The Journal of Horticultural Science and Biotechnology

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Influence of endogenous IAA levels and exogenous

IBA on rooting and quality of leafy cuttings of
Prunus ‘GiSelA 5’

M. Ătefančič, F. Ătampar & G. Osterc

To cite this article: M. Ătefančič, F. Ătampar & G. Osterc (2006) Influence of endogenous
IAA levels and exogenous IBA on rooting and quality of leafy cuttings of Prunus
‘GiSelA 5’, The Journal of Horticultural Science and Biotechnology, 81:3, 508-512, DOI:
10.1080/14620316.2006.11512095

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 Journal of Horticultural Science & Biotechnology (2006) 81 (3) 508–512

Influence of endogenous IAA levels and exogenous IBA on rooting

and quality of leafy cuttings of Prunus ‘GiSelA 5’

By M. S̆TEFANC̆IC̆*, F. S̆TAMPAR and G. OSTERC

University of Ljubljana, Biotechnical Faculty, Jamnikarjeva 101, 1000 Ljubljana, Slovenia
(e-mail: mateja.trobec@bf.uni-lj.si) (Accepted 20 December 2005)

SUMMARY
The influence of exogenously applied indole-3-butyric acid (IBA) on root and shoot development of leafy cuttings was
analysed in Prunus cerasus
P. canescens ‘GiSelA 5’, a dwarfing cherry rootstock, in two successive years. Compared
to control cuttings, IBA application (4 g l–1 in 2003; 2.5 g l–1 in 2004) caused higher indole-3-acetic acid (IAA)
accumulation in the cutting bases, but that did not influence the percentage of rooted cuttings, nor their survival in
either year. However, IBA inhibited callus formation and, consequently, influenced the quality of the developed
The Journal of Horticultural Science and Biotechnology 2006.81:508-512.

cuttings. Callus formation impeded root development, reducing the number of main roots, and inhibited the growth of
the cuttings, reducing the average total length of shoots formed by individual cuttings. Callus formation was most
reduced in the cuttings in the second experimental year, with high initial IAA concentrations.

R ooting responses of cuttings are determined by

interactions between endogenous and
environmental factors (Davis et al., 1988). The success of
propagation is affected by the plant material (its
physiological age and nutrition), by the propagation
system and conditions (water temperature, propagation
substrate) and by seasonal timing and annual ecological
conditions (Hübl et al., 1984; Hartmann et al., 1997).
Among endogenous factors, the greatest impact on root
formation is ascribed to auxins (Davis et al., 1988;
Hartmann et al., 1997; Spethmann, 2000).
Auxins induce division and elongation of meristematic
cells and influence the differentiation of root primordia
(Blakesley et al., 1991; Davis et al., 1988; Hartmann et al.,
1997). They are produced mainly in active growing
organs, such as shoot apices and young leaves. The
percentage of rooted cuttings is positively correlated
with the concentration of applied auxin, but only to a
certain value; at high concentrations of applied auxin
rooting ceases or even decreases (Rosier et al., 2004;
Gulen et al., 2004). Exogenous auxin treatment is
especially important in difficult-to-root plant species that
suffer heavy losses due to the poor quality of their root
systems (or shoots) as well as poor or slow rooting
(De Klerk, 1999). There is no direct evidence that
synthetic auxins could substitute for natural auxins
found in cells (Davis et al., 1988), but they can add to the
endogenous indole-3-acetic acid(IAA)-pool in a plant
(Bartel et al., 2001); and, if IAA deficiency or slow
basipetal auxin transportation is the reason for poor
rooting, thereby promote adventitious root formation
(Spethmann and Hamzah, 1988; De Klerk et al., 1999;
Ludwig-Müller, 2000).
IAA is generally regarded as the major auxin and is
found universally in higher plants. Although it plays a
central role in adventitious rooting (Davis et al., 1988;
*Author for correspondence.
De Klerk et al., 1999), indole-3-butyric acid (IBA) has
proved more efficient in promoting adventitious root
formation by exogenous application (De Klerk et al.,
1999; Ludwig-Müller, 2000; Spethmann and Hamzah,
1988). Biochemical studies on numerous plants, and
genetic studies of IBA-mutants of Arabidopsis indicate
that IBA stimulates adventitious root formation
through conversion to IAA, which occurs by a
mechanism similar to peroxisomal fatty acid -
oxidation (Nordström et al., 1991). On the other hand,
some evidence suggests that IBA can act as an auxin
itself or modulate the activity of IAA (Ludwig-Müller,
2000; Nordström et al., 1991).
Exogenously applied auxins are not always essential
for root formation, but they can cause earlier root
emergence and increase the number of adventitious
roots (Hartmann et al., 1997; Ludwig-Müller, 2000;
Spethmann, 2000). Callus formation is also reduced by
growth hormone treatment (Spethmann and Hamzah,
1988; Spethmann, 2000). These parameters determine the
quality of root systems, which is a decisive factor for the
further development of cuttings.
This work investigates the rooting of ‘GiSelA 5’, a
commercially important dwarfing cherry rootstock
clone. Prunus species are generally classified as difficult-
to-root plant species (Spethmann, 2000). Therefore,
applied auxin is thought to be necessary for a better
rooting capacity and cuttings should be prepared during
the optimal propagating season. The optimal timing for
Prunus species is propagation by softwood cuttings (just
before the beginning of wood ripening) (Hartmann et al.,
1997; Spethmann, 2000). In our experiments, the
influence of IBA on rooting and development of ‘GiSelA
5’ leafy cuttings was investigated in two successive years.
Besides obtaining rooting results, endogenous basal IAA
level measurements were performed in the first 5 d after
sticking of cuttings to evaluate the influence of IBA on
the endogenous pool of the active auxin.
 M. S̆TEFANC̆IC̆, F. S̆TAMPAR and G. OSTERC
MATERIALS AND METHODS
Experimenal design
Leafy cuttings (15 cm) were taken from stock plants of
P. cerasus
P. canescens ‘GiSelA 5’ on 16 June in 2003
and 2004. The stock plants were 5–6 years-old, yearly-
hedged plants. For these experiments, terminal cuttings
with apical meristems were used. Half of the cuttings
were dipped for approx. 5 s in IBA solution [4.0 g l–1 (19.7
mM) in 2003, and 2.5 g l–1 (12.3 mM) in 2004], while half
were stuck directly into a wet peat:sand (3:1) substrate
with slow release fertiliser (Osmocote® Exact
16+11+11+3Mg+Te; Scotts International, Heerlen, The
Netherlands). All treatments were replicated three-times
with 40 cuttings per plot. In total, 240 cuttings were used
in each experiment, each year. Six extra cuttings for
auxin analysis were also used per plot for each day of
data collection. The experiment was carried out in an
unheated glasshouse under a fog system (Plantfog-
Befeuchtungsanlagen Nebelsysteme, Fischamend,
The Journal of Horticultural Science and Biotechnology 2006.81:508-512.
Austria). Fogging was regulated manually to obtain on
average of 90–95% relative humidity. Fogging intervals
lasted approx. 30 s, followed by a 60 s pause. Fogging was
not carried out during the night (19.00 – 07.00h). The
pause intervals were extended in late-August and
fogging was stopped completely in late-September.
At the end of the experiments, the rooting rate (%),
callus formation (%), number of main roots and shoot
growth [total length of shoots grown after cutting
establishment (cm)] were measured.
Extraction and analysis of indole-3-acetic acid
Cuttings were collected immediately and 1, 2, 3, 4 and
5 d after excision. The basal 3 cm-portions of cuttings
were stored at –80°C until analysis. Other parts of the
cuttings were rejected. Each sample contained the bases
of six cuttings in order to obtain enough material for
auxin extraction.
Auxin extraction was performed using a slightly
modified method of Kovac̆ et al. (2003). Samples were
ground to a powder in a mortar and pestle. Each sample
was divided into three portions, each of 0.15 g and
extracted separately with 5 ml BHT-MeOH solution
[0.5 g l–1 BHT (2,6-di-tert-butyl-4-methylphenol) in
methanol] in 2003; or with 1 ml BHT-MeOH solution
and 4 ml 5 mM potassium phosphate buffer pH 6.5, in
2004. In one of three repetitions, IAA was added. After
1 h at 4°C, the extract was filtered and 3 ml 5 mM
potassium phosphate buffer, pH 6.5 was added to each
sample. To purify auxins, Strata C18-E columns (pore
size 55 µm, retention capacity 500 mg, tube size 6 ml;
Phenomenex, Torrance, CA, USA) were used. Complete
sample extracts were first run through the column pre-
conditioned with potassium phosphate buffer, pH 6.5
and washed with 4 ml 5 mM potassium phosphate buffer,
pH 6.5. The eluate was acidified to pH 2.5 with 20 mM
HCl (in 2003) or with 0.5 M H3PO4 (in 2004), then
applied to a second column pre-conditioned with
potassium phosphate buffer, pH 2.5. The column was
rinsed with 2 ml double-distilled water and eluted with 2
ml 80% (v/v) methanol.
The concentrated eluate (1 ml) was separated by TSP
(Thermo Separation Products) HPLC (Spectra Physics,
San Jose, CA, USA), using a Chromsep column [SS 250

4.6 mm, Hypersil 5 ODS (Octa Decyl Silica; Varian,
509
Palo Alto, CA, USA)] and analysed by fluorescence
(Spectrasystem FL2000; Spectra Physics) and a UV-VIS
detector (K-2500; Knauer, Berlin, Germany). The
mobile phase consisted of solvent A (1% acetic acid)
and solvent B (methanol). The gradient was linear from
10–55% of solvent B over 40 min, and the flow rate was
1 ml min–1. Excitation of the fluorescence detector was
set at 254 nm and emission at 360 nm. The UV-VIS
detector was adjusted to measure absorption at 280 nm.
IAA was quantified by fluorimetry, comparing peak
areas with those of known amounts of IAA. Losses
were evaluated by standards that had gone through the
extraction and purification processes. Calculated auxin
contents were the means of two replicate
measurements.
Statistical analyses
Statistical analyses were carried out with the
programme Statgraphics Plus (version 4.0), using
analysis of variance (ANOVA). Statistically significant
differences between treatments were tested with the
LSD test at the 0.95 confidence level.
RESULTS AND DISCUSSION
As seen in Table I, there was no difference in rooting
response between cuttings in the two experimental years.
The percentage of rooted cuttings ranged from 80–87%,
regardless of treatment. Rooting values were better than
in previous studies on leafy cuttings of ‘GiSelA 5’ in 2002
(Trobec et al., 2005) and were comparable to results
reported by Osterc and Spethmann (1998) for the same
rootstock. Auxin treatment did not enhance the
frequency of rooted cuttings. Neither the higher IBA
concentration in 2003, nor the lower concentration of
IBA in 2004, increased the percentage of rooting
compared to control treatments. These results are
contrary to the view that application of auxins is essential
for better rooting, especially in difficult-to-root plant
species. Gulen et al. (2004) obtained the highest rooting
percentage for ‘GiSelA 5’ leafy cuttings with 5 mM IBA
treatment. According to their results, our IBA solutions
were rather high for maximum rooting success.
Compared to the controls in both years, IBA
treatment enlarged the endogenous pool of IAA in
cuttings (Figure 1). This indicates that IBA was taken up
successfully by ‘GiSelA 5’ cuttings and that some of the
absorbed plant growth regulator was metabolised into
TABLE I
Development of root systems and bare callus formation (%) in
IBA-treated and untreated (control) Prunus ‘GiSelA 5’ leafy cuttings at
the end of the growing season.
Treatment
Year Development of roots/callus Control IBA
2003 Roots 8 ± 1† * 44 ± 4
Roots+callus 72 ± 7 * 38 ± 3
Callus 18 ± 8 NS 10 ± 2
2004 Roots 64 ± 6 NS 81 ± 8
Roots+callus 23 ± 3 * 3±1
Callus 0±1 NS 1±1
2003/2004 Roots * *
Roots+callus * *
Callus NS *
*, Statistically significant differences (P = 0.05; LSD test) between
treatments and between experimental years; NS, not significant.
†
Means (± SE) are shown for individual treatments and years.
 510 Rooting of ‘GiSelA 5’ leafy cuttings

250 250

A B
control control
200 IBA (4 g l¯¹)
200 IBA (2.5 g l¯¹)

IAA (ng g¯¹ FW)

IAA (ng g¯¹ FW)

150 150

100 100

50 50

0 0
0 1 2 3 4 5 0 1 2 3 4 5

Days after excision Days after excision

FIG. 1
IAA contents in the basal parts of (4 g l–1 or 2.5 g l–1) IBA-treated or untreated (control) Prunus ‘GiSelA 5’ leafy cuttings in 2003 (Panel A) and 2004
(Panel B). Bars represent ± SE (n = 3).
The Journal of Horticultural Science and Biotechnology 2006.81:508-512.

the active auxin (IAA). Enlargement of the basal IAA
pool was most noticeable on day-1 when accumulation
was observed in all treatments. In control treatments, this
was a consequence of the interrupted polar flow of IAA
by cutting, while in IBA-treated cuttings IAA pool
enlargement occured for the same reason and, in
addition, because of conversion of absorbed IBA into
free IAA (Bartel et al., 2001; Nördstrom et al., 1991). The
period of increasing IAA coincided with the induction
phase of the rooting process, whereas during the root
initiation phase, IAA levels decreased (Nag et al., 2001).
Levels of free IAA in the root formation zone at the
beginning of the initiation phase were higher in IBA-
treated cuttings (147.8 ng g–1 FW in 2003; 182.8 ng g–1 FW
in 2004) than in the controls (30.9 ng g–1 FW in 2003;
131.3 ng g–1 FW in 2004).
An important difference in rooting success was noticed
when the manner of root system development was
observed. Exogenous IBA application lowered the
percentage of cuttings with callus formation in both
experimental years (Table I). Similar results were
obtained by Spethmann and Hamzah (1988). But more
than IBA application, it was the high initial concentration
of IAA that was unfavourable for callus development.
The endogenous concentration of free IAA in the basal
parts of leafy cuttings of ‘GiSelA 5’ on 16 June 2004 was
higher than on the same date in 2003 (Figure 1). The
concentration of endogenous IAA was lower in 2003
(3.0 ng g–1 FW) than in the majority of other experiments
[e.g., Ford et al. (2002) for lilac cuttings; Nördstrom et al.
(1991) for pea cuttings; and Auderset et al. (1994) for
apple rootstock cuttings]. The low IAA concentrations in
stock plants caused a high percentage callus formation in
the first experimental year (90% of control, and 48% of
IBA-treated cuttings formed calli in 2003) compared to
the low callus production with 14-times higher IAA levels
in 2004 (when 23% of control and 4% of IBA-treated
cuttings formed calli). The importance of endogenous
auxin levels at the time when cuttings are prepared has
been reported by other authors (Blakesley et al., 1991;
Davis et al., 1988).
Callus development is common in wound healing,
where it appears as a thin layer around the wounded-
ends of cuttings. The development of a thick callus
usually indicates irregularities in root formation
(Spethmann, 2000). Hübl et al. (1984) investigated root
formation in Prunus species and established that root
initiation does not have its origin in the callus tissue.
Spethmann and Hamzah (1988) also stated that callus is
not the point of origin for roots, rather it stops or delays
root formation. As can be seen in Figure 2 and Figure 3,
‘GiSelA 5’ cuttings which formed roots in the presence
of callus, were of lower quality. They developed
statistically fewer roots per rooted cutting, and average
shoot growth was weaker than in cuttings that did not
form callus.

70
14
60
Shoot growth (cm)

12
Number of main roots

50
10
40 roots-callus
8 roots-callus
roots+callus
roots+callus 30
6
20
4
10
2
0
0
2003 2004
2003 2004
FIG. 3
FIG. 2 Effect of callus formation on average shoot growth (= average total
Influence of callus formation on the number of main roots in all rooted length of all shoots formed on an individual cutting) for all rooted leafy
leafy cuttings of Prunus ‘GiSelA 5’ at the end of the growing season in cuttings of Prunus ‘GiSelA 5’ at the end of the growing season in 2003
2003 and 2004. Bars represent ± SE (n = 3). and 2004. Bars represent ± SE (n = 3).
 M. S̆TEFANC̆IC̆, F. S̆TAMPAR and G. OSTERC
TABLE II
Percentage survival or decay of rooted Prunus ‘GiSelA 5’ leafy cuttings
treated with IBA or left untreated (control) at the end of the growing
season.
Treatment
Year Survival Control IBA
2003 Living rooted cuttings (%) 63 ± 7 NS 59 ± 6
Failed rooted cuttings (%) 17 ± 0 NS 23 ± 8
2004 Living rooted cuttings (%) 45 ± 5 NS 56 ± 4
Failed rooted cuttings (%) 42 ± 3 * 28 ± 2
2003/2004 Living rooted cuttings (%) NS NS
Failed rooted cuttings (%) * NS
*, Statistically significant differences (P = 0.05; LSD test) between
treatments and between experimental years; NS, not significant.
Means (± SE) are shown for individual treatments and years.
According to previous findings (Hartmann et al., 1997;
Ludwig-Müller, 2000; Spethmann, 2000), IBA-treated
cuttings developed higher average numbers of
adventitious roots (6.5 in 2003; 13.1 in 2004) compared to
controls (3.3 in 2003; 7.4 in 2004). The mean number of
The Journal of Horticultural Science and Biotechnology 2006.81:508-512.
roots was negatively correlated with the percentage of
callus formation, which indicates that callus impeded
root formation.
Callus formation inhibited shoot development, as seen
in Figure 3, through impeding root development. The
number of adventitious roots induced per cutting had a
visible influence on subsequent shoot growth. Shoot
growth from cuttings at the end of the season was better
following IBA-treatment. On average, shoots grew 29 cm
in 2003 and 26 cm in 2004, while control cuttings grew on
average by 17.9 cm in 2003 and 16.4 cm in 2004.
Parameters which define better cutting quality (e.g.,
higher numbers of roots for a more compact root system,
and more intensive growth of cuttings for earlier
grafting) thus depend on callus formation. The
development of calli was decreased by application of
IBA and by higher endogenous basal levels of free IAA
at the time of preparing the cuttings.
However, cutting quality had no effect on its survival
at the end of the propagating season (Table II). Survival
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