Chrysoidine

For microscopy, the main stem of field-grown WT trees was harvested 1 m above soil level in August 2017 of the second growth season. At that time, the WT tetraploids already showed the brittle apex phenotype. Harvested parts were fixed in 70% (v/v) ethanol. Multiple transversal stem sections of 20 μm thickness were made per plant using a Reichert-Jung 2040 Autocut Microtome (Leica) and stained with 0.5% (w/v) astra blue, 0.5% (w/v) chrysiodine and 0.5% (w/v) acridine red for 10 min. To prepare the triple staining solution, 0.5% (w/v) astra blue (Santa Cruz Biotechnology) in 2% tartaric acid was mixed with 0.5% (w/v) chrysoidine (Sigma Aldrich) in 5% (w/v) ammonium aluminum sulfate and 0.5% (v/v) glacial acetic acid and 0.5% (w/v) acridine red (Santa Cruz Biotechnology) in 5% ammonium aluminum sulfate and 0.5% (v/v) glacial acetic acid in a 4:1:1 ratio. After dehydration in isopropyl alcohol, the stained sections were mounted in Euparal mounting medium (Carl Roth). Images were acquired using a Zeiss Axioskop 2 microscope and processed using automated software described by Andriankaja et al. (2012 (link)) to quantify the average number and average area of vessels and fibers per selected area. The number of vessels was divided by the number of fiber cells to provide a ratio. The proportion of vessel lumen was defined as the total vessel area per selected area.

Lignification of the shell was shown via Wiesner and Fuchsin-Chrysoidine-Astra blue (FCA) staining on whole nuts and thin sections. For the Wiesner solution, 20 mg/ml phloroglucinol (Sigma Aldrich) was first mixed with 20% ethanol and then with concentrated 12 M hydrochloric acid (v:v = 80:20). FCA solution was prepared by mixing 0.1 mg/mL of New Fuchsin (Roth), 0.143 mg/mL of Chrysoidine (Sigma Aldrich), 1.25 mg/mL of Astra blue (Roth) with acetic acid (v:v = 1:50). For the Wiesner staining, all nuts were halved with a razor blade (developing nuts) or a band saw (mature nuts) and placed in the Wiesner solution for at least 30 min. Immediately thereafter cross-sections were photographed. Thin sections of shell tissue cut by a cryo-microtome (CM3050 S, Leica) were stained for 5 min. For the FCA staining, thin sections were cut from shell tissues by cryo- (developing) and rotary (RM225, Leica) microtomes (mature) and stained for 30 min, followed by successive washing with distilled water and an ethanol series (30, 70, 30%). The images of the stained tissues were acquired using a Labophot-2 microscope (Nikon) in a bright field.