Plant Cell Lines in Cell Morphogenesis Research - Plant Cell Morphogenesis: Methods and Protocols - page 210

2.4 Transformation
of Cell Cultures

1. Sterile cell fi ltration set (Nalgene “Filter Holder with Receiver”
or similar) with 20 m nylon mesh fi lter, sterile Petri dishes

(60 and 90 mm diameter), sterile regular pipette tips, and
pipette tips cut off by ca. 1-1.5 cm (suitable for 1, 5, or 10 ml
pipettes).
2. Liquid YEP medium for cultivation of Agrobacterium tumefa-
ciens : 10 g/l select yeast extract, 10 g/l peptone, 5 g/l NaCl,
adjust pH to 7.0-7.5 with 3 M KOH.
3. Acetosyringone stock solution (20 mM in ethanol): weigh
39.2 mg of acetosyringone (Fluka-Sigma) in a Falcon tube.
Add 10 ml of 96 % ethanol and let dissolve. Filter through
Millipore MF membrane fi lter for sterilization of aqueous
solutions (0.22

μ

m) and pipette 1 ml aliquots into sterile epi-
tubes under sterile conditions. Store at −20 °C.
4. Overnight culture (approximately 16 h) of the A. tumefaciens
strain carrying binary vector with your gene construct
(GV2260, C58C1, LBA1115, LBA1100 or others).
5. Stock solution of cefotaxime (Claforan, 100 mg/ml): under
sterile conditions add sterilized distilled water into the original
Claforan bottle (i.e., 10 ml water to 1 g of cefotaxime) and
pipette 1 ml aliquots into sterile epi-tubes. Store at −20 °C.
6. Plates with solid modifi ed MS or V4 medium ( see
Subheading 2.2 ) containing 100

μ

g/ml cefotaxime (Claforan)
and appropriate selection antibiotics ( see Note 3 ).
7. Sterile stock solutions of appropriate antibiotics (depending
on the construct, i.e., mostly hygromycin or kanamycin; see
Note 4 ), sterile 3 % sucrose solution (0.5 l for each construct).

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2.5 Description,
Phenotyping, and
Cytological Analysis
of Suspension
Cultures

1. Pasteur pipette, rack for test tubes, and glass or plastic test
tubes.
2. Microscope slides and cover glasses.
3. Fuchs-Rosenthal counting chamber (size of the chamber
4 × 4 × 0.2 mm, total volume 3.2 mm 3 ).
4. Fluorescein diacetate (FDA) solution. Stock solution: dissolve
20 mg of FDA in 10 ml of acetone, prepare aliquots and store
at −20 °C. Working solution to be prepared freshly before
each experiment: mix 40 l of FDA acetone stock solution

with 15 ml of culture medium.
5. Trypan blue solution (0.4 %).
6. Upright and inverted light microscope equipped with epifl uo-
rescence and camera, image analysis software (e. g., ImageJ,
NIS-Elements).
7. Hoechst fl uorescent dye for nuclei staining (stock solution
1 mg/ml dissolved in H 2 O, aliquots should be stored at −20 °C).
8. 10 % Triton X-100 solution (dissolved in H 2 O).

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