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 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

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

 

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.

 

Material required

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)

 

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 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

 

Material required

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

E Forceps (autoclave)

E Parafilm

 

Procedure

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.

 

 

Key points

E Use selection medium containing antibiotics for preservation of transformants if possible.@ In selection medium, transformants can survive for longer time under storage condition.