An official website of the United States government
Summary Integration ofAgrobacterium tumefacienstransferred 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 andpolQmutantArabidopsisand 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 ofpolQmutants was c.20% that of wild‐type plants. T‐DNA/plant DNA junctions from these transformed rice andArabidopsis polQmutants closely resembled those from wild‐type plants, indicating that loss of PolQ activity does not alter the characteristics of T‐DNA integration events.polQmutant 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.
more »
« less
Transformation and regeneration of DNA polymerase Θ mutant rice plants
Abstract AgrobacteriumT‐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 thePolQgene, for T‐DNA integration is controversial, with some groups claiming it is essential whereas others claim T‐DNA integration inArabidopsisand ricepolQmutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenicpolQmutant plants. We describe a protocol for regenerating transgenicpolQmutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.
more »
« less
- Award ID(s):
- 2006668
- PAR ID:
- 10457845
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Plant Direct
- Volume:
- 7
- Issue:
- 9
- ISSN:
- 2475-4455
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Abstract BackgroundTransgenic plants are essential for both basic and applied plant biology. Recently, fluorescent and colorimetric markers were developed to enable nondestructive identification of transformed seeds and accelerate the generation of transgenic plant lines. Yet, transformation often results in the integration of multiple copies of transgenes in the plant genome. Multiple transgene copies can lead to transgene silencing and complicate the analysis of transgenic plants by requiring researcher to track multiple T-DNA loci in future generations. Thus, to simplify analysis of transgenic lines, plant researchers typically screen transformed plants for lines where the T-DNA inserted in a single locus — an analysis that involves laborious manual counting of fluorescent and non-fluorescent seeds for screenable markers. ResultsTo expedite T-DNA segregation analysis, we developed SeedSeg, an image analysis tool that uses a segmentation algorithm to count the number of transformed and wild-type seeds in an image. SeedSeg runs a chi-squared test to determine the number of T-DNA loci. Parameters can be adjusted to optimize for different brightness intensities and seed sizes. ConclusionsBy automating the seed counting process, SeedSeg reduces the manual labor associated with identifying transgenic lines containing a single T-DNA locus. SeedSeg is adaptable to different seed sizes and visual transgene markers, making it a versatile tool for accelerating plant research.more » « less
-
Summary Cuscuta campestris, a stem parasitic plant, has served as a valuable model plant for the exploration of plant–plant interactions and molecular trafficking. However, a major barrier toC. campestrisresearch is that a method to generate stable transgenic plants has not yet been developed.Here, we describe the development of aCuscutatransformation protocol using various reporter genes (GFP, GUS, or RUBY) and morphogenic genes (CcWUS2andCcGRF/GIF), leading to a robust protocol forAgrobacterium‐mediatedC. campestristransformation.The stably transformed and regenerated RUBYC. campestrisplants produced haustoria, the signature organ of parasitic plants, and these were functional in forming host attachments. The locations of T‐DNA integration in the parasite genome were confirmed through TAIL‐PCR. TransformedC. campestrisalso produced flowers and viable transgenic seeds exhibiting betalain pigment, providing proof of germline transmission of the RUBY transgene. Furthermore, RUBY is not only a useful selectable marker for theAgrobacterium‐mediated transformation, but may also provide insight into the movement of molecules fromC. campestristo the host during parasitism.Thus, the protocol for transformation ofC. campestrisreported here overcomes a major obstacle toCuscutaresearch and opens new possibilities for studying parasitic plants and their interactions with hosts.more » « less
-
Abstract High‐precision genome editing tools, such as programmable nucleases, are poised to transform crop breeding and significantly impact fundamental plant research. Among these tools, the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR‐associated 9) system is a programmable, RNA‐guided nuclease that introduces targeted, site‐specific double‐stranded breaks in the target DNA loci. When these breaks are repaired, it often results in a frame‐shift mutation via short insertion/deletion (indel), leading to gene knockout. Since its first successful use in plants, CRISPR/Cas9 has been widely adopted for targeting genes of agronomic and scientific importance in multiple crops, including rice, maize, wheat, and sorghum. These cereal crops ensure global food security, provide essential nutrition, and support economic stability. Additionally, such crops support biofuel production, livestock feed, and sustainable farming practices through crop rotation. This article outlines the strategies for implementing CRISPR/Cas9 genome editing in plants, including a step‐by‐step process of guide RNA target selection, oligonucleotide design, construct development, assembly, and analysis of genome edits. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: CRISPR/Cas9 guide RNA target selection Support Protocol 1: Genomic DNA extraction in‐house protocol Basic Protocol 2: Construction of a binary plasmid vector Support Protocol 2:Agrobacteriumtransformation with a binary vector construct and stability check Support Protocol 3: Plant transformation Basic Protocol 3: Genotyping of edited eventsmore » « less
-
Abstract Cytosine base editors (CBEs) and adenine base editors (ABEs) enable precise C-to-T and A-to-G edits. Recently, ABE8e, derived from TadA-8e, enhances A-to-G edits in mammalian cells and plants. Interestingly, TadA-8e can also be evolved to confer C-to-T editing. This study compares engineered CBEs derived from TadA-8e in rice and tomato cells, identifying TadCBEa, TadCBEd, and TadCBEd_V106W as efficient CBEs with high purity and a narrow editing window. A dual base editor, TadDE, promotes simultaneous C-to-T and A-to-G editing. Multiplexed base editing with TadCBEa and TadDE is demonstrated in transgenic rice, with no off-target effects detected by whole genome and transcriptome sequencing, indicating high specificity. Finally, two crop engineering applications using TadDE are shown: introducing herbicide resistance alleles inOsALSand creating synonymous mutations inOsSPL14to resistOsMIR156-mediated degradation. Together, this study presents TadA-8e derived CBEs and a dual base editor as valuable additions to the plant editing toolbox.more » « less
