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.

Efficient Targeted Gene Insertion in Maize using Agrobacterium-Mediated Delivery

CRISPR-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 knock-outs in multiple crops, precise nucleotide replacement and especially gene insertion into a pre-defined genomic location remain highly challenging. Here we report an efficient, heritable, selectable marker-free site-specific gene insertion in maize using Agrobacterium-mediated delivery. Advancements in maize transformation and new vector design enabled targeted insertion with frequencies as high as 8–10%. Importantly, these advancements allowed not only an 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.

An efficient gene excision system in maize

ABSTRACTUse 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. In addition, altering the ectopic 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 53.3%-68.4%; however, the transformation frequency was lower (12.9%-49.9%). 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 (3.6%-14%), 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.

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.”

Expression of Oryza sativa galactinol synthase gene in maize (Zea may L.)

Galactinol synthase (GolS) is a key biological catalyst for the synthesis of the raffinose oligosaccharides (RFOs) which play important roles in abiotic stress adaptation of plants, especially drought tolerance. GolS gene has been isolated on a variety of plants in order to create material resources for generating transgenic plants resistant to adverse environmental factors. In our previous research, we have isolated a GolS gene from drought stress cDNA library of Oryza sativa L. Moctuyen (named OsGolS). In this study, the expression vector pCAM-Rd/OsGolS carrying the isolated OsGolS gene under the control of stress-inducible Rd29A promoter was constructed and introduced into Agrobacterium tumefaciens LBA4404, which was used for maize transformation. PCR and Real-time PCR assay indicated that transgene was integrated in the genome of the regenerated Zea mays plants. Reverse transcription-PCR showed that the OsGolS was transcribed into mRNA in Zea mays and was highly expressed. These results provide a basis for the study of the function of OsGolS in drought responses and for the development of drought stress tolerant crops.