PROCEDURE NO. 49622

L I F E C Y C L E O F T H E MOSS, PHYSCOMITRELLA PATENS,

Paula A. Collier and Km'en W. Hughes

Department of Botany University of Tennessee

Knoxville, TN37916

I N C U L T U R E

SUMMARY: A method employing tissue culture techniques for growth of mosses is described. This method allows for the completion of the sexual life cycle of the moss Physcomitrella patens (Hedw.) BSG under controlled conditions in a two month period. The moss system is useful for class demonstrat ion or for research in the areas of development, genetics and plant physiology.

Key words: Moss; Physcomitrella paten« plant tissue culture.

I. INTRODUCTION

The bryophytes are good organisms for s tudying many types of biochemical, genetic and developmental processes. They are haploid, the cells are totipotent, they will grow on a de- fined medium under controlled environmental conditions, and several will complete their life cycle in culture (1-3). The experimental organ- ism described here is the moss PhyscomitreUa patens (Hedw.) BSG. I t was originally isolated from material collected in Gransden wood, Huntingshire, England, by H . L . K . White- house. This species represents an especially promising tool in that several classes of bio- chemical mutants have been isolated and stud- ied genetically (1,2,4). Protoplasts have been obtained (5-7) and used for somatic hybridiza- tion (4,6). The following procedure outlines the methods used to culture Physcomitrella patens and the conditions necessary for the completion of the life cycle. The procedure has been used successfully with two other moss species of the Funariaceae, Physcomitrium pyriforme (Hedw.) Hampe and Funaria hygrometrica (Hedw.). With minor alterations in the growth cycle, the procedure should be applicable to a wide variety of moss species.

II. MATERIALS

A. Equipment

Constant temperature environmental chamber with fluorescent lighting, No. E-54U Percival'

Laminar flow hood, Baker Edge-Gard 2 pH meter, No. 13-637-650 Fisher 3 Hot plate with magnetic stirrer, No. 11-496-83 Autoclave, No. 14-488-203, or pressure cooker Dissecting microscope, Stereo, AO 43RT ~

Journal of Tissue Culture Methods Vol. 7, No. 1, 1982 19

B. Medium and chemicals tAll chemicals from Fisher'~

1. Knops Stock Solution (8), Modified

2.

3.

are

Ca(NO3h 4H20 0.5 g/1 KN03 0.125 g/l M g S O , 7H~O 0.125 g/1 KH~PO~ 0.125 g/1

Nitch's Minor Elements Stock Solution (8)

4. Agar, SP 4

H2SO« sg 1.83 0.5 ml/1 MnC12 4H20 2.5 g/l H3BO4 2.0 g/1 ZnSO4 7H~O 0.05 g/1 CoC12 6H~O 0.03 g/1 CuCL 2H~O 0.015 g/1 NaMoO4' 2H~O 0.025 g/1

Ferric Citrate Stock Solution FeCuH~O7 5H~O 25.0 g/l (raise pH

to get into solution)

8.0 g/1 (optional, see Procedure, A.2)

C. Glasswareandother

Petri dishes, 15 x 150 mm, plastic, No. 08-757- 14 ~

Erlenmeyer flasks, 125 ml, No. 10-040D ~ Glass funnels, No. 10-346-5A ~ Coil cotton, 25 mm, No. 07-885D ~ Aluminum foil, purchased locally Pasteur pipettes, No. 13-678-20A ~ Forceps, No. 08-8803 Dissecting needles, No. 08-960A ~ Parafilm, No. 13-374-5 ~ Nitex cloth, 64 micron pore size, Tetko Inc. » Test tubes, No. 14-9563 Blender, No. 14-509-173 Blender container, No. 14-509-18A 3 Magnetic stir bar, No. 14-511-663 Clorox, purchased locally Ethanol, 95% ~

0271-8057/82/0019-0022 $1.50/0 ~~ 1982 Tissue Culture Association, Inc.

C O L L I E R A N D H U G H E S - - P R O C E D U R E NO. 49622

D. Moss cultures

Available from Dr. Karen W. Hughes. is the result of meiosis; consequently, each is haploid and capable of germinating directly into a new haploid plant.

III. PROCEDURES

A.

1.

2.

Preparation of materials

Use s tandard sterilization techniques for all glassware, instruments used for transfers, and media.

Preparation of medium

a. Combine 500 ml of Knops Stock, 0.5 ml of Nitsch's Minor elements, 0.4 ml of ferric citrate solution and 500 ml of double dis- tilled water. Bring to pH 5.5. If solid cul- tures are desired, heat the medium and add 8.0 g/1 agar. Bring to a boil while stir- ring to dissolve agar. Dispense to con- tainers and autoclave to sterilize.

b. For liquid cultures, dispense 50 ml of li- quid medium (without agar) into 125 Erlenmeyer flasks. Plug the flasks with cotton, cap with aluminum foil and autoclave.

C. For the isolation of spores, dispense 10 ml of liquid medium into 25 ml test tubes. Plug the tubes with cotton, cap with aluminum foil and autoclave.

d. For solid culture, dispense 25ml of medium containing agar into 150 ml glass petri plates, and autoclave. For plastic plates, autoclave the medium and cool to 50 ° C prior to dispensing to the plates.

B. Establishment of moss cultures

Initial cultures are established from spores because of the difficulty in decontaminating moss gametophyt ic tissues; however, once the plants are established in culture, tissue from the plants may be used for subculture as de- scribed below.

The leafy plant bears gametangia in which gametes, non-motile egg and flagellated sperm, are produced by mitosis. Fertilization is ac- complished by the sperm swimming to the egg in a drop or thin film of water. The resultant zygote germinates into the sporophyte (2N) which produces the spore capsule. Spores of Physcomitrella patens are produced in a cleisto- carpus (non-dehiscent) capsule which is not ele~ vated above the leafy gametophyte. Each spore

1.

2.

3.

4.

5.

6.

7.

8.

With the aid of a dissecting microscope, select an unopened (undehisced) capsule from a mature culture or plant or from a dried specimen.

Surface sterilize the capsule by dipping it in 70% ethanol to wet the surface then place it into commercial Clorox for 5 minutes.

Place the capsule on the inside wall of a small test tube containing 10 ml of sterile liquid medium. Break the capsule open with a dissecting needle to liberate the spore mass.

Replug the test tube with cotton and tip the tube to allow the liquid medium to pick up and disperse the spores. The resultant spore suspension should contain approximately 1-2 x 10 3 spores/ml of medium (3).

Spores may be germinated in this medium for 2-3 days or used as an inoculum immediately.

Liquid cultures are established by adding ali- quots of the spore suspension to 50 ml of medium in Erlenmeyer flasks. Liquid cul- tures will grow primarily as protonema and will produce gametophytes only when the cultures are old.

Solid cultures are established by plating ali- quots of the spore suspension onto agarized medium in petri dishes. The dishes are sealed with strips of parafilm to prevent dehydra- tion. Solid cultures will briefly grow as protonema then will become gametophyte colonies. E ach gametophyte is the product of a single protonemal cell.

Spores may be plated at low densities from an initial spore isolate in order to obtain gametophyte colonies with a single spore ori- gin. Such colonies are genetically uniform. Subsequent isolations from a single gameto- phyte colony, either from spores or from blended plant tissue, will be genetically uni- form. All of our cultures are clonal and are genetically uniform.

C. Subcul tureofmoss

Since cells of the moss are totipotent, any por- tion of the green plant may be used as inocu-

20 J o u r n a l of T i s s u e C u l t u r e M e t h o d s Vol. 7, No. 1, 1982

COLLIER AND H U G H E S -- PROCEDURE NO. 49622

lum. Plants may be removed from culture and directly subcultured or blended to produce a vegetat ive cell suspension. For experimental purposes where large numbers of cells are re- quired (e.g., mutagenesis and selection for mutant phenotypes), the use of blended proto- nemal cultures is preferable to the use of spores. Blending techniques quickly provide thousands of cells, but obtaining and dissecting spore capsules to obtain an equivalent number of spores is tedious and time consuming.

1. Place 50 ml of sterile liquid medium into a small blender container. If cultures from solid medium are used, remove the leafy gametophores from the agar surface with forceps and place in the blender container. If liquid cultures are used, pour the liquid me- dium containing the cultures into the blender. Cap the blender.

2. Blend the moss in the blender for approxi- mately 40 seconds alternating between low and high speeds. The resulting slurry con- tains fragments of various sizes (usually 4-5 cells each) and cellular debris.

3. Filter the resulting slurry through nitex cloth and wash the retained material with 50 - 100 ml of sterile medium to remove the cell debris.

4. Resuspend the material collected in 50 ml of sterile liquid medium. This suspension may be used directly as the inoculum and may be plated onto plates or aliquots may be added to liquid medium as outlined for spores. Fragments will develop one or more protonemal strands, each strand arising from a single fragment cell. Gametophytes will develop from the protonemal cells.

D. Culture conditions

The cultures are maintained under controlled conditions of temperature and light in environ- mental growth chambers. The temperature for vegetat ive growth should be 20 - 30 ° C. Light is provided by G .E . Cool White fluorescent bulbs (wave length 330 - 750 nm) at a fluence of 36 Joules/cm 2 with a daylength of 14 hours.

capsules are mature in three weeks from the ini- tiation of induction conditions. Fertilization oc- curs in drops of water. Fertilization will occur naturally under the surface of the agar or where water has condensed bu t may be encouraged by irrigating the cultures with sterile distilled water. If a sexual life cycle is needed for genetic analysis, it is important to induce the sexual cycle every few months. If cultures are main- tained as gametophytes for long periods of time, they rend to become sterile, probably as a result of accumulated chromosomal aberrations.

IV. DISCUSSION

The moss PhyscomitreUa patens is a useful organism for both research and classroom use. The moss cells are haploid (an advantage in the isolation of mutants); the generation time is short; large numbers of cells may be grown in culture under controlled conditions in the dif- ferentiated or undifferentiated stare; and the cells are totipotent. Moss culture provides a system whereby a large number of cells may be employed for developmental and genetic stud- ies under controlled environmental and nutri- tional conditions. Developmental studies may include analyses of spore germination require- ments, phase change from protonemal to gametophore growth form, conditions neces- sary for the initiation of a sexual life cycle (3) or the effects of environmental factors such as light on development (8). Additionally, the or- ganism may be used for the isolation and char- acterization of morphological and biochemical variant lines from mutagenized cultures. The organisms may be grown on inorganic medium or supplements may be added to establish selective conditions for mutant isolation. Moss plants express many characteristics of higher plants and may be used as a convenient model system to examine aspects of plant biology.

V. REFERENCES

1. Engel, P. The induction of biochemical and morphological mutants in the moss, Physcomitrella patens. Amer. Jour. Bot. 55: 438-446; 1968.

E. Sexual life cycle in culture

The sexual life cycle of the moss in culture may be induced by lowering the temperature to 17 ° C. For Physcomitrella patens, mature gametangia are produced before two weeks and

2. Nakosteen, P.C. Studies on the induction, selection and inheritance of biochemical mutants in mosses. Ph.D. Dissertation; University of Tennessee, pp. 228, 1978.

3. Nakosteen, P. C.; Hughes, K. W. Sexual life cycle of three species of Funariaceae in culture. The Bryologist 81: 307-314; 1978.

Journal of Tissue Culture Methods Vol. 7, No. 1, 1982 21

COLLIER AND HUGHES -- PROCEDURE NO. 49622

4. Ashton, N.W.; Cove, D.J . The isolation and preliminary characterization of auxotrophic and analogue resistant mutants of the moss, Physcomitrella patens. Molec. Gen. Genet. 154: 87-95; 1977.

5. Grimsley, N. H.; Ashton, N. W.; Cove, D. J. The production of somatic hybrids by protoplast fusion in the moss, Physcomitrella paten. Molec. Gen. Genetic. 154: 97-100; 1977a.

6. Grimsley, N. H.; Ashton, N. W.; Cove, D. J. Complementa- tion analysis of auxotrophic mutants of the moss,

Physcomitrella patens, using protoplast fusion. Molec. Gen. Genet. 155: 103-107; 1977b.

7. Stumm, I.; Meyer, Y.; Abel, W. O. Regeneration of the moss Physcomitrella patens (Hedw.) from isolated protoplasts. Plant Sci. Lett. 5: 113-118; 1975.

8. Steeves, T. A.; Sussex, I. M.; Partanen, C. R. In vitro stud- ies in abnormal growth of prothalli of the brachen fern. Amer. J. Bot. 42: 232-245; 1955.

' Percival Manufacturing Co., Boone, IA. Baker Co., Sanford, ME.

' Fisher Scientific, Norcross, GA.

Difco Bacto agar. Scientific Products, Stone Mt., GA. Tecto, Elmsford, NY.

~ j ~ Approved by Author

. s/Il Z~....X~.J B -- J ' - 9-X.2~, Da~"

22 Journal of Tissue Culture Methods Vol. 7, No. 1, 1982