Directory UMM :Data Elmu:jurnal:S:Scientia Horticulturae:Vol86.Issue1.Sept2000:
Scientia Horticulturae 86 (2000) 23±32
Stem segments of apple microcuttings take up
auxin predominantly via the cut surface
and not via the epidermal surface
Huiyuan Guana,b, Geert-Jan De Klerka,*
a
Centre for Plant Tissue Culture Research, PO Box 85, 2160 AB Lisse, Netherlands
b
Woody Plants Research Station of Hebei Province, No. A99, Canganxi Road,
Shijiazhuang, Hebei 050081, PR China
Accepted 10 January 2000
Abstract
In conventional cuttings, auxin applied to achieve rooting is taken up predominantly via the cut
surface and not via the epidermal surface of the stem. Even though in tissue-cultured plants the
cuticle is poorly developed and the stomata do not function properly, stem segments of apple
microcuttings took up labelled indoleacetic acid also predominantly via the cut surface. Stem
segments with an epidermis with gaps caused by excision of the petioles required a lower
exogenous concentration of auxin to achieve rooting than segments with an intact epidermis,
indicating that the gaps facilitated uptake of auxin from the medium. This was con®rmed in an
experiment on uptake of labelled indoleacetic acid. # 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Adventitious root formation; Auxin uptake; Cuttings; Epidermis; Malus; Micropropagation
1. Introduction
Conventional cuttings and microcuttings are rooted by application of auxin. In
conventional cuttings, auxin is taken up predominantly via the cut surface.
Uptake via the epidermal surface is negligible (Kenney et al., 1969). Because in
Abbreviations: IAA, Indoleacetic acid; NAA, Naphthaleneacetic acid
Corresponding author. Tel.: 31-252-462130; fax: 31-252-417762.
E-mail address: geert-jan.de.klerk@lbo.agro.nl (G.-J. De Klerk)
*
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 3 2 - 1
24
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
microcuttings cuticular waxes are reduced (Johansson et al., 1992) and stomatal
functioning is disturbed (Santamaria and Kerstiens, 1994), their epidermis may be
more permeable. Previously, we reported that in apple microcuttings auxins are
taken up very rapidly from the medium (Guan et al., 1997). In this paper, we
examine how much uptake occurs either via the epidermal or via the cut surface.
2. Materials and methods
2.1. Plant material
Shoot production of Malus `Jork 9' was maintained as described previously
(De Klerk et al., 1995). Shoots 1 cm in length were subcultured in tubes with
15 ml of a modi®ed Murashige±Skoog medium (Murashige and Skoog, 1962)
with 4.4 mM benzylaminopurine and 0.5 mM indolebutyric acid. After ®ve weeks
of proliferation at 258C and a 16 h photoperiod (35 mmol mÿ2 sÿ1 provided by
cool-white, ¯uorescent lamps), clusters consisting of 5±10 shoots had been
formed by axillary branching. Shoots were excised and either used for further
shoot proliferation or for experiments.
2.2. Preparation of segments
From defoliated stems, segments of 1, 2, 3, 4 or 6 mm were cut with a special
device consisting of razor blades separated by the appropriate number of 1 mm
metal plates. It was carefully noted whether the epidermis of a segment had gaps
because of excision of the petioles. Adjacent segments from a shoot were
distributed over several treatments because it has been previously found that there
exists a weak correlation between the capability to root of adjacent segments (De
Klerk and Caillat, 1994). There is no correlation between the capability to root of
nonadjacent segments. Furthermore, in the middle part (1 cm) of stems there is no
gradient with respect to the capability to root (De Klerk and Caillat, 1994).
2.3. Uptake and oxidation of IAA
To determine uptake of IAA, we added tracer amounts of 1-14C-IAA. When
IAA is oxidized chemically or enzymatically, the carboxyl group is removed
(Bandurski et al., 1995). Thus, from 1-14C-IAA, 14CO2 is released. Apple stem
slices massively oxidize IAA (Guan et al., 1997). This oxidation probably occurs
at the surface of the explants and not within them.
The experiments were performed in 2 cm high 9 cm Petri dishes with 20 ml
solidi®ed rooting medium (De Klerk et al., 1995), or in 100 ml Erlenmeyer ¯asks
with 10 ml liquid rooting medium (the same composition but without agar) on a
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
25
rotary shaker (40 rpm). The speci®c conditions of each incubation are indicated
with the tables and ®gures. 1-14C-IAA (Sigma, 1.48106 kBq mmolÿ1) was
added as indicated.
The amounts of label in the explants, in the headspace (14CO2 formed by
oxidation of 1-14C-IAA), and in the medium were measured. To determine label
in the explants, samples of three segments were taken at random and digested
overnight at 408C in 200 ml Soluene (Packard). Then 4.5 ml Hionic ¯uor
(Packard) was added. 14CO2 was trapped in 300 ml 1 M KOH in a small vial with
a piece of ®lter paper. To determine the amount of radioactivity, 4.5 ml Aqua gold
(Packard) was added to the ®lter paper and the 300 ml KOH. Radioactivity in
solid medium was determined by adding 4.5 ml Aqua gold to an excised piece of
solidi®ed medium (ca. 100 mg). In this case, radioactivity was counted after 48 h.
Radioactivity in liquid medium was determined in a sample of 100 ml to which
4.5 ml Aqua gold had been added.
2.4. Rooting conditions
The segments were cultured with their apical cut surface down (Guan and De
Klerk, 2000) on 30 ml of solidi®ed rooting medium in a 2 cm high 9 cm Petri
dish. The Petri dish was incubated in a culture room at 258C. After ®ve days, the
segments were transferred to hormone-free medium and to the light (16 h
photoperiod; 35 mmol mÿ2 sÿ1). Roots were counted after 21 days under a
dissecting microscope.
2.5. Statistics
For each treatment, two Petri dishes with 30 segments each were used, or two
Erlenmeyer ¯asks with 10 shoots and/or 20 segments each. Thus, the root counts
were obtained with 60 segments per treatment, and the data on 14CO2 with two
Petri dishes. Uptake of label was determined by taking two samples of three
segments from each dish/Erlenmeyer (so four samples in total). In the graphs and
tables, the means are given S.E.
3. Results
3.1. Uptake of IAA via the epidermis and the cut surface
In our research program on the mechanisms of rooting in apple microcuttings,
we examine shoots that are stuck in rooting medium so that 3±4 mm of the stem
is submerged, and 1 mm stem slices are cultured on top of the rooting medium
(De Klerk et al., 1997). In shoots and slices, maximal rooting is achieved at
26
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
different auxin concentrations (De Klerk et al., 1997). This may be caused by
differences in auxin accumulation in the basal part of the stem (in this section of
the stem adventitious roots are formed) and in slices. To examine this, shoots and
1 mm slices were cultured on medium with 10 mM IAA and a tracer amount of
1-14C-IAA. In shoots, almost all label occurred in the basal part of the stem and
reached the same level as in slices, ca. 0.4 nmol/day/slice or per shoot. Since in
shoots not only the cut surface (the diameter of a stem is ca. 1 mm, so the cut
surface is ca. 1 mm2) but also the epidermis (ca. 9 mm2) was in contact with the
medium, this indicates that uptake via the epidermis was negligible. This
prompted us to examine the uptake via the epidermis in more detail.
The contribution of the epidermis in uptake of auxin was ®rst studied in a
similar experiment in which 3 mm segments were cultured vertically on medium
with 10 mM IAA either with the cut surface on top of the medium (so only the cut
surface was in contact with the medium) or with ca. half of the segment
submerged in the medium (with the basal half emerging from the medium; note
that the segments were cultured with the apical side down on the medium). Even
though in the latter segments the surface in contact with the medium was ®ve
times larger (ca. 5 mm2 instead of ca. 1 mm2) in both cases, the uptake was ca.
0.8 nmol/segment for 2 days (Table 1).
We determined uptake via the epidermis and the cut surface in various other
ways. Segments of 4 mm were cultured on 1±2 mm wide and 3 mm high strips of
solidi®ed medium that contained 10 mM IAA with 1-14C-IAA. The length of the
strips was 3±5 cm. Either the cut surface or the epidermal surface touched the
medium. In the latter case, the segments were cultured transversely on the strips
in such a way that only the epidermis touched the strips. Fig. 1 shows that when
the cut surface touched the medium, the uptake of IAA was ca. 3.5-fold higher.
This difference was probably an underestimate, since only little medium was
available for each segment and IAA-depletion of the medium occurred. Loss of
label from the medium close to the segments (determined in an excised piece of
the strip of 6 mm in length; the segment had been in contact with the centre of
this piece) after 24 h of culture was 13 or 65% for the medium in contact with the
Table 1
Uptake of IAA by apple stem segments cultured on the surface of solid medium or partially
submerged in solid mediuma
Not submerged in medium
Submerged in medium for 1.5 mm
a
Estimated surface of the explant that
has contact with the medium (mm2)
Uptake (nmol IAA/
segment for 2 days)
1
5
0.850.06
0.820.06
Segments of 3 mm were cultured for two days vertically on top of solidi®ed medium or
vertically with the apical half submerged in the solidi®ed medium. The medium contained 10 mM
IAA and 1 kBq 1-14C-IAA. Uptake was determined after two days.
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
27
Fig. 1. Uptake of IAA by apple stem segments that were either with the epidermal or with the cut
surface in contact with medium. Segments of 4 mm were cultured on 1±2 mm wide strips of
solidi®ed medium with 3 mM IAA and 0.05 kBq mlÿ1 1-14C-IAA. Uptake of label in the segments
was determined at 4 or 24 h after the start of culture.
epidermis or the cut surface, respectively. It should be noted that the loss of label
was not only caused by uptake, but also by oxidation (data not shown, compare
Guan et al., 1997).
We also determined the uptake via the epidermal surface by culturing segments
of 1, 2, 3, or 4 mm in liquid medium on a shaker. In this way, various pitfalls (see
Section 4) related to culture on solid medium were avoided. Both after 24 and
72 h, the uptake was very similar for all segments (Fig. 2). After 24 or 72 h, 65 or
Fig. 2. Uptake of IAA by apple stem segments of various lengths in liquid medium. Segments of 1,
2, 3, and 4 mm were cultured for 24 or 72 h in liquid medium with 10 mM IAA to which 1 kBq
1-14C-IAA had been added. Uptake was also determined in the presence of ferulic acid (FA). This
phenolic compound inhibits the oxidation of IAA almost completely so that more IAA was
available for uptake, and the medium was more slowly depleted. Note that the estimated surface of
the 1 or 4 mm segments was 4 mm2 (1 mm2 cut surface3 mm2 epidermal surface) or 14 mm2
(1 mm2 cut surface13 mm2 epidermal surface), respectively.
28
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Table 2
Uptake of IAA by apple shoots, stem segments and defoliated stems in liquid mediuma
Shoots
Shootssegments
Defoliated stems
Uptake 0±24 h
(nmol IAA/explant)
Uptake 0±24 h in
basal 2 mm portion
(nmol IAA/explant)
Uptake 0±72 h in
basal 2 mm portion
(nmol IAA/explant)
7.00.2
7.60.2 (shoots);
0.80.1 (segments)
4.50.2
0.290.03
0.320.04 (shoots)
0.720.04
n.d.b
0.580.16
1.730.09
a
Ten shoots, 10 shoots20 2 mm stem segments, or 10 defoliated stems were cultured in 50 ml
Erlenmeyer ¯asks with 20 ml liquid medium supplemented with 10 mM IAA1 kBq 1-14C-IAA. At
24 and 72 h, uptake in the complete explants and, in the case of shoots and defoliated stems, also in
the basal 2 mm of the stem were determined.
b
Not determined.
90% of the label had been lost from the medium, respectively. This was not only
caused by uptake, but also by massive oxidation of IAA (Guan et al., 1997).
Depletion of the medium might have masked differences in the rate of uptake.
Therefore, we measured uptake from medium to which 300 mM ferulic acid had
been added. This phenolic carboxylic acid with a phenolic hydroxyl plus an
adjacent methoxyl stops oxidation of IAA (Bandurski et al., 1995). After 24 and
72 h, 30 and 60% of the label had been lost from the medium. Fig. 2 shows that
uptake in the presence of ferulic acid was similar in all segments.
Finally, the rapid uptake via the cut surface was demonstrated in a competition
experiment in which segments and shoots were cultured together in liquid
medium (Table 2). The uptake by the complete shoots was only 10 times larger
than the uptake in segments, even though the surface of the shoots was 100±200
times larger than that of the segments. It should be noted that the amount of label
in the basal part of the stem (0.32 nmol/explant/day) was about half of that in
segments (0.8 nmol/explant/day) and that in segments uptake occurs via both the
apical and the basal cut surface and in shoots only via the basal cut surface.
3.2. Effect of damage of the epidermis
In the experiments described in the previous sections, care was taken that only
segments with an intact epidermis were used (unless indicated otherwise). In the
following experiment, we examined segments with an intact epidermal surface
and segments from which the epidermal surface had gaps because of excision of
the petioles. Uptake of IAA up to 72 h was only 30% higher in segments with a
damaged epidermis (Table 3). However, when only uptake during the inductive
phase of rooting was considered (24±72 h, cf. De Klerk et al., 1995), there was an
29
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Table 3
Uptake and oxidation of IAA in 3 mm apple stem segmentsa
0±24 h
24±72 h
0±72 h
Uptake (nmol IAA/segment)
Without gaps
With gaps
0.180.01
0.210.02
0.130.03 (calc.)
0.220.03 (calc.)
0.320.03
0.430.02
Oxidation (nmol IAA/segment)
Without gaps
With gaps
0.090.01
0.140.01
0.210.04
0.280.01
0.300.04 (calc.)
0.410.02 (calc.)
a
Segments were cultured vertically with ca. 1.5 mm of the apical side submerged in 20 ml
medium with 3 mM IAA and 1 kBq 1-14C-IAA. Segments with an intact epidermis or with gaps in
the epidermis because of excision of petioles were selected. At 24 or 72 h after the start of culture,
samples were taken and uptake and oxidation of IAA were determined. Uptake over the period
24±72 h and oxidation over the period 0±72 h were calculated.
almost 2-fold difference in uptake between the two types of segments. Damage of
the epidermis also resulted in increased oxidation of IAA (Table 3).
Segments with gaps in the epidermis required less auxin to obtain the same
rooting response than segments with an intact epidermis. In Fig. 3, data are shown
for segments either cultured with the apical half submerged in the medium
(Fig. 3a) or horizontally on top of the medium (Fig. 3b). To exclude a possible
effect of oxidation, we used the auxin NAA that is not oxidized (Smulders et al.,
1990). For NAA, ca. 2 times lower concentration was required in the segments
with damaged epidermis (Fig. 4).
Fig. 3. Rooting of apple stem segments with intact or damaged epidermis. Segments of 3 mm were
cultured vertically with the apical half put in the medium (A) or 4 mm segments horizontally on the
surface of the medium (B). The epidermis of the segments was either intact or had gaps caused by
excision of the petioles. After ®ve days, the segments were transferred to basal medium.
30
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Fig. 4. Rooting of 6 mm apple stem segments cultured for 3 mm submerged in solidi®ed medium.
The submerged portion was the apical half and had either an intact epidermis or an epidermis with
gaps. Segments were cultured for ®ve days at a range of NAA concentrations and after that
transferred to basal medium. Note that the optimal NAA concentration for segments with a
damaged epidermis had shifted to the left.
4. Discussion
In intact normal plants, water loss via the epidermis is reduced by 95% when
the stomata are closed (Kerstiens, 1996). Apparently, the cuticle is relatively
impermeable to water. The cuticle is also impermeable to polar compounds
dissolved in water such as auxins. In normal cuttings, uptake of IAA occurs
predominantly via the cut surface. This was shown by Kenney et al. (1969) for
Avena mesocotyls, Pisum epicotyls and Gossypium hypocotyls. By scanning
electron microscopy, it has been observed that tissue-cultured plants have a
poorly developed cuticle (Johansson et al., 1992). Furthermore, the stomata of
tissue-cultured plants do not function properly (Santamaria and Kerstiens, 1994).
Therefore, we examined whether the epidermal surface of microcuttings is still
impermeable to auxin.
In the uptake experiments, various pitfalls may occur. IAA might penetrate in
or adhere to the cuticle and be retained there (Baker, 1987). Thus, uptake via the
epidermis may well be overestimated. In the experiments in which the segments
were partially submerged in the medium, anaerobiosis might have affected
uptake. These experiments might also have suffered from IAA depletion of the
medium close to the explants (Guan et al., 1997). In the experiment in which the
cut surfaces did not touch the medium (Fig. 1), label might have diffused along
the epidermis to the cut surface. All pitfalls with the exception of the ®rst one
(adherence to the epidermis) were avoided in an experiment in which segments
with different lengths were incubated in liquid medium (Fig. 2). All experiments
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
31
indicate that the uptake via the epidermal surface is small. For example, in the
experiment in liquid medium (Fig. 2) the total surface of 1 or 4 mm segments was
4 or 14 mm2, respectively, but uptake of IAA was almost the same. These data
agree with the ®ndings that the cuticle of tissue-cultured plants function
suf®ciently in spite of its reduced thickness (Santamaria and Kerstiens, 1994).
When segments with a damaged epidermis (because of excision of the petiole)
were cultured partially submerged in medium, they required less auxin for rooting
than segments with an intact epidermis. This demonstrates that auxin taken up via
gaps in the epidermis contributed to the rooting response. The shift in the doseresponse curve corresponds with the additional uptake during the inductive phase
of rooting, i.e., the period of rhizogenic action of auxin, 24±72 h (De Klerk et al.,
1995). Thus, auxin taken up via the gaps was transported basipetally to the cells
from which the adventitious roots are formed.
In conclusion, our data show that, just as conventional cuttings, microcuttings
take up auxin predominantly via the cut surface and that uptake via the epidermal
surface is both absolutely (contribution to total uptake) and relatively (uptake per
mm2) negligible. However, via gaps that are formed by excision of leaves,
considerable uptake may occur.
Acknowledgements
We thank Dr. Ton Croes for critical reading of the manuscript.
References
Baker, E.A., 1987. Penetration studies with sprays applied to isolated cuticles and leaf segments.
Asp. Appl. Biol. 14, 141±151.
Bandurski, R.S., Cohen, J.D., Slovin, J.P., Reinecke, D.M., 1995. Auxin biosynthesis and
metabolism. In: Davies, P.J. (Ed.), Plant Hormones. Kluwer Academic Publishers, Dordrecht,
pp. 39±65.
De Klerk, G.J., Caillat, E., 1994. Rooting responses of stem-disks excised from the same M9 Jork
microcutting. Adv. Hortic. Sci. 8, 15±18.
De Klerk, G.J., Keppel, M., Ter Brugge, J., Meekes, H., 1995. Timing of the phases in adventitious
root formation in apple microcuttings. J. Exp. Bot. 46, 965±972.
De Klerk, G.J., Ter Brugge, J., Marinova, S., 1997. Effectiveness of indoleacetic acid, indolebutyric
acid and naphthaleneacetic acid during adventitious root formation in vitro in Malus Jork 9. Org.
Cult. 49, 39±44.
Guan, H., De Klerk, G.J., 2000. Regeneration of roots from apple stem segments in vitro: effect of
orientation on the medium. Acta Hortic. 520, 171±182.
Guan, H., Huisman, P., De Klerk, G.J., 1997. Rooting of apple stem slices in vitro is affected by
rapid decline of indoleacetic acid in the medium. J. Appl. Bot. 71, 80±85.
Johansson, M., Kronestedt-Robards, E.C., Robards, A.W., 1992. Rose leaf structure in relation to
different stages of micropropagation. Protoplasma 166, 165±176.
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Kenney, G., Sudi, J., Blackman, G.E., 1969. The uptake of growth substances XIII. Differential
uptake of indole-3yl-acetic acid through the epidermal and cut surfaces of etiolated stem
segments. J. Exp. Bot. 20, 820±840.
Kerstiens, G., 1996. Diffusion of water vapour and gases across cuticles and through stomatal pores
presumed closed. In: Kerstiens, G. (Ed.), Plant Cuticles, An Integrated Approach. BIOS
Scienti®c Publishers, Oxford, pp. 121±134.
Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco
tissue cultures. Physiol. Plant 15, 473±497.
Santamaria, J.M., Kerstiens, G., 1994. The lack of control of water loss in micropropagated plants is
not related to poor cuticle development. Physiol. Plant 91, 191±195.
Smulders, M.J.M., Van De Ven, E.T.W.M., Croes, A.F., Wullems, G.J., 1990. Metabolism of
1-naphthaleneacetic acid in explants of tobacco: evidence for release of free hormone from
conjugates. J. Plant Growth Regul. 9, 27±34.
Stem segments of apple microcuttings take up
auxin predominantly via the cut surface
and not via the epidermal surface
Huiyuan Guana,b, Geert-Jan De Klerka,*
a
Centre for Plant Tissue Culture Research, PO Box 85, 2160 AB Lisse, Netherlands
b
Woody Plants Research Station of Hebei Province, No. A99, Canganxi Road,
Shijiazhuang, Hebei 050081, PR China
Accepted 10 January 2000
Abstract
In conventional cuttings, auxin applied to achieve rooting is taken up predominantly via the cut
surface and not via the epidermal surface of the stem. Even though in tissue-cultured plants the
cuticle is poorly developed and the stomata do not function properly, stem segments of apple
microcuttings took up labelled indoleacetic acid also predominantly via the cut surface. Stem
segments with an epidermis with gaps caused by excision of the petioles required a lower
exogenous concentration of auxin to achieve rooting than segments with an intact epidermis,
indicating that the gaps facilitated uptake of auxin from the medium. This was con®rmed in an
experiment on uptake of labelled indoleacetic acid. # 2000 Elsevier Science B.V. All rights
reserved.
Keywords: Adventitious root formation; Auxin uptake; Cuttings; Epidermis; Malus; Micropropagation
1. Introduction
Conventional cuttings and microcuttings are rooted by application of auxin. In
conventional cuttings, auxin is taken up predominantly via the cut surface.
Uptake via the epidermal surface is negligible (Kenney et al., 1969). Because in
Abbreviations: IAA, Indoleacetic acid; NAA, Naphthaleneacetic acid
Corresponding author. Tel.: 31-252-462130; fax: 31-252-417762.
E-mail address: geert-jan.de.klerk@lbo.agro.nl (G.-J. De Klerk)
*
0304-4238/00/$ ± see front matter # 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 1 3 2 - 1
24
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
microcuttings cuticular waxes are reduced (Johansson et al., 1992) and stomatal
functioning is disturbed (Santamaria and Kerstiens, 1994), their epidermis may be
more permeable. Previously, we reported that in apple microcuttings auxins are
taken up very rapidly from the medium (Guan et al., 1997). In this paper, we
examine how much uptake occurs either via the epidermal or via the cut surface.
2. Materials and methods
2.1. Plant material
Shoot production of Malus `Jork 9' was maintained as described previously
(De Klerk et al., 1995). Shoots 1 cm in length were subcultured in tubes with
15 ml of a modi®ed Murashige±Skoog medium (Murashige and Skoog, 1962)
with 4.4 mM benzylaminopurine and 0.5 mM indolebutyric acid. After ®ve weeks
of proliferation at 258C and a 16 h photoperiod (35 mmol mÿ2 sÿ1 provided by
cool-white, ¯uorescent lamps), clusters consisting of 5±10 shoots had been
formed by axillary branching. Shoots were excised and either used for further
shoot proliferation or for experiments.
2.2. Preparation of segments
From defoliated stems, segments of 1, 2, 3, 4 or 6 mm were cut with a special
device consisting of razor blades separated by the appropriate number of 1 mm
metal plates. It was carefully noted whether the epidermis of a segment had gaps
because of excision of the petioles. Adjacent segments from a shoot were
distributed over several treatments because it has been previously found that there
exists a weak correlation between the capability to root of adjacent segments (De
Klerk and Caillat, 1994). There is no correlation between the capability to root of
nonadjacent segments. Furthermore, in the middle part (1 cm) of stems there is no
gradient with respect to the capability to root (De Klerk and Caillat, 1994).
2.3. Uptake and oxidation of IAA
To determine uptake of IAA, we added tracer amounts of 1-14C-IAA. When
IAA is oxidized chemically or enzymatically, the carboxyl group is removed
(Bandurski et al., 1995). Thus, from 1-14C-IAA, 14CO2 is released. Apple stem
slices massively oxidize IAA (Guan et al., 1997). This oxidation probably occurs
at the surface of the explants and not within them.
The experiments were performed in 2 cm high 9 cm Petri dishes with 20 ml
solidi®ed rooting medium (De Klerk et al., 1995), or in 100 ml Erlenmeyer ¯asks
with 10 ml liquid rooting medium (the same composition but without agar) on a
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
25
rotary shaker (40 rpm). The speci®c conditions of each incubation are indicated
with the tables and ®gures. 1-14C-IAA (Sigma, 1.48106 kBq mmolÿ1) was
added as indicated.
The amounts of label in the explants, in the headspace (14CO2 formed by
oxidation of 1-14C-IAA), and in the medium were measured. To determine label
in the explants, samples of three segments were taken at random and digested
overnight at 408C in 200 ml Soluene (Packard). Then 4.5 ml Hionic ¯uor
(Packard) was added. 14CO2 was trapped in 300 ml 1 M KOH in a small vial with
a piece of ®lter paper. To determine the amount of radioactivity, 4.5 ml Aqua gold
(Packard) was added to the ®lter paper and the 300 ml KOH. Radioactivity in
solid medium was determined by adding 4.5 ml Aqua gold to an excised piece of
solidi®ed medium (ca. 100 mg). In this case, radioactivity was counted after 48 h.
Radioactivity in liquid medium was determined in a sample of 100 ml to which
4.5 ml Aqua gold had been added.
2.4. Rooting conditions
The segments were cultured with their apical cut surface down (Guan and De
Klerk, 2000) on 30 ml of solidi®ed rooting medium in a 2 cm high 9 cm Petri
dish. The Petri dish was incubated in a culture room at 258C. After ®ve days, the
segments were transferred to hormone-free medium and to the light (16 h
photoperiod; 35 mmol mÿ2 sÿ1). Roots were counted after 21 days under a
dissecting microscope.
2.5. Statistics
For each treatment, two Petri dishes with 30 segments each were used, or two
Erlenmeyer ¯asks with 10 shoots and/or 20 segments each. Thus, the root counts
were obtained with 60 segments per treatment, and the data on 14CO2 with two
Petri dishes. Uptake of label was determined by taking two samples of three
segments from each dish/Erlenmeyer (so four samples in total). In the graphs and
tables, the means are given S.E.
3. Results
3.1. Uptake of IAA via the epidermis and the cut surface
In our research program on the mechanisms of rooting in apple microcuttings,
we examine shoots that are stuck in rooting medium so that 3±4 mm of the stem
is submerged, and 1 mm stem slices are cultured on top of the rooting medium
(De Klerk et al., 1997). In shoots and slices, maximal rooting is achieved at
26
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
different auxin concentrations (De Klerk et al., 1997). This may be caused by
differences in auxin accumulation in the basal part of the stem (in this section of
the stem adventitious roots are formed) and in slices. To examine this, shoots and
1 mm slices were cultured on medium with 10 mM IAA and a tracer amount of
1-14C-IAA. In shoots, almost all label occurred in the basal part of the stem and
reached the same level as in slices, ca. 0.4 nmol/day/slice or per shoot. Since in
shoots not only the cut surface (the diameter of a stem is ca. 1 mm, so the cut
surface is ca. 1 mm2) but also the epidermis (ca. 9 mm2) was in contact with the
medium, this indicates that uptake via the epidermis was negligible. This
prompted us to examine the uptake via the epidermis in more detail.
The contribution of the epidermis in uptake of auxin was ®rst studied in a
similar experiment in which 3 mm segments were cultured vertically on medium
with 10 mM IAA either with the cut surface on top of the medium (so only the cut
surface was in contact with the medium) or with ca. half of the segment
submerged in the medium (with the basal half emerging from the medium; note
that the segments were cultured with the apical side down on the medium). Even
though in the latter segments the surface in contact with the medium was ®ve
times larger (ca. 5 mm2 instead of ca. 1 mm2) in both cases, the uptake was ca.
0.8 nmol/segment for 2 days (Table 1).
We determined uptake via the epidermis and the cut surface in various other
ways. Segments of 4 mm were cultured on 1±2 mm wide and 3 mm high strips of
solidi®ed medium that contained 10 mM IAA with 1-14C-IAA. The length of the
strips was 3±5 cm. Either the cut surface or the epidermal surface touched the
medium. In the latter case, the segments were cultured transversely on the strips
in such a way that only the epidermis touched the strips. Fig. 1 shows that when
the cut surface touched the medium, the uptake of IAA was ca. 3.5-fold higher.
This difference was probably an underestimate, since only little medium was
available for each segment and IAA-depletion of the medium occurred. Loss of
label from the medium close to the segments (determined in an excised piece of
the strip of 6 mm in length; the segment had been in contact with the centre of
this piece) after 24 h of culture was 13 or 65% for the medium in contact with the
Table 1
Uptake of IAA by apple stem segments cultured on the surface of solid medium or partially
submerged in solid mediuma
Not submerged in medium
Submerged in medium for 1.5 mm
a
Estimated surface of the explant that
has contact with the medium (mm2)
Uptake (nmol IAA/
segment for 2 days)
1
5
0.850.06
0.820.06
Segments of 3 mm were cultured for two days vertically on top of solidi®ed medium or
vertically with the apical half submerged in the solidi®ed medium. The medium contained 10 mM
IAA and 1 kBq 1-14C-IAA. Uptake was determined after two days.
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
27
Fig. 1. Uptake of IAA by apple stem segments that were either with the epidermal or with the cut
surface in contact with medium. Segments of 4 mm were cultured on 1±2 mm wide strips of
solidi®ed medium with 3 mM IAA and 0.05 kBq mlÿ1 1-14C-IAA. Uptake of label in the segments
was determined at 4 or 24 h after the start of culture.
epidermis or the cut surface, respectively. It should be noted that the loss of label
was not only caused by uptake, but also by oxidation (data not shown, compare
Guan et al., 1997).
We also determined the uptake via the epidermal surface by culturing segments
of 1, 2, 3, or 4 mm in liquid medium on a shaker. In this way, various pitfalls (see
Section 4) related to culture on solid medium were avoided. Both after 24 and
72 h, the uptake was very similar for all segments (Fig. 2). After 24 or 72 h, 65 or
Fig. 2. Uptake of IAA by apple stem segments of various lengths in liquid medium. Segments of 1,
2, 3, and 4 mm were cultured for 24 or 72 h in liquid medium with 10 mM IAA to which 1 kBq
1-14C-IAA had been added. Uptake was also determined in the presence of ferulic acid (FA). This
phenolic compound inhibits the oxidation of IAA almost completely so that more IAA was
available for uptake, and the medium was more slowly depleted. Note that the estimated surface of
the 1 or 4 mm segments was 4 mm2 (1 mm2 cut surface3 mm2 epidermal surface) or 14 mm2
(1 mm2 cut surface13 mm2 epidermal surface), respectively.
28
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Table 2
Uptake of IAA by apple shoots, stem segments and defoliated stems in liquid mediuma
Shoots
Shootssegments
Defoliated stems
Uptake 0±24 h
(nmol IAA/explant)
Uptake 0±24 h in
basal 2 mm portion
(nmol IAA/explant)
Uptake 0±72 h in
basal 2 mm portion
(nmol IAA/explant)
7.00.2
7.60.2 (shoots);
0.80.1 (segments)
4.50.2
0.290.03
0.320.04 (shoots)
0.720.04
n.d.b
0.580.16
1.730.09
a
Ten shoots, 10 shoots20 2 mm stem segments, or 10 defoliated stems were cultured in 50 ml
Erlenmeyer ¯asks with 20 ml liquid medium supplemented with 10 mM IAA1 kBq 1-14C-IAA. At
24 and 72 h, uptake in the complete explants and, in the case of shoots and defoliated stems, also in
the basal 2 mm of the stem were determined.
b
Not determined.
90% of the label had been lost from the medium, respectively. This was not only
caused by uptake, but also by massive oxidation of IAA (Guan et al., 1997).
Depletion of the medium might have masked differences in the rate of uptake.
Therefore, we measured uptake from medium to which 300 mM ferulic acid had
been added. This phenolic carboxylic acid with a phenolic hydroxyl plus an
adjacent methoxyl stops oxidation of IAA (Bandurski et al., 1995). After 24 and
72 h, 30 and 60% of the label had been lost from the medium. Fig. 2 shows that
uptake in the presence of ferulic acid was similar in all segments.
Finally, the rapid uptake via the cut surface was demonstrated in a competition
experiment in which segments and shoots were cultured together in liquid
medium (Table 2). The uptake by the complete shoots was only 10 times larger
than the uptake in segments, even though the surface of the shoots was 100±200
times larger than that of the segments. It should be noted that the amount of label
in the basal part of the stem (0.32 nmol/explant/day) was about half of that in
segments (0.8 nmol/explant/day) and that in segments uptake occurs via both the
apical and the basal cut surface and in shoots only via the basal cut surface.
3.2. Effect of damage of the epidermis
In the experiments described in the previous sections, care was taken that only
segments with an intact epidermis were used (unless indicated otherwise). In the
following experiment, we examined segments with an intact epidermal surface
and segments from which the epidermal surface had gaps because of excision of
the petioles. Uptake of IAA up to 72 h was only 30% higher in segments with a
damaged epidermis (Table 3). However, when only uptake during the inductive
phase of rooting was considered (24±72 h, cf. De Klerk et al., 1995), there was an
29
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Table 3
Uptake and oxidation of IAA in 3 mm apple stem segmentsa
0±24 h
24±72 h
0±72 h
Uptake (nmol IAA/segment)
Without gaps
With gaps
0.180.01
0.210.02
0.130.03 (calc.)
0.220.03 (calc.)
0.320.03
0.430.02
Oxidation (nmol IAA/segment)
Without gaps
With gaps
0.090.01
0.140.01
0.210.04
0.280.01
0.300.04 (calc.)
0.410.02 (calc.)
a
Segments were cultured vertically with ca. 1.5 mm of the apical side submerged in 20 ml
medium with 3 mM IAA and 1 kBq 1-14C-IAA. Segments with an intact epidermis or with gaps in
the epidermis because of excision of petioles were selected. At 24 or 72 h after the start of culture,
samples were taken and uptake and oxidation of IAA were determined. Uptake over the period
24±72 h and oxidation over the period 0±72 h were calculated.
almost 2-fold difference in uptake between the two types of segments. Damage of
the epidermis also resulted in increased oxidation of IAA (Table 3).
Segments with gaps in the epidermis required less auxin to obtain the same
rooting response than segments with an intact epidermis. In Fig. 3, data are shown
for segments either cultured with the apical half submerged in the medium
(Fig. 3a) or horizontally on top of the medium (Fig. 3b). To exclude a possible
effect of oxidation, we used the auxin NAA that is not oxidized (Smulders et al.,
1990). For NAA, ca. 2 times lower concentration was required in the segments
with damaged epidermis (Fig. 4).
Fig. 3. Rooting of apple stem segments with intact or damaged epidermis. Segments of 3 mm were
cultured vertically with the apical half put in the medium (A) or 4 mm segments horizontally on the
surface of the medium (B). The epidermis of the segments was either intact or had gaps caused by
excision of the petioles. After ®ve days, the segments were transferred to basal medium.
30
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
Fig. 4. Rooting of 6 mm apple stem segments cultured for 3 mm submerged in solidi®ed medium.
The submerged portion was the apical half and had either an intact epidermis or an epidermis with
gaps. Segments were cultured for ®ve days at a range of NAA concentrations and after that
transferred to basal medium. Note that the optimal NAA concentration for segments with a
damaged epidermis had shifted to the left.
4. Discussion
In intact normal plants, water loss via the epidermis is reduced by 95% when
the stomata are closed (Kerstiens, 1996). Apparently, the cuticle is relatively
impermeable to water. The cuticle is also impermeable to polar compounds
dissolved in water such as auxins. In normal cuttings, uptake of IAA occurs
predominantly via the cut surface. This was shown by Kenney et al. (1969) for
Avena mesocotyls, Pisum epicotyls and Gossypium hypocotyls. By scanning
electron microscopy, it has been observed that tissue-cultured plants have a
poorly developed cuticle (Johansson et al., 1992). Furthermore, the stomata of
tissue-cultured plants do not function properly (Santamaria and Kerstiens, 1994).
Therefore, we examined whether the epidermal surface of microcuttings is still
impermeable to auxin.
In the uptake experiments, various pitfalls may occur. IAA might penetrate in
or adhere to the cuticle and be retained there (Baker, 1987). Thus, uptake via the
epidermis may well be overestimated. In the experiments in which the segments
were partially submerged in the medium, anaerobiosis might have affected
uptake. These experiments might also have suffered from IAA depletion of the
medium close to the explants (Guan et al., 1997). In the experiment in which the
cut surfaces did not touch the medium (Fig. 1), label might have diffused along
the epidermis to the cut surface. All pitfalls with the exception of the ®rst one
(adherence to the epidermis) were avoided in an experiment in which segments
with different lengths were incubated in liquid medium (Fig. 2). All experiments
H. Guan, G.-J. De Klerk / Scientia Horticulturae 86 (2000) 23±32
31
indicate that the uptake via the epidermal surface is small. For example, in the
experiment in liquid medium (Fig. 2) the total surface of 1 or 4 mm segments was
4 or 14 mm2, respectively, but uptake of IAA was almost the same. These data
agree with the ®ndings that the cuticle of tissue-cultured plants function
suf®ciently in spite of its reduced thickness (Santamaria and Kerstiens, 1994).
When segments with a damaged epidermis (because of excision of the petiole)
were cultured partially submerged in medium, they required less auxin for rooting
than segments with an intact epidermis. This demonstrates that auxin taken up via
gaps in the epidermis contributed to the rooting response. The shift in the doseresponse curve corresponds with the additional uptake during the inductive phase
of rooting, i.e., the period of rhizogenic action of auxin, 24±72 h (De Klerk et al.,
1995). Thus, auxin taken up via the gaps was transported basipetally to the cells
from which the adventitious roots are formed.
In conclusion, our data show that, just as conventional cuttings, microcuttings
take up auxin predominantly via the cut surface and that uptake via the epidermal
surface is both absolutely (contribution to total uptake) and relatively (uptake per
mm2) negligible. However, via gaps that are formed by excision of leaves,
considerable uptake may occur.
Acknowledgements
We thank Dr. Ton Croes for critical reading of the manuscript.
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