ORIGINAL PAPER

Quantification of arabinogalactan proteins during in vitromorphogenesis induced by b-D-glucosyl Yariv reagentin Centaurium erythraea root culture

Milana Trifunovic • Vojin Tadic • Marija Petric •

Danijela Jontulovic • Sladana Jevremovic •

Angelina Subotic

Received: 11 October 2013 / Revised: 21 January 2014 / Accepted: 22 January 2014

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2014

Abstract Arabinogalactan proteins (AGPs) are a family

of highly glycosylated cell surface proteins located at the

plasma membrane and plant cell wall. AGPs play important

roles in plant growth and development. Yariv phenylgly-

coside (bGlcY), synthetic red-brown dye that specifically

binds and precipitates AGPs, has been used for detection

and quantification of AGPs in plant tissue. Graded con-

centrations of bGlcY (0–75 lM) were used to investigate

the effect of this synthetic dye on induction of in vitro

morphogenesis in Centaurium erythraea root culture on

two nutrient media: �MS and �MS ? IBA 1.0 lM.

Regeneration of C. erythraea shoots on root explants was

stimulated on both media supplemented with 25 lM

bGlcY after 8 weeks in culture. Quantification of AGPs in

different tissues of C. erythraea was determinate with

single radial diffusion method. This work emphasizes clear

effect of bGlcY on induction of morphogenesis in vitro in

C. erythraea root culture.

Keywords Centaury � In vitro morphogenesis �Organogenesis � Hydroxyproline-rich glycoproteins �Single radial gel diffusion

Introduction

Arabinogalactan proteins (AGPs) belong to the class of

plant cell wall proteoglycans analogous to animal proteo-

glycans. These proteoglycans consist of a hydroxyproline-

rich protein and covalently attached carbohydrate rich in

arabinose and galactose-rich polysaccharide units (Show-

alter 2001; Yang et al. 2007). AGPs are widely expressed

throughout the plant kingdom (Seifert and Roberts 2007).

AGPs were isolated from the leaves, roots, flower parts and

seeds. At the subcellular level they are always located on

the cell surface of the plant cells or associated with plant

cell wall and plasma membrane. However, specific distri-

bution of AGPs of different families is present in various

plant organs and tissues (Schowalter 2001). These proteins

play an important role in the growth and development of

plants. AGPs belong to the important group of extracellular

proteins involved in cell differentiation (Pennel and Rob-

erts 1990), cell divison (Serpe and Nothangel 1994; Cos-

kun et al. 2010), formation of cell wall (Schindler et al.

1995), programmed cell death (Gao and Showalter 1999;

Mashiguchi et al. 2008), pollen tube growth (Jauh and Lord

1996; Roy et al. 1998; Lee et al. 2008), zygote division

(Qin and Zhao 2006), androgenesis (Tang et al. 2006),

pollen formation (Coimbra et al. 2009), cotyledons matu-

ration (Zhong et al. 2011), and somatic embryogenesis

(Egertsdotter and Van Arnold 1995; Kreuger and Van

Holst 1995; Wu et al. 2000; Pan et al. 2011). Despite

widely known numerous roles of AGPs, regulatory mech-

anisms of these functions are still completely uncertain and

unknown (Chapman et al. 2000; Seifert and Roberts 2007).

Very important characteristic of AGPs is specific binding

and precipitating to a synthetic chemical, a phenylglycoside

red-brown dye [1,3,5-tris (4-b-D-glycopyranosylox-

yphenylazo)-2,4,6-trihydroxy-benzene], commonly known

Communicated by E. Schleiff.

M. Trifunovic (&) � V. Tadic � M. Petric � D. Jontulovic �S. Jevremovic � A. Subotic

Institute for Biological Research ‘‘Sinisa Stankovic’’, University

of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade,

Serbia

e-mail: milanag@ibiss.bg.ac.rs

123

Acta Physiol Plant

DOI 10.1007/s11738-014-1495-y

as Yariv phenylglycoside reagent (bGlcY) (Yariv et al.

1962, 1967). Although AGPs represent a group of numerous

and diverse amino acids and carbohydrates it cannot be

generalized that all AGPs react with Yariv reagent (Show-

alter 2001). Precise mechanism of the interacion between

AGPs and Yariv reagent is not fully understood yet (Gao and

Showalter 1999; Pettolino et al. 2006). Notwithstanding,

precipitation via Yariv reagent is used for determination,

purification and quantification of AGPs (Van Holst and

Clarke 1985; Willats and Knox 1996). The use of Yariv

reagent presents a very suitable method to study the role of

AGPs during plant morphogenesis (Chapman et al. 2000).

While b-D-glucosyl and b-D-galactosyl Yariv reagents pre-

cipitate AGPs, a-D-galactosyl and a-D-mannosyl Yariv

reagents do not bind and precipitate AGPs and often are used

as negative control (Yariv et al. 1962; Seifert and Roberts

2007).

Centaurium erythraea Rafn. is an short-lived annual or

biennial medicinal plant belonging to the Gentianaceae

family. This cosmopolitan plant species, known as common

centaury, inhabits mountain slopes, dry grasslands, scrubs,

as well as saline soils throughout the Europe and North

America (Chevallier 2000). Centaury has been used for

centuries for medical purposes to reduce fever; regulate

blood sugar; treat anemia, jaundice and gout; and to

increase appetite, stimulate digestion and strengthen the

digestive system. Also, centaury has long been used as

bittering agents and in traditional medicine. Because of the

numerous biologically active pharmacological compounds

with therapeutic properties, herbal extract of centaury is

officially recognized as a drug in a number of world’s

pharmacopoeias. The most important secondary metabo-

lites of the genus Centaurium are bitter secoiridoid gluco-

sides such as swertiamarin, gentiopicrin and sweroside (van

der Sluis et al. 1983; Jensen and Schripsema 2002) and

xanthones such as eustomin and demethyleustomin (van der

Sluis 1985; Valentao et al. 2002). Generally, centaury is the

most investigated plant species among numerous plant

species of genus Centaurium. Uncontrolled and intensive

collection of centaury from natural habitat listed this plant

species as an endangered and species pretty rarely found in

nature. Plant tissue culture represents a very suitable

method for efficient multiplication and conservation of this

pharmacologically valuable and endangered plant species.

The aim of this work was (1) to investigate the effect of

bGlcY on induction of in vitro morphogenesis in C. ery-

thraea root culture and (2) isolation and quantification of

AGPs from centaury shoots and roots by precipitation of

bGlcY. In the present study we used root culture, well-

established model system for efficient induction of in vitro

morphogenesis of C. erythraea, to determine the effect of

graded bGlcY concentrations on morphogenesis itself and

also endogenous AGP secretion in centaury tissue.

Materials and methods

Plant material and culture conditions

Mother stock shoot culture of C. erythraea was initiated

and maintained as described previously by Subotic et al.

(2003/4). In brief, seeds of centaury, after surface decon-

tamination, were transferred to half-strength MS medium

(�MS, Murashige and Skoog 1962) solidified with 0.7 %

agar and supplemented with 3 % sucrose and 100 mg l-1

myo-inositol. The medium was adjusted to pH 5.8 with 1 N

NaOH/1 N HCl and autoclaved at 121 �C for 25 min. The

same, hormone-free medium, was used for regeneration

procedure. The obtained seedlings (15–20 mm) were

transferred and further cultivated on solid �MS. To

establish a root culture, root explants (&10-mm long)

isolated from seedlings obtained under sterile conditions

were transferred on two different nutrient media supple-

mented with �MS mineral solution or �MS supplemented

with IBA 1.0 lM. Different concentrations of bGlcY (0, 5,

15, 25, 50 and 75 lM), a synthetic phenylglycoside that

specifically binds AGPs, were added to the both nutrient

media for the induction of in vitro morphogenesis in C.

erythraea root culture. Different concentrations of the

bGlcY were added to the culture medium after autoclaving.

The frequency of shoot regeneration and the average

number of shoots per root explant were recorded after

8 weeks in culture. All in vitro cultures were maintained at

temperature of 25 ± 2 �C under fluorescent light of

47 lM s-1m-2 and 16-h/8-h light/dark photoperiod.

Histochemical localization of AGPs with bGlcY

During in vitro morphogenesis of C. erythraea in solid root

culture, AGPs were histochemically localized using posi-

tive as well as negative probe, bGlcY and a-D-galactosyl

Yariv reagent (Biosupplies Australia Pty Ltd, Parkville,

Victoria, Australia). Hand cross sections of centaury root

explants were treated with a solution of both reagents

(according to the manufacturer’s recommendations, 2 mg

of dye was dissolved in 1 ml of 0.15 M NaCl) and incu-

bated for 1 h at room temperature. All hand sections

(treated with bGlcY and a-D-galactosyl Yariv reagent)

were washed three times with distilled water to remove

over color and prepared for light microscopy study. All

root sections were examined with a LEICA DMLB light

microscope (Leica, Germany).

Isolation of AGPs from root cultures of C. erythraea

and quantification by single radial gel diffusion

AGPs were isolated and quantified through single radial gel

diffusion by combination and modification of two protocols

Acta Physiol Plant

123

described by van Holst and Clarke (1985) and Popper

(2011). C. erythraea shoots and roots cultured 8 weeks on

media supplemented with bGlcY (0–75 lM) were ground

in liquid nitrogen in a mortar and then extracted with

extraction buffer (50 mM Tris–HCl, 10 mM EDTA, 1 M

DTT, 1 % Triton X-100, pH 8.0). Plant tissue then was

homogenized by ultrasonic homogenizer and incubated

overnight at 4 �C together with extraction buffer. Cell

debris was removed from the extract by centrifugation at

4 �C (4,000g, 10 min). The supernatant was analyzed

directly by single radial diffusion method. A solution

containing 1 % w/v agarose, 0.15 M NaCl, 0.02 M NaN3

and 10 lg/ml bGlcY was heated to boiling. Aliquots were

poured onto preheated level glass plates and allowed to

cool to room temperature to form gels of uniform thickness

(1 mm). The end of a glass Pasteur pipette was used to cut

out wells of uniform diameter. Wells were filled with 30 ll

test solution and plates incubated for at least 48 h at room

temperature in a moist chamber. A standard curve was

constructed using series of a standard gum arabic solutions.

As a negative control a-D-galactosyl Yariv was used.

Defined red halos were formed around the test wells. The

outher diameter of these halos was measured using cali-

brated eyepiece. Total protein content was determined

according to the method of Bradford (1976), using bovine

serum albumin as a standard.

Statistical analysis

The experiments using bGlcY were repeated three times

with 30 root explants per treatment. Each determination of

AGPs was also repeated three times and results are pre-

sented as mean ± SE. Statistical analyses were performed

using StatGrafics software version 4.2 (STSC Inc. and

Statistical Graphics Corporation, 1985–1989, USA). The

data were subjected to analysis of variance (ANOVA) and

comparisons between the mean values were made using the

least significant difference (LSD) test calculated at a con-

fidence level of p B 0.05.

Results

Effect of bGlcY on morphogenesis in root culture

of C. erythraea

Different concentrations of bGlcY were used to investigate

the effect of this synthetic dye on induction of in vitro

morphogenesis in C. erythraea root culture. The addition

of graded concentration of bGlcY into the culture medium

did not inhibited in vitro morphogenesis of C. erythraea.

Fully developed centaury regenerants were observed after

8 weeks on �MS hormone-free medium and on �MS

medium supplemented with IBA (1.0 lM), both in the

presence of 0–75 lM bGlcY. Identically as well as in

control root explants first changes in the basal part of the

initial root explants were noticed only after 7 days on

�MS media supplemented with graded concentration of

bGlcY. Root tissue started to thicken and begun to overshot

alongside the entire length of the basal part of root explant.

Somatic embryos and adventitious buds of centaury were

developed along whole root explants. Centaury plants,

typical rosette form, regenerated on all �MS media sup-

plemented with different concentrations of bGlcY devel-

oped normally after 8 weeks of subculture and also showed

a similar morphology as to plants regenerated in root cul-

tures without bGlcY used as control (Fig. 1).

We noticed that frequency of shoot regeneration was

significantly increased on root explants cultured on �MS

hormone-free medium in the presence of 15 and 25 lM

bGlcY and on �MS medium supplemented with 1.0 lM

IBA in the presence of 25 lM bGlcY (Table 1). We also

noticed that the average number of regenerated C. eryth-

raea shoots on root explants on �MS hormone-free med-

ium in the presence of 25 lM bGlcY after 8 weeks in

culture was 2.18 ± 0.10 while on �MS medium supple-

mented with 1.0 lM IBA in the presence of 15 and 25 lM

bGlcY the average number of regenerated shoots was

2.08 ± 0.09 and 2.13 ± 0.07. Decreased and increased

concentrations of bGlcY in both culture media, after

8 weeks in culture, did not show statistically significant

increment of the average number of regenerated shoots

compared to the control (Table 2).

Histochemical localization of AGPs with bGlcY

The precipitation of AGPs was observed in hand sections

of C. erythraea root explants during induction of mor-

phogenesis in vitro using bGlcY. A synthetic dye bGlcY

intensively stained AGPs in the surface cell layers of

centaury root explants. Intensive red-brown color of epi-

dermal cells and also vascular tissue was noticed (Fig. 2a).

Hand cross sections of centaury root explants showed no

precipitation of AGPs (Fig. 2b). These results confirmed

that bGlcY specifically stained and precipitated AGPs

while a-D-galactosyl Yariv reagent did not bind AGPs and

could serve as a negative control in quantification of AGPs

in centaury tissue.

Quantification of AGPs in root cultures of C. erythraea

during in vitro morphogenesis

We investigated the content of AGPs in regenerated shoots

and roots of C. erythraea after 8 weeks in culture on �MS

hormone-free medium and on �MS medium supplemented

with IBA 1.0 lM, both in the presence of graded

Acta Physiol Plant

123

concentrations of bGlcY. The amount of AGPs in plant

tissue was quantified by single radial gel diffusion (Fig. 3).

Increased content of AGPs in regenerated shoots is

achieved on both media in the presence of 25, 50 and

75 lM bGlcY. Lower concentration of bGlcY (5 and

15 lM) showed no statistically significant difference in

content of AGPs in comparison to the control (Fig. 4a).

Toward shoots, AGPs content increased in all roots cul-

tured on hormone-free medium while in roots cultured on

medium supplemented with IBA AGPs content increased at

Fig. 1 Induction of in vitro

morphogenesis in root culture of

Centaurium erythraea.

Regeneration of shoots and

roots on �MS hormone-free

medium (a) and on �MS

medium supplemented with

IBA 1.0 lM (b). Eight-week-

old C. erythraea plants

developed on �MS hormone-

free medium in the presence of

25 lM bGlcY (c) and on �MS

medium supplemented with

IBA 1.0 and 25 lM bGlcY (d)

Table 1 The effects of different concentration of bGlcY on fre-

quency of shoot regeneration after 8 weeks in solid root culture of

Centaurium erythraea

Concentration of bGlcY Yariv

reagent (lM)

Regeneration (%)

�MS �MS ? IBA

1.0 lM

0 71.67 ± 9.82a 85.00 ± 5.69a

5 80.00 ± 7.70ab 85.00 ± 4.86a

15 93.89 ± 2.39b 86.11 ± 6.11a

25 92.22 ± 3.46b 97.22 ± 2.24b

50 83.33 ± 7.76ab 85.55 ± 6.13a

75 77.78 ± 7.03ab 91.67 ± 4.11a

Each value represents the mean ± SE, n = 3. Differences between

values marked with the same letter are significant at the p B 0.05

level according to the LSD test

Table 2 Average number of regenerated Centaurium erythraea

shoots per root explant after 8 weeks in solid root culture on media

supplemented with graded concentration of bGlcY

Concentration of bGlcY (lM) Average no. of regenerated shoots

�MS �MS ? IBA

1.0 lM

0 1.39 ± 0.10ab 1.63 ± 0.09a

5 1.25 ± 0.07a 1.77 ± 0.08a

15 1.62 ± 0.07b 2.08 ± 0.09b

25 2.18 ± 0.10c 2.13 ± 0.07b

50 1.40 ± 0.07ab 1.49 ± 0.07a

75 1.26 ± 0.07a 1.60 ± 0.07a

Each value represents the mean ± SE, n = 3. Differences between

values marked with the same letter are significant at the p B 0.05

level according to the LSD test

Acta Physiol Plant

123

concentration of bGlcY above 50 lM. The presence of 5,

15 and 25 lM bGlcY on �MS medium supplemented with

IBA drastically increased the amount of AGPs in roots

(Fig. 4b). In nontreated shoots and roots we determined the

same amount of AGPs. Against this, AGPs content in

nontreated roots was about four times higher than in shoots.

Beside AGPs content, total protein content in all treated

cultures after 8 weeks was detected. Also, the rate of AGPs

in total protein content was calculated. Different rates of

AGPs in shoots and roots cultured on �MS medium sup-

plemented with graded concentration of bGlcY were

noticed (Fig. 5). In centaury shoots the highest rate of

AGPs was detected on higher concentration of bGlcY (25

and 50 lM). Contrarily, the lowest concentrations of

bGlcY (5 and 15 lM) on �MS hormone-free medium

were affected on the highest rate of AGPs in total protein

content in centaury roots. Unlike previously mentioned, the

results obtained on another culture medium, �MS medium

supplemented with IBA 1.0 lM and graded concentration

of bGlcY, showed clearly exponential dependence. The

highest concentrations of bGlcY Yariv (25, 50 and 75 lM)

in the culture medium were affected on the highest rate of

AGPs of total protein content in centaury shoots as well as

in roots.

Fig. 2 Histochemical

localization of AGPs in root

explants of Centaurium

erythraea. Detail of hand cross

section of root explants after

staining with bGlcY; intensive

staining of epidermal cells and

vascular tissue, bar 40 lm (a).

Detail of hand cross section of

root explants after staining with

a-D-galactosyl Yariv reagent

with no precipitation of AGPs,

bar 40 lm (b)

Fig. 3 Single radial gel diffusion quantification of AGPs in Centau-

rium erythraea plants regenerated in solid root culture. Quantification

of AGPs in 8-week old C. erythraea shoots (top halos) and roots

(bottom halos) cultured on: �MS hormone-free medium (a), �MS

medium supplemented with IBA 1.0 lM (b), �MS hormone-free

medium in the presence of 25 lM bGlcY (c) and on �MS medium

supplemented with IBA 1.0 and 25 lM bGlcY (d). a-D-galactosyl as

a negative control (e). Serial dilutions (0.1–1.0 mg/ml) of a standard

gum arabic solution (f)

Acta Physiol Plant

123

Discussion

Due to possibility of direct shoot regeneration and/or

somatic embryos of centaury, root culture represents very

suitable model system for studying morphogenesis in vitro.

Root culture, beside easy manipulation, characterizes many

other advantages such as small differences in the physio-

logical response, small variability, as well as high potential

for growth and metabolic activities. After testing a few

different explants it was shown that the root and leaf

explants are the most suitable explants for centaury buds

regeneration (Piatczak and Wysokinska 2003). Plant

Fig. 4 AGPs content in

regenerated shoots (a) and roots

(b) of Centaurium erythraea

after 8 weeks in culture on

�MS hormone-free medium

and on �MS medium

supplemented with IBA 1.0 lM

both in the presence of bGlcY.

Data represent mean ± SE.

Means marked with an asterisk

are significantly different from

the control according to LSD

test (p B 0.05)

Fig. 5 The rate of AGPs in

total protein content in shoots

(a) and in roots (b) of

Centaurium erythraea after

8 weeks in culture on �MS

hormone-free medium and on

�MS medium supplemented

with IBA 1.0 lM both in the

presence of bGlcY. Data

represent mean ± SE. Means

marked with an asterisk are

significantly different from the

control according to LSD test

(p B 0.05)

Acta Physiol Plant

123

regeneration by direct somatic embryogenesis and organ-

ogenesis in root culture originated from seedlings of C.

erythraea has previously reported 10 years ago when root

explants from mother stock plants were cultured on �MS

hormone-free medium (Subotic et al. 2003/4). Spontaneous

regeneration of adventitious buds in centaury root cultures

was previously described also by Subotic et al. (2006,

2009). Authors showed that the average number of spon-

taneously regenerated buds in centaury root culture was

higher in the light than in the dark on �MS hormone-free

medium. Authors also showed that IBA, probably due to

rapid metabolism in the cell, effectively induce in vitro

morphogenesis in relation to other plant growth regulators.

Later, histological analyses confirmed centaury plant

regeneration through simultaneously somatic embryogen-

esis and/or organogenesis on the same root explant in vitro

(Subotic and Grubisic 2007). Somatic embryogenesis and/

or adventitious buds formation were asynchronous because

somatic embryos and buds were observed at different

stages of development on same explants. Fully developed

regenerants have occurred after 4 weeks in culture.

We investigated the presence of AGPs during induction

of morphogenesis in vitro in centaury root culture. It is

already known that bGlcY specifically binds and precipi-

tates AGPs, while a-D-galactosyl Yariv reagent does not

bind AGPs and serves as a negative control (Showalter

2001). Application of bGlcY in hand cross sections of C.

erythraea root explants showed the specific staining pattern

of AGPs (Fig. 2a) while sections treated with a-D-galac-

tosyl Yariv reagent showed no characteristic brown–red

stain indicating of AGPs (Fig. 2b). Intensive brown-red

color was also observed in root epidermal cells during

somatic embryogenesis in root culture of Cichorium

(Chapman et al. 2000). To date the most literature data

regarding the role of AGPs in somatic embryogenesis and

androgenesis (Seifert and Roberts 2007). Histochemical

localization of AGPs presented in this work could imply

the possible role of AGPs during in vitro morphogenetic

pathway induced in centaury root culture.

Literature data shows different effects of bGlcY on plant

tissues depending on explant and culture medium used in

investigations. The effect of bGlcY on different plant

development pathways was investigated for the first time

about 20 years ago (Serpe and Nothnagel 1994). It was

shown that rose cell divisions were inhibited in suspension

culture in a concentration-dependent manner. It was also

shown that addition of 50 lM bGlcY into the culture

medium completely inhibits cell division in Brassica sp.

microspores (Tang et al. 2006). Reduction of cell growth

was also obtained in cell cultures of Cucurbita pepo (Ben

Amar et al. 2010). The highest inhibition of growth was

noticed at 50 lM bGlcY. The presence of bGlcY reduced

cell growth even if there were no changes in viability of

cell suspension cultures of Beta vulgaris L. (Capataz-Tafur

et al. 2010). Oppositely previously mentioned, cell divi-

sions of Marchantia polymorpha protoplasts were

increased in addition of bGlcY (Shibaya and Sugawara

2007). Recent data showed that the presence of Yariv

reagent affected callus development rate and morphology

of somatic embryos but there were no effects on fresh

weight increment of peach palm in vitro (Steinmacher et al.

2012). In the presence of extremely high concentration of

bGlcY (250 lM) somatic embryogenesis in root cultures of

Chicorium sp. was completely inhibited (Chapman et al.

2000).

In our study for induction of in vitro morphogenesis

in centaury root culture, a concentration-dependent

response was investigated. Significant stimulation of

centaury shoots regeneration was observed only in the

presence of lower concentrations of bGlcY (15 and

25 lM). It was shown that, in this model system, bGlcY

definitively stimulate centaury shoots regeneration.

Higher concentration of bGlcY did not promote either

inhibited or stimulated regeneration of shoots compared

to control. Almost all previously mentioned reports

explained the effect of bGlcY in cell suspension cul-

tures but we investigated the effect of this synthetic dye

in solid centaury root cultures. Bearing on mind that

bGlcY is high molecular weight synthetic phenylgly-

coside with low diffusion potential between cells it is

possible that only continuous contact plant tissue with

the bGlcY is necessary for blocking the developmenthal

pathways. Willats and Knox (1996) showed that effect

of bGlcY was observed only in the root epidermal cells.

Zhong et al. (2011) showed that bGlcY only in early

days of treatment inhibited embryo germination. When

the culture time was extended Yariv reagent did not

have such inhibition effect. In this work the effect of

Yariv reagent on morphogenesis in vitro in solid root

culture of C. erythraea was investigated only after

8 weeks of subculture. It is possible that impact of

bGlcY become weaker and weaker when cultured in a

relatively long period.

Results from this investigation showed a possible cor-

relation between the amount of AGPs, determined in shoots

and roots regenerated on root explants, and concentration

of applied bGlcY. We noticed increased AGPs content in

shoots as well as in roots cultured on medium supple-

mented with higher concentration of bGlcY (25, 50 and

75 lM). These data correspond with the reports of

increased amount of AGPs in embryogenic cultures during

development of Euphorbia pulcherrima somatic embryos

(Saare-Surminski et al. 2000). In this report, we also

showed that higher concentration of bGlcY (25, 50 and

75 lM) influenced the highest rate of AGPs in total protein

content of roots as well as in regenerated shoots. It seems

Acta Physiol Plant

123

that higher concentration of Yariv reagent increased the

amount of AGPs and also increased the rate of AGPs in

total protein content in centaury plant tissue. High accu-

mulation of AGPs was also detected in the conditioned

medium during somatic embryogenesis of Cichorium sp.

(Chapman et al. 2000). Due to the fact that centaury shoots

regeneration was stimulated on root explants on both used

nutrient media supplemented only with lower concentra-

tions of bGlcY (15 and 25 lM) and correlation between of

the amount of AGPs precipitated with the higher concen-

tration of bGlcY, it could be indicated that Yariv reagent

affected the described centaury morphogenetic pathway.

Either way the role of AGPs remains to be further

elucidated.

To summarize, we used previously developed good

model system for induction of morphogenesis in vitro in C.

erythraea solid root culture. The current paper, to our

knowledge, presents first report about effect of bGlcY on

induction of morphogenesis in vitro in C. erythraea root

culture and at same time on the AGPs content in regener-

ated shoots and roots of any plant species belonging to the

genus Centaurium.

Author contribution M. Trifunovic contributed to all

experimental work, statistical analyses and manuscript

preparation. V. Tadic contributed to optimization of pro-

tocol for quantification of AGPs using single radial gell

diffusion method. M. Petric and D. Jontulovic contributed

to all experimental work considering AGPs analyses.

S. Jevremovic and A. Subotic designed the experiments.

S. Jevremovic contributed to data analyses and obtained

results interpretation. A. Subotic, research team leader,

supervised the whole study and also contributed in pre-

paring the final manuscript.

Acknowledgments This work was supported by the Ministry of

Education, Science and Technological Development of the Republic

of Serbia (Grant No. ON173015).

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