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

Upon incubation of Agrobacterium tumefaciens A348 with acetosyringone, the vir genes encoded by the Ti (tumor-inducing) plasmid are induced. The addition of certain opines, including octopine, nopaline, leucinopine, and succinamopine, enhanced this induction 2-to 10-fold. The compounds mannopine, acetopine, arginine, pyruvate, and leucine did not stimulate the induction of the vir genes to such an extent. The enhancement of vir gene induction by opines depended on acetosyringone and the genes virA and virG. Opines stimulated the activity of the vir genes, the double-stranded cleavage of the T (transferred)-DNA at the border repeat sequences, and the production of T-strands by the bacterium. The transformation efficiency of cotton shoot tips was markedly increased by the addition of acetosyringone and nopaline at the time of infection.Phenolic compounds secreted by wounded plant cells can induce the activity of certain genes encoded by the Ti (ttmor-inducing) plasmid of Agrobacteriiin tuimeficiens (3,37,39 (42, 51) for such phenolic inducers as acetosyringone (37). The virG locus, which is expressed at a low basal level, is subsequently induced to higher levels of expression (38). This gene presumably encodes a transcriptional activator that induces its own expression and the expression of the genes 1i'rB, virC, virD, virE, and pinF (27,42,51). The virD locus encodes a T-DNA border-specific endonuclease (1,17,41,49,52,53) that nicks the T-DNA border repeat sequences on the bottom strand, generating single-stranded T-DNA molecules called T-strands (1,40,48). T-strands can be coated with a virE-encoded singlestranded-DNA-binding protein (6,8,11,15) and have been hypothesized to be the form of the T-DNA transferred to the plant cell (43). The virD endonuclease also generates doublestranded scissions at the T-DNA borders (17,47,48). The regulation of the genes *irC and virD by the chromosomal locus ros has also been described (9,10,44).Following insertion into plant nuclear DNA, T-DNA genes are transcribed and direct the synthesis of enzymes involved in the production of phytohormones and unusual low-molecular-weight compounds termed opines (for reviews, see references 2, 16, and 31). The phytohormones stimulate the uncontrolled proliferation of plant cells, resulting in tumors called crown gall tumors. Opines, which are crown gall tumor-specific compounds, are secreted into the rhizosphere where they can be used as an energy source by the inciting bacterium to the exclusion of most other soil microorganisms. It (45,46).We are interested in the relationship, if any, between opines and vir gene induction. We initially hypothesized that opines may inhibit the induction of these genes by acetosyringone. The rationale was that the bacterium would not induce the T-DNA transfer machinery if it sensed that a tumor, recognized by the presence of opines, had already been formed. The results presented below suggest that many opines do affect the acetosyringone induction of the vir genes. The effect, however, is to enhance r...

A method of regenerating cotton plants from the shoot apical meristem of seedlings was developed for use with particle gun and Agrobacterium-mediated transformation. This method was developed to circumvent the problems of genotype restriction and chromosomal damage frequently encountered in cotton regeneration in tissue culture through somatic embryogenesis. In this procedure, the cells of the shoot meristem are targeted for transformation. Normal and fertile plants of Gossypium barbadense Pima S-6, and 19 cultivars of G. hirsutum were regenerated using this method. Shoot regeneration from these tissues was direct and relatively rapid. A MS based, hormone-free medium could be used with all the varieties tested.

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