Plastid transformation of liverwort Marchantia polymorpha L.
suspension-cultured cell and immature thalli*6 to 10 transformants by a shot of particle bombardment were obtained for suspension-cultured
cell. 10 to 20 transformants by 10 shots of particle bombardment can be expected for immaturethalli with this protocol.

Materials(Plant)
Maintain liverwort suspension-cultured cells in 70 ml of 1M51C medium in a 300 ml flask on
rotary shaker (130 rpm) under continuous white light (50-60 µmol photons m-2 s-1) at 22°C. Use
cells from 7- to 10-day-old cultures for plastid transformation. For immature thalli, use mature
sporangia (the authors use F1 sporangia of Takaragaike-1 × Takaragaike-2).
(Transformation vector)
pCS31 vector
Approximately 1 kb each of the trnI and trnA sequences were cloned into pBluescript II as
homologous sequences*1. The aadA expression cassette was integrated between the trnI andtrnA sequences. The aadA expression cassette consists of the promoter of the ribosomal
RNA operon from the tobacco plastid genome, the rbcL ribosome binding site, the aadA
gene, and the tobacco psbA 3’-UTR (Fig. 1).

Plasmid DNA was prepared by QIAGEN Plasmid Midi Kit, and adjusted to 1 µg/µl.

For transformation of immature thalli, pCS31 was linearized by EcoRI and ScaI prior tobombardment.

*1: DNA fragments of approx. 1 kb is sufficient for homologous recombination.

Fig. 1 Schematic illustration of pCS31.
Gray boxes represent homologous
sequences. Arrows indicate PCR primers
for selection of plastid transformants
and checking of homoplastomic state.

(Equipment)
Filter (Nalgene: 134 mm x 230 mm, #300-4050) *2

Filter disks (Whatman: No. 1φ55 mm) *2



Cellophane *1 (washed with boiled water before autoclaving)AspiratorParticle delivery system (Bio-Rad: PDS-1000/He Particle delivery system)

0.6 µm gold particle (Bio-Rad: #165-2262)



900-psi Rupture Disk (Bio-Rad: #165-2328)*3

Macrocarrier (Bio-Rad, #165-2335)*3

Stopping Screen (Bio-Rad, #165-2336)*3*2: Autoclaved*3: Sterilized with 70% ethanol and air dried in clean hood(Reagents)
2.5 M CaCl 2



0.1 M SpermidineSpectinomycin dihydrochloride hexahydrate (SIGMA, #S9007-5G). Stock 50 mg/mLDNA extraction buffer

50 mM Tris-HCl (pH 8.0), 20 mM EDTA (pH 8.0), 0.3 M NaCl, 0.5% SDS, 5 M urea, 5% (v/v)
phenol (pH 7.0)
0M51C/1M51C media (1 L)

10× 0M51C stock solution 100 ml
sucrose 20 g, L-glutamate 0.3 g, Casamino acid 1.0 g, (+ 2,4-dichlorophenoxyacetic acid 1mg/L for 1M51C media). Adjust to pH 5.5 with 1N KOH. Add 1.2% agar for plate.
10 × 0M51C stock solution (4 L)

KNO 3 80 g, NH 4 NO 3 16 g, MgSO 4 ·7H 2 O 14.8 g, CaCl 2 ·H 2 O 12 g, KH 2 PO 4 11 g,EDTA-NaFe (III) 1.6 g, B5 micro components 40 ml, B5 vitamin 40 ml, 0.75% KI 4 ml


B5 micro components (100 ml)

NaMoO 4 ·2H 2 O 25 mg, CuSO 4 ·5H 2 O 2.5 mg, CoCl 2 ·6H 2 O 2.5 mg, ZnSO 4 ·7H 2 O 200 mg,MnSO 4 ·7H 2 O 1 g, H 3 BO 300 mg


B5 vitamin (100 ml)

Inositol 10 g, nicotinic acid 100 mg, pyridoxine hydrochloride 100 mg
thiamine hydrochloride 1 gMethods(Preparation of plant material)

For suspension-cultured cell
1: Layer suspension-culture cells onto the center of a filter disk using vacuum filtration (1-2 mmthickness).
2: Place filter disk with the cells onto a 1M51C plate, and incubate over night (22°C, 50-60 µmol
photons m-2 s-1) (Fig. 2A)
For immature thalli:
1: Suspend sporangia in sterile water (100 µl/sporangium)
2: Drop the spore suspension on the center of 0M51C plate covered with cellophane, and culture
spores for a week (22°C, 50-60 µmol photons m-2 s-1) (Fig. 2C).

Fig. 2 Preparation of plant materials for particle
bombardment and selection for spectinomycin
resistant lines. (A) Suspension-cultured cells
layered on a filter disk. (B) Spectinomycinresistant calli on selective medium. (C) Immaturethalli grown for 1 week. (D) Spectinomycinresistant thalli selected on selective medium.

(Particle bombardment)
1: Successively add 250 µl of 2.5 M CaCl2, 25 µL of 1 µg/µl DNA solution, and 50 µl of 0.1 M
spermidine into 230 µl of 10 mg/ml 0.6-µm diameter gold particle in 1.5 ml plastic tube.
2: Incubate on ice for 10 min with vigorous mixing for 10 seconds once in a minute.
3: Centrifuge at 2,000 × g for 3 min at 4°C.
4: Discard supernatant.
5: Add 500 µl ethanol and vortex thoroughly.
6: Centrifuge at 2,000 × g for 3 min at 4°C.
7: Repeat steps 4-6.
8: Suspend gold particles in 60 µl ethanol.
9: Use 5.4 µL aliquot of the gold particle suspension for each bombardment.
10: Perform particle bombardment according to manufacturer’s instructions. Parameters: vacuum= 28 inHg, the distance between the target stage and the stopping screen = 120 mm.

11: Culture bombarded cells overnight (22°C, 50-60 µmol photons m-2 s-1).
12: Spread cells evenly onto four selective 1M51C plates containing 500 mg/L spectinomycin
dihydrochloride without sucrose.
13: Transfer spectinomycin-resistant calli to fresh selective media after 4 weeks of culture (Figs.
2B and 2D).

(Establishment of homoplastomic transformant)
1: Transfer ~10 mg of cells or tissue (2 × 2 mm) in 1.5 ml plastic tubes.*4
2: Add 100 µl of DNA extraction buffer and disrupt cells by pestle.
3: Add 400 µl of DNA extraction buffer and vigorously mix.
4: Add 500 µl of phenol/chloroform, vigorously mix and centrifuge (10,000 ×g, 5 min).
5: Transfer 200 µl of supernatant and add 500 µl of ethanol.
6: Centrifuge (10,000 ×g, 4°C, 15 min).
7: Add 1 ml of 70% ethanol and centrifuge (10,000 ×g, 4°C, 15 min).
8: Suspend the pellet to 100-200 µl of Tris-HCl (pH 8.0) containing 10 µg/ml RNase A.
9: Select plastid transformants by PCR using primers P1 and P2 for the aadA cassette (Fig. 1) .
10: Subculture the candidate plastid transformants on selective media.
11: Check homoplastomic state by PCR using primers P1 and P3 which anneal to the regions
outside the homologous sequences (Fig. 1).*5*4: For DNA extraction, QIAGEN DNeasy Plant Mini Kit also works well.
*5: Homoplastomic transformants are readily obtained after the primary selection.

Reference
Chiyoda,S., Linley, P.J., Yamato, K.T., Fukuzawa,H., Yokota,A. and Kohchi,T (2007)
Simple and efficient plastid transformation system for the liverwort Marchantia
polymorpha L. suspension-culture cells. Transgenic Res., 16, 41-49.