Fig. 3

Illustration of the ADI method. The figures are modified with permission [17]. The anthers are treated with enzyme mixtures and incubated at 37 °C for 1–2 h. After incubation, the enzyme surrounding the anther is cleaned using a Kimwipe tissue. Fixative I solution (5-10 μl) is applied directly onto the anther (a). The anther is macerated as quickly as possible using dissected needles (b). Most of fixative I spreads out to the edge of the slide but partially remains around the cells. After a few minutes, the fixative I evaporates, leaving behind a thin layer, and the spherical cells floating in the fixative adhere to the slide by surface tension (c-f). If the humidity is less than 50%, most of the fixative evaporates before the cell becomes flat, and the chromosomes aggregate together (g). If the temperature is > 25 °C and humidity is > 80%, the fixative retracts again to the center of the slide (f). If the temperature is 20 °C with 65%–70% humidity, the evaporation speed of the fixative and flattening of the cell by surface tension are balanced (h). When the breakage of the fixative membrane covers approximately half of the area of the spreading cells (i), 5–10 μl of freshly prepared fixative II is added (j). Fixative I (60%, 1:1) then moves to the edge of the slide immediately (k) and is replaced by fixative II (absolute, 1:1). Fixative II should be applied before the breakage of the fixative membrane to allow expansion of the chromosomes. Fixative I accumulation at the edge of the slide can be removed with rolled filter paper (l). When the breakage of the fixative II membrane covers approximately half of the area of the spreading cells, 5–10 μl of freshly prepared fixative III is added (m), and fixative II moves to the edge of the slide (n). Fixative III is effective in removing cytoplasm and cleaning up the background of metaphase spreads. Fixative II accumulation at the edge of the slide can be removed with rolled filter paper (o) to dry the slide completely (p)