Kan Wang

• Iowa State University

The spatial characteristics and structure of an axisymmetric turbulent boundary layer under strong adverse pressure gradient and weak transverse curvature are investigated using incompressible large-eddy simulation. The boundary layer is on a $20^{\circ}$ tail cone of a body of revolution at a length-based Reynolds number of $1.9\times10^6$. The si...

Maize is one of the major crops that are susceptible to Aspergillus flavus infection and subsequent aflatoxin contamination, which poses a serious health threat to humans and domestic animals. Here, an RNA interference (RNAi) approach called Host-Induced Gene Silencing (HIGS) was employed to suppress the O-methyl transferase gene (omtA, also called...

Plant genetic transformation is essential for understanding gene functions and developing improved crop varieties. Traditional methods, often genotype‐dependent, are limited by plants' recalcitrance to gene delivery and low regeneration capacity. To overcome these limitations, new approaches have emerged that greatly improve efficiency and genotype...

The introduction of maize genetic transformation in the 1990s brought forth a powerful tool for crop improvement and a deeper understanding of plant genetics. Despite decades of genetics research, however, and the promise of CRISPR-mediated gene editing, maize transformation currently faces several challenges, such as genotype dependence and limita...

Maize genetic transformation is a critical tool for functional genomics and crop improvement. Many laboratories, however, continue to face multiple challenges in attempting to achieve routine genetic transformation of maize inbred genotypes. Here, we describe a rapid and robust maize B104 transformation method using immature embryos as explants. Th...

Conventional maize transformation has largely relied on immature embryos as explants, and is thus often hampered by the limited access to high-quality immature embryos year-round. Here, we present a detailed protocol using seedling leaf whorls as alternative explants for tropical maize inbred transformation. This approach involves the use of a cass...

The RUBY reporter system has demonstrated great potential as a visible marker to monitor gene expression in both transiently and stably transformed plant tissues. Ectopic expression of the RUBY reporter leads to bright red pigmentation in plant tissues that do not naturally accumulate betalain. Unlike traditional visual markers such as β-glucuronid...

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 additional vir gene helper plasmid into disarmed Agrobacter...

Compressible direct numerical simulations are employed to elucidate the low-wavenumber behaviour of wall-pressure fluctuations in turbulent channel flow and the effect of flow Mach number in the nearly incompressible regime. Simulations are conducted at bulk Mach numbers 0.4, 0.2 and 0.1, and friction Reynolds number 180. In addition to the convect...

The acoustic response of a five-bladed rotor to an axisymmetric turbulent boundary layer at the tail end of a body of revolution (BOR) is investigated numerically to elucidate the physical sources of acoustics, particularly the role of coherent structures in sound generation. The BOR is at a length-based Reynolds number of $1.9 \times 10^6$ and fre...

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...

Aspergillus flavus is an opportunistic fungal pathogen that infects maize and produces aflatoxins. Using biocontrol or developing resistant cultivars to reduce aflatoxin contamination has only achieved limited success. Here, the A. flavus polygalacturonase gene (p2c) was targeted for suppression through host-induced gene silencing (HIGS) to reduce...

Recent advances in the genome-editing tools have demonstrated a great potential for accelerating functional genomics and crop trait improvements, but the low efficiency and genotype dependence in plant transformation hinder practical applications of such revolutionary tools. Morphogenic transcription factors (MTFs) such as Baby boom, Wuschel2, GROW...

Biolistic transfection is a popular and versatile tool for plant transformation. A key step in the biolistic process is the binding of DNA to the heavy microprojectile using a delivery agent, usually a positively charged molecule containing amine groups. Currently, the choice of the commercial delivery agent is mostly limited to spermidine. In addi...

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 made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, man...

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 ye...

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...

Switchgrass (Panicum virgatum) is an excellent feedstock for biofuel production. While genetic transformation is routinely done in lowland switchgrass, upland cultivars remain recalcitrant to genetic transformation. Here we report the establishment of an efficient and reproducible transformation protocol for two upland cultivars, ‘Summer’ and ‘Blac...

Delivery of genome editing reagents using CRISPR-Cas ribonucleoproteins (RNPs) transfection offers several advantages over plasmid DNA-based delivery methods, including reduced off-target editing effects, mitigation of random integration of non-native DNA fragments, independence of vector constructions, and less regulatory restrictions. Compared to...

Modern maize exhibits a significantly different phenotype than its wild progenitor teosinte despite many genetic similarities. Of the many subspecies of Zea mays identified as teosinte, Zea mays ssp. parviglumis is the most closely related to domesticated maize. Understanding teosinte genes and their regulations can provide great insights into the...

Aspergillus flavus is a fungal pathogen that infects maize and produces aflatoxins. Host-Induced Gene Silencing (HIGS) has been shown to reduce host infection by various fungal pathogens. Here, the A. flavus alkaline protease (alk) gene was targeted for silencing through HIGS. An RNAi vector carrying a portion of the alk gene was incorporated into...

Maize functional genomics research and genetic improvement strategies have been greatly accelerated and refined through the development and utilization of genetic transformation systems. Maize transformation is a composite technology based on decades’ efforts in optimizing multiple factors involving microbiology and physical/biochemical DNA deliver...

While optical aberrations caused by atmospheric turbulence have been extensively investigated and well characterized, recent research has identified structural differences in optical phase distortions caused by aircraft-induced, compressible turbulence. These so-called aero-optical distortions can be a critical obstacle in the development of airbor...

Developed over thousands of years largely through human intervention, the modern maize genome can now be precisely modified for agricultural improvement and scientific research. This chapter focuses on progress made in recent decades utilizing site-specific nuclease (SSN) technologies in maize genome engineering. Many SSNs, such as meganucleases, z...

Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel dev...

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 ofte...

Biolistic delivery is widely used for genetic transformation but inconsistency between bombardment samples for transient gene expression analysis often hinders quantitative analyses. We developed a methodology to improve the consistency of biolistic delivery results by using a double-barrel device and a cell counting software. The double-barrel dev...

Large-eddy simulation is combined with the Ffowcs Williams–Hawkings equation to investigate the noise generation by a 10-bladed rotor ingesting the turbulent wake of a circular cylinder in a low-Mach-number flow. Two rotor advance ratios corresponding to zero thrust and a thrusting condition are considered. The computed sound pressure levels agree...

Background: Delivery of CRISPR reagents into cells as ribonucleoprotein (RNP) complexes enables transient editing, and avoids CRISPR reagent integration in the genomes. Another technical advantage is that RNP delivery can bypass the need of cloning and vector construction steps. In this work we compared efficacies and types of edits for three Cas9...

Agrobacterium-mediated transformation is a widely used gene delivery method for fundamental researches and crop trait improvement projects. Auxotrophic Agrobacterium tumefaciens strains are highly desirable for plant transformation because they can be easily removed from the explants after co-cultivation due to their dependence on essential nutrien...

CRISPR–Cas technology now enables chromosome structural engineering in plants.

Maize (Zea mays L.) is one of the major crops susceptible to Aspergillus flavus infection and subsequent contamination with aflatoxins, the most potent naturally produced carcinogenic secondary metabolites. This pathogen can pose serious health concerns and cause severe economic losses due to the Food and Drug Administration (FDA) regulations on pe...

Genetic improvement of rice is crucial to achieve global food security as rice is an important staple crop for more than half of the global population. One of the methodologies for genetic improvement is biolistic delivery of genetic components into plant cells. In this chapter, we describe steps involved in introducing plasmid DNA carrying gene of...

Intracellular protein delivery in plant tissues is becoming an important tool for addressing both basic and applied research questions by plant biologists, especially in the era of genome editing. The ability to deliver proteins or protein/RNA complexes into cells allows for producing gene-edited plants that are free of transgene integration in the...

One of the key factors for ensuring a successful genetic transformation is to effectively introduce genetic materials, such as plasmid DNA, into plant cells. A biolistic gun is one of the two best established and most popular tools for delivery of DNA into maize cells. It is the method that generated the first fertile transgenic maize plant. In thi...

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 fa...

Key message Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome. Abstract Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially inte...

Optical distortions caused by turbulent airflow surrounding an aircraft, known as aero-optical phenomena, are a major impediment to applications of airborne laser systems. To better understand the spectral properties of aero-optical distortions, a general expression for the wavenumber spectrum of the refractive index is derived from the ideal-gas l...

Table S1 Summary of Cas‐OFFinder prediction. Table S2 List of oligonucleotides used in this study. Table S3 Summary of genotyping results of T0 and T1 lines. Table S4 Summary of CIRCLE‐seq‐identified off‐targets.

Data S1 Off‐target mutagenesis analysis.

Background CRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nuc...

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...

CRISPR‐Cas9 and Cas12a (Cpf1) nucleases are two of the most powerful genome editing tools in plants. In this work, we compared their activities by targeting maize glossy2 gene coding region that has overlapping sequences recognized by both nucleases. We introduced constructs carrying SpCas9‐guide RNA (gRNA) and LbCas12a‐CRISPR RNA (crRNA) into maiz...

Main conclusion: Expressing an RNAi construct in maize kernels that targets the gene for alpha-amylase in Aspergillus flavus resulted in suppression of alpha-amylase (amy1) gene expression and decreased fungal growth during in situ infection resulting in decreased aflatoxin production. Aspergillus flavus is a saprophytic fungus and pathogen to sev...

During the last decade, small noncoding RNAs (ncRNAs) have emerged as essential post-transcriptional regulators in bacteria. Nearly all important physiological and stress responses are modulated by ncRNA regulators, such as riboswitches, trans-acting small RNAs (sRNAs), and cis-antisense RNAs. Recently, three RNA-seq studies identified a total of 1...

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell’s ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for...

We describe a non-DNA-based system for delivering Cre recombinase protein into maize tissue using gold-plated mesoporous silica nanoparticle (Au-MSN). Cre protein is first loaded into the pores of Au-MSNs and then delivered using the biolistic method to immature embryos of a maize line (Lox-corn), which harbors loxP sites flanking a selection and a...

In Agrobacterium tumefaciens-mediated plant transformation, the promoter chosen to drive a selectable marker gene has an effect on transformation frequency. The objective of this work was to compare the effect on soybean transformation of different promoter and regulator combinations driving a selectable marker gene using mature seeds as explants....

Figure S1 a1 and a4 Genotypes of CRISPR line 24‐3. Table S1 Primers and sequences used in this study.

The CRISPR-Cas9 system (clustered regularly interspaced short palindromic repeats with associated Cas9 protein) has been used to generate targeted changes for direct modification of endogenous genes in an increasing number of plant species; but development of plant genome editing has not yet fully considered potential off-target mismatches that may...

Significance One of the most important agronomic traits in crop breeding is yield, which includes increased seed size and weight in grain crops and leaf biomass in forage crops. In this work, we demonstrate that a transcription regulator encoded by the BIG SEEDS1 ( BS1 ) gene from the model legume Medicago truncatula , negatively regulates primary...

Genome editing with engineered nucleases (GEEN) is a highly efficient means of generating useful traits in crops. The application of GEEN for multiple traits and crops is resulting in products that are already entering the marketplace. Genome editing represents diverse and robust techniques targeting highly specific genome locations to cause double...

CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease-based genome editing tools, optimization requires consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve a...

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than thirty years of technological advances. Genome editing provides new opportunities to enhance crop productivity, but relies on genetic t...

Key message: Bacterial phosphite oxidoreductase gene and chemical phosphite can be used as a selection system for Agrobacterium -mediated maize transformation. Application of phosphite (Phi) on plants can interfere the plant metabolic system leading to stunted growth and lethality. On the other hand, ectopic expression of the ptxD gene in tobacco...

Genome editing with engineered nucleases (GEEN) represents a highly specific and efficient tool for crop improvement with the potential to rapidly generate useful novel phenotypes/traits. Genome editing techniques initiate specifically targeted double strand breaks facilitating DNA-repair pathways that lead to base additions or deletions by non-hom...

Key message: Oral administration of maize-expressed H3N2 nucleoprotein induced antibody responses in mice showing the immunogenicity of plant-derived antigen and its potential to be utilized as a universal flu vaccine. Influenza A viruses cause influenza epidemics that are devastating to humans and livestock. The vaccine for influenza needs to be...

Transcription activator-like effector nuclease (TALEN) technology has been utilized widely for targeted gene mutagenesis, especially for gene inactivation, in many organisms, including agriculturally important plants such as rice, wheat, tomato and barley. This report describes application of this technology to generate heritable genome modificatio...

The importance of rice, as a food crop, is reflected in the extensive global research being conducted in an effort to improve and better understand this particular agronomic plant. In regard to biotechnology, this has led to the development of numerous genetic transformation protocols. Over the years, many of these methods have become increasingly...

In this chapter we describe an Agrobacterium tumefaciens transformation method of soybean that utilizes mature half seeds and regeneration from the cotyledonary node region. This method results in fertile transformed soybean plants and transgenic seed in approximately 9 months. Using mature half seeds as starting material has proven to be a reliabl...

In this work, an intracellular protein delivery methodology termed "proteolistics" is described. This method utilizes a biolistic gun apparatus and involves a simple protein/projectile preparation step. The protein to be delivered is mixed with a gold particle microprojectile suspension and is placed onto a gene gun cartridge, where it is dehydrate...

The invention provides an article having a mesoporous silicate matrix, such as a particle, having one or more pores; and one or more releasable caps obstructing one or more of the pores for delivery of one or more agents to plant cells or other chlorophyll containing cells, or fungi.

Rapid changes and significant progress have been made in the Agrobacterium field, such as genetically transforming plants for both basic research purposes and agricultural development. In Agrobacterium Protocols, Third Edition, Volumes 1 and 2, a team of leading experts and veteran researchers describe in detail techniques for delivering DNA to pla...

Delivery of proteins instead of DNA into plant cells allows for transient presence of the protein or enzyme that can be useful for biochemical analysis or genome modifications. This may be of particular interest for genome editing because it can avoid DNA (transgene) integration into the genome and generate precisely modified "non-transgenic" plant...

Of the more than 50,000 edible plant species in the world, at least 10,000 species are cereal grains. Three major cereal crops, rice (Oryza sativa), maize (Zea mays), and wheat (Triticum sp.), provide two-thirds of the world’s food energy intake. Although crop yields have improved tremendously thanks to technological advances in the past 50 years,...

Agrobacterium tumefaciens is a plant pathogen that has the natural ability of delivering and integrating a piece of its own DNA into plant genome. Although bacterial non-coding RNAs (ncRNAs) have been shown to regulate various biological processes including virulence, we have limited knowledge of how Agrobacterium ncRNAs regulate this unique inter-...

Table S1, Comparision of expression fold change of vir genes in a microarray study and two RNA-seq studies; Table S2, TSS-mapping; Table S3, List of identified candidate ncRNAs on all four replicons; Table S4, Differentially expressed candidate ncRNAs; Table S5, Selected ncRNAs for 5′ and 3′ RACE; Table S6, Oligonucleotides used in this study. (PDF...

Figure S1, Effects of primary transcript enrichment by terminator 5′-phosphate-dependent exonuclease; Figure S2, 5′ and 3′ RACE for ncRNAs on Ti plasmid; Figure S3, Expression profiling of virD4* internal transcript with primary transcript enrichment (+TEX); Figure S4, Expression profiling of C3 and Ti2; Figure S5, Effects of two antisense RNAs (pT...

Key message: Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain. Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch...

The synthesis and characterization of a gold nanoparticle functionalized mesoporous silica nanoparticle (Au-MSN) platform for codelivery of proteins and plasmid DNA to plant tissues using a biolistic particle delivery system is reported. The in vitro uptake and release profiles of fluorescently labeled bovine serum albumin (BSA) and enhanced green...

The cell wall of plant cells is a physical barrier for nanoparticle uptake that limits nanotechnology development in plant sciences. On page 3576, Brian G. Trewyn, Kan Wang, and co-workers report using gold-plated mesoporous silica nanoparticles, which have increased performance as projectiles through the biolistic method, to co-deliver proteins an...

Here we present a routine and efficient protocol for year-round production of fertile transgenic maize plants. Type II callus derived from maize Hi II immature zygotic embryos was transformed using the PDS 1000/He biolistic gun and selected on bialaphos. In an effort to improve the transformation protocol, the effects of gold particle size and call...

The non-toxic B subunit (CT-B) of cholera toxin from Vibrio cholerae is a strong immunogen and amplifies the immune reaction to conjugated antigens. In this work, a synthetic gene encoding for CT-B was expressed under control of a γ-zein promoter in maize seeds. Levels of CT-B in maize plants were determined via ganglioside dependent ELISA. The hig...

Applying nanotechnology to plant science requires efficient systems for the delivery of nanoparticles (NPs) to plant cells and tissues. The presence of a cell wall in plant cells makes it challenging to extend the NP delivery methods available for animal research. In this work, research is presented which establishes an efficient NP delivery system...

In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the m...

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in...

Collagens require the hydroxylation of proline (Pro) residues in their triple-helical domain repeating sequence Xaa-Pro-Gly to function properly as a main structural component of the extracellular matrix in animals at physiologically relevant conditions. The regioselective proline hydroxylation is catalyzed by a specific prolyl 4-hydroxylase (P4H)...

An in vitro continuous endosperm callus culture derived from developing endosperm of transformation-amenable maize Hi-II genotype was obtained. The endosperm callus was composed of cells that differentiated into aleurone-like and starchy endosperm-like cell types. This callus has been maintained for 4 yr. Endosperm callus cells transcribe and produ...

Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex prot...

Epidermal and subepidermal cells in the abaxial, basal region of the maize (Zea mays L.) immature zygotic embryo (IZE) scutellum can be induced by exogenous auxin to proliferate and undergo somatic embryogenesis. Successful genetic transformation of IZEs depends not only on optimizing transformation parameters for these totipotent cells, but also o...