N. tabacum cultured cell line BY-2 (Ikeda et al.
Agric. Biol. Chem. 40 1765 (1976)). This cell is grown in a
medium described by Nagata et al. (MGG 184 161 (1981)).
Cells were subcultured weekly at 1.2-1.5 % inoculum (1.5-3 ml) in 95 ml
flesh medium in 300-ml-vol Erlenmeyer flask. The flask is shaken at
130-135 rpm with rotory shaker (the radius of the shaker is 3.5 cm) at 26
to 26.5 oC, dark. With this condition, the cell doubling time
is 12 to 14 hours. The higher the cell growth rate of the cell cause the
higher transformation frequency and higher level of transient expression.
At log phase (2 to 4 days after subculturing), single cell clump should
contain less than 16 cells and most of the clump contain 8 cells. The
suspension of this cells can be handled with normal pipett. If the cell
clump is too large and cells grow slowly, check the culute condition of the
cells or to get the cells from other sources.
Mediumfor the growth of tobacco cells.
MS salts suppliment with 200 mg/l KH2PO4, 0.1 mg/l
thiamin-HCl, 10 mg/l myo-inositol, 3% sucrose, 0.2 mg/l 2,4-D. pH 4.8-5.2
before autoclaving. Sterilize by autoclaving at 121oC for 15
For the selection of transformants, cultue medium is
solidified by 0.4 % gelan gum (GeliteTM) and 0.1% MgSO4 7H2O. Cefotaxim
(500 mg/l, e.g. Cefotax bland) and appropriate antibitics for the selection
of transformed tobacco cells (e.g. Kanamycin, 200 mg/l or hybromycin B, 50
mg/l) should be add to the media after autoclaving.
Growth chamber for co-cultivation and selection of
Growth chamber or of 26-27oC, dark or dim
light is recomended for co-cultivation. Do not exceed the temperature
above 28oC. For the selection of tobacco cell transformants,
use 28oC growth chamber (temperature below 26 oC
inhibit the growth of BY-2 cells). The chamber should be kept dark, since
light is inhibitory for the growthand light enhance the degradation of
Plastic ware for co-cultivation
Falcon dish No. 1029 (diameter = 100 mm) or other
gamma-ray sterilized culture dish.
Do not use gas-sterilized dish, as there is a
possibility that ethylen remained in the dish.
Seal tape of dishes during the co-cultivation periods
Pylon E-G CUT tape, width 18 mm. (Kyowa Ltd. Tachibana
3-20-28, Nishinari-ku, Osaka 557 JAPAN). (maybe insufficient gas exchange
during the culture).
4 ml of 72 h-old
culture of BY-2 in 9-cm petri dish (gamma-ray sterilized).
Add 100 ul of agrobacteria (1 A660/ml to 3 A660/ml).
Mix, seal dish by E-G CUT tape.
Incubate for 42 to 60 hours at 25-27oC dark,
on a completely vertical place. (This is quite important)
24-36 hours after: Harvest and transfer the co-culture
by pipet into 15 ml sterilized disposable conical tube. Wash cells by
culture medium for 3 to 4 times.
Washing steps are as follows;
add culture medium to 12 ml
mix cells and culture medium by pipet
centrifuge at 1000 rpm for 1 min by bench-top centrifuge
Suspend cells to 0.1 ml pcv*/ml for the selection of transformant
0.5 to 1 ml of this suspension is transferred on solid
culture medium containing antibiotics and sporead the cell suspension to
cover the whole surface of the medium. Remove the liquid meedium on the
plate as much as possible. Seal the plate with E-G cut tape (or parafilm)
and incubate at 28$B!k(BC, dark for 2 to 4 weeks. Within 20 days, small
colonies of the transformants appear on the plates. Usually, 100 to 3000
coloneis appear on a single plate.