Abstract
Efficient methods for multigene transformation are important for developing novel crop varieties. Methods based on random integrations of multiple genes have been successfully used for metabolic engineering in plants. However, efficiency of co‐integration and co‐expression of the genes could present a bottleneck. Recombinase‐mediated integration into the engineered target sites is arguably a more efficient method of targeted integration that leads to the generation of stable transgenic lines at a high rate. This method has the potential to streamline multigene transformation for metabolic engineering and trait stacking in plants. Therefore, empirical testing of transgene(s) stability from the multigene site‐specific integration locus is needed. Here, the recombinase technology based on Cre‐lox recombination was evaluated for developing multigenic lines harboring constitutively‐expressed and inducible genes. Targeted integration of a five genes cassette in the rice genome generated a precise full‐length integration of the cassette at a high rate, and the resulting multigenic lines expressed each gene reliably as defined by their promoter activity. The stable constitutive or inducible expression was faithfully transmitted to the progeny, indicating inheritance‐stability of the multigene locus. Co‐localization of two distinctly inducible genes by heat or cold with the strongly constitutive genes did not appear to interfere with each other's expression pattern. In summary, high rate of co‐integration and co‐expression of the multigene cassette installed by the recombinase technology in rice shows that this approach is appropriate for multigene transformation and introduction of co‐segregating traits.Significance StatementRecombinase‐mediated site‐specific integration approach was found to be highly efficacious in multigene transformation of rice showing proper regulation of each gene driven by constitutive or inducible promoter. This approach holds promise for streamlining gene stacking in crops and expressing complex multigenic traits.
Highlights
The demand for resilient, productive, and value-added crops mandates breeding with multiple genes
This study used the Cre-lox mediated site-specific integration approach based on the use of mutant lox sites, lox75 and lox76, to stabilize the integration locus as described earlier (Albert et al, 1995; Srivastava and Ow, 2002)
T5 target locus consists of a lox76 placed between the maize ubiquitin-1 promoter (ZmUbi1) and cre coding sequence (Fig. 1a), and pNS64 contains a loxP and lox75 flanked gene construct consisting of a promoter-less selectable marker (NPT II) and 4 genes-of-interest (GFP, GUS, DREB1a, pporRFP), each expressed by their dedicated promoters (Fig. 1b)
Summary
The demand for resilient, productive, and value-added crops mandates breeding with multiple genes. Agrobacterium-mediated T-DNA transfer is an excellent method of multigene transformation as TDNA harboring many genes enters into the plant cell and integrates into the genome (Collier et al, 2018; Li et al, 2003; Ruiz-Lopez et al, 2015). Integration of tandem repeats or truncated copies of TDNA, and disruption of critical genomic regions cannot be ruled out. These features of random transformation methods pose major challenges for multigene transformation, rendering a high number of the recovered events unsuitable for product development (Anand and Jones, 2018; Halpin, 2005)
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