rpc00065-H440-2.2 (2021.3.15; revised 2024.8.28)
RIKEN BRC plant cell line documentation

rpc00065

Amaranthus H440 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, 0.25% (w/v) gellan gum, pH 5.7 (medium no.
44) [Materials III]
• Culture conditions: 25℃, continuous light [Methods II]• Subculture: 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: “Amaranthus H440 cell line
(rpc00065) 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
Amaranth H440 cell line was established from a leaf of Amaranthus sp. (Asano 2011,Asano and Otobe 2011). The H440 callus cells are dark red and produce betacyanin. The
H440 cells are grown on a phytohormone-free Murashige and Skoog (MS) medium solidiﬁed with 0.25% (w/v) gellan gum, pH 5.7. Our H440 cell culture has been maintained
under the continuous light at 25℃ 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 ChemicalCorporation (#392-00591)
B) SucroseC) MS_VT
Nicotinic acid
Pyridoxine·HCl
Thiamine·HClGlycine

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

40 mg/mL

E) Gellan gum
Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)
F) KOH (1 N)
Glassware and equipment
A) Erlenmeyer ﬂask (200 mL), capped with two layers of aluminum foil
B) Forceps, sterilized before use
Preparation of MS medium (medium no. 44)
1. Dissolve the following chemicals in approximately 800 mL of distilled water.

2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

1 mL
2.5 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 60 mL of the medium into a 200-mL ﬂask containing 0.15 g of gellan gum.
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 MS medium.
2. Incubate cell cultures under the continuous light condition (photosynthetic photon
ﬂux density 45–50 µmol m−2 s−1 ) at 25℃.

Notes
• We send H440 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 H440 callus culture stably, it is essential to observe the growth ofcells carefully. Because proliferation of H440 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 ﬁve pieces of H440 callus (about 3–10-mm in diameter) on 60 mL of MS medium in a 200-mL ﬂask, and culture themfor 28 days.
• Bright pink H440 cells are suitable for subculture.

References
Asano S (2011) Research into pigment production using cultured plant cells grown without
phytohormones. PhD thesis, University of Tsukuba, Japan (in Japanese). http://hdl.
handle.net/2241/114680

3Asano S, Otobe K (2011) Production of phytochemicals by using habituated and long-term
cultured cells. Plant Biotechnology 28: 51–62. DOI: 10.5511/plantbiotechnology.
10.1109a4

Appendix A: Formulation of culture medium
Table A.1. Murashige and Skoog medium(medium no. 44)
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 O8.6KI

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

Gellan gum

25005