2.3 Stem anatomy
Branches were classified according to both diameter and stiffness (see Figure 1): < 2 mm (primary growth, flexible), < 2 mm (stiff and twisted around a support), 6-10 mm (green shoots with secondary growth), 11-15 mm (stems at the base of the vine with highly twisted-irregular bark). First, we evaluated the general anatomy of the stems in transverse sections at the top three axial locations (the widest stems could not be cut transversely to preserve the plants): flexible searchers (2mm diameter), stiff thin branches (2mm diameter), and mature stems (6-10mm diameter). Stem material was maintained in 1X Tris-buffered saline (TBS), sectioned transversally at 50 μm with a Reichert-Jung Hn-40 sliding microtome (Austria), mounted onto glass slides, and stained with aniline blue that binds to callose of the sieve tubes, or with 0.1% of acridine orange in TBS buffer, which yields fluorescence of the lignified tissues (Robertson et al., 1992). To sample the phloem tissue, the external side of stems containing the bark were extracted with a sharp knife at the different positions described above, kept in buffer, sectioned longitudinally with a microscalpel, and stained with aniline blue. We measured the length and width of at least 30 sieve tubes per sampling location (total n=107), and the number of sieve areas per compound plate (total n=45).