Construction and behavior of engineered minichromosomes in maize

Abstract

Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes. By using telomere-mediated chromosomal truncation, it was demonstrated that such an approach is feasible for the generation of minichromosomes of normal A chromosomes by selection of spontaneous polyploid events that compensate for the deficiencies produced. B chromosomes are readily fractionated by biolistic transformation of truncating plasmids. Foreign genes were faithfully expressed from integrations into normal B chromosomes and from truncated miniB chromosomes. Site-specific recombination between the terminal transgene on a miniA chromosome and a terminal site on a normal chromosome was demonstrated. It was also found that the miniA chromosome did not pair with its progenitor chromosomes during meiosis, indicating a useful property for such constructs. The miniB chromosomes are faithfully transmitted from one generation to the next but can be changed in dosage in the presence of normal B chromosomes. This approach for construction of engineered chromosomes can be easily extended to other plant species because it does not rely on cloned centromere sequences, which are species-specific. These platforms will provide avenues for studies on plant chromosome structure and function and for future developments in biotechnology and agriculture.artificial chromosomes ͉ FISH ͉ genetic engineering ͉ telomere truncation A rtificial chromosomes involving de novo centromere formation on an independently assembled unit and engineered minichromosomes produced by telomere truncation provide striking advantages over traditional methods of gene transformation in yeast and mammalian cells (1-5). The development of such chromosomes in plants would provide these advantages for many applications in basic studies, biotechnology, and agriculture. These chromosomes could be used as independent platforms for foreign gene expression without random integration into the normal chromosomes. Further additions of unlimited amounts of DNA could be added to these platforms in a sequential manner via different site-specific recombination cassettes. Genes introduced in this way would be present in a defined context and thus could be expressed at a more predictable level than through random integration (6). Hence, additional genes, multigene complexes, or even whole metabolic pathways could potentially be added to a genotype. Moreover, engineered or artificial chromosomes could be easily introduced or removed from a genotype by genetic crosses and would facilitate introgression of transgenes to different genetic backgrounds.To extend engineered chromosome technology to plants, we developed a method of telomere-mediated chromosomal truncation in maize by Agrobacterium-mediated transformation of constructs with multiple copies of the telomere sequence (7). Here, we report the use of this technology to produce minichromosome platforms by truncating both normal A and supernumerary B maize chromosomes and at the same time introducing site-spe...

Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors

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Molecular Plant Breeding as the Foundation for 21st Century Crop Improvement

“…yieldgardvt.com/VTScience/Default.aspx), or the combination of transgenes that simultaneously increase synthesis and decrease catabolism of Lys in maize seeds (Frizzi et al, 2008). Recent reports of improvements in gene targeting technology (Ow, 2007) and the construction of meiotically transmissible plant minichromosomes (Carlson et al, 2007;Yu et al, 2007) pave the way for introducing more traits with increasing complexity. With such advances, biotechnology is now poised to assemble useful genetic diversity from essentially any source into constructs that concentrate favorable gene action and maximize heritability for a greatly expanded set of traits.…”

Bioactive Beads-Mediated Transformation of Rice with Large DNA Fragments Containing Aegilops tauschii Genes, with Special Reference to Bead-Production Methodology

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