Agrobacterium-mediated transformation

Original reference; G. An Meth. Enzymol. 153 292-305. Extensively modified and edited by Ken Matsuoka. Ver. 20030904

Materials

Agrobacteria

Agrobacterium EHA101 (or other agrobacterial strains, such as LBA4404, C58C1Rif(pGV2260), A4RS, can be used for the transformation, however, the efficiency for transformation varies with each strain. See Yoshioka et al., Plant Cell Physiol. 37 782-789, 1996) containing appropriate binary plasmid. For control experiment, EHA101/pIG121-Hm is used. Agrobacteria is grown at 30oC in YEB plate with antibiotics (in the case of pIG121-Hm, 50 mg/l of Hygromycin B, for pGA type plasmids, 5 mg/l of Tetracycline). Two or three-day old culture was suspended into BY-2 culture medium at a concentration of 1-3 OD600. (Higher concentration up to 10 OD600 increase transient expression but decrease the number of stable transformant).

EHA101: Hood E. E. et al. J. Bacteriol. 168 1291 (1986). This strain contain pEHA101, which is a derivative of a supervirlent Ti plasmid pTiBo542. The entire T-DNA region of pTiBo542 was replaced to Tn5 Kanamycin resistance gene to yeild pEHA101. The chromosomal background of EHA101 is A136, which is a Rifr, Nalr and Ti plasmid cured derivative of C58. pIG121-Hm, Ohta et al. Plant and Cell Physiol. 31: 805-813, 1990)

Tobacco cell

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 min.

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 transformants

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 cefotaxim.

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).

Methods

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

discard supernatant

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°C, 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.

: Time depends on the conditon of cells and the growth rate.

Usually, cell concentration is 0.04-0.06 ml packed cell volume/ml culture.

# : Co-culture time depends on the construct and purposes.