Aust. J. Bot., 1984, 32, 439-47

Surgical Experiments on Eucalypt Lignotubers

D. J. CarrA, R. JahnkeB and S. G. A[. CarrA

A Department of Environmental Biology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601.

Department of Neurobiology, Research School of Biological Sciences, Australian National University, P.O. Box 475, Canberra City, A.C.T. 2601.

Abstract

Experiments involving surgery such as ring-barking or excision of lignotubers of seedling Eucalyptus st johnii plants are described. Removal of the strip of stem bark and cambium from between two lignotubers at the same node delays but does not prevent their lateral fusion. Ring-barking results in outgrowth of lignotuber buds below the ring and a check in growth, as compared with the untreated lignotuber at the same node. Excision of one lignotuber results in the formation of a 'replacement lignotuber' which lacks lignotuber buds; excision of both lignotubers at a node results in the formation of a pair of replacement lignotubers. Anatomical investigation shows that replacement lignotubers arise following conversion of stem cambial initials to lignotuber-type initials. The results lead to the following conclusions: outgrowth of lignotuber buds is suppressed by the seedling shoot; lignotuber buds do not form adventitiously. At a lignotuberous node, removal of stem or lignotuber cambium results in its replacement from the flanks of remaining lignotuber cambium. Stem cambium is not converted to lignotuber-type cambium by homeogenetic induction since it takes place also in the absence of lignotuber cambium.

Introduction

The lignotubers which develop at the cotyledonary and a few succeeding nodes of certain eucalypts are, as their name implies, expansions of woody tissue produced by a cambium developed initially across the cotyledonary or other leaf gap in the vascular cylinder of the primary stem (Carr et al. 1984). The seedling lignotuber cambium differs from the stem cambium in a number of ways, including the size and frequency of ray initials, the lengths of the fusiform initials and the composition of the xylem produced (Carr et al. 1984). The lignotuber bears many dormant buds that owe their origin to proliferation from accessory buds of the node (Carr et al. 1984); some authors (Kerr 1925; Chattaway 1958) have concluded, however, that some of the lignotuber buds are formed de novo from tissues of the cortex or secondary phloem.

After initiation of the lignotuber, conditions favouring persistence and continued growth of the seedling stem tend to depress further expansion of the lignotuber but loss of or injury to the seedling stem favours outgrowth of lignotuber buds and further expansion of the lignotuber itself. Outgrowth of the lignotuber buds thus appears to be dominated by the seedling shoot. A similar set of dominance relations appears to regulate the relationship between growth of the stem between the lignotubers at a given node and growth of the lignotubers themselves. If the activity of the stem cambium predominates, the lignotubers may cease expanding and eventually seem to regress. If, however, the activity of the lignotuber cambium predominates, the lignotubers may fuse laterally, surrounding the stem which bore them (Carr et al. 1984). Downwards and lateral expansion of the

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D. J. Carr et al.

lignotuber appear to take place by conversion of stem cambial initials into lignotuber cambial initials, possibly by homeogenetic induction (Carr et al. 1984).

Experiments involving surgical treatments were carried out using seedlings of Eucalyptus sf john11 to test these various hypotheses.

Materials and Methods

The plants were raised from seeds collected from a tree of E. st johnii on the A.N.U. campus. Seedlings were raised in soil in a greenhouse and plants were selected for experimental treatments when 18 months old and having well developed lignotubers at the cotyledonary node and at the node cf the first pair of !eaves. The plants wcrc ;€potted peiiodical!j;, e\;eniua::j; to pots 25 cm iii diam. Additional nutrients were supplied periodically. When 30 months old, the plants were transferred to a shadehouse. For the purposes of continued observation and measurement, lignotubers at the cotyledonary node were classified arbitrarily as 'right' or 'left'. Apart from untreated controls, three treatments were applied to the lignotubers: (1) removal of a horizontal strip, 4 mm wide, of cortex and bark down to the wood, i.e. ring-barking, from one of a pair of lignotubers; (2) total removal, i.e. amputation, of one or both of a pair of lignotubers; (3) removal of the cortex and bark, down to the wood, from both sides of the stem between a pair of lignotubers. Vernier calipers were used to measure to the nearest millimetre the growth of the lignotubers.

Table 1 summarizes the increases in growth of lignotubers during c. 13 months from the time of treatment, 31 March 1980-21 April 1981.

Table 1. Growth in lignotuber length during 13 months following surgical treatments: comparison with untreated controls

Treatment No. of Mean growth s.d. d.f. P replicates (cm) (t test)

Control Left lignotuber 20 1 20 0 18 Right lignotuber 1 .18 0 .39

Ring-barking of one lignotuber (B) of a pair (A) 10 1 60 0 .55

(B) 10 1 20 0 38 Removal of stem cambium 20 1 57 0 .65

between lignotubers Excision of one 10 2 07 0 .44

lignotuber of a pair

Results

In the controls (Fig. l), the pair of lignotubers at the first leaf node fused laterally around the node and with the laterally fused lignotubers at the cotyledonary node. The latter also grew downwards, assimilating the hypocotyl.

As expected, the lignotubers of a pair in the untreated controls showed no statistically significant differences in downward growth (Table 1). Within 2 weeks of ring-barking one of a pair of lignotubers. lignotuber buds grew out on its lower half, just below and central to the ring (Figs 2 and 4): none of the buds on the upper half grew out. The buds withered and abscised within 4 weeks. The upper part of the lignotuber, above the ring, subsequently grew much less (Figs 3, 5 and 6) than the lower part, which usually but not invariably (Figs 5 and 6) maintained a growth rate comparable to that of the controls (Fig. 3). On the other hand. ring-barking one lignotuber significantly increased the growth rate of its opposite partner, as compared with the controls (Table 1, Figs 3 and 5 ) . The wounded surface became covered with tissue derived from the adjacent lignotuber cambium. The

Surgical Experiments on Eucalypt Lignotubers

Fig. 1. Lignotubers of control plant 18 months after experimental treatments. S, seedling stem; N, lignotuber at first seedling node.

Figs 2 and 4. Buds developed on lower halves of ringed cotyledonary lignotubers (arrows).

Figs 3 and 5. Lignotubers 18 months after ring-barking. R, scar left by ring-barking.

Fig. 6. Median longitudinal section through the lignotuber of Fig. 5. Arrow shows site of ring-barking. t , Vascular traces of lignotuber buds. Scales: 1 cm.

D. J. Carr et al.

Surgical Experiments on Eucalypt Lignotubers

connection between the cambia of the upper and lower halves of the lignotuber was eventually restored and continuity of the xylem and phloem across the wound reestablished, but growth over the wounded area was slow.

Removal of the strips of stem cambium lying between the lignotubers at a node resulted in a significant increase in the rate of growth of the lignotubers, as compared with controls (Table I, Fig. 7). On the other hand, judging by the constriction evident on cross sectioning, lateral fusion of the lignotubers was delayed by the treatment (Fig. 8).

Excision of one lignotuber of a pair resulted in a twofold compensatory growth of the opposite lignotuber. A year or so after the treatment, the wound surface had been covered completely, or mostly, by scar tissue (Fig. 10). Some of the plants so treated, in which the wounded surface had become compieteiy covered with new tissue, were decapitated above the node to see whether buds would grow out of the newly formed surface. Buds grew out from the remaining lignotuber but not from the tissue over the wound surface. In some cases, once the wounded surface had become covered by callus tissue, little or no further outward expansion of the tissue over the site took place (Figs 12 and 13). However, in other cases, some further expansion (e.g. Fig. 9) was produced by the activity of a new cambial layer contiguous with and of the same nature as the cambium of the intact lignotuber which had incorporated the intervening stem cambium (Fig. 11). This development completed the encirclement of the node by lignotuber tissue; the lignotuber also expanded downwards over the hypocotyl (Fig. 9).

Following amputation of both lignotubers. the wounds completely or almost completely healed over and in a few cases little or no further development took place. In most cases, however, wound healing was followed by the formation of a bulbous swelling, simulating a laterally fused pair of lignotubers (Figs 14, 16 and 17). Transverse sections of such swellings (Figs 15 and 18) demonstrate their origin and development. The stem cambium proliferates, its derivatives extending over the wounds and eventually covering them (Fig. 15). At the same time, the nature of the cambium itself changes; it comes to resemble lignotuber cambium, producing xylem indistinguishable from that of a lignotuber. Buds are not formed and bud traces are not apparent within the xylem. In the case illustrated in Figs 16, 17 and 19, accidental damage to the stem on one side at the time of amputation of both lignotubers led to an eccentric swelling in which the lignotuber-like cambium produced from the stem cambium on the undamaged side eventually extended all around the cotyledonary node. That the new tissue added at the cotyledonary node following lignotuber amputation is clearly lignotuberous in nature is clearly revealed in its ability to fuse smoothly with the downward growth of the lignotubers at the first leaf node (Figs 20 and 21) as well as to take part in the lignotuberous swelling of the hypocotyl (Figs 20 and 2 1).

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Figs 7 and 8. Cotyledonary lignotubers 18 months after removal of the stem cambium (arrows in Fig. 7). Fig. 8. TS showing scars left by the operation (arrows).

Figs 9-13. Development following removal of one of a pair of lignotubers. N, lignotuber at first leaf node; H, thickened hypocotyl.

Figs 9 and 10. Regrowth of lignotuber (*) observed 18 months (Fig. 9) or 6 months (Fig. 10) after the operation.

Fig. 11. TS of lignotuber shown in Fig. 9: arrows indicate the scar left by the operation. Beyond the scar, fresh tissue has developed from a cambium originating at the flanks of the cambium of the intact lignotuber.

Figs 12 and 13. Scar (* in Fig. 12, arrows in Fig. 13) left by amputation of a single lignotuber, but little or no further overgrowth (Fig. 13). Scales: 1 cm.

D. J. Carr et al.

Figs 14-18. Development following amputation of both cotyledonary lignotubers. Swellings or 'replacement lignotubers' (*) 18 months after the operation.

Figs 14 and 15. Symmetrical replacement of amputated lignotubers. Fig. 14. One side of the cotyledonary node showing the replacement lignotuber. Fig. 15. TS across the cotyledonary node. Black arrows, scars left by the operation; white arrows, lignotuber-like tissue produced by a continuous cambial cylinder formed from the original stem cambium (c). The two arcs of cambium met at about the positions of the white arrows.

Surgical Experiments on Eucalypt Lignotubers

Discussion

The results support the hypothesis of a suppression of outgrowth of lignotuber buds by the seedling shoot, interrupted temporarily by ring-barking. Tissue continuity is quickly restored to an extent at least sufficient to restore the inhibition. Removal of the strip of stem cambium from between lignotubers of a pair delays their lateral fusion. This supports the view (Carr et al. 1984) that lateral fusion takes place by conversion of the stem cambium into lignotuber cambium; where the stem cambium is removed, the adjacent lignotuber cambia first produce tissue that covers the wound, then fuse laterally over it. It could be supposed that conversion of the stem cambium to lignotuber cambium takes place gradually by homeogenetic induction from the flanks of the lignotuber cambium, contiguous with the stem cambium. This hypothesis is not supported by the amputation experiments. When both lignotubers of a node are amputated, effectively removing all the lignotuber cambium, the stem cambium still converts to lignotuber cambium which then produces a woody swelling, identical in constitution to a laterally fused pair of lignotubers except for the complete absence of lignotuber buds.

There are two important corollaries. (1) We have previously (Carr et al. 1984) failed to find evidence to support the hypothesis (Chattaway 1958) that lignotuber buds arise adventitiously from cells of the cortex or secondary phloem. The failure of buds to form on replacement lignotubers is evidence against the hypothesis. (2) The activity of the lignotuber cambium in producing the woody swelling that constitutes the mass of the lignotuber is not itself dependent on the presence of buds, although, in the seedling, lignotuber initiation is dependent on the presence of a suite of accessory buds (Carr et al. 1983, 1984). It may well be that once a lignotuber has been initiated at a node its continued growth is independent of the presence or proliferation of buds. This could be tested by excision of the suites of accessory buds from sufficiently young seedlings, an operation which presents considerable difficulties. The lignotuberous node may also be 'programmed' to have a 'lignotuber' develop even if the original lignotuber cambium is totally removed, another instance of the 'position effect' of lignotuber production (Carr et al. 1982). Since the replacement lignotuber formed following amputation of the original lignotuber lacks buds, it also lacks one (perhaps the prime) function of a real lignotuber, the ability to produce replacement shoots. The question as to whether the lignotuber swellings consist merely of callus tissue can be answered in the negative: similar wounds on the stem do not result in such swellings, whether they are at a lignotuberous node (Figs 7 and 8) or on an internode (Kerr 1925; Carr et al., unpublished observations).

Removal of the stem phloem does not interfere with translocation, which still takes place in the secondary phloem of the lignotuber and, until an age of about 18 months at least, in the internal phloem (Jahnke 1983). Removal of one lignotuber of a pair, or its ring-barking, results in extra growth of the opposite lignotuber. This compensatory growth is undoubtedly due to the redirection of materials previously shared between both lignotubers to the remaining intact one. There must therefore be some competition between the lignotubers at a node for nutrients for their growth. The decussate phyllotaxis of early seedlings of eucalypts such as E. st johnii implies that the two lignotubers at a node are likely to be of equal initial status and growth capacity but frequently the members of a pair do not develop equally (Figs 1, 10, 12, 17 and 20).

Figs 16 and 17. Asymmetrical replacement (*) of amputated lignotubers. Fig. 16. Replace- ment lignotuber at the cotyledonary node). Fig. 17. Same plant photographed at right angles to Fig. 16. Regions indicated by arrowheads in Figs 17 and 18 correspond.

Fig. 18. TS of the replacement lignotuber in Figs 16 and 17. Accidental damage to the stem cambium on one side during the operation (arrows). Extent of tissue replacement on the damaged side (arrowheads) from a continuous cambium ring derived almost entirely from the stem cambium on the undamaged side. Extent of outgrowth of the replacement cambium at an early stage (*). Scales: 1 cm.

D. J. Carr et a1

Figs 19-21. Replacement lignotubers at the cotyledonary node 18 months after amputation of both the original lignotubers.

Fig. 19. Replacement lignotubers (*) fused with the (single) lignotuber at the first leaf node ( N ) .

Figs 20 and 21. LS of the plant shown in Fig. 19. Fig. 20. Scars (smallarrows) left by amputation of both cotyledonary lignotubers. Extent of replacement lignotubers (large arrows); note that they

Surgical Experiments on Eucalypt Lignotubers

References Carr, D. J., Carr, S. G. M., and Jahnke, R. (1982). The eucalypt lignotuber: a position-dependent

organ. Ann. Bot. (London) 50, 523-50. Can; D. J., Jahnke, R., and Carr, S. G. M. (1983). Development of the lignotuber and plant form

in Lehrnannianae. Aust. J. Bot. 31, 629-43. Cam, D. J., Jahnke, R., and Carr, S. G. M. (1984). Initiation, development and anatomy of lignotubers

in some eucalypts. Aust. J. Bot. 32, 415-37. Chattaway, M. M. (1958). Bud development and lignotuber formation in Eucalyptus. Aust. J. Bot.

6 , 103-15. Jahnke, R. S. (1983). Studies on the eucalypt lignotuber. M.Sc. Thesis, Australian National University. Kerr, L. R. (1925). The lignotubers of eucalypt seedlings. Proc. R. Soc. Victorla 37, 79-97.

Manuscript received 7 November 1983, accepted 16 March 1984

have fused below with the lignotuber-like thickening of the hypocotyl. t , Vascular traces of lignotuber buds. Fig. 21. Detail of Fig. 20. Lignotuber-like thickening produced from the newly developed cambium (white arrow), following overgrowth of scars by tissue (*) derived from the stem cambium. The replacement lignotuber tissue is continuous with that of the down-growing lignotuber at the first leaf node (black arrow). Scales: 1 cm.