**7. Slide preparation**

Before starting this step, it is important to treat the slides, as in 6 N HCl for at least 6 h, for instance. Then, wash the slides under flowing water for 15 min, dip in distilled water and store in absolute ethanol until use.


**7. Slide preparation**

12 Plant Breeding from Laboratories to Fields

microscope.

slip (Fig. 5J).

store in absolute ethanol until use.

Before starting this step, it is important to treat the slides, as in 6 N HCl for at least 6 h, for instance. Then, wash the slides under flowing water for 15 min, dip in distilled water and

**2.** Digest the root tips in a 2% cellulase and 20% pectinase solution at 37 °C for 30-90 min, depending on the potential of the enzymatic activity. Digest only one root tip per slide by using high quality enzymes. Use a stereoscopic microscope to remove the root cap,

**3.** Wash the digested meristems twice with distilled water for 5 min each. For each wash, dry the root tips with filter paper without touching the digested material to avoid dam‐

**4.** Add a drop of 45% acetic acid for, at least, 20 min (Fig. 5E). Afterwards, remove the ace‐ tic acid with filter paper and add a drop of distilled water either for 20 min (at least) at room temperature or overnight in a moisture chamber at 4-10 °C (in the refrigerator). Then, dry the root tips and add again a drop of 45% acetic acid and maintain the slides

**5.** With the aid of a stereoscopic microscope, disrupt completely the meristem with two histological needles, (Fig. 5F). Do not let the material dry. If this starts to happen, add carefully a little more 45% acetic acid. It is important to notice that the quantity of acetic acid used during this step is very critical for obtaining high quality preparations. Too much acid will lead to the loss of material; if too little acid is used, there will be air bub‐

**6.** Put an 18×18 mm glass coverslip over the material and tap gently with a blunt tip nee‐ dle (Fig. 5G-H). While tapping, hold the coverslip with a piece of folded filter paper to avoid cell damage caused by slippage of the coverslip. In a bright field optical micro‐ scope, always observe the distribution of the material after and before tapping. This measure is important to determine how intense the tapping must be. A too intense tap‐ ping may break drastically the cells and cause chromosome losses. On the other hand, if the tapping is too weak, the material will not spread properly. The ideal condition is when the cells are broken, but all chromosomes are still in a same field of view in the

**7.** Heat the preparation in an alcohol Bunsen burner ca. three times, carefully to prevent

**8.** Then smash the material thoroughly. Put the slide-coverslip set within two sheets of folded filter paper and press with a thumb always taking precaution not to move cover‐

**9.** Dip the slide-coverslip set in liquid nitrogen for approximately 3 min (Fig. 5K).

aging it (Fig. 5D). Add a drop of distilled water carefully with a Pasteur pipette.

in a moisture chamber until use. Follow the step 5 for each slide individually.

bles, which will negatively affect the quality of the slides.

boiling (Fig. 5I). Fell the temperature with the back of the hand.

**1.** Wash fixed root tips twice in a Petri dish with distilled water for 5 min each.

add a drop of the enzymatic solution (ca. 5-10 μL) and incubate at 37 °C.

**12.** Store the slides at -20 °C (or, if possible, at -80 °C) for an indefinite period until the *in situ* hybridization procedures. The results of the GISH will be better when newly pre‐ pared slides are used.

**Figure 5.** Preparation of slides for genomic *in situ* hybridization. (A) Full length root tips after washing in distilled wa‐ ter. (B) Root meristem to be digested. (C) Enzymatic digestion. (D) Removal of the enzymatic solution. (E) Addition of 45% acetic acid. (F) Disruption of the root meristem, with histological needles in a stereoscopic microscope. (G) Addi‐ tion of an 18×18 mm coverslip. (H) Gentle tapping with a blunt tip needle. (I) Quick heating of the preparation in an alcohol Bunsen burner. (J) Squashing of the slide-coverslip set with two sheets of filter paper. (K) Dipping the slidecoverslip set in liquid nitrogen. (L) Quick removal of the coverslip. A, B, C, F, H, J, K and L: Sandra P. Brammer; D, E, G and I: Ana R. Oliveira & Ana C. Brasileiro-Vidal.
