Transformation of Zea mays L. Using Agrobacterium tumefaciens and the Shoot Apex

Abstract

Agrobacterium tumefaciens is established as a vector for gene transfer in many dicotyledonous plants but is not accepted as a vector in monocotyledonous plants, especially in the important Gramineae. The use of Agrobacterium to transfer genes into monocot species could simplify the transformation and improvement of important crop plants. In this report we describe the use of Agrobacterium to transfer a gene into corn, the regeneration of plants, and detection of the transferred genes in the F1 progeny.Shoot apices of Zea mays L. variety Funk's G90 were cocultivated with A. tumefaciens EHAl, which harbored the plasmid pGUS3 containing genes for kanamycin resistance (NPT II) and ,B-glucuronidase (GUS). Plants developed from these explants within 4 to 6 weeks. Fluorometric GUS assays of leaves and immature seeds from the plants exhibited low GUS activity. Both NOS and GUS gene fragments were amplified by polymerase chain reaction in the DNA isolated from the F1 generations of one of the original transformed plants. Southern analysis showed both GUS and NPT probes hybridized to DNA in several of the F1 progeny, demonstrating the incorporation of GUS and NPT II genes into high molecular weight DNA. These data establish successful gene transfer and sexual inheritance of the genes.Until recently, the monocotyledons and particularly the graminaceous crop species have been considered to be outside the Agrobacterium host range (1, 5). In the past, a general definition of host species range has been based on tumor or gall formation in inoculated plants. Gene transfer methods developed for economically important species considered to be outside of the Agrobacterium host range have previously been restricted to the direct transfer of DNA into protoplasts and to the few cultivars which can be regenerated from protoplasts. With the development of the particle discharge or acceleration methods of direct DNA transfer, intact cells of embryogenic callus and cell suspensions can be used. Recently, this approach resulted in the successful transformation and regeneration of corn (7,10). This approach will be applicable to maize genotypes which form embryogenic cultures.The host-range ofAgrobacterium has been under continual revision since the original review by DeCleene (5). Upon

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Fertile Transgenic Asparagus Plants Produced by Agrobacterium-mediated Transformation.

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