We have developed a new plant vector system for repeated transformation (called MAT for multi-autotransformation) 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 ؋ 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.

SummaryWe represent here the GST-MAT vector system. The R recombinase gene of the site-speci®c recombination system R/RS from Zygosaccharomyces rouxii was fused to the chemical inducible promoter of the glutathione-S-transferase (GST-II-27) gene from Zea mays. Upon excision, the isopentenyltransferase (ipt) gene that is used as a selectable marker gene is removed. When the cauli¯ower mosaic virus 35S promoter (CaMV 35S) was used to express R recombinase, 67% of the marker-free transgenic plants had more than three transgene copies. Because the CaMV 35S promoter transiently and ef®ciently excised the ipt gene before callus and adventitious bud formation, the frequency of emergence of the ipt-shooty explants with a single T-DNA copy might be reduced. In this study we show that the GST-MAT vector ef®ciently produced transgenic ipt-shooty explants from 37 (88%) out of 42 differentiated adventitious buds and marker-free transgenic plants containing the GUS gene from ®ve (14%) out of 37 ipt-shooty lines. Furthermore, the GST-MAT vector also induced two marker-free transgenic plants without the production of ipt-shooty intermediates. Southern blot analysis showed that six (86%) out of seven marker-free transgenic plants had a single GUS gene. This result suggests that the GST-MAT vector is useful to generate high frequency, marker-free transgenic plants containing a single transgene.

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We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii ϫ Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.Peroxidases (EC 1.11.1.7, donor: hydrogen peroxide oxidoreductase) are widely found in animals, plants, and microbes and oxidize a vast array of compounds (electron donors) in the presence of hydrogen peroxide (H 2 O 2 ). Forty-two expected sequence tags encoding different peroxidase isoenzymes are found in rice (Oryza sativa; Hiraga et al., 2001). Hoyle (1977) found 42 isoenzymes and/or isoforms in commercial preparations of horseradish (Armoracia rusticana) peroxidase (HRP). The plant peroxidase superfamily is divided into three classes based on differences in primary structure (Welinder, 1992). Class I plant peroxidases include the intracellular enzymes in plants, bacteria, and yeast (Saccharomyces cerevisiae), such as microbial cytochrome c peroxidase (EC 1.11.1.5), bacterial catalase-peroxidase (EC 1.11.1.6), and ascorbate peroxidase (EC 1.11.1.11). Class II plant peroxidases are extracellular peroxidases from fungi, including lignin peroxidase (EC1.11.1.14) and Mn 2ϩ -independent peroxidase (EC 1.11.1.13). Class III plant peroxidases (EC 1.11.1.7) were originally described as peroxidases and are secreted outside of the cells or transported into vacuoles. HRP prxC1a of this study is a member of the class III peroxidases. To date, four genomic DNAs that encode HRP (Fujiyama et al., 1990) and four cDNAs have been isolated (Fujiyama et al., 1988; Bartnek-Roxa et al., 1991). All of the genes consist of four exons and three introns, and the number of amino acid residues deduced from nucleotide sequences varies from 327 to 353. Nucleotide sequence homologies in the coding regions were found to be 90% between prxC1a and prxC1b, 71% between prxC1a and prxC2, and 66% between prxC1a and prxC3. The prxC2 gene is induced by wounding and functional analysis of the prxC2 promoter has been reported previou...