Advancing in planta Transformation and Gene Editing in Plants and Deciphering the Molecular Complexities of Somatic Cell Regeneration

Plants, being sessile, have a remarkable ability to self-repair and regenerate into whole new tissue, organ, or individual plants. Plant cells are generally highly pluripotent (ability to attend stem cell activity) and totipotent (ability to divide until it creates an entire fertile organism). Pluripotent cells are primarily associated with the cells found in the meristem. In natural conditions plants, regeneration response can be initiated at a single cell, tissue or whole-organ level. Each type of regeneration process involves complex reprogramming mechanisms that enable the plant to restore lost or damaged structures or continue normal growth. But, under artificial (in vitro) conditions, reprogramming can be achieved by phytohormonal treatments. In general, the regeneration process hinges on responses to growth hormones, tissue repair after wounding, or environmental cues. But importantly, these triggers collaborate with developmental regulators (DRs) or morphogenic regulators (MRs) to initiate distinct responses that facilitate differentiation, dedifferentiation, and regeneration. We exploit this phenomenon in plants to ectopically express wound induced developmental programs and morphogenic regulators to develop a robust and genotype flexible in planta transformation and regeneration pipeline. This will bypass lengthy tissue culture intermediates thereby efficiently regenerating transgenic and genome-edited plants. It is envisioned that development of this technology will advance our understanding of plant regeneration and current plant transformation technologies. On the other hand, Protoplast is an excellent model and serves as a resourceful and high-throughput system in functional plant biology. However, the ultimate challenge lies in protoplast regeneration, a significant obstacle that hinders its broader utility beyond in vitro assays. How a single somatic cell gains pluripotency remains elusive at the cellular and molecular level, hindering its utility in diverse plant species. Furthermore, the signals important for initiating cellular programming and gaining pluripotency in “regenerable” plants, such as tobacco (via direct organogenesis) or carrot (via somatic embryogenesis) are yet unknown. The mechanism of iPSC in animals and humans is well studied and supported by extensive data, but research on plant iPSC is significantly lagging. As per a recent review article in The Plant Cell [29] and Science [30], “how regeneration is achieved from a differentiated single cell” remains an important yet unsolved question in plant biology. Therefore, a systematic comparison and understanding (as proposed in this study) is necessary to understand these processes. With the development of robust single cell transcriptomics technology, it has been possible to understand molecular insights of development at single cell resolution. Therefore, using this technology, we attempted to uncover molecular complexities/mechanisms of somatic cell regeneration in plants. For this, we used protoplast from model organism, N. benthamiana and used several stages of protoplast regeneration for single cell transcriptomics. Chapter 1; Plant cells are totipotent, meaning they can regenerate into whole plant. Due to the sessile nature of the plant, they frequently encounter wounding. Wounding is the primary trigger to organ regeneration. However, the understanding of wounding signal triggering molecular programs leading to regenerative response is not very well understood. It has been observed that plant somatic cells dedifferentiate, proliferate, and acquire new fates to repair damaged tissues and forms callus that have regenerative potential. Along with the activation of a battery of molecular programs to reprogram and give cells a new fate, it also activates endogenous hormonal biosynthetic pathways. Understanding of wound induced callus formation and activation of other transcriptional programs involved in organ regeneration is evolving. WOUND INDUCED DEDIFFERENTIATION1 (WIND1) has been identified as a master-regulator of wound-induced cellular reprogramming in plants. Plants lacking functional WIND1 lacks in callus formation. More recently, it has been found that plant regeneration at the wounding site via Regeneration factor1 (REF1)-PEPR1/2 ORTHOLOG RECEPTOR-LIKE KINASE1 (PORK1) (REF1-PORK1)-mediated WIND1 signaling. It has also been well elucidated that wound-induced regeneration of plants is based on the transcriptional activation of ENHANCER OF SHOOT REGENERATION1 (ESR1). It has been characterized that WIND1 upon wounding peaks expression at the site of injury within one hour of wounding and it binds to wound-responsive cis-regulatory motif of the ESR1 and its expression picks at three hours suggesting that ESR1 expresses downstream of WIND1 after its binding. In this study, I have revalidated that the ectopic expression of WIND1 induces in vitro callus formation in Nicotiana benthamiana explants (internode and petioles). The area of the callus induced by the construct carrying WIND1 induces callus up to six-fold larger in area compared to the controls. We also further revalidated the activation of WIND1 binding to ESR1 promoter by measuring the expression of RUBY gene by driven by under ESR1 promoter. Our data suggests that the relative expression of RUBY gene 6-fold higher than the controls. Our results corroborate with the previous findings and therefore WIND1-ESR1 work together to form wound-induced callus as a prerequisite for organ regeneration in plants. Chapter 2, Development of transgenic and/or gene-edited plants largely depends on tedious, lengthy, and costly in vitro regeneration protocols. While plants have remarkable regeneration ability, not all species, genotype or even explants exhibit the same transformation and regeneration potential under in vitro conditions. Tackling this bottleneck, we developed a seamless and user-friendly system to induce transgenic and gene-edited de novo meristems via a synthetic cascade comprising wound-induced regeneration pathway, plant developmental regulators and gene-editing reagents. WOUND INDUCED DEDIFFERENTIATION 1 (WIND1) is used as transcriptional regulator to control the expression of various developmental regulators (DRs) through ENHANCER OF SHOOT REGENERATION 1 (ESR1). This cascade was strategically applied to the non-meristematic internode of N. benthamiana to develop de novo shoots with knock-out mutations of phytoene desaturase (PDS) gene. This synthetic toolkit was further applied to tomato with ~20% transgenesis efficiency. This methodology not only advances our understanding of plant regeneration but also offers a transformative approach to overcome barriers in plant biotechnology, potentially revolutionizing the generation of transgenic and gene-edited plants without reliance on conventional tissue-culture intermediates. Chapter 3, Protoplasts are the plant single cells that have the potential to regenerate cell wall, dedifferentiate, divide mitotically and proliferate to form callus, a mass of unlimited cell clones. These cell clones are the precursors for the regeneration of a complete fertile plant. Regeneration of complete plants from a differentiated single somatic cell is a very complex process and involves molecular reprogramming of these cells that triggers plant cells to reenter into cell cycle and divide. Regeneration of new organs at the wound site in plants is an outcome of this developmental plasticity of plant cells. Exogenous supplement of growth regulating hormones like auxin and cytokinin highly promotes regenerative responses in plants in vitro by activating developmental programs required for regeneration. Molecular mechanisms governing regeneration of new organs from the wound site in an existing tissue is increasingly well defined. However, regeneration from a single somatic cell, a unique developmental plasticity exhibited by plant cell and underlying molecular mechanisms remain largely undefined and unexplored. Regeneration of an entire plant from a single somatic plant cell is a very complex process and therefore requires a very powerful high throughput technology to uncover the genetic basis of somatic cell regeneration in plants. With the development of advanced molecular-genetic and computational technologies like single-cell RNA sequencing (ScRNA-seq), it is possible to track cells and the cell types that have regenerative potential thereby defining morphological and cellular changes by acquiring transcriptomic status coupled with high throughput imaging technologies. Here in this study, I attempted to decode genetic basis of somatic cell regeneration in plants by using single cell transcriptomic approach in N. benthamiana, a model organism with shorter lifespan and relatively easy to isolate somatic cells (protoplasts) with higher regenerative capacity. Seven different developmental stages during somatic cell regeneration were considered for nuclei isolation from tissues for single cell transcriptomics. It was envisioned that this study will uncover molecular markers and cell types required for plant regeneration at single-cell resolution. The molecular information uncovered in this study could provide an unprecedented view of tissue regeneration in plants that can be applied in other plant species recalcitrant to regeneration.