An official website of the United States government
For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16–22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7–10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.
more »
« less
Enhancing Maize Transformation and Targeted Mutagenesis through the Assistance of Non-Integrating Wus2 Vector
Efficient genetic transformation is a prerequisite for rapid gene functional analyses and crop trait improvements. We recently demonstrated that new T-DNA binary vectors with NptII/G418 selection and a compatible helper plasmid can efficiently transform maize inbred B104 using our rapid Agrobacterium-mediated transformation method. In this work, we implemented the non-integrating Wuschel2 (Wus2) T-DNA vector method for Agrobacterium-mediated B104 transformation and tested its potential for recalcitrant inbred B73 transformation and gene editing. The non-integrating Wus2 (NIW) T-DNA vector-assisted transformation method uses two Agrobacterium strains: one carrying a gene-of-interest (GOI) construct and the other providing an NIW construct. To monitor Wus2 co-integration into the maize genome, we combined the maize Wus2 expression cassette driven by a strong constitutive promoter with a new visible marker RUBY, which produces the purple pigment betalain. As a GOI construct, we used a previously tested CRISPR-Cas9 construct pKL2359 for Glossy2 gene mutagenesis. When both GOI and NIW constructs were delivered by LBA4404Thy- strain, B104 transformation frequency was significantly enhanced by about two-fold (10% vs. 18.8%). Importantly, we were able to transform a recalcitrant inbred B73 using the NIW-assisted transformation method and obtained three transgene-free edited plants by omitting the selection agent G418. These results suggest that NIW-assisted transformation can improve maize B104 transformation frequency and provide a novel option for CRISPR technology for transgene-free genome editing.
more »
« less
- Award ID(s):
- 1917138
- PAR ID:
- 10448801
- Date Published:
- Journal Name:
- Plants
- Volume:
- 12
- Issue:
- 15
- ISSN:
- 2223-7747
- Page Range / eLocation ID:
- 2799
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Goetz, H (Ed.)Agrobacterium-mediated transformation is an essential tool for functional genomics studies and crop improvements. Recently developed ternary vector systems, which consist of a T-DNA vector and a compatible virulence (vir) gene helper plasmid (ternary helper), demonstrated that including an additionalvirgene helper plasmid into disarmedAgrobacteriumstrains significantly improves T-DNA delivery efficiency, enhancing plant transformation. Here, we report the development of a new ternary helper and thymidine auxotrophicAgrobacteriumstrains to boostAgrobacterium-mediated plant transformation efficiency. AuxotrophicAgrobacteriumstrains are useful in reducingAgrobacteriumovergrowth after the co-cultivation period because they can be easily removed from the explants due to their dependence on essential nutrient supplementation. We generated thymidine auxotrophic strains from publicAgrobacteriumstrains EHA101, EHA105, EHA105D, and LBA4404. These strains exhibited thymidine-dependent growth in the bacterial medium, and transientGUSexpression assay using Arabidopsis seedlings showed that they retain similar T-DNA transfer capability as their original strains. Auxotrophic strains EHA105Thy- and LBA4404T1 were tested for maize B104 immature embryo transformation using our rapid transformation method, and both strains demonstrated comparable transformation frequencies to the control strain LBA4404Thy-. In addition, our new ternary helper pKL2299A, which carries thevirAgene from pTiBo542 in addition to othervirgene operons (virG,virB,virC,virD,virE, andvirJ), demonstrated consistently improved maize B104 immature embryo transformation frequencies compared to the original version of pKL2299 (33.3% vs 25.6%, respectively). Therefore, our improvedAgrobacteriumsystem, including auxotrophic disarmedAgrobacteriumstrains and a new ternary helper plasmid, can be useful for enhancing plant transformation and genome editing applications.more » « less
-
Summary Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter‐kingdom gene transfer capability madeAgrobacteriuman essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such asAgrobacterium‐host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA‐guided integrase system forAgrobacteriumgenome engineering. In this work, we demonstrate thatINsertion ofTransposableElements byGuideRNA–AssistedTargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre‐loxPrecombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies forAgrobacteriumspecies and their gene functional analyses.more » « less
-
null (Ed.)Maize ( Zea mays ssp. mays ) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium -mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.more » « less
-
Abstract Until recently, precise genome editing has been limited to a few organisms. The ability of Cas9 to generate double stranded DNA breaks at specific genomic sites has greatly expanded molecular toolkits in many organisms and cell types. Before CRISPR‐Cas9 mediated genome editing,P. patenswas unique among plants in its ability to integrate DNA via homologous recombination. However, selection for homologous recombination events was required to obtain edited plants, limiting the types of editing that were possible. Now with CRISPR‐Cas9, molecular manipulations inP. patenshave greatly expanded. This protocol describes a method to generate a variety of different genome edits. The protocol describes a streamlined method to generate the Cas9/sgRNA expression constructs, design homology templates, transform, and quickly genotype plants. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Constructing the Cas9/sgRNA transient expression vector Alternate Protocol 1: Shortcut to generating single and pooled Cas9/sgRNA expression vectors Basic Protocol 2: Designing the oligonucleotide‐based homology‐directed repair (HDR) template Alternate Protocol 2: Designing the plasmid‐based HDR template Basic Protocol 3: Inducing genome editing by transforming CRISPR vector intoP. patensprotoplasts Basic Protocol 4: Identifying edited plants.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/plants12152799
