Methods for The Generation of Genetically Engineered Dicotyledonous Plants Using Developmental Regulators

Abstract

There is an expanding need to engineer plant lines with genetic modifications that will help advance both basic and applied plant research. A growing human population, loss of arable land, and global fluctuations in biotic and abiotic stresses (such as drought and pest activity) have created an increasing demand for novel agricultural germplasm. In recent years, basic plant biological research has made great headway in generating large volumes of data that may hold the keys to elucidate novel gene regulatory networks and gene functions to meet these pervasive demands; however, translating these insights into plant lines for validation and application has been hampered by bottlenecks in the creation of necessary germplasm.Creating novel germplasm is typically performed by delivering plant gene editing tools, such as Cas9 and single guide RNAs, to explants in sterile culture. Edited cells are then induced to differentiate into whole plants by exposure to various hormones regimes in tissue culture. These methods are often arduous and inefficient, as they are technically challenging, expensive, time consuming, cause confounding unintended changes to the genome/epigenome, and work in limited plant species and genotypes. Broadly, these bottlenecks have limited the capacity of basic researchers to decipher molecular function of biological processes and have thus throttled the translation of plant biological knowledge to innovations in the field. Herein, we present novel methods to generate gene-edited dicotyledonous plants through de novo meristem induction. These Agrobacterium-based methods involve the delivery of genes encoding developmental regulators (DRs), factors involved in development and patterning of the meristem, to induce de novo meristems. DRs and gene-editing reagents are delivered to somatic cells on whole plants, and developmental pathways are elicited to produce meristems with coincident DNA modifications without the need for tissue culture. In Chapter 1 of this thesis, I describe how all plant organs are derived from the indeterminate growth of the meristem, a highly ordered and regulated cluster of plant stem cells. I further describe the prevailing methods for editing existing meristems or creating them de novo, as well as inherent challenges and drawbacks. I further present DRs as the driving force in meristem formation and regulation and describe how they have previously been utilized to enhance transformation and regeneration efficiencies. Lastly, I present theories on how these regulatory genes may be co-opted in non-sterile plants to enable the efficient generation of novel plant germplasm. In Chapter 2, I present published proof-of-concept experiments that employ two methods utilizing DRs to generate de novo meristems in planta in dicot species. The first method applies the expression of DRs in a high-throughput variant of a previously published in vitro Agrobacterium co-culture protocol, AGROBEST (Agrobacterium-mediated Enhanced Seedling Transformation).1 This modified co-culture method utilizing DRs, termed Fast Agrobacterium Treated Co-Culture (Fast-TrACC), was used to quickly test reagent expression and DR activities in germinating seedlings, as well as to create novel germplasm. The second method, which I developed, utilized DRs to generate de novo meristems directly on non-sterile, soil grown plants, here called the Direct Delivery (DD) method. In DD experiments, I demonstrated that DRs may not only induce de novo meristems on non-sterile, soil grown plants, but that these modified meristems may at some frequency transmit gene edits to the next generation, independent of the DR transgene. This initial body of work was performed in Nicotiana benthamiana and additionally shows promising translation to Vitus vinifera (grape) and Solanum tuberosum (potato). In Chapter 3, I present comprehensive protocols for the application of the DD and Fast-TrACC methods. I offer novel protocols for the molecular assembly of DR vectors into binary T-DNA vectors and provide descriptions of additional vectors we have manufactured and disseminated to the broader plant science community. We additionally provide detailed protocols describing methodology and best practices in applying the DD and Fast-TrACC methods inferred from published and unpublished experiments to enable researchers to translate these learnings to other plant species of interest. The final chapter, Chapter 4, describes the deeper theory and rationale driving our approaches for de novo meristem induction, including vector features and novel procedures utilized in the DD method. I then reflect on functional outcomes of de novo meristem formation and how this may affect downstream tissues. I close this chapter by exploring potential avenues for future development and optimization of the DD method, as well as other DR-based meristem induction methods. Methods utilizing DRs to enhance plant genome engineering have begun appearing at an accelerated pace, and these advances have opened exciting avenues for quickly generating novel plant germplasm of interest. Using our DD method, we repeatedly derived fixed, gene edited tissues within the first generation without detectable DR transgenes. Further, these edits were heritably transmitted to the next generation. As a result, DD offers an alternative approach to generate novel plant germplasm that is less expensive, less technically challenging, and often takes less time than standard tissue culture approaches. Additionally, the DD methodology provides for accelerated genome engineering by sidestepping the use of plant tissue culture and sterile plant handling, which significantly decreases the cost and technical expertise necessary. This method has been demonstrated in the model plant species, Nicotiana benthamiana, as well as grape and potato and has the potential to expand further to other species. Together, the DD method provides promise to overcome a bottleneck in plant gene editing and to advance basic research and applied plant biotechnology.