Agrobacterium-mediated transformation of Arabidopsis thaliana T87 cultured cells

Materials

rpc00008: Arabidopsis thaliana T87 cell suspension culture
Agrobacterium tumefaciens strain GV3101, harboring a vector with an appropriate transformation construct, growing on LB agar plates

Glassware and equipment used for culturing T87 cells (rpc00008: Arabidopsis thaliana T87 cell suspension culture)
Cell culture dish, plastic, 9 cm
Cell culture dish, plastic, 15 cm
Multi-well cell culture plate, 6-well
Conical centrifuge tube, plastic, 15 mL
Nylon membrane: Product. Cut into a circle with a diameter of about 14 cm. Sterilize by autoclave at 121°C for 20 min.
Forceps: Sterilize before use

Gamborg’s B5 Medium Salt Mixture: 399-00621, FUJIFILM Wako Pure Chemical Corporation
MES hydrate: 2-(N-Morpholino)ethanesulfonic acid hydrate, M8250, Sigma-Aldrich
Gamborg’s B5 vitamin solution, 1000×: G1019, Sigma-Aldrich
1-Naphthaleneacetic acid (NAA): Potassium 1-naphthylacetate, 161-04021, FUJIFILM Wako Pure Chemical Corporation
Agar, powder: BD BACTO Agar, 214010, Becton Dickinson
Claforan: Dissolve in distilled water, sterilize by filtration, and store at −30°C
Kanamycin: Dissolve in distilled water, sterilize by filtration, and store at −30°C

Pass 7-day-old T87 suspension cell culture through a 1-mm Stainless test sieve (rpc00008: Arabidopsis thaliana T87 Figure 3).
Subculture the T87 cell suspension into JPL medium in an Erlenmeyer flask (10-fold dilution).
Incubate the T87 cell culture under continuous light at 22 with rotary shaking at 120 rpm for 2–3 days.

Inoculate a colony of Agrobacterium in LB liquid medium containing appropriate antibiotics.
Incubate the Agrobacterium culture at 30 for two days.
Transfer the Agrobacterium culture to a larger volume of LB medium containing appropriate antibiotics.
Incubate the Agrobacterium culture at 30 overnight.
Transfer 500 µL of the Agrobacterium culture to a 1.5-mL microcentrifuge tube.
Centrifuge the tube.
Wash the Agrobacterium cells with JPL medium three times.
Resuspend the Agrobacterium cells in 500 µL of JPL medium.

Transfer 5 mL of the T87 suspension cell culture into a well of a 6-well cell culture plate.
Add 50 µL of Agrobacterium culture to the T87 cell culture.
Incubate the co-cultivation culture under continuous light with rotary shaking at 120 rpm at 22 for two days (Notes 1).

Transfer the co-cultivation culture into a 15-mL conical centrifuge tube.
Centrifuge the tube at 600 rpm for 30 sec.
Wash the T87 cells with 3 mL of JPL medium three times.
Resuspend the T87 cells in 5 ml of JPL medium supplemented with 300 g/mL Claforan.
Transfer the T87 cell suspension into a well of a 6-well cell culture plate.
Incubate the T87 cell culture under continuous light with rotary shaking for 3–4 days.

Transfer the T87 suspension cell culture into a 15-mL conical centrifuge tube.
Wash the T87 cells with JPL medium one time.
Resuspend the T87 cells in JPL medium (total volume 2 mL).
Spread the T87 cells over a nylon membrane placed on CIM (15-cm dish) supplemented with 200 g/mL Claforan.
Incubate the dish under continuous light for 3 days.
Transfer the nylon membrane to CIM (15-cm dish) supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan.
Incubate the dish for further 2 weeks.
Transfer an individual green callus onto CIM (9-cm dish) supplemented with 30 g/mL Kanamycin and 200 g/mL Claforan.
Incubate the callus cultures.

Transfer a piece of green callus into JPL liquid medium supplemented with 5 g/mL Kanamycin and 200 g/mL Claforan.
Maintain the transgenic culture according to rpc00008: Arabidopsis thaliana T87 cell suspension culture.

Agrobacterium grow in the co-cultivation culture. Overgrowth of Agrobacterium damages T87 cells.
Some research papers also report transformation methods of the Arabidopsis T87 cell line. [1] [2]

Chemical/Stock solution	For 1 L
Gamborg’s B5 Medium Salt Mixture	1 bag
Sucrose	30 g
MES hydrate	0.5 g
Gamborg’s B5 vitamin solution, 1000×	1 mL
NAA (1 mM)	1 mL
H₂O	—

CIM	1 L
Agar, powder	6 g