Basta Selection on Soil

The major advantage of Basta selection is that it can be performed on plants growing in soil and does not require the use of sterile techniques. Basta (glufosinate ammonium) can be purchased as a herbicide solution from garden supply stores. In the United States, it is sold as Finale by Scotts Company, which has a long-term supply agreement with the manufacturer, AgrEvo. It can also be ordered on-line (http://www.biconet. com/lawn/finale.html). The pure chemical can be ordered in the United States from Crescent Chemical Co. (1-800-877-3225).

The concentration of glufosinate ammonium (C5H15N204P; FW = 198.16) in commercial preparations varies from product to product. Work out how much is present in the chosen product and use the formula weight 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 lOOOx, it provides a 300 jim working solution.

To perform Basta selection on plates, follow the procedure for kanamycin selection above, but include 25-50 jim Basta instead of kanamycin in the medium.

Other selection markers include hygromycin and gentamycin resistance. However, hygromycin is quite harmful even to transformed plants and is therefore best avoided. In addition, hygromycin is very toxic to humans, and special care must be exercised when handling it or any media containing hygromycin.

Materials

Basta (200-300 [LM) Seeds

Procedure

1. Sow seeds: Mix -0.5 g of seeds with 10 g of fine sand in a salt shaker. 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 4°C, and then move it to a growth room under lights on a 16-hour light/8-hour dark cycle.

3. After 5 days, spray the seedlings with Basta using a regular spray bottle that produces a fine mist (available from garden supply stores). Spray at least twice more at 2-3-day intervals. If germination is very uneven, continue spraying to eliminate late-germinating, nontransformed seedlings.

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

5. To avoid false positives, which may appear as a consequence of uneven herbicide application or when plants are grown at very high densities, 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.

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