Automated Microscopy in Forward Genetic Screening of Arabidopsis - Plant Cell Morphogenesis: Methods and Protocols - page 54

2 Materials

Unless otherwise specifi ed, solutions are prepared using ultrapure
double-distilled water (ddH 2 O). The chemical compounds used
were of pro analysi (p.a.) purity or higher.

2.1 Plant Seeds
and Sterilization

1. Seeds of individual M2 families (progeny of self-pollinated M1
plants, grown from EMS-mutagenized M1 seeds) harvested
separately and seeds from a non-mutagenized control ( see
Note 1 ).
2. 70 % (v/v) ethanol: mix 70 ml ethanol and 30 ml water.
3. Sterile fi lter papers, 1.5 ml tubes, gas-permeable (textile or
paper) tape such as a surgical paper tape, and sterile Petri dishes.

2.2 Plant Cultivation

1. MS media (pH 5.7-5.9): Murashige and Skoog medium
4.3 g/l (Duchefa Biochemie); MES monohydrate 2.35 mM
(Duchefa Biochemie), sucrose 1 %, plant agar 1 % (Duchefa
Biochemie). Weigh 4.3 g of MS salts, 0.5 g of MES monohy-
drate, and 10 g of sucrose, and then adjust volume to 1,000 ml
using ddH 2 O. Adjust pH to 5.7-5.9 using 1 M KOH (approx-
imately 1.25 ml).
2. Petri dishes: cylindrical, with diameter of 9 cm (or of similar size).
3. Cultivation growth chamber: e.g., Percival Scientifi c, Inc. or
similar, allowing plant cultivation under selected growth
conditions ( see Note 2 ).

2.3 GUS Staining

1. 0.1 M Pi buffer: for preparation of 500 ml of 0.5 M
NaH 2 PO 4 H 2 O and adjust

volume to 500 ml by adding ddH 2 O. For preparation of
500 ml of 0.5 M Na 2 HPO 4

⋅ H 2 O, weigh 34.49 g of NaH 2 PO 4 ⋅

⋅ 2H 2 O, weigh 44.99 g of

Na 2 HPO 4 2H 2 O and adjust volume to 500 ml using ddH 2 O.

Mix 39 ml of 0.5 M NaH 2 PO 4

⋅

⋅ H 2 O and 61 ml 0.5 M

Na 2 HPO 4 ⋅ 2H 2 O and adjust volume to 400 ml by ddH 2 O.

2. 10 % (w/v) X-GlcA/DMF: weigh 100 mg of X-GlcA, sodium
trihydrate (Duchefa Biochemie), and dissolve it in 1 ml DMF
(N,N-dimethylformamide). 100 mg of X-GlcA is suffi cient for
staining approximately 100 samples ( see Note 3 ).
3. 10 % (v/v) Triton X-100: measure 10 ml of Triton X-100 and
adjust to 100 ml using ddH 2 O ( see Note 4 ).
4. 50 mM Fe salts: prepare 10 ml of 50 mM salts of K 3 [Fe(CN) 6 ]
and of K 4 [Fe(CN) 6 ] in ddH 2 O. Weigh 164.6 mg of K 3 [Fe(CN) 6 ]
and dissolve in 10 ml of ddH 2 O. Weigh 211.2 mg of K 4 [Fe(CN) 6 ]
and dissolve in 10 ml of ddH 2 O ( see Notes 5 and 6 ).

5. GUS staining buffer: 0.1 M Pi buffer, pH 7, 0.1 % X-GlcA in
DMF, 0.05 % Triton X-100, 0.1 mM Fe salts. To prepare