rpc00098: Nicotiana tabacum topless3-GFP transgenic callus culture
Note
Components
A 9-cm plastic Petri dish, containing cells placed on semi-solid medium
Notice
Subculture the cells to fresh medium immediately after arrival.
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, 0.2 mg/L 2,4-D, 0.5% (w/v) gellan gum, pH 5.8 (medium no. 59)
Culture conditions: 27°C, dark
Subculture: 21-day intervals
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: “Nicotiana tabacum topless3-GFP cell line (rpc00098) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”
Introduction
Tobacco topless3-GFP cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with NtTPL3 (Nagashima et al. 2019 [1]). GFP fluorescence is observed in nuclei by using a fluorescence microscope. The parent cell line BY-2 (rpc00001) was established from a callus induced from a seedling of Nicotiana tabacum L. cultivar Bright Yellow 2 (Nagata et al. 1992 [2]). The topless3-GFP cells are grown on a modified Linsmaier and Skoog (mLS) medium supplemented with 0.2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D), and solidified with 0.5% (w/v) gellan gum, pH 5.8. Our topless3-GFP cell culture has been maintained in the dark at 27°C and subcultured at 21-day intervals.
Materials
Chemicals and stock solutions
(All stock solutions are stored at 4°C)
MS salt mix
Murashige and Skoog Plant Salt Mixture, FUJIFILM Wako Pure Chemical Corporation (#392-00591)
Sucrose
BY2_P
Chemical
Concentration (mg/mL)
KH2PO4
80
LS_VT_modified
Chemical
Concentration (mg/mL)
Thiamine·HCl
0.4
myo-Inositol
40
2,4-D (0.2 mg/mL)
Chemical
Concentration (mg/mL)
2,4-D sodium monohydrate
0.236
(2,4-Dichlorophenoxy)acetic acid sodium salt monohydrate, Sigma-Aldrich (D6679)
Gellan gum
Gellan gum, FUJIFILM Wako Pure Chemical Corporation (#073-03071)
KOH (1 N)
Glassware and equipment
Petri dish (9 cm diameter, 2 cm height), sterile
Forceps, sterilized before use
Surgical tape
3M™ Micropore™ Surgical Tape, 12.5 mm × 9.1 m, 3M Japan Limited (#1530-0)
Preparation of mLS medium (medium no. 59)
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical
Amount
MS salt mix
1 bag (1 L)
Sucrose
30 g
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution
Volume (mL)
BY2_P
2.5
LS_VT_modified
2.5
2,4-D (0.2 mg/mL)
1
Adjust the pH of the solution to 5.8 with KOH (1 N), and fill up to 1 L with distilled water.
Add 5 g of gellan gum to the medium.
Autoclave the medium at 121°C for 20 min.
Pour 35 mL of the medium into a 9-cm Petri dish.
Methods
Pick up an appropriate amount of callus cells from a 21-day-old culture with a forceps and place the cells onto fresh mLS medium.
Incubate cell cultures under the dark condition at 27°C.
Seal the Petri dishes using two rounds of surgical tape.
Notes
We send topless3-GFP cells on semi-solid mLS medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh mLS medium immediately after arrival.
In order to maintain topless3-GFP callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of topless3-GFP cells is affected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, an amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate sixteen pieces of topless3-GFP callus (about 1–2-mm in diameter) on 35 mL of mLS medium in a 9-cm Petri dish, and culture them for 21 days.