Maize Transformation Research Articles

An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos.

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.

Optimized Transformation and Gene Editing of the B104 Public Maize Inbred by Improved Tissue Culture and Use of Morphogenic Regulators.

Plant transformation is a bottleneck for the application of gene editing in plants. In Zea mays (maize), a breakthrough was made using co-transformation of the morphogenic transcription factors BABY BOOM (BBM) and WUSCHEL (WUS) to induce somatic embryogenesis. Together with adapted tissue culture media, this was shown to increase transformation efficiency significantly. However, use of the method has not been reported widely, despite a clear need for increased transformation capacity in academic settings. Here, we explore use of the method for the public maize inbred B104 that is widely used for transformation by the research community. We find that only modifying tissue culture media already boosts transformation efficiency significantly and can reduce the time in tissue culture by 1 month. On average, production of independent transgenic plants per starting embryo increased from 1 to 4% using BIALAPHOS RESISTANCE (BAR) as a selection marker. In addition, we reconstructed the BBM-WUS morphogenic gene cassette and evaluated its functionality in B104. Expression of the morphogenic genes under tissue- and development stage-specific promoters led to direct somatic embryo formation on the scutellum of zygotic embryos. However, eight out of ten resulting transgenic plants showed pleiotropic developmental defects and were not fertile. This undesirable phenotype was positively correlated with the copy number of the morphogenic gene cassette. Use of constructs in which morphogenic genes are flanked by a developmentally controlled Cre/LoxP recombination system led to reduced T-DNA copy number and fertile T0 plants, while increasing transformation efficiency from 1 to 5% using HIGHLY-RESISTANT ACETOLACTATE SYNTHASE as a selection marker. Addition of a CRISPR/Cas9 module confirmed functionality for gene editing applications, as exemplified by editing the gene VIRESCENT YELLOW-LIKE (VYL) that can act as a visual marker for gene editing in maize. The constructs, methods, and insights produced in this work will be valuable to translate the use of BBM-WUS and other emerging morphogenic regulators (MRs) to other genotypes and crops.

Optimization of Agrobacterium tumefaciens-Mediated Genetic Transformation of Maize

Immature embryos of inbred maize (Zea mays) lines (H8183, H8184, and H8185) were used for Agrobacterium infection. We used the β-glucuronidase gene (GUS) as the target gene and the glufosinate resistance gene (bar) as the selection marker. We conducted research on several aspects, such as different genotypes, coculture conditions, screening agent concentrations, and concentrations of indole-3-butytric acid (IBA), 6-benzylaminopurine (6-BA), and ascorbic acid (Vc) in the differentiation medium. We optimized the genetic transformation system, and the obtained results indicated that among the three lines studied, the induction rate of H8185 was the highest at 93.2%, followed by H8184, with H8183 having the lowest induction rate (80.1%). The best coculture method was that using the N6 coculture medium layered with a sterile filter paper. Using orthogonal analysis, we found that the optimal combination of the three factors in the differentiation medium was A3 (1 mg mL−1 IBA), B3C1 (1.6 mg mL−1 6-BA), and D3 (1.5 mg mL−1 Vc). Through GUS staining analysis, Bar test-strip analysis, and polymerase chain reaction, five transgenic plants were finally obtained. This study established the optimal conditions for genetic transformation in maize.

Genetic variations in ZmSAUR15 contribute to the formation of immature embryo-derived embryonic calluses in maize.

The ability of immature maize (Zea mays) embryos to form embryonic calluses (ECs) is highly genotype dependent, which limits transgenic breeding development in maize. Here, we report the association map-based cloning of ZmSAUR15 using an association panel (AP) consisting of 309 inbred lines with diverse formation abilities for ECs. We demonstrated that ZmSAUR15, which encodes a small auxin-upregulated RNA, acts as a negative effector in maize EC induction. Polymorphisms in the ZmSAUR15 promoter that influence the expression of ZmSAUR15 transcripts modulate the EC induction capacity in maize. ZmSAUR15 is involved in indole-3-acetic acid biosynthesis and cell division in immature embryo-derived callus. The ability of immature embryos to induce EC formation can be improved by the knockout of ZmSAUR15, which consequently increases the callus regeneration efficiency. Our study provides new insights into overcoming the genotypic limitations associated with EC formation and improving genetic transformation in maize.

Maize Transformation: From Plant Material to the Release of Genetically Modified and Edited Varieties.

Over the past decades, advances in plant biotechnology have allowed the development of genetically modified maize varieties that have significantly impacted agricultural management and improved the grain yield worldwide. To date, genetically modified varieties represent 30% of the world’s maize cultivated area and incorporate traits such as herbicide, insect and disease resistance, abiotic stress tolerance, high yield, and improved nutritional quality. Maize transformation, which is a prerequisite for genetically modified maize development, is no longer a major bottleneck. Protocols using morphogenic regulators have evolved significantly towards increasing transformation frequency and genotype independence. Emerging technologies using either stable or transient expression and tissue culture-independent methods, such as direct genome editing using RNA-guided endonuclease system as an in vivo desired-target mutator, simultaneous double haploid production and editing/haploid-inducer-mediated genome editing, and pollen transformation, are expected to lead significant progress in maize biotechnology. This review summarises the significant advances in maize transformation protocols, technologies, and applications and discusses the current status, including a pipeline for trait development and regulatory issues related to current and future genetically modified and genetically edited maize varieties.

Establishment of a Bivector Genetic Transformation System in Recalcitrant Maize Inbred Lines

Maize is an important grain crop with high nutritional value. An effective transformation system is crucial for the genetic improvement of maize traits, but many important maize inbred lines remained recalcitrant to transformation. In this study, we developed a bivector transformation system that worked well in two recalcitrant maize inbred lines. This system included an induction vector (ZmBBM-ZmWUS) and an indicator vector (GFP), using microprojectile bombardment technology combined with Agrobacterium-mediated transformation. We found that the Zheng58 and Mo17 recalcitrant inbred lines could be transformed with this system. The whole transformation cycle lasted only 52 days, 38 days less than the traditional transformation cycle. Additionally, it was possible to eliminate inference of the induction vector and obtained progenies with only the target gene. Our results suggested that the bivector system was an optimization of the current maize transformation methods and could potentially be used in genetic improvement of maize inbred lines.

Advances in Agrobacterium transformation and vector design result in high-frequency targeted gene insertion in maize.

SummaryCRISPR‐Cas is a powerful DNA double‐strand break technology with wide‐ranging applications in plant genome modification. However, the efficiency of genome editing depends on various factors including plant genetic transformation processes and types of modifications desired. Agrobacterium infection is the preferred method of transformation and delivery of editing components into the plant cell. While this method has been successfully used to generate gene knockouts in multiple crops, precise nucleotide replacement and especially gene insertion into a pre‐defined genomic location remain highly challenging. Here, we report an efficient, selectable marker‐free site‐specific gene insertion in maize using Agrobacterium infection. Advancements in maize transformation and new vector design enabled increase of targeted insertion frequencies by two orders of magnitude in comparison to conventional Agrobacterium‐mediated delivery. Importantly, these advancements allowed not only a significant improvement of the frequency, but also of the quality of generated events. These results further enable the application of genome editing for trait product development in a wide variety of crop species amenable to Agrobacterium‐mediated transformation.

Maize tissue culture, transformation, and genome editing

Maize tissue culture, transformation, and genome editing

Maize transformation: history, progress, and perspectives.

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 delivery, as well as cellular and molecular biology. This review provides a historical reflection on the development of maize transformation technology including the early failures and successful milestones. It also provides a current perspective on the understanding of tissue culture responses and their impact on plant regeneration, the pros and cons of different DNA delivery methods, the identification of a palette of selectable/screenable markers, and most recently the development of growth-stimulating or morphogenic genes to improve efficiencies and extend the range of transformable genotypes. Steady research progress in these interdependent components has been punctuated by benchmark reports celebrating the progress in maize transformation, which invariably relied on a large volume of supporting research that contributed to each step and to the current state of the art. The recent explosive use of CRISPR/Cas9-mediated genome editing has heightened the demand for higher transformation efficiencies, especially for important inbreds, to support increasingly sophisticated and complicated genomic modifications, in a manner that is widely accessible. These trends place an urgent demand on taking maize transformation to the next level, presaging a new generation of improvements on the horizon. Once realized, we anticipate a near-future where readily accessible, genotype-independent maize transformation, together with advanced genomics, genome editing, and accelerated breeding, will contribute to world agriculture and global food security.

A sugarcane mosaic virus vector for rapid in planta screening of proteins that inhibit the growth of insect herbivores.

SummarySpodoptera frugiperda (fall armyworm) is a notorious pest that threatens maize production worldwide. Current control measures involve the use of chemical insecticides and transgenic maize expressing Bacillus thuringiensis (Bt) toxins. Although additional transgenes have confirmed insecticidal activity, limited research has been conducted in maize, at least partially due to the technical difficulty of maize transformation. Here, we describe implementation of a sugarcane mosaic virus (SCMV) vector for rapidly testing the efficacy of both endogenous maize genes and heterologous genes from other organisms for the control of S. frugiperda in maize. Four categories of proteins were tested using the SCMV vector: (i) maize defence signalling proteins: peptide elicitors (Pep1 and Pep3) and jasmonate acid conjugating enzymes (JAR1a and JAR1b); (ii) maize defensive proteins: the previously identified ribosome‐inactivating protein (RIP2) and maize proteinase inhibitor (MPI), and two proteins with predicted but unconfirmed anti‐insect activities, an antimicrobial peptide (AMP) and a lectin (JAC1); (iii) lectins from other plant species: Allium cepa agglutinin (ACA) and Galanthus nivalis agglutinin (GNA); and (iv) scorpion and spider toxins: peptides from Urodacus yaschenkoi (UyCT3 and UyCT5) and Hadronyche versuta (Hvt). In most cases, S. frugiperda larval growth was reduced by transient SCMV‐mediated overexpression of genes encoding these proteins. Additionally, experiments with a subset of the SCMV‐expressed genes showed effectiveness against two aphid species, Rhopalosiphum maidis (corn leaf aphid) and Myzus persicae (green peach aphid). Together, these results demonstrate that SCMV vectors are a rapid screening method for testing the efficacy and insecticidal activity of candidate genes in maize.

SPATIAL DISTRIBUTION, EVOLUTION AND STRUCTURE OF MAIZE AND SOYBEAN PRODUCTION SYSTEMS IN STATE OF PARANÁ

The Brazilian state of Paraná is one of the leading producers of maize. However, expansion of soybean cropping has caused a drop in maize production and could have impacted production systems. The aim of the study was to verify the evolution and identify the structure, spatial dynamics and transformation of maize and soybean production systems in the state of Paraná. Municipal Agricultural Production data from the Brazilian Institute of Geography and Statistics (IBGE) provided the basis for this study. The Location Quotient was analyzed to identify microregions specialized in the production of soybean and first and second crops of maize. Principal components and groups were analyzed in order to characterize the structure and dynamics of maize and soybean production systems in specialized microregions. The results show how maize and soybean production systems have been transformed; soybean is now cropped in areas previously occupied by first crop maize, and the area occupied by second crop maize has increased. This has led to the predominant use of the crop rotation system with first crop soybean followed by second crop maize.

An Efficient Gene Excision System in Maize.

Use of the morphogenic genes Baby Boom (Bbm) and Wuschel2 (Wus2), along with new ternary constructs, has increased the genotype range and the type of explants that can be used for maize transformation. Further optimizing the expression pattern for Bbm/Wus2 has resulted in rapid maize transformation methods that are faster and applicable to a broader range of inbreds. However, expression of Bbm/Wus2 can compromise the quality of regenerated plants, leading to sterility. We reasoned excising morphogenic genes after transformation but before regeneration would increase production of fertile T0 plants. We developed a method that uses an inducible site-specific recombinase (Cre) to excise morphogenic genes. The use of developmentally regulated promoters, such as Ole, Glb1, End2, and Ltp2, to drive Cre enabled excision of morphogenic genes in early embryo development and produced excised events at a rate of 25–100%. A different strategy utilizing an excision-activated selectable marker produced excised events at a rate of 53–68%; however, the transformation frequency was lower (13–50%). The use of inducible heat shock promoters (e.g. Hsp17.7, Hsp26) to express Cre, along with improvements in tissue culture conditions and construct design, resulted in high frequencies of T0 transformation (29–69%), excision (50–97%), usable quality events (4–15%), and few escapes (non-transgenic; 14–17%) in three elite maize inbreds. Transgenic events produced by this method are free of morphogenic and marker genes.

Glyphosate selection of maize transformants containing CP4epsps gene

Aim. To study the selection conditions of maize transformants containing the CP4epsps gene using glyphosate as a selective agent. Methods. Tissue culture in vitro, Agrobacterium-mediated transformation, selection of transgenic plants, isolation of total plant DNA, analysis of plant DNA by polymerase chain reaction (PCR). Results. The morphogenic maize callus of immature embryos of the hybrid (PLS61)R2×PLS61 was produced, which had a high regeneration rate (up to 95%), that persisted over long cultivation. Agrobacterium mediated transformation of the morphogenic callus and selection of the transgenic material using glyphosate yielded maize transformants containing the CP4epsps gene at a frequency of 1%. Conclusions. Maize genotype (PLS61)R2×PLS61 is promising for studies on the maize genetic transformation, in particular for the production of transgenic maize resistant to glyphosate herbicide. The use of morphogenic maize callus (PLS61)R2×PLS61 and glyphosate as a selective agent at a concentration of 0.1 mM and 0.25 mM in media for callusogenesis and 0.01 mM in the medium for regeneration was effective for the selection of transgenic plants with the gene CP4epsps.
 Keywords: Zea mays L., morphogenic callus, Agrobacterium-mediated transformation, PCR, genetic engineering.

The potential and efficacy of Allium sativum leaf lectin (ASAL) against sap-sucking insect pests of transgenic maize

The gene sequence encoding the mannose-binding homodimeric protein Allium sativum leaf agglutinin (ASAL) was introduced into maize inbred lines to achieve resistance against sap-sucking corn leaf aphids in transgenic lines. Maize transformants were generated after the co-cultivation of immature maize embryos with the Agrobacterium strain LB4404 harbouring a recombinant Ti-binary ASAL construct driven by the polyubiquitin promoter. The preliminary screening of maize transformants was performed via GUS histochemical analysis, and PCR and Southern blotting confirmed the insertion of the transgene and its stable integration in five transgenic maize lines. Transcript abundance was quantified by a quantitative real-time PCR assay, which revealed variable expression of the ASAL transgene among five transgenic maize lines. The highest mRNA expression of the ASAL gene was found in the A23 transgenic maize line, while the lowest expression was found in the AU1 transgenic maize line. In planta bioassays in the T1 progeny of the transgenic maize lines revealed high resistance against corn leaf aphids compared to the control non-transgenic line. The mortality of the infesting aphids (Rhopalosiphum maidi) was found to vary from 40 to 71% compared to that of the non-transgenic control maize line.

Use of non-integrating Zm-Wus2 vectors to enhance maize transformation

The use of Baby boom (Bbm) and Wuschel2 (Wus2) has made maize transformation more efficient across an increasingly wide range of inbreds. However, the benefits have come with the requirement of excising these transformation helper components to enable plant regeneration, which adds size to the T-DNA, and complexity to the transformation system. A new system with the advantages of smaller size and simplicity for the selectable marker gene-containing T-DNA is described. First, expression of Zm-Wus2 alone driven by the maize Pltp promoter (Zm-Pltppro), was determined to be sufficient to induce rapid somatic embryo formation from the scutella of maize immature embryos. It was also demonstrated that co-infecting with two strains of Agrobacterium, one with a Wus2 expression cassette, and the other with a combination of both selectable and visual marker cassettes, produced transformed T0 plants that contained only a single copy of the selectable marker T-DNA, without the integration of Wus2. Furthermore, the process was optimized by varying the ratio of the two Agrobacterium strains, and by modulating Wus2 expression to enable high-frequency recovery of selectable marker-containing T0 plants that did not contain Wus2. Several factors may have contributed to this outcome. Wus2 expression in localized cell(s) appeared to stimulate somatic embryogenesis in neighboring cells, including those that had integrated the selectable marker. In addition, in cells in which the Wus2 T-DNA did not integrate but the selectable marker T-DNA did, transient Wus2 expression stimulated somatic embryo formation and regeneration of stable T0 plants that contained the selectable marker. In addition, augmenting the Pltp promoter with three viral enhancer elements to increase Wus2 expression stimulated embryogenesis while precluding their regeneration. The phenomenon has now been designated as “altruistic transformation.”

Transcriptome Profiling Predicts New Genes to Promote Maize Callus Formation and Transformation.

Maize transformation is highly based on the formation of embryonic callus, which is mainly derived from scutellum cells of the immature maize embryo. However, only a few genes involved in callus induction have been identified in maize. To reveal the potential genes involved in the callus induction of maize, we carried out a high-throughput RNA sequencing on embryos that were cultured for 0, 1, 2, 4, 6, and 8 days, respectively, on a medium containing or lacking 2,4-dichlorophenoxyacetic acid. In total, 7,525 genes were found to be induced by 2,4-dichlorophenoxyacetic acid and categorized into eight clusters, with clusters 2 and 3 showing an increasing trend related to signal transmission, signal transduction, iron ion binding, and heme binding. Among the induced genes, 659 transcription factors belong to 51 families. An AP2 transcription factors, ZmBBM2, was dramatically and rapidly induced by auxin and further characterization showed that overexpression of ZmBBM2 can promote callus induction and proliferation in three inbred maize lines. Therefore, our comprehensive analyses provide some insight into the early molecular regulations during callus induction and are useful for further identification of the regulators governing callus formation.

Restriction Release: Improved Maize Transformation Efficiency.

Improvement of crops using traditional breeding is too slow to ensure food production able to sustain the growing human population, especially in the face of climate change ([Hickey et al., 2019][1]). Transformation methods for monocot crops depend on the availability of immature embryos and are

Application of Beta-Glucuronidase Transient Expression for Selection of Maize Genotypes Competent for Genetic Transformation

Genetic transformation of inbred maize lines and F1 hybrids registered in Ukraine has been carried out. The study employed a biolistic method for genetic transformation of immature maize embryos that formed callus tissue and the pAHC25 vector containing the genes of phosphinothricin-N-acetyltransferase (bar) and β-glucuronidase (uidA). As a result of the transformation of callus tissue of maize genotypes, lines resistant to phosphinothricin and regenerated plants were obtained. The activity of β-glucuronidase in herbicide-resistant calli was detected. The presence of the bar gene in callus DNA was demonstrated by the PCR method. The rate of stable transformation ranged from 2.2 to 30% depending on the genotype. The relationship between the results of transient expression of the β-glucuronidase gene and stable genetic transformation was observed. The proposed protocol for genetic transformation of maize using the study of transient expression of the β-glucuronidase gene makes it possible to significantly simplify the process of selecting genotypes competent for genetic transformation and create transgenic organisms with new traits.

A Novel Ternary Vector System United with Morphogenic Genes Enhances CRISPR/Cas Delivery in Maize.

The lack of efficient delivery methods is a major barrier to clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas)-mediated genome editing in many plant species. Combinations of morphogenic regulator (MR) genes and ternary vector systems are promising solutions to this problem. In this study, we first demonstrated that MR vectors greatly enhance maize (Zea mays) transformation. We then tested a CRISPR/Cas9 MR vector in maize and found that the MR and CRISPR/Cas9 modules have no negative influence on each other. Finally, we developed a novel ternary vector system to integrate the MR and CRISPR/Cas modules. Our ternary vector system is composed of new pGreen-like binary vectors, here named pGreen3, and a pVS1-based virulence helper plasmid, which also functions as a replication helper for the pGreen3 vectors in Agrobacterium tumefaciens The pGreen3 vectors were derived from the plasmid pRK2 and display advantages over pGreen2 vectors regarding both compatibility and stability. We demonstrated that the union of our ternary vector system with MR gene modules has additive effects in enhancing maize transformation and that this enhancement is especially evident in the transformation of recalcitrant maize inbred lines. Collectively, our ternary vector system-based tools provide a user-friendly solution to the low efficiency of CRISPR/Cas delivery in maize and represent a basic platform for developing efficient delivery tools to use in other plant species recalcitrant to transformation.

Comparing microbial transformation of maize residue‐N and fertilizer‐N in soil using amino sugar‐specific 15N analysis

AbstractIn agroecosystems, both fertilizer‐nitrogen (N) and crop residue‐N make important contributions to soil‐N retention. However, how different N sources are dynamically involved in microbial processes and how they govern soil‐N transformations is poorly understood. A field experiment with half‐yield maize residue mulching was conducted in an Alfisol. The 15N‐labelled fertilizer and maize (Zea mays L.) residue were applied in the first year and then unlabelled ones were used in the following 4 years. The origins of newly formed fungal and bacterial residues were investigated by amino sugar‐specific 15N analysis. The aim was to gain insights into the microbial transformation dynamics of fertilizer‐N and maize residue‐N, as well as the maintenance of these exogenous N sources in soil. The initial transformation of fertilizer‐N into amino sugars was much more rapid than that of maize residue‐N. However, the eventual accumulation of maize residue‐N in amino sugars was significantly greater than that of fertilizer‐N. The transformation of maize residue‐15N into amino sugars was 3.5–6.7 times higher than that of fertilizer‐15N over the course of 5 years, implying a higher immobilization of maize residue‐derived N than fertilizer‐derived N in the soil. The effective transformation of maize residue‐N into microbial residues and the low level of maize residue‐derived mineral N accumulation suggested that a direct route probably dominated the microbial process. Moreover, the maize residue‐derived fungal glucosamine was more apt to accumulate than muramic acid, compared with that derived from fertilizer, suggesting that fungi contributed more to the direct assimilation of maize residue‐derived N. Such a pattern was mostly attributed to fungal decomposition of less bioavailable components of maize residue. Thus, maize residue‐N played a significant role in building soil organic N reserves, being a sustaining foundation for the effective utilization of fertilizer‐N by crops.Highlights Fungal and bacterial residues derived from maize residue‐N and fertilizer‐N were investigated. Fungi dominated maize residue‐N immobilization mostly via direct route. Maize residue‐N had higher microbial immobilization efficiency than fertilizer‐N. Maize residue‐N contributed dominantly to the building of soil organic N pool.