rpc00029-LcyD7-2.2 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00029

Luﬀa cylindrica LcyD7 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: LS medium, 0.1 µM 2,4-D, 0.2 mg/L BAP, 1.2% (w/v) agar,
pH 5.6 (medium no. 25) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 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: “Luﬀa cylindrica LcyD7cell line (rpc00029) 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
LcyD7 cell line was established from a seedling of Luﬀa cylindrica (L.) M.Roem. The
LcyD7 cells are grown on a Linsmaier and Skoog (LS) medium supplemented with 0.1µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/L 6-benzylaminopurine (BAP),and solidiﬁed with 1.2% (w/v) agar, pH 5.6. Our LcyD7 cell culture has been maintainedin the dark at 27℃ and subcultured at 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) D(+)-GlucoseC) LS_VTThiamine·HCl
myo-Inositol

0.16 mg/mL
40 mg/mLD) 2,4-D (1 mM)
2,4-D sodium monohydrate
0.261 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)E) BAP (1 mg/mL)
6-Benzylaminopurine
1 mg/mL
Dissolve 6-benzylaminopurine 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 (200 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use

2Preparation of LS medium (medium no. 25)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.
MS salt mix
D(+)-Glucose

1 bag (1 L)
30 g

2. Add following stock solutions, and ﬁll up to approximately 950 mL with distilledwater.
LS_VT
2,4-D (1 mM)
BAP (1 mg/mL)

2.5 mL
0.1 mL0.2 mL

3. Adjust the pH of the solution to 5.6 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 80 mL of the medium into a 200-mL ﬂask containing 0.96 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 28-day-old culture with a forceps and place the cells onto fresh LS medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send LcyD7 cells on semi-solid LS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh LS medium immediately after arrival.
• In order to maintain LcyD7 callus culture stably, it is essential to observe the growthof cells carefully. Because proliferation of LcyD7 cells is aﬀected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, anamount of cells transferred to fresh medium and the subculture intervals may varyfrom one lab to another. We usually inoculate three pieces of LcyD7 callus (about8-mm in diameter) on 80 mL of LS medium in a 200-mL ﬂask, and culture them for
28 days.

3Appendix A: Formulation of culture medium
Table A.1. Linsmaier and Skoog medium(medium no. 25)
ChemicalConcentration (mg/L)

KNO3

1900

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

KI0.83Na2 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·HCl0.4

myo-Inositol

100

D(+)-Glucose

30000

2,4-D sodium monohydrate

0.0261

6-Benzylaminopurine

0.2

Agar12000

4