Selection of marker-free transgenic plants using the isopentenyl transferase gene

Abstract

We have developed a new plant vector system for repeated transformation (called MAT for multi-auto-transformation) in which a chimeric ipt gene, inserted into the transposable element Ac, is used as a selectable marker for transformation. Selectable marker genes conferring antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants, have three major problems: (i) the selective agents have negative effects on proliferation and differentiation of plant cells; (ii) there is uncertainty regarding the environmental impact of many selectable marker genes; (iii) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The MAT vector system containing the ipt gene and the Ac element is designed to overcome these difficulties. When tobacco leaf segments were transformed and selected, subsequent excision of the modified Ac produced marker-free transgenic tobacco plants without sexual crosses or seed production. In addition, the chimeric ipt gene could be visually used as a selectable marker for transformation of hybrid aspen (Populus sieboldii x Populus grandidentata). The chimeric ipt gene, therefore, is an attractive alternative to the most widely used selectable marker genes. The MAT vector system provides a promising way to shorten breeding time for genetically engineered crops. This method could be particularly valuable for fruit and forest trees, for which long generation times are a more significant barrier to breeding and genetic analysis.

Figure 1

Diagram of MAT vector pNPI106. Plasmid pNPI106 has a “hit and run” cassette in which the chimeric ipt gene with ³⁵S promoter is inserted into Ac as a selectable marker. The gusA and nptII genes are unselected markers in these experiments. Arrows, PCR primers (see Fig. 3); 35S-P, CaMV ³⁵S promoter; ipt, isopentenyl transferase gene; T, isopentenyl transferase terminator; N-P, nopaline synthase promoter; N-T, nopaline synthase terminator; nptII, neomycin phosphotransferase gene; gusA, β-glucuronidase gene.

Figure 2

Visible selection of marker-free transgenic tobacco plants. (a) Regeneration of adventitious shoots from leaf segments on nonselective medium. (b) Differentiation of ESP from adventitious shoots. (c) Appearance of “normal” morphological shoots from ESP. (d) Normal rooted plant.

Figure 3

PCR analysis of ESP shoots and normal plants obtained from ESP shoots. (a) Amplification of the DNA fragments using primers c and d flanking the ipt gene (Fig. 1). The arrow indicates a fragment of approximately 800 bp. (b) Amplification of the fragments using primers a and b (Fig. 1) flanking the position of hit and run cassette. The arrow indicates amplified fragments of 3 kb that result from empty donor sites. Lanes: 1, HindIII size marker (TAKARA shuzo); 2, plasmid pNPI106 in a and plasmid pBI121 in b; 3 and 4, DNA from two independent ESP shoot-derived clones, in which normal shoots did not reappear; 5, DNA from a ESP shoot-derived clone, in which normal shoots reappeared; 6–11, DNA from two independent normal shoots from each of three ESP shoot-derived clones. Lanes 6 and 7 represent 2 shoots from line 2, lanes 8 and 9 represent 2 shoots from line 3, and lanes 10 and 11 represent 2 shoots from line 4.

Figure 4

Transformation of hybrid aspens by the MAT vector system. (a) Differentiation of the ESP from adventitious shoots. (b) Appearance of morphologically normal shoots from ESP.