rpc00003-VR-2.2 (2017.10.19; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00003

Vitis VR callus culture

Components• 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 ﬂow
cabinet.
Method• Culture medium: mLS medium, 0.05 mg/L 2,4-D, 0.2 mg/L kinetin, 1.2%(w/v) agar, pH 6.1 (medium no. 3) [Materials III]
• Culture conditions: 27℃, dark [Methods II]• Subculture: 56-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: “Vitis VR cell line (rpc00003)
was provided by the RIKEN BRC through the National BioResource Projectof 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/

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Introduction
Grape VR cell line was established from anthers of Vitis interspeciﬁc hybrid cultivar Bailey Alicante A (Yamakawa et al. 1983a, b). The VR callus cells accumulate higher amountof anthocyanins than VW callus cells (rpc00004). The VR cells are grown on a modiﬁed
Linsmaier and Skoog (mLS) medium supplemented with 0.05 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/L kinetin, and solidiﬁed with 1.2% (w/v) agar, pH 6.1. Our
VR cell culture has been maintained in the dark at 27℃ and subcultured at 56-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_modiﬁed
Thiamine·HCl
myo-Inositol

0.4 mg/mL
40 mg/mLD) 2,4-D (0.2 mg/mL)
2,4-D sodium monohydrate
0.236 mg/mL(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich(D6679)
E) Kinetin (0.2 mg/mL)
Kinetin
0.2 mg/mL
Dissolve 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)

2Glassware and equipment
A) Erlenmeyer ﬂask (300 mL), capped with two layers of aluminum foilB) Forceps, sterilized before use
Preparation of mLS medium (medium no. 3)
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_modiﬁed
2,4-D (0.2 mg/mL)
Kinetin (0.2 mg/mL)

2.5 mL0.25 mL
1 mL

3. Adjust the pH of the solution to 6.1 with KOH (1 N), and ﬁll up to 1 L with distilledwater.
4. Pour 100 mL of the medium into a 300-mL ﬂask containing 1.2 g of agar.
5. Autoclave the ﬂask at 121℃ for 20 min.

Methods
1. Pick up an appropriate amount of callus cells from a 56-day-old culture with a forceps and place the cells onto fresh mLS medium.
2. Incubate cell cultures under the dark condition at 27℃.

Notes
• We send VR cells on semi-solid mLS medium in a 9-cm disposable Petri dish. Thecells should be subcultured to fresh mLS medium immediately after arrival.
• In order to maintain VR callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of VR 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 onelab to another. We usually inoculate two to three pieces of VR callus (about 8-mmin diameter) on 100 mL of mLS medium in a 300-mL ﬂask, and culture them for 56days.

3• It is important to subculture good healthy cells. Yellow and pink VR cells are themost suitable, but red cells are too aged.

References
Yamakawa T, Ishida K, Kato S, kodama T, Minoda Y (1983a) Formation and identiﬁcationof anthocyanins in cultured cells of Vitis sp. Agricultural and Biological Chemistry47: 997–1001. DOI: 10.1080/00021369.1983.10865764
Yamakawa T, Kato S, Ishida K, Kodama T, Minoda Y (1983b) Production of anthocyanins by Vitis cells in suspension culture. Agricultural and Biological Chemistry
47: 2185–2191. DOI: 10.1080/00021369.1983.10865938

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Appendix A: Formulation of culture medium
Table A.1. modiﬁed Linsmaier and Skoog medium(medium no. 3)
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 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

1

myo-Inositol

100

Sucrose30000

2,4-D sodium monohydrate

0.059

Kinetin

0.2

Agar

12000

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