Axillary meristems in the Wollemi pine: a response to Geoffrey E. Burrows

We appreciate the opportunity, provided by the Chief Editor, to respond hermeneutically to the critique by Burrows (2012) of our paper on branch regeneration in plagiotropic axes of Wollemia nobilis, where we claim that reiterated axes do not necessarily arise from axillary growth centres referred to as ‘axillary meristems’ (Tomlinson and Huggett, 2011). Our observations did not indicate that these existed in our material prior to manipulative techniques that generated new shoots behind decapitated axes. This statement does run counter to the description by Burrows (1999) in material he examined. Here he claims that such growth centres are ubiquitous in Araucariaceae as he has extensively documented in other papers. Burrows suggests that our techniques (we used thick sections of unembedded material) are inadequate to reveal groups of cells at most 100 µm across, although it is admitted that our source material could differ from his. The high starch content of our material could be a contributory factor. But we see no reason why our method should not reveal axillary meristems in view of their recognition in orthotropic axes by Tomlinson and Murch (2009) using this approach. Fink (1983) also used sections up to 80 µm thick in his initial discoveries. The claim that ‘axillary meristems’, because they have been demonstrated in a diversity of axis types of other Araucariaceae (Agathis and Araucaria), must be universally present in all leaf axils in Wollemia is not an unreasonable assumption, but it is still an assumption in need of further testing, especially in view of the distinctive architecture and reiterative ability of Wollemia contrasted with Agathis and Araucaria (Grosfeld et al., 1999; Tomlinson, 2009). We also emphasize that our observations have been made on juvenile material of the type we illustrate and do not necessarily apply to adult structures, which are still in need of detailed description. We believe that our cautious assessment is justified. Therefore we adopted a novel approach based on the demonstration that decapitated shoots could readily generate axillary branches, often in a basipetal sequence that suggests the universality of nodal behaviour along an axis. An aspect of the plagiotropic branch in Wollemia is the apparent preformation of all leaves in one unit within the terminal bud, which is a possible developmental contribution to nodal consistency. Our approach was then to section serially in such a way that we worked backwards along decapitated shoots of differing ages so that a sequence from older to younger stages could be observed, continuing into more proximal regions where branch initials were not externally visible and where the earliest stages could be recognized. One must emphasize that the leaf insertion becomes twisted as the shoot extends, so the node is very oblique to the long axis of the shoot, requiring many sections. The earliest stages should have led to the recognition of the source of induced branch meristems. It is important to recognize that despite the growing literature on the existence of these ‘axillary’ or ‘detached’ meristems, no detailed effort has gone into charting in detail the vascular connection that is eventually made between parent axis and the subsequent branch types once a putative meristem is reactivated by some mechanism. We did this for plagiotropic axes in Wollemia, thereby demonstrating how a superficial growth centre, induced by our manipulation, could make a vascular connection across a rather complex cortex to the parent vascular system. This began from the group of apparently de-differentiated cells that become divided into a distal outer group of meristematic cells, which originate the new shoot, and a proximal inner group of contrasted vacuolated cells from which procambium-like strands originate the ultimate connection. In Wollemia the ingrowth is necessarily complex and indirect because of the unusual histology of the cortex, consisting of a network of small living cells, easily recognized in our material by their starch content, within a matrix of large empty cells of unknown function. How this process relates to the anatomy of other Araucariaceae remains to be explored. The tendency for a ‘one size fits all’ attitude towards branching in conifers should be resisted. From the earliest literature (Henry, 1846) it has been recognized that conifers produce relatively few axillary buds on a shoot. But unexplained was their ability to elaborate adaptive architectural features by axis reiteration. It required the precise and meticulous observations of Fink (1983, 1984) to demonstrate the existence of axillary groups of meristematic cells in conifers (and some angiosperms) by means of which the distinction between ‘preventitious’ and ‘adventitious’ buds (Hartig, 1878) could be recognized. This approach has been well extended by Burrows in Araucariaceae. However, there is some evidence, as we cited, that developmental processes in the leaf axils of conifers can involve both differentiation and de-diffentiation of cells, as well as the persistence of ‘detached’ elements originating from the shoot apex. Furthermore, we cannot totally dismiss the fact that Holthusen (1940) could not find them in the material he examined, because they might not have been there! He did, after all, use paraffin sections 10–15 µm thick, well within the reported range used in identifying them subsequently. Branching of many kinds is the most important component in the development of canopy structure in modern forests, but its diversity and especially the attachment of branch to parent axis is an almost completely neglected field of plant morphology and anatomy. We congratulate Burrows on his critique of the apparent deficiences of our paper as it should surely help to inform botanists not merely of controversial matters, but of a field of endeavour in which there is unlimited scope for original research using innovative and diverse methods. The primary distinction between preventitious and adventitious needs to be made, the contrast between proleptic and sylleptic branches (Halle et al., 1978) clearly recognized, and the contrast between architectural and reiterative features discerned. The relation between parent and branch axis type must be consistently recognized. We cannot but agree with Fink (1983) when he says ‘Only detailed ontogenetic study can hence clarify the origin of a bud in every single case’ (our italics).