RNA interference-based silencing of the alpha-amylase (amy1) gene in Aspergillus flavus decreases fungal growth and aflatoxin production in maize kernels.

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 several important food and feed crops, including maize. Once the fungus colonizes lipid-rich seed tissues, it has the potential to produce toxic secondary metabolites, the most dangerous of which is aflatoxin. The pre-harvest control of A. flavus contamination and aflatoxin production is an area of intense research, which includes breeding strategies, biological control, and the use of genetically-modified crops. Host-induced gene silencing, whereby the host crop produces siRNA molecules targeting crucial genes in the invading fungus and targeting the gene for degradation, has shown to be promising in its ability to inhibit fungal growth and decrease aflatoxin contamination. Here, we demonstrate that maize inbred B104 expressing an RNAi construct targeting the A. flavus alpha-amylase gene amy1 effectively reduces amy1 gene expression resulting in decreased fungal colonization and aflatoxin accumulation in kernels. This work contributes to the development of a promising technology for reducing the negative economic and health impacts of A. flavus growth and aflatoxin contamination in food and feed crops.

Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred.

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 gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10-14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16-21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

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 the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium-delivered CRISPR/Cas9 for high-frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4-reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono- or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi-II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target-specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

Heritable site-specific mutagenesis using TALENs in maize.

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 modifications in maize. TALENs were employed to generate stable, heritable mutations at the maize glossy2 (gl2) locus. Transgenic lines containing mono- or di-allelic mutations were obtained from the maize genotype Hi-II at a frequency of about 10% (nine mutated events in 91 transgenic events). In addition, three of the novel alleles were tested for function in progeny seedlings, where they were able to confer the glossy phenotype. In a majority of the events, the integrated TALEN T-DNA segregated independently from the new loss of function alleles, producing mutated null-segregant progeny in T1 generation. Our results demonstrate that TALENs are an effective tool for genome mutagenesis in maize, empowering the discovery of gene function and the development of trait improvement.

Maize (Zea mays L.).

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

Rice, Japonica (Oryza sativa L.).

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 straightforward, rapid, and efficient, thereby making rice valuable as a model crop for scientific research and functional genomics. The focus of this chapter is on one such protocol that uses Agrobacterium-mediated transformation of Oryza sativa L. ssp. Japonica cv. Nipponbare with an emphasis on tissue desiccation. The explants consist of callus derived from mature seeds which are cocultivated on filter paper postinfection. Hygromycin selection is used for the recovery of subsequent genetically engineered events.

Genetic transformation using maize immature zygotic embryos.

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 on achieving high embryogenic callus induction frequency (ECIF) in a population of targeted explants. In maize, ECIF is strongly influenced by genotype, the tissue culture media used, and the interaction of these two factors. Altering tissue culture media components to increase ECIF and/or transformation frequency (TF) has been one approach used to expand the range of maize genotypes amenable to genetic transformation using the IZE. This chapter outlines such an approach--an Agrobacterium-mediated transformation protocol is used for direct-targeting IZEs of the hybrid Hi Type II and inbred B104 lines. Two different media regimes are used for successful culture and transformation of two distinct genotypes.

Generation of backbone-free, low transgene copy plants by launching T-DNA from the Agrobacterium chromosome.

In both applied and basic research, Agrobacterium-mediated transformation is commonly used to introduce genes into plants. We investigated the effect of three Agrobacterium tumefaciens strains and five transferred (T)-DNA origins of replication on transformation frequency, transgene copy number, and the frequency of integration of non-T-DNA portions of the T-DNA-containing vector (backbone) into the genome of Arabidopsis (Arabidopsis thaliana) and maize (Zea mays). Launching T-DNA from the picA locus of the Agrobacterium chromosome increases the frequency of single transgene integration events and almost eliminates the presence of vector backbone sequences in transgenic plants. Along with novel Agrobacterium strains we have developed, our findings are useful for improving the quality of T-DNA integration events.

Biolistic gun-mediated maize genetic transformation.

Biolistic gun-mediated transformation is one of the two most effective and popular methods for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype, Hi II. The protocol uses 0.6-microm gold particles as microcarriers and the herbicide resistance bar gene as a selective marker. Both immature zygotic embryos and immature embryo-derived callus cultures can be transformed using this protocol. To ensure successful reproduction of this protocol, we provide step-by-step laboratory transformation procedures as well as details on growing and caring for transgenic plants in the greenhouse.

Novel plant transformation vectors containing the superpromoter.

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2' (mas2') transcriptional activating element plus minimal promoter. We tested a superpromoter-beta-glucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2', and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.

Maize (Zea mays L.).

Agrobacterium tumefaciens-mediated transformation is an effective method for introducing genes into maize. In this chapter, we describe a detailed protocol for genetic transformation of the maize genotype Hi II. Our starting plant material is immature embryos cocultivated with an Agrobacterium strain carrying a standard binary vector. In addition to step-by-step laboratory transformation procedures, we include extensive details in growing donor plants and caring for transgenic plants in the greenhouse.

Gene expression patterns during somatic embryo development and germination in maize Hi II callus cultures.

Gene expression patterns were profiled during somatic embryogenesis in a regeneration-proficient maize hybrid line, Hi II, in an effort to identify genes that might be used as developmental markers or targets to optimize regeneration steps for recovering maize plants from tissue culture. Gene expression profiles were generated from embryogenic calli induced to undergo embryo maturation and germination. Over 1,000 genes in the 12,060 element arrays showed significant time variation during somatic embryo development. A substantial number of genes were downregulated during embryo maturation, largely histone and ribosomal protein genes, which may result from a slowdown in cell proliferation and growth during embryo maturation. The expression of these genes dramatically recovered at germination. Other genes up-regulated during embryo maturation included genes encoding hydrolytic enzymes (nucleases, glucosidases and proteases) and a few storage genes (an alpha-zein and caleosin), which are good candidates for developmental marker genes. Germination is accompanied by the up-regulation of a number of stress response and membrane transporter genes, and, as expected, greening is associated with the up-regulation of many genes encoding photosynthetic and chloroplast components. Thus, some, but not all genes typically associated with zygotic embryogenesis are significantly up or down-regulated during somatic embryogenesis in Hi II maize line regeneration. Although many genes varied in expression throughout somatic embryo development in this study, no statistically significant gene expression changes were detected between total embryogenic callus and callus enriched for transition stage somatic embryos.

Improved Agrobacterium-mediated transformation of three maize inbred lines using MS salts.

Transformation technology as a research or breeding tool to improve maize is routinely used in most industrial and some specialized public laboratories. However, transformation of many inbred lines remains a challenging task, especially when using Agrobacterium tumefaciens as the delivery method. Here we report success in generating transgenic plants and progeny from three maize inbred lines using an Agrobacterium-mediated standard binary vector system to target maize immature embryos. Eleven maize inbred lines were pre-screened for transformation frequency using N6 salts. A subset of three maize inbred lines was then systematically evaluated for frequency of post-infection embryogenic callus induction and transformation on four media regimes: N6 or MS salts in each of two distinct media backgrounds. Transgenic plants recovered from inbred lines B104, B114, and Ky21 were analyzed for transgene integration, expression, and transmission. Average transformation frequencies of 6.4% (for B104), 2.8% (for B114), and 8% (for Ky21) were achieved using MS salts. Availability of Agrobacterium-mediated maize inbred line transformation will improve future opportunities for maize genetic and functional genomic studies.

Leaf senescence is delayed in maize expressing the Agrobacterium IPT gene under the control of a novel maize senescence-enhanced promoter.

We have genetically modified maize plants to delay leaf senescence. A senescence-enhanced promoter from maize (P(SEE1)) was used to drive expression of the Agrobacterium cytokinin biosynthesis gene IPT in senescing leaf tissue. Three maize lines expressing IPT from P(SEE1), Sg1, Sg2 and Sg3, were analysed in detail, representing mild, intermediate and extreme expression, respectively, of the delayed-senescence phenotype. Backcross populations segregating for the presence or absence of the P(SEE1Xba)IPTNOS transgene also simultaneously segregated for the senescence phenotype. At the time of ear leaf emergence, individuals of lines Sg1 and Sg2 segregating for the presence of the transgene carried about three fewer senescing leaves than control (transgene-minus) segregants, and IPT transcript levels were higher in leaves at incipient senescence than in young leaves. Leaves of transgenic Sg3 plants were significantly greener than controls and progressed directly from fully green to bleached and dead without an intervening yellowing phase. IPT transcript abundance in this line was not related to the initiation of senescence. Extended greenness was accompanied by a delay in the loss of photosynthetic capacity with leaf age. The delayed-senescence trait was associated with relatively minor changes in morphology and development. The phenotype was particularly emphasized in plants grown in low soil nitrogen. The reduced ability of the extreme transgenic line Sg3 to recycle internal nitrogen from senescing lower leaves accounted for significant chlorosis in emerging younger leaves when plants were grown in low nutrient conditions. This study demonstrates that the agronomically important delayed-senescence ('stay-green') trait can be engineered into a monocot crop, and is the first example outside Arabidopsis of senescence modification using a homologous senescence-enhanced promoter.

Agrobacterium tumefaciens-mediated transformation of maize embryos using a standard binary vector system.

We have achieved routine transformation of maize (Zea mays) using an Agrobacterium tumefaciens standard binary (non-super binary) vector system. Immature zygotic embryos of the hybrid line Hi II were infected with A. tumefaciens strain EHA101 harboring a standard binary vector and cocultivated in the presence of 400 mg L-1 L-cysteine. Inclusion of L-cysteine in cocultivation medium lead to an improvement in transient beta-glucuronidase expression observed in targeted cells and a significant increase in stable transformation efficiency, but was associated with a decrease in embryo response after cocultivation. The average stable transformation efficiency (no. of bialaphos-resistant events recovered per 100 embryos infected) of the present protocol was 5.5%. Southern-blot and progeny analyses confirmed the integration, expression, and inheritance of the bar and gus transgenes in R0, R1, and R2 generations of transgenic events. To our knowledge, this represents the first report in which fertile, stable transgenic maize has been routinely produced using an A. tumefaciens standard binary vector system.