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Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens   

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Original research article

A brief version of this protocol appeared in:
Journal of Experimental Botany
May 2014

Abstract

Moss spores germinate to form an alga-like filamentous structure called the protonemata. Protonemata are the earliest stage (the haploid phase) of a bryophyte life cycle and eventually give rise to a mature gametophyte. Protonemata of the moss Physcomitrella patens (P. patens) are important not only in their life cycle, but also for research. Protonemata are used for various things such as RNA/DNA extractions and protoplast isolation. We can obtain high yield of intact protoplasts from protonemata. Protoplasts can be used to study a variety of cellular processes, such as subcellular localization of proteins, isolation and analyses of intact organelles and DNA transformation. In addition, the completed sequence of the P. patens genome facilitates the use of genetic and molecular approaches to identify genes and the ability of the moss to undergo homologous recombination at appreciable frequency offers a powerful way to determine gene function. Therefore, culture of P. patens protonemata is critical.

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.
Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Wang, X. and He, Y. (2015). Tissue Culturing and Harvesting of Protonemata from the Moss Physcomitrella patens. Bio-protocol 5(15): e1556. DOI: 10.21769/BioProtoc.1556.
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