rpc00033: Lotus japonicus LjA 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: MS medium, 1 mg/L 2,4-D, 0.1 mg/L kinetin, 0.9% (w/v) agar, pH 5.7 (medium no. 29)
Culture conditions: 27°C, dark
Subculture: 28-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: “Lotus japonicus LjA cell line (rpc00033) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”
Introduction
LjA cell line was established from a shoot apex of a seedling of Lotus japonicus (Regel) K.Larsen accession Gifu B-129. The LjA cells are grown on a Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.1 mg/L kinetin, and solidified with 0.9% (w/v) agar, pH 5.7. Our LjA cell culture has been maintained in the dark at 27°C and subcultured at 28-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
MS_VT
Chemical
Concentration (mg/mL)
Nicotinic acid
0.5
Pyridoxine·HCl
0.5
Thiamine·HCl
0.1
Glycine
2
MS_inositol
Chemical
Concentration (mg/mL)
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)
Kinetin (0.2 mg/mL)
Chemical
Concentration (mg/mL)
Kinetin
0.2
Dissolve kinetin in small volume of KOH (1 N), and fill up with distilled water
Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
KOH (1 N)
Glassware and equipment
Erlenmeyer flask (100 mL), capped with two layers of aluminum foil
Forceps, sterilized before use
Preparation of MS medium (medium no. 29)
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)
MS_VT
1
MS_inositol
2.5
2,4-D (0.2 mg/mL)
5
Kinetin (0.2 mg/mL)
0.5
Adjust the pH of the solution to 5.7 with KOH (1 N), and fill up to 1 L with distilled water.
Pour 40 mL of the medium into a 100-mL flask containing 0.36 g of agar.
Autoclave the flask at 121°C for 20 min.
Methods
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.
Incubate cell cultures under the dark condition at 27°C.
Notes
We send LjA cells on semi-solid MS medium in a 9-cm disposable Petri dish. The cells should be subcultured to fresh MS medium immediately after arrival.
In order to maintain LjA callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of LjA 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 three pieces of LjA callus (about 5-mm in diameter) on 40 mL of MS medium in a 100-mL flask, and culture them for 28 days.
The parent accession Gifu B-129 is available from National BioResource Project Lotus/Glycine (https://www.legumebase.brc.miyazaki-u.ac.jp/).
Genotyping
Maintenance history
Protocols
Genotyping of plant cell lines: Lotus japonicus cell lines