Materials and Reagents

1. 90 mm plastic Petri dish
   
2. 50 ml tube
   
3. 1.5 ml tube
   
4. Physcomitrella patens protonemal cells (cultivated for 4 days)
   
5. BCDAT medium (Nishiyama et al., 2000)
   
6. Methyl methanesulfonate (MMS) (Sigma-Aldrich, catalog number: 129925 )
   
7. Chloroform (Sigma-Aldrich, catalog number: V800117 )
   
8. Ethanol (Sigma-Aldrich, catalog number: 09-0770 )
   
9. RNase A
   
10. LA Taq (TAKARA BIO, catalog number: RR002A )
    
11. Agarose powder (Promega, catalog number: V3125 )
    
12. 0.7% agarose gel
    
13. Ethidium bromide (EtBr)
    
14. Power SYBR^® green PCR master mix (Thermo Fisher Scientific, Applied Biosystems^TM, catalog number: 4367659 )
    
15. Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H-5882 )
    
16. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 28-2270 )
    
17. Tris-HCl, pH 8.0 (Wako Pure Chemical Industries, catalog number: 206-07884 )
    
18. Ethylenediamine-N,N,N’,N’-tetraacetic acid, disodium salt, dihydrate (EDTA) (DOJINDO, catalog number: 345-01865 )
    
19. PEG8000 (MP Biomedicals, catalog number: 02194839 )
    
20. Oligonucleotide DNA primers for long amplicon quantitative PCR:
    Nuclear DNA (5′-GTGGAAGTGAGATGCAGTTTGG-3′ and 5'-GTGGCTCTGGACAGTGAAATTG-3′)
    Chloroplast DNA (5′-AATTGGAGTCGGTCCTTCCATA-3′ and 5′-TGGCAAATACAATGGCAAAAAG-3′)
    Mitochondrial DNA (5′-GACTGCCCTCACTAGGATGCTT-3′ and 5′-TGGGTGATTTACTCCATTGACG-3′)
    
21. Oligonucleotide DNA primers for short amplicon quantitative PCR:
    Nuclear DNA (5′-CAACCGTCTTCTGTGTCTAGGTC-3′ and 5′-GAAACCGGCCTGCATTACATG-3′)
    Chloroplast DNA (5′-AGTAATCCCATCGCGTGACAT-3′ and 5′-AGGTATGGAAAAAATCGCTGAAAA-3′)
    Mitochondrial DNA (5′-CGTGCTAAAAATCCAGTCCATTC-3′ and 5′-AGCAAAGAAGTCAAGACCTAACAAAAC-3′)
    
22. 2x CTAB buffer (see Recipes)
    
23. 10% CTAB (see Recipes)
    
24. CTAB ppt buffer (see Recipes)
    
25. NaCl-TE (see Recipes)
    
26. TE buffer (see Recipes)
    
27. PEG solution (see Recipes)
    

Equipment

1. Forceps
   
2. Multi-beads shocker (Yasui Kikai)
   
3. Vacuum pump (Tokyo Rikakikai, catalog number: AS-3 )
   
4. Bench top centrifuge for 1.5 ml tubes
   
5. Heat block for 1.5 ml tubes (TAITEC, catalog number: DTU-2CN )
   
6. Standard thermal cycler (Bio-Rad Laboratories, catalog number: 170-8720JA )
   
7. Real-time PCR thermal cycler (Thermo Fisher Scientific, Applied Biosystems^TM, model: Applied Biosystems^® 7500 Fast Real-Time PCR Systems )
   

Software

1. ImageJ (https://imagej.nih.gov/ij/)
   
2. Excel (Microsoft)

Procedure