IAWA Journal, Vol. 16 (4),1995: 433-442

WOUND RESPONSE IN THE STEM OF THE ROYAL PALM by Gudrun Weiner & WaIter Liese Institute for Wood Biology, University ofHamburg, Leuschnerstrasse 91, D-2l 031 Hamburg, Germany

SUMMARY

Mechanical damage of a palm stern endangers physiological functions, such as water transport, and induces quality reduction due to dis­ colouration. The wound response of a 35-year-old Royal palm (Roystonea regia) has been investigated in order to understand the protective mech­ anisms and the origin of the staining. Wounds were induced in the basal and top regions 21, 14, 7 and 1 day(s) before the palm was felled. As initial reaction phenolic compounds are deposited in few of the smaller ground parenchyma cells. Subsequently slime fills up metaxylem vessels and protoxylem tracheids ofboth regions, whereas tyloses only develop in vessels at the top. Additionally, phenolic compounds originate also in phloem, vascular parenchyma, ground parenchyma and fibres. Key words: Monocotyledons, palmae, Roystonea regia, wound response, parenchyma, vascular bundles, light microscopy

INTRODUCTION

The anatomical structure of palm sterns originates from the apical meristem during axial growth. The cells developed must serve their function for a li fe time of several decades and maintain the physiological requirements of transport and storage. If the transport of water within the stern is interrupted, e.g. by wounding or by ageing, palms as weIl as other monocotyledons must have an effective defense system to prevent the inflow of air with a subsequent blockage of the vessels and followed by an invasion of microorganisms. The need for an efficient wound response to protect the existing trans­ port system is emphasized by the lack of a secondary meristem for the production of additional pathways. Whereas for trees the defense mechanisms against wounding have been extensively investigated in recent years (Shigo & Marx 1977; Shigo 1984, 1994; Dujesiefken et al. 1989; Liese & Dujesiefken 1989, 1995; Bonsen & Kucera 1990; Schmitt & Liese 1990; Blanchette & Biggs 1992), only little corresponding information exists about the wound response in monocotyledons, like the reactions in the rhizome of banana (Van der Molen et al. 1977). The presence of such mechanisms is indicated by the restricted discoloration around the 'harvesting steps' in coconut palms, as reported by Killmann (1989). Wound reactions are mostly associated with a darkening of the affected tissue which has a negative influence on the quality of any product produced from the stern.

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With the increasing utilization of palrn sterns as an additional resource for industrial use quality aspects becorne irnportant. Therefore a better knowledge of the origin and nature of such discolorations should be obtained. Investigations have been under­ taken with few palrns and also with barnboo, as both groups differ in their axial devel­ oprnent with consequences for the age and the vitality of the affected tissue. Palrns grow in height for decades, barnboo for only a few months. In the following sorne observations on Roystonea regia are reported.

Fig. 1. Wounds at the stern base of Roystonea regia.

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MATERIAL AND METHODS

Roystonea regia (Kunth) O.F. Cook originates from the Caribbean Islands, and is widely cultivated in both hemispheres. With its tall, stout stern up to 25 m height and a promi­ nent crownshaft reaching up to 8 m in length, R. regia belongs to the most elegant of the large palms. Royal palm is its common name. For the investigations a 35-year-old palm with a height of about 11 m was available at the tropical greenhouse from the 'Old Botanical Garden', Hamburg. Wounds of 4 mm width and 40 mm depth were set with a drilling machine in the basal region of the stern at about 1.5 m height and in the top region at 9 m height, directly below the oldest leaves. With the difference in age between the tissue of the base and the top being at least 25 years possible differences in response activity could be indicated. In order to follow the reactions over three weeks, wounds were induced 21,14,7 and 1 day(s) before the palm was felled (Fig. 1). Sampies were taken immedi­ ately after felling. For light microscopy small pieces (3 x 3 x 5 mm3) around the wound edge (Fig. 2) were fixed for 2 days in 4% formaldehyde, dehydrated in a rising series of ethanol and transferred into a glycol-methacrylate-monomere (GMA, Technovit 7100). After 7 days of infiltration the sampies were placed into Histoform moulds with a mixture of GMA and acuring agent (15: 1) for polymerization. The plastic blocks from the moulds were mounted on specimen holders (Histobloc) with a two-component glue (Technovit 3040). Sections of 9 ).lIll thickness were cut on a rotary microtome (Histoknife), stretched on a waterbath and attached to microscope slides. Since GMA-sections stick to the glass surface after drying on a warming tray (35°C), no further adhesive was necessary. Section staining was performed without removing the plastic matrix. For general observation the sections were stained with 20% Giemsa for 5 min, dipped in acidified water, dehydrated twice in 96% ethanol and in isopropanol with subsequent clearance in xylene. For fluorochroming an aqueous 0.1 % solution of Acridine orange was used for 15 min. Slime (pectionophile-substances) were easily made visible by staining with

------apical... Ic 0 r 4 ,center t -~N!>~~a e x 1.0 ....- basal L ----- Fig. 2. Mode of wounding; units in mm.

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Ruthenium red (Gerlach 1977). Stained and Eukitt-embedded sections were examined with an Olympus BH-2 microscope equipped for fluorescence (UV-excitation wave length between 370-420 nm).

RESULTS

Stem anatomy of Roystonea regia In order to observe the wound response by the various cell types an information of the general stern anatomy of R. regia appears beneficial, the more as only little knowl­ edge exists (Tomlinson 1961). The cortex as the tissue between epidermis and completely developed vascular bundles is very narrow, with a smooth transition to the central cylinder; the vascular bundles are embedded in the cortex ground parenchyma which consists of small pa­ renchyma cells. In the central cylinder the vascular bundles are diffusely oriented, within a soft, unlignified ground parenchyma. Their cells are thin-walled, mainly tangentially ex­ panded with large intercellular spaces. Parenchyma cells adjoining the vascular bun­ dIes and also smaller ones interspersed between the tangentially stretched cells evince an isodiametric shape and little intercellular spaces. Raphids often occur between pa­ renchyma cells. The conducting elements of a vascular bundle consist of one phloem field, one sim­ ply perforated metaxylem vessel and protoxylem tracheids. The phloem contains sieve tubes with companion cells. Stegmata are arranged in longitudinal, continuous strings adjacent to the randomly located fibres of a vascular bundle.

Wound response Macroscopy - On the surface of the palm stern only a very narrow discoloration around the wound becomes evident 21 days after wounding (Fig. 1). A distinct brown­ ing effect surrounds the radial split through the wound canal already after one day and is more pronounced after 7 and 14 days (Fig. 3a, b, c). After 21 days the discoloration has only slightly extended whereby the affected tissue shows a structural disintegra­ tion (Fig. 3d). Microscopy - First wound reactions become obvious already after one day only, as phenolic compounds are deposited within a few of the smaller ground parenchyma cells, both at the top and basal region (Fig. 4). No reactions appeared in the vascular bundle elements. After 7 days phenolic compounds are irregularly distributed in many of the smaller ground parenchyma cells, but not in the larger, tangentially stretched ones. The meta­ xylem vessels ofthe basal and top regions contain slime (Fig. Sb). Tyloses develop in some of the vessels in the top region only (Fig. Sa), while the protoxylem tracheids contain ty loses also at the base. In sieve tubes of the younger region slime is present as a thin layer, but much less than in the vessels. After 14 days, the large intercellular spaces within the ground parenchyma at the top are filled with slime. In both regions some parenchyma cells tangentially extend

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further while septa-like walls are formed, thusdividingthe en­ larged cells (Fig. 6b). The meta­ xylem vessels appear now en­ tirely occluded with slime. Ty­ loses develop in the metaxylem vessels around the wound, but only in the younger tissue at the top and not at the base (Fig. 6a) according to observations made duringearlierstages.Sieve tubes also show slime as deposits and some of their companion cells phenolic compounds. In sampies taken after 21 days necrotic parenchyma cells are found. These cells as weIl as fibres of vascular bundles near the wound are occluded with phenolic compounds (Fig. 7). Within a zone of up to 10 mm to the wound edge some of the smaller parenchyma cells are entirely filled, others appear thicker-walled than in earlier stages. Thetangentially expand­ ed parenchyma cells and also some of the smaller ones devel­ op septa-like walls. Metaxylem vessels and also protoxylem tracheids of the top region show both slime and tyloses. How­ ever, at the base the vessels con­ tain onlyslime, whereasthe pro­ toxylem tracheids additionally reveal slime and tyloses. Sieve tubes and companion cells ex­ hibit phenolic compounds in both regions.

Fig. 3. Browning around the wound canal after a: 1 day - b: 7 days - c: 14 days - d: 21 days.

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Fig. 4-7. Longitudinal seetions of Roy­ stonea regia. - 4: After 1 day: ground parenchyma cells with phenolic com­ pounds (arrows); scale bar = 50 Iffil. - 5: After 7 days: a: metaxylem vessel with tyloses (top area); b: metaxylem vessel with slime-like substances(basal area); scale bar = 100 Iffil. - 6: After 14 days; a: meta­ xylem vessel with tyloses and slime-like substances (top area); scale bar = 50 Iffil; ·b: expanded ground parenchyma cells with septa (arrows) in contact with a vascular bundle (basal area); scale bar = 100 Iffil. - 7: After 21 days: wound edge at basal area: fibres, vascular bundle parenchyma and ground parenchyma with phenolic compounds; scale bar = 100 Iffil.

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Table 1. Wound reactions in astern of Roystonea regia.

basal, older region top, younger region

ceII type 1 day 7 days 14 days 21 days 1 day 7 days 14 days 21 days

sieve tubes - slime slime slime+ - slime slime slime+ -comp. ceIIs - slime slime phen. c. - slime phen. c. phen. c.

metaxylem - slime slime slime+ - slime + slime + slime + vessel phen. c. tyloses tyloses tyloses

protoxylem - slime+ slime+ slime+ - slime+ slime+ slime + tracheids tyloses tyloses tyloses tyloses tyloses tyloses

vascular parenchyma -- - phen. c. - phen. c. phen. c. phen. c.

ground- parenchyma phen. c. phen. c. phen. c.+ phen. c.+ phen. c. phen. c. phen. c.+ phen. c.+ septa septa + septa septa + necrotic necrotic

- interceIIular spaces ------slime slime

fibres --- phen. c. --- phen. c.

comp. ceIIs = companion ceIIs; phen. c. = phenolic compounds.

The reactions of the different cell types in their sequence during the four dates of exarnination are summarized in Table 1. The cellular changes and their occurrence within the response time correspond to each other at the older, basal region and the younger, top region. Slime, tyloses and septa-like walls are general response mecha­ nisms. As a distinct difference only the absence of tyloses in metaxylem vessels at the base has been noted. This deficiency by the surrounding parenchyma is possibly due to the age difference of about 25 years compared with the reaction potential at the top region. It should be emphasized that cells at the base still contain their nuclei, as ob­ served within the vascular bundle parenchyma, the ground parenchyrna, the lignified fibres and the companion cells. In order to evaluate the efficiency of the cellular response for the protection of physiological functions its spatial expansion within the tissue is of interest. The reac­ tions of the ground parenchyma and the one surrounding the metaxylem vessels and protoxylem tracheids are rather closely confined to the periphery of the wound canal. The reaction zone is restricted to 10 mm maximum in width and about 20 rnrn in length after three weeks. Even at the end of exposure in the humid greenhouse no fungal hyphae were ob­ served in the discoloured wound area.

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DISCUSSION

The observations on Roystonea regia have shown that cellular reactions in the ad­ jacent tissue occur as protection against physical damage of the functional system. Although microscopical observations about the protective mechanisms of palms in general hardly exist so far, the observations indicate that generally palms respond sim­ ilarly to hardwoods when wounded: tyloses develop into the metaxylem vessels and protoxylem tracheids, additionally slime and phenolic compounds are accumulated in vessels, parenchyma and also in fibres. Such slime was also observed in the rhizome of banana (Musa sp.) by VanderMolen et al. (1977) and referred to as gels. These gels were identified by staining with Ruthenium red, "indicating that they were acidic and in part pectinaceous." Since the slime in R. regia reacted also to Ruthenium red, a chemical similarity between the two appears obvious and the terms 'slime' and 'gels' are synonymous. However, no suberization as a cellular response around a wound has been identified so far in R. regia. Suberin provides a general protective mechanism in the phloem and xylem of hardwoods (Trockenbrodt & Liese 1991; Schmitt & Liese 1993). Corresponding investigations on the wound reactions of bamboo revealed a distinct suberization as wound response. Palms as monocotyledons do not develop a barrier zone as a protective tissue and no callus in response to mechanical wounding, as typical for dicotyledons. Neither can ray cells in monocotyledons contribute structurally to the spatial restriction of the dis­ colored tissue, i.e., wall 2 ofthe CODIT-model by Shigo and Marx (1977). Moreover a structural boundary in tangential direction for wall 3, either by parenchyma, growth rhythms or heartwood does not exist. The well-known term 'compartmentalization' appears therefore not adequate for the defense system of palms. Shigo (1994) com­ ments on wound reactions in palms with the terms 'wall off' or 'seal off' which can be supported by the present results. In the discolored tissue of R. regia no fungal infection was observed after three weeks in the warm-humid atmosphere of a greenhouse. However, the tissue of Acacia mangium Willd., wounded similary in a tropical c1imate, showed fungal infection al­ ready after two weeks (Schmitt et al. 1995).

Further wounding-experiments with other palms and with bamboo will contribute more observations. While the mechanisms of wound response are probably quite similar within various plants, the spatial extent of the reactions depends on a number of fac­ tors, inc1uding structural differences. According to aremark by Shigo (1994) Cocos nucifera reacts stronger than R. regia.

ACKNOWLEDGEMENT

The investigation has been supported by the Deutsche Forschungsgemeinschaft (DFG). We thank Dr. Uwe Schmitt for his contributions.

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