Minichromosomes In Plants Research Articles

Synthetic minichromosomes in plants: past, present, and promise.

The status of engineered mini-chromosomes/artificial chromosomes/synthetic chromosomes in plants is summarized. Their promise is that they provide a means to accumulate foreign genes on an independent entity other than the normal chromosomes, which would facilitate stacking of novel traits in a way that would not be linked to endogenous genes and that would facilitate transfer between lines. Centromeres in plants are epigenetic, and therefore the isolation of DNA underlying centromeres and reintroduction into plant cells will not establish a functional kinetochore, which obviates this approach for in vitro assembly of plant artificial chromosomes. This issue was bypassed by using telomere-mediated chromosomal truncation to produce mini-chromosomes with little more than an endogenous centromere that could in turn be used as a foundation to build synthetic chromosomes. Site-specific recombinases and various iterations of CRISPR-Cas9 editing provide many tools for the development and re-engineering of synthetic chromosomes.

Engineered minichromosomes in plants.

Engineered minichromosomes have been produced in several plant species via telomere-mediated chromosomal truncation. This approach bypasses the complications of the epigenetic nature of centromere function in plants, which has to date precluded the production of minichromosomes by the re-introduction of centromere sequences to a plant cell. Genes to be added to a cleaved chromosome are joined together with telomere repeats on one side. When these constructs are introduced into plant cells, the genes are ligated to the broken chromosomes but the telomere repeats will catalyze the formation of a telomere on the other end cutting the chromosome at that point. Telomere-mediated chromosomal truncation is sufficiently efficient that very small chromosomes can be generated consisting of basically the endogenous centromere and the added transgenes. The added transgenes provide a platform onto which it should be possible to assemble a synthetic chromosome to specification. Combining engineered minichromosomes with doubled haploid breeding should greatly expedite the transfer of transgenes to new lines and to test the interaction of transgenes in new background genotypes. Potential basic and applied applications of synthetic chromosomes are discussed.

Engineered Minichromosomes in Plants: Structure, Function, and Applications.

Engineered minichromosomes are small chromosomes that contain a transgene and selectable marker, as well as all of the necessary components required for maintenance in an organism separately from the standard chromosome set. The separation from endogenous chromosomes makes engineered minichromosomes useful in the production of transgenic plants. Introducing transgenes to minichromosomes does not have the risk of insertion within a native gene; additionally, transgenes on minichromosomes can be transferred between lines without the movement of linked genes. Of the two methods proposed for creating engineered minichromosomes, telomere-mediated truncation is more reliable in plant systems. Additionally, many plants contain a supernumerary, or B chromosome, which is an excellent starting material for minichromosome creation. The use of site-specific recombination systems in minichromosomes can increase their utility, allowing for the addition or subtraction of transgenes in vivo. The creation of minichromosomes with binary bacterial artificial chromosome vectors provides the ability to introduce many transgenes at one time. Furthermore, coupling minichromosomes with haploid induction systems can facilitate transfer between lines. Minichromosomes can be introduced to a haploid-inducing line and crossed to target lines. Haploids of the target line that then contain a minichromosome can then be doubled. These homozygous lines will contain the transgene without the need for repeated introgressions.

Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants

Telomere-mediated Chromosomal Truncation via Agrobacterium tumefaciens or Particle Bombardment to Produce Engineered Minichromosomes in Plants

Engineered plant minichromosomes

Minichromosomes offer an enormous potential for plant breeding and biotechnology, because they may simultaneously transfer and stably express multiple genes. Segregating independently of their host chromosomes, they provide a platform for accelerating plant breeding. Minichromosomes can be established from cloned components in vivo (bottom up) or via engineering of natural chromosomes (top down). When they possess functional centromeres and telomeres, they should be stably inherited, but their meiotic transmission rate is below that of endogenous chromosomes. To achieve the customized generation and control the regular transmission of minichromosomes are important challenges for applied research in chromosome biology. Here, construction and biology of plant minichromosomes are compared with data available for yeast and animal systems.

Engineered Minichromosomes in Plants

Engineered minichromosomes provide the ability to target transgenes to a defined insertion position for predictable expression on an independent chromosome. This technology promises to provide a means to add many genes to a synthetic chromosome in sequential manner. An additional advantage is that the multiple transgenes will not be inserted into the normal chromosomes and thus will not exhibit linkage drag when converging the transgenes to different germplasm nor will they be mutagenic. Telomere truncation coupled with the introduction of site-specific recombination cassettes has proven to be an easy method to produce minichromosomes. Telomere truncation results from the transformation of plasmids carrying a block of telomere repeats at one end. Minichromosomes consisting of little more than a centromere have been produced for B chromosomes of maize. Such small chromosomes have been studied for their meiotic behavior, which differs from normal sized chromosomes in that homologue pairing is rare or nonexistent and sister chromatid cohesion fails at meiosis I. Potential modifications of the minichromosomes that can address these issues are discussed. Minichromosomes can be recovered from transformed plants that are polyploid or that carry an additional chromosome as the preferred target for truncation. Site-specific recombination has been demonstrated to operate on these terminally located sites. By introducing normal B chromosomes into lines with engineered mini-B chromosomes, the latter can be increased in copy number, which provides the potential to augment the expression of the introduced genes. Because the vast majority of plant species have the same telomere sequence, the truncating transgenes should be effective in most plants to generate engineered minichromosomes. Such chromosomes establish the means to add or subtract multiple transgenes, multigene complexes, or whole biochemical pathways to plants to change their properties for agronomic applications or to use plants as factories for the production of foreign proteins or metabolites.

Engineered Plant Minichromosomes: A Bottom-Up Success?

Engineered minichromosomes offer an enormous opportunity to improve crop performance, as recently discussed ([Houben and Schubert, 2007][1]). Unlike conventional gene transformation technologies, minichromosomes can be used simultaneously to transfer and to express stably (multiple) sets of genes.

Engineered minichromosomes in plants

Genetic engineering for complex or combined traits requires the simultaneous expression of multiple genes, and has been considered as the bottleneck for the next generation of genetic engineering in plants. Minichromosome technology provides one solution to the stable expression and maintenance of multiple transgenes in one genome. For example, minichromosomes can be used as a platform for efficient stacking of multiple genes for insect, bacterial and fungal resistances together with herbicide tolerance and crop quality traits. All the transgenes would reside on an independent minichromosome, not linked to any endogenous genes; thus linkage drag can be avoided. Engineered minichromosomes can be easily constructed by a telomere-mediated chromosomal truncation strategy. This approach does not rely on the cloning of centromere sequences, which are species-specific, and bypasses the any complications of epigenetic components for centromere specification. Thus, this technique can be easily extended to all plant species. The engineered minichromosome technology can also be used in combination with site-specific recombination systems to facilitate the stacking of multiple transgenes.

Engineered Plant Minichromosomes: A Resurrection of B Chromosomes?

A number of crop species of commercial interest have been transformed using either Agrobacterium -mediated, biolistic, or other systems. However, these methods have several limitations. For example, they allow insertion of single or a few genes at random genomic positions, but complex traits cannot

Using Arabidopsis to understand centromere function: progress and prospects.

Arabidopsis thaliana has emerged in recent years as a leading model for understanding the structure and function of higher eukaryotic centromeres. Arabidopsis centromeres, like those of virtually all higher eukaryotes, encompass large DNA domains consisting of a complex combination of unique, dispersed middle repetitive and highly repetitive DNA. For this reason, they have required creative analysis using molecular, genetic, cytological and genomic techniques. This synergy of approaches, reinforced by rapid progress in understanding how proteins interact with the centromere DNA to form a complete functional unit, has made Arabidopsis one the best understood centromere systems. Yet major problems remain to be solved: gaining a complete structural definition of the centromere has been surprisingly difficult, and developing synthetic mini-chromosomes in plants has been even more challenging.

Nuclear extrachromosomal DNA of higher plants.

Recent reports indicate that minichromosomes and other small genetic entities may occur in the nuclei of uninfected higher plants. They become especially abundant under some special growth conditions and, sometimes, resemble extrachromosomal genes of ciliated protozoa. An example of such gene-sized DNA species was isolated from resting wheat embryos. The presence of telomeric sequences at its termini and the ability to replicate autonomously in wheat nuclei made it possible to distinguish this nuclear minichromosome from chromosomal DNA fragmentation products. The biological significance of plant minichromosomes remains to be elucidated.

A minichromosome-like structure in wheat embryos

A crude nuclear fraction of resting wheat embryos was used as the source of putative plant minichromosomes: unique DNA sequences the size of genes and flanked by telomere-type repeats. Preliminary separation of low-molecular-weight DNA species from chromosomal DNA (Hirt's method), velocity sedimentation, and isopycnic centrifugation were followed by PCR amplification of minichromosome-like sequences. The most abundant PCR product was cloned and sequenced. In addition to telomeric repeats (defined by a PCR primer), which were the expected sequences, the linear DNA molecule (637 pb) contained an ARS-like element, RAP1-binding site, and two relatively long ORFs. The whole sequence seems to represent a naturally occurring plant minichromosome.