THE ROLE OF OSMOTIC STRESS DURING in vitro

THE ROLE OF OSMOTIC STRESS DURING in vitro

ZESZYTY PROBLEMOWE POSTĘPÓW NAUK ROLNICZYCH 2008 z. 524: 221-230
THE ROLE OF OSMOTIC STRESS
DURING in vitro REGENERATION OF Triticum aestivum L.
AND Vicia faba ssp. minor 1*
1
1
2
Edyta Skrzypek , Magdalena Szechyńska-Hebda , GraŜyna Dąbrowska ,
2
Anna Goc
1
2
Institute of Plant Physiology Polish Academy of Sciences, Kraków
Department of Genetics, Nicolaus Copernicus University, Toruń
Introduction
Developmental processes during in vitro culture are influenced by many factors
such as the plant genotype and explant type, the composition of the culture medium
including the amino acids, carbohydrates and growth regulators, or water stress
treatment. Changes in some physico-chemical conditions are required to start
regeneration programs encoded in the genome. Carbon source in the regeneration
induction medium affected the efficiency of that process. In most cases, sucrose as a
major carbohydrate synthesized by plants or its constituent monosaccharides, glucose
and fructose, were used in culture media as the carbon source to support cell growth and
differentiation. However, several reports indicated that sucrose may not be the most
suitable carbohydrate for the induction of somatic embryogenesis and plant regeneration
in cell cultures [STRICKLAND et al. 1987; CHU et al. 1990; NAVARRO-ALVAREZ et al. 1994;
BLANC et al. 1999, 2002]. There are data confirming the important role of sugar content
and osmotic potential of medium for somatic embryo formation. A high osmotic potential of medium efficiently induces cucumber somatic embryogenesis [LOU, KAKO
1995]. Furthermore, it was suggested that osmotic stress induced by a high concentration
of sugars started somatic embryogenesis in carrot [KAMADA et al. 1989]. Osmotic
treatment with a high concentration of sucrose was found to enhance the single-cell
embryo formation in ginseng [CHOI, SOH 1997]. Some reports [JAIN et al. 1996, 1997;
IANTCHEVA et al. 2005; LI et al. 2005] showed that not only media with elevated
concentrations of carbohydrates, but also media containing additional carbohydrates,
such as mannitol, stimulate plant differentiation in cultured cells. Carbohydrates, as
regulators of osmotic potential of medium during in vitro cultures could modify the
regeneration process. SHEN et al. [1997] suggest that the exogenously applied mannitol
can additionally act as free radical-scavenger and reduce oxidative damage of cells
caused by hydroxyl radicals.
This work reports the effects of oxidative stress caused by osmotic potential of
medium on the in vitro culture regeneration. Changes in the activity of antioxidative
1
* The work was supported by the Ministry of Science and Higher Education, grant no 3P06A
00925.
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E. Skrzypek et al.
enzymes and endogenous level of hydrogen peroxide and plant regeneration frequency
in response to the osmotic stress created by higher concentrations of sucrose and
mannitol in shoot regeneration media were measured.
Material and methods
Experiments were carried out on callus of wheat (Triticum aestivum L.) cv.
Kamila and field bean (Vicia faba ssp. minor) cv. Nadwiślański Immature and mature
embryos of wheat were cultured on MS medium [MURASHIGE, SKOOG 1962] with 2
mg⋅dm-3 2,4-dichlorophenoxyacetic acid (2,4-D), 30 g⋅dm-3 sucrose, and 6 g⋅dm-3 agar
(pH 5.8), whereas field bean on MS medium containing 1 mg⋅dm-3 casein hydrolysate,
750 mg⋅dm-3 myo-inositol, 1 mg⋅dm-3 6-benzyl-aminopurine (BAP), 0.5 mg⋅dm-3
naphthalene acetic acid (NAA), 0.25 mg⋅dm-3 gibberellic acid (GA3), 30 g⋅dm-3 sucrose,
and 6 g⋅dm-3 agar (pH 5.8). Callus initiated from immature embryos, unlike that from
mature embryos, was capable of shoot regeneration.
Four-week-old callus was placed for three weeks on media which modify callus
differentiation: 3% sucrose (ok. -0.42 MPA), 6% sucrose (ok. -0.71 MPA) and 3%
sucrose together with 7% mannitol (ok. -1.33 MPA). Material for analyses was
collected 3 h and 1, 2, 4, 7, and 14 days after subculture. For the determination of
regeneration potential calluses were transferred onto MS media without growth
regulators.
The total H2O2 content was measured according to ISHIKAWA et al. [1993], after
callus homogenization with 5% trichloroacetic acid (TCA). For enzymes analysis
calluses were homogenized with 1% PVP400 and 0.05 mol⋅dm-3 phosphate buffer (pH
7.0) containing 0.1 mmol⋅dm-3 ethylenediaminetetraacetic acid (EDTA) at 4°C. Total
superoxide dismutase (SOD, EC 1.15.1.1.) activity was assayed according to the
modified method of MCCORD and FRIDOVICH [1969]. Catalase (CAT, EC 1.11.1.6) activity
was determined according to AEBI [1984] and total peroxidases (POD) to LÜCK [1962].
For SOD isoenzyme analysis, tissue samples were homogenized in 0.05 M phosphate
buffer (pH 6.8) containing 0.1 mmol⋅dm-3 EDTA, 0.1% N,N,N’,N’tetramethylenediamine (TEMED), 1 mmol⋅dm-3 dithiotreitol (DTT), and 0.1% ascorbic
acid. SOD isozymes were separated by native PAGE using 11.5% polyacrylamide gels
and 0.l mol⋅dm-3 Tris-glycine as an electrode buffer. Gels were stained by the method of
BEAUCHAMP and FRIDOVICH [1971]. Proteins were assayed by BRADFORD’S dye-binding
technique [1976] with bovine serum albumin as a protein standard.
Results and discussion
Little information is available regarding the effect of carbohydrates on in vitro
embryogenesis and plant regeneration. Carbohydrates, besides their role as a carbon
source, are known to function as osmotic regulators in culture media and this function
was found to be critical for regeneration. Several reports showed that osmotic stress
induced by carbohydrates, when used at elevated concentrations or by additional
carbohydrates, such as mannitol, stimulate plant differentiation in cultured cells [LOU,
KAKO 1995; JAIN et al. 1996; CHOI, SOH 1997; JAIN et al. 1997; BLANC et al. 1999, 2002; LI et al.
2005]. Mannitol acts mainly as osmotic regulators and, unlike sucrose, neither supports
in vitro callus growth nor is metabolized in higher plants.
In our experiment the regeneration of shoots was observed only in callus
THE ROLE OF OSMOTIC STRESS DURING in vitro ...
223
originated from immature embryos of both species. Regeneration efficiency depended
on osmotic potential of the medium. After tree weeks culture on medium with 3%
sucrose (control condition) there was no meristematic centers on the callus surface (Fig.
1), however further subculture on medium without growth regulators induced shoot
formation. High concentration of sucrose in medium (6%) decreased the callus growth
and blocked the regeneration even after passage on the regeneration medium. BLANC et
al. [2002] suggested the participation of carbohydrates in synthesis and accumulation of
starch reserves in callus cells, which were followed by the differentiation process:
elongation and vacuolation. These processes could result in blocking regeneration
potential in our culture, because the maintenance of small cell volumes is believed to be
one of the factors needed to enable the onset of regeneration and to represent a
dedifferentiated cell state with the potency to initiate a new developmental program
[PASTERNAK et al. 2002].
In contrast, the presence of mannitol in medium caused the formation of meristematic centers and shoots on wheat callus much earlier as compared to other
conditions. Shoots appeared on that medium one week before transfer onto regeneration
media (Fig. 1). Moreover, regeneration efficiency was greatly enhanced. Wheat callus
grown on medium with 7% mannitol regenerate approximately 50% more shoots as
comparing with callus grown on medium without mannitol. Similarly to our experiment
IANTCHEVA et al. [2005] reported that the osmotic pre-treatment had a primary effect of
shortening the regeneration phase, and promoting the embryogenic potential of tissues.
However, field bean callus did not regenerate any shoots during sucrose and
mannitol treatment. Shoots were observed 3 weeks later in regeneration conditions on
calluses after 3% sucrose and 3% sucrose with 7% mannitol treatments. Therefore it is
not clear how osmotic stress or reduction in cell water content influences the process of
plant regeneration. Our experiment suggested that osmotic stress did not act directly,
but could be a trigger of mechanisms leading to changes in developmental program.
WETHERELL [1984] suggested that the osmotic stress may disrupt the plasmodesmatal
connections between the pre-embryonic cells, thus making the cells physiologically
isolated and allowing a greater number of cells to differentiate.
Changes in physico-chemical conditions of in vitro cultures could induce
oxidative stress in cells. In our experiment, modification of medium osmotic potential
and sugar composition (6% sucrose and 3% sucrose together with 7% mannitol) caused
changes in endogenous H2O2 content and response of antioxidative enzymes system.
Regenerating callus of wheat showed a lower content of H2O2 in all conditions in
comparison to non-regenerating one and a higher activity of SOD, CAT and POD (Fig.
2).
Fig. 1.
Regeneration of wheat and field bean callus after three weeks on the medium with
3% sucrose (3% S), 6% sucrose (6% S) and 3% sucrose with 7% mannitol (3% S +
7% M)
Rys. 1.
Regeneracja kalusa pszenicy i bobiku po trzech tygodniach wzrostu na poŜywkach z
3% sacharozą (3% S), 6% sacharozą (6% S) oraz z 3% sacharozą i 7% mannitolem
(3% S + 7% M)
E. Skrzypek et al.
224
Field bean
Wheat
Pszenica
Bobik
4000
1000
H2O2 [ng g FW-1]
H2O2 [ng g ś.m.-1]
1d
800
3000
600
2000
400
1000
200
0
CAT [∆A µg protein-1 min.-1]
CAT [∆A µg białka-1 min.-1]
SOD [U µg protein-1]
SOD [U µg białka-1]
0,2
POD [∆A µg protein-1 min.-1]
POD [∆A µg białka-1 min.-1]
2d
R 3% S
R 6% S
R 3% S + 7% M
NR 3% S
NR 6% S
4 d 7 d 14 d
NR 3% S + 7% M
0
1/8 d
1d
2d
4d
7d
14 d
0,04
0,15
0,03
0,1
0,02
0,05
0,01
0
0
0,04
1/8 d
1d
2d
4d
7d
14 d
0,04
0,03
0,03
0,02
0,02
0,01
0,01
0
0
1,6
1/8 d
1d
2d
4d
7d
14 d
1,6
1,2
1,2
0,8
0,8
0,4
0,4
0
0
1/8
1
2
4
7
14
1/8 d
1d
2d
4d
7d
1/8 d
1d
2d
4d
7d
14 d
1/8 d
1d
2d
4d
7d
14 d
1/8
1
2
4
7
14
Days of culture after 3 weeks growth of callus on media with different osmotic potential
Dni prowadzenia kultury po 3 tygodniach wzrostu kalusa na poŜywkach o róŜnym potencjale
osmotycznym
Fig. 2.
Rys. 2.
Endogenous amount of H2O2 and activity of SOD, CAT and POD in regenerable
(R) and non-regenerable (NR) callus of wheat and field bean after 3 week growth
on the medium with 3% sucrose (3% S), 6% sucrose (6% S) and 3% sucrose with
7% mannitol (3% S + 7% M). Means of three replicates ± SE
Ilość endogennego H2O2 oraz aktywność SOD, CAT i POD w regenerującym (R)
i nieregenerującym (NR) kalusie pszenicy i bobiku po 3 tygodniach wzrostu na
poŜywkach z 3% sacharozą (3% S), 6% sacharozą (6% S) oraz z 3% sacharozą i
7% mannitolem (3% S + 7% M). Średnie z trzech powtórzeń ± SE
14 d
THE ROLE OF OSMOTIC STRESS DURING in vitro ...
225
It suggests that the antioxidative enzymes effectively scavenge reactive oxygen species
induced by high concentration of carbohydrates in medium. Endogenous H2O2 content
showed a decrease in the early stages of wheat callus regeneration and then increase
after 7th day. The maximum of SOD and CAT activities in wheat callus were observed
on 7th day, then on 14th day of culture the SOD activity decreased. We suggest that a
high production of endogenous H2O2 on 7th day, despite of high efficiency of enzymes,
might act as a signal for initiation of regeneration mechanisms, because from that time
the development of shoots was observed. POD activity was almost at the same level
during whole wheat culture, both regenerating and non-regenerating, however higher
POD activity was detected in regenerating callus in 14 days after treatment. Similarly to
KRSNIK-RASOL [1991] report indicating peroxidase as a developmental marker in plant
tissue we could suggest the role of POD in regeneration processes.
Wheat
Pszenica
Mn
Cu/Zn 1
Cu/Zn 2
Field bean
Bobik
Mn
Cu/Zn 1
Fig. 3.
2 of wheat and field bean after 3 week growth on
Isoforms of SOD in regenerating (R) and non-regeneratingCu/Zn
(NR) callus
Rys. 3.
Izoformy SOD na Ŝelu akrylamidowym w regenerującym (R) i nieregenerującym (NR) kalusie pszenicy i bobiku po 3 t
Field bean callus, potentially able to regeneration, showed a higher concentration
of endogenous H2O2 in comparison with non-regenerating one and with wheat callus.
Endogenous H2O2 content greatly increased from 7th day of culture. Enzymes activities
in field bean were different from activities in wheat callus, too. The activity of SOD in
field bean was almost ten times lower than in wheat. The highest values were detected
on 14th day of culture. The fluctuations of CAT activity were observed during 14 days
of culture. POD activity in field bean was higher than in wheat and decreased gradually
during the subsequent days of culture. These data suggest that in field bean cells the
mechanisms involved in antioxidative defense against free radicals generated during
osmotic stress were different. Field bean calluses easily turned brown during culture as
a result of polyphenol oxidation caused by peroxidases. Carbohydrates additionally
might modulate polyphenol accumulation in cell culture [BLANC et al. 2002]. These processes generate H2O2 and free radicals. Probably too high level of reactive oxygen
species could not be efficiently reduced by enzymes and results in tissue darkening and
inhibition of field bean regeneration.
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E. Skrzypek et al.
Gel electrophoresis showed one Mn and two Cu/Zn isoforms of SOD in wheat
and field bean callus (Fig. 3). Activities of SOD increased together with the increase of
osmotic potential of medium, however the most sensitive in wheat callus was the
cytosolic isoform Cu/ZnSOD 1 (bound with lower mobility), whereas in field bean
mitochondrial isoform MnSOD. These data revealed different mechanism and acting
place of antioxidative enzymes.
Conclusions
The results of the experiment indicated that the oxidative stress and the osmotic
potential play an important role in the regulation of regeneration processes. The
mechanism involved in antioxidative defense during in vitro culture and the place of
free radicals scavenging could determine the regeneration potential of cells. We suggest
the function of SOD, CAT and POD as an indicator of tissue potential for regeneration.
Activities of these enzymes and level of H2O2 can also be used in the prognosis of the
ability of tissues to differentiate and regenerate.
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Key words:
osmotic potential, oxidative stress, regeneration, Triticum aestivum L.,
Vicia faba L. minor
Summary
Developmental processes during in vitro culture are influenced by many
environmental factors, which are required to start regeneration programs encoded in the
genome. Isolation of explants and their in vitro culture might cause oxidative imbalance
in cells. The aim of the experiment was to determine the effects of osmotic potential of
medium on the in vitro regeneration focusing on the oxidative stress. Regenerating
callus of Triticum aestivum and Vicia faba ssp. minor were obtained from immature
embryos and non-regenerating from mature embryos. Callus after four week induction
was placed for three weeks on the media with 3% sucrose (ok. -0.42 MPA), 6% sucrose
(ok. -0.71 MPA), and 3% sucrose together with 7% mannitol (ok. -1.33 MPA). After
that, the amount of endogenous H2O2 and activity of SOD, CAT and POD were
measured during the subsequent days of callus culture. 6% sucrose inhibited
regeneration of wheat callus, and addition of 7% mannitol stimulated shoot regeneration
E. Skrzypek et al.
228
in comparison with 3% sucrose, whereas did not change regeneration potential of field
bean callus. Regenerating callus of wheat accumulated less endogenous H2O2 than nonregenerating one and showed a higher activity of SOD, CAT and POD. Regenerating
callus of field bean showed a higher amount of endogenous H2O2 and antioxidative
enzymes. The results of the experiments indicated that the oxidative stress caused by
changes of osmotic potential of medium and type of carbohydrates play an important
role in regulation of regeneration processes. The activities of antioxidative enzymes and
endogenous level of H2O2 can be used in prognosis for the ability of tissues to
differentiate and regenerate.
ROLA STRESU OSMOTYCZNEGO PODCZAS
REGENERACJI in vitro Triticum aestivum L. I Vicia faba ssp. minor 2*
Edyta Skrzypek 1, Magdalena Szechyńska-Hebda 1,
GraŜyna Dąbrowska 2, Anna Goc 2
1
Instytut Fizjologii Roślin im. F. Górskiego Polskiej Akademii Nauk w Krakowie
2
Zakład Genetyki, Uniwersytet Mikołaja Kopernika w Toruniu
Słowa kluczowe:
potencjał osmotyczny, regeneracja, stres oksydacyjny, Triticum
aestivum L., Vicia faba L. ssp. minor
Streszczenie
Na procesy rozwojowe w kulturach in vitro wpływa wiele czynników środowiskowych, które mogą indukować proces regeneracji zakodowanego w genomie.
Izolacja eksplantatów i indukcja kalusa w warunkach in vitro moŜe powodować zmiany
potencjału oksydo-redukcyjnego komórki. Celem eksperymentu było określenie
wpływu potencjału osmotycznego poŜywki na regenerację w kulturach in vitro z
uwzględnieniem stresu oksydacyjnego. Regenerujący kalus Triticum aestivum i Vicia
faba ssp. minor uzyskano z niedojrzałych zarodków, a nieregenerujący z dojrzałych
zarodków. Kalus po 4-tygodniowej indukcji rósł przez trzy tygodnie na poŜywkach z
3% sacharozą (ok. -0,42 MPA), 6% sacharozą (ok. -0,71 MPA) i 3% sacharozą z
dodatkiem 7% mannitolu (ok. -1,33 MPA). Po tym czasie, ilość endogennego H2O2 i
aktywność SOD, CAT i POD była mierzona w kolejnych dniach regeneracji kalusa. 6%
sacharoza hamowała regenerację kalusa pszenicy, a 7% mannitolu stymulował
regenerację pędów w porównaniu z 3% sacharozą, ale nie zmieniał zdolności
regeneracyjnych kalusa bobiku. Regenerujący kalus pszenicy akumulował mniej
endogennego H2O2 niŜ nieregenerujący i wykazywał większą aktywność SOD, CAT i
POD. Regenerujący kalus bobiku akumulował większą ilość H2O2 i wykazywał wyŜszą
aktywność enzymów antyoksydacyjnych. Wyniki eksperymentu wykazują, Ŝe stres
oksydacyjny indukowany przez zmiany potencjału osmotycznego poŜywki oraz rodzaj
zastosowanych węglowodanów odgrywają rolę w regulacji procesu regeneracji. Aktywności enzymów antyoksydacyjnych i endogenny poziom H2O2 mogą być stosowane do
prognozowania zdolności regeneracyjnych tkanek.
2
* Badania finansowane przez Ministerstwo Nauki i Szkolnictwa WyŜszego w ramach grantu nr 3
PO6A 009 25.
THE ROLE OF OSMOTIC STRESS DURING in vitro ...
Dr Edyta Skrzypek
Instytut Fizjologii Roślin im. F. Górskiego
Polska Akademia Nauk
ul. Niezapominajek 21
30-239 KRAKÓW
e-mail: [email protected]
229