Efficient genetic transformation methods like Agrobacterium-mediated as well as direct delivery methods have been used successfully in crop plants for integration of various useful genes. However, these methods have some disadvantages, especially in the transfer of large genes, gene complexes and gene stacking. The recent method developed to overcome such issues is the development of Plant artificial chromosomes (PACs). Engineered minichromosomes, artificial chromosomes or synthetic chromosomes are chromosomebased vectors that are now being proposed as a next-generation vehicle in plant genetic engineering. They are small versions of a chromosome that include the basic components necessary for replication (centromere, telomeres and origin of replication). Since they contain no or few genes of their own, they allow the addition, deletion and replacement of genes on them. Generally two approaches are made use for minichromosome construction: Bottom-up approach (de novo assembly) and Top-down approach (chromosomal truncations). In the first approach, components of chromosome centromere repeats, telomeres and origins of replication are cloned, assembled in vitro and transformed into cells to direct mini-chromosome assembly and has been successful in yeast and mammalian cells, but has not reported in plants so far. In contrast, minichromosome construction by use of telomeremediated chromosomal truncation (TMCT) has been successful in plants1. Engineered minichromosomes were constructed in maize by modifying natural A and supernumerary B chromosomes by using telomere-mediated chromosomal truncation. FISH analysis of the primary transgenic plants (T0) identified a minichromosome with a transgene signal. The minichromosomes R2 and miniB were derived from truncation of the long arm of chromosome 7 and B chromosome, respectively and were found stable during meiosis and mitosis2.

Production of artificial chromosomes in Brassica napus carried out by using a 0.35 kb direct repeat of the Arabidopsis telomeric sequence and was analysed by multi fluorescence in-situ hybridization (multi- FISH) and Southern hybridization. C2 and C4 were the two truncated chromosomes developed by chromosomal truncation and were stably inherited to the next generation. Later, these artificial chromosomes were tracked over different generations3.

The development of PACs has opened a new platform for the next generation of molecular breeding and molecular farming. Using PACs along with gene editing approaches can potentially design and manipulate genomes for many applications. Although attempts to create PACs have succeeded, several barriers need to be overcome before application.

REFERENCES:

1DHAR, M. K., KAUL, S., AND KOUR, J., 2011, Towards the development of better crops by genetic transformation using engineered plant chromosomes. Plant cell reports, 30:799-806.

2YU, W., HAN, F., GAO, Z., VEGA, J. M., AND BIRCHLER, J. A., 2007, Construction and behaviour of engineered minichromosomes in maize. Proc. Natl. Acad. Sci., 104(21): 8924-8929.

3YIN, X., ZHANG, Y., CHEN, Y., WANG, J., WANG, R. R. C., FAN, C. AND HU, Z., 2021, Precise characterization and tracking of stably inherited artificial mini-chromosomes made by telomere mediated chromosome truncation in Brassica napus. Front. Plant Sci, 12(4): 743-792.