A mini-B chromosome generated by chromosomal telomere truncation. A normal B chromosome, also present, and the mini-chromosome are labeled with a B chromosome specific probe (green). The truncating transgene is labeled red on the truncated B. Arrow indicates the mini-B chromosome. At right, top to bottom, are the the merged image of the minichromosome, the B specific label alone and the truncating transgene image alone.
The development of plant artificial chromosome technology will establish minichromosomes to build new chromosomes to pre-determined specifications. This ability will result in more predictable gene expression than is possible with current minichromosomes of plant genetic engineering. The nature of the minichromosomes produced will permit continued additions to the chromosome so that its composition can be continually modified with future manipulations. Thus, this technology will open the possibility to add whole biochemical pathways to plants that will confer new properties to them for improved agricultural practices or to use plants as factories for foreign proteins or metabolites that have medicinal value. Advantageous new properties for plants would include those that would reduce or eliminate the use of chemical fertilizers and herbicides, provide insect or microbial resistance, allow adaptation to new environments, improve cultivation techniques, foster production of biofuels and increase yield.
The engineered artificial chromosome platforms were produced by introduction of the specialized ends of chromosomes, called telomeres, during transformation of maize. In this process, the chromosome is truncated and the distal fragments are eliminated. Such truncations closely flanking a centromere result in the production of a minichromosome. The introduced truncating transgenes carry sites that can be used for future additions to the minichromosomes.
The specific goals to be pursued during this project are as follows. The procedure of telomere truncation will be optimized by examining the impact of telomere array number and position in the truncating transgene to determine the best method to achieve a high number of chromosomal fractionation events. Secondly, minichromosomes with visible marker genes will be generated to facilitate the tracking of the chromosomes from one generation to the next and to examine the developmental stability of the small chromosomes. Thirdly, procedures will be developed for perfecting the addition of new DNA sequences to the minichromosomes. Optimization of procedures to add large fragments of DNA to the minichromosomes will be performed. Fourthly, minichromosomes derived from the maize B chromosome, an extra nearly inert chromosome, will be increased in copy number to test the maximum number that can be achieved and whether the newly introduced genes present on the minichromosomes become silenced under these circumstances. This objective will establish whether artificial chromosomes can be used to increase the output from the foreign genes on the minichromosomes. Lastly, because minichromosomes do not have perfect transmission from one generation to the next, a gene will be added to a minichromosome that will only allow pollen grains carrying it to function. When this pollen is applied to the silks of other maize plants, this genetic cross will produce a new generation with all individuals carrying the engineered minichromosome with the desired foreign genes.
Collectively, the completion of these goals will establish engineered chromosome technology in maize and guide the adaptation of this technology to other plant species.