Gene targeting and transgene stacking using intra genomic homologous recombination in plants
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Plant Methods Open Access
REVIEW
Gene targeting and transgene stacking using intra genomic homologous recombination in plants Sandeep Kumar*, Pierluigi Barone and Michelle Smith
Abstract Modern agriculture has created a demand for plant biotechnology products that provide durable resistance to insect pests, tolerance of herbicide applications for weed control, and agronomic traits tailored for specific geographies. These transgenic trait products require a modular and sequential multigene stacking platform that is supported by precise genome engineering technology. Designed nucleases have emerged as potent tools for creating targeted DNA double strand breaks (DSBs). Exogenously supplied donor DNA can repair the targeted DSB by a process known as gene targeting (GT), resulting in a desired modification of the target genome. The potential of GT technology has not been fully realized for trait deployment in agriculture, mainly because of inefficient transformation and plant regeneration systems in a majority of crop plants and genotypes. This challenge of transgene stacking in plants could be overcome by Intra-Genomic Homologous Recombination (IGHR) that converts independently segregating unlinked donor and target transgenic loci into a genetically linked molecular stack. The method requires stable integration of the donor DNA into the plant genome followed by intra-genomic mobilization. IGHR complements conventional breeding with genetic transformation and designed nucleases to provide a flexible transgene stacking and trait deployment platform. Keywords: Gene targeting, Plant transformation, Transgene stacking, Designed nuclease, Intra genomic homologous recombination, Somatic recombination Background The Green Revolution in the 1960s combined advances in breeding and agricultural practice, and provided food security to millions of people [1]. Given an increasing global population, there is a projected need to increase world food production by 40 % in the next 20 years [2]. In addition to a growing population, climate change, degrading natural resources and changing food preferences have raised food and nutritional security to the level of the biggest challenge of the twenty-first century [3]. Genetically modified (GM) trait technology in the mid-1990s made a major impact in meeting the world food demand and there has been a rapid adoption of the technology. These first generation trait products involved *Correspondence: [email protected] Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, IN 46286, USA
simple herbicide and insect traits that required introduction of a single gene. Control of the broad range of insect pests and weeds desired today requires multiple insect and herbicide tolerance genes [4]. In addition, modern genomics and gene networking tools have revealed that many agronomic traits depend on different genes and complex interactions of proteins reacting to various external stimuli [1]. The next generation trait products, therefore, require integration of multiple transgenes and would also
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