Genetic engineering has revolutionized plant breeding by allowing precise modifications of genetic material, overcoming the limitations of traditional breeding. Since most agronomic traits are quantitative and controlled by multiple genes, single-gene modifications often fall short of achieving significant improvements. While Agrobacterium-mediated transformation with one or a few genes is routine, introducing and maintaining large constructs carrying multiple genes remains a challenge (Halpin, 2005).

Multigene Engineering (MGE) and Its Significance

Multigene engineering (MGE) refers to the introduction of multiple genes into a plant genome to enhance resistance against biotic and abiotic stresses and improve quality traits such as nutrient biosynthesis. Unlike single-gene transformation, MGE enables the manipulation of entire metabolic pathways, allowing for comprehensive improvements in plant traits.

Methods of Multigene Engineering

Several approaches have been developed to introduce multiple genes into plant genomes:

  • Crossing Transgenic Lines: Breeding separate transgenic lines carrying different genes to produce plants with multiple traits.
  • Sequential Transformation: Introducing genes in successive transformation events.
  • Co-transformation: Simultaneous delivery of multiple genes using a single transformation vector.
  • Chloroplast Transformation: Integration of multiple genes into the chloroplast genome to ensure stable inheritance.
  • Site-Specific Integration via Homologous Recombination: Directing gene insertion at specific genomic loci to minimize gene silencing and positional effects (Urwin et al., 2002).

Advances in Multigene Engineering

Gateway Cloning is a widely used technique that employs site-specific recombination systems, such as att sites, to transfer multiple genes precisely. This method simplifies the assembly of complex gene constructs, making it an efficient choice for multigene projects.

GAANTRY (Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technology) is another breakthrough system that facilitates the stable maintenance of multi-gene constructs. This system has successfully generated high-quality transgenic potato plants carrying stacked transgenes (McCue et al., 2019).

Successful Applications of MGE

A notable example of MGE is Xiaoyan22 (XY22), a medium-gluten wheat variety developed by stacking resistance genes such as Yr26 (yellow rust), ML91260 (powdery mildew), and high-quality glutenin subunits. This variety demonstrates high yield potential even under biotic and abiotic stress conditions (Zheng et al., 2020).

Challenges and Future Prospects

Despite its potential, MGE faces several challenges:

  • Gene Silencing: The introduction of multiple transgenes may lead to unpredictable gene interactions and transcriptional silencing.
  • Promoter Design: Efficient expression of multiple genes under a single promoter remains a challenge, requiring advanced regulatory elements.
  • Stable Integration: Ensuring that all introduced genes are stably inherited across generations.

With continuous advancements in promoter engineering, synthetic biology, and gene editing technologies, MGE is expected to play a crucial role in the future of crop improvement. By integrating multiple resistance genes, breeders can develop crops with superior traits that surpass the limitations of conventional breeding techniques.

References

  1. Halpin, C., 2005, Gene stacking in transgenic plants–the challenge for 21st-century plant biotechnology. Plant Biotechnol. J., 3(2): 141-155.
  2. Urwin, P. E., Zubko, E. I., & Atkinson, H. J., 2002, The biotechnological application and limitation of IRES to deliver multiple defense genes to plant pathogens. Physiol. Mol. Plant Pathol., 61(2): 103-108.
  3. McCue, K. F., Gardner, E., Chan, R., Thilmony, R., & Thomson, J., 2019, Transgene stacking in potato using the GAANTRY system. BMC Res. Notes, 12(1): 1-6.
  4. Zheng, W., Li, S., Liu, Z., Zhou, Q., Feng, Y., & Chai, S., 2020, Molecular marker-assisted gene stacking for disease resistance and quality genes in the dwarf mutant of an elite common wheat cultivar Xiaoyan22. BMC Genet., 21(1): 14-28.