Troublesome material: how to deal with?

1. Troublesome material: how to deal with? Jan Suda Department of Botany, Faculty of Science, Charles University Institute of Botany, Academy of Sciences of the Czech Republic

2. Steps towards the improvement 1) choosing of another isolation / staining buffer 2) inclusion of antioxidants or other preservatives 3) selection of different plant tissue 4) employment of different technique (image cytometry)

3. B u f f e r s e l e c t i o n O t t o b u f f e r s Otto I Citric acid . H2O 4.2 g Tween 20 1 ml Otto II Na2HPO4 . 12H2O 28.65 g Water to 200 ml L B 0 1 b u f f e r Tris 363.4 mg Na2EDTA 148.9 mg spermine . 4HCl 34.8 mg KCl 1.193 g NaCl 233.8 mg Triton X-100 200 μl Water to 200 ml, pH 7.5 Tris - MgCl2 b u f f e r Tris 4.84 g MgCl2 . 6H20 162.64 mg Triton X-100 1 ml Water to 200 ml, pH 7.5 M a t z k b u f f e r Tris 1.211 g MgCl2 . 6H20 0.107 g NaCl 0.5 g Triton X-100 0.1 ml Water to 200 ml, pH 7.5

4. C h e m i c a l p r e s e r v a t i v e s Antioxidants 2-mercaptoethanol (2μl / ml) Different fluorochrome DAPI generally less problematic Preservatives PVP-10, PVP-40 (min. 10 mg / ml) Rosaceae (Rosa, Rubus)

5. Plant group tannins (Marcetella) mucilaginous compounds (Viola, Lythrum, Lavatera, Ocotea) organic acids (Aeonium, Monanthes) ??? (Boraginaceae - Echium) Reynoutria (tannins, organic acids?, only with PI) Teline, Spartocytisus, Chamaecytisus Helianthemum (mucilaginous compounds) Geranium (tannins) Improvement cotyledons petioles, young stems, inflorescence axes, cotyledons stems, very young leaves cotyledons youngest (in-rolled) leaves young stems ??? (seeds) T i s s u e s e l e c t i o n

6. leaves cotyledons Echium leaves young stems Lythrum H i s t o g r a m s d e m o n s t r a t i o n

7. T r o u b l e s o m e h i s t o g r a m Dactylorhiza