5.4.1.3.1 Macroscopic features of transition zone
At the time of first sampling, the above-ground component of seedlings of G. straminea consisted of a rosette of leaves (Figure 5.3A). Based on the fact that none of the seedlings had leaf scars or intact leaves on the stem-like structure below the rosette, this compressed and thickened structure in-between the root and the rosette (Figure 5.3A, B & D) was tentatively identified as the transition zone, i.e. region between the true stem and root (refer Section 5.4.1.3.2 for histological confirmation). Towards the end of the second and third growth cycle, many crown buds were visible in the transition zone, as both individual buds and clusters (Figure 5.3B, C, E & F). During the course of this study, some of these crown buds developed into shoots (Figure 5.3E). Although crown buds arising from the transition zone were the primary source of buds evident, axillary buds were visible occasionally within the axils of the earliest formed leaves, and were different to the crown buds arising from the transition zone in both size and shape (Figure 5.3C).
Longitudinal sections along the shoot/stem axis of intact seedlings of G. straminea, permitted the rosette, transition zone, and primary root, to be visible (Figure 5.3G & H) macroscopically. As described in the following section, the region indicated by an ‘x’ within Figure 5.3H was used for further microscopy. In terms of the visual appearance of macroscopic features, such as the transition zone and presence of crown buds, seedlings of G. lutea, G. triflora and G. scabra (Appendix III) were similar to that noted for the seedlings of G. straminea (Figure 5.3A-G). One additional feature noted with plants of G. scabra and G. triflora however, was that they also developed crown buds on the proximal region of storage roots (Appendix III).
Figure 5.1 Transverse section of (A-C) floral shoot and (D-F) storage root of ‘Spotlight’ at increasing magnification; (A) full section indicating vascular ring, (B) magnified view of the vascular ring (red coloured square in Figure 5.1A), (C) arrangement of xylem vessels and phloem fibers, (D) full section of secondary storage root, (E) magnified view of secondary storage root indicating xylem of vascular tissues in the center, (F) full section of a primary storage root. (G) Radial arrangement of xylem vessels. As indicated by staining with Toluidine Blue, tissue stained light blue are xylem vessels: c – cortex, e – epidermis, p – pith, ph – phloem, vc - vascular cambium, vr - vascular ring, vy – vascular rays, x – xylem.
Figure 5.2 Floral shoot of ‘Spotlight’ sampled in Nov 2007 (late spring); (A) the region highlighted by red coloured square from which (B - I) present an acropetal sequence of transverse sections, including the node and associated axillary buds, indicating; (B-E) changes in vasculature of the main shoot and, (F-G) development of leaf base and bud primordia. As indicated by staining with Toluidine Blue, tissue stained light blue are xylem vessels: ab - vascular tissue of axillary bud, c – cortex, L – leaf, lb – vascular tissue of leaf base, lp - leaf primordia, ms - vascular tissue of main shoot , p - petal primordia, sp - sepal primordia, v - vascular ring.
A B C cb ab cb E ns F cbc Beginning of second growth
cycle
Beginning of third growth cycle D H X Shoot Rosette Primary root G
First growth cycle
tz
Figure 5.3 Seedlings of Gentiana straminea; (A-C) in the beginning of the second
growth cycle from germination, (D-F) in the beginning of the third growth cycle, and (G and H) in their first growth cycle; (A) whole plant with region highlighted by orange coloured parenthesis magnified in subsequent images, (B) magnified view of transition zone with buds, (C) buds within leaf axils and transition zone, (D) whole crown region with an axillary bud above transition zone, and magnified in (E) showing the base of an emerging new shoot from transition zone, and (F) a crown bud cluster arising from the transition zone. (G) intact and, (H) longitudinal, hand-cut sections through the stem axis of juvenile seedlings in their first growth cycle indicating, area taken for microscopic sections presented in Figure 5.4, Figure 5.5, Figure 5.6 denoted by ‘X’. ab – axillary bud, cb – crown buds, cbc - bud cluster, ns - new shoot from transition zone, tz - transition zone.
5.4.1.3.2 Histological features of transition zone
As evident in other plants, the transition zone of gentians has vascular characteristics between those observed in a shoot/stem and root, in that vascular bundles gave the appearance of gradually diverging from the centre with progressive sampling positions of transverse sections from the root towards the shoot (Clegg and Cox, 1978; Roland and Roland, 1980). In seedlings of G. straminea, at the root end vascular tissue were
centrally located (Figure 5.4B-D), with scattered xylem vessels occupying the central stele (Figure 5.4D). For sections of the transition zone from the shoot-end, the vascular tissue was visible surrounding the pith (Figure 5.4E, F, & K). Based on the details of histological features presented below, it is proposed that the vascular tissues were beginning to diverge from the centre with progression towards the stem (Clegg and Cox, 1978). Compared to transverse sections from the shoot of ‘Spotlight’ (e.g. Figure 5.1A), and as further evidence of morphological differences of the transition zone, the vascular bundles within this region of the transition zone were not arranged as a ring, but more in the shape of a square (Figure 5.4F). Arrangement of xylem vessels as four separate groups was visible in the central column of vascular bundles (Figure 5.4E & K). There were outward extensions of xylem vessels from the four corners of these groups of xylem tissue (indicated by ‘X’ in Figure 5.4E & K), each terminating in clusters of unidentified cells of greater density close to the epidermis (Figure 5.4E-J). These results were therefore similar to those reported within the transition zone for Helianthus sp. where six vascular groups were observed (Clegg and Cox, 1978) and, in Arabidopsis, two groups (Busse and Evert, 1999). In G. straminea, bud initiation was visible arising from groups of cells in each of the four corners of the square-shaped vascular cambium (Figure 5.4K-R and Figure 5.5B-D). In some cases, several bud initials were being initiated from a single corner (Figure 5.5B). Within the transition zone, where these buds/bud initials were present, vascular traces arising from the primordia or scales were not visible however. At any one time, in transverse sections of the transition zone, while not usually macroscopically visible, buds were found to be at differing degrees of development, from bud initials through to buds with well- developed primordia (Figure 5.4K-R and Figure 5.5B-D). As evident from acropetal sectioning of one bud which was also visible macroscopically, bud clusters were clearly attached as an external outgrowth to the transition zone (Figure 5.4L - Q). Near the rosette of leaves in the transition zone of G. straminea, the vascular bundles were spread among the parenchyma cells of the shoot (Figure 5.4S - U) as observed in Arabidopsis (Busse and Evert, 1999), and are likely to be the traces of leaves that form the rosette (Busse and Evert, 1999). Based on the histological observations therefore, the region below the leaf whorl in juvenile seedlings of gentians can be considered to be the transition zone.
Within longitudinal sections of young seedlings of G. lutea and G. straminea the undulations visible on the external surface of the transition zone (Figure 5.6A) were
visible as protrusions from the outer, corky periderm (Figure 5.6B). Buds with leaf primordia (Figure 5.6F, H and Figure 5.5F), and undifferentiated cell masses (Figure 5.6C-E, G & J), were present along the periderm of the transition zone. On the assumption that dividing cells are smaller in diameter (Chiatante et al., 2008), it is hypothesised that the undifferentiated cell masses observed within the current study are potential sites for initiation of crown buds. In longitudinal sections, these putative meristematic zones could be identified as regions of high cell density, arranged on the periderm of the transition zone, each at different stages of development (Figure 5.6C-J). Some bud initials were embedded within the periderm (Figure 6E, G & J), while others protruded (Figure 5.6C-D, F & H). Within microtome and hand-cut sections, vascular traces could be seen extending from well-developed buds to the central vascular tissue (Figure 5.3H & Figure 5.5F). In some plants flattened axillary buds were sometimes visible at the basal node of the shoot, whereas crown buds were located just below, originating within the transition zone (Figure 5.3C).
The confirmation of the presence of the transition zone and the understanding of the histological features present and associated with bud initials, can now be used for contrasts between true seedlings and gentian plants propagated from tissue culture, in subsequent discussions involving the origin and development of crown bud clusters.