Abstract
Genetic transformation has become a promising tool for improvement of a variety of crop species. However, transferring genes across species, the presence of selectable marker genes, and bacteria-derived vector backbone sequences have raised considerable health and environmental concerns. Intragenic vector system-based intragenesis/cisgenesis is a new method using transgenic approach to achieving traditional breeding objectives but circumventing many of the associated shortcomings. We report here the development of an intragenic vector by assembling a T-DNA-like fragment and a buffering sequence following the left border from Citrus clementina into the backbone of the binary vector pCB302. Recovery of citrus regenerants is performed under non-selective conditions and positive intra-/cisgenic regenerants were identified through PCR analysis. Transformation efficiencies obtained in Arabidopsis and “Duncan” grapefruit were ~3% and ~0.67%, respectively, demonstrating the potential of the system for development of “foreign DNA-free” intra-/cisgenic citrus cultivars.
Highlights
Genetic improvement of citrus (Citrus spp.) by conventional breeding is hindered by incompatibility, apomixes, heterozygosity, and lengthy juvenile period
When a gene cassette of interest is ligated into pUFCI and introduced into recepient citrus genome, positive regenerants can be identified through PCR amplification using a gene specific primer in combination with a primer annealing to the T-DNA-like region
To decrease the possibility of the presence of non-citrus sequences in the regenerants, we added a ~3 kb C. clementina-originated DNA fragment outside of the left border (LB) to serve as a buffering sequence
Summary
Genetic improvement of citrus (Citrus spp.) by conventional breeding is hindered by incompatibility, apomixes, heterozygosity, and lengthy juvenile period. Currently preferred Agrobacterium-mediated plant genetic transformation and elimination of selectable marker genes largely rely on prokaryotederived vector systems [5]. Intragenic vector system-based intra-/cisgenesis combines the benefits of traditional breeding and genetic engineering, but circumvents many of their problematic issues [5,6]. It involves identifying functional equivalents of vector components from target or crossable plant species and using these DNA sequences to assemble vector for plant transformation. The transformation efficiencies obtained in both Arabidopsis and “Duncan” grapefruit indicate its great potential for citrus genetic improvement
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