2.2@ Culture and storage of protonemata and gametophores
1. Culture
conditions
Introduction
BCDATG or BCDAT medium is used for sub-culture
of P. patens protonemata. Protonemata for PEG-mediated transformation
are sub-cultured on BCDATG agar medium every 4 - 5 day. In BCDATG or BCDAT,
growth of chloronemata is enhanced by ammonium as nitrogen source.@ Glucose in BCDATG medium makes it easier to
monitor contamination.@
Protonemata cultured in BCD medium supplemented
with 1 mM Ca (we routinely call BCD medium) grow slower than in BCDATG and
BCDAT medium. This medium is used for induction of sporophytes.@
1)
Growth medium
Stock
medium
All stock
media are stored at 4˚C. Stock D should be used within 2-3 month before
iron precipitates.
Stock A (x
100)@ Autoclave
Ca(NO3)2
4H2O |
118 g@ @(0.5 M) |
FeSO4
7H2O |
1.25 g@@ (4.5 mM) |
|
Fill up to
1000 ml with H2O |
Stock B (x
100) )@ Autoclave
MgSO4
7H2O |
25 g@@ (0.1 mM) |
|
Fill up to
1000 ml with H2O |
Stock C (x
100)@@ Autoclave
KH2PO4 |
25 g@@ (1.84 mM) |
|
Adjust to
pH6.5 with 4M KOH |
|
Fill up to
1000 ml with H2O |
Stock D (x
100)@ DO NOT autoclave
KNO3 |
|
FeSO4
7H2O |
1.25 g@@ (4.5 mM) |
|
Fill up to
1000 ml with H2O |
Alternative
TES (x 1000)@@ Autoclave
CuSO4
5H2O |
55 mg@@ (0.22 mM) |
H3BO3 |
614 mg@@ (10 mM) |
CoCl2
6H2O |
55 mg@@ (0.23 mM) |
Na2MoO4
2H2O |
25 mg@@ (0.1 mM) |
ZnSO4
7H2O |
55 mg@@ (0.19 mM) |
MnCl2
4H2O |
389 mg@@ (2 mM) |
KI |
28 mg@@ (0.17 mM) |
|
Fill up to
1000 ml with H2O |
500mM
Ammonium Tartrate (x 100)@@ Autoclave
Ammonium
Tartrate |
92.05 g |
|
Fill up to
1000 ml with H2O |
50mM CaCl2
(x 50)@ Autoclave
CaCl2
2H2O |
7.35 g |
|
Fill up to
1000 ml with H2O |
Medium
routinely used
BCD+1mM Ca
medium (we call BCD)@ 1000 ml
H2O |
900 ml |
Stock B |
10 ml |
Stock C |
10 ml |
Stock D |
10 ml |
Alternative
TES |
1 ml |
50mM CaCl2
2H2O (powder) |
20 ml (1 mM) (0.15 g) |
Agar (Sigma:
A6924) |
8 g (0.8%) |
|
Fill up to
1000 ml with H2O |
After autoclave, pour into 9 cm-Petri dish to solidify, then
in dry for 30 min in a clean bench. Store at room temperature.
BCDAT
medium @1000 ml
H2O |
900 ml |
Stock B |
10 ml |
Stock C |
10 ml |
Stock D |
10 ml |
Alternative
TES |
1 ml |
500mM Ammounim
tartrate |
10 ml (= 5
mM) |
50mM CaCl2
2H2O (powder) |
20 ml (= 1
mM) @(0.15 g) |
Agar (Sigma:
A6924) |
8 g (= 0.8%) |
|
Fill up to
1000 ml with H2O |
After
autoclaved, pour into 9 cm-dish and solidify for 30 min in a clean bench. Store
at room temperature.
BCDATG
medium@
BCDAT medium
are supplemented with 5 g/l glucose.
After
autoclave, pour into 9 cm-dish to solidify, then dry up for 30 min in a clean
bench. Store at room temperature.
Other
reagents
Ethiamine HCl @(MW.337.3)@@@ (1.5 µM)
0.5mg/ 1L
medium
Ep-aminobenzoid acid (MW.137.1)@@ (1.8 µM)
247 µg/
1L medium
2). Light
For routine
culture, continuous light or long day (16L8D) condition is used.@ We use the following fluorescent tube at an
intensity of between 30 and 80 µmol/m2/s.
daylight@NEC FL40SD@@@@
40 µmol/m2/s1
daywhite@NEC FL40SEX-N-HG@@@@@ 80 µmol/m2/s1
For induction
of sporophyte, short day (8L16D) condition is used.@
3). Temperature
For routine
culture, P. patens grows on solid medium in temperature below
28˚C. We usually set temperature at 25˚C.
For induction
of sporophyte, temperature between 15 and 16˚C is used.@
EHumidity@
condition
We do not
control humidity for culture of P. patens. P. patens grow well at
humidity between 30 and 50% of an incubators.@
2. Routine
sub-culture
For routine
sub-culture, protonemal tissue is used. Growth of protonemata is enhanced when
ammonium is provided as nitrogen source.@
A part of protonema (1-2 mm) is inoculated on new medium to establish a
new culture. For a large-scale culture, we use a polytron homogenizer.
The 7-days
culture on 9 cm-dish is harvested with forceps, suspended in 20-30 ml of
sterile water and blended by using homogenizer.@ A part (1/10 vol.) of suspension is inoculated in a 9 cm dish
overlaid with a cellophane. After 7 days incubation at 25˚C under
continuous white light (40 µmol /m2/s), growing protonemal tissue,
consisting mainly of chloronemata, is obtained on the dish.@
The dish may
be sealed with medical surgical tape or parafilm to prevent contamination.
Surgical tape is recommended to keep appropriate moisture and to prevent
contamination. Grows of protonemata become slower with parafilm without air
change.
E Solid medium (BCDAT or
BCDATG) – 9 cm-petri dish
E Sterile water
E Cellophane (autoclave)
– Wash as the follows
1) Cut
cellophanes to a little bit smaller than the size of a 9 cm-dish.
2) Place
cellophane in a glass Petri dish and then add 5 mM EDTA solution (pH8.0).
Autoclave.
3) Wash with
MilliQ water several times.
4) Add MilliQ
water in the glass plate, then autoclave.
E Pipette (autoclave)
E Forceps (autoclave)
E Polytron@ PT2110
E Polytron generator shaft
DA2121/2 (autoclave)
E Test tube (autoclave)
1. Overlay the
solid medium with cellophane.
2. Add 20-30 ml
of sterile water in a test tube.
3. Recover
propagated protonemata from one 9 cm-petri dish (7-day-old) with a forceps and
add into the test tube.
4. Blend for 10
sec at minimum speed (Polytron PT2100) or at 4-5 level speed (Polytron model
K).
5. Inoculate 2-3
ml of suspension into a new 9 cm-petri dish
6. Incubate at
25˚C under continuous white light without sealing plates.
3.
Storage of protonemata
E Solid medium (BCD or
selection medium) @9 cm-petri dish
E Forceps (autoclave)
E Parafilm
1. Inoculate a
part of protonemata on the solid medium.
2. Seal the
plate with surgical tape and incubate at 25˚C for 2-3 weeks.
3. Seal the
plate with parafilm and store at 4˚C.
E Use selection medium
containing antibiotics for preservation of transformants if possible.@ In selection medium, transformants can
survive for longer time under storage condition.