There are several emerging genetic tools that will have increasingly important roles in the future of plant biotechnology and crop genetics.
Precise transgene or endogenous gene expression can be regulated at the transcriptional level by novel synthetic promoters, as well as synthetic transcriptional activators and repressors, for increased spatio-temporal control.
The recent development of several advanced DNA construction and assembly methods will allow the production of long DNA constructs and vectors that are needed for multigene transformation into plants.
Plant transformation with large constructs that are needed for metabolic pathway engineering is enabled by several techniques, including plant artificial chromosomes. There is no clear 'winner' among the several techniques presented.
Plant-genome editing using a host of new tools, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs), is poised to have, perhaps, the greatest effect on precisely changing DNA sequences in crops in novel ways.
Tools for transgene removal and confinement are crucial for the commercialization of certain crops and crop–transgene combinations to ensure biosafety and government regulatory compliance.
Basic research has provided a much better understanding of the genetic networks and regulatory hierarchies in plants. To meet the challenges of agriculture, we must be able to rapidly translate this knowledge into generating improved plants. Therefore, in this Review, we discuss advanced tools that are currently available for use in plant biotechnology to produce new products in plants and to generate plants with new functions. These tools include synthetic promoters, 'tunable' transcription factors, genome-editing tools and site-specific recombinases. We also review some tools with the potential to enable crop improvement, such as methods for the assembly and synthesis of large DNA molecules, plant transformation with linked multigenes and plant artificial chromosomes. These genetic technologies should be integrated to realize their potential for applications to pressing agricultural and environmental problems.
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The authors thank J. Haseloff, who provided much feedback on earlier drafts. They appreciate their respective universities for the freedom and resources to undertake this Review, including funding by Advanced Research Projects Agency-Energy to J.S.Y. and by the BioEnergy Science Center to C.N.S. The BioEnergy Science Center is a US Department of Energy (DOE) Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The authors appreciate the work of multiple anonymous reviewers. They thank J. Hinds and M. Rudis for their assistance on various drafts of the paper.
J.S.Y. is a principal in a start-up company. C.N.S.Jr and J.S.Y. have received grants and contracts from the private sector, which has an interest in plant biotechnology.
- Agrobacterium tumefaciens-mediated transformation
The most common plant transformation method. It involves the transfer of genes of interest from A. tumefaciens vectors and the subsequent integration of these genes into plant nuclear genomes.
A commonly used transformation method in which high velocity microprojectiles coated with gene constructs are used to deliver genes into cells and tissues.
- Targeted genome modification
The fusion of engineered DNA-binding proteins or domains with sequence specificities to effector domains that modify genetic sequences and/or gene expression.
- Genome editing
Genome modification achieved by the induction of a double-strand break in a specific genome site, followed by DNA-break repair and the generation of desired modifications (gene disruption, addition or correction).
- Homology-dependent gene silencing
A gene silencing phenomenon induced by homologous sequences at the transcriptional or post-transcriptional levels.
- Zinc-finger proteins
(ZFPs). DNA-binding protein domains that consist of a tandem array of 2–9 zinc-fingers, each of which recognizes approximately three bases of DNA sequence.
- Transcription activator-like effectors
(TALEs). Major virulence factors (containing an amino terminus, a unique type of central DNA-binding domain and a carboxyl terminus with the activation domain) that are secreted by the pathogenic Xanthomonas spp. bacterium when it infects plants. Their DNA-binding domains can be custom-designed to specifically bind to any DNA sequences.
- Seamless assembly
The precise joining of DNA fragments without the addition of intervening or unwanted nucleotides at the junctions.
- De novo DNA synthesis
The synthesis of continuous strands of DNA molecules using a laboratory instrument without the presence of pre-existing templates.
- Plant artificial chromosome
An engineered non-integrating vector that harbours large amounts of DNA (including telomeres, origins of replication, a centromere and genes of interest) and is transmissible in cell division after transformation into plant cells.
- Cre–loxP recombination system
A site-specific recombination system mediated by the Cre recombinase in a genome that contains pre-existing or pre-engineered loxP sites which are recognized by the Cre recombinase.
- Gene stacking
The accumulation of multiple trangenes of interest into the same plant genome for stacked traits.
- FLP–FRT systems
A recombination system in which the FLP recombinase specifically recognizes the FRT site and mediates excision of any sequence that is flanked by the FRT sites.
- B chromosomes
Supernumerary or accessory chromosomes that are heterochromatic. They do not contain functional genes and do not to pair with A chromosomes at meiosis.
- Zinc-finger nucleases
(ZFNs). Fusions of engineered zinc-finger arrays (that consist of 3–6 C2H2 fingers) to a non-specific DNA-cleavage domain of the FokI endonuclease.
- TALE nucleases
(TALENs). Fusions of truncated TALEs (containing an amino terminus, a custom-designed DNA-binding domain and a carboxyl terminus with the activation domain being removed) to a non-specific DNA-cleavage domain of the FokI endonuclease.
- Gene targeting
The incorporation of a transgene (or transgenes) of interest into one or more desired specific genomic loci for the permanent modification of plant genomes using homologous recombination (or another method) followed by the selection for a rare recombination event.
The most common transient transformation method in plants which uses injection or vacuum infiltration to transform genes into cells and tissues using Agrobacterium tumefaciens.
The replacement of normal sexual reproduction by asexual reproduction without fertilization.
A phenomenon in which certain plants propagate using non-opening, self-pollinating flowers.
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Liu, W., Yuan, J. & Stewart Jr, C. Advanced genetic tools for plant biotechnology. Nat Rev Genet 14, 781–793 (2013). https://doi.org/10.1038/nrg3583
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