Annals of Botany 93: 53±60, 2004

doi:10.1093/aob/mch014, available online at www.aob.oupjournals.org

Graft Union Formation in Tomato Plants: Peroxidase and Catalase Involvement

NIE V ES FE RNA Â N D E Z - G A R C ÂI A , M I C A E L A C A R V A J A L and E N R I Q U E O L M O S *
Departamento de FisiologõÂa y NutricioÂn Vegetal, Centro de EdafologõÂa y BiologõÂa Aplicada del Segura, CSIC, Aptdo.
Correos 164, 30100 Murcia, Spain

Received: 2 June 2003 Returned for revision: 12 September 2003 Accepted: 3 October 2003 Published electronically: 20 November 2003

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d Background and Aims The use of grafted plants in vegetable crop production is now being expanded greatly.

However, few data are available on the formation of graft unions in vegetables. In this work, the structural
development of the graft union formation in tomato plants is studied, together with the possible relationship
with activities of peroxidases and catalases.
d Methods Tomato (Lycopersicon esculentum Mill.) seedlings of cultivar Fanny were grafted on the rootstock of

cultivar AR-9704 using the `tongue approach grafting' method, and were grown in a crop chamber. A study of
the structural development of the graft union and the involvement of peroxidases and catalases in the process
of graft formation was carried out during the ®rst stages of the graft union (4, 8 and 15 d after grafting).
d Key Results Observation of the structure of the graft union showed formation of xylem and phloem vessels

through the graft union 8 d after grafting. In addition, root hydraulic conductance, L0, indicate that the graft
union is fully functional 8 d after grafting, which coincided with an increase of peroxidase and catalase activ-
ities.
d Conclusions These results suggest that increased peroxidase and catalase activities might be implicated in graft

development in tomato plants. ã 2004 Annals of Botany Company

Key words: Catalase, graft, Lycopersicon esculentum, peroxidase, tomato.

INTRODUCTION present in all vascular plants. Apart from cellulose, lignin is

the most abundant organic natural product known.
The use of grafted plants in vegetable crop production is still
Deposition of lignin in plants is normally located in the
rare compared with the use of grafting for tree crops.
sclerenchyma of the ground tissues, as well as in tracheary
However, this technique is now being expanded greatly to
elements and ®bres of the vascular tissues. Lignin is
reduce infections caused by pathogens (Biles et al., 1989;
synthesized mainly in cells to become part of the transport
Padgett and Morrison, 1990), to increase the resistance to
system. The last catalytic step in the synthesis of lignin is
drought (White and Castillo, 1989) and to enhance nutrient the oxidation of cinnamyl alcohols, and this step is catalysed
uptake (Ruiz-Sifre et al., 1997). Few data are available on by a peroxidase (Whetten et al., 1998). Peroxidases catalyse
the formation of graft unions in vegetables. oxyreduction between H2O2 and various reductants. The
The investigation of water relations by measurement of range of electron donors is wide and includes phenols,
parameters such as root hydraulic conductance (L0) could amines and alcohols. Catalases are similar to peroxidases
indicate the nutrient and water uptake status of grafted but, as in the case of catalase H2O2, can act as both electron
plants (Turquois and Malone, 1996). In a previous inves- donor and acceptor. Catalase is implicated in destruction of
tigation (Fernandez-GarcõÂa et al., 2002), it was observed harmful H2O2 generated in excess by different subcellular
that measurements carried out above and below the graft processes, and by biotic and abiotic stresses.
union gave similar L0 values, suggesting that the graft union In the present work, the structural development of the
cannot be considered a barrier to water ¯ow. These results graft union formation is studied in tomato (Lycopersicon
are in agreement with those reported by Turquois and esculentum Mill.) plants together with the possible
Malone (1996), who used a non-destructive measurement of relationship with peroxidase and catalase activities.
functional hydraulic connections within the intact plant,
providing a useful indication of the progress of graft
development. They showed that the graft union acts like a MATERIALS AND METHODS
continuous unit with respect to water movement. Plant material and culture conditions
In grafted plants, vascular regeneration can re-establish
the continuity of the transport system by means of a Tomato seedlings, cultivars Fanny and AR-9704 (root-
complex developmental process involving structural and stock), were germinated for 3±4 d in vermiculite moistened
physiological differentiation of parenchyma into xylem and with 0´5 mM CaSO4 in a germination chamber at 30 °C.
phloem elements (Jeffree and Yeoman, 1983). Lignin is After this, plants were put in a controlled environment
chamber at a day/night temperature of 25/20 °C, a day/night
* For correspondence. Fax 0034 968 39 62 11, e-mail eolmos@cebas. relative humidity of 65/85 % and a 16-h photoperiod.
csic.es Photon ¯ux density was adjusted to 400 mmol m±2 s±1. Light
Annals of Botany 93/1, ã Annals of Botany Company 2004; all rights reserved
54 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato
was provided by a combination of ¯uorescent tubes (Philips determined by following the decomposition of H2O2 at
TLD 36 W/83, Sylvania F36 W/GRO) and metal halide 240 nm. The extinction coef®cent was 36 mM±1 cm±1.
lamps (Osram HQL. T 400 W). After 9 d under these
conditions, plants were grown hydroponically in aerated
Tissue-printing of peroxidases
Hoagland nutrient solution: KNO3 (6 mM), Ca(NO3)2
(4 mM), NH4H2PO4 (1 mM), MgSO4 (1 mM), KCl Nitrocellulose membranes were soaked in distilled H2O
(50 mM), H3BO3 (25 mM), MnSO4 (2 mM), ZnSO4 (2 mM), and blotted dry with a tissue. Stem sections were placed on
CuSO4 (0´5 mM), (NH4)2MoO4 (0´5 mM), Fe-EDDHHA the membrane and lightly pressed on to it. The sections were
(20 mM). removed carefully and the membrane rinsed with distilled
water. Membranes were soaked in a 10 mM hydrogen
peroxide/9 mM guaiacol solution as described above.
Grafting procedure
Membranes were rinsed as soon as colour development
Twenty-®ve days after germination, cultivar Fanny (the was optimal (Spruce et al., 1987). Eight plants, grafted and

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scion) was grafted onto cultivar AR-9704 (the rootstock) ungrafted, were sampled at each time point (4, 8 and 15 d).
using the procedure of `cleft grafting' described by Lee
(1994). Grafted plants were covered with a transparent
H2O2 determination
plastic bag for 5 d to increase the relative humidity and
avoid leaf dehydration by water loss. Measurements and Total H2O2 was measured in fresh material by a
samples were taken 4, 8 and 15 d after grafting. peroxidase-coupled assay, using 4-aminoantipipyrine and
phenol as substrate donors (Frew et al., 1983). Five plants,
grafted and ungrafted, were sampled at each time point (4, 8
Root hydraulic conductance
and 15 d).
Hydraulic conductance (L0) was measured by natural
exudation of the roots. This method was based on volume
Histochemical stain for lignin
¯ow through detached root systems. The aerial parts of a
plant were removed, leaving a cylinder of leaf bases, and Lignin was detected using the method described by Ros-
this was sealed with silicone grease into a tapered glass tube. Barcelo (1998). The middle of the graft union was
After 2 h, the exuded xylem sap was collected using a determined using a stereomicroscope and sectioned with a
Pasteur pipette and transferred to an Eppendorf tube. The scalpel. Fresh cut sections (100±200 mm thick) were
sap was weighed and the roots removed and weighed. Sap obtained using a hand-microtome and were then soaked in
¯ow was expressed as mg (g root f. wt)±1 h±1. Samples of sap 1´0 % (w/v) phloroglucinol in 25Ã:Ã75 (v/v) HCl±ethanol for
(100 mL) were placed in Eppendorf tubes and the osmotic 10±15 min. The stained sections were observed and photo-
potentials of the sap and the root nutrient solution were graphed under a stereomicroscope (MZ8, Leica). A full
measured using an osmometer (Digital Osmometer, section of the graft union was dif®cult to obtain at day 4 due
Roebling, Berlin, Germany). The osmotic potential differ- to the limited development of the graft. Ten plants, grafted
ence between the xylem sap and the external solution, DyP, and ungrafted, were sampled at each time point (4, 8 and 15 d).
was calculated from their osmolarity values. The hydraul-
ic conductance, L0, which has units mg (g root
Scanning electron microscopy
f. wt)±1 h±1 MPa±1, was determined as: L0 = Jv/Dy P
(Fernandez-GarcõÂa et al., 2002). Six replicate plants per Stems were frozen in liquid N2 and fractured with a pre-
treatment were sampled at each time point (4, 8 and 15 d). cooled knife. The samples were then freeze-dried before
mounting and coating with gold. Samples were studied
using a JEOL JSM-6100 scanning electron microscope. The
Enzyme activities
acceleration voltage used was 15 kV (Olmos and Hellin,
Stems (1´0 g f. wt) were homogenized in 50 mM 1998). Ten plants, grafted and ungrafted, were sampled at
potassium phosphate buffer containing 1 % (w/v) polyvinyl each time point (4, 8 and 15 d).
pyrrolidone (pH 7´2) at 4 °C. The homogenate was ®ltered
through a nylon net and centrifuged at 12000 g for 10 min.
The supernatant was used to determine peroxidase and RESULTS
catalase activities. Five plants per treatment were analysed
Root hydraulic conductance
at each sampling time (4, 8 and 15 d).
Peroxidase activity was determined spectrophotometri- Root hydraulic conductance, L0, was measured at 4, 8, 12
cally by following the increase in absorbance at 470 nm. and 15 d after grafting (Fig. 1). L0 increased linearly from 4
The reaction mixture contained 50 mM potassium phosphate to 12 d, starting from zero, and attained a constant value
(pH 6´8), 10 mM hydrogen peroxide, 9 mM guaiacol and between 12 and 15 d.
enzyme extract in a total volume of 3 mL, as described by
Olmos et al. (1997).
Scanning electron microscopy
Catalase was assayed according to Aebi (1984). The
reaction mixture contained 50 mM potassium phosphate Figure 2 shows the morphological differentiation of
(pH 7´8), 10 mM H2O2 and the enzyme extract. Activity was grafted tomato plants. Figure 2A shows the stem cross-
 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato 55
union (Fig. 3F) showed a well-developed, stained xylem.
Similarly, the section 250 mm above the graft showed an
intense stain in the vascular system (Fig. 3I).

Peroxidase and catalase activities

The activities of peroxidase and catalase were analysed in
both parts of the graft (scion and rootstock) to differentiate
between the involvement of each in graft formation.
The increase of peroxidase activity is shown in Fig. 4.
Peroxidase activity increased during plant development in
both control and grafted plants. However, it was always
higher in grafted than in control plants. At 8 and 15 d, a

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signi®cant difference between rootstock and scion was
observed, with activities being higher in the rootstock than
F I G . 1. Values of root hydraulic conductance (L0). Measurements were in the scion.
made using natural exudation (see text for details). Data are means 6 s.e. Peroxidase tissue-printing (Fig. 5) showed an increase in
(n = 6). peroxidase activity as samples were taken nearer the region
of the graft union. Four days after grafting (Fig. 5A),
peroxidase activity was mainly localised in the graft union
section from control plants before grafting. The graft union between the rootstock and scion, and 250 mm above the graft
at day 4 is presented in Fig. 2B, where a necrotic layer can union (in the scion) (Fig. 5A, iii and iv). Peroxidase activity
be observed. Figure 2C (scion cross-section at the graft was much lower when measured more than 2 mm above or
level) shows detail of hypertrophic cells in the graft union at below the graft union (Fig. 5A, i and v). The control plants
4 d. At day 8, in many grafted plants, the parenchymatic showed very slight peroxidase activity, similar to that found
cells of the central pith were dead, leaving a central hole in more than 2 mm from the graft union (Fig. 5A, vi). At 8 d
which the surface was covered by a necrotic layer (Fig. 2D, after grafting, peroxidase activity was higher than at 4 d
scion cross-section at the level of the graft union and Fig. 2E, (Fig. 5B). However, the results were similar to those at 4 d,
stem longitudinal section at the level of the graft union). in that peroxidase was localized mainly in the graft union
However, the vascular system was not damaged and, with (Fig. 5B, iii). At this stage, peroxidase activity was absent in
new xylem strands, connected the rootstock and scion. At the central parenchyma, which correlates with the dead
day 15, the graft union was fully developed (Fig. 2F, stem region observed by microscopy (Figs 2E and 3H). In the
longitudinal section at the level of the graft union and vascular region more than 2 mm above the graft union,
Fig. 2H, surface of the graft union). Xylem vessels intense points of peroxidase activity were observed. At 15 d
connecting the rootstock and scion could be observed in after grafting (Fig. 5C), peroxidase activity was higher than
the graft union (Fig. 2G, detail of xylem vessels of the graft at 8 d. Peroxidase was also mainly distributed at the graft
union). union (Fig. 5C, iii). A high activity in the new vascular
region of the graft union was also observed (Fig. 5C). In
common with previous days, peroxidase activity was lower
Lignin localization (Wiesner test)
above and below the graft union.
The Wiesner test is based on the reaction of Figure 6 shows catalase activity during graft develop-
phloroglucinol with the aromatic aldehyde fraction con- ment. Catalase, an ef®cient scavenger of H2O2, showed
tained in lignin, the reaction yielding a pink stain. Figure 3 some small variation during development of control plants.
shows the progress of ligni®cation during graft develop- At 4 d after grafting, grafted plants showed the same
ment. catalase activity as control plants. However, activity was
Four days after grafting, the stain was mainly localized in greatly increased at day 8, being about three times greater
the xylem of the rootstock (Fig. 3A) and scion (Fig. 3J). In than for control plants. At day15, catalase activity was the
the graft union (Fig. 3D), small spots of pink stain could be same in control and grafted plants. No differences in
observed. Similarly, the section 250 mm above the graft catalase activity between the rootstock and scion were
union showed a pink stain near the vascular strand of the observed.
scion (Fig. 3G). Table 1 shows quanti®cation of H2O2 in scion and
At 8 d, a signi®cant increase in staining was observed in rootstock. Total H2O2 increased during plant development
the vascular system in both the rootstock (Fig. 3B) and in the in both control and grafted plants. However, there was a
scion (Fig. 3K). The graft union (Fig. 3E) showed a notable signi®cant increase in the amount of H2O2 at 8 d in grafted
increase in staining compared with day 4. The section plants.
250 mm above the union showed a large increase in staining
(Fig. 3H) compared with day 4. It can also be observed that
the lignifying cells exhibited a radial distribution. DISCUSSION
At 15 d, the vascular systems of the rootstock (Fig. 3C) A number of developmental stages can be recognized in the
and scion (Fig. 3L) showed an intense pink colour. The graft formation of a graft union. The early stage begins within 4 d,
56 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato
and is characterized by the death of cell layers at the graft grafted tomato plants, the callus is formed by all living,
interface as a wound reaction (Moore, 1984; Tiedemann, undamaged cells at the graft union, probably from cambial,
1989), and by generation of a parenchymatous wound callus ray parenchyma and phloem parenchyma cells. Living cells
that ®lls the gap between the two graft components. In from the surface quickly began to grow in size (hypertrophic

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 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato 57

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F I G . 3. Wiesner stain during graft development at day 4 (A, D, G and J), day 8 (B, E, H and K) and day 15 (C, F, I and L). Positive staining is shown
as a pink colour. (A±C) Sections of the rootstock at 250 mm below the graft union. (D±F) Sections of the graft union. (G±I) Sections of the scion
250 mm above the graft union. (J±L) Sections of the scion at 1 mm above the graft union.

F I G . 2. Scanning electron microscopy of grafted plants. (A) Stem cross-section from control plants before grafting. Bar = 2 mm. (B) Scion cross-
section from graft union at day 4, showing a necrotic layer at the graft interface. Bar = 1 mm. (C) Scion cross-section, detail of callus cells in a graft
union at 4 d, showing hypertrophic cells (arrow). Bar = 500 mm. (D) Transverse section of a scion at 8 d showing the pith totally destroyed, although
the vascular system was unaltered (arrows). Bar = 2 mm. (E) Longitudinal section of the graft at 8 d showing the graft union (arrow). Bar = 2 mm.
(F) Longitudinal section of the graft at 15 d (arrow = graft union). Bar = 2 mm. (G) Detail of xylem vessels from the graft union. Bar = 50 mm. (H)
An external view of the graft union at 15 d after grafting. Bar = 2 mm. * Cavity of the pith.
58 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato

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F I G . 4. Peroxidase activity during graft development. Data are means 6
s.e. (n = 5). Columns with the same letters are not signi®cantly different
for each parameter (P < 0´05, Tukey test).

cells) and to divide. Similar results have been obtained

previously in auto-grafted tomato plants (Jeffree and
Yeoman, 1983). It was observed that, in many of the
grafted plants, the pith of the rootstock exhibited a cavity of
about four or ®ve cell layers, although the vascular system
was unaltered. This could indicate a slight incompatibility
between scion and rootstock, or a wound response of the
scion during the ®rst days of grafting. Yeoman et al. (1978)
found that the necrotic layer was produced in both
compatible and incompatible Solanaceous grafts, which
supports the latter hypothesis.
The differentiation of callus parenchyma to form new
cambial initials and the subsequent union of the newly
formed vascular strand with the original vascular bundle in
both rootstock and scion begins between days 4 and 8 and is
fully developed after 15 d. Stoddard and McCully (1979)
showed mature xylem and phloem connections in pea root
auto-grafts 8 d after grafting. Most authors consider a graft
union to be successful and complete when several phloem
and xylem connections cross the graft interface. Turquois
and Malone (1996) observed that the major hydraulic
connections within the graft union of tomato became
functional 5 d after grafting. The observations described F I G . 5. Peroxidase tissue-printing of graft development at (A) day 4,
are consistent with these results, the gradual increase of (B) day 8 and (C) day 15. Sections for tissue-printing were taken
L0 4±8 d after grafting indicating the establishment of new sequentially at (i) 1 mm and (ii) 0´25 mm from the graft union in the
connections in the vascular bundle in the graft union. rootstock, (iii) at the graft union, (iv) 0´25 mm and (v) 1 mm above the
graft union in the scion and (vi) control sections from ungrafted plant.
After the graft assemblage between the cells of the
rootstock and scion was developed, differentiation of the
new vascular system began. For this to occur, xylem
differentiation and ligni®cation are necessary. Many studies demonstrated that peroxidase activity was located mainly in
have suggested that peroxidases play a role in ligni®cation the graft union (Fig. 5A, iii, B, iii and C, iii). These results
(Whetten et al., 1998; Quiroga et al., 2000). During growth are in accordance with the increase of ligni®cation observed
development of the stem of herbaceous plants, xylem cells in the graft union. Histochemical analysis in the grafted
are highly ligni®ed. The results given here show that total region was carried out to analyse lignin synthesis. Weisner
peroxidase activity increased during development of control staining can be used for speci®c detection of the
and grafted plants. However, grafted plants showed more hydroxycinnamyl aldehyde end units contained in lignin,
activity than controls. Tissue printing of the grafted region which are assembled during the early stages of xylem cell
 FernaÂndez-GarcõÂa et al. Ð Graft Union in Tomato 59
concentration of H2O2 could be toxic for the parenchymatic
cells, inducing the cellular death observed. Guan et al.
(2000) have recently observed, in maize plants, that H2O2
mediated catalase gene expression in response to wounding.
Finally, it is considered that the results suggest that
peroxidase and catalase activities are spatially and tempo-
rally coordinated. However, further study would be required
to appreciate the full implications of this for graft devel-
opment in tomato plants.

L I T E RA TU R E C I TE D

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