ON the ORIGIN of INTERCELLULAR CANALS in the SECONDARY XYLEM of SELECTED MELIACEAE SPECIES* Oliver Dünisch1,2 & Pieter Baas

Home , Carapa, Carapa guianensis, Carapa procera, Cedrela, Cedrela fissilis, Entandrophragma

IAWA Journal, Vol. 27 (3), 2006: 281–297

ON THE ORIGIN OF INTERCELLULAR CANALS IN THE SECONDARY XYLEM OF SELECTED MELIACEAE SPECIES*

Oliver Dünisch1,2 & Pieter Baas3

SUMMARY The anatomy, frequency, and origin of intercellular canals in the xylem of ten Meliaceae species (Carapa guianensis Aubl., Carapa procera DC., Cedrela odorata L., Cedrela fissilis Vell., Entandrophragma cilindricum Sprague, Entandrophragma utile Sprague, Khaya ivorensis A. Chev., Khaya senegalensis (Desr.) A. Juss., Swietenia macrophylla King, Swietenia mahagoni (L.) Jacq.) were investigated using 327 samples from institutional wood collections, 398 plantation grown trees, and 43 pot cultivated plants. Tangential bands of intercellular canals and single canals were found in the xylem of all ten species. Staining of microtome sections indicated that the chemical composition of the secretion is similar to that of “wound-gums”. Studying the origin of the intercellular canals along the stem axis, it became obvious that the formation of the canals can be induced by wounding of the primary meristems (in particular by insect attacks of Hypsipyla spp., wounding of root tips) and by wounding of the cambium (formation of 43–100% of the intercellular canals). In fast growing trees of Carapa spp., Entandrophragma utile, and Khaya ivorensis, planted at an experimental site near Manaus, Brazil, numerous canals were found which were not induced by wounding of the meristems. In these trees an out of phase sequence of xylem cell development and high growth stresses were observed, which are hypothesised to be a further trigger for the traumatic formation of intercellular canals. Key words: Traumatic canals, mechanical injury, meristem, xylem cell development, growth stresses.

INTRODUCTION Most studies on structure and formation of canals in wood have concentrated on resin canals in gymnosperms. In gymnosperms resin canals are classified into two groups, namely, non-traumatic and traumatic resin canals (Richter et al. 2004). The formation of non-traumatic resin canals is considered to be under genetic control (Werker &

1) Institute of Applied Botany, University of Hamburg, Ohnhorststr. 18, D-22609 Hamburg, Germany. 2) Institute for Wood Biology and Wood Preservation, Federal Research Centre for Forestry and For- est Products, Leuschnerstr. 91, D-21031 Hamburg, Germany. – Corresponding address [E-mail: [email protected]]. 3) Nationaal Herbarium Nederland, Leiden University branch, P.O. Box 9514, 2300 RA Leiden, The Netherlands. *) Dedicated to Prof. Dr. J. Bauch on the occasion of his 70th birthday.

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Fahn 1969), while the formation of traumatic resin canals is induced by exogenous factors, in particular by wounding (Lenely & Moore 1977; Fahn et al. 1979) and other environmental stressors (Wimmer & Grabner 1997). In contrast, information on the structure, the distribution, and factors influencing the formation of intercellular canals in the wood of dicotyledonous trees is very fragmen- tary, although in some dicotyledonous species intercellular canals are so frequent that they are important diagnostic features for wood identification (e.g., Dipterocarpaceae, Gottwald & Parameswaran 1966; Gottwald 1980; Wheeler et al. 1989). Intercellular canals are frequent in the Meliaceae (Record 1926; Wagenführ 2000; Dünisch et al. 2002a), but there is no standard term for them (terms used have been: resin ducts, gum ducts, traumatic resin canals and others). Because there are many high quality timber species within the Meliaceae (mahogany group), special attention has been given to the occurrence of intercellular canals with regard to wood quality and utilisation (e.g., Gottwald 1961; Wagenführ & Steiger 1963). Information on factors influencing the formation of these intercellular canals is rare. The first anatomical descriptions of intercellular canals in Meliaceae wood are in the pioneering works of Moeller (1876), Janssonius (1908), and Groom (1926). These studies indicated that the secretion in the canals is of a “wound-gum type” and that the “distances between suc- cessive intercellular canals or arcs is uneven and the formation is not periodic” (Groom 1926); yet these authors stated that the origin of this secretory tissue is unknown. Other publications considered intercellular canals in the xylem of Meliaceae species to be traumatic (Normand & Sallenave 1958; Normand & Paquis 1976). We studied the structure, frequency, and origin of the intercellular canals in the xylem of ten selected Meliaceae species (five genera). Special attention was given to 1) the impact of wounding, 2) the development and differentiation of xylem cells during the formation of the intercellular canals, and 3) growth stresses and their possible effect on the formation of intercellular canals.

MATERIAL AND METHODS Selected species and plant material Ten tree species from five genera of the family Meliaceae were selected: Carapa guianensis Aubl., Carapa procera DC., Cedrela odorata L., Cedrela fissilis Vell., Entandrophragma cilindricum Sprague, Entandrophragma utile Sprague, Khaya ivorensis A. Chev., Khaya senegalensis (Desr.) A. Juss., Swietenia macrophylla King, and Swietenia mahagoni (L.) Jacq. The occurrence of intercellular canals in the secondary xylem of all ten species is documented in the literature (Richter & Dallwitz 2000; Wheeler et al. [www.insidewood.lib.ncsu.edu/search]). 327 samples from wood collections (Table 1), stem and branch samples of 398 plantation grown trees (age: 4 years; Table 2) and of 43 pot cultivated plants (age: 2 years; Table 3) were examined. Samples from wood collections — Wood samples and samples from slide collections of the selected species were available from the collections of the National Herbarium of the Netherlands (Leiden - Lw / Utrecht - Uw), of the Federal Research Centre for Forestry and Forest Products, Hamburg, Germany (RBHw), and of the Federal Uni-

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Table 1. Number of wood samples from the wood collections of the National Herbarium of the Netherlands (Leiden/Utrecht), the Federal Research Centre for Forestry and Forest Products, Hamburg, Germany, and the Federal University of Paraná State, Curitiba, Brazil examined for this study. Number and portion (%) of samples with intercellular canals. Frequency (number per cm2 cross section area) of canals (local or in tangential bands) in the wood samples. Values followed by different letters differ significantly between species atp < 0.05 (Fisherʼs F-test).

Species No. of samples No. of samples Frequency of inter- with intercellular cellular canals canals (%) (no. cm-2)

Carapa guianensis 53 30 (57) 0.0160 a Carapa procera 43 26 (60) 0.0225 b Cedrela odorata 69 18 (26) 0.0180 a Cedrela fissilis 30 12 (40) 0.0143 c Entandrophragma cilindricum 23 6 (26) 0.0092 d Entandrophragma utile 23 5 (22) 0.0125 c Khaya ivorensis 16 15 (94) 0.0375 e Khaya senegalensis 10 6 (60) 0.0371 e Swietenia macrophylla 28 5 (18) 0.0090 d Swietenia mahagoni 32 8 (25) 0.0060 f

versity of Paraná State, Curitiba, Brazil. As far as possible the samples were cross- checked for duplication within and between the collections. For the statistical analyses on the occurrence of intercellular canals only one of duplicated samples was included. Plantation grown trees — Trees from 4-year-old plantations located in the region of Manaus, Amazonas (03° 08' S, 59° 52' W; Carapa guianensis, Carapa procera, Cedrela odorata, Khaya ivorensis, Khaya senegalensis, Swietenia macrophylla, Swietenia mahagoni), in the region of Santarem, Para (02° 52' S, 54° 45' W; Entan- drophragma cilindricum, Entandrophragma utile), and in the region of Ponta Grossa, Paraná, Brazil (25° 15' S, 50° 45' W; Cedrela fissilis) were selected. The Manaus site (tropical) is located at approximately 30–50 m above sea level with an annual precipitation of about 2500 mm (min. 110 mm [August], max. 295 mm [February] per month), a mean air temperature of 26.4 °C, and a mean air humidity of 87%. The soil is a poor oxisol (FAO-UNESCO 1990) with a low cation exchange capacity (Schroth et al. 2000). Edaphic factors of the Santarem region (30 m above sea level) correspond to the Manaus region, but the soil of the plantation near Santarem is more fertile than that in the plantation near Manaus (Dünisch et al. 2002b). In addition, the drier period is more distinct on the Santarem site than on the Manaus site. The subtropical site near Ponta Grossa is located 870 m above sea level. The annual precipitation is about 1570 mm and the mean air temperature is 19.1 °C. July and August are the driest and coldest months of the year with a monthly precipitation of about 70 mm and a mean air temperature of about 14 °C. The soil is of the cambisol type (EMBRAPA-SNLS 1984). The plantations near Manaus and Santarem /Belterra were installed at the research stations of the Brazilian Federal Research Centre EMBRAPA, while the plantation near

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Ponta Grossa was installed by private owners (Fazenda Bitumirim). All species were planted in open plots (spacing: 3 × 3 m) of 12 to 100 trees each (Bauch et al. 1999; experiment 1). In addition Carapa guianensis and Khaya ivorensis were planted in a line enrichment of a secondary vegetation and in a mixed agroforestry plantation, re- spectively (Dünisch 2001; Schroth & Sinclair 2003; experiment 2). In these plantations the growth rate of the trees was reduced compared to trees grown in open plantations (Table 2). For the study on the occurrence and on the origin of intercellular canals, 4-year-old trees of the plantations were completely harvested by excavation (Fig. 1). The trees were divided into segments of 1 m length. After that each root/shoot segment was cut in the longitudinal direction into 2 parts (radial cut, Fig. 3b).

Table 2. Mean annual radial increment (mm) of plantation grown trees examined. Plant age: 4 years. Number of trees with intercellular canals in the stem, total number of intercellular canals found, number and portion (% total intercellular canals) of traumatic canals induced by wounding of primary meristems, number and portion (% total intercellular canals) of traumatic canals induced by wounding of the cambium. Number of trees with high growth stresses (released strain (radial) > 500 × 10-6). Experiment 1: Open plantation; Experiment 2: Line enrichment (Carapa guianensis), agroforestry system (Khaya ivorensis). -6 ) -1

Species

(number of trees) Radial growth (mm year No. of trees with intercellular canals No. of intercellular (%) Total canals. No. of canals-wounding primary meristem (%) No. of canals-wounding cambium (%) No. of trees-released strain (radial) > 500 × 10

Carapa guianensis Experiment 1 (10) 11.2 ± 0.9 10 37 (100) 9 (24) 7 (19) 10 Experiment 2 (10) 4.9 ± 1.3 4 7 (100) 0 (0) 5 (71) 1 Carapa procera (10) 6.2 ± 0.7 10 24 (100) 14 (58) 2 (8) 7 Cedrela odorata (12) 9.9 ± 0.9 12 19 (100) 8 (42) 5 (26) 0 Cedrela fissilis (7) 7.1 ± 1.1 2 3 (100) 2 (67) 1 (33) 0 Entandrophragma cilindricum (5) 4.9 ± 1.7 5 13 (100) 9 (69) 2 (15) 0 Entandrophragma utile (5) 7.5 ± 3.1 5 11 (100) 4 (36) 4 (36) 3 Khaya ivorensis Experiment 1 (10) 15.0 ± 1.2 10 16 (100) 0 (0) 2 (12) 9 Experiment 2 (10) 7.3 ± 1.4 2 2 (100) 2 (100) 0 (0) 0 Khaya senegalensis (10) 13.5 ± 1.9 2 2 (100) 1 (50) 1 (50) 0 Swietenia macrophylla (17) 4.3 ± 2.2 17 46 (100) 23 (50) 23 (50) 0 Swietenia mahagoni (6) 2.9 ± 0.6 1 1 (100) 0 (0) 1 (100) 0

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Fig. 1. Sample collection (whole tree) in a 4-year-old plantation of Meliaceae species near Manaus (03° 08' S, 59° 52' W), Brazil.

Plants grown in pot cultures — High-resolution timing of the kinetics of xylem cell development by laserscan measurements and microscopical methods (see below) was restricted to 2-year-old plants (Dünisch & Bauch 1994; Dünisch et al. 2003). After a one-month germination period in a standard soil substrate, plants were cultivated in the greenhouses of the Federal Research Centre for Forestry and Forest Products, Hamburg and of the Brazilian Federal Research Centre EMBRAPA in Manaus.

Quantification of the frequency of longitudinal intercellular canals The number of intercellular canals in the secondary xylem was counted on the polished surfaces of the wood samples with a hand lens (×10). The dimensions of the wood samples were measured and the frequency of intercellular canals was calculated as the number of canals per cross section area. In this statistic, tangential bands as well as clusters of canals were considered as one intercellular canal.

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Microscopic characterization of intercellular canals Serial transverse, radial and tangential sections were prepared with a sliding microtome (Reichert, Austria; section thickness: 15 μm; Fig. 4b). The sections were stained with 1% safranine /1% astrablue (1 : 1), phloroglucin / HCl, and with the “Mäule” colour reaction (1% KMnO4 [5 min.], 3% HCl [1 min.], mounted on glass slides in drops of a 25% solution of NH4OH; Watanabe et al. 2004). The sections were covered with coverslips and were analysed using a light microscope.

Determination of the kinetics of xylem cell development The kinetics of xylem cell development were studied according to Dünisch et al. (2003) and Dünisch and Rühmann (2006) by high-resolution laser measurements in combination with microscopical observations. Light microscopical observations reveal- ed no periderm formation, dilatation of rays or a collapse of sieve tubes in the phloem (Dünisch & Bauch 1994; Dünisch et al. 2003). It was therefore assumed that the increase in shoot radius detected by the measuring device was due to the formation and expansion of cambium derivatives. Three laserscanners (MEL laserscanner M2D, MEL GmbH, Eching, Germany) were installed at the same height at an angle of 120° to each other around the shoot. Each scanner measured the distance between the scanner head and the shoot surface of a shoot segment of 120° with a spatial resolution of each laser line of approximately 1° (~150 laser lines per scanner, ~120 lines for a shoot segment of 120°, ~30 lines overlapping between two scanner heads). The corresponding optical resolution of the laser measurements along the shoot circumference was 12.1 to 38.4 μm. From the simultaneous measurement of 3 shoot segments, the profile of the shoot circumference/cross section was calculated and visualised by the MEL software package. The measuring device allows the quantification of the cross-sectional area of the shoot within a circle area of 4.52 m2 as a maximum without movements of the shoot affecting the accuracy of the measurements. The data and the cross-sectional profiles were stored in a computer (i-Control, MEL GmbH) at 1 to 60 s intervals. During each measuring interval, the laser light was switched on for 20 ms only in order to avoid an impact of the laser light on the cambial activity of the plants. The radial diameter of the phloem and xylem cells of the shoot portion analysed by the laser measurements were quantified in order to explain the significance of the increment curves of each laser line in terms of cell production and radial expansion. After harvest the samples were fixed in a FAA solution. The relevant shoot portions were embedded in polyethylene glycol (PEG 1500) with increasing concentration (PEG 1500: H2O, 1 : 2, 1:1, 2 :1, 1: 0). For identification and the histometrical analyses of the phloem and xylem cells, transverse sections (5 μm) were prepared from the shoot portions analysed by the laser measurements. For identification of the phases of primary wall formation, secondary wall formation, and lignification, the sections were stained with safranine (1%). For the comparison of the laser measurements and the histometrical data, the microphotographs of the sections and the shoot profiles obtained from the scanning unit were overlapped and adjusted. Histometrical measurements were carried out with an image analyser. The histometrically determined radial diameters

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of the xylem and phloem cells were compared with the radius increment obtained from the laser measurements during a distinct growth period along the radial transects through the tissue using the technique of Dünisch and Bauch (1994) and Dünisch et al. (2003). Maximum conformity between the two data sets was correlated with the program SYSTAT by means of Boolean algebra. If one laser line did not fit with a radial cell row (overlap in the tangential direction < 80%) or if during a distinct growth period the radius increment differed more than 2 μm from the radial diameter of the cell, it was not possible to time the enlargement of the individual cell. The duration of radial cell expansion of cambium derivative cells was calculated as the histometrically determined radius increase of the derivative cells (compared to the cambium cells) per time period (obtained from the laser measurements). The period for the formation of secondary cell walls and their lignification was calculated as the time period between the date of sample collection and the date of the offset of radial cell expansion.

Measurement of growth stresses The growth stresses in the stem of the 4-year-old plantation grown trees expressed in terms of released strain were quantified according to Kübler (1959; modified by Dünisch & Rühmann 2006). Stem cores (outermost 2 cm of the stem xylem) were sam- pled from the north and the south radii by means of an increment borer. Immediately after collection, the dimensions of the samples were measured in the three cardinal directions with an accuracy of ± 1 μm by the laserscanner. Longitudinal sections were then immediately prepared from the shoot segments by means of a cryo-microtome (section thickness: 40 ± 2 μm). The dimensions of the sections were quantified under the stereo-microscope by means of an image analyser. The amount of released strain in the longitudinal, radial, and tangential direction of the shoot was calculated as the

Table 3. Time period for complete radial cell expansion (hours) of vessels, fibres, axial parenchyma cells, and ray parenchyma cells. Plant age: 2 years. Mean values ± standard deviation. Values followed by different letters differ significantly between species (first letter) and cell types (second letter) at p < 0.05 (Fisherʼs F-test).

Species Vessels Fibres Axial parenchyma Ray parenchyma (hours) (hours) (hours) (hours)

Carapa guianensis 3.37 ± 0.92 a,a 7.02 ± 3.07 a,b 21.38 ± 6.10 a,c 1.42 ± 0.84 a,d Carapa procera 4.04 ± 2.37 a,a 6.95 ± 3.36 a,a 14.57 ± 6.21 b,b 1.65 ± 0.61a,c Cedrela odorata 0.88 ± 0.37 b,a 3.27 ± 1.46 b,b 3.92 ± 2.04 c,b 1.75 ± 0.82 a,c Cedrela fissilis 0.95 ± 0.49 b,a 3.05 ± 1.63 b,b 2.89 ± 1.33 c,b 0.93 ± 0.47 b,a Entandrophragma cilindricum 1.73 ± 0.68 c,a 3.61 ± 2.07 b,b 7.03 ± 2.73 d,c 1.08 ± 0.57 b,d Entandrophragma utile 2.04 ± 1.46 c,a 4.06 ± 1.84 c,b 10.90 ± 4.6 e,c 0.49 ± 0.27 c,d Khaya ivorensis 0.79 ± 0.34 b,a 2.93 ± 1.03 b,b 12.03 ± 5.13 e,c 0.52 ± 0.20 c,a Khaya senegalensis 2.79 ± 1.92 d,a 5.02 ± 2.07 d,b 7.04 ± 2.51 d,b 1.05 ± 0.47 b,c Swietenia macrophylla 0.57 ± 0.51b,a 3.81 ± 1.88 b,b 4.70 ± 2.63 c,b 1.71 ± 0.62 a,c Swietenia mahagoni 1.03 ± 0.43 b,a 4.57 ± 2.05 d,b 5.15 ± 2.74 d,b 0.36 ± 0.09 c,d

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Table 4. Amount of released strain (× 10-6) in the longitudinal, radial, and tangential direction of shoot microsamples from plantation grown samples. Tree age: 4 years. Mean values ± standard deviation. Values followed by different letters differ significantly between species at p < 0.05 (Fisherʼs F-test).

Species Longitudinal Radial Tangential (number of trees) direction direction direction (× 10-6) (× 10-6) (× 10-6)

Carapa guianensis (78) 713 ± 212 a 602 ± 63 a 497 ± 82 a Carapa procera (20) 909 ± 206 a 437 ± 131b 509 ± 73 a Cedrela odorata (62) 183 ± 43 b 82 ± 19 c 55 ± 24 b Cedrela fissilis (20) 206 ± 71b 92 ± 27 c 83 ± 20 b Entandrophragma cilindricum (20) 328 ± 53 c 193 ± 58 d 197 ± 73 c Entandrophragma utile (20) 719 ± 217 a 538 ± 193 b 416 ± 170 a Khaya ivorensis (40) 812 ± 323 a 573 ± 148 b 367 ± 194 a Khaya senegalensis (37) 847 ± 242 a 499 ± 219 b 205 ± 57 c Swietenia macrophylla (91) 238 ± 31b 67 ± 41e 73 ± 48 b Swietenia mahagoni (10) 251 ± 79 b 53 ± 35 e 91 ± 40 b

difference between the dimensions of the intact shoot segment obtained from the laser measurements and the sum of the dimensions of the microtome sections prepared from the shoot segment.

Statistical analysis The mean value and the standard deviation of each parameter were calculated. The significance of differences between species and anatomical parameters was assessed by ANOVA at p ≤ 0.05 by Fisherʼs F-test. Values followed by different letters differ significantly (Tables 3 & 4).

RESULTS

Anatomical characteristics of the intercellular canals Longitudinal intercellular canals were found in the secondary xylem of all selected species, while radial canals were absent. In samples from wood collections which predominantly came from old trees from natural sites, intercellular canals were more frequent (in terms of no. cm-2 cross section area) in the wood of Carapa sp., Cedrela odorata, and Khaya sp. (Table 1) compared to the other species. In contrast, in the 4-year-old plantation grown trees, intercellular canals were more frequent in the secondary xylem of Swietenia macrophylla, Entandrophragma sp., Carapa guianensis (Experiment 1) and Carapa procera (Table 2). In all species, two types of intercellular canals were present: 1) single or small clusters of canals (Fig. 2a); 2) thin bands of canals parallel to the circumference of the axis (Fig. 2b). In some cases the bands were continuous around the entire stem (Fig. 2b). The length of the intercellular canals extend from millimetres to a couple

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(a) 100 μm

(b) 200 μm

Fig. 2. – a: Local intercellular canal in Khaya ivorensis wood. – b: Band of intercellular canals in Khaya ivorensis wood.

of metres, and in some cases extended down the entire axis of the tree. The secretory tissue is produced directly by the cambium or by differentiating axial parenchyma cells. Rounded parenchyma cells are frequent at the edges of the intercellular canals and sometimes project into the secretion (Fig. 2a, b). The secretion is mainly homogeneous, but partly granular. The secretion is not soluble in cold water, ethanol, and acetone. Unstained the secretion has a yellow to dark orange colour. Staining by safranine and phloroglucin gave the secretion a reddish orange to dark red colour, characteristic for wound-gums.

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3 mm

Fig. 3. Induction of the formation of intercellular canals in the secondary xylem of Swietenia macrophylla by wounding of the primary meristem. – a: Insect attack by Hypsipyla grandella. – b: Larval tunnel in the pith of the upper part of the shoot. – c: Band of traumatic canals in the secondary xylem of the damaged shoot (53 cm distant from the shoot apex).

(a)

Primary meristem

Callus tissue Transition zone: Resin canals Primary/secondary meristem Accessory compounds Thick-walled fibres Resin canals

Accessory compounds Resin canals Secondary meristem: Cambium

Axial parenchyma

Accessory compounds Thick-walled fibres Resin canals

Axial parenchyma

Fig. 4. – a: Scheme and anatomical characteristics of the formation of traumatic intercellular canals in the secondary xylem of Meliaceae species by wounding of the primary meristem (de- rived from studies on the attack by Hypsipyla grandella). – b: Series of microtome sections for the investigation on the formation of intercellular canals along the shoot axis.

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Induction of traumatic canal formation by wounding of primary meristems By following the intercellular canals along the stem axis, it became obvious that some canals in the secondary xylem of the trees originated in injuries of the primary meristems (Table 2, Fig. 3a–c). Insect attacks of the apical meristems by the shoot borer (larvae) Hypsipyla spp. induced the formation of traumatic intercellular canals in the cambium of the trees. Wounding of root tips (e.g. by soil fauna, infection by fungi, silvicultural treatments) also induced the formation of the secretory tissue. Often, wounding of the primary meristem induced the formation of a continuous band of canals around the entire stem (Fig. 3c). Near the wounding, callus tissue of parenchymatic cells and intercellular canals filled with reddish to dark red coloured accessory compounds are formed (Fig. 4). Further away from the wound, there were thick-walled fibres and an increased production of accessory compounds. With increasing distance from the wounded primary meristem, the formation of intercellular canals disappeared and the

800 μm 20 μm

Fig. 5. Induction of the formation of traumatic intercellular canals in the secondary xylem of Carapa guianensis by wounding of the cambium. – a: Wounding of the cambium by thinning activities in a 4-year-old plantation near Manaus, Brazil. – b: Cross section, formation of traumatic canals approx. 7 cm above the injury. – c: Tangential section, injury and local formation of intercellular canals.

Downloaded from Brill.com10/01/2021 03:45:57AM via free access 292 IAWA Journal, Vol. 27 (3), 2006 Dünisch & Baas — Intercellular canals in Meliaceae 293 wound reaction ended in the formation of a tangential parenchyma band (Fig. 4). The longitudinal extension of the traumatic canals varied strongly between species and with the intensity of the wounding of the primary meristem. In the plantations, Cedrela spp., Entandrophragma cilindricum and Swietenia macrophylla were more sensitive to wounding in terms of the intensity of traumatic canal formation (Table 2) than the other species.

Induction of traumatic canal formation by wounding of the cambium A second important trigger for the formation of traumatic intercellular canals was identified as wounding of the cambium (Table 2). In the plantations, trees were often intensively wounded during thinning (Fig. 5a). Wounding of the cambium induced the local formation (single or cluster) of traumatic canals (Fig. 5b). Traumatic canals were formed as part of the barrier zone after wounding of the cambium approximately 0.5 to 2 mm distant from the injury (Fig. 5c).

Induction of intercellular canal formation by non-chronological xylem cell development and high growth stresses Although wounding was identified as an important trigger for the formation of traumatic intercellular canals in these Meliaceae species, there was still a high number

Phase of cell development Out of phase sequence Expansion SW formation lignification

0 10 100 1000 Time [h] Start 30 μm

Fig. 6. Out of phase sequence of xylem cell development in a very fast growing tree of Carapa guianensis in a 4-year-old plantation near Manaus, Brazil. – a: Shorter primary wall phase of fibres compared to axial parenchyma (polarised light). – b: Shorter primary wall phase of vessels compared to axial parenchyma (polarised light). – c: Kinetics of cell development of an ___ axial parenchyma cell ( ) and a subsequently formed fibre cell (---) in a fast growing tree of Carapa guianensis.

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of canals of unknown origin in fast grown trees, in particular in Carapa guianensis, Carapa procera, and Khaya ivorensis (Table 2). Microscopical studies and analyses on the kinetics of cell development showed that in these species in periods of high radial growth, the sequence of xylem cell development was not in phase (Fig. 6). Radial expansion of differentiating fibre cells was two (Carapa procera) to four (Khaya ivorensis) times faster, of vessels up to approximately ten times faster than radial expansion of axial parenchyma cells (Table 3). Consequently, during periods of fast radial growth, expanding axial parenchyma cells in the primary wall phase of cell development were embedded in fibres and vessels, in which secondary cell wall formation and lignification had started (Fig. 6). In addition, increased rates of xylem cell formation in these species was correlated with the formation of high growth stresses (Table 4), which are here hypothesised to induce the formation of intercellular canals in the immature parenchyma cells. DISCUSSION

In terms of the anatomical structure of the intercellular canals and their origin, the pattern of canal formation was similar in the ten Meliaceae species studied. This indicates that there is a high genetic uniformity between these species related to their wood formation and the metabolism for the production of secondary plant compounds. This is sup- ported by investigations of Muellner et al. (2003) indicating a high uniformity within this family also on the molecular level, although genetic markers for the formation of intercellular canals in Meliaceae are not identified. Molecular biological studies may contribute to answering the question: why are traumatic intercellular canals formed in some species of Meliaceae (Record & Hess 1943; Hess 1946) and not in others? Com- paring our results with studies on the formation of traumatic intercellular canals in the secondary xylem of other angiosperms, there seems to be evidence for a general ontoge- netic pattern of traumatic axial canal formation related to callus formation throughout those dicotyledonous species that have the ability to form them (Ghosh & Purkayastha 1960; Nikitin 1962; Zhang et al. 1992). The frequency of traumatic canals in the secondary xylem of the species differed significantly between wood samples from old trees grown in natural forests (wood from wood collections) and wood samples from younger trees grown in plantations. This shows that tree age and site conditions significantly influence the formation of the intercellular canals, although a genetic predisposition for the sensitivity to exogenous input with regard to the induction of canal formation is present in each species (Zobel & Jett 1995). With regard to plantation forestry, these results indicate that the successful cultivation of Meliaceae, in particular of Swietenia macrophylla and of Khaya ivorensis for high quality timber production, depends on careful management of the plantations, avoiding strong insect attacks of Hypsipyla sp. and wounding of the cambium (Mayhew & Newton 1998). The study on the origin of the canals was restricted in most cases to young trees grown in plantations. Therefore, the statistics on the origin of the intercellular canals (Table 2) might not be completely representative for the species (Zobel 1985). Because the shoot borer Hypsipyla sp. more frequently attacks the apical meristems of younger

Downloaded from Brill.com10/01/2021 03:45:57AM via free access 294 IAWA Journal, Vol. 27 (3), 2006 Dünisch & Baas — Intercellular canals in Meliaceae 295 trees with a height lower than 6–8 m (Whitmore 1976), the number and portion of traumatic canals induced by wounding of the primary meristem might be overestimated for the whole species. Despite these statistical uncertainties, the results showed that wounding is an important trigger for the induction of intercellular canal formation in the selected species. If the primary meristems are wounded, the formation of intercellular canals is not induced locally in the cambium, but over the whole cambium cylinder. As a result, continuous tangential bands of canals are formed, which, depending on the distance from the injury, may resemble increment zones not unlike the situation in Dipterocarpaceae, where the intercellular canals are assumed to be non-traumatic (Dünisch et al. 2002a). The relationship between the formation of intercellular canals and wounding of the cambium confirmed that exogenous influences are important factors for the development of secretory tissue in the xylem of Meliaceae. The results indicate that the formation of the secretory canals are part of the wound reaction, in particular of the formation of the barrier zone according to the concept of compartmentalisation of damage and decay in trees (Shigo 1984; Schmitt & Liese 1993). In agreement with observations of Groome (1926) staining of microtome slides indicated a more polyphenolic-like character of the secretion (reddish to orange staining by phloroglucin /HCl), characteristic for wound- gums, which gives further evidence for the significance of mechanical stress for the induction of intercellular canal formation in the secondary xylem of Meliaceae. In agreement with observations of Catesson (1962, 1994) and Oribe et al. (2001), the results on the kinetics of xylem cell development showed that the speed of cell formation also depends on the cell type. Very slow radial expansion of axial parenchyma cells was observed in Carapa guianensis, Carapa procera, and Khaya ivorensis. This was the reason for the “out of phase” sequence of xylem formation in fast growing trees of these species. As a consequence, bands of axial parenchyma with a weak primary cell wall structure are embedded in fibres/vessels in more advanced phases of cell development (secondary wall formation, lignification). Following Yamamoto (1998), the high growth stresses observed in the stem of these species presumably originate during the formation of the secondary cell wall and during lignification. Under these conditions high mechanical stress could be generated in the parenchyma bands during cell development, which in turn is hypothesised to trigger the formation of resin canals within these developing parenchyma bands. This might also explain the occurrence of continuous tangential bands in some older trees (growth stresses often increase with tree age). Whether intercellular canals induced by this pathway can be considered as traumatic canals might be questioned, because they originate during regular xylem formation (Ohbayashi & Shiokura 1989; Dupuy & Koua 1993). From the results it is concluded that the formation of axial intercellular canals in the xylem of these ten Meliaceae species was induced in the cambium or in developing axial parenchyma cells by mechanical stimuli. Due to the strong relationship between exogenous factors and the formation of intercellular canals, studies on the formation of intercellular canals in highly valued wood of Meliaceae species is important for forest management and wood quality.

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ACKNOWLEDGEMENTS

We thank the European Community (SYNTHESYS Fellowship Program), the CNPq/CAPES, Brasilia, the Federal Ministry for Education and Research (BMBF), the German Research Foundation (DFG), and the German Academic Exchange Service (DAAD), Bonn for financial support. We also thank the Empresa Brasileira de Pesquisa Agropecuaria, Manaus, the Ervateira Bitumirim, Ivai, Dr. L. Gasparotto, Dr. G. Schroth, Dr. E. Neves, A. Olizeski, and O. Rühmann for providing the experimental plants. We thank V. Ribeiro Montoia and V. Seabra for technical assistance. We are indebted to M. Sack for the preparation of microtome sections and microphotographs.

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