rpc00052-Cl-2.2 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00052

Curcuma longa Cl callus cultureComponents• A 9-cm plastic Petri dish, containing cells placed on semi-solid medium
Notice• Subculture the cells to fresh medium immediately after arrival [Notes I].
• 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: MS medium, 1 mg/L NAA, 0.1 mg/L kinetin, 0.9% (w/v)
agar, pH 5.7 (medium no. 37) [Materials III]
• Culture conditions: 23℃, continuous light [Methods II]• Subculture: 21–28-day intervals [Methods I]
Citation of cell line
When results obtained by using this cell line are published in a scientiﬁc journal, it should be cited in the following manner: “Curcuma longa Cl cell line(rpc00052) 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
Turmeric Cl cell line was established from a root of Curcuma longa L. Some of the Cl
cells accumulate yellow pigment. The Cl cells are grown on a Murashige and Skoog (MS)medium supplemented with 1 mg/L 1-naphthaleneacetic acid (NAA) and 0.1 mg/L kinetin,and solidiﬁed with 0.9% (w/v) agar, pH 5.7. Our Cl cell culture has been maintained under
the diﬀuse ﬂuorescent light at 23℃ and subcultured at 21–28-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) MS_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
Glycine

0.5 mg/mL
0.5 mg/mL
0.1 mg/mL
2 mg/mLD) MS_inositol
myo-Inositol

40 mg/mL

E) NAA (1 mg/mL)
NAA·K
1.2 mg/mL
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation(#161-04021)
F) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mLDissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterG) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
H) KOH (1 N)

2Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of MS medium (medium no. 37)
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.
MS_VT
MS_inositol
NAA (1 mg/mL)
Kinetin (0.2 mg/mL)

1 mL
2.5 mL1 mL
0.5 mL

3. Adjust the pH of the solution to 5.7 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 40 mL of the medium into a 100-mL ﬂask containing 0.36 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 21–28-day-old culture with aforceps and place the cells onto fresh MS medium.
2. Incubate cell cultures under the diﬀuse ﬂuorescent light condition (photosynthetic
photon ﬂux density 7–12 µmol m−2 s−1 ) at 23℃.

Notes
• We send Cl cells on semi-solid MS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh MS medium immediately after arrival.
• In order to maintain Cl callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of Cl cells is aﬀected by culture conditions,such as a room temperature, aeration conditions of the culture and so on, an amountof cells transferred to fresh medium and the subculture intervals may vary from one3lab to another. We usually inoculate three to ﬁve pieces of Cl callus (about 7–10mm in diameter) on 40 mL of MS medium in a 100-mL ﬂask, and culture them for
21–28 days.

4Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 37)
ChemicalConcentration (mg/L)

KNO3

1900

NH4 NO3

1650

CaCl2 ·2H2 O

440

MgSO4 ·7H2 O

370

KH2 PO4170

H3 BO3

6.2

MnSO4 ·4H2 O

22.3

ZnSO4 ·7H2 O8.6

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

Nicotinic acid

0.5

Pyridoxine·HCl

0.5

Thiamine·HCl

0.1

Glycine2

myo-Inositol

100

Sucrose

30000

NAA·K

1.2

Kinetin

0.1

Agar9000

5