Heidi F. Kaeppler

SUMMARY: Plant transformation is an important part of plant research and crop improvement. Transformation methods remain complex, labor intensive, and inefficient. PlantGENE is a community of scientists from academia, industry, non‐profit research institutes, and government organizations working to improve plant transformation. PlantGENE hosts virtual training, interactive webinars, and a website with career opportunities, directories, and more. The plant science community has shown great interest and support for PlantGENE.

SUMMARY: Genome‐wide association study (GWAS) with single nucleotide polymorphisms (SNPs) has been widely used to explore genetic controls of phenotypic traits. Alternatively, GWAS can use counts of substrings of length k from longer sequencing reads, k‐mers, as genotyping data. Using maize cob and kernel color traits, we demonstrated that k‐mer GWAS can effectively identify associated k‐mers. Co‐expression analysis of kernel color k‐mers and genes directly found k‐mers from known causal genes. Analyzing complex traits of kernel oil and leaf angle resulted in k‐mers from both known and candidate genes. A gene encoding a MADS transcription factor was functionally validated by showing that ectopic expression of the gene led to less upright leaves. Evolution analysis revealed most k‐mers positively correlated with kernel oil were strongly selected against in maize populations, while most k‐mers for upright leaf angle were positively selected. In addition, genomic prediction of kernel oil, leaf angle, and flowering time using k‐mer data resulted in a similarly high prediction accuracy to the standard SNP‐based method. Collectively, we showed k‐mer GWAS is a powerful approach for identifying trait‐associated genetic elements. Further, our results demonstrated the bridging role of k‐mers for data integration and functional gene discovery.

The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro‐propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype‐independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype‐flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype‐flexible morphogenic gene‐based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well‐established, work‐horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research.

Summary: The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation‐based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non‐responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.

Plant breeding relies on the presence of genetic variation, which is generated by a random process of mutagenesis that acts on existing gene pools. This variation is then recombined into new forms at frequencies impacted by the local euchromatin and heterochromatin environment. The result is a genetic lottery where plant breeders face increasingly low odds of generating a “winning” plant genotype. Genome editing tools enable targeted manipulation of the genome, providing a means to increase genetic variation and enhancing the chances for plant breeding success. Editing can be applied in a targeted way, where known genetic variation that improves performance can be directly brought into lines of interest through either deletion or insertion. This empowers approaches that are traditionally difficult such as novel domestication and introgression of wild accessions into a germplasm pool. Furthermore, broader editing-mediated approaches such as recombination enhancement and targeted random mutagenesis bring novel ways of variation creation to the plant breeding toolbox. Continued development and application of plant genome editing tools will be needed to aid in meeting critical global crop improvement needs.

The highly embryogenic and transformable maize inbred line A188 is an attractive model for analyzing maize gene function. Here we constructed a chromosome-level genome assembly of A188 using long reads and optical maps. Genome comparison of A188 with the reference line B73 identified pervasive structural variation, including a 1.8 Mb duplication on the Gametophyte factor1 locus for unilateral cross-incompatibility and six inversions of 0.7 Mb or greater. Increased copy number of the gene, carotenoid cleavage dioxygenase 1 ( ccd1 ) in A188 is associated with elevated expression during seed development. High ccd1 expression together with low expression of yellow endosperm 1 ( y1 ) condition reduced carotenoid accumulation, which accounts for the white seed phenotype of A188 that contrasts with the yellow seed of B73 that has high expression of y1 and low expression of the single-copy ccd1 . Further, transcriptome and epigenome analyses with the A188 reference genome revealed enhanced expression of defense pathways and altered DNA methylation patterns of embryonic callus.

The USDA’s Animal and Plant Health Inspection Service (APHIS) is one of three agencies that govern the importation, interstate movement, or environmental release of certain genetically engineered (GE) organisms. APHIS regulations are in 7 CFR part 340, originally issued in 1987 ( https://www.aphis.usda.gov/biotechnology/downloads/7_cfr_340.pdf ) and based on the premise that a GE crop could pose a plant pest risk. A major revision was proposed June 6, 2019 ( https://www.aphis.usda.gov/brs/fedregister/BRS_20190606.pdf ), with public comments accepted for 60 d. Members of the Society for In Vitro Biology (SIVB)—especially within the plant section—are impacted by the current rules and will be impacted by changes. Therefore, the public policy committee studied the changes and prepared a response that was reviewed by the board of directors and submitted to APHIS. APHIS received 6186 comments overall. In general, the SIVB welcomes the proposed revisions, which recognize established scientific guidelines and principles for plant pest risk assessment. In addition, they recognize the safety record accumulated over multiple decades of current GE mechanisms of action (MOA), so the proposed rules include an exemption for new events with established MOA’s in a previously reviewed crop. Further, the proposed rules focus on DNA function and impact rather than its source organism with regulatory review triggered by potential for increased plant pest risk. Most importantly, the proposed revisions codify a light regulatory approach for many applications of genome editing, because these applications result in plants that could otherwise have been developed through traditional breeding techniques or found in nature. If implemented appropriately in the final rule, we find these changes remove many current barriers, and thus are likely to stimulate university and business “startup” innovation. The SIVB recognizes the need for exemption for further model species and gene delivery organisms. Such concerns were adequately expressed by the submitted response. Remaining hurdles for innovative plant incorporated protectants are still problematic. Nevertheless, the SIVB applauds the efforts to update APHIS rules in a scientifically defensible manner. The detailed response is shared below.

Maize stover is an important source of crop residues and a promising sustainable energy source in the United States. Stalk is the main component of stover, representing about half of stover dry weight. Characterization of genetic determinants of stalk traits provide a foundation to optimize maize stover as a biofuel feedstock. We investigated maize natural genetic variation in genome-wide association studies (GWAS) to detect candidate genes associated with traits related to stalk biomass (stalk diameter and plant height) and stalk anatomy (rind thickness, vascular bundle density and area). Using a panel of 942 diverse inbred lines, 899,784 RNA-Seq derived single nucleotide polymorphism (SNP) markers were identified. Stalk traits were measured on 800 members of the panel in replicated field trials across years. GWAS revealed 16 candidate genes associated with four stalk traits. Most of the detected candidate genes were involved in fundamental cellular functions, such as regulation of gene expression and cell cycle progression. Two of the regulatory genes (Zmm22 and an ortholog of Fpa) that were associated with plant height were previously shown to be involved in regulating the vegetative to floral transition. The association of Zmm22 with plant height was confirmed using a transgenic approach. Transgenic lines with increased expression of Zmm22 showed a significant decrease in plant height as well as tassel branch number, indicating a pleiotropic effect of Zmm22. Substantial heritable variation was observed in the association panel for stalk traits, indicating a large potential for improving useful stalk traits in breeding programs. Genome-wide association analyses detected several candidate genes associated with multiple traits, suggesting common regulatory elements underlie various stalk traits. Results of this study provide insights into the genetic control of maize stalk anatomy and biomass.

Embryogenic and regenerable tissue cultures are widely utilized in plant transformation, clonal propagation, and biological research applications. Germplasm utilized in those applications are limited, however, due to genotype‐dependent culture response. The goal of this study was to identify genomic regions controlling embryogenic and regenerable tissue culture response in the globally important crop, maize ( Zea mays L.), toward the long‐term objective of developing approaches for genotype‐independent plant genetic engineering and clonal propagation systems. An inbred maize line, WCIC2, nearly‐isogenic to reference inbred B73, was developed by phenotypic selection and molecular marker analysis. WCIC2 has over 50x increase in tissue culture response relative to the recurrent parent, B73. This line was used to genetically fine‐map a region on chromosome 3 controlling embryogenic and regenerable tissue culture response to a 23.9 Mb region. WCIC2 and derivatives will be useful materials to enable maize research in a genetic background similar to B73, and our genetic mapping results will advance research to identify causal genes controlling somatic embryo formation and plant regeneration in maize.

The cell wall polymer lignin provides structural support and rigidity to plant cell walls, and therefore to the plant body. However, the recalcitrance associated with lignin impedes the extraction of polysaccharides from the cell wall to make plant-based biofuels and biomaterials. The cell wall digestibility can be improved by introducing labile ester bonds into the lignin backbone that can be easily broken under mild base treatment at room temperature. The FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) enzyme, which may be naturally found in many plants, uses feruloyl-CoA and monolignols to synthesize the ester-linked monolignol ferulate conjugates. A mutation in the first lignin-specific biosynthetic enzyme, CINNAMOYL-CoA REDUCTASE (CCR), results in an increase in the intracellular pool of feruloyl-CoA. Maize (Zea mays) has a native putative FMT enzyme, and its ccr mutants produce an increased pool of feruloyl-CoA that can be used for conversion to monolignol ferulate conjugates. The decreased lignin content and monomers did not, however, impact the plant growth or biomass. The increase in monolignol conjugates correlated with an improvement in the digestibility of maize stem rind tissue. Together, increased monolignol ferulates and improved digestibility in ccr1 mutant plants suggests that they may be superior biofuel crops.