In situ Hybridization in Apple Roots

Harvested roots of GL-3 with a diameter of 0.5 mm were cut into 1-mm segments and fixed immediately in 4% (w/v) paraformaldehyde in 0.1 m phosphate-buffered saline buffer (pH = 7) for 4 h at room temperature and then overnight at 4°C. Root segments were then rinsed three times with water, dehydrated in a graded ethanol series (75%, 85%, 95%, and 100%, v/v), embedded in paraffin, and then processed into 10-µm sections using a microtome (RM2125RTS, Leica, Germany). Finally, sections were collected onto po-Lys slides, dried on a hot plate at 45°C for 3 h, and then overnight at 37°C, for complete drying. Prepared slides were store at −80°C until used.

cDNA fragment of MdMYB88 and MdMYB124 was cloned into a pST19 vector with the primers listed in Supplemental Table S1, resulting in MdMYB88/124-pST19. MdMYB88/124-pST19 plasmid DNA was transcribed in vitro and labeled with DIG (Digoxin) using a DIG labeling kit (Roche, Switzerland). For hybridization, the probes were hydrolyzed in carbonate buffer (0.04 mm NaHCO₃ and 0.06 mm Na₂CO₃) to 100- to 150-bp fragments, precipitated in 70% (v/v) ethanol, and dissolved in DEPC (Diethylpyrocarbonate)-treated water to a final concentration of 1 ng/μL. In situ hybridization was performed as described by Omori et al. (2009).