rpc00019: Luffa cylindrica Lcy-1 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, 0.1 µM NAA, 1.2% (w/v) agar, pH 5.7 (medium no. 14)
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: “Luffa cylindrica Lcy-1 cell line (rpc00019) was provided by the RIKEN BRC through the National BioResource Project of the MEXT, Japan.”
Introduction
Lcy-1 cell line was established from a seedling of Luffa cylindrica (L.) M.Roem. The Lcy-1 cells are grown on a modified Linsmaier and Skoog (mLS) medium supplemented with 0.1 µM 1-naphthaleneacetic acid (NAA), and solidified with 1.2% (w/v) agar, pH 5.7. Our Lcy-1 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)
D(+)-Glucose
LS_VT
Chemical
Concentration (mg/mL)
Thiamine·HCl
0.16
myo-Inositol
40
NAA (1 mM)
Chemical
Concentration (mg/mL)
NAA·K
0.224
Potassium 1-naphthylacetate, FUJIFILM Wako Pure Chemical Corporation (#161-04021)
Agar, powder
Agar, powder, Junsei Chemical (#24440-1201)
NaOH (1 N)
Glassware and equipment
Erlenmeyer flask (100 mL), capped with two layers of aluminum foil
Forceps, sterilized before use
Preparation of mLS medium (medium no. 14)
Dissolve the following chemicals in approximately 800 mL of distilled water.
Chemical
Amount
MS salt mix
1 bag (1 L)
D(+)-Glucose
30 g
Add following stock solutions, and fill up to approximately 950 mL with distilled water.
Stock solution
Volume (mL)
LS_VT
2.5
NAA (1 mM)
0.1
Adjust the pH of the solution to 5.7 with NaOH (1 N), and fill up to 1 L with distilled water.
Pour the medium into a 100-mL flask containing 1.2% (w/v) 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 mLS medium.
Incubate cell cultures under the dark condition at 27°C.
Notes
We send Lcy-1 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 Lcy-1 callus culture stably, it is essential to observe the growth of cells carefully. Because proliferation of Lcy-1 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 Lcy-1 callus (about 10-mm in diameter) on mLS medium in a 100-mL flask, and culture them for 28 days.