2.2 Culture and storage of protonemata and gametophores

Yuji Hiwatashi

 

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 mediumh) 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

101 g@@ (1 M)

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.

 

Material required

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)

 

Procedure

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

 

Material required

E Solid medium (BCD or selection medium) @9 cm-petri dish

E Forceps (autoclave)

E Parafilm

 

Procedure

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.

 

 

Key points

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.