Maintenance of Tobacco TBY2-AtRER1B Callus Culture

Transcription

1 Introduction C-TBY2-AtRER1B-005 Maintenance of Tobacco TBY2-AtRER1B Callus Culture The Tobacco TBY2-AtRER1B cell line is a transgenic BY-2 cell line expressing Green Fluorescent Protein (GFP) fused with AtRer1B (Takeuchi et al and 2002). Because the Golgi can be visualized by using a fluorescent microscope, TBY2-AtRER1B cell line is used for studying detailed structures of plant cells. The parent cell line BY-2 was derived from a callus induced from a stem of Nicotiana tabacum L. cultivar Bright Yellow 2 in the presence of a phytohormone, 2,4-dichlorophenoxyacetic acid (2,4-D) (Nagata et al. 1992). Our TBY2-AtRER1B cells have been maintained on a modified Linsmaier and Skoog (mls) medium solidified with 4 g/l gellan gum in dark at 27 C and subcultured at four-week intervals. Materials I. Stock Solution and Chemicals A) MS salt mixture Murashige and Skoog Plant Salt Mixture, Wako Pure Chemical Industries (catalog # ) B) Sucrose C) Modified LS_VT (100 ml) Thiamine HCl 40 mg myo-inositol 4 g D) BY2_P (100 ml) KH 2 PO 4 8 g E) 0.2 mg/ml 2,4-D (100 ml) 2,4-D 20 mg F) Gellan gum, powder G) 200 mg/ml Kanamycin (1 ml) Kanamycin 200 mg Sterilize the solution by membrane filtration. H) 500 mg/ml Carbenicillin (1 ml) Carbenicillin 500 mg Sterilize the solution by membrane filtration. Glassware and Equipment (All are sterilized by autoclaving at 121 C for 20 min.) A) Erlenmeyer flask (100 ml), capped with two layers of aluminum foil B) Forceps 1

2 I Preparation of mls Medium Methods A) Dissolve one bag (1 L) of the MS salt mixture and 30 g of sucrose in approximately 800 ml of distilled water. B) Add following stock solutions to A), and fill up to approximately 950 ml with distilled water. Modified LS_VT 2.5 ml BY2_P 2.5 ml 0.2 mg/ml 2,4-D 1 ml C) Adjust the ph of the solution to 5.8 with 0.2 N KOH, and fill up to 1 L with distilled water. D) Pour 40 ml of mls medium into a flask containing 0.16 g of gellan gum. E) Autoclave the flask at 121 C for 20 min. F) Add following antibiotics to the medium just before agar solidification and swirl the medium gently to mix. 200 mg/ml Kanamycin 40 μl 500 mg/ml Carbenicillin 40 μl I. Pick up an appropriate amount of callus cells from a four-week-old culture with a forceps and place the cells onto fresh mls medium. Incubate callus cultures under dark condition at 27 C. Notes I. We transport the TBY2-AtRER1B callus on semi-solid mls medium in a 90-mm Petri dish. The callus should be transferred to fresh mls medium immediately after arrival. The whole callus may be transferred without dissection. To maintain the TBY2-AtRER1B cell culture stably, the growth of cells should be observed carefully. Because proliferation of TBY2-AtRER1B cells is affected by culture conditions, such as a room temperature, aeration conditions of the culture and so on, an adequate amount of cells transferred to fresh medium and the subculture intervals may vary from one lab to another. We usually inoculate three pieces of TBY2-AtRER1B callus (about 5-mm in diameter) on 40 ml of semi-solid mls medium in a 100-ml flask, and culture them for four weeks (Figure 1). I It is essential to use good and healthy cells. Brownish cells near the upper part of a callus are not suitable for subculturing. IV. Very occasionally, GFP fluorescence of the TBY2-AtRER1B cells can be weakened during long-term maintenance. 2

3 References Nagata T, Nemoto Y, Hasezawa S (1992) Tobacco BY-2 cell line as the HeLa cell in the cell biology of higher plants. International Review of Cytology 132: 1-30 Takeuchi M, Ueda T, Sato K, Abe H, Nagata T, Nakano A (2000) A dominant negative mutant of sar1 GTPase inhibits protein transport from the endoplasmic reticulum to the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant J. 23: Takeuchi M, Ueda T, Yahara N, Nakano A (2002) Arf1 GTPase plays roles in the protein traffic between the endoplasmic reticulum and the Golgi apparatus in tobacco and Arabidopsis cultured cells. Plant J. 31:

4 (A) (B) Figure 1: Tobacco TBY2-AtRER1B callus culture TBY2-AtRER1B cells were cultured in a 100-mL flask containing 40 ml of semisolid mls medium under dark condition at 27 C for 0 (A) and 4 (B) weeks. 4

5 Appendix 1 Composition of modified Linsmaier and Skoog medium Chemical Concentration (mg/l) KNO NH 4 NO CaCl 2 2H 2 O 440 MgSO 4 7H 2 O 370 KH 2 PO H 3 BO MnSO 4 4H 2 O 22.3 ZnSO 4 7H 2 O 8.6 KI 0.83 Na 2 MoO 4 2H 2 O 0.25 CuSO 4 5H 2 O CoCl 2 6H 2 O FeSO 4 7H 2 O 27.8 Na 2 EDTA 37.3 Thiamine HCl 1 myo-inositol 100 Sucrose ,4-Dichlorophenoxyacetic acid 0.2 Gellan gum, powder 4000 Kanamycin 200 Carbenicillin 500 5