MINICHROMOSOMES: THE NEXT GENERATION TECHNOLOGY FOR PLANT GENETIC ENGINEERING
Weichang Yu & James A. Birchler
August, 2007

Genetic transformation occurs frequently in nature in prokaryotes. The transfer of genes from one organism to another is termed horizontal gene transfer1. For example, bacteria can acquire virulence factors, as well as antibiotic resistance genes, which may lead to the breakdown of the efficacy of antibiotics. Horizontal gene transfers are rare in higher eukaryotes, but years ago scientists found that a pathogenic bacterium, Agrobacterium, could transfer genes from its genome to its plant hosts, where expression of the transferred genes caused crown gall disease2.

The development of Agrobacterium-mediated genetic transformation, and direct transformation by biolistics, i.e., the high velocity delivery of DNA attached to metal particles, led to the first generation of transgenic plants and the rapid application of this technology to crop improvement.

Genetic engineering as a driving force for modern agriculture

Genetic engineering is a powerful tool for improving crop quality and productivity, and reducing labor and resource utilization of farming. For example, farmers saved up to an estimated 60% of costs for pest control by growing Bt (insect resistance) cotton in certain regions of the US in 1997, according to a USDA report3. The reduction of pesticide spray has other safety benefits for both the environment and humans. Because of these and other benefits, the adoption of three primary GE crops (corn, cotton and soybean) for two traits, HT (herbicide tolerance) and Bt, has dramatically increased since the first commercial introduction in 1996. According to recent USDA survey data (http://www.ers.usda.gov/Data/biotechcrops/), the acreage of GE soybeans reached 89% in the US in 2006, followed by 83% for cotton, and 61% for corn. Although most of the current GE crops are either herbicide tolerant or insect resistant, there is a trend toward a small but steadily increasing percentage of GE crops with stacked genes (containing both HT plus Bt traits). For example, 39% of GE cotton planted last year were varieties with stacked genes, compared with only 20% in 2000; 15% of GE corn had stacked genes, compared with 1% in 2000.

The next generation engineered minichromosomes

Despite many successes, genetic engineering does have limitations. For example, the technology used to create stacked gene varieties is not efficient. When stacking genes in inbred lines, the two genes located on different chromosomes will segregate at a ratio of 1:2:1 following first a genetic cross, and then self-pollinate to make each homozygous. This means that only one-sixteenth of the progeny will maintain the two homozygous stacked transgenes in the next generation. Gene stacking or pyramiding is also widely used in conventional crop breeding programs to combine multiple characteristic traits.

Another difficulty is linkage drag, which refers to the reduction in fitness of a cultivar due to deleterious genes introduced along with the beneficial gene during backcrossing. Linkage drag may occur because genes closely linked to the desired genes or transgenes are usually difficult to recombine during the transfer from one line to another. Many generations of backcrosses are usually required to introduce a good character gene to an elite cultivar without linkage drag. This problem has created a bottleneck that limits the advancement of plant genetic engineering technology from the first generation of GE crops, characterized as 'input traits', to the next generation, characterized as 'output traits'4.

In a recent paper5, Yu et al. describe an engineered minichromosome system that can be used as a genetic engineering platform for the expression of an almost unlimited number of genes that can be inherited together as an independent unit. This engineered minichromosome technology should remove the bottleneck described above, and may lead to the second generation of genetic engineered crops.

Chromosome engineering and minichromosomes

Minichromosomes have been produced in yeast and mammalian cells through either de novo construction using the minimum constituent parts of chromosomes or telomere-mediated chromosomal truncation of existing chromosomes6,7 (Fig. 1).

Figure 1. Minichromosomes produced from maize B chromosome truncation. Arrows denote minichromosomes. B chromosome centromeres are labeled green with a B specific repeat probe. Transgenes carrying the truncating sequences and site specific recombination cassettes, as detected with the transgene probe, are red. Chromosomes are stained blue with DAPI. A) A minichromosome without the transgene (a copy of a stably integrated transgene is present on an A chromosome). B) Two minichromosomes with the truncating terminal transgenes (red) occurring in the same cell are shown, as well as two normal B chromosomes.

However, these technologies for minichromosome construction were not successful in plants until recently when telomere-mediated chromosomal truncation in maize was reported8, which causes chromosome fracture at the telomere sequence insertion site. An array consisting of 2.6 kb telomere repeats efficiently produced chromosomal truncations in maize. However, due to deficiencies in truncated chromosomes, recovery of chromosomes with large truncations is difficult to attain through the genetic transformation process. Moreover, genetic transmission of truncated chromosomes is unlikely; they are non-viable in the haploid gametophyte generation because they lack essential genes. However, the lethality of truncated chromosomes can be overcome either by using tetraploid materials in which the gametes will be diploid and the deficiency of truncated chromosomes would be compensated by additional copies of the homologs, or by using a non-essential chromosome, such as the maize B chromosome5.

Yu et al. recovered a minichromosome formed by the truncation of chromosome 7 in a tetraploid plant. This minichromosome was transferred to a diploid background by repeated backcrossing and was stably maintained. By using the same set of constructs, they targeted the maize B chromosome with biolistic-mediated gene transformation. Truncated B chromosomes were recovered with much greater efficiency. The sizes of the mini-B chromosomes ranged from very small (Fig. 1) to almost the full size of the normal B chromosome. These minichromosomes from both A and B chromosomes of maize were studied for their utility as artificial chromosomes for future genetic engineering in plants.

Attaching genes to minichromosomes

Minichromosomes generated by telomere truncation can have different configurations: those with transgenes and those without. This difference depends on the orientation of construct integration relative to the centromere of the recipient chromosome, which is subsequently broken at the site of telomere integration. The transgene can segregate with either fragment of the broken chromosome. However, if the transgene is retained on the chromosomal fragment without a functional centromere, which is essential for normal chromosome segregation during cell division, it will be lost after cell division. In this circumstance, minichromosomes lacking a transgene will be formed and could be selected if there are other transgenes in the same cell able to produce resistance to the selection agent in the transformation process (Fig. 1A). If the transgene is present on the fragment containing a centromere, it is then present on a minichromosome (Fig. 1B). In fact, both types of minichromosomes were recovered. Minichromosomes with attached transgenes were characterized in detail for mitotic and meiotic behaviors, transmission frequencies, gene expression, and the ability to use site-specific recombination.

Figure 2. GUS gene expression from a targeted gene on a miniB chromosome. A) shows no GUS expression in the absence of the transgene. B) shows GUS gene expression in both embryo (em) and endosperm (en) of a mature kernel with a copy of the transgene on a miniB chromosome.

All minichromosomes in the experiment were transmissible from generation to generation. They never paired with other chromosomes during meiosis, which minimized the chance of recombination with other normal chromosomes in the genome; thus all transgenes on the minichromosome can transmit as a unit. In addition, the research demonstrated that foreign genes could be expressed from maize B chromosomes, which otherwise are inert chromosomes with no essential genes (Fig. 2).

Minichromosomes from maize B chromosomes have other properties that make them exceptionally useful as artificial chromosome platforms. For example, mini-B chromosomes can accumulate to high copy numbers in the presence of normal B chromosomes, but remain stable in the absence of normal B chromosomes. This property might allow one to manage the copy number of the chromosome in order to increase gene expression from transgenes on the chromosome under appropriate circumstances.

Manipulation of genes on minichromosomes

Yu et al. also demonstrated that additional transgenes could be targeted to the minichromosome by site-specific recombination, which allowed the addition of subsequent genes. Yu et al. crossed a minichromosome containing a promoterless lox-DsRed gene, which encodes a red fluorescent protein, with another transgenic plant that expresses Cre, the recombinase for Cre/lox recombination, in a 35S promoter-lox-Cre cassette. Recombination occurred between the two chromosomes at the lox site, thus activating DsRed gene expression, and at the same time placing all genetic elements distal to the 35S-lox-Cre gene onto the minichromosome5. The demonstration of the Cre/lox recombination system in the minichromosome facilitates further manipulation of foreign genes onto minichromosomes. By using other site-specific recombination systems9,10, Yu et al. predict they will be able to add, delete, or replace genes on minichromosomes in order to redesign existing engineered chromosome platforms. This new technology has numerous advantages over the current genetic engineering technology.

As noted above, an immediate application of minichromosome technology is to enable stacking of genes involved with herbicide tolerance and pest resistances. Future developments could facilitate any application that requires the inheritance of multiple foreign genes as a unit. One can now contemplate adding entire biochemical pathways to plants to confer new properties or to synthesize novel metabolites in mass quantities. With mini-B chromosomes, the quantity can be increased to maximize output from foreign genes present on the minichromosome. Because the production of minichromosomes relies on the conserved telomere structure, they can be produced in most plant species for a wide spectrum of new applications in most agricultural crops.

References:

1. Richardson AO & Palmer JD. 2007. Horizontal gene transfer in plants. J Exp Bot 58, 1-9

2. Chilton MD et al. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11, 263-71

3. Price GK et al. 2003.The size and distribution of marker benefits from adopting agricultural biotechnology. U.S. Department of Agriculture, Economic Research Service, Technical Bulletin No. 1906

4. Halpin C. 2005. Gene stacking in transgenic plants - the challenge for 21st century plant biotechnology. Plant Biotechnol J 3, 141-55

5. Yu W et al. 2007. Construction and behavior of engineered minichromosomes in maize. Proc Natl Acad Sci U S A, 104, 8924-9

6. Farr C et al. 1991. Functional reintroduction of human telomeres into mammalian cells. Proc Natl Acad Sci USA 88, 7006-10

7. Harrington JJ et al. 1997. Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat Genet 15, 345-55

8. Yu W et al. 2006. Telomere-mediated chromosomal truncation in maize. Proc Natl Acad Sci USA 103, 17331-6

9. Ow DW. 2007. GM maize from site-specific recombination technology, what next? Curr Opin Biotechnol 18, 115-20

10. Wright DA et al. 2005. High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44, 693-705

Weichang Yu and James A. Birchler
University of Missouri Columbia
Division of Biological Sciences
BirchlerJ@Missouri.edu