1,720,963 research outputs found
Advancing in planta Transformation and Gene Editing in Plants and Deciphering the Molecular Complexities of Somatic Cell Regeneration
No full textPlants, 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
Functional Characterization of Induced and Natural Variations in Sorghum Water Transport and Grain Quality
No full textAbstract
Sorghum (Sorghum bicolor), a resilient cereal crop from grass family, exhibits strong tolerance to abiotic stresses such as heat and drought, making it an excellent model for stress biology. However, the genetic mechanisms underlying these traits remain insufficiently understood. In this study, we characterize hs1, a heat-sensitive EMS-induced mutant that displays a pronounced leaf scorching phenotype under elevated field temperatures. Physiological analyses and anatomical observations reveal that this phenotype arises from defective protoxylem development at the leaf base, impairing water transport and reducing water use efficiency. Using Bulk Segregant Analysis (BSA), we identified hs1 as a gene encoding a myosin protein, which further confirmed by the complementation tests with an allelic mutant. This discovery highlights a novel link between cytoskeletal regulation, xylem development, and abiotic stress responses in sorghum. Ongoing work focuses on dissecting the gene’s regulatory network and exploring its potential for improving stress resilience in other crops like maize.
In addition to induced mutants, we also explored natural genetic variations at the GRANULE-BOUND STARCH SYNTHASE (GBSS) locus, which governs the waxy endosperm trait characterized by low amylose content and a glutinous starch profile. While waxy sorghum is valued for its enhanced starch digestibility in food, feed, and biofuel applications, conventional breeding has been limited by the need to phenotype mature grain and the dependence on PCR-based markers not optimized for high-throughput genotyping. To address this limitation, we identified and validated the competitive allelic PCR markers for the two most prevalent wx alleles and fully resolved a previously uncharacterized 5.6 kb Copia retrotransposon insertion. These resources enable accurate genotyping of Wx alleles via short-read sequencing and support early-generation selection. Functional markers were developed and validated across public and private breeding programs, enhancing the efficiency of waxy sorghum hybrid development.
Together, this work demonstrates the value of integrating induced and natural genetic diversity in sorghum to uncover gene function, elucidate developmental mechanisms, and develop scalable molecular tools that accelerate genomics-assisted breeding
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
koamabayili/VECTRON-author-checklist: VECTRON author checklist
No full textWe have done our best to complete the author checklist relating to the use of animals in the hut study. Note that the objective for the hut study was to evaluate the IRS treatment applications for residual efficacy against Anopheles mosquitoes, including the local An. coluzzii mosquito population. Cows were only used to attract mosquitoes into the huts and no tests were carried out directly on the cows. The author checklist is intended for use with studies where experiments are carried out on animals, which is why we have had such difficulty in completing this for the hut study, as many of the questions do not relate to how the cows were used
Author-wise bibliometric analysis based on entropy.
No full textAuthor-wise bibliometric analysis based on entropy.</p
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