Celltram microinjector

The anthers were crushed in 2 µL of 6% sorbitol on a glass slide, as described above, and 30 microspores per cultivar were individually isolated in 0.2‐mL centrifuge tubes through a pulled glass capillary (Harvard Apparatus, Holliston, Massachusetts, USA) using a Cell‐Tram micro‐injector (Eppendorf, Hamburg, Germany) mounted on a Märzhäuser (HS‐6) micromanipulator (Narishige International, Tokyo, Japan). Each tube was then centrifuged at 5000 rpm for 5 min after phosphate‐buffer saline (8 g/L NaCl, 200 mg/L KCl, 1.44 g/L Na₂HPO₄, and 240 mg/L KH₂PO₄) was added to a final volume of 4 µL. Next, 6 µL of reconstituted extraction buffer from the REPLI‐g single cell kit (Qiagen) was added into the microcentrifuge tubes before a 10‐min incubation at 65°C. Afterward, 3 µL stop solution (Qiagen) was added, and then the WGA reaction was performed. The final 50‐µL reaction volume comprised 9 µL H₂O, 29 µL REPLI‐g Reaction Buffer (Qiagen), and 2 µL DNA polymerase (Qiagen). The tubes were then centrifuged and incubated at 30°C for 8 h in a thermocycler, followed by 3‐min inactivation of the polymerase at 65°C. The resulting MDA products were then quantified using a NanoDrop One spectrophotometer (Thermo Fisher Scientific).