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Troublesome material:how to deal with?

Jan SudaDepartment of Botany, Faculty of Science, Charles University

Institute of Botany, Academy of Sciences of the Czech Republic

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Steps towards the improvementSteps towards the improvementSteps towards the improvementSteps 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)

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B u f f e r s e l e c t i o n B u f f e r s e l e c t i o n B u f f e r s e l e c t i o n 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

C h e m i c a l p r e s e r v a t i v e sC h e m i c a l p r e s e r v a t i v e sC h e m i c a l p r e s e r v a t i v e sC 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)

T i s s u e s e l e c t i o n T i s s u e s e l e c t i o n T i s s u e s e l e c t i o n 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

cotyledons

stems, very young leaves

??? (seeds)

youngest (in-rolled) leaves

young stems

H i s t o g r a m s d e m o n s t r a t i o nH i s t o g r a m s d e m o n s t r a t i o nH i s t o g r a m s d e m o n s t r a t i o nH i s t o g r a m s d e m o n s t r a t i o n

Lythrum

Echium

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leaves

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leaves

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cotyledons

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young stems

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

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G 2

? ? ?

D acty lo rh iza

Dactylorhiza