Autonomous chromosomes are generated in yeast (yeast artificial chromosomes) and human fibrosarcoma cells (human artificial chromosomes) by introducing purified DNA fragments that nucleate a kinetochore, replicate, and segregate to daughter cells. These autonomous minichromosomes are convenient for manipulating and delivering DNA segments containing multiple genes. In contrast, commercial production of transgenic crops relies on methods that integrate one or a few genes into host chromosomes; extensive screening to identify insertions with the desired expression level, copy number, structure, and genomic location; and long breeding programs to produce varieties that carry multiple transgenes. As a step toward improving transgenic crop production, we report the development of autonomous maize minichromosomes (MMCs). We constructed circular MMCs by combining DsRed and nptII marker genes with 7–190 kb of genomic maize DNA fragments containing satellites, retroelements, and/or other repeats commonly found in centromeres and using particle bombardment to deliver these constructs into embryogenic maize tissue. We selected transformed cells, regenerated plants, and propagated their progeny for multiple generations in the absence of selection. Fluorescent in situ hybridization and segregation analysis demonstrated that autonomous MMCs can be mitotically and meiotically maintained. The MMC described here showed meiotic segregation ratios approaching Mendelian inheritance: 93% transmission as a disome (100% expected), 39% transmission as a monosome crossed to wild type (50% expected), and 59% transmission in self crosses (75% expected). The fluorescent DsRed reporter gene on the MMC was expressed through four generations, and Southern blot analysis indicated the encoded genes were intact. This novel approach for plant transformation can facilitate crop biotechnology by (i) combining several trait genes on a single DNA fragment, (ii) arranging genes in a defined sequence context for more consistent gene expression, and (iii) providing an independent linkage group that can be rapidly introgressed into various germplasms.
The production of transgenic maize has traditionally used techniques that integrate DNA fragments into a host chromosome. This can disrupt important native genes or can lead to poor expression of the added gene; consequently, large numbers of transgenic plants must be screened to find one suitable for commercial use. Further, there is a limit to the amount of DNA that can be integrated, making it difficult to add multiple genes at one time. Here, we describe a new system for delivering genes to maize. We constructed a minichromosome vector that remains separate, or autonomous, from the plant's chromosomes when introduced into maize cells. These minichromosomes were constructed from DNA sequences that naturally occur in maize centromeres, the chromosomal regions needed for inheritance. We characterized the behavior of Maize Minichromosome 1 (MMC1) through four generations, showing that it is efficiently inherited and that the genes it carries are expressed. This work makes it possible to design minichromosomes that carry several genes, enhancing the ability to engineer plant processes, including improving disease resistance, drought tolerance, or the production of complex biochemicals.