If you leave to one side the public acceptance of genetically modified crops as a means to overcome constraints on agriculture, a significant problem with genetically engineered crops is that few genes have been successfully transferred and expressed in commercial crops and most only contain one gene addressing one particular issue eg herbicide resistance.
Although this improvement has resulted in considerable gains in productivity, yield, sustainable chemical use and the like, research into other genes that confer advantages like resistance to drought and salinity stress are making real the possibility that we could benefit from the ability to transfer multiple genes into a crop with confidence of inheritance and the ability to swap in and swap out different genes.
For multiple genes to be inserted into a chromosome, directing both genes to be located in close proximity to each other in order for the gene set to passed to subsequent generations is a tool still under development. Even so, the randomness of meiosis can disrupt our best work.
Could the development of Plant Artificial Chromosomes (“PACs”) be a means to overcome these issues?
Plant Artificial Chromosomes
A review article in Plant Biotechnology Journal suggests that recent advances in genetic engineering such as specific site-directed breaking of DNA with CRISPR/Cas9 systems and subsequent homologous recombination processes could be combined with PACs to construct synthetic chromosomes conferring a stack of advantageous genes into crops..
PACs, or ‘minichromosomes’, are described in the paper as “super vectors for foreign gene organisation, expression and manipulation”, with properties such as:
- they are small with few genes of their own, leaving them capable of accepting large gene inserts;
- they are separate chromosomes to the plant’s, resulting in minimal interference with and from the plant’s own genetic material;
- they are stable during mitosis and meiosis, leading to their stability through growth and into subsequent generations;
- they can be constructed to allow subsequent manipulation whilst in the plant.
There are two methods of constructing PACs, the first being to clone the essential components like a centromere, origin of replication and telomeres, assembly the gene outside the plant cell and then transfer them into the plant cell. However, this approach is yet to be demonstrated unequivocally and plant centromeric repeats are species specific, adding a further complexity to their use at all.
A second method called ‘telomere-mediated chromosomal truncation’ however has been demonstrated as a way of creating minichromosomes in crops such as maise and rice. The process works on past observations that if telomere repeat sequences, conserved throughout most plant species, is inserted in to a plant chromosome, the chromosome will truncate itself by seeding new telomeres at the site of transformation.
Telomere-Mediated Chromosomal Truncation. Credit DWilliams4, Wikipedia.
Although this truncation process has been known for some time, this review talks-up the potential use of the tool in conjunction with site specific engineering tools that have recently been developed. These tools can direct sites of truncation with precision and create minichromosomes that can be easily re-engineered with stacks of genes covering a number of traits with a level of stability that cant be achieved if the stack were inserted directly into the genome. Further, unlike insertions into the genome, inserting an artificial genome into a crop avoids linkage drag, a detrimental process where the inserted gene is linked to a deleterious gene on the chromosome that, when expressed, harms the plant.
PACs and the recent advances in site specific genetic engineering tools
Producing these minichromosomes currently requires the generation and screening of numerous plants to find those successfully transformed. Tools such as CRISPR/Cas9 can improve our productivity given they can
- direct the insertion of telomere sequences to specific sites on the chromosome to be transformed;
- simplify the process of removing the selectable marker genes from the chromosome after the screening process (to free up more room on the chromosome, avoid antibiotic resistance genes being expressed in crop or avoid us having green fluorescing pea plants!); and
- easily add, remove or change single genes or stacks of genes either after creation or whilst in the plant they are desired to be expressed in.
The article discusses previous research performed by the authors demonstrating this last point. The selectable marker gene (SMG) and the Gene of Interest (GOI) in a particular minichromosome were removed and replaced by those on a donor plasmid using specifically added restriction sites on the minichromsome and homologous recombination.
Site specific recombination swapping out SMG1 and GOI1 for SMG2 and GOI2 in a Plant Artificial Chromosome. Reproduced from the article.
Insertion in to recipient plant genomes
PACs, after being generated in plants, can be cloned in vitro and transferred back into plants of the same species they were generated in. A different method, fusing protoplasts of the recipient plant to a protoplast containing the PAC is suggested as way of overcoming this species specificity and allowing the insertion of the minichromosome into alien species.
Plant Artificial Chromosomes combined with the advanced genetic engineering tools could be a pathway to stable, inheritable stacks of traits into crops with the ability to change the required genes as needed. As we expand our catalogue of genes conferring advantages on crops through research, PACs may start being used in research to test combinations of genes and stable products specifically tailored to particular environments may see general application.