BTI Tomato Transformation Protocol – last updated 8/1/06

From Joyce van Eck, Boyce Thompson Institute, Cornell University. If questions, email Joyce: jv27@cornell.edu.

A) Preparation of Plant Material

1)    Sterilize seed

a.   Immerse seed in 20% Clorox for 20 min. (100 seeds~380mg.)

b.   Rinse well with sterile Milli-Q water (2 or more times).

2)   Sow seed in Magenta boxes containing 1/2 MSO (approximately 30 seeds/box).

3)    Prepare feeder layer (one day prior to cutting cotyledons).

a.     Pipet 2 ml of a one-week- old NT1 suspension culture onto a KCMS medium plate.  (NT1 cells are subcultured weekly (2:48) in KCMS liquid medium).

b.     Cover suspension with a sterile 7 cm Whatman filter

c.     Culture in dark, overnight

4)   One day prior to inoculation with Agrobacterium, cut cotyledons from 6 – 8-day-old seedlings or the appropriate germination time for your material.  It is very important that the first true leaves have not emerged.

a.     Place seedling on a sterile paper towel moistened with sterile water

b.   Excise cotyledon at petiole and cut tips off.  Cut in half if size

of cotyledon is > 1 cm.  Use curved scalpel blades because they cause less damage than straight blades.

c.   Place explants on feeder plates adaxial side down

d.     Culture 25C, 16 hr photoperiod.

B) Agrobacterium

1)    Streak Agrobacterium onto LB selective media.  Incubate 24 - 48 hrs at 28C.

2)    Select 4, single well-formed colonies from the plate and transfer to 50 ml of YM selective medium.  Culture in a shaking incubator 250 rpm at 28^oC for 24 hours.

3)    After 24 hrs, check the OD₆₀₀. Optimum OD₆₀₀ = 0.6 –0.7.  If OD₆₀₀ > 1.0, dilute the culture until the OD₆₀₀ reading is below 0.5 and grow for another hour. Check the OD₆₀₀ reading periodically.

4)    Centrifuge at 8000 rpm (Sorvall centrifuge, SS34 rotor) for 10 minutes at 20C.

5)    Pour the supernatant into a graduated cylinder and record the volume.  Discard the supernatant.

6)    Resuspend the pellet in the same amount of MS-0,2% liquid medium by vortexing.  Inoculum is ready to use.

C) Transformation

1)    Incubate explants in Agrobacterium culture/MS-O,2%

a.     Pipette 25 ml of Agrobacterium culture into a sterile Magenta box.

b.     Transfer explants from 2 to 3 plates into inoculum in Magenta box.

c.     Incubate for 5 min with occasional shaking.

d.     Remove explants to a sterile paper towel.

e.     Return explants to feeder plates, adaxial side down.

f.      Seal plates with Nesco film.

2)    Cocultivate explants in the dark at 19C or 25C if 19C not available, for 48 hrs.

3)    Transfer explants to selection media (2Z) adaxial side up.  Culture only 20 – 25 explants per plate.  Seal plates with micropore tape.  Culture at 25C, 16-hr photoperiod.  Transfer weekly to fresh 2Z selection medium for 3 weeks, then transfer to 1Z selection medium.

4)    Transfer explants to new 1Z selection medium plates every 2 weeks until all the shoots you need are harvested.  When shoots begin to appear, transfer explants to 1Z selection medium in Magenta boxes.

D) Regeneration/Rooting

1)    Initial shoots should appear within 4 - 6 weeks.

2)    Excise shoots from explants when shoots are at least 2 cm and include at least 1 node.  Place in Magenta boxes (4/box) containing Tomato Rooting Media with selective agent and antibiotic used to prevent Agrobacterium growth.

3)    Roots should begin to appear in about 2 weeks.  When plants are large enough, they can be propagated to Tomato Rooting medium + selection agent + antibiotic.

4)   For propagation of confirmed lines, transfer to RM-0 -- no antibiotics and no selection agent.  Antibiotic- and selection agent-free medium allows the plants to grow faster and will provide a check to determine if Agrobacterium is present.  However, do not transfer to antibiotic-free medium until the shoots have gone through at least 3 propagations onto medium with antibiotics and selection agent as described under #3. 

This protocol is a modified version of Fillatti et al. 1987, Biotechnology 5:726-730.

Media

1/2 MSO

                                                                        KCMS

LB

YM

Alternatively, YM in powder form can be purchased (Gibco BRL; catalog #10090-011).  To make liquid culture medium, add 11.1 g to 1 liter water.

MS 0, 2% Liquid Medium

2Z

Add zeatin after autoclaving.  Use a filter sterilized stock solution to give a final concentration of 2 mg/l.  We make our stock at 1 mg/ml and add 2 ml per liter.  The selective agent and timentin should also be added after autoclaving.  Use a concentration of selection agent appropriate for your tomato line.  We use timentin at a final concentration of 300 mg/l/.  Selection agents we have used include: kanamycin (50, 75, 100 mg/l), bialaphos (see  protocol for bialaphos selection), hygromycin (6 mg/l).  We had to modify the protocol for bialaphos selection.  If interested, contact Joyce Van Eck (jv27@cornell.edu) for the modified protocol.

1Z medium is the same basic recipe as 2Z, however, the final concentration of zeatin is 1 mg/l.

Selective Rooting Medium

Add the following filter-sterilized components per liter after autoclaving:

Kanamycin: Depending on the tomato line and what works best for your tomato, use kanamycin at either a final concentration of 50, 75, or 100 mg/l.

Other selection agents we have used: Bialaphos: (see protocol for bialaphos selection), hygromycin (6 mg/l).

Timentin: 3 ml of a 100 mg/ml stock solution

Nitsch Vitamins Stock (1000x)

Adjust pH to 7.0 to clear solution.

Aliqout into 1 ml volumes in tubes, and freeze.