ORIGINAL PAPER

The effect of low temperature and GA3 treatments on dormancybreaking and activity of antioxidant enzymes in Fritillariameleagris bulblets cultured in vitro

Marija Petric • Sladana Jevremovic •

Milana Trifunovic • Vojin Tadic • Snezana Milosevic •

Milan Dragicevic • Angelina Subotic

Received: 16 April 2013 / Revised: 18 June 2013 / Accepted: 14 August 2013 / Published online: 29 August 2013

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2013

Abstract We investigated the effect of low temperature

and gibberellic acid (GA3) treatment on dormancy in

Fritillaria meleagris L. bulbs. Also, we studied the effect

of dormancy breaking on the antioxidant enzymes activity.

To overcome dormancy, bulbs require a period

(4–8 weeks) of exposure to low temperature. Bulbs

regenerated in vitro were grown in the dark on medium

without growth regulators at the standard (24 �C) or at low

temperatures (4 and 15 �C) for 4, 6, 8 and 10 weeks. Bulbs

were collected after 3, 4 and 5 weeks of cooling at 4 �C. To

investigate the influence of GA3 on dormancy, bulbs were

treated for 24 h with GA3 solutions with 1, 2 and 3 mg l-1

concentrations. During the period of growth of bulbs at

4 �C, regeneration of bulbs was very weak, while at 15 �C

the number of regenerated bulbs increased significantly.

Improved bulb sprouting was achieved by a short treatment

with gibberellin. Low temperature also represents a kind of

oxidative stress for the plant. The activity of superoxide

dismutase, catalase (CAT) and peroxidase (POX) in bulbs

of F. meleagris L. grown in vitro and ex vitro increased

with decreasing temperature in contrast to glutathione

reductase. POX showed generally lower activity than CAT

which indicates that major role in the breaking dormancy

and preparing bulbs for sprouting have catalases.

Keywords Snake’s had fritillary � Cold treatment �Gibberellin � Superoxide dismutase � Catalase �Peroxidase � Glutathione reductase

Abbreviations

GA3 Gibberellic acid

SOD Superoxide dismutase

CAT Catalase

POX Peroxidase

GR Glutathione reductase

TDZ Thidiazuron

MS Murashige and Skoog medium (1962)

PVP Polyvinylpyrrolidon

DTT Dithiothreitol

PMSF Phenyl methyl sulfonyl fluoride

BSA Bovine serum albumin

GSSH Glutathione disulfide

EDTA Ethylenediaminetetraacetic acid

NBT Tetrazolium chloride

NADPH Dinucleotide phosphate

Introduction

Fritillaria meleagris L. (Liliaceae), snake’s had fritillary,

is a very valuable, bulbous plant used in horticultural

purposes. Genus Fritillaria is mainly distributed through-

out temperate climates of the Northern Hemisphere and

spends a particular period of the year in the form of dor-

mant bulbs under the ground. Production of fritillaries by

conventional methods is very slow and it takes several

years to produce a whole plant (Paek and Murthy 2002). In

cases when conventional methods are ineffective, in vitro

plant propagation of F. meleagris L. can lead to effective

Communicated by A. Krolicka.

M. Petric (&) � S. Jevremovic � M. Trifunovic � V. Tadic �S. Milosevic � M. Dragicevic � A. Subotic

Department for Plant Physiology, Institute for Biological

Research ‘‘Sinisa Stankovic’’, University of Belgrade,

Bulevar Despota Stefana 142, 11000 Belgrade, Serbia

e-mail: marija.nikolic@ibiss.bg.ac.rs

123

Acta Physiol Plant (2013) 35:3223–3236

DOI 10.1007/s11738-013-1357-z

and rapid multiplication of this species through various

morphogenetic pathways (Kukulczanka et al. 1989; Sub-

otic et al. 2010).

Bulbous species develop dormancy to survive unfa-

vorable environmental conditions. These species respond

to different environmental factors which determine when

to enter or exit dormancy. These factors include temper-

ature, photoperiod and drought (Vegis 1964). Dormancy

occurs during the life cycle of geophytes and is required

for their normal development (Kamenetsky et al. 2003).

High temperatures cause dormancy, while low tempera-

tures break it. If there is no cold period for extended time

during the year, plant growth is very slow, and the flowers

do not develop or are deformed (De Hertogh and Le Nard

1993). In dormant seeds and organs, the germination and

growth are stopped in a way that has not yet been clarified

(Bewley 1997). External conditions are responsible for

breaking dormancy at some point. In dormant seeds and

bulbs no external morphological changes can be detected,

while in the bulbs various physiological and morpholog-

ical changes are happening, such as the differentiation of

floral buds or roots (Le Nard 1983). Many species of the

genus Fritillaria begin dormancy in early summer and

during the winter months, and low temperature breaks

dormancy and leads to sprouting in the spring of next year

(Zhu et al. 1980; Sun and Wang 1991). Several factors

define the duration and termination of dormancy in bulbs:

the presence of specific proteins (Higuchi and Sisa 1967),

changes in the level of gibberellins and abscisic acid

(Rakhimbaev et al. 1978; Aung and De Hertogh 1979;

Gorin and Heidema 1985; Rebers et al. 1995), amylase-

dependent degradation of starch (Nowak et al. 1974;

Hobson and Davies 1977; Banasik et al. 1980) and

binding water with large molecules and its release from

hydrated molecules (Yamazaki et al. 1995; Okubo et al.

1997; Zemah et al. 1999). In many cases, breaking dor-

mancy leads to increased cell division (Okagami 2003;

Rohde and Bhallerao 2007).

The main factors that influence the growth and ger-

mination of newly formed bulbs in vitro are dormancy,

bulb size and maturity of bulbs (Langens-Gerrits et al.

2003b). Bulbs regenerated in vitro often become dormant

and stop growing and forming leaves (Li and Qin 1987).

Low-temperature treatment can have a positive impact on

the percentage of regenerating new bulbs (Paek 1996).

Improved sprouting of bulbs can be achieved by a short

treatment with gibberellins that can be used with or

without exposing the plants to low temperatures (Niimi

et al. 1988). Langens-Gerrits et al. (1997) proved the

stimulating role of gibberellins in the process of lily bulb

germination.

Factors that have negative effect on the growth and

development of plants reducing their productivity level,

often cause the release of large amounts of reactive oxygen

species (ROS; Bowler et al. 1994). ROS can react with

DNA, proteins and lipids which results in cell damage.

Plants have a very efficient enzymatic antioxidant system

that catalyzes the removal of ROS (Inze and Montagu

1995). Enzymes such as catalase (CAT), peroxidase

(POX), superoxide dismutase (SOD) and glutathione

reductase (GR) are part of plant enzymatic antioxidative

defense system (Apel and Hirt 2004). Low temperature,

which is necessary to overcome dormancy, also represents

a kind of oxidative stress for plants. The amount of stress,

to which the plant is exposed, can be measured indirectly

by measuring the activity of antioxidant enzymes that are

part of plant antioxidant defense system. Oxidative stress

has been studied in many plants, but the mechanisms of its

action are still not completely understood (Ziv 1991).

The aim of the study was to determine the effect of

temperature and gibberellic acid on formation of adventi-

tious bulbs and to overcome their dormancy stage. Also,

the relationship between the activity of antioxidant

enzymes and plant dormancy was analyzed.

Materials and methods

Plant material

Bulb cultures of F. meleagris L. were established accord-

ing to previously published procedures (Petric et al. 2011).

Cultures were maintained on Murashige and Skoog (MS)

medium (1962) supplemented with 3 % sucrose, 0.7 %

agar, 250 mg l-1 casein hydrolysate, 250 mg l-1L-proline

and 1.0 mg l-1 thidiazuron for shoot multiplication. Stock

cultures were maintained at temperature of 24 ± 2 �C and

16 h light/8 h dark photoperiod with irradiance of

40 lmol m-2 s-1. After a month, regenerated bulbs, of

approximately 100 mg, were used for further experiments.

Bulbs regenerated in vitro were further grown in the

dark on medium without growth regulators at the standard

(24 �C) or at low temperatures (4 and 15 �C) for 4, 6, 8 and

10 weeks. Bulbs were stored at 4 �C in the cold room,

while the bulbs at 15 ± 1 �C were kept in growth chamber.

To investigate the influence of gibberellic acid (GA3) on

the process of dormancy, bulbs were treated for 24 h with

GA3 solutions with 1, 2 and 3 mg l-1 concentrations.

Bulbs were collected after 3, 4 and 5 weeks of cooling at

4 �C to study the influence of cold treatment on dormancy

breaking. The number of bulbs, as well as weight increase

and percentage of sprouting were noted after the end of

treatment.

As a starting material for analyzing the activities of

antioxidant enzymes, bulbs grown on MS medium without

growth regulators were used. Antioxidant enzymes were

3224 Acta Physiol Plant (2013) 35:3223–3236

123

studied from bulbs regenerated in vitro and grown at three

different temperatures (24, 15 and 4 �C) for 8 weeks, at the

beginning of exposure to low temperature (4 �C), 3 and

7 days after the end of exposure to this temperature.

During the winter period (November–May), bulbs were

grown in natural conditions (ex vitro) to investigate the

activities of antioxidant enzymes. Analysis of enzyme

activities was performed on bulbs grown 1, 5 and 7 months

in natural conditions or at the end of November, March and

April.

Acclimatization of plants

Regenerated plants were planted in a mixture of humus and

sand (3:1) and grown under greenhouse conditions.

Enzyme extraction

As starting material for antioxidant enzymes activity

analyses, bulbs grown on MS medium without growth

regulators were used.

To determine activity of antioxidant enzymes, whole

bulbs grown in different treatments described above were

used.

Frozen (-70 �C) bulbs (500 mg) were homogenized in

1 ml of 0.1 M potassium phosphate (K–P) extraction buffer

(pH 7, containing 1.5 % insoluble polyvinylpyrrolidon,

10 mM dithiothreitol (DTT) and 1 mM phenyl methyl

sulfonyl fluoride. The homogenate was centrifuged for

5 min at 12,000g at 4 �C. Protein content of supernatants

was determined according to Bradford (1976). Based on

the standard curve, made using bovine serum albumin

solution, total protein concentration was calculated.

Quantification of SOD (EC 1.15.1.1.), CAT (EC 1.11.1.6.),

GR (EC 1.8.1.7.) and POX (EC 1.11.1.x) was performed

spectrophotometrically (Agilent 8453, Life Science, USA).

Quantification of SOD activity

SOD activity was determined spectrophotometrically by a

modified method of Beyer and Fridowich (1987). The

reaction mixture (1 ml) contained 100 mM K–P buffer (pH

7.8), 2 mM ethylenediaminetetraacetic acid, 260 mM

methionine, 1.5 mM nitroblue tetrazolium chloride (NBT)

and 0.04 mM riboflavin. For each sample, six dilutions

were prepared (sample volume: 0, 5, 10, 15, 20 and 25 ll;

K–P buffer volume: 800, 795, 790, 785, 780 and 775 ll)

and placed in a microtiter plate. The reaction mixture was

then illuminated for 30 min at 25 �C. The measurement

was done at 540 nm. One unit of SOD activity is the

amount of sample required for 50 % inhibition of NBT

photoreduction and is presented as specific activity (U/mg).

All measurements were repeated three times.

Quantification of CAT activity

Catalase activity was determined spectrophotometrically

by monitoring the kinetics of disappearance of hydrogen

peroxide by the method of Aebi (1984) which can be

detected by measuring the decrease in absorbance at

240 nm of reaction mixture consisting of 50 mM K–Na-P

buffer (pH 7), 20 mM hydrogen peroxide and enzyme

extract. Catalase activity was measured at a temperature of

20 �C, every 20 s for 3 min. Unit of catalase activity is

defined as the amount of enzyme that degrades 1 lmol of

hydrogen peroxide in 1 min and is indicated as lmol

min-1 mg-1 (U/mg).

Quantification of GR activity

Spectrometric analysis of GR activity was performed using

modified method of Carlberg and Mannervik (1985) by

monitoring the decrease of absorbance at 340 nm due to

oxidation nicotinamide adenine dinucleotide phosphate

(NADPH). The reaction mixture contained 1.5 ml

0.1 M K–P buffer, 150 ll 20 mM glutathione disulfide,

1 ml of water and 150 ll of 2 mM NADPH (diluted in

Tris–HCl buffer, pH 7). Enzyme activity is defined as the

amount of enzyme that oxidizes 1 lmol NADPH per

minute at 25 �C and is indicated as lmol min-1

mg-1 (U/mg).

Quantification of POX activity

Activity of POX was determined spectrophotometrically by

measuring the change in absorbance at 430 nm (Kukavica

and Veljovic-Jovanovic 2004). The reaction mixture con-

tained 2.9 ml of 0.05 M K–P buffer (pH 6.5), 60 ll 1 M

pirogallol (Sigma) as enzyme substrate. The reaction was

started by addition of 30 ll of 30 % hydrogen peroxide

after the first 20 s. The POX catalyzed oxidation of pyro-

gallol with hydrogen peroxide to purpurogallin which was

monitored at 430 nm. Enzyme activity is indicated as

lmol min-1 mg-1 (U/mg).

Statistical analysis of data

The results of all experiments are presented as mean

values ± standard errors. Statistical analyses were per-

formed using StatGrafics software version 4.2. Data were

subjected to analysis of variance and comparisons

between the mean values of treatments were made by the

least significant difference test calculated at the confi-

dence level of P B 0.05. Population which was used in

all treatments was 30 bulbs. All measurements were

repeated three times.

Acta Physiol Plant (2013) 35:3223–3236 3225

123

Results

The effects of low temperature (4 and 15 �C)

and duration of bulbs exposure to this temperature

on multiplication and growth of bulbs

Control bulbs of F. meleagris L. were grown on standard

temperature (24 �C) in culture. Number and weight

increase of newly formed bulbs are shown in Fig. 1a, b.

Bulbs grown at 4 �C did not multiply, while temperatures

between 15 and 24 �C led to an increase in the number of

newly formed bulbs (Fig. 1a). The highest average number

of bulbs (4.8) was obtained for bulbs grown during 8 and

10 weeks at 15 and 24 �C. During the same period,

regeneration of bulbs, grown in culture at 4 �C was sig-

nificantly lower.

The temperature and the duration of exposure resulted in

a weight increase of newly formed bulbs in culture

(Fig. 1b). When bulbs were grown at the lower (4 and

15 �C) temperatures for a period of 6 weeks, an increase in

weight was observed compared to control cultures grown at

24 �C. The largest increase in bulb weight (104.6 %) was

observed in cultures that were grown for 6 weeks at 4 �C.

A trend in weight increase was noted when bulbs were

continually grown at 15 �C.

The effect of pretreatments with low temperature

(4 and 15 �C) on bulbs further grown at standard

temperature

All bulbs were further grown in vitro for another 4 weeks

at standard temperature (24 �C). From the results shown in

Fig. 1c, it can be seen that the previous growth at lower

temperatures (4 and 15 �C) led to multiplication of bulbs.

The largest number of bulbs (8.7) for bulbs previously

grown at 4 �C was achieved after 8 weeks and this is

almost eight times more than the number of bulbs regen-

erated during cold period. Cold period of 10 weeks at 4 �C,

had a negative effect on the average number of bulbs.

Bulbs grown for 10 weeks at 15 �C showed the highest

morphogenetic potential and the number of newly formed

bulbs was 10.3.

The great weight increase was noted after 4, 6 and

8 weeks of growing bulbs at 4 �C. Growing bulbs for

10 weeks at 4 �C had a negative effect on weight increase

(Fig. 1d). Growing bulbs at lower temperatures (4 and

15 �C) had a positive impact on their sprouting compared

to those grown continuously at standard temperature

(Fig. 2). Pretreatment of bulbs at 4 �C led to a decrease in

the percentage of sprouting bulbs. So, growing bulbs at

4 �C during 10 weeks leads to a decrease in sprouting

percentage by 20 %, compared to those grown at the same

temperature for 4 weeks.

Effect of GA3 on the growth and development of bulbs

grown at 4 �C

Pretreatment with different concentration of GA3 led to

regeneration of a lower number of bulbs than in the control

bulbs (Fig. 3a).

Besides the effect on multiplication of bulbs, GA3

negatively affected the weight increase (Fig. 3b).

Effect of GA3 on the growth and development of bulbs

grown further at 24 �C

All bulbs grown at 4 �C and treated with different con-

centrations of GA3 were further grown at standard tem-

perature for 4 weeks. Pretreatment with GA3 positively

affected the number of new bulbs in comparison to the

control bulbs that were not treated with that growth regu-

lator (Fig. 3c). Growing bulbs at 4 �C at all concentrations

of GA3 had a positive effect on their multiplication. The

highest number of regenerated bulbs (*7) was formed

after 5 weeks growing at 4 �C (Fig. 3c).

Increasing concentrations of GA3 during the pretreat-

ment led to weight increase of bulbs grown at 4 �C

(Fig. 3d). Effect of GA3 on weight increase depended on

the duration of growth at 4 �C.

Sprouting of bulbs

GA3 had a positive effect on the sprouting of bulbs that

were not previously grown at 4 �C (Fig. 4). Bulbs that have

sprouted in the standard temperature did not continue with

further growth as compared to those grown at 4 �C.

Acclimation of plants

Completely formed plants F. meleagris L. (Fig. 5a) were

grown under greenhouse conditions to acclimatize to ex

vitro conditions. A total of 64 plants were planted, of which

30 successfully acclimated so that the total percentage of

acclimatization was 46.8 % (Fig. 5b, c).

Antioxidant enzyme activities in bulbs grown in vitro

When the bulbs were grown at 15 �C, the lowest SOD

activity was observed and it was 15.70 ± 0.14 lmol

min-1 mg-1 (Fig. 6a). On bulbs grown at 4 �C during the

same time period, we observed an increased activity of

SOD which was seven times higher than in bulbs grown at

15 �C. The increase in SOD activity was observed already

as soon as 3 days after cultivation at 4 �C and it was

50.03 ± 0.01 lmol min-1 mg-1.

Catalase activity decreased with lowering the tempera-

ture at which the bulbs were grown (Fig. 6b). Bulbs grown

3226 Acta Physiol Plant (2013) 35:3223–3236

123

continuously at 24 �C had a relatively high activity of

catalase, which was 208.97 ± 44.69 lmol min-1 mg-1,

while the bulbs grown at 15 �C showed the lowest catalase

activity (3.52 ± 0.88 lmol min-1). The highest activity of

catalase was observed on bulbs a week after the termina-

tion of growth at 4 �C and it was 412.45 ± 26.75

lmol min-1 mg-1.

Glutathione reductase activity was reduced when the

bulbs were grown at lower temperatures and this reduction

in activity was proportional to the temperature decrease.

The highest activity of glutathione reductase was observed

in bulbs grown at 24 �C (263.69 ± 21.20 lmol

min-1 mg-1), while the lowest activity of glutathione

reductase was observed 7 days after exposure to a

Fig. 1 The effect of

temperature (4 and 15 �C) and

duration of growing

(4–10 weeks) on the

regeneration of bulbs (a) and

their weight increase (b) during

low temperature treatment and

after 4 weeks (?4) growing at

standard temperature (c, d)

Acta Physiol Plant (2013) 35:3223–3236 3227

123

Fig. 2 The effect of

temperature (4 and 15 �C) and

duration of growing

(4–10 weeks) on sprouting of

F. meleagris L. bulbs after

4 weeks (?4) growing at

standard temperature (24 �C)

in vitro

Fig. 3 The effect of gibberellic

acid pretreatment on

regeneration of bulbs (a) and

their weight increase (b) during

cold treatment (3–5 weeks) and

after 4 weeks of growing at

standard temperature (c, d)

3228 Acta Physiol Plant (2013) 35:3223–3236

123

temperature of 4 �C and it was 146.82 ± 32.42 lmol

min-1 mg-1 (Fig. 6c).

Bulbs of F. meleagris L. grown in vitro at standard

temperature (24 �C) had the lowest peroxidase activity

(2.12 ± 0.69 lmol min-1) (Fig. 6d). Much higher perox-

idase activity was measured in bulbs grown at 15 �C

(37.43 ± 2.17 lmol min-1). Increased peroxidase activity

was observed after 3 days of growing bulbs at 4 �C (more

than 20 times).

Antioxidant enzyme activities in bulbs grown ex vitro

The increase in SOD activity was observed after 1 month

of cultivation under natural conditions, in late November,

when it was 108.53 ± 3.17 lmol min-1 mg-1. The high-

est SOD activity (340.05 ± 15.11 lmol min-1 mg-1) was

in bulbs that were grown during the 5 months of winter

(Fig. 7a).

Catalase activity was relatively constant and in the end

of March it was 60.15 lmol min-1 mg-1 compared to

November when it was 89.52 lmol min-1. During April,

catalase activity in bulbs increased 14 times and it was

841.80 ± 23.39 lmol min-1 mg-1 (Fig. 7b).

Glutathione reductase activity showed less oscillation

compared to the other enzymes that were measured in

bulbs grown in natural conditions as well as in bulbs grown

in vitro (Fig. 7c).

Bulbs grown in natural conditions showed the highest

peroxidase activity (632.12 ± 22.54 lmol min-1 mg-1)

during the first months of growth ex vitro. Peroxidase

activity decreased and reached a minimum value

(109.30 ± 9.08 lmol min-1 mg-1) after 5 months of

growing bulbs ex vitro, while at the end of April, after

7 months of ex vitro growth POX activity increased and

reached 325.72 ± 29.23 lmol min-1 mg-1 (Fig. 7d).

Discussion

The effects of low temperatures on overcoming dormancy

in bulbs regenerated in vitro were investigated in Lilium

(Shin et al. 2002; Langens-Gerrits et al. 2003a), Lachenalia

(Slabbert and Niederwieser 1999), Allium (Specht and

Keller 1997; Yamazaki et al. 2002), Narcissus (Hulscher

et al. 1992). Bulbs of F. meleagris L. regenerated in vitro

were also dormant and the low temperature period was the

main factor in dormancy breaking. During the period of

growing bulbs at 4 �C, regeneration of bulb was very weak,

while on temperature of 15 �C the number of regenerated

bulbs increased significantly. In most cases, the need for a

longer period of low temperatures depends on the species,

while the optimum length of exposure to cold temperatures

results in better germination and faster growth of roots and

leaves. Further prolonging the cooling time, which would

Fig. 4 The effect of gibberellic

acid pretreatment on sprouting

of F. meleagris L. bulbs in vitro

Fig. 5 Acclimatization of in vitro regenerated plants F. meleagris a plants regenerated in vitro after 8 weeks of growing at 4 �C b completely

formed in vitro plants before planting c in vitro regenerated plants acclimatized to the greenhouse conditions

Acta Physiol Plant (2013) 35:3223–3236 3229

123

be longer than optimal, has no effect on the above

parameters, and often has a negative impact. Low-tem-

perature treatment had a positive effect on the formation of

narcissus bulbs (Hulscher et al. 1992). Bulbs of F. melea-

gris regenerated at 15 �C had a smaller weight increase

after 4 weeks similar to bulbs of lilies where the dry weight

of bulbs regenerated at 15 �C showed lower values than the

bulbs that were exposed to low (4 �C) temperature (De

Klerk 2009). The duration of cooling varies in different

species, and even among different varieties of the same

species (Langens-Gerrits et al. 2001). Bulbs of F. melea-

gris that were cooled sprout to a higher percentage than

050

100150200250300350400450500

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enzy

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treatments

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treatments

050

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a

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Fig. 6 The activity of

antioxidant enzymes in

F. meleagris L. bulbs growing

in vitro at different treatments

a superoxide dismutase,

b catalase, c glutathione

reductase, d peroxidase

3230 Acta Physiol Plant (2013) 35:3223–3236

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0

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1 month (November) 5 months (November-April) 7 months (the end of April)

time spend in natural condition

1 month (November) 5 months (November-April) 7 months (the end of April)

time spend in natural condition

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a

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Fig. 7 The activity of

antioxidant enzymes in

F. meleagris L. bulbs growing

ex vitro during different periods

of the year a superoxide

dismutase, b catalase,

c glutathione reductase,

d peroxidase

Acta Physiol Plant (2013) 35:3223–3236 3231

123

those grown on standard temperature. Ledesma et al.

(1980) published that the bulbs of garlic did not sprout after

planting, due to dormancy, but the sprout increased rapidly

if the bulbs were kept at low temperatures for a specified

time. Sprouting of garlic bulbs formed in vitro was also

weak, and they were dormant (Moriconi et al. 1990).

Growing bulbs Dioscorea polystachya at low tempera-

ture (12–18 weeks) in culture as well as in natural condi-

tions positively affected their sprouting (Walck et al.

2010), as well as the termination of dormancy (Okagami

and Tanno 1991).

Lily bulbs that sprouted without cold treatment formed

only poorly developed leaves without stem (Langens-

Gerrits et al. 1997). Langens-Gerrits et al. (2003b) found

that a low temperature is required for the transition from

juvenile into the adult state when plants can form stem and

flower. For the transition from the juvenile stage, a deter-

mined size of the bulbs was required which was species

specific (Le Nard and De Hertogh 1993).

After treatment of bulbs with GA3, it was determined

that the largest number of regenerated bulbs as well as a

highest weight increase occurred in control bulbs that have

been chilled for 5 weeks and have not been influenced by

gibberellins. The largest number of bulbs sprouted was

observed 1 month after GA3 treatment of bulbs which were

previously grown at 4 �C. This is similar to the result with

Lilium speciosum (Kim 1991) where the optimal concen-

tration of GA4 ? 7, which led to the interruption of dor-

mancy, were 1 mg l-1 but in that case GA3 had no effect.

Sprouting of bulbs can be hastened with short gibber-

ellins treatment (Niimi et al. 1988). Bulbs of F. meleagris

treated with gibberellins without cold treatment did not

continue to grow after germination or grew very slowly.

Bulbs of L. speciosum also did not grow after sprouting

when treated with gibberellins (Langens-Gerrits et al.

1997), although they sprouted if they were previously

cooled (Kim 1991). Sprouting percentage increased in

bulbs treated with gibberellic acid, in Allium sativum

(Rahman et al. 2006), where the highest percentage of

germination was achieved at the concentration of 250 ppm

GA3. The slow growth of non-cooled bulbs suggests that

the effect of gibberellic acid on the sprouting of onion

bulbs is useless unless the bulbs were grown for a specific

time period at low temperatures. GA3 can compensate for

the lack of low-temperature specific time period, but not

completely. It can be concluded that GA3 affects the ter-

mination of dormancy in terms of early sprouting bulbs, but

for their further growth and formation of leaves, low

temperatures are necessary. It is believed that the transport

of gibberellins during dormancy stops (Rohde and Bhall-

erao 2007), but the ways in which it is re-established are

still unclear. From what is stated above, it can be concluded

that the termination of dormancy in F. meleagris L. bulbs

(sprouting bulbs) and their further growth are two separate

processes. Similar results were reported in lily (Langens-

Gerrits et al. 2003b). The same author state that in the bulbs

treated with gibberellic acid, hydrolysis of sugar does not

normally happen, supporting the theory that gibberellins

are not the only factor required for the termination of

dormancy in bulbs.

Certain amount of hydrolyzed sugar must be stored in the

bulb during the minimum period of low temperatures,

which in the case of F. meleagris L. bulb is 4 weeks, to have

enough energy to sprout and grow after a dormancy break.

The process of seed germination and seedling growth of

Arabidopsis are also two separate processes regulated by

different metabolic pathways (Pritchard et al. 2002). The

fact that only gibberellins which are applied in the short

term could not break dormancy properly and lead to growth

after sprouting bulbs, can be explained by the short time

during which not enough sugars can be stored to support the

future growth of bulbs. Chilling period of several weeks

provides enough time for the hydrolysis of sugar.

The role of antioxidant enzymes in overcoming

dormancy in bulbs

Mechanisms underlying plant growth at low temperatures

are unknown. It is believed to be the biggest damage ROS

causes to cell membranes (Steponkus 1984). Many plants

become more or less tolerant for periods of low tempera-

tures through the process of acclimatization. Plants which

activate antioxidative system faster and whose antioxida-

tive system quickly returned to initial levels after the stress

are considered more resistant to low temperatures (Lukat-

kin 2002).

Antioxidant enzymes are the most common markers of

oxidative stress. However, a clear correlation between the

activity of antioxidant enzymes and oxidative stress has not

been shown in all investigated species (Seppanen and

Fagerstedt 2000). In tomato, no relationship was shown

between the activities of antioxidant enzymes in varieties

resistant to cold and those that are sensitive (Walker and

McKersie 1993), while for example in maize, it was

demonstrated that antioxidant enzyme had a key role in the

elimination of ROS generated during the action of low

temperatures (Jahnke et al. 1991).

The activity of all antioxidant enzymes of F. meleagris

L., except GR, increased with decreasing temperature.

SOD showed the highest activity after 8 weeks of bulbs

growth at 4 �C. Some isoforms of SOD might be associated

with dormancy and appear only during cold periods. It has

been documented that genes for specific SOD isoforms can

be activated by low temperatures (Tsang et al. 1991;

Kaminaka et al. 1999; Lee and Lee 2000; Lee et al. 1999).

There is evidence that the Mn-SOD isoforms were induced

3232 Acta Physiol Plant (2013) 35:3223–3236

123

in the bulbs of F. meleagris L. during period of growth at

low temperatures (Jevremovic et al. 2010). It is believed

that this isoform of SOD eliminates ROS that accumulate

during oxidative stress caused by low temperatures (Vyas

and Kumar 2005). The highest SOD activity in F. melea-

gris L. was in bulbs that were grown in the natural con-

ditions for 5 months during the winter, which may be

associated with increased activity of Mn-SOD. The

increased activity of SOD can be attributed to the other

types of stress that occur when the plant is grown under

natural conditions (McKersie et al. 1999). In potato vari-

eties that are tolerant to low temperatures, a higher toler-

ance to superoxide anion radical and increased SOD

activity were observed compared to the genotypes that are

sensitive to low temperatures (Seppanen et al. 1998). Dif-

ferent results and interpretation for the role of SOD in

oxidative stress caused by low temperatures can be

explained by the different isoforms that have been inves-

tigated by many researchers, different duration of cold

treatment to which the plants were subjected and the dif-

ference in temperature applied (Lukatkin 2002).

The highest CAT activity was shown after the treatment

with low temperatures. It was shown that the CAT activity

increased rapidly at the beginning of the cold period,

especially in genotypes that are well adapted to low tem-

peratures as in maize (Hodges et al. 1997a, b), cucumber

(Shen et al. 1999) and rice (Fadzillah et al. 1996). In

transgenic tomato plants with decreased CAT activity,

resistance to low temperature was reduced (Kerdnai-

mongkol and Woodson 1999) indicating that catalase plays

an important role in oxidative stress induced by low tem-

peratures. The increased activity of catalase in bulbs of

F. meleagris after treatment with low temperatures may

mean that these enzymes play an important role in

repairing damage caused by low temperatures, similar to

tomato and wheat (Lukatkin 2002).

Nir et al. (1986) concluded that the dormancy in grape is

positively correlated with the activity of catalase and

hydrogen peroxide production. Function of catalase

inhibitors such as thiourea, sodium nitrite and hydroxyla-

mide led to rapid interruption of dormancy in lettuce seeds

(Hendricks and Taylorson 1975) and potato tubers (Beuk-

ema and van der Zaag 1990). Increases in catalase activity

during periods of low temperatures can be explained by

their low affinity for hydrogen peroxide (Mizuno et al.

1998). Hydrogen peroxide levels need to reach a certain

concentration that would be sufficient to activate catalase

and that happens during low temperature effect. Increased

accumulation of hydrogen peroxide can be associated with

increased activity of SOD, which is highest in bulbs of

F. meleagris L. after 8 weeks at 4 �C. As SOD activity in

F. meleagris L. bulbs increases, so does CAT probably

with a role in the elimination of SOD, produced hydrogen

peroxide.

GR activity was highest in the bulbs grown at a constant

24 �C in vitro and was lowest when the bulbs were grown

under natural conditions for 5 months of winter. It is

believed that GR indirectly affects photosynthesis, via

production of NADP, which is most intense when the plant

grows at an optimal temperature (Steffen and Palta 1987).

Fontaine et al. (1994) noted that dormancy break in oat

leads to increased production of glutathione, which is the

product of GR activity. Slight increases in GR gene

expression were shown during the first week of exposure to

low temperatures and the expression level decreased after

that (Baek and Skinner 2003). Glutathione as a component

of the antioxidant defense system may be required in larger

amounts after dormancy for rehabilitation of any damage

caused by oxidative stress. Metabolic processes that are

intensely activated after dormancy, or when bulb is pre-

paring for sprouting, lead to the formation of large amounts

of ROS, which must be eliminated in this case with

glutathione.

Peroxidase activity in bulbs of F. meleagris L. is the

greatest 3 days after the beginning of low-temperature

treatment in vitro. Slight changes in peroxidase activity

were observed in potato tubers which were dormant (Rojas-

Beltran et al. 2000). Under natural conditions, POX activity

was highest during the month of November i.e., at the

beginning of the winter period.

At the beginning of low temperature exposure, hydrogen

peroxide could be formed in increased amounts as a

response to stress caused by these temperatures. The first

enzyme to be activated in the case of F. meleagris L. is

peroxidase. During the cold treatment, the role of hydrogen

peroxide removal is assumed by catalase activity which

reaches a maximum 7 days after the end of cold treatment.

Peroxidase had the lowest activity at the time when CAT

activity was highest, i.e., 7 days after the removal of plants

at 24 �C. Because these two enzymes are in competition

for the same substrate, hydrogen peroxide, and since they

have different affinities regarding hydrogen peroxide, these

enzymes are activated at different times. Peroxidase

activity was generally lower than catalase which indicates a

major role of CAT in breaking dormancy and preparing

bulbs for sprouting, which is similar to the results obtained

in potato tubers (Rojas-Beltran et al. 2000). Increased

accumulation of hydrogen peroxide during the cold treat-

ment of bulbs can reach a certain concentration that is

required to activate the cells, which leads to break dor-

mancy. This hypothesis was confirmed in species such as

potato (Rojas-Beltran et al. 2000), soybean (Puntarulo et al.

1988) and oats (Cakmak et al. 1993) which were treated

with certain concentrations of hydrogen peroxide and

Acta Physiol Plant (2013) 35:3223–3236 3233

123

because of that had a shorter period of dormancy than a

control plant.

Author contribution MP produced and maintained

in vitro cultures, designed and supervised the whole

experiment. SJ provided plant material. MT performed

statistical analysis of date and help for experimental

design. VT, SM and MD were involved in detection of

antioxidant enzymes activity. AS helped with the inter-

pretation of data and supervised writing the manuscript.

Acknowledgments This work was supported by the Serbian Min-

istry of Education, Science and Technological Development (Projects

No. ON173015 and TR31019).

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