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 days. In BCDATG or BCDAT, growth
of chloronemata is enhanced by including ammonium as the 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 this gBCD
mediumh) 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 months,
before iron precipitates.
Stock
A (x 100)@ DO NOT 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@@ (100 mM)
|
|
Fill up to 1000 ml with H2O |
Stock
C (x 100)@@ Autoclave
KH2PO4 |
25 g@@ (184 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 |
500
mM Ammonium Tartrate (x 100)@@ Autoclave
Ammonium Tartrate |
92.05 g |
|
Fill up to 1000 ml with H2O |
50
mM 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, Nacalai Tesque: cat. no. 01028-85) |
8 g (0.8%) |
|
Fill up to 1000 ml with H2O |
After autoclaving, pour into 9 cm-petri dishes to solidify, then dry for 30 min in a Clean bench / Laminar flow cabinet. 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 2H+O (powder) |
20 ml (= 1 mM) @(0.15 g) |
Agar (Sigma: A6924, Nacalai Tesque: cat. no. 01028-85) |
8 g (= 0.8%) |
|
Fill up to 1000 ml with H2O |
After
autoclaving, pour into 9 cm-petri dishes and solidify for 30 min in a Clean
bench / Laminar flow cabinet. Store at room temperature.
BCDATG medium@
BCDAT
medium is supplemented with 5 g/l glucose.
After
autoclave, pour into 9 cm-petri dish to solidify, then dry up for 30 min in a
Clean bench / Laminar flow cabinet. Store at room temperature.
Other reagents
Ethiamine
HCl @(MW.337.3)@@@ (1.5 µM)
0.5mg/
1L medium
Ep-aminobenzoic
acid (MW.137.1)@@ (1.8 µM)
247
µg/ 1L medium
2). Light
For
routine culture, continuous light or long day (16L8D) conditions are 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 sporophytes, short day (8L16D) condition are used.@
3). Temperature
For
routine culture, P. patens grows on
solid medium at temperatures below 28˚C. We usually set temperature at 25˚C.
For
induction of sporophytes, a lower temperature, between 15 and 16˚C, is
used.@
4). Humidity condition
We
do not control humidity for culture of P.
patens. P. patens grows well at a
humidity between 30% and 50% in the incubator.@
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 small explant of
protonemal tissue (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 a 9
cm-petri dish is harvested with forceps, suspended in 20-30 ml of sterile water
and blended using the homogenizer. A part (1/10 vol.) of the suspension is
inoculated into a 9 cm-petri dish overlaid with cellophane. After 7 days
incubation at 25˚C under continuous white light (40 µmol /m2/s),
a mat of growing protonemal tissue, consisting mainly of chloronemata, is obtained
on the petri dish.
For
sub-culture of multiple samples, we use 6-well tubes with ceramic balls
(KURABO). Protonemata are put into a well of the tube and autoclaved water is
added in the well. Then the tube is mixed by vortexing. The suspended protonemata
are inoculated into a 9 cm-petri dish overlaid with cellophane.@
The
petri dish may be sealed with medical surgical tape or parafilm to prevent
contamination. Surgical tape is recommended to reduce dehydration and to
prevent contamination. Growth of protonemata is slower with parafilm as air
exchange is reduced.
E Solid
medium (BCDAT or BCDATG) – 9 cm-petri dish
E Sterile
water
E Cellophane (autoclave) – Quality of cellophane is different
depending on companies. For regular cellophane, it is better to be pre-treated
as follows. If you use P-5 cellophane (Futamura Chemical Co., Ltd, Nagoya), we
can use without EDTA treatment. However, other cellophanes may be better to be
treated as follows:
1) Cut
cellophanes to a little bit smaller than the size of a 9 cm-petri dish.
2) Place
cellophane in a 500 ml beaker (usually ** seats) and then add 5 mM EDTA
solution (pH8.0). Autoclave.
3) Wash
with MilliQ water several times. Add MilliQ water in the beaker. Autoclave.
4) Place
cellophane in a glass petri dish and add MilliQ water, then autoclave
When
use P-5 Cellophane
1)
Cut cellophanes to a little bit smaller than the size of a 9 cm-petri dish.
2)
Place cellophane in a 500 ml beaker and then autoclave. 3) Place cellophane in
a glass petri dish and add MilliQ water, then autoclave
E Pipette
(autoclave)
E Forceps
(autoclave)
E Polytron@ PT2110
E Polytron generator shaft DA2121/2 (autoclave)
E Test tube
(autoclave)
(if
you use a KURABO tube)
E 6-well
tube (KURABO, RT-5000) containing a ball (KURABO, Z-07) per well, covered with
a cap (KURABO, C-600) (autoclave)
E 3 ml
transfer pipette (Falcon no.7575)
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 forceps and
add into the test tube.
4. Blend for
10 sec at minimum speed (Polytron PT2100) or at speed level 4-5 (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 the plates.
If you use KURABO tubes,,
1. Overlay
the solid medium with cellophane.
2. Add 1 ml of
sterile water in a KURABO tube.
3. Recover
propagated protonemata or a colony from one 9 cm-petri dish with forceps and
place in the test tube.
4. Vortex for
15 sec at maximum speed.
5. Add 5 ml of
sterile water to the KURABO tube.
6. Inoculate 1-2
ml of suspension into a new 9 cm-petri dish
7. Incubate
at 25˚C under continuous white light.
3. Storage
of protonemata
E Solid
medium (BCD or selection medium) @9 cm-petri
dish
E Forceps
(autoclave)
E Parafilm
1. Inoculate
a piece of protonemal tissue 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 a longer time
under storage conditions.