Protocol

Root Transformation of Arabidopsis

This protocol was adapted from "How to Transform Arabidopsis," Chapter 5, in Arabidopsis by Detlef Weigel and Jane Glazebrook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2002.

INTRODUCTION

Arabidopsis can be stably transformed using Agrobacterium tumefaciens-mediated transfer of T-DNA. We describe the generation of transgenic plants via root transformation in tissue culture, which can be useful for transforming sterile mutants.

RELATED INFORMATION

Root transformation of Arabidopsis has largely been superseded by the vacuum-infiltration method, which is described in In Planta Transformation of Arabidopsis.

MATERIALS

Reagents

Agrobacterium culture (fresh, saturated, grown for 24-48 hours) carrying a suitable vector (see Transformation of Agrobacterium Using Electroporation or Transformation of Agrobacterium Using the Freeze-Thaw Method)

See Vectors and Agrobacterium Hosts for Arabidopsis Transformation for considerations regarding Agrobacterium strains and T-DNA vectors.

Arabidopsis seeds (250-500)

Bleach (50% [v/v]) containing Tween-20 (0.05% [v/v])

Callus-inducing medium (CIM), liquid and plates

CIM supplemented with 0.5 mg/ml timentin or carbenicillin

Ethanol (70%)

Germination medium (GM) plates

Metro-mix 200 (Grace Sierra Horticultural Products Company) or similar medium (see Step 26)

Root-inducing medium (RIM) plates

Root-inducing medium (RIM), slanted plates (see Step 24)

Selection plates

Shoot-inducing medium (SIM) plates

Equipment

Filter paper, sterile (wrap 5 x 5-cm squares in aluminum foil and autoclave)

Forceps, fine-point

Growth chamber with lights, preset to 23°C

Microcentrifuge tubes, 1.5-ml

Petri dishes, various sizes

Pipette, wide-bore

Polypropylene tubes, 50-ml sterile

Plastic wrap

Porous tape (e.g., 3M)

Scalpels, sterile

Scissors, sterile

Squirt bottle

METHOD

The original protocol for root transformation was developed by Valvekens et al. (1988). The following protocol is based on that published by Huang and Ma (1992) and on those posted by Roger Innes and Pablo Scolnik on the electronic Arabidopsis bulletin board. Several parameters that improve transformation have been investigated by Marton and Browse (1991).

Sterilization of Seeds and Growth of Seedlings

1. Aliquot 250-500 seeds into a 1.5-ml microcentrifuge tube, and then add 1 ml 70% ethanol. Let stand on the bench for 5 minutes.

2. Wash the seeds twice with H₂O and then add 1 ml of 50% (v/v) bleach/0.05% Tween-20.

3. Incubate the seeds for 10 minutes, agitating occasionally. Wash the seeds three times with double-distilled water and resuspend them in ~0.5 ml H₂O.

4. Use a wide-bore pipette to sow ~50 seeds in a dense, straight line near the edge of a GM plate (as shown in Figure 1 ). Wrap the plates with porous tape.

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Figure 1. Sowing seeds for root transformation

5. Stratify the seeds by incubating for 48 hours at 4ºC, and then stand the plates vertically in a 23ºC growth chamber. Make sure the line of seeds is horizontal.

6. Allow the seeds to grow for 2-3 weeks under constant light or for long days, preferably in a growth chamber used only for tissue culture, at 22ºC-23ºC. Roots should be white and healthy.

Callus Induction and Agrobacterium Inoculation

Use sterile working techniques for the following steps.

7. Transfer the plants from GM plates to a sterile Petri dish. Sever the roots. It is important to remove any remaining green parts as these will inhibit regeneration. Place roots on CIM without antibiotics. Use two CIM plates for each GM plate. Make sure that the entire root system contacts with medium surface.

8. Seal plates with porous tape, and incubate for 3 days in a growth chamber under continuous light or long days at 22°C-23°C.

9. After preculture on CIM, transfer the roots into a sterile 50-ml polypropylene tube containing 20 ml of liquid CIM.

10. Add 2 ml of a fresh, saturated Agrobacterium culture grown for 24-48 hours, and swirl or vortex gently.

11. Allow the roots to settle for ~2 minutes.

12. Decant the liquid and blot the roots on several layers of sterile filter paper to remove excess liquid.

13. Transfer the roots to CIM plates without antibiotics.

14. Incubate the roots for 48 hours, after which root explants should be overgrown by Agrobacterium.

Selection and Regeneration

15. Transfer root explants to a 50-ml polypropylene tube containing 20 ml of liquid CIM supplemented with timentin or carbenicillin.

16. Shake or vortex the roots gently. Allow the root fragments to settle and decant the liquid. Transfer any escaped root fragments back into the tube.

17. Repeat Step 16.

18. Blot the roots on sterile filter paper to remove excess liquid, and then cut the roots into 5- to 10-mm-long fragments using a sterile scissors or scalpel.

19. Place the root fragments in clumps on SIM plates with appropriate antibiotics.

20. Seal plates with porous tape and incubate in a growth chamber as in Step 6.

21. Green calli (initially apparent as dark flecks) will appear after about 6 days. When calli reach 1-2 mm in diameter, use a sterile scalpel to sever the roots 1-2 mm away from each callus and place on a fresh SIM plate.

22. After ~2 weeks, shoots should begin to appear. Excise shoots from callus, and transfer calli and shoots to separate, fresh SIM plates. Calli usually go on to produce more shoots. Break large calli (>5-mm diameter) into smaller pieces.
Keep track of multiple shoots originating from single calli--these will normally not constitute independent events. However, because of the vagaries of the regeneration procedure, it is useful to keep several shoots from each callus.

23. When shoots are 4-10 mm in length, remove callus with fine-point forceps and transfer shoots to RIM plates. Avoid shoots that do not have a largely normal rosette (i.e., those that consist only of disorganized leaves).

24. Place shorter shoots (4-6 mm) on a 60 x 20-mm Petri dish, and longer ones (7-10 mm) on a 100 x 15-mm Petri dish with slanted RIM agar (Fig. 2 ).

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Figure 2. Slanted plates and regenerating plantlets

25. Place large RIM plates with the longer shoots vertically in an incubator. Regenerating roots will grow on or near the surface of the agar, making it easy to remove plants without damaging the roots.

Transfer of Transformants to Soil

Although flowers can develop under tissue culture conditions, those that do produce very few, if any, seeds. The critical step in transferring transformants to soil is the regeneration of roots, which Huang and Ma (1992) found to be promoted by elevated -napththylphthalamic acid (NAA) concentrations. High shoot density on RIM plates also promotes root generation, with a dozen or more shoots on each RIM plate being advisable.

26. Lightly pack Metro-mix 200 or similar medium into small plastic pots. Moisten soil thoroughly with tap water.

27. Carefully remove robust plantlets with several millimeters of root growth from the agar. Rinse with water using a squirt bottle to remove residual agar and vitreous tissue from the base of the shoot. Place the plantlets in soil, and use a squirt bottle to water them.

28. Cover the pot with plastic wrap to maintain high humidity and return it to the growth chamber. After ~5 days, or as soon as stems have straightened, puncture the plastic wrap. Remove the plastic wrap completely when the plants have further adjusted to lower humidity. Keep soil damp, but not wet, and allow the plants to set seeds.

29. Confirm the presence of transgenes by germinating seeds on kanamycin-containing selection plates (see Kanamycin Selection of Transformed Arabidopsis).

DISCUSSION

There are large differences in the efficiency with which different ecotypes are transformed by this method. No-0 is one of the more efficient ecotypes, but C24, RLD, and Col-0 also work. Landsberg erecta is notoriously difficult to transform using this method.

REFERENCES

Huang H. and Ma H. 1992. An improved procedure for transforming Arabidopsis thaliana (Landsberg erecta) root explants. Plant Mol. Biol. Rep. 10: 372–383.

Marton L. and Browse J. 1991. Facile transformation of Arabidopsis. Plant Cell Rep. 10: 235–239.

Valvekens D., Van Montagu M., Van Lijsebettens M. 1988. Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. 85: 5536–5540.[Abstract/Free Full Text]