Bar gene as a selection marker for maize transformation
--Z. Zhao, K. Lowe and W. Marsh

The Bar gene has been widely used as a selectable marker for plant transformation. Transgenic cells and plants expressing this gene are resistant to the herbicides Basta (registered in Europe), Bialaphos (registered in Japan) and Ignite (registered in the USA).

A reliable selectable marker is essential for the recovery of transgenic plants at the present time. When a transgenic plant is crossed with a non-transgenic plant, segregation of the transformed gene(s) in the next generations is expected. A screenable selectable marker linked to an agronomic gene(s) can make the identification of transformed progeny efficient and fast. The data presented here indicate that the Bar gene is ideal for this purpose. Large or small scale screening to identify transformants can easily be done in greenhouse and field settings. Herbicide screening has been used to successfully identify transformants, and these results have been confirmed by Southern analysis.

The herbicide Basta is considered a non-selective herbicide, and is toxic to all non-transgenic plants. To determine the correct dose of Basta, seedlings from three maize genotypes (B73, Mo17 and W22) were treated at the 3-5 leaf stage. The seedlings were sprayed with different concentrations of Basta solution and scored for herbicide damage as shown in Figure 1. From these results, it has been noticed that three major factors, genotype, herbicide concentration, and the environment, influence the response of maize plants to Basta.

Based on these results, seedlings carrying the Bar gene, grown as above, were sprayed with Basta solutions ranging from 4.5 L/ha. to 8 L/ha. These seedlings were resistant to all of the treatments (Fig. 1).

Seed was produced from Bar-containing plants pollinated with non-transgenic pollen representing three independent transformation events (making up three families). The original plants representing these three events indicated varying levels of Bar enzyme activity, from high (1,518 units) to low (635 units, control=476 units). However, the resistant seedlings representing these three transformation events (families) showed equal resistance to these herbicides at all concentrations. When older plants (7-10 leaves) were sprayed in the same manner occasional lesions were observed but these did not affect plant development. Bar-containing adult plants painted with 1% Basta solution to an area of their leaves usually showed very good resistance with minimal damage, correlating to Bar enzyme activity. However, painting 3 inches of a leaf tip of a non-transgenic adult plant with 1% Basta solution will result in necrosis of the painted area or in some cases death of the entire plant.

Since young plants (3-5 leaf stage) carrying the Bar gene are completely resistant to herbicide spraying we recommend screening plants at this stage of growth.

To confirm the reliability of herbicide screening for identification of Bar containing plants, genomic Southern hybridization was done on randomly selected plants segregating for the Bar gene. Southern analysis (using the Bar coding region as a probe) from a random sampling of 45 out of 1447 segregating seedlings confirmed 33 out of 45 plants contained the Bar gene, and only these plants were resistant to Basta.

In summary, the herbicide spraying (Basta/Ignite) of young maize seedlings is a very dependable and economic method that can successfully identify Bar-containing plants.

Figure 1.


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