used in our experiments with the
electroporation of the licorice suspension protoplasts.
In this report we optimized electroporation efficiency
to study the activity of the reporter chloramphenicol
acetyltransferase (CAT) gene in electroporated lico
rice cells and the total flavonoids production from
these electroporated cells. The transient expression of
the reporter CAT gene under the control of cauli
flower mosaic virus promoter has been demonstrated
to be a powerful tool to study the introduction of
exogenous DNA into plant protoplasts [6] . The
testing of marker genes, usually under the 35S
promoter control, is also of great interest for the
licorice biotechnology. In this case the CAT assay was
used as a sensitive method to detect the functional
activity of the root-specific (pDNt23) promoter se
quences in electroporated licorice cells. Second, the
successful genetic manipulation with licorice suspen
sion cells depends significantly from the physiological
conditions of the freshly isolated protoplasts.
However, up to now there is limited information
on the relationship between electroporation conditions
and the CAT expression in the licorice protoplasts
cells. The electroporation method used by us, inclu
ded the rectangular (RC) pulse generating system
and the capacitor discharge to deliver pulses of
exponentially decaying voltage.
The general aim of this work was to characterize
the factors affecting the CAT expression in the
electroporated licorice cells, while using the RC pulse
generating system. First, we have optimized elec
troporation efficiency to study the activity of the
reporter CAT gene in electroporated licorice cells.
The CAT expression is shown to be influenced by
interactions between electrical parameters of the sys
tem and physiological state of the isolated cells.
Second, these electroporated licorice cells were tested
for the total flavonoids production. Third, electro
porated cell lines have been treated by elicitors and
which were found to increase the total flavonoids level
(2—5 fold) in comparison with the control (i. e.
non-electroporated and non-elicitated) licorice cells.
Materials and Methods. Plant material and cell
culture. The callus culture obtained from seeds, stolon
and root of licorice, G. glabra L., was used for
subsequent initiation of a cell suspension culture. The
callus was cultured in the modified MS (Murashige-
Skoog) [7] medium with addition of 0.2—1.0 mg/1
benzyladenin (BA), 0.5—5.0 mg/1 naphthalen acetic
acid (NAA), 0.9 % agar. Approximately 67 % of the
explants, used in this work, initiated callus growth
during 3 weeks. Growth of callus was slightly better
at pH 5.7—6.0. The well-growing callus cell lines
were used for further experiments in the modified
liquid MS medium with addition of 0.2—2.0 mg/1 BA,
0.2—2.0 mg/1 NAA. In this medium, cell suspensions,
consisted of clumps of 10—30 mostly spherical cells,
were produced. The cultures were agitated in a
reciprocal shaker (110 agitation/min) in the dark at
25 °С, and subcultured at 24 days intervals. The 3
months old actively growing suspension cells, were
sieved using stainless steel mesh (500—1000 ,wm
opening) to exclude large cell clusters and washed.
Cells were then treated for 9 h in incubation buffer
containing 0.5 % cellulase R-10 «Onozuka» (Yakult
Honsha Co, Japan), 0.5 % macerozyme R-10 («Sigma
Chemical* Co, USA), and 0.3 % dricelase («Sigma
Chemical*) dissolved in 0.6 M mannitol, with MS
salts. During the enzymes treatment, cells were
incubated in the dark at 25 °С. Following enzymes
treatment, protoplasts were washed once with 10 ml
of sucrose-mannitol gradient and followed by three
washes with electroporation buffer. The protoplasts
density was determined by counting on a haemocyto-
meter and their viability was measured by the exc
lusion of phenosafranine («Aldrich», USA) according
to Widholm [8] . Typical yield was (0.7—1.0) • 10 7 of
protoplasts per 1 g with viability of 70—80 %.
Electroporation, The electroporator Invitrogen-4
was used for electroporation of the licorice suspension
protoplasts. This electroporator has the capacitor
discharge system (capacitance from 50 to 200 fiF).
The protoplasts of density from 4-Ю 5 to 10" cells
per 1 ml were electroporated in buffer: 20 mM KC1,
6 mM MgCl 2 ) 0.6 M sorbitol, pH 6.0, and then mixed
with plasmid DNA and carrier DNA. 340 /лі of
protoplasts suspension in a cylindrical chamber (tem-
422
TRANSIENT G E N E E X P R E S S I O N A N D T O T A L FLAVONOID P R O D U C T I O N
perature 4 °С, resistance 1.7 kQ) were electroporated
using stainless steel electrodes, placed 1 cm apart.
For the optimization of the electroporation pro
cess, the protoplasts were subjected to three electrical
pulses (RC pulse duration 80 ms at 0—350 V/cm,
with 30 s intervals between the pulses). After the
electroporation, the protoplasts samples were kept on
ice for 8 min to extend the open-pore status [14] .
Then the protoplasts were diluted with an equal
volume of the modified MS medium and cultured at
25 °С in darkness.
Plasmid construction. The pDNt-35SCaMV-
CAT-nos-3'-NPT-II gene construction was obtained
from the Department of Bioengineering, Institute of
Bioorganical and Oil Chemistry NAS of Ukraine
(Kyiv) and used for the direct gene transfer into the
licorice protoplasts by electroporation [9] .
Both linearized and supercoiled forms of the
plasmid were used in the experiments.
The plasmids were propagated in Escherichia coli
and purified according to the standard protocol. The
plasmid DNA was linearized by means of digestion
with appropriate restriction enzymes. After phenol-
chloroform extraction and ethanol precipitation DNA
was resuspended in water under sterile conditions.
Non-linearized plasmid DNA was treated in the same
way as linearized DNA except for the digestion step.
DNA concentration was measured by spectropho
tometry at 260 nm. The carrier DNA (calf thymus
DNA, «Sigma Chemical*) in concentrations of 0, 20,
50 or 150 ,Mg/ml was added. The plasmid was
sterilized by ethanol precipitation and dissolved in the
electroporation buffer.
Protoplasts viability and CAT assay. After 48—
52 h of cultivation the number of survived protoplasts
was counted in ten random fields using the light field
microscopy («LOMO», Russia). Dead protoplasts were
clearly distinguishable and appeared as condensed
and twisted structures. The protoplasts viability was
also confirmed by staining with fluorescein diacetate
[8 ]. The study on CAT activity was performed in
general as described by Gorman et al. [10] .
Briefly, protoplasts (3 -Ю 5 cells per 1 ml) were
collected by centrifugation and the pellets were resus
pended in 500 pi of buffer (0.225 M Tricine, pH 7.8,
5 mM phenylmethyl sulphonyl fluoride and 5 mM
EDTA). The extracts were clarified by centrifugation
for 10 min, and the supernatant was transferred to a
fresh tube and heated for 10 min at 65 °С. Acetyl-
CoA (final concentration of 1 mM) and '^-chloram
phenicol (1 /uCi, 60 juCi-mmof') were added and the
reaction was allowed to proceed for 1 h at room
temperature being terminated by the addition of 10
volumes of ethyl acetate. 0.5 unit of commercial CAT
(«Sigma Chemical*) was used as a control. After
clarification by centrifugation the pellet was dried,
redissolved in ЗО ц\ of ethyl acetate and submitted to
ascending chromatography in chloroform/methanol
(95:5 v/v) on silicagel plates. Separated spots of
'^-chloramphenicol and its acetylated forms were
identified by the autoradiography percent conversion
on TASMA (Ru) film after 7 days of exposure. The
results are expressed as percent conversion of chlo
ramphenicol to the acetylated forms. The calibration
experiments with commercial CAT showed that there
was a linear relationship between the enzyme amount
(up to 1 unit) and the percent conversion obtained
under the conditions used (1 unit gave 93 % con
version) .
The CAT activity in «negative controls* was
determined either after incubation of the protoplasts
with the plasmid and carrier DNA without electro
poration, or after electroporation in the presence of
carrier, but not the plasmid DNA, as described by
Jones et al. [11 ].
Selection of transformants. After electroporation,
cells were diluted with the fresh modified liquid MS
medium with 0.2—0.5 mg/1 BA, 0.5—1.0 mg/1 NAA,
0.05 mg/1 2.4D (dichlorophenoxyacetic acid), and
stored for 2 days at 22 °С in the dark. After this time,
the electroporated protoplasts were transferred to a
fresh modified MS medium supplemented with
100 mg/1 kanamycin sulfate and cultivation at 25 °С
in the dark at 7-days. The fresh medium with the half
reduce concentration of kanamycin sulphate were
used on the every new passage. Well-growing suspen
sion protoplasts were selected to produce lines of
kanamycin-resistant cell aggregates and which were
used as a source for the total flavonoids production.
Non-electroporated (control) licorice protoplasts were
cultured on the modified MS medium without kana
mycin sulphate.
Production of flavonoids by electroporated licorice
cells. Concentration of the accumulated flavonoids in
the electroporated licorice suspension cells was ana
lyzed after 4 weeks in culture using spectropho-
tometric analysis («Specord UV», Germany) at
295 nm.
The flavonoids were extracted with ten volumes
of hot water (90 °С) over the harvested cells for 3 h.
The calibration curve was constructed for liquiri-
tigenin as described in [12] and modified in [13] . All
the experiments were triplicated and average values
were analysed.
Results and Discussion. Electroporation of the
licorice suspension protoplasts G. glabra with the
pDNt-35SCaMV-CAT-nos-3'-NPT-II plasmid resul
ted in transient expression of the CAT reporter gene
423
KOVALENKO P. G.
in the electroporated cells as an indicator of successful
plasmid delivery. In control, the licorice protoplasts
were electroporated in the absence of plasmid DNA.
The reporter CAT gene expression in these cells was
never observed.
For the obtain the optimal level by the CAT
assay in the electroporated licorice cells, the electrical
parameters were optimized. It has been shown earlier
that the electroporation can be successfully used for
the variety of protoplasts transformation. Two electro
poration systems are commonly in use: based either
on low-voltage/long pulses or on high-voltage/short
pulses [14] . We used the square-wave pulse generator
with capacitor discharge system, which delivers expo
nentially decaying voltage pulses. The typical values
for the low-voltage/long pulse method are 200—450
V/cm and 10—90 ms (exponential decay) [15] . This
method results in obtaining stably transformed cell
lines and high cell viability after electroporation [15,
16]. The optimal voltage and time constant depend on
the protoplast diameter, the plasmid concentration
and form, cell density and physiological properties of
the isolated protoplasts.
Electroporation, therefore, requires a balance
between conditions, which either lead to the increase
in membrane permeability or result in membrane
breakdown and loss of protoplast viability. The me
chanisms of the electrically induced permeability were
based on specific changes in membranes. However,
the processes of lipid bilayer configuration and the
penetration of cell surface adsorbed plasmid DNA
into the cell interior are not well known yet. Transient
membrane pores are caused by an increase in the
dipole moment of hydrophilic phospholipid heads,
which move in the same direction as the applied
electric field and provoke highly localized dielectric
breakages in membrane structure [17] .
Our initial experiments were focused on the
establishing of licorice cell lines (suspension proto
plasts) and determination of effective voltage for the
transient CAT gene expression. The results obtained
correspond to those described by other authors [11]
where transient CAT activity was shown to depend on
the balance between conditions favoring membrane
permeabilization and those leading to irreversible
membrane breakdown.
Using the capacitor discharge system, Fromm et
al. have found that the CAT activity in the carrot
protoplasts and their survival after electroporation
may be influenced by the salt composition in the
electroporation buffer [18] . The intensity of electrical
pulse (voltage, pulse length) and pulse number re
quired for the transformation of protoplasts were
inversely proportional to the protoplast diameter [22 ].
Transient CAT activity in G. glabra suspension protoplasts elec
troporated with plasmid pDNt23-3SS-nos in linear form. Lane / and
6 — controls: CAT assay with commercial enzyme; voltage not
applied and viability was 6 4 ± 1 . 2 %; 2 — 250 V/cm and 38 .5±1 .3
% viability; 3 — 400 V/cm and viability 9 . 4 ± 1 . 5 %; 4 — 350 V/cm
and viability were 2 7 . 2 ± 3 . 3 %
The average cell size of the licorice suspension
protoplasts used in our experiments was 29—36 //M,
the protoplasts being isolated from exponentially
dividing suspension cells (3—4 days culture). This
population of suspension cells obviously contains the
largest proportion of mitotic cells, and freshly isolated
protoplasts appeared to be the most suitable for the
electroporation. The older (5—7 days) licorice sus
pension cells do not divide synchronously, therefore
their use for electroporation is insufficient. It has
been shown previously that the suspension culture
protoplasts can be permeabilised at a range of voltage,
presumably because of the cell size heterogeneity in
the population. The voltage required for membrane
breakdown depends on the cell size [11] , the larger
protoplasts being permeabilized at lower field
strengths, while the smaller cells at higher field
strengths.
Viability of the electroporated and non-electropo-
rated licorice suspension protoplasts was 38.5±1.3 %
at 250 V/cm; 27 .2±3.3 % at 350 V/cm, 64±1.2 %
in control (non-electroporated cells) (Figure).
To achieve better viability of electroporated lico
rice protoplasts and to increase the flavonoid produc
tion by electroporated cells, we have transferred the
electroporated cells to the fresh modified MS medium
with addition of phytohormones and bioregulators.
Maximum effect of the field strength on reporter CAT
gene expression of pDNt-CaMV35S-nos-NPT-II plas
mid was observed within 50 h after electroporation.
The optimal field strength was: 300 V/cm with
30 s intervals between 3 pulses (RC pulse duration =
80 ms), and with the constant capacitance 95,«F. This
424
*pDNt35S-nos-NPT-II; " c o u n t of cpm-chloramphenicol (CM). The
field strength was constant as 350 V/cm.
is comparable to the field strength used in other plant
cell transformation systems [6, 11, 15] . The plasmid
DNA concentration of 100 ^/g/rnl was sufficient for
routine measurement of the CAT activity. The effect
of the field strength on the CAT expression in the
licorice protoplasts with linearized and supercoiled
DNA forms is shown in Table 1.
The concentration of the pDNt-CaMV35Scat-nos-
NPT-II plasmid used during electroporation was an
important factor influencing the transient CAT ac
tivity in the licorice suspension protoplasts. The
addition of 0, 20, 50, or 150 ^g /ml of the carrier
DNA to the licorice protoplasts (in the presence of 0,
50, or 100 uglml of the plasmid DNA) did not cause
any increase in the CAT expression (Table 2) .
From the results presented in this paper, it is
apparent that the transient CAT expression in the
licorice suspension protoplasts depends on two cate
gories of parameters: 1) the use of a rectangular pulse
generating system and electroporation optimized pro
tocol have given as a simple and effective approach for
the CAT reporter gene activity in electroporated
protoplasts, and 2) the maximal transient CAT exp
ression depended on the physiological characteristics
(after enzymes treatment) of the freshly isolated
licorice protoplasts.
Thus, the licorice suspension protoplasts con
tained many large starch granules and these sus
pension cells were highly cytoplasmic. The successful
of the plasmid penetration in the electroporated
suspension protoplasts were depended from the elec
troporation conditions, and when relative optimum of
low field strengths and long pulse duration was
applied. It is possible that such combination of
electrical parameters induces more pores in the plas
ma membrane, allowing more plasmid DNA to be
taken into the nucleus; or perhaps a longer pulse
duration facilitates the electrophoretic movement of
the plasmid DNA molecules to the nucleus.
The level of transient CAT expression (percent
conversion of chloramphenicol to its acetylated pro
ducts) were very similar to the results obtained for
the suspension of sorghum, wheat, bean, sugar beet
and conifer plant species [19—23] .
The pulse generator has delivered RC pulses, the
duration of which was under precise control.
These results demonstrated that the highest CAT
activity per 10 4 surviving suspension protoplasts was
obtained at 350 V/cm, when the linearized plasmid of
lOO^g/l concentration was used (a mean of 72 CM).
Similarly, using the linearized plasmid DNA
resulted in higher levels of the transient expression as
compared to the supercoiled DNA. Higher trans
formation efficiency with the linear plasmid DNA has
been reported for bacteria, yeasts, filamentous fungi,
protoplasts and intact tissue [6, 8, 14] . Due to the
inner twist of the molecule and the absence of the
double helix distortions, the linear plasmid DNA is
highly mobile in the electric field. Macromolecular
movement across pores is facilitated when the mole
cules are linear because the absence of tertiary and
quaternary structures reduces their volume and allows
425
KOVALENKO P. G.
Table 3
Flavonoids production in electroporated licorice suspension cells
(4 weeks of culture)
Plasmid DNA i/ig/m\)
pDNt35S- t ios -NPTII-CAT
Field strength, V / c m
Total flavonoids content,
% (calculated on the
dry weight)
a more uniform superficial polarization to be induced
by the electric field [17, 2 2 ] .
The transient nature of both membrane pores
and structural alterations in the cell wall, induced by
the electric field, imposes conformational restrictions
on the type of macromolecules which can be in
troduced to the cell.
The linear DNA has been found to be better than
the circular one for the plasmid intake and integra
tion, leading to the increased gene expression; and
these results were shown with various important plant
species [6, 19, 21, 2 2 ] . We have also found that, in
order to obtain high expression level, the licorice
protoplasts must be prepared from rapidly growing
suspension culture cells.
The increase in the cell wall permeability to DNA
depended on the treatment of the licorice suspension
culture cells with the pectolytic enzymes at the
concentration of 0.5 % (w/v) .
Furthermore, the CAT expression can be enhan
ced by these pectolytic enzymes treatment. Our data
indicate that this effect, at least in part, may be due
to the breakdown of cell clumps into smaller aggre
gates, in which a larger proportion of cells may be
permeabilized, and presumably to the removal of
pectin from the wall itself. The cold pretreatment of
the suspension protoplasts and cell lines is known to
be important for the achievement of the highest
degree of electroporation and the protoplast viability
after electroporation.
The electroporated cell lines were transferred
into the modified MS medium with the addition of
phytohormones, such as BA — 0.5 mg/1 and NAA —
0.2 mg/1. The level of flavonoids production was
studied in the cells electroporated at various electric
field strengths. The amount of flavonoids in the
electroporated cell lines (Table 3) increased by 1.2 to
2.0 times, as compared to the control (non-electro-
porated cells) and depended on the field strenght and
from the root-specific promoter (pDNt23) and which
regulate the process in secondary metabolites pro
duction pathways. The highest level of flavonoids
production (14.8 %) was found in the licorice cells
electroporated at the field strength of 350 V/cm.
Our results have shown that the optimized pro
tocol for protoplasts isolation and the optimal electro
poration conditions are important for the trans
formation rates of the licorice species. The movement
of plasmid penetration into a freshly isolated proto
plasts by electroporation occurs by electrophoresis
through pores formed in the membrane by the
electrical breakdown of the cells membrane. With a
highly viable suspension protoplasts, the membrane
breakdown is reversible. A good indicator to assess
the suitability of a particular licorice protoplasts
preparation for electroporation is yield. Only iso
lations suspension protoplasts yield exceeded 1-Ю 7
per gram fresh weight of suspension-cultured cells
were used.
In our experiments, we have used plasmid with
utilized the double 35S-pDNt (root-specific) pro
moters. The root-specific promoter is suitable for the
increase the total flavonoids production in electro
porated licorice cell lines.
Further investigation is needed to determine the
factors which cause an increase in intracellular^
stored secondary metabolites production, especially in
the cell lines culture.
Besides, the optimal mode of electroporation of
the licorice protoplasts plays important role too.
After three weeks of incubation the protoplasts
were transferred to the fresh modified MS medium.
Subsequently this material was treated with fungal
extract (FB) obtained from Acremonium sp., endo-
phyte mycorrhizal fungus, and diluted before the use
to 10~5 per 1 1 of medium. This homogenate of an
Acremonium sp., fungal strain has been shown to be
an effective elicitor for the total flavonoids production
in the suspension culture of G. glabra. To achieve the
maximum stimulation of flavonoids synthesis, we have
used combination with the other elicitor as a yeast
extract (YE) added to the nutrient medium at the
concentration of 500 mg/1. The electroporated sus
pension licorice cells were additionally treated with
these both biotic elicitors on the time prolong period
as 1 week. Than, these treated cell lines were
transferred on the fresh medium. Such elicitated
procedure could stimulate obtaining maximal level (as
426
TRANSIENT G E N E E X P R E S S I O N A N D T O T A L FLAVONOID P R O D U C T I O N
Table 4
Flavonoids production in electroporated (350 V/cm) and elicited
licorice suspension cells (4 weeks of culture)
*Control — non-electroporated cells.
high as 19.1 % of dry weight) of the total flavonoids
production on the 28th day of culturing. Both the
elicitors doses and the age of electroporated proto
plasts to be elicited needed to be optimized. The
optimal time for elicitor addition may be specific to
the elicitor and the culture system, in general it was
in the mid- to late exponential-growth phase.
The existence of an optimal dose may suggest
that, at elicitor doses smaller than the optimum, the
elicitor-binding sites in the electroporated cells were
still not fully utilized for activating the flavonoids
synthesis, while excessive doses caused a deleterious
effect on the suspension cells biosynthetic activity.
However, it is still not well understood how these
elicitors mediated the pathway of the flavonoids
biosynthesis in electroporated plant cells. These res
ponses may include the synthesis and incorporation of
hydroxyproline-rich glycoproteins, cellulose and poly
mers, the production of phytoalexins, and the enhan
ced expression of gene encoding enzymes such as
phenylalanine ammonium-lyase and which is a gene
ral in flavonoids biosynthesis pathway. To further
improve the effect of the fungal elicitor on flavonoids
production, we may need to use more purified of the
fungal homogenate. Furthermore, the dosage, timing
and frequency of elicitation and the medium renewal
strategy should be optimized systematically.
It has been shown in several reports [24—28] ,
that the use of yeast and fungi as elicitors provokes
accumulation of phytoalexins. In our system with the
licorice electroporated cells the biggest effect on the
flavonoids production was obtained in the modified
MS medium with both elicitors; the percent of viable
cells was 78.3±3.3 % as compared to the control cell
lines (the same medium, but without elicitors) was
67 .3±4 .1%.
Many previous studies have shown positive effects
the some elicitors on a variety the important se
condary metabolites as well too [25, 26, 28 ].
When elicitors, bioextract from fungus Acremo
nium sp. and YE, were used, total flavonoids content
increased 2—5 times (Table 4).
Experiments are now aimed at the obtaining of
stable transformation and subsequent cell prolife
ration of the licorice protoplasts and at the selection
of the transformed over-productive cell strains in the
medium with these elicitors.
In summary, the results presented in this paper
demonstrate the first example of direct gene transfer
in suspension protoplasts of the licorice cells as well
as describe electroporation conditions for successful
transient gene expression and flavonoids production
from electroporated suspension cells.
П. Г. Коваленко
Транзієнтна генетична експресія в електропорованих
суспензійних протопластах солодцю Glycyrrhiza glabra L.
і їхній вплив на вихід флавоноїдів
Резюме
Суспензійні протопласти солодцю Glycyrrhiza glabra L. транс
формували химерною плазмідою pDNt23-CaMV35S-nos мето
дом електропорації, для чого було підібрано оптимальні пара
метри цього процесу. Експресію підтверджували за допомогою
репортерного гена хлорамфеніколацетилтрансферази (CAT).
Максимальна його активність у трансформованих клітинах
солодки спостерігалася через 50 год. Отримані трансформо
вані клітинні лінії продовжували культивувати протягом
місяця для визначення сумарної фракції флавоноїдів. Транс
формовані клітинні агрегати солодцю додатково обробляли
біотичними еліситорами. Вміст флавоноїдів в оброблених
клітинних агрегатах у 2—5 разів перевищував контроль.
П. Г. Коваленко
Транзиентная генетическая экспрессия и накопление
флавоноидов в электропорированных суспензионных
протопластах солодки Glycyrrhiza glabra L.
Резюме
Суспензионные протопласты солодки G. glabra L. трансформи
ровали методом электропорации химерной плазмидой pDNt23-
CaMV35S-nos, для чего были подобраны оптимальные пара
метры этого процесса. Экспрессию подтверждали с помощью
репортерного гена хлорамфениколацетилтрансферазы (CAT).
Максимальная его активность в трансформированных клет
ках солодки наблюдалась спустя 50 ч. Полученные трансфор
мированные клеточные линии продолжали культивировать в
течение месяца для определения суммарной фракции флавоно
идов. Трансформированные клеточные агрегаты солодки до
полнительно обрабатывали биотическими элиситорами. Со
держание флавоноидов в обработанных агрегатах в 2—5 раз
превышало контроль.
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УДК 575.22:581.143.6
Надійшла до редакції 20.02.03
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| id | nasplib_isofts_kiev_ua-123456789-157979 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 0233-7657 |
| language | English |
| last_indexed | 2025-12-07T18:16:30Z |
| publishDate | 2004 |
| publisher | Інститут молекулярної біології і генетики НАН України |
| record_format | dspace |
| spelling | Kovalenko, P.G. 2019-06-22T05:33:04Z 2019-06-22T05:33:04Z 2004 Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts / P.G. Kovalenko // Біополімери і клітина. — 2004. — Т. 20, № 5. — С. 421-428. — Бібліогр.: 28 назв. — англ. 0233-7657 DOI:http://dx.doi.org/10.7124/bc.0006C5 https://nasplib.isofts.kiev.ua/handle/123456789/157979 575.22:581.143.6 The bacterial chloramphenicol acetyltransferase (CAT) gene was expressed in licorice G. glabra L. suspension protoplasts by electroporation after introduction of the chimeric plasmid pDNt23-CaMV35S-nos-cat (pDNt23-root-specific and CaMV 35S promoters, nopaline synthase nos-terminator, and selectable NPT-II gene). Maximum of CAT activity in the licorice protoplasts was observed in 50 h after electroporation. Together with recent advances in the cell isolation and electroporation procedures, this system allows to study expression of the root-specific promoter introduced into the licorice cells. The level of total flavonoids production in 4 weeks culture was tested in the electroporated cell lines. Additionally, these electroporated cells were treated by elicitors (yeast extract and fungal bioextract). The total flavonoids production increased by 2–5 times in comparison with the non-electroporated licorice cells. Суспензійні протопласти солодцю Glycyrrhiza glabra L. трансформували химерною плазмідою pDNt23-CaMV35S-nos методом електропорації, для чого було підібрано оптимальні параметри цього процесу. Експресію підтверджували за допомогою репортерного гена хлорамфеніколацетилтрансферази (CAT). Максимальна його активність у трансформованих клітинах солодки спостерігалася через 50 год. Отримані трансформовані клітинні лінії продовжували культивувати протягом місяця для визначення сумарної фракції флавоноїдів. Трансформовані клітинні агрегати солодцю додатково обробляли біотичними еліситорами. Вміст флавоноїдів в оброблених клітинних агрегатах у 2–5 разів перевищував контроль Суспензионные протопласты солодки G. glabra L. трансформировали методом электропорации химерной плазмидой pDNt23-CaMV35S-nos, для чего были подобраны оптимальные параметры этого процесса. Экспрессию подтверждали с помощью репортерного гена хлорамфениколацетилтрансферазы (CAT). Максимальная его активность в трансформированных клетках солодки наблюдалась спустя 50 ч. Полученные трансфорированные клеточные линии продолжали культивировать в течение месяца для определения суммарной фракции флавоноидов. Трансформированные клеточные агрегаты солодки до полнительно обрабатывали биотическими элиситорами. Содержание флавоноидов в обработанных агрегатах в 2–5 раз превышало контроль. en Інститут молекулярної біології і генетики НАН України Біополімери і клітина Молекулярна та клітинна біотехнології Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts Транзієнтна генетична експресія в електропорованих суспензійних протопластах солодцю Glycyrrhiza glabra L. і їхній вплив на вихід флавоноїдів Транзиентная генетическая экспрессия и накопление флавоноидов в электропорированных суспензионных протопластах солодки Glycyrrhiza glabra L. Article published earlier |
| spellingShingle | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts Kovalenko, P.G. Молекулярна та клітинна біотехнології |
| title | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts |
| title_alt | Транзієнтна генетична експресія в електропорованих суспензійних протопластах солодцю Glycyrrhiza glabra L. і їхній вплив на вихід флавоноїдів Транзиентная генетическая экспрессия и накопление флавоноидов в электропорированных суспензионных протопластах солодки Glycyrrhiza glabra L. |
| title_full | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts |
| title_fullStr | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts |
| title_full_unstemmed | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts |
| title_short | Transient gene expression and total flavonoids production in the electroporated licorice Glycyrrhiza glabra L. suspension protoplasts |
| title_sort | transient gene expression and total flavonoids production in the electroporated licorice glycyrrhiza glabra l. suspension protoplasts |
| topic | Молекулярна та клітинна біотехнології |
| topic_facet | Молекулярна та клітинна біотехнології |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/157979 |
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