Biology Reference
In-Depth Information
2
Materials
2.1 Plant Material
and Growth Conditions
1. Infl orescence meristems of
Arabidopsis thaliana
: seeds are
sowed on soil and plants are usually kept at 22 °C in short day
conditions (8 h light/16 h dark) for at least 3 weeks to increase
the size of the meristem. Plants are then transferred to long
day conditions (16 h light/8 h dark;
see
Note 1
). For light, we
use a combination of white and gro-lux neons.
2. NPA-treated Arabidopsis seedlings: sterile seeds are sowed on
the Arabidopsis medium supplemented with 10
M NPA (
see
below). Seedlings are grown in tall petri dishes, in long day
conditions, at 22 °C.
3. Vegetative meristems: 11- to 12-day-old tomato (
Solanum
lycopersicum
/
Lycopersicon esculentum,
e.g.,
cv M82 or
Moneymaker)
seedlings are used. Seedlings are grown in soil in
long day conditions (16 h light per day, 110
μ
μ
Em
2
/s) in
65 ± 10 % humidity, at 20 ± 2 °C.
2.2 Media
1. Medium used to grow Arabidopsis shoot apices (Arabidopsis
apex culture medium, ACM): dissolve 2.2 g/l MS medium
without vitamins (Duchefa Biochemie—MS basal salt mix-
ture), add 1 % sucrose, and adjust pH to 5.8 with KOH. The
solid medium is obtained by adding 0.8 % agarose. Vitamins
are added to the lukewarm solution from the stock listed below.
2. 1,000× vitamin stock solution: for 50 ml, dissolve 5 g myoino-
sitol, 0.05 g nicotinic acid, 0.05 g pyridoxine hydrochloride,
0.05 g thiamine hydrochloride, 0.1 g glycine in millipore
water. Filter the solution at 22
μ
m under laminar hood, ali-
quot, and store at −20 °C.
3. Medium for growing NPA-treated seedlings (whole plantlets
with naked meristems): we use the Duchefa “Arabidopsis”
medium (DU0742.0025). Dissolve 47.28 g in 4 l of water,
add 8 ml of 1 M Ca(NO3)
2
4H
2
O, adjust pH to 5.8 with
KOH, and add 1 % of agar (Merck). We germinate the seeds
on 10
M NPA (
N
-(1-naphthyl) phthalamic acid) using a
0.1 M stock solution (10,000×) in DMSO and stored for no
more than 3 months at −20 °C. There is more chance to get
naked stems if seedlings are densely sowed.
4. Medium used to grow tomato shoot apices (tomato apex cul-
ture medium): dissolve 1× MS minimal organic powder
medium; add 2 % (fi nal conc.) sucrose, 1.5 % (fi nal conc.) aga-
rose; adjust pH to 5.8 with KOH; and add hormones—
gibberellic acid A3 (0.01
μ
M). To avoid
medium contamination, add preservative for plant tissue cul-
ture; we use 1
μ
M) and kinetin (0.01
μ
l/ml PPM (Preservative for Plant Tissue
Culture Media, Plant Cell Tech).
μ
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