SI : TISSUE CULTURE

Changes in endogenous cytokinin profiles in micropropagatedHarpagophytum procumbens in relation to shoot-tip necrosisand cytokinin treatments

Michael W. Bairu • Ondrej Novak •

Karel Dolezal • Johannes Van Staden

Received: 12 April 2010 / Accepted: 15 December 2010 / Published online: 1 January 2011

� Springer Science+Business Media B.V. 2010

Abstract Changes in cytokinin (CK) profiles and their

physiological implications in micropropagated Harpago-

phytum procumbens [(Burch.) DC. ex Meisn.] tissues in

relation to shoot-tip necrosis (STN) and CK treatments were

studied. Total CK content was quantified in benzyladenine

(BA)-treated necrotic and normal plantlets and in plantlets

treated with the CKs BA, meta-topolin (mT) and meta-top-

olin riboside (mTR) with and without the auxin indole-

3-acetic acid (IAA). Generally necrotic shoots yielded more

total CK compared to normal shoots. Cytokinin accumula-

tion was higher at the basal section (basal[middle[ top).

Further analysis of the CKs based on structural and func-

tional forms revealed excessive accumulation of 9-gluco-

sides (deactivation products—toxic metabolites) and limited

amounts of O-glucosides (storage forms—re-utilizable) in

necrotic and BA-treated shoots compared to normal and

topolin-treated cultures. The addition of IAA enhanced the

formation of 9-glucosides in BA-treated cultures but reduced

it in topolin-treated cultures. The symptom of STN could

therefore be attributed to conversion of active cytokinins to

other forms such as 9-glucosides which are neither active nor

reversibly sequestrated to active forms. Literature shows that

metabolites like 9-glucosides ofBAhave a detrimental effect

in plant tissue culture.

Keywords Auxin cytokinin interaction � Cytokinin

purification and analysis � Devil’s claw � Glucosylation �

Harpagophytum procumbens � Micropropagation �

Pedaliaceae � Shoot-tip necrosis

Abbreviations

BA N6-benzyladenine

BA9G N6-benzyladenine-9-glucoside

BAR N6-benzyladenosine

BAR50MP N6-benzyladenosine-50-monophosphate

CK Cytokinin

cZ cis-zeatin

cZ9G cis-zeatin-9-glucoside

cZOG cis-zeatin-O-glucoside

cZR cis-zeatin riboside

cZR50MP cis-zeatin riboside-50-monophosphate

cZROG cis-zeatin-O-glucoside riboside

DHZ Dihydrozeatin

DHZ9G Dihydrozeatin-9-glucoside

DHZOG Dihydrozeatin-O-glucoside

DHZR Dihydrozeatin riboside

DHZR50MP Dihydrozeatin riboside-50-monophosphate

DHZROG Dihydrozeatin-O-glucoside riboside

iP N6-isopentenyladenine

iP9G N6-isopentenyladenine-9-glucoside

iPR N6-isopentenyladenosine

iPR50MP N6-isopentenyladenosine-50-

monophosphate

M. W. Bairu � J. Van Staden (&)

Research Centre for Plant Growth and Development,

School of Biological and Conservation Sciences,

University of KwaZulu-Natal Pietermaritzburg,

Private Bag X01, Scottsville 3209, South Africa

e-mail: rcpgd@ukzn.ac.za

M. W. Bairu

e-mail: bairum@ukzn.ac.za

O. Novak � K. Dolezal

Laboratory of Growth Regulators, Palacky University & Institute

of Experimental Botany AS CR, Slechtitelu 11,

783 71 Olomouc, Czech Republic

e-mail: ondrej.novak@upol.cz

K. Dolezal

e-mail: karel.dolezal@upol.cz

123

Plant Growth Regul (2011) 63:105–114

DOI 10.1007/s10725-010-9558-6

IAC Immunoaffinity chromatography

MRM Multiple reaction monitoring

MS medium Murashige and Skoog (1962) basal medium

mT meta-topolin

mT9G meta-topolin-9-glucoside

mTOG meta-topolin-O-glucoside

mTR meta-topolin riboside

mTR50MP meta-topolin-50-monophosphate

mTROG meta-topolin-O-glucoside riboside

oT ortho-topolin

oT9G ortho-topolin-9-glucoside

oTOG ortho-topolin-O-glucoside

oTR ortho-topolin riboside

oTR50MP ortho-topolin-50-monophosphate

oTROG ortho-topolin-O-glucoside riboside

pT para-topolin

pTOG para-topolin-O-glucoside

pTR para-topolin riboside

pTR50MP para-topolin-50-monophosphate

pTROG para-topolin-O-glucoside riboside

STN Shoot-tip necrosis

tZ trans-zeatin

tZ9G trans-zeatin-9-glucoside

tZOG trans-zeatin-O-glucoside

tZR trans-zeatin riboside

tZR50MP trans-zeatin riboside-50-monophosphate

tZROG trans-zeatin-O-glucoside riboside

UPLC Ultra performance liquid chromatography

Introduction

Harpagophytum procumbens (Pedaliaceae), commonly

known as Devil’s Claw, belong to some of the most studied

medicinal plants with pharmacological activity analysis

and clinical tests dating back to the 1960s. Extracts of

tubers of Harpagophytum spp. are active in the treatments

of degenerative rheumatoid arthritis, osteoarthritis, ten-

donitis, kidney inflammation and heart disease (Stewart

and Cole 2005). Despite a wealth of knowledge on its

pharmacology, very little published data is available on the

growth and biology of this species. Attempts to propagate it

from seeds failed due to low germination rates. Plants

propagated by cuttings failed to produce primary roots

resulting in a single harvest (Kathe et al. 2003). While

attempting to develop a micropropagation protocol we

encountered a serious problem of STN (Fig. 1).

The development of plant tissue culture techniques and

the utilization of plant hormones revolutionized the science

of plant propagation. Among other things, the development

of tissue culture (collective name used for in vitro culture

of cells, tissues and organs) protocols relies largely on

optimizing the type and concentrations of auxins and CKs.

This is mainly due to the fact that plants respond differently

to sub- or supra-optimal concentrations of plant growth

regulators (PGR). This variation in response ranges from

failure to grow, to various types of growth disorders (genetic

and physiological) such as STN, hyperhydricity, diminished

rooting, somaclonal variation and tissue browning.

Shoot-tip necrosis is one of the most common problems in

the micropropagation industry affecting a wide range of plant

species. It is caused by many factors in the tissue culture

system (Bairu et al. 2009a). Cytokinins are one of the main

factors contributing to the problem of STN. There are, how-

ever, different opinions as to how they affect the problem

which can be categorized into three groups. Some suggest that

the cause is due to eliminatingor reducing the concentrationof

CKs in the media (Kataeva et al. 1991; Piagnani et al. 1996).

On the other hand there are reports to the contrary. Bairu et al.

(2009b) for example, observedgradual recovery fromnecrotic

symptoms when Harpagophytum procumbens cultures were

transferred to cytokinin-free rooting medium. Others suggest

that the effect of CKs on STN is influenced by the type and

concentration of CKs used (Mackay et al. 1995; Bairu et al.

2009b) and genotype dependent responses (Grigoriadou et al.

2000). This investigation was aimed at providing a better

explanation and understanding of the effect of CKs on STN.

We undertook a metabolic study by analyzing CK profiles of

micropropagatedH. procumbens based on the following three

hypotheses in two separate experiments;

1. If STN is affected by CK metabolism, there must be

variation in the CK profiles of normal and necrotic

shoots of the various sections of the plant;

Fig. 1 Examples of normal (right) and severely affected necrotic

shoots (left) of Harpagophytum procumbens plantlets. Note the basal

callus-like tissue on the necrotic shoots

106 Plant Growth Regul (2011) 63:105–114

123

2. If STN is affected by the type of CK used, there must be

variation in the CK profiles of plants treated with the

different types of CK in relation to the untreated controls;

and

3. If the presence or absence of auxin in the multiplica-

tion medium affects STN, this might be reflected in a

variation in CK profiles.

We acknowledge the fact that the cause and effect of the

findings presented in this manuscript could have been better

understood in conjunction with some growth and physiolog-

ical data. Comprehensive growth and physiological data from

the same experiment, however, can be found in our previous

report (Bairu et al. 2009b). It is the observations of these

experiments that led to the current analysis. Hence results are

discussed aligned with previous findings on the same species.

Materials and methods

Micropropagation conditions

Maintenance cultures of H. procumbens, derived from

nodal explants, were used as a source of samples for

experiment one and as a source of explants for experiment

two. Cultures were maintained by sub-culturing to fresh

MS medium (Murashige and Skoog 1962) containing

2.5 lM of BA supplemented with 0.9% (w/v) agar and 3%

(w/v) sucrose on a monthly basis. Cultures were kept in a

growth room with cool white fluorescent tubes (Osram

L75 W/20X) at a light intensity of 45 lmol m-2 s-1 and a

temperature of 25 ± 1�C in a 16 h photoperiod. For details

of the tissue culture protocol see Bairu et al. (2009b).

Experiment one

Jars containing one-month-old normal and necrotic cultures

of H. procumbens were randomly taken from maintenance

cultures.Whole plantlets were removed from the jars and the

agar on the basal section of the plantlets removed. Plantlets

for both normal and necrotic shoots were then cut into three

sections (basal, medium and top). Sections were cut, frozen

in liquid nitrogen and ground to fine powders using a mortar

and pestle (one sample at a time). Ground samples (three

replicates) were transferred to 1.5 ml Eppendorff tubes,

frozen in liquid nitrogen and stored in-70�C until analysis.

Experiment two

To study the effects of CK types on CK profiles of H.

procumbens, plants were cultured on MS medium con-

taining 5 lM of BA, mT or mTR with and without IAA

(2.5 lM). Two sets of controls, one with no PGR and one

with 2.5 lM IAA were included. Medium supplements and

growth conditions were the same as for the maintenance

cultures. One-month-old whole-plant-samples were col-

lected and prepared for CK analysis as above.

Cytokinin purification and analysis

For cytokinin purification, a modified method described by

Faiss et al. (1997) was used. Deuterium-labelled CK internal

standards (Olchemim Ltd, Czech Republic) were added,

1 pmol of each per sample, to check recovery during puri-

fication and to validate the determination (Novak et al.

2008). The samples were purified using a combination of

cation (SCX-cartridge), anion [DEAE-Sephadex-C18-car-

tridge] exchangers and immunoaffinity chromatography

(IAC) based on wide-range specific monoclonal antibodies

against cytokinins (Novak et al. 2003). The eluates from the

IAC columns were evaporated to dryness and dissolved in

20 ll of the mobile phase used for quantitative analysis. The

samples were analysed by ultra performance liquid chro-

matography (UPLC) (Acquity UPLCTM; Waters, Milford,

MA, USA) coupled to a Quatro microTM API (Waters,

Milford, MA, USA) triple quadrupole mass spectrometer

equipped with an electrospray interface. The purified sam-

ples were injected onto a C18 reversed-phase column (BEH

C18; 1.7 lm; 2.1 9 50 mm; Waters). The column was

eluted with a linear gradient (0 min, 10% B; 0–8 min, 50%

B; flow-rate of 0.25 ml/min; column temperature of 40�C)

of 15 mM ammonium formate (pH 4.0, A) and methanol

(B). Quantification was obtained by multiple reaction

monitoring (MRM) of [M ? H]? and the appropriate

product ion. For selective MRM experiments, optimal con-

ditions, dwell time, cone voltage, and collision energy in the

collision cell corresponding to exact diagnostic transition

were optimized for each cytokinin (Novak et al. 2008).

Quantification was performed byMasslynx software using a

standard isotope dilution method. The ratio of endogenous

cytokinin to appropriate labeled standard was determined

and used to quantify the level of endogenous compounds in

the original extract, according to the known quantity of

added internal standard (Novak et al. 2003).

Results and discussion

Results of the CK analysis of the experiments are presented

in Tables 1 and 2 respectively. Tables 3, 4, 5, 6, 7, and 8

are pooled from Tables 1 and 2 respectively to appraise the

profiles of the specific CK groups and their metabolites

(presented as the sum total) in the plant tissue in relation to

STN and CK applications.

To the best of our knowledge, this report is the first on

endogenous CK analysis of H. procumbens. The type and

Plant Growth Regul (2011) 63:105–114 107

123

Table 1 Cytokinins detected (pmol g-1 FW) after 4 weeks of growth in culture

Cytokinins detected Experiment one

Nor. B Nec. B Nor. M Nec. M Nor. T Nec. T

BA 121.32 ± 8.4 150.09 ± 9.2 5.96 ± 0.79 6.55 ± 0.19 1.01 ± 0.04 8.01 ± 0.14

BA9G 886.17 ± 104 1,223.6 ± 53.34 507.16 ± 41.12 544.65 ± 42.14 185.27 ± 13.54 203.26 ± 17.22

BAR 0.75 ± 0.04 1.65 ± 0.05 0.07 ± 0.004 0.13 ± 0.01 0.06 ± 0.008 0.05 ± 0.001

BAR50MP 0.23 ± 0.08 0.22 ± 0.03 0.03 ± 0.006 0.03 ± 0.01 0.05 ± 0.01 0.02 ± 0.007

cZ 2.32 ± 0.1 2.72 ± 0.2 4.29 ± 0.07 5.26 ± 0.04 5.25 ± 0.24 7.13 ± 0.27

cZ9G 26.08 ± 2.09 32.19 ± 0.8 55.54 ± 4.7 51.48 ± 2.53 46.85 ± 3.56 44.67 ± 1.96

cZOG 2.03 ± 0.1 4.05 ± 0.5 3.13 ± 0.08 4.06 ± 0.12 5.87 ± 0.67 4.07 ± 0.48

cZR 6.32 ± 0.3 7.22 ± 0.3 25.6 ± 0.75 26.62 ± 0.15 50.28 ± 3.59 54.9 ± 0.74

cZR50MP 2.01 ± 0.61 2.27 ± 0.58 11 ± 3.18 9.33 ± 2.24 19.66 ± 7.29 14.26 ± 1.53

cZROG 3.57 ± 0.13 5.86 ± 0.72 6.2 ± 0.19 7.23 ± 0.2 9.35 ± 1.23 8.64 ± 0.91

DHZ 0.02 ± 0.002 0.03 ± 0.003 \LOD 0.01 ± 0.001 0.01 ± 0.001 0.05 ± 0.002

DHZ9G 0.28 ± 0.03 0.34 ± 0.04 0.37 ± 0.02 0.36 ± 0.01 0.28 ± 0.01 0.43 ± 0.02

DHZOG 0.12 ± 0.03 \LOD 0.1 ± 0.02 \LOD 0.03 ± 0.009 0.04 ± 0.01

DHZR 0.12 ± 0.01 0.15 ± 0.01 0.17 ± 0.003 0.19 ± 0.005 0.33 ± 0.02 0.45 ± 0.02

DHZR50MP 0.04 ± 0.04 0.06 ± 0.05 0.04 ± 0.01 0.07 ± 0.009 0.13 ± 0.03 0.15 ± 0.04

DHZROG 0.26 ± 0.01 0.28 ± 0.001 0.1 ± 0.006 0.14 ± 0.008 0.17 ± 0.01 0.38 ± 0.02

iP 0.13 ± 0.003 0.13 ± 0.008 0.51 ± 0.007 0.72 ± 0.01 0.32 ± 0.01 0.75 ± 0.03

iP9G 45.36 ± 2.61 49.11 ± 1.96 93.34 ± 0.15 105.95 ± 2.76 38.54 ± 1.33 81.93 ± 2.38

iPR 0.23 ± 0.02 0.15 ± 0.01 3.94 ± 0.09 4.27 ± 0.04 6.15 ± 0.32 7.92 ± 0.15

iPR50MP 0.07 ± 0.02 0.03 ± 0.02 0.41 ± 0.12 0.26 ± 0.07 0.86 ± 0.19 0.58 ± 0.04

mT 0.1 ± 0.004 0.19 ± 0.06 0.33 ± 0.01 0.05 ± 0.008 0.12 ± 0.01 0.09 ± 0.02

mT9G 3.82 ± 1.07 5.09 ± 03 0.6 ± 0.05 0.06 ± 0.02 0.11 ± 0.003 0.18 ± 0.005

mTOG 0.05 ± 0.007 0.07 ± 0.001 0.03 ± 0.009 0.02 ± 0.004 0.02 ± 0.005 0.05 ± 0.007

mTR 0.05 ± 0.002 0.09 ± 0.002 0.03 ± 0.003 0.04 ± 0.004 0.02 ± 0.002 0.05 ± 0.001

mTR50MP \LOD \LOD \LOD \LOD \LOD \LOD

mTROG 0.04 ± 0.007 0.07 ± 0.002 0.03 ± 0.004 0.05 ± 0.006 \LOD 0.02 ± 0.001

oT 0.26 ± 0.01 0.62 ± 0.04 0.51 ± 0.007 0.06 ± 0.01 0.03 ± 0.008 0.06 ± 0.02

oT9G 13.45 ± 1.27 25.67 ± 1.82 1.57 ± 0.1 1.86 ± 0.15 0.2 ± 0.009 0.36 ± 0.02

oTOG 0.03 ± 0.003 0.07 ± 0.02 0.01 ± 0.003 0.01 ± 0.001 \LOD \LOD

oTR 0.17 ± 0.007 0.42 ± 0.03 0.08 ± 0.007 0.04 ± 0.008 0.02 ± 0.002 0.03 ± 0.007

oTR50MP 0.03 ± 0.007 0.04 ± 0.008 0.01 ± 0.008 \LOD \LOD \LOD

oTROG \LOD \LOD \LOD \LOD \LOD \LOD

pT 0.13 ± 0.01 0.2 ± 0.04 0.09 ± 0.005 0.17 ± 0.02 0.05 ± 0.006 0.05 ± 0.006

pTOG 0.7 ± 0.02 1.52 ± 0.28 0.43 ± 0.14 0.63 ± 0.13 0.17 ± 0.02 0.21 ± 0.03

pTR 0.4 ± 0.01 0.72 ± 0.11 0.27 ± 0.03 0.53 ± 0.01 0.15 ± 0.003 0.21 ± 0.02

pTR50MP \LOD \LOD \LOD \LOD \LOD \LOD

pTROG 0.63 ± 0.05 1.1 ± 0.05 0.26 ± 0.03 1.01 ± 0.04 0.14 ± 0.008 0.26 ± 0.006

tZ 0.1 ± 0 0.11 ± 0 0.67 ± 0 1.68 ± 0.1 1.31 ± 0.1 2.95 ± 0

tZ9G 1.93 ± 0.1 0.89 ± 0.05 2.42 ± 0.05 5.41 ± 0.05 2.71 ± 0.15 7.45 ± 0.03

tZOG 1.84 ± 0.04 1.81 ± 0.08 4.91 ± 0.16 7.06 ± 0.13 7.56 ± 0.5 9.4 ± 0.26

tZR 0.36 ± 0.05 0.3 ± 0.06 1.11 ± 0.01 2.29 ± 0.07 2.8 ± 0.3 5.46 ± 0.06

tZR50MP 0.94 ± 0.2 2.5 ± 0.8 2.28 ± 0.7 1.58 ± 0.2 9.16 ± 2.9 1.97 ± 0.69

tZROG 1.27 ± 0.05 1.06 ± 0.03 1.07 ± 0.03 2.44 ± 0.04 1.34 ± 0.12 3.46 ± 0.11

Samples were taken from maintenance cultures grown on media containing 2.5 lM BA. Nor normal shoots, Nec necrotic shoots, B bottom part,

M middle part, T top part of the plant. Results are mean ± SD, n = 3.\LOD indicates values below the detection limit

108 Plant Growth Regul (2011) 63:105–114

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Table 2 Cytokinins detected (pmol g-1 FW) after 4 weeks of growth in culture

Cytokinins detected Experiment two

Control Control ? IAA BA BA ? IAA mT mT ? IAA mTR mTR ? IAA

BA 1.31 ± 0.2 2.46 ± 0.08 61.06 ± 2.7 58.03 ± 3.9 1.55 ± 0.12 0.54 ± 0.1 1.40 ± 0.1 2.52 ± 0.2

BA9G 30.68 ± 0.9 116.7 ± 13.8 5,478.66 ± 209.2 6,948.43 ± 202.6 306.64 ± 23.5 69.12 ± 5.6 167.5 ± 7.08 243.74 ± 20.9

BAR \LOD \LOD 2.73 ± 0.08 3.69 ± 0.03 \LOD \LOD \LOD \LOD

BAR50MP \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

cZ 3.88 ± 0.2 5.27 ± 0.5 2.91 ± 0.02 2.32 ± 0.1 3.13 ± 0.3 2.07 ± 0.05 3.82 ± 0.06 2.33 ± 0.05

cZ9G 33.41 ± 3.8 79.72 ± 14.7 43.72 ± 11.4 24.04 ± 2.9 75.71 ± 13.7 14.58 ± 1.8 56.81 ± 9.9 29.52 ± 4.4

cZOG 2.52 ± 0.6 7.44 ± 1.8 6.87 ± 0.4 6.27 ± 0.5 7.39 ± 0.7 5.22 ± 0.5 11.37 ± 0.6 7.41 ± 1.4

cZR 35.24 ± 0.8 44.54 ± 6.6 40.46 ± 1.3 33.67 ± 1.8 44.4 ± 2.36 32.69 ± 2.08 53.75 ± 2.05 38.45 ± 1.02

cZR50MP 30.14 ± 3.6 42.03 ± 10.6 32.89 ± 9.8 22.87 ± 4.2 24.85 ± 6.4 19.87 ± 5.9 31.88 ± 7.4 22.05 ± 6.4

cZROG 7.61 ± 1.8 12.22 ± 2.7 12.20 ± 1.8 11.45 ± 0.4 13 ± 1.7 9.4 ± 1.2 17.12 ± 2.5 12.76 ± 1.1

DHZ 0.04 ± 0.01 0.035 ± 0.01 \LOD \LOD \LOD \LOD \LOD \LOD

DHZ9G 0.44 ± 0.03 0.95 ± 0.1 0.26 ± 0.08 \LOD 0.79 ± 0.2 \LOD 0.83 ± 0.2 \LOD

DHZOG \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

DHZR 0.04 ± 0.0 0.043 ± 0.01 0.34 ± 0.004 0.027 ± 0.003 0.044 ± 0.01 0.03 ± 0.003 0.07 ± 0.01 0.04 ± 0.01

DHZR50MP \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

DHZROG 0.15 ± 0.04 0.27 ± 0.02 0.13 ± 0.02 0.09 ± 0.004 0.26 ± 0.05 0.07 ± 0.02 0.36 ± 0.08 0.15 ± 0.003

iP 1.09 ± 0.07 0.76 ± 0.05 0.39 ± 0.02 0.30 ± 0.01 0.36 ± 0.04 018 ± 0.005 0.41 ± 0.01 0.31 ± 0.02

iP9G 115.87 ± 1.6 126.45 ± 8 71.44 ± 0.9 53.88 ± 4 79.5 ± 7.8 28.53 ± 1.5 152.18 ± 10.5 47.53 ± 3

iPR 6.66 ± 0.2 7.88 ± 0.9 4.76 ± 0.11 3.97 ± 0.1 3.97 ± 0.05 3.35 ± 0.22 5.6 ± 0.13 5.34 ± 0.2

iPR50MP \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

mT \LOD \LOD \LOD \LOD 81.51 ± 5.6 85.65 ± 3.2 114.27 ± 4.4 53.34 ± 1

mT9G \LOD \LOD \LOD \LOD 744.43 ± 111.4 513.9 ± 9.5 550.5 ± 31.5 242.2 ± 20.6

mTOG \LOD \LOD \LOD \LOD 830.54 ± 213.7 816.93 ± 65.2 1,262.4 ± 227 664.73 ± 185.3

mTR \LOD \LOD \LOD \LOD 25.61 ± 2.3 27.14 ± 2.8 45.16 ± 4.4 25.93 ± 2.3

mTR50MP \LOD \LOD \LOD \LOD 32.63 ± 8 39.73 ± 1.7 71.12 ± 11.6 31.08 ± 2.29

mTROG \LOD \LOD \LOD \LOD 235.48 ± 17.7 206.21 ± 24.4 379.9 ± 17 181.82 ± 19.4

oT \LOD \LOD 0.22 ± 0.06 0.28 ± 0.03 \LOD \LOD \LOD \LOD

oT9G \LOD \LOD 6.64 ± 0.8 9.13 ± 1.6 \LOD \LOD \LOD 2.93 ± 0.8

oTOG \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

oTR \LOD \LOD 0.24 ± 0.05 0.24 ± 0.06 \LOD \LOD \LOD \LOD

oTR50MP \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

oTROG \LOD \LOD \LOD \LOD \LOD \LOD \LOD \LOD

pT \LOD \LOD 0.34 ± 0.04 0.85 ± 0.23 \LOD \LOD \LOD \LOD

pTOG \LOD \LOD 6.09 ± 0.28 19.40 ± 3.95 \LOD \LOD \LOD \LOD

pTR \LOD \LOD 0.69 ± 0.07 0.99 ± 0.04 \LOD \LOD \LOD \LOD

Plan

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109

123

concentration of the CKs detected varied among the

treatments (Tables 1, 2). Samples from the maintenance

cultures (cultured in 2.5 lM BA) yielded smaller total

CK pools compared to samples from the CK treatments

(cultured in 5.0 lM CKs). This indicates that exogenous

application of CK does have an effect on the endogenous

CK pool. The positive effects of applied CK on increasing

the endogenous CK pool has been reported (Auer et al.

1999; Vandemoortele et al. 2001; Ivanova et al. 2006).

This increase in total CK pool was mainly due to

high levels of 9-glucosides and to a lesser extent on

O-glucosides in topolin-treated cultures (Tables 5, 8). Of

the isoprenoid type of CKs detected, cZ types were found

in relatively high levels. Little or no ribotides were

detected except for cZR50MP in both experiments and

mTR50MP in experiment two. The variation in the cyto-

kinin types detected could be due to the presence of more

than one CK biosynthetic pathway (Taylor et al. 2003)

being affected by the various CK applications.

Higher levels of CKs were detected in necrotic tissues

compared to the normal tissues in all three plant parts

analysed. One would presume that this is due to the drier

nature of necrotic shoots since quantification was made as

pmol g-1 FW. This, however, was not the case as indicated

by the basal section of the samples where there was no

difference in moisture content (data not presented) but

significant difference in the CK pool size. Cytokinin

accumulation was significantly higher at the basal section

Table 3 Total cytokinin pool (pm g-1 FW) on the different sections

of normal (Nor.) and necrotic (Nec.) shoots

Treatments Isoprenoid CKs Aromatic CKs Total CK

Nor. B 95.36 1,028.32 1,123.68

Nec. B 111.27 1,411.42 1,522.69

Nor. M 217.2 517.46 734.66

Nec. M 236.43 556.46 792.89

Nor. T 208.97 187.44 396.41

Nec. T 257.04 212.91 469.95

B basal section, M middle section, T top section

Table 4 Total aromatic cytokinin pool (pm g-1 FW) on the different

sections of normal (Nor.) and necrotic (Nec.) shoots

Treatments BA mT oT pT Total

Nor. B 1,008.47 4.05 13.94 1.86 1,028.32

Nec. B 1,375.56 5.5 26.83 3.53 1,411.42

Nor. M 513.21 1.01 2.18 1.05 517.45

Nec. M 551.36 0.79 1.97 2.34 556.46

Nor. T 186.39 0.27 0.25 0.52 187.43

Nec. T 211.33 0.39 0.43 0.73 212.88

B basal section, M middle section, T top section

Table

2continued

Cytokininsdetected

Experim

enttwo

Control

Control?

IAA

BA

BA?

IAA

mT

mT?

IAA

mTR

mTR?

IAA

pTR50MP

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

pTROG

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

tZ1.14±

0.04

1.28±

0.15

0.7±

0.06

0.75±

0.07

0.78±

0.09

0.32±

0.03

0.98±

0.14

0.73±

0.14

tZ9G

5.65±

0.4

8.33±

12.93±

0.8

1.48±

0.5

7.24±

1.8

0.55±

0.1

7.06±

1.8

2.28±

0.5

tZOG

5.33±

1.02

14.65±

4.2

12.32±

0.6

5.22±

0.6

7.1±

0.6

1.26±

0.3

11.16±

0.5

3.84±

0.2

tZR

2.45±

0.1

2.82±

0.5

2.34±

0.1

1.38±

0.1

3.1±

0.1

0.77±

0.02

2.17±

0.2

1.99±

0.1

tZR50MP

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

\LOD

tZROG

4.86±

0.3

4.61±

0.4

2.73±

0.3

1.79±

0.2

5.01±

0.2

1.39±

0.1

6.57±

0.9

2.51±

0.2

Whole

plantsamplesweretaken

from

thevariouscytokinin

(5lM)treatm

entswith(2.5lM)orwithoutIA

Aas

indicated.Thecontrolsampleswerefrom

cytokinin-freemedia.Resultsare

mean±

SD,n=

3.\

LOD

indicates

values

below

thedetectionlimit

110 Plant Growth Regul (2011) 63:105–114

123

followed by the middle and top sections (Table 3). The

presence of a larger CK pool in necrotic plants explains

partly that the problem of STN in H. procumbens is asso-

ciated with distribution rather than the availability of CKs

in the shoots and/or in the medium. Shoot-tip necrosis in

chestnut was controlled by localized application of CK to

the shoot-tip (Vieitez et al. 1989; Piagnani et al. 1996)

suggesting CK transport from the site of synthesis or

storage to the meristematic tissues in necrotic plants as the

limiting factor. Measures improving the uptake and trans-

port of CK may therefore help alleviate the problem. In

necrotic grape cultures for instance, axillary branching and

vigorous rooting rendered STN relatively harmless (Tho-

mas 2000). We have also observed (Bairu et al. 2009b)

recovery from necrotic symptoms in H. procumbens

cultures in the presence of roots. These reports suggest that

the development of new shoots and roots might have

triggered some physiological processes in plants lacking

roots and shoot-tips due to STN thereby reinstating the

physiological cross talk including CK transport, metabo-

lism and signaling between the different plant parts.

Positive correlation between an higher CK pool and

hyperhydricity has been reported (Kataeva et al. 1991; I-

vanova et al. 2006). These authors attributed the effect to

either overproduction of CK and/or their accumulation

resulting from an inability to use or metabolize them due to

failed physiological processes such as CK signaling. The

presence of elevated levels of 9-glucosides in this study

shows the latter to be a more likely explanation.

The relatively high level of aromatic CKs detected

(Tables 3, 6) could be the result of the exogenous appli-

cation of CKs. The presence of the isoprenoid type of CKs

(Tables 3, 6) in the samples indicates the presence of de

novo CK synthesis by the plantlets. This was more evident

in samples derived from media without exogenous CKs

(Table 6) where the isoprenoid type of CKs was higher

than the aromatic types. In the absence of roots, the pres-

ence of isoprenoid CKs suggests CK synthesis in shoots.

Compared to the isoprenoid CKs, the amounts of non-BA

aromatic CKs detected in the samples were very low

(Tables 4, 7) which indicates a very low level of endoge-

nous synthesis of topolins.

Further observation of the CKs detected based on the

various structural and functional groups revealed that

necrotic samples consistently yielded more 9-glucosides

compared to their normal counterparts in the three sections

of the plants analysed (Table 5). Abnormalities in BA-

treated tissue cultures have often been associated with a

relatively high level of BA9G (Werbrouck et al. 1995;

Bairu et al. 2007). This metabolite, the result of glucosy-

lation of the adenine ring at the N9-position, is neither

active nor readily convertible to other usable forms of CKs

(Sakakibara 2006). Similar to Werbrouck et al. (1995) we

detected an extremely high level of this metabolite in the

basal portion of the plant. The amounts detected in the

middle and upper sections of the plant were also consis-

tently higher than the other CK forms viz free bases,

ribosides, O-glucosides and ribotides (Table 5). Werbrouck

Table 5 Total cytokinin pool

(pm g-1 FW) based on

structural and functional forms

on the different sections of

normal (Nor.) and necrotic

(Nec.) shoots

B basal section, M middle

section, T top section

Treatments Free base Ribosides O-Glucosides 9-Glucosides Ribotides Total

Nor. B 124.38 8.4 10.54 977.09 3.32 1,123.73

Nec. B 154.09 10.7 15.89 1,336.89 5.12 1,522.69

Nor. M 12.36 31.27 16.27 661 11.77 732.67

Nec. M 14.5 34.11 22.65 710.34 11.01 792.61

Nor. T 8.1 59.81 24.97 273.96 29.86 396.7

Nec. T 19.09 69.07 26.53 338.28 16.98 469.95

Table 6 Whole-plant total cytokinin pool (pm g-1 FW) detected

from the various treatments

Treatments Isoprenoid CKs Aromatic CKs Total

Control 256.52 31.99 288.51

Control ? IAA 359.3 119.16 478.46

BA 237.39 5,556.72 5,794.11

BA ? IAA 169.92 7,041.15 7,211.07

mT 276.65 2,258.2 2,534.85

mT ? IAA 129.1 1,758.66 1,887.76

mTR 362.2 2,591.9 2,954.1

mTR ? IAA 177.24 1,448.29 1,625.53

Table 7 Whole-plant aromatic cytokinin pool (pm g-1 FW) detected

from the various treatments

Treatments BA mT oT pT Total

Control 31.99 \LOD \LOD \LOD 31.99

Control ? IAA 119.16 \LOD \LOD \LOD 119.16

BA 5,542.5 \LOD 7.1 7.12 5,556.72

BA ? IAA 7,010 \LOD 9.65 21.2 7,040.85

mT 308.2 1,950 \LOD \LOD 2,258.2

mT ? IAA 69.66 1,689 \LOD \LOD 1,758.66

mTR 168.94 2,423 \LOD \LOD 2,591.94

mTR ? IAA 246.26 1,199.1 2.93 \LOD 1,448.29

\LOD indicates values below the detection limit

Plant Growth Regul (2011) 63:105–114 111

123

et al. (1995, 1996) highlighted that the excessive presence

of this metabolite coupled with its pronounced stability in

plant tissue causes various disorders during acclimatization

and rooting. The same could be said for STN in H. proc-

umbens cultures with slightly different physiological pro-

cesses. We assume that when plants were cultured in BA-

containing media, the majority of the BA undergoes N-

glucosylation as part of detoxification/inactivation efforts.

With time, when the plantlets need CK to elicit various

physiological processes such as cell division, in the

absence of O-glucosides, the available active forms (free

bases and ribosides) may not be sufficient leading to ces-

sation of cell division and necrosis of meristematic regions,

resulting in STN.

In a previous report (Bairu et al. 2009b) it was noted that

plants treated with mTR showed reduced incidence of STN.

To test one of our hypotheses, we analysed plants treated

with various types of CKs to assess the various metabolites

formed. Plants treated with topolins (mT and mTR) con-

sistently yielded higher amounts of both the active forms

(free bases and ribosides) and the storage forms (O-glu-

cosides) compared to their BA-treated counterparts

(Table 8). This would ensure sufficient active CK when

needed. This result explains why the topolins consistently

out-performed BA in our tissue culture experiments (Bairu

et al. 2007, 2008, 2009b, c). Furthermore, high amounts of

BA9G still persisted in topolin-cultivated explants even

after 1 month of cultivation on BA-free media (Table 2).

On the contrary, these explants contained only very low

levels of BA itself and no BAR and/or BAR50MP, high-

lighting the stability and/or extended effect of BA9G in

plant culture systems. This scenario is in agreement with a

previous report (Werbrouck et al. 1996). To ascertain this

phenomenon experiments are in progress on cultures with

no previous exposure to BA.

It has been 52 years since Skoog and Miller (1957)

demonstrated the significance of auxin:cytokinin ratios in

organogenesis. Since then various experiments were done

on the interaction of these two hormones. Coenen and

Lomax (1997) concluded that these interactions could be

synergistic, antagonistic and/or additive. In this study the

addition of IAA enhanced the formation of 9-glucosides in

necrotic and BA-treated cultures but reduced it in topolin-

treated cultures. Better O-glucosylation was also observed

in topolin-treated cultures when IAA was omitted

(Table 8). The presence of an –OH group gives the topolins

a structural advantage over BA since this allows them to

form O-glucosides which can not occur with BA. The

generally low levels of the free bases and ribosides can be

attributed to them being actively utilized in the regulation

of the cell cycle and various developmental processes

(Schmulling 2004).

The addition of IAA also showed a marked differential

effect on the total cytokinin pool; IAA-treated samples

showed an increase in total cytokinin in BA-treated and the

control samples but had an opposite effect on topolin-

treated samples (Table 8). Furthermore, there was a sig-

nificant decrease of the isoprenoid cytokinin pool in all

IAA-treated samples (grown on BA as well as on topolins)

in comparison with non IAA-treated samples (Tables 2, 6).

Auxin-mediated negative control of the CK pool size, by

suppressing isopentenyladenosine-50-monophosphate-inde-

pendent biosynthetic pathways, was reported (Nordstrom

et al. 2004). These authors also indicated the lateral roots to

be the likely sites of isopentenyladenosine-50-monophos-

phate-dependent CK synthesis. The ability of auxin to

impose regulation of CK biosynthesis and the ability of

these hormones to interact at metabolic level (Nordstrom

et al. 2004) could also have attributed to the change in total

CK pools between the treatments. In view of this report the

increase in total cytokinin by the IAA-control plants

compared to the controls without IAA, could likely be

attributed to root development. Likewise a reduction in

total CK by the topolin-treated samples when IAA was

included could be due to this auxin-mediated negative

control on the CK pool size.

What caused an increase in total CK when IAA was

included in BA-treated samples is subject to speculation. In

our previous experiments on the same plant (Bairu et al.

2009b) an increase in basal-callus-like tissue (Fig. 1) was

Table 8 Whole-plant total cytokinin pool (p mg-1 FW) based on structural and functional forms detected from the various treatments

Treatments Free bases Ribosides O-Glucosides 9-Glucosides Ribotides Total

Control 7.46 44.39 20.47 186.05 30.14 288.51

Control ? IAA 9.81 55.28 39.19 332.15 42.03 478.46

BA 65.62 51.32 40.34 5,603.69 32.89 5,793.86

BA ? IAA 62.5 44 44.25 7,036.51 22.9 7,210.16

mT 87.33 77.12 1,099.02 1,214.24 57.48 2,535.19

mT ? IAA 106.58 60.63 1,040.48 626.68 59.6 1,893.97

mTR 120.91 106.75 1,688.48 934.9 103 2,954.04

mTR ? IAA 59.23 71.75 873.22 568.2 53.13 1,625.53

112 Plant Growth Regul (2011) 63:105–114

123

observed when IAA was added to the multiplication

medium. This phenomenon and STN were also more

pronounced in BA-treated cultures in general and in

BA-treated cultures containing IAA in particular. Based on

growth parameters, we suspected that this callus-like tis-

sue, by virtue of being a sink to essential growth require-

ments and/or an obstruction to root development might

have caused physiological disorders. It was these obser-

vations that led us to the current study and it is on these

grounds that we speculate that the physiological effect of

IAA on BA-treated plants might have been altered due to

other factors which are not known at the moment.

Alternatively, it is possible that the following sequence

of events could have occurred; the inability of BA to form

O-glucosides coupled with the excessive deactivation of

the active forms to BA9G caused a CK deficiency in the

growing tissue unlike in the topolin-treated plants. This CK

deficiency could have caused up-regulation of CK syn-

thesis. But, since the presence of IAA aggravates the for-

mation of basal callus-like tissue (a sink), the total CK

(both the applied and synthesized) might have been trapped

in this callus-like tissue making it unavailable to the plant.

A possible repeat of these events could be the cause for the

larger CK pool in BA-treated samples from IAA containing

medium. This was more apparent from the relatively large

quantity of BA9G in BA-IAA-treated samples compared to

BA-treated samples (Table 2). However, increased CK

biosynthesis is a less likely scenario since there was a

consistent decrease in the isoprenoid CK pool size in the

presence of IAA (Table 6). Instead, an increase in the CK

pool of exogenous CKs and their direct metabolites in

IAA-treated plants (Table 7) may suggest that the high CK

pool size in BA-IAA-treated plants is the result of the sink

effect of the basal callus-like tissue.

Conclusions

Exogenously applied CKs affected STN by altering the

endogenous CK pool. The change in endogenous CK pool

affects physiological processes related to CK metabolism,

signaling and transport. In H. procumbens the problem is

aggravated by the formation by the plant of basal callus-

like tissue which acts as a sink. This was evident by the

over-accumulation of CKs in these tissues. The type of CK

applied had an effect on the total CK pool. The presence of

an hydroxyl group in topolins gives them a structural

advantage over BA. This was reflected by the presence of a

generous amount of O-glucosides in topolin-treated sam-

ples and hence little or no CK shortage. The contrasting

effects observed when IAA was used shows the complexity

of auxin-cytokinin interaction. Molecular characterization

and explanation of these interactions are largely based on

Arabidopsis mutants. The application of such findings on

plants with a more complex genome and physiology are a

subject for debate. In the absence of roots, presence of a

sink and poorly developed anatomical structures of in vitro

plants, STN in H. procumbens may be attributed to a poor

CK transport system in topolin-treated cultures and both

CK metabolism and transport in BA-treated cultures. The

presence of oT and pT and their derivatives on BA treated

cultures needs further investigation. Moreover, the role of

mineral elements such as calcium and boron which play an

important role in auxin and CK transport and signaling

should not be overlooked.

Acknowledgments The financial support of the National Research

Foundation-South Africa, the Grant Agency of the Czech Republic

(GA 206/07/0570) and Czech Ministry of Education (MSM

6198959216, 1M06030). We also wish to thank Hana Martınkova for

technical assistance.

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