Stem segments of apple microcuttings take up
auxin predominantly via the cut surface
and not via the epidermal surface
Huiyuan Guan
a,b, Geert-Jan De Klerk
a,* aCentre 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; Micropropa-gation
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)
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 4.4mM benzylaminopurine and 0.5mM indolebutyric acid. After ®ve weeks of proliferation at 258C and a 16 h photoperiod (35mmol mÿ2sÿ1 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.
rotary shaker (40 rpm). The speci®c conditions of each incubation are indicated with the tables and ®gures. 1-14C-IAA (Sigma, 1.48106kBq 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 200ml Soluene (Packard). Then 4.5 ml Hionic ¯uor (Packard) was added.14CO2was trapped in 300ml 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 300ml 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 100ml 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; 35mmol mÿ2sÿ1). 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 14CO2with 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 givenS.E.
3. Results
3.1. Uptake of IAA via the epidermis and the cut surface
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 10mM 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 10mM 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 mm2instead 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 10mM 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
Estimated surface of the explant that has contact with the medium (mm2)
Uptake (nmol IAA/ segment for 2 days) Not submerged in medium 1 0.850.06 Submerged in medium for 1.5 mm 5 0.820.06
aSegments of 3 mm were cultured for two days vertically on top of solidi®ed medium or
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. 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 3mM IAA and 0.05 kBq mlÿ11-14C-IAA. Uptake of label in the segments was determined at 4 or 24 h after the start of culture.
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 300mM 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
Table 2
Uptake of IAA by apple shoots, stem segments and defoliated stems in liquid mediuma Uptake 0±24 h Shootssegments 7.60.2 (shoots);
0.80.1 (segments)
0.320.04 (shoots) n.d.b
Defoliated stems 4.50.2 0.580.16 1.730.09
aTen 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 10mM 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
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).
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 0.180.01 0.130.03 (calc.) 0.320.03 With gaps 0.210.02 0.220.03 (calc.) 0.430.02
Oxidation(nmol IAA/segment)
Without gaps 0.090.01 0.210.04 0.300.04 (calc.) With gaps 0.140.01 0.280.01 0.410.02 (calc.)
a
Segments were cultured vertically with ca. 1.5 mm of the apical side submerged in 20 ml medium with 3mM 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.
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
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 dose-response 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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