protocol-overseas-BY-2-1.0 (2024.3.13)
RIKEN BRC plant cell line documentation

Protocol for overseas customers

Re-establishment of suspension cultures of tobacco BY-2 and related
cell lines

Cell lines• rpc00001: BY-2
• rpc00039: GV7• rpc00040: GF11
• rpc00041: GT16
• rpc00062: BY-TIPG• rpc00091: TBY2-31/ST• rpc00093: TBY2-41/ST• rpc00095: TBY2-R31
Culture medium• medium no. 1: modiﬁed Linsmaier and Skoog (mLS) medium, 0.2 mg/L
2,4-dichlorophenoxyacetic acid, pH 5.8Experimental 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Methods
Regrowth of BY-2 cells
1. Recieve an 250-mL Erlenmeyer ﬂask containing BY-2 cells.

2. Loosen the screw cap slightly to keep good aeration.
CAUTION

Avoid microbial contamination.

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3. Incubate the BY-2 cells in the dark at 27℃ for 1–2 weeks.

After 8 days.
The red arrowheads indicate the growing BY-2 cells.

BY-2 cells around the edge are alive.

BY-2 cells around the center are dead.

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4. Use the growing BY-2 cells for the induction of suspension cell cultures and for themaintenance of stock agar cultures.
Induction of suspension cell cultures
1. Transfer the BY-2 cells into a 100-mL Erlenmeyer ﬂask containing 20 mL of a liquid
culture medium.
CAUTIONUse at least the amount of BY-2 cells shown below.

2. Incubate the BY-2 cells in the dark at 27℃ with rotary shaking at 130 rpm for 5–10days.
CAUTION

Avoid microbial contamination.

CAUTIONCover the ﬂask mouth loosely with an aluminum foil cap for good aeration.

NOTEA silicone sponge plug can be used instead of an aluminum foil cap(e.g., Cap-type Silicosen; Shin-Etsu Polymer, Tokyo, Japan).

After 7 days

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3. Allow the Erlenmeyer ﬂask to stand for a few minutes just before the ﬁrst subcultureof suspension cell culture.
4. Transfer 1–2 mL of the sedimented cells into 95 mL of a fresh culture medium in a
300-mL Erlenmeyer ﬂask.
NOTESee Appendix A for the BY-2-related cell lines.

5. Culture the BY-2 cells by the usual method.

After 7 days
6. After 3–4 subculturing, the BY-2 suspension cell cultures grow stably.

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Maintenance of stock agar cultures
1. Prepare cell culture dishes containing 30 mL of a culture medium solidiﬁed with
0.8% (w/v) agar.
NOTE

BY-2 cells can also be cultured on the media solidiﬁed with gellangum.

2. Transfer small pieces (3–5 mm in diameter) of BY-2 cells onto agar culture medium.

3. Seal culture dishes by using surgical tape to keep good aeration.
4. Incubate the BY-2 cells in the dark at 27℃ for 2–4 weeks.

After 4 weeks
5. Subculture the BY-2 cells on fresh agar medium at 2–4-week intervals.

6Appendix A: The ﬁrst subculture of suspension cell cultures of BY-2related cell lines
BRC No.

Cell line

Transfer volume*(mL)

Culture medium(mL)

Erlenmeyer ﬂask(mL)

rpc00001

BY-2

1–295300

rpc00039

GV7

1–395300

rpc00040

GF11

3–695300

rpc00041

GT16

3–695300

rpc00062

BY-TIPG

1–395300

rpc00091

TBY2-31/ST

0.4–0.830100

rpc00093TBY2-41/ST

0.4–0.830100

rpc00095

TBY2-R31

0.4–0.830100* Subculture sedimented cells to a fresh culture medium.
CAUTIONFor the transgenic BY-2 cell lines, check the expression of ﬂuorescent proteins in re-established suspension cells before use.

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Appendix B: Preparation of BY-2 cell cultures for transportation
Preparation of a cell culture
1. An agar-solidiﬁed culture medium was prepared in a 250-mL disposable Erlenmeyer
ﬂask.
NOTEmLS medium (medium no. 1) solidiﬁed with 1.4% (w/v) agar, 80 mL2. BY-2 cell suspension culture was prepared on day 7 of the culture.
3. BY-2 cells were spread on the agar culture medium in the 250-mL disposable Erlenmeyer ﬂask.
4. The screw cap was loosely closed to keep good aeration.
5. The BY-2 cells were pre-cultured in the dark at 27℃ for 6 days.
6. The screw cap was tightly closed and sealed with thermoplastic sealing ﬁlm.
Transportation test
1. The BY-2 cells was stored in the dark at 27℃ for 7 days (simulating transport).
2. The BY-2 cells were tested for regrowth.

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