Engineered minichromosomes provide efficient platforms for stacking transgenes in crop plants. Methods for modifying these chromosomes in vivo are essential for the development of customizable systems for the removal of selection genes or other sequences and for the addition of new genes. Previous studies have demonstrated that Cre, a site-specific recombinase, could be used to modify lox sites on transgenes on maize minichromosomes; however, these studies demonstrated somatic recombination only, and modified minichromosomes could not be recovered. We describe the recovery of an engineered chromosome composed of little more than a centromere plus transgene that was derived by telomere-mediated truncation. We used the fiber fluorescence in situ hybridization technique and detected a transgene on the minichromosome inserted among stretches of CentC centromere repeats, and this insertion was large enough to suggest a tandem insertion. By crossing the minichromosome to a plant expressing Cre-recombinase, the Bar selection gene was removed, leaving behind a single loxP site. This study demonstrates that engineered chromosomes can be modified in vivo using site-specific recombinases, a demonstration essential to the development of amendable chromosome platforms in plants.

Synthetic chromosomes provide the means to stack transgenes independently of the remainder of the genome. Combining them with haploid breeding could provide the means to transfer many transgenes more easily among varieties of the same species. The epigenetic nature of centromere formation complicates the production of synthetic chromosomes. However, telomere-mediated truncation coupled with the introduction of site-specific recombination cassettes has been used to produce minichromosomes consisting of little more than a centromere. Methods that have been developed to modify genes in vivo could be applied to minichromosomes to improve their utility and to continue to increase their length and genic content. Synthetic chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agricultural applications or to use plants as factories for the production of foreign proteins or metabolites.

Barbara McClintock first showed that transposable elements in maize can induce major chromosomal rearrangements, including duplications, deletions, inversions, and translocations. More recently, researchers have made significant progress in elucidating the mechanisms by which transposons can induce genome rearrangements. For the Ac/Ds transposable element system, rearrangements are generated when the termini of different elements are used as substrates for transposition. The resulting alternative transposition reaction directly generates a variety of rearrangements. The size and type of rearrangements produced depend on the location and orientation of transposon insertion. A single locus containing a pair of alternative transposition-competent elements can produce a virtually unlimited number of genome rearrangements. With a basic understanding of the mechanisms involved, researchers are beginning to utilize both naturally occurring and in vitro-generated configurations of transposable elements in order to manipulate chromosome structure.

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