Centre of Plant Structural and
Functional Genomics
of the Institute of Experimental Botany AS CR

use deionized or double-distilled water in all recipes

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Hoaglands Nutrient Solution (Gamborg and Wetter 1975)

Solution A

H3BO3	280 mg
MnSO4 . H2O	340 mg
CuSO4 . 5H2O	10 mg
ZnSO4 . 7H2O	22 mg
(NH4)6Mo7O24 . 4H2O	10 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution B

H2SO4

0.5 ml

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution C

Na2EDTA	3.36 g
FeSO4	2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the colour turns yellow-brown 
Cool down, adjust the volume to 500 ml 
Store at 4°C

Hoaglands Stock Solution (10x)

Ca(NO3)2 . 4H2O	4.7 g
MgSO4 . 7H2O	2.6 g
KNO3	3.3 g
NH4H2PO4	0.6 g
solution A	5 ml
solution B	0.5 ml

Adjust volume to 500 ml with deionized H₂O 
Store at 4°C

Hoaglands Nutrient Solution (1x)

10x stock solution	100 ml
solution C	5 ml

Adjust volume to 1000 ml with deionized H₂O 
Prepare just before use

Hoaglands Nutrient Solution (0.1x)

10x stock solution	10 ml
solution C	0.5 ml

Adjust volume to 1000 ml with deionized H₂O 
Prepare just before use

Amiprophos-Methyl Solutions 
Stock Solutions (20 mM)

amiprophos-methyl

60.86 mg

Adjust volume to 10 ml with cold acetone 
Store at - 20°C in 1 ml aliquots 

Treatment Solution 
Prepare the treatment solution by adding specified volume of amiprophos-methyl stock solution to Hoaglands Nutrient Solution

B5 nutrient medium (Gamborg et al. 1968)

Solution A

H3BO3	300 mg
MnSO4 . H2O	758 mg
ZnSO4 . 7H2O	200 mg

Adjust volume to 1000 ml with deionized H₂O 
Store at 4°C

Solution B

Na2MoO4 . 2H2O	25 mg
CoCl2 . 6 H2O	2.5 mg
KI	75 mg
CuSO4 . 5 H2O	2.5 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution C

thiamine	100 mg
pyridoxine	10 mg
nicotinic acid	10 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution D

m-inositol

100 mg

Adjust volume to 100 ml with deionized H₂O 
Store at 4°C

Solution E

Na2EDTA	3.36 g
FeSO4	2.79 g

Adjust volume to approximately 400 ml 
Heat the solution to 70°C while stirring until the color turns yellow-brown 
Cool down and adjust the volume to 500 ml 
Store at 4°C

B5 Stock Solution (10x)

KNO3	30 g
MgSO4 . 7H2O	5 g
(NH4)SO4	1.34 g
CaCl2 . 2H2O	1.5 g
NaH2PO4 . H2O	1.5 g
solution A	100 ml
solution B	10 ml

Adjust volume to 500 ml with deionized H₂O 
Store at 4°C

B5 Nutrient Solution (1x)

10x stock solution	100 ml
solution C	10 ml
solution D	10 ml
solution E	5 ml

Adjust volume to 1000 ml with deionized H₂O 
Adjust the final pH to 5.5 
Sterilise by autoclaving

Tris Buffer

10 mM Tris	0.606 g
10 mM Na2EDTA	1.861 g
100 mM NaCl	2.922 g

Adjust volume to 500 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Prepare just before use

Formaldehyde Fixative

10 mM Tris	0.303 g
10 mM Na2EDTA	0.931 g
100 mM NaCl	1.461 g
0.1% v/v Triton X-100	250 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Add formaldehyde solution (37%, Catalog No. 1.04003, Merck, Darmstadt, Germany):

Species	Volume	Conc.
Field Bean	27 ml	4%
Pea	20 ml	3%
Chickpea	13.5 ml	2%
Vicia sativa	13.5 ml	2%
Barley	13.5 ml	2%
Oat	13.5 ml	2%
Rye	13.5 ml	2%
Wheat	13.5 ml	2%
Durum Wheat	13.5 ml	2%

Finally, adjust volume to 250 ml with deionized H₂O 
Prepare the fixative just before use

LB01 Lysis Buffer (Dolezel et al. 1992)

15 mM Tris	0.363 g
2 mM Na2EDTA	0.149 g
0.5 mM spermine . 4HCl	0.035 g
80 mM KCl	1.193 g
20 mM NaCl	0.234 g
0.1 % v/v Triton X-100	200 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH*) 
Filter through a 0.22 ľm filter to remove small particles 
Add 220 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 10 ml aliquots 
*) Please note that the original LB01 buffer was adjusted to pH 7.5. However, we have found later that the optimal pH differs for different applications. To distinguish the variants, we use the following abbreviations: 
LB01/7.5  pH adjusted to 7.5 using 1N HCl 
LB01/9  pH adjusted to 9 using 1M NaOH

LB01T Lysis Buffer (for sorting)

22.5 mM Tris	0.545 g
3 mM Na2EDTA	0.223 g
0.75 mM spermine . 4HCl	0.052 g
120 mM KCl	1.790 g
30 mM NaCl	0.351 g
0.15% v/v Triton X-100	300 µl

Adjust volume to 200 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N HCl 
Filter through a 0.22 ľm filter to remove small particles 
Add 330 ľl ß-mercaptoethanol and mix well 
Store at - 20°C in 5 ml aliquots

3:1 Fixative 
Mix 3 volumes of 96% ethanol with 1 volume of glacial acetic acid 
Prepare the fixative just before use

Schiff's reagent

1N HCl	30 ml
Parafuchsin (Serva, C.I.42500)	2 g
K2S2O5	3.8 g
deionized H2O	170 ml

Stir for 2 hours in a tightly closed bottle and leave to stay overnight 
Add 2 g of active charcoal 
mix 1 min and filter through a paper filter moistened with 1N HCl 
Repeat the filtration if the solution is not colourless 
Store in a tightly closed bottle at 4°C

Fructose syrup

fructose	30 g
deionized H2O	20 ml

Incubate the mixture at 37°C overnight 
Add a crystal of thymol 
store at 4°C

5N HCl

HCl - 35% (v/v)

419.8 ml

Adjust volume to 1000 ml with deionized H₂O

45% (v/v) acetic acid

acetic acid - 99% (v/v)

227 ml

Adjust volume to 500 ml with deionized H₂O

DAPI Stock Solution (0.1 mg/ml)

Dissolve 5 mg DAPI in 50 ml deionized H₂O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Mithramycin Stock Solution (1 mg/ml) 

Dissolve 50 mg mithramycin A in 50 ml deionized H₂O by stirring for 60 min. 
Filter through a 0.22 ľm filter to remove small particles 
Store at - 20°C in 0.5 ml aliquots

Sheath Fluid SF50

50 mM NaCl

7.31 g

Adjust volume to 2500 ml with deionized H₂O 
Sterilise by autoclaving

Magnesium Sulfate Stock Solution (100 mM)

Dissolve 1.23 g of MgSO₄ . 7H₂O in 50 ml of deionized H₂O. 
Filter through a 0.22 ľm filter to remove small particles 
Store at 4°C

PRINS Buffer

10 mM Tris base	0.605 g
50 mM KCl	1.864 g
2mM MgCl2.6H2O	0.203 g

Adjust volume to 500 ml with deionized H₂O 
Adjust the final pH to 8.0 using 1N HCl 
Sterilise by autoclaving 
Store at 4°C

PRINS Reaction Mix

10x DNA polymerase buffer*	5 µl
25 mM MgCl2 (4 mM final)	5 µl
2 mM dCTP, dGTP (0.1 mM each final)	2.5 µl
0.2 mM fluorescein-12-dUTP (2 ľM final)	2 µl
0.2 mM fluorescein-15-dATP (2 ľM final)	2 µl
0.2 mM dTTP (34 ľM final)	4.25 µl
0.2 mM dATP (34 ľM final)	4.25 µl
20 ľM forward primer (2 ľM final)	5 µl
20 ľM reverse primer (2 ľM final)	5 µl
5U/ľl Taq DNA polymerase (3 U / 50 ľl final)	1.5 µl

*) The buffer contains 15 mM MgCl₂ 
Add sterile deionized H₂O to 55 ľl (includes 5 ľl for evaporation) 
Actual composition of the mix (e.g., MgCl₂, concentration and ratio of labelled and unlabeled nucleotides, primer concentration) should be optimised for given primer pair and species

Stop Buffer for PRINS

0.5 M NaCl	2.923 g
50 mM Na2EDTA	1.861 g

Adjust volume to 100 ml with deionized H₂O 
Adjust the final pH to 8.0 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

Wash Buffer for PRINS

100 mM maleic acid	1.161 g
150 mM NaCl	0.876 g
0.05% v/v Tween-20	0.5 ml

Adjust volume to 100 ml with deionized H₂O 
Adjust the final pH to 7.5 using 1N NaOH 
Sterilise by autoclaving 
Store at 4°C

PCR Premix

(for a 50 ľl reaction mixture)

10x Taq DNA polymerase buffer*	5 µl
25 mM MgCl2 (1.5 mM final)	3 µl
10 mM dNTPs (0.2 mM each final)	1 µl
50 ľM forward primer (1 ľM final)	1 µl
50 ľM reverse primer (1 ľM final)	1 µl
5U/ľl Taq DNA polymerase (2.5 U/50 ľl final)	0.5 µl
sterile deionized H2O	18.5 µl

*) The buffer does not contain MgCl₂ 
Mix well and centrifuge briefly 
Prepare shortly before the use

TAE buffer 

Stock Solution (50 x)

Tris (2M final)	242 g
glacial acetic acid (1 M final)	57.1 ml
0.5 M EDTA (pH 8.0), (100 mM final)	200 ml

Adjust volume to 1000 ml with deionized H₂O 
Store at room temperature 

Working Solution (1x) 
Dilute TAE stock solution 1:50 in deionized H₂O (final concentrations: 40 mM Tris, 20 mM acetic acid, 2 mM EDTA)

Loading Buffer

0.5M EDTA (pH 8.0) (100 mM final)	2 ml
SDS (1% w/v final)	100 mg
bromophenol blue (0.05% w/v final)	5 mg
xylenecyanol (0.05% w/v final)	5 mg
gycerol (50% v/v final)	5 ml

Adjust volume to 10 ml with deionized H₂O 
Store at room temperature

Ethidium Bromide Solution

Stock Solution (0.5 mg/ml)

Dissolve 5 mg ethidium bromide in 10 ml deionized H₂O by stirring for 60 min

Ethidium Bromide Working Solution (0.5 µg/ml)

Dilute stock solution 1:1000 in deionized H₂O 
The solution may be used several times 
Store at room temperature

References 

• Dolezel J, Cihalikova J, Lucretti S. A high-yield procedure for isolation of metaphase chromosomes from root tips of Vicia faba. L. Planta 188: 93 - 98 (1992) 
• Gamborg OL, Wetter LR. Plant Tissue Culture Methods. Saskatoon: National Research Council of Canada (1975) 
• Gamborg OL, Miller RA, Ojima K. Nutrient requirement of suspension cultures of soybean root cells. Experimental Cell Research 50: 151 - 158 (1968)