Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens

Materials and Reagents

1. A vigorously growing tissue which is about 10 d old (the earliest stage of gametophyte; Physcomitrella patens subspecies patens (Gransden) was used as the tissue and it was obtained from Ralph S. Quatrano (Department of Biology, Washington University in St. Louis, MO 63130, USA)
   
2. 200 ml sterile distilled water
   
3. 70% alcohol in a spray bottle (for surface sterilization)
   
4. Growth medium (see Recipes)
   

Equipment

1. 1 L flask in which to prepare the growth medium
   
2. Sterile petri dishes (90-mm)
   
3. Sterile cellophane discs
   
4. Sterile tweezers
   
5. Sterile test tubes (25 x 150 mm)
   
6. Micropore surgical tape
   
7. Sterile pipettes (1 ml)
   
8. Sterile tips (1 ml)
   
9. Dispensing instrument (e.g., IKA T 10 basic ULTRA-TURRAX®)
   
10. Laminar flow cabinet
    
11. Autoclave (e.g., Sanyo, model: MLS-3780 )
    

Procedure

Note: Steps 2-12 should be carried out under sterile conditions.

1. BCDA medium is prepared according to Table 1 and sterilized by an autoclave for 20 min at 121 °C;
   
2. Surface sterilization is performed with 70% alcohol in the Laminar flow hood;
   
3. Media was cooled to 60 °C and approximately 30 ml BCDA medium was poured into sterile petri dishes (90 mm). This was allowed to cool until the media solidified; Place a piece of sterile cellophane discs onto the BCDA medium (Video 1);
   
   
   
   Video 1. Place a piece of sterile cellophane discs onto the BCDA medium
   
   
4. Place one petri dish tissue (vigorously growing tissue) into one test tube (Video 2);
   
   
   
   Video 2. Place one petri dish tissue into one test tube
   
   
5. Add 6-8 ml of H₂O to one sterile test tube (containing one petri dish tissue), and cut it into fragments using dispensing instrument for 1-2 min at a speed of about 15,000 rpm;
   
6. Pipette 1-2 ml of the protonemata suspension from step 6 onto each petri dish from step 4. Spread the suspension evenly by gently swirling the plates (Video 3);
   
   
   
   Video 3. Pipette 1-2 ml of the protonemata suspension from step 6 onto each petri dish from step 4. Spread the suspension evenly by gently swirling the plates
   
   
7. Seal the Petri dishes with micropore surgical tape;
   
8. Incubate these fragments for 6-7 d under standard conditions (25 °C) with a light cycle of 16 h of light/8 h of darkness and a light intensity of 70-80 μmol/s/m²;
   
9. Transfer 1-2 dishes of the tissue (over cellophane) onto BCD solid media (Table 2) growth 3-4 d under the same culture conditions to step 9 for next cycle (this is a vigorously growing tissue which is about 10 d old tissues; Video 4);
   
   
   
   Video 4. Transfer 1-2 dishes of the tissue onto BCD solid media growth 3-4 d under the same culture conditions to step 9 for next cycle
   
   
10. Harvest the tissue (protonemata of P. patens from step 9) for downstream analysis by scraping them from the cellophane using sterile tweezers.
    
11. For tissue stock, take a tiny amount (about 2 mm²; using sterile tweezers) of 10 d old tissues into sterile test tubes with BCD medium. Wrap the tubes in foil and store them at 4 °C. They remain viable for about six months.

Recipes

1. Growth medium
   
   Table 1. BCDA medium recipe
   

   Reagent	Quantity (for 1 L)	Final concentration
   Solution B	10 ml	1 mM MgSO4
   Solution C	10 ml	1.84 mM KH2PO4
   Solution D	10 ml	10 mM KNO3
   CaCl2	111 mg	1 mM
   FeSO4.7H2O	12.5 mg	45 μM
   (NH4)2C4H4O6	0.92 g	5 mM
   Agar	7.5 g	0.75% (w/v)
   Glucose	5 g	0.5% (w/v)
   Hoagland’s A-Z trace	1 ml	Trace element solution
   H2O	To 1 L

   
   Table 2. BCD medium recipe
   

   Reagent	Quantity (for 1 L)	Final concentration
   Solution B	10 ml	1 mM MgSO4
   Solution C	10 ml	1.84 mM KH2PO4
   Solution D	10 ml	10 mM KNO3
   CaCl2	111 mg	1 mM
   FeSO4.7H2O	12.5 mg	45 μM
   Agar	7.5 g	0.75% (w/v)
   Glucose	5 g	0.5% (w/v)
   Hoagland’s A-Z trace	1 ml	Trace element solution
   H2O	To 1 L

   
   Table 3. Recipe of Solution B, C, D and Hoagland’s A-Z trace
   

   	Reagent	Quantity (for 1 L)	Final concentration
   Solution B	MgSO4.7H2O	25 g	0.1 M
   	H2O	To 1 L
   Solution C	KH2PO4	25 g	184 mM
   	H2O	To 1 L	Adjust the pH to 6.5 using KOH
   Solution D	KNO3	101 g	1 M
   	H2O	To 1 L
   Hoagland’s A-Z trace	Al2(SO4)3.K2SO4.24H2O	55 mg	0.006% (w/v)
   	CoCl2.6H2O	55 mg	0.006% (w/v)
   	CuSO4.5H2O	55 mg	0.006% (w/v)
   	H3BO3	614 mg	0.061% (w/v)
   	KBr	28 mg	0.003% (w/v)
   	KI	28 mg	0.003% (w/v)
   	LiCl	28 mg	0.003% (w/v)
   	MnCl2.4H2O	389 mg	0.039% (w/v)
   	SnCl2.2H2O	28 mg	0.003% (w/v)
   	ZnSO4.7H2O	55 mg	0.006% (w/v)
   	H2O	To 1 L

   Solution B, C, D and Hoagland’s A-Z trace are sterilized and then stored at 4 °C or room temperature.
   

Acknowledgments

This work was supported by grants from Beijing Natural Science Foundation (No. 5132004), China Postdoctoral Science Foundation and State Education Ministry Scientific Research Foundation for the Returned Overseas Chinese Scholars to Dr. Wang.

References

1. Ashton, N. W., and Cove, D. J. (1977). The isolation and preliminary characterisation of auxotrophic and analogue resistant mutants in the moss Physcomitrella patens. Mol Gen Genet 154, 87-95.
   
2. Boyd, P. J., Hall, J., and Cove, D. J. (1988). An airlift fermenter for the culture of the moss Physcomitrella patens. In: Glime, J. M. (ed). Methods in bryology. Hattori Botany Laboratory, 41-45.
   
3. Cove, D. J., Perroud, P. F., Charron, A. J., McDaniel, S. F., Khandelwal, A. and Quatrano, R. S. (2009). The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harb Protoc 2009(2): pdb emo115.