Protocol

Kanamycin Selection of Transformed 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

The most commonly used markers for selection of transgenic Arabidopsis are resistance to the antibiotic kanamycin and to the herbicide glufosinate ammonium. Resistance to kanamycin is conferred by a bacterial gene encoding the enzyme neomycin phosphotransferase (NPT). In this protocol, kanamycin-resistant seedlings are selected on solid medium.

RELATED INFORMATION

For a protocol describing the selection of transgenic Arabidopsis using glufosinate ammonium, see Glufosinate Ammonium Selection of Transformed Arabidopsis.

MATERIALS

Reagents

Agarose (0.1%), sterile

Ethanol

Reagents for PCR screening of transformed plants (optional; see Step 7)

Seed sterilization solution

Seeds transformed with the vector of interest (see In Planta Transformation of Arabidopsis)

Selection plates

Triton X-100 (optional; see Step 3)

Tween-20 (optional; see Step 3)

Equipment

Filter paper, sterile (optional; see Step 3)

Petri dishes, 150 mm

Standard equipment for growing Arabidopsis (see Cultivation of Arabidopsis)

Tape, microporous (e.g., 3M)

METHOD

1. Surface-sterilize the appropriate quantity of seeds by soaking them in ethanol for 1 minute and then soaking them in seed sterilization solution for an additional 10 minutes.
Use 100 mg of dry seed per 150-mm Petri dish. At higher seed densities, the kanamycin selection is less effective.

2. Wash the seeds in four changes of H₂O, and suspend them in the appropriate volume of 0.1% agarose (5 ml/Petri dish or 100 mg of seed).

3. Spread the seed suspension over the selection plates and allow the plates to dry a little so that the seeds do not float when the plate is moved.
Alternatively, surface-sterilize the appropriate quantity of seeds by soaking in 70% ethanol with 0.05% Triton X-100 or Tween-20 for 5 minutes and then rinsing in 95% (v/v) ethanol. Air-dry the seed on sterile filter paper and sprinkle the seed on the selective plates.

4. Seal the plates with microporous tape and incubate at 4ºC to break dormancy.

5. After 2 days, transfer the plates to a plant tissue culture room with adequate light.

6. After 7 days, check the plates for transformants, which will be visible as green seedlings with long roots. Untransformed seedlings will be yellowish with short roots.

7. Transfer the transformants to soil. Gently pull seedlings out of the agar, and remove residual agar with a forceps or fingers.
Once the plants start to flower, it is advisable to verify by PCR that the transformants contain the construct of interest.