The ability to edit plant genomes through gene targeting (
- NSF-PAR ID:
- 10371763
- Publisher / Repository:
- Wiley-Blackwell
- Date Published:
- Journal Name:
- Plant Biotechnology Journal
- Volume:
- 20
- Issue:
- 10
- ISSN:
- 1467-7644
- Page Range / eLocation ID:
- p. 1916-1927
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Summary GT ) requires efficient methods to deliver both sequence‐specific nucleases (SSN s) and repair templates to plant cells. This is typically achieved usingAgrobacterium T‐DNA , biolistics or by stably integrating nuclease‐encoding cassettes and repair templates into the plant genome. In dicotyledonous plants, such asNicotinana tabacum (tobacco) andSolanum lycopersicum (tomato), greater than 10‐fold enhancements inGT frequencies have been achieved usingDNA virus‐based replicons. These replicons transiently amplify to high copy numbers in plant cells to deliver abundantSSN s and repair templates to achieve targeted gene modification. In the present work, we developed a replicon‐based system for genome engineering of cereal crops using a deconstructed version of the wheat dwarf virus (WDV ). In wheat cells, the replicons achieve a 110‐fold increase in expression of a reporter gene relative to non‐replicating controls. Furthermore, replicons carryingCRISPR /Cas9 nucleases and repair templates achievedGT at an endogenousubiquitin locus at frequencies 12‐fold greater than non‐viral delivery methods. The use of a strong promoter to express Cas9 was critical to attain these highGT frequencies. We also demonstrate gene‐targeted integration by homologous recombination (HR ) in all three of the homoeoalleles (A, B and D) of the hexaploid wheat genome, and we show that with theWDV replicons, multiplexedGT within the same wheat cell can be achieved at frequencies of ~1%. In conclusion, high frequencies ofGT usingWDV ‐basedDNA replicons will make it possible to edit complex cereal genomes without the need to integrateGT reagents into the genome. -
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Abstract Agrobacterium T‐DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non‐homologous end‐joining (NHEJ) protein DNA polymerase Θ, encoded by thePolQ gene, for T‐DNA integration is controversial, with some groups claiming it is essential whereas others claim T‐DNA integration inArabidopsis and ricepolQ mutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenicpolQ mutant plants. We describe a protocol for regenerating transgenicpolQ mutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species. -
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Summary Integration of
Agrobacterium tumefaciens transferred DNA (T‐DNA) into the plant genome is the last step required for stable plant genetic transformation. The mechanism of T‐DNA integration remains controversial, although scientists have proposed the participation of various nonhomologous end‐joining (NHEJ) pathways. Recent evidence suggests that inArabidopsis , DNA polymerase θ (PolQ) may be a crucial enzyme involved in T‐DNA integration.We conducted quantitative transformation assays of wild‐type and
polQ mutantArabidopsis and rice, analyzed T‐DNA/plant DNA junction sequences, and (forArabidopsis ) measured the amount of integrated T‐DNA in mutant and wild‐type tissue.Unexpectedly, we were able to generate stable transformants of all tested lines, although the transformation frequency of
polQ mutants wasc. 20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQ mutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQ mutant plants show growth and developmental defects, perhaps explaining previous unsuccessful attempts at their stable transformation.We suggest that either multiple redundant pathways function in T‐DNA integration, and/or that integration requires some yet unknown pathway.
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Abstract The arrival to the
U nitedS tates of theA fricanized honey bee, a hybrid betweenE uropean subspecies and theA frican subspeciesA pis mellifera scutellataA frican andE uropean lineages in a feral population inS outhT exas. An 11‐year survey of this population (1991–2001) showed that mitochondrial haplotype frequencies changed drastically over time from a resident population of eastern and western European maternal ancestry, to a population dominated by theA frican haplotype. A subsequent study of the nuclear genome showed that theA fricanization process included bidirectional gene flow between European and Africanized honey bees, giving rise to a new panmictic mixture ofA . m. scutellata‐A. m. scutellata maternal ancestry, and three were of western European maternal ancestry. No colonies of eastern European maternal ancestry were detected, although they were present in the earlier samples. NuclearDNA revealed little change in the introgression ofA . m. scutellata -
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Abstract Background Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency.
Results We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1generation of any crop transformed via
Agrobacterium . We present an example using the allohexaploid oil-seed plantCamelina sativa to determine insertion site location of two transgenes.Conclusion This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.
