rpc00058-GlV-2.4 (2018.3.23; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00058

Glehnia littoralis GlV 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: B5 medium, 1 mg/L NAA, 0.01 mg/L kinetin, 0.8% (w/v)
agar, pH 5.8 (medium no. 48) [Materials III]
• Culture conditions: 27℃, dark [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 scientiﬁcjournal, it should be cited in the following manner: “Glehnia littoralis GlV
cell line (rpc00058) 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
GlV cell line was established from a petiole of Glehnia littoralis F.Schmidt ex Miq. (Miuraet al. 1998). The GlV callus culture is violet, and produces anthocyanins (Miura et al.
1998) and coumarin derivatives (umbelliferone) (Ishikawa et al. 2005). The GlV cells aregrown on a Gamborg’s B5 medium supplemented with 1 mg/L 1-naphthaleneacetic acid(NAA) and 0.01 mg/L kinetin, and solidiﬁed with 0.8% (w/v) agar, pH 5.8. Our GlV cell
culture has been maintained in the dark at 27℃ and subcultured at 21–28-day intervals.

Materials
Chemicals and stock solutions(All stock solutions are stored at 4℃)
A) B5 salt mix
Gamborg’s B5 Medium Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#399-00621)
B) SucroseC) B5_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HCl
myo-Inositol

0.4 mg/mL
0.4 mg/mL
4 mg/mL
40 mg/mLD) NAA (1 mg/mL)
NAA·K

1.2 mg/mLE) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mLDissolve kinetin in small volume of KOH (1 N), and ﬁll up with distilled waterF) Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
G) KOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (100 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use

2Preparation of B5 medium (medium no. 48)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
B5 salt mix
Sucrose1 bag (1 L)
20 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
B5_VT
NAA (1 mg/mL)
Kinetin (0.2 mg/mL)

2.5 mL
1 mL
0.05 mL

3. Adjust the pH of the solution to 5.8 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 0.32 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 B5 medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send GlV cells on semi-solid B5 medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh B5 medium immediately after arrival.
• In order to maintain GlV callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of GlV 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 one
lab to another. We usually inoculate three to four pieces of GlV callus (about 3–5mm in diameter) on 40 mL of B5 medium in a 100-mL ﬂask, and culture them for
21–28 days.

ReferencesIshikawa A, Kitamura Y, Ozeki Y, Itoh Y, Yamada A, Watanabe M (2005) Post-stressmetabolism involves umbelliferone production in anthocyanin-producing and non-3

producing cells of Glehnia littoralis suspension cultures. Journal of Plant Physiology 162: 703–710. DOI: 10.1016/j.jplph.2004.11.014
Miura H, Kitamura Y, Ikenaga T, Mizobe K, Shimizu T, Nakamura M, Kato Y, Yamada
T, Maitani T, Goda Y (1998) Anthocyanin production of Glehnia littoralis callus
cultures. Phytochemistry 48: 279–283. DOI: 10.1016/S0031-9422(97)01115-1

4Appendix A: Formulation of culture medium
Table A.1. Gamborg’s B5 medium(medium no. 48)
ChemicalConcentration (mg/L)

KNO32500(NH4 )2SO4134

CaCl2 ·2H2 O

150

MgSO4 ·7H2 O

250

NaH2 PO4 ·H2 O

150

H3 BO33MnSO4 ·H2 O

10ZnSO4 ·7H2 O2KI0.75

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 acid1

Pyridoxine·HCl1

Thiamine·HCl10

myo-Inositol

100

Sucrose

20000

NAA·K

1.2

Kinetin

0.01

Agar8000

5