Biology Reference
In-Depth Information
Table 2
Stock solutions for testing various media for pollen in vitro cultures
Solution
MW (g/l)
Grams per 50 ml
1 M KCl
74.56
3.7280
100 mM CaCl
2
147.02
0.7351
100 mM Ca(NO
3
)
2
236.15
1.1801
100 mM MgCl
2
203.31
1.0166
100 mM MgSO
4
246.48
1.2324
100 mM H
3
BO
3
61.83
0.3092
100 mM KH
2
PO
4
136.09
0.6805
amount of biological material which are important for pollen
proteomic studies were obtained for lily pollen.
When testing various media conditions for optimizing pollen
grain germination and tube growth, prepare stock solutions of the
media ingredients (Table
2
). The culture media listed below have
been tested in the authors' lab. However, other media composi-
tions are reported in the literature.
1. Medium B (lily): 10 % (w/v) sucrose, 1.6 mM H
3
BO
3
, 1 mM
KCl, 0.1 mM CaCl
2
. Adjust to pH 5.6 with tiny amounts of
MES or Tris (
see
Note 5
).
2. Medium M (lily): 300 mM mannitol, 1.6 mM H
3
BO
3
, 1 mM
KCl, 0.1 mM CaCl
2
, 25 mM MES adjusted with BTP to pH
5.6.
3. Medium P (lily): 20 % (w/v) PEG (3350), 1.6 mM H
3
BO
3
.
Adjust to pH 5.6 with tiny amounts of Tris or BTP.
4. Arabidopsis pollen medium (solidifi ed, [
4
]): 18 % (w/v)
sucrose, 0.01 % boric acid, 1 mM MgCl
2
, 1 mM CaCl
2
, 1 mM
Ca(NO
3
)
2
, 0.5 % (w/v) agar, pH 7 adjust with HCl or KOH.
5. Arabidopsis pollen medium (solidifi ed, [
3
]): 10 % (w/v)
sucrose, 0.01 % (w/v) boric acid, 5 mM CaCl
2
, 5 mM KCl,
1 mM MgSO
4
, pH 7.5 adjust with ca. 30
l of 0.1 M NaOH
(non-buffered), 1.5 % (w/v) low-melting agarose (Sigma, type
VII, A4018). Add sucrose after pH was adjusted.
6. Arabidopsis pollen medium (liquid, [
3
]): 0.01 % boric acid,
5 mM CaCl
2
, 5 mM KCl, 1 mM MgSO
4
, 10 % (w/v) sucrose,
pH 7.5 (non-buffered).
7. Arabidopsis pollen medium (solidifi ed, [
5
]): 20 % (w/v)
sucrose, 0.001 % (w/v) H
3
BO
3
, 20 mM CaCl
2
, 0.1 mM
KH
2
PO
4
, 12 % (w/v) PEG 4000, 0.5 % (w/v) agar, pH 7.
μ
