rpc00069-156-S-2.2 (2021.3.15; revised 2024.1.29)
RIKEN BRC plant cell line documentation

rpc00069

Coptis japonica 156-S transgenic cell suspension cultureComponents• Domestic delivery: A 50-mL plastic conical centrifuge tube, containing cellsuspension• Overseas delivery: A 250-mL plastic Erlenmeyer ﬂask, containing cells
placed on semi-solid medium
Notice• Subculture the cells to fresh medium immediately after arrival [Notes I, II].
• Do not store the cell culture in a refrigerator and a freezer.
• Maintain aseptic conditions of the cell culture, and work in a laminar ﬂowcabinet.
Method• Culture medium: LS medium, 10 µM NAA, 0.01 µM BAP, pH 5.7 (mediumno. 54) [Materials III]
• Culture conditions: 25℃, dark, 90 rpm [Methods II]• Subculture: 21-day intervals [Methods I]
Citation of cell line
When results obtained by using this cell line are published in a scientiﬁcjournal, it should be cited in the following manner: “Coptis japonica 156S cell line (rpc00069) was provided by the RIKEN BRC through the National
BioResource Project of the MEXT, Japan.”Experimental Plant Division
RIKEN BioResource Research Center (BRC)
Koyadai 3-1-1, Tsukuba, Ibaraki 305-0074
JapanFAX: +81 29 836 9053
E-mail: plant.brc@riken.jp
http://epd.brc.riken.jp/en/

1

Introduction
Coptis japonica 156-S cell line is a transgenic cell line over-expressing Coptis (S)-scoulerine 9-O-methyltransferase (Sato et al. 2001). The 156-S is a yellow high berberineproducing cell line. The parent cell line 156-1 was established from a petiole-derived root
culture of Coptis japonica (Thunb.) Makino var. dissecta (Yatabe) Nakai ex Satake (Yamada and Sato 1981, Sato and Yamada 1984). The 156-S cells are grown in a Linsmaierand Skoog (LS) medium supplemented with 10 µM 1-naphthaleneacetic acid (NAA) and
0.01 µM 6-benzylaminopurine (BAP), pH 5.7. Our 156-S cell culture has been maintainedin the dark at 25℃ with rotary shaking at 90 rpm and subcultured at 21-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical
Corporation (#392-00591)
B) SucroseC) LS_VT
Thiamine·HCl
myo-Inositol

0.16 mg/mL
40 mg/mLD) NAA (1 mM)
NAA·K
0.224 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
E) BAP (1 mM)
6-Benzylaminopurine
0.225 mg/mL
Dissolve 6-benzylaminopurine in small volume of KOH (1 N), and ﬁll up with
distilled waterF) KOH (1 N)
GlasswareA) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foil, sterilized byautoclaving at 121℃ for 20 min2Preparation of LS medium (medium no. 54)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
Sucrose1 bag (1 L)
30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
LS_VT
NAA (1 mM)
BAP (1 mM)

2.5 mL10 mL
0.01 mL

3. Adjust the pH of the solution to 5.7 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Autoclave the medium at 121℃ for 20 min.

Methods
1. Leave to stand a ﬂask containing a 21-day-old culture until the cells settle to thebottom of the ﬂask.
2. Discard culture medium by decantation.
3. Wash the cells with a fresh LS medium two times. If the cell amount is too much,discard some cells with the medium.
4. Add 1.5 times the volume of fresh LS medium to the cells (up to a total volume ofapproximately 100 mL).
5. Incubate cell cultures on a rotary shaker at 90 rpm under the dark condition at 25℃.

Notes
• For domestic customers: We send 156-S cell suspension in a 50-mL disposable conical centrifuge tube. The cells should be transferred to fresh LS medium immediatelyafter arrival.
• For overseas customers: We send 156-S cells placed on semi-solid LS medium
in a 250-mL disposable Erlenmeyer ﬂask. The cells should be transferred tofresh LS medium immediately after arrival. Collect the cells from the semi-solid
medium with a spatula and transfer them to Erlenmeyer ﬂasks containing fresh liquid medium.

3• In order to maintain 156-S cell suspension cultures stably, it is essential to transfer
an adequate amount of cells to fresh LS medium in every subculture. The amountof cells may vary from one lab to another, because proliferation of 156-S cells is
aﬀected by culture conditions, such as a room temperature, rotation speed of a rotary
shaker, and aeration condition of the culture.
• In order to obtain good aeration of a suspension culture, a silicone sponge plug
may be used instead of the aluminum foil cap (e.g., cap-type Silicosen; Shin-Etsu
Polymer, Tokyo, Japan; https://www.shinpoly.co.jp/en/product/product/medical/plugs.html).
• Growth of 156-S is very slow. Do not signiﬁcantly reduce the cell amount in theliquid medium.

References
Sato F, Hashimoto T, Hachiya A, Tamura K, Choi K-B, Morishige T, Fujimoto H, Yamada Y (2001) Metabolic engineering of plant alkaloid biosynthesis. Proceedingsof National Academy of Sciences of USA 98: 367–372. DOI: 10.1073/pnas.98.1.3
67
Sato F, Yamada Y (1984) High berberine-producing cultures of Coptis japonica cells.
Phytochemistry 23: 281–285. DOI: 10.1016/S0031-9422(00)80318-0
Yamada Y, Sato F (1981) Production of berberine in cultured cells of Coptis japonica.
Phytochemistry 20: 545–547. DOI: 10.1016/S0031-9422(00)84193-X

4Appendix A: Formulation of culture medium
Table A.1. Linsmaier and Skoog medium(medium no. 54)
ChemicalConcentration (mg/L)

KNO31900

NH4 NO31650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O

8.6

KI

0.83

Na2 MoO4 ·2H2 O

0.25

CuSO4 ·5H2 O

0.025

CoCl2 ·6H2 O

0.025

FeSO4 ·7H2 O

27.8

Na2 -EDTA

37.3

Thiamine·HCl

0.4

myo-Inositol

100

Sucrose30000

NAA·K

2.24

6-Benzylaminopurine

0.002255