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.
Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic ...traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6 × 10
cells from pennycress and 3 × 10
cells from camelina per gram of fresh leaf tissues. Using a 7.3 kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.
An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence ...of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Precise genome engineering can be efficiently made using the revolutionary tool named CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein) systems. Adapted ...from the bacterial immune system, CRISPR/Cas systems can generate highly specific double-strand breaks (DSBs) at the target site, and desired sequence modifications can be introduced during the DSB repair process, such as nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways. CRISPR/Cas9 is the most widely used genome editing tool for targeted mutagenesis, precise sequence modification, transcriptional reprogramming, epigenome editing, disease treatment, and many more. The ease of use and high specificity make CRISPR/Cas9 a great tool not only for basic researches but also for crop trait improvements, such as higher grain yield, better tolerance to abiotic stresses, enhanced disease resistance, and better nutritional contents. In this protocol, we present a step-by-step guide to the CRISPR/Cas9-mediated targeted mutagenesis in maize Hi II genotype. Detailed procedures will guide through the essential steps including gRNA design, CRISPR/Cas9 vector construction, Agrobacterium-mediated maize immature embryo transformation, and molecular analysis of the transgenic plants to identify desired mutant lines.
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.
Endophytic fungi represent diverse taxa that inhabit plant hosts without causing disease symptoms. We used endophytic isolates of Fusarium verticillioides (Sacc.) Nirenberg to understand how ...endophytic fungi interact with pathogens, in this case, the corn smut pathogen, Ustilago maydis DC (Corda). Endophytic F. verticillioides strains were inoculated onto maize seedlings before, simultaneously, or after inoculation with U. maydis, and the effects on smut disease severity and on plant growth were assessed. When F. verticillioides is simultaneously coinoculated with U. maydis, smut disease severity is significantly decreased and plant growth is increased, compared with other treatments. Controls show that F. verticillioides by itself does not have measurable effects on plant growth. Together, our results suggest that a commonly occurring fungal endophyte on maize, F. verticillioides, ameliorates the effects of a host-specific pathogen, U. maydis, by interfering with the early infection process and limiting disease development, resulting in increased plant growth.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Key message
Combining with a CRISPR/Cas9 system,
Agrobacterium
-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.
Agrobacterium
-mediated T-DNA integration ...into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard
Agrobacterium
binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene
Os01g04020
. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (
hptII
) gene. One version of the vector had
hptII
expression driven by an
OsAct2
promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless
hptII
gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the
hptII
gene would allow
hptII
expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50−80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that
Agrobacterium
-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OBVAL, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
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