Protocol

Glufosinate Ammonium 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

One of the most commonly used markers for the selection of transgenic Arabidopsis is resistance to glufosinate ammonium, an herbicide that is sold under a variety of trade names including Basta and Finale. Resistance to glufosinate ammonium is conferred by the bacterial bialophos resistance gene (BAR) encoding the enzyme phosphinotricin acetyl transferase (PAT). This protocol describes the use of glufosinate ammonium to select transformed Arabidopsis plants. The major advantage of glufosinate ammonium selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques.

RELATED INFORMATION

To perform glufosinate ammonium selection on plates, follow the protocol Kanamycin Selection of Transformed Arabidopsis, but include 25-50 µM glufosinate ammonium instead of kanamycin in the medium.

MATERIALS

Reagents

Glufosinate ammonium (200-300 µM)

Glufosinate ammonium can be purchased as an herbicide solution (e.g., as Finale from Scotts Company) or as a pure chemical (Crescent Chemical Co.). The concentration of glufosinate ammonium in commercial preparations varies from product to product, so determine how much glufosinate ammonium is present in the chosen product and use its formula weight (FW = 198.16) to calculate the required quantity. For example, one of the commercially available Finale preparations sold in garden centers contains 5.78% of glufosinate ammonium. When diluted 1000X, it provides a 300-µM working solution.

Reagents for PCR screening of transformed plants (see Step 6)

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

Equipment

Growth room with lights on a 16-hour light/8-hour dark cycle

Pots

Salt shaker

Sand, fine

Soil

Spray bottle that produces a fine mist

Tray, 12 x 24-inch

METHOD

1. Sow seeds:

i. Mix ~0.5 g seeds with 10 g of fine sand in a salt shaker.

ii. Sprinkle the seeds evenly over the surface of a 12 x 24-inch tray filled with soil.

2. Cover the tray and keep it for 2-3 days at 4ºC .

3. Move the tray to a growth room under lights on a 16-hour light/8-hour dark cycle, and allow the seedlings to grow for another 5 days.

4. Spray the seedlings with glufosinate ammonium using a regular spray bottle that produces a fine mist. Spray at least twice more at 2- to 3-day intervals. If germination is uneven, continue spraying to eliminate late-germinating, nontransformed seedlings.

5. When healthy green plants are clearly distinguishable from moribund seedlings, transplant them to new pots.

6. False positives may appear as a consequence of uneven herbicide application or when plants are grown at very high densities, so it is advisable to carry out PCR assays for the transgene.
Alternatively, progeny of the primary transformants can be subjected to selection to make sure that the presumed transformants were real.