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

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Presentation on theme: "Troublesome material: how to deal with? Jan Suda Department of Botany, Faculty of Science, Charles University Institute of Botany, Academy of Sciences."— Presentation transcript:

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. H 2 O4.2 g Tween 201 ml Otto II Na 2 HPO 4. 12H 2 O28.65 g Water to 200 ml L B 0 1 b u f f e r Tris363.4 mg Na 2 EDTA148.9 mg spermine. 4HCl34.8 mg KCl1.193 g NaCl233.8 mg Triton X μl Water to 200 ml, pH 7.5 Tris - MgCl 2 b u f f e r Tris 4.84 g MgCl 2. 6H mg Triton X-1001 ml Water to 200 ml, pH 7.5 M a t z k b u f f e r Tris g MgCl 2. 6H g NaCl0.5 g Triton X 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 T i s s u e s e l e c t i o n 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 petioles, young stems, inflorescence axes, cotyledons cotyledons stems, very young leaves ??? (seeds) youngest (in-rolled) leaves young stems

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

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

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