Article

Physical, mechanical, and microstructural characterization of novel, 3D-printed, tunable, lab-grown plant materials generated from Zinnia elegans cell cultures

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

To date, wood has been viewed as an attractive commodity because of its low relative cost and widespread availability. However, supply is increasingly strained, and, in many ways, trees make a non-ideal feedstock—with slow, climate and seasonally dependent growth, low yields of high-value products, and susceptibility to pests and disease. Recent research offered an approach to generate plant-based materials in vitro without needing to harvest or process whole plants, thereby enabling: localized, high-density production, elimination of energy-intensive collection and hauling, reduced processing, and inherent climate resilience. This work reports the first physical, mechanical, and microstructural characterization of 3-D printed, lab-grown, and tunable plant materials generated with Zinnia elegans cell cultures using such methodology. The data show that the properties of the resulting plant materials vary significantly with adjustments to hormone levels present in growth medium. In addition, configuration of the culture environment via bioprinting and casting enables the production of net-shape materials in forms and scales that do not arise naturally in whole plants. Finally, new comparative data on cell development in response to hormone levels in culture medium demonstrates the repeatability of growth trends, clarifies the relationship between developmental pathways, and helps to elucidate the relationships between cellular-level culture characteristics and emergent material properties.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... On the other hand, in vitro systems have been proven capable of synchronously inducing not only TEs, but also phloem-like cells, which will enable more targeted questions to be asked about xylogenesis. Answering these targeted questions will enable us to unlock the full power of xylogenic differentiation, thus enabling us to apply this knowledge practically to produce biomaterial products, and alleviate some of the strain placed on natural forest ecosystems (Beckwith et al. 2022). ...
... In addition to improving our fundamental understanding of wood formation, TE systems have also been proposed as a tool for ex planta biomaterial production, with the first proof-of-concept studies being conducted recently (Beckwith et al. 2021). Applying the Z. elegans model, 3D-printable and tuneable plant materials are produced in culture, and offer the potential to increase targeted biomaterial yields while simultaneously reducing the waste and environmental implications associated with harvesting full live plants (Beckwith et al. 2022). Although still rather premature, we propose that this system could be adapted to operate in woody plant species, using callus rather than mesophyll cells, where various PGR combinations can stimulate the production of TEs with properties suited to any given biomedical or structural application. ...
Article
Full-text available
Tracheary elements (TEs), including vessels and tracheids, occur as a product of xylogenesis and are highly adapted for the transportation of water and solutes. Xylogenesis or wood formation encompasses various stages of cellular development, which requires stringent temporal and spatial regulation. To further complicate matters, TEs are polymorphous and associated with other complex tissues. These complexities have necessitated the development of in vitro culture systems that are capable of synchronously inducing TEs on demand. In this review, we cover the challenges associated with inducing TEs in vitro and how this has been overcome using mesophyll and callus culture systems in herbaceous plants, yielding transdifferentiation efficiencies of up to 76% and 90%, respectively. We postulate that when equipped with such information, a great opportunity exists to optimise these culture systems in commercially valuable woody genera that currently display lower efficiencies in the range of 15.8–65%. Although both the mesophyll and callus induction cultures have proven essential for uncovering the fundamental processes associated with secondary growth, the mesophyll-based systems have recently become much less prominent (2.8x) in the literature compared to the callus-based systems. This is largely due to ease of application of the callus system to other plant species, paving the way for applications ranging from fundamental research in economically valuable woody genera to the 3D-printing of biomaterial products in vitro.
... Likewise, plant cells have been bioprinted into wood analogue to explore a solution to wood shortage. 12 In fact, these developments encompass a broad spectrum of applications, including bioelectronics, 13 soft robotics, 14 space exploration, 15 plant biology, 16 and sustainability. 17 All the above have changed our perspective and understanding of bioprinting fundamentally, and we see bioprinting becoming more and more separated from tissue engineering in terms of goals and capabilities. ...
Article
Full-text available
Our understanding of bioprinting originated from tissue engineering and 3D printing. Over the last two decades, 3D printing has been serving tissue engineering to fulfill its goal of solving the worldwide challenge of human organ shortage. Fundamental research and translation to clinical settings are currently the mainstream and have led to the emergence of translational bioprinting. However, as bioprinting evolves with more refined capabilities such as spatial and temporal controls of multiple biological materials at the microscale, perhaps it is time to reflect on a reciprocal approach, i.e., tissue engineering, to serve 3D printing with the hope to address problems more than or other than organ shortage. Recent examples of cultivated meat, digital bioprinting, bioelectronics, and space exploration may have just revealed the tip of the iceberg, though some still overlap with the goal of tissue engineering. Whenever a new material is introduced to the realm of 3D printing and becomes printable, it always leads to versatile or even unforeseen applications. This has happened to metals, ceramics, composite, and electronics. Although it has been known for a long time that biological cells are 3D-printable, the application of such concept has been always perceived from the angle of tissue engineering with human organ shortage as the sole motivation. Therefore, in this review based on existing evidence, we offer an innovative perspective of bioprinting, termed “transformative bioprinting,” to emphasize the role of an enabling tool of bioprinting and its alternative motivations.
... Recently, the researchers reported the development of tunable, lab-grown plant materials generated from a Zinnia elegans cell culture, 142 which respond to various concentrations of hormones in the growth medium. 143 The 3DbioP and casting of this bioink resulted in net-shaped structures with varying hardnesses at scales and forms that do not occur naturally in plants. The PSCs have found widespread application in the cosmetic industry due to their ability to provide the tissues of plant origin (TPOs) with antioxidant, antibacterial, and antifungal properties. ...
Article
Full-text available
The scope of three-dimensional printing is expanding rapidly, with innovative approaches resulting in the evolution of state-of-the-art 3D bioprinting (3DbioP) techniques for solving issues in bioengineering and biopharmaceutical research. The methods and tools in 3DbioP emphasize the extrusion process, bioink formulation, and stability of the bioprinted scaffold. Thus, 3DbioP technology augments 3DP in the biological world by providing technical support to regenerative therapy, drug delivery, bioengineering of prosthetics, and drug kinetics research. Besides the above, drug delivery and dosage control have been achieved using 3D bioprinted microcarriers and capsules. Developing a stable, biocompatible, and versatile bioink is a primary requisite in biofabrication. The 3DbioP research is breaking the technical barriers at a breakneck speed. Numerous techniques and biomaterial advancements have helped to overcome current 3DbioP issues related to printability, stability, and bioink formulation. Therefore, this Review aims to provide an insight into the technical challenges of bioprinting, novel biomaterials for bioink formulation, and recently developed 3D bioprinting methods driving future applications in biofabrication research.
... Cell culture techniques can also be applied to plant cells to grow plant-based alternatives such as artificial wood without the need for cutting down mature trees. Researchers at Massachusetts Institute of Technology (MIT) successfully cultured Zinnia elegans cell, present in a flower, and have since studied the potential for growing wood-like materials with tenable mechanical properties (Beckwith, Borenstein and Velásquez-García, 2022). The culture that the cells were grown within is a sugar-based solution pointing to a demand for either a waste stream compatible with this particular species or the need for agricultural produce that places pressures on the environment. ...
Book
https://www.routledge.com/Designers-Guide-to-Lab-Practice/Crawford/p/book/9781032426846 This book explores the growing field of bio-design through interdisciplinary creative practice. The volume illustrates a range of experimental working techniques while offering a foundational understanding of lab practice principles. The book highlights the myriad of opportunities presented by microorganisms that have reshaped the planet and made it habitable. The book provides an account of the creation of living materials from the point of view of an architectural design practitioner. The transition from traditional design practice to laboratory investigation is captured, highlighting strategies of creating partnerships across a range of fields. The book demonstrates laboratory methods and ways of investigating the development of living materials and celebrates the growing body of practitioners, scientists, activists and anthropologists who are reimagining new strategies for addressing contemporary environmental challenges. Designer's Guide to Lab Practice looks at ways in which integrating living components with needs of their own would not only help offset the environmental impact that we have on our planet but could also create a closer relationship with nature. It is a working manual as well as a guide to emerging practitioners seeking to transition into a field that is yet to be defined and that offers the promise of a new era of human habitat making as a direct response to the looming ecological crisis.
... The MIT spinoff Foray Biosciences 545 is developing a platform to produce wood, cellulose pulp, and wood-derived chemicals from plant cell cultures. 546,547 In contrast with meat cultivation, equivalent tissue culture technologies in plant sciences have long been overlooked and underdeveloped. Hopefully, with the recent technological advances in plant synthetic biology, more and more startups such as Foray will join the effort to create cultured plant materials and drive the cost down to reach price parity. ...
Article
Recent advances in synthetic biology and materials science have given rise to a new form of materials, namely engineered living materials (ELMs), which are composed of living matter or cell communities embedded in self-regenerating matrices of their own or artificial scaffolds. Like natural materials such as bone, wood, and skin, ELMs, which possess the functional capabilities of living organisms, can grow, self-organize, and self-repair when needed. They also spontaneously perform programmed biological functions upon sensing external cues. Currently, ELMs show promise for green energy production, bioremediation, disease treatment, and fabricating advanced smart materials. This review first introduces the dynamic features of natural living systems and their potential for developing novel materials. We then summarize the recent research progress on living materials and emerging design strategies from both synthetic biology and materials science perspectives. Finally, we discuss the positive impacts of living materials on promoting sustainability and key future research directions.
Article
Recent developments in factory-grown foods suggest that factory-grown wood (FGW) may be on the horizon. In fact, recent work at Massachusetts Institute of Technology introduces tunable plant-based materials, an early indicator of what may evolve into a new source of raw material for forest sector companies, and others. Industry and academia would be wise to monitor developments in this field as they may present significant opportunities and/or adjustments for both. We explore the state-of-the-art in this budding area of science and contemplate implications of successfully growing wood or other lignocellulosic materials in factories. Given a changing climate and focus on carbon emissions, the pressure to drastically reduce CO2 production will continue climb. Could reduction of their footprint via FGW be an important part of this equation for forest sector companies, going beyond the need to “make every tree count”? In other words, might FGW present an environmental and climate protection breakthrough? Or might it simply trade forest-based environmental impacts for others? What other consequences does FGW promise for companies? And, what might it mean for wood science programs, critical suppliers of research & development and skilled employees for the industry? We explore each of these questions and contemplate potential actions and outcomes.
Article
Full-text available
Current systems for plant-based biomaterial production are inefficient and place unsustainable demands on environmental resources. This work proposes a novel solution to these shortcomings based on selective cultivation of tunable plant tissues using scalable, land-free techniques unconstrained by seasonality, climate, or local resource availability. By limiting biomass cultivation to only desirable plant tissues, ex planta farming promises to improve yields while reducing plant waste and competition for arable land. Employing a Zinnia elegans model system, this work provides the first proof-of-concept demonstration of isolated, tissue-like plant material production in vitro by way of gel-mediated cell culture. Parameters governing cell development and morphology including hormone concentrations, medium pH, and initial cell density are optimized and implemented to demonstrate the tunability of cultured biomaterials at cellular and macroscopic scales. Targeted deposition of cell-doped, nutrient-rich gel scaffolds via casting and 3D bioprinting enable biomaterial growth in near-final form, reducing downstream processing requirements. These investigations demonstrate the implementation of plant cell culture in a new application space, propose novel methods for quantification and evaluation of cell development, and characterize morphological developments in response to critical culture parameters—illustrating the feasibility and potential of the proposed techniques.
Article
Full-text available
In this paper we afford a quantitative analysis of the sustainability of current world population growth in relation to the parallel deforestation process adopting a statistical point of view. We consider a simplified model based on a stochastic growth process driven by a continuous time random walk, which depicts the technological evolution of human kind, in conjunction with a deterministic generalised logistic model for humans-forest interaction and we evaluate the probability of avoiding the self-destruction of our civilisation. Based on the current resource consumption rates and best estimate of technological rate growth our study shows that we have very low probability, less than 10% in most optimistic estimate, to survive without facing a catastrophic collapse.
Article
Full-text available
Plant cell walls constitute the extracellular matrix surrounding plant cells and are composed mainly of polysaccharides. The chemical makeup of the primary plant cell wall, and specifically, the abundance, localization, and arrangement of the constituting polysaccharides are intimately linked with growth, morphogenesis, and differentiation in plant cells. Visualization of the cell wall components is, therefore, a crucial tool in plant cell developmental studies. In this technical update, we present protocols for fluorescence visualization of cellulose and pectin in selected plant tissues and illustrate examples of some of the available labels that hold promise for live imaging of plant cell wall expansion and morphogenesis.
Article
Full-text available
Systematic analysis of the extrusion process in 3D bioprinting is mandatory for process optimization concerning production speed, shape fidelity of the 3D construct and cell viability. In this study, we applied numerical and analytical modelling to describe the fluid flow inside the printing head based on a Herschel-Bulkley model. The presented analytical calculation method nicely reproduces the results of Computational Fluid Dynamics (CFD) simulation concerning pressure drop over the printing head and maximal shear parameters at the outlet. An approach with dimensionless flow parameter enables the user to adapt rheological characteristics of a bioink, the printing pressure and needle diameter with regard to processing time, shear sensitivity of the integrated cells, shape fidelity and strand dimension. Bioinks consist of a blend of polymers and cells, which lead to a complex fluid behavior. In the present study, a bioink containing alginate, methylcellulose and agarose (AMA) was used as experimental model to compare the calculated with the experimental pressure gradient. With cultures of an immortalized human mesenchymal stem cell line and plant cells (basil) it was tested how cells influence the flow and how mechanical forces inside the printing needle affect cell viability. Influences on both sides increased with cell (aggregation) size as well as a less spherical shape. This study contributes to a systematic description of the extrusion-based bioprinting process and introduces a general strategy for process design, transferable to other bioinks.
Article
Full-text available
Background Cultured meat forms part of the emerging field of cellular agriculture. Still an early stage field it seeks to deliver products traditionally made through livestock rearing in novel forms that require no, or significantly reduced, animal involvement. Key examples include cultured meat, milk, egg white and leather. Here, we focus upon cultured meat and its technical, socio-political and regulatory challenges and opportunities. Scope and approach The paper reports the thinking of an interdisciplinary team, all of whom have been active in the field for a number of years. It draws heavily upon the published literature, as well as our own professional experience. This includes ongoing laboratory work to produce cultured meat and over seventy interviews with experts in the area conducted in the social science work. Key findings and conclusions Cultured meat is a promising, but early stage, technology with key technical challenges including cell source, culture media, mimicking the in-vivo myogenesis environment, animal-derived and synthetic materials, and bioprocessing for commercial-scale production. Analysis of the social context has too readily been reduced to ethics and consumer acceptance, and whilst these are key issues, the importance of the political and institutional forms a cultured meat industry might take must also be recognised, and how ambiguities shape any emergent regulatory system.
Article
Full-text available
Many plant tissues fluoresce due to the natural fluorophores present in cell walls or within the cell protoplast or lumen. While lignin and chlorophyll are well-known fluorophores, other components are less well characterized. Confocal fluorescence microscopy of fresh or fixed vibratome-cut sections of radiata pine needles revealed the presence of suberin, lignin, ferulate, and flavonoids associated with cell walls as well as several different extractive components and chlorophyll within tissues. Comparison of needles in different physiological states demonstrated the loss of chlorophyll in both chlorotic and necrotic needles. Necrotic needles showed a dramatic change in the fluorescence of extractives within mesophyll cells from ultraviolet (UV) excited weak blue fluorescence to blue excited strong green fluorescence associated with tissue browning. Comparisons were made among fluorophores in terms of optimal excitation, relative brightness compared to lignin, and the effect of pH of mounting medium. Fluorophores in cell walls and extractives in lumens were associated with blue or green emission, compared to the red emission of chlorophyll. Autofluorescence is, therefore, a useful method for comparing the histology of healthy and diseased needles without the need for multiple staining techniques, potentially aiding visual screening of host resistance and disease progression in needle tissue.
Article
Full-text available
Plant cell cultures produce active agents for pharmaceuticals, food and cosmetics. However, up to now process control for plant cell suspension cultures is challenging. A positive impact of cell immobilization, such as encapsulation in hydrogel beads, on secondary metabolites production has been reported for several plant species. The aim of this work was to develop a method for bioprinting of plant cells in order to allow fabrication of free-formed three-dimensional matrices with defined internal pore architecture for in depth characterization of immobilization conditions, cell agglomeration and interactions. By using extrusion-based 3D plotting of a basil cell-laden hydrogel blend consisting of alginate, agarose and methylcellulose (alg/aga/mc), we could demonstrate that bioprinting is applicable to plant cells. The majority of the cells survived plotting and crosslinking and the embedded cells showed high viability and metabolic activity during the investigated cultivation period of 20 days. Beside its compatibility with the plant cells, the novel alg/aga/mc blend allowed fabrication of defined 3D constructs with open macropores both in vertical and horizontal direction which were stable under culture conditions for several weeks. Thus, Green Bioprinting, an additive manufacturing technology processing live cells from the plant kingdom, is a promising new immobilization tool for plant cells that enables the development of new bioprocesses for secondary metabolites production as well as monitoring methods.
Article
Full-text available
Gellan gum (GG) is one of the natural hydrogels showing potential for tissue engineering. In this study, we investigate GG for wound dressing and cartilage applications through 3D printing which allows for the creation of complex structures and scaffolds with different porosities. Degradation of two different GG scaffold designs and one solid sample were performed using both simulated body fluid and phosphate buffered saline. It was found that the scaffolds with a higher surface area to mass ratio have a higher degradation rate, and that the compressive modulus and strength increase after degradation in simulated body fluid.
Article
Full-text available
Bioprinting has emerged as a novel technological approach with the potential to address unsolved questions in the field of tissue engineering. We have recently shown that Laser Assisted Bioprinting (LAB), due to its unprecedented cell printing resolution and precision, is an attractive tool for the in situ printing of a bone substitute. Here, we show that LAB can be used for the in situ printing of mesenchymal stromal cells, associated with collagen and nano-hydroxyapatite, in order to favor bone regeneration, in a calvaria defect model in mice. Also, by testing different cell printing geometries, we show that different cellular arrangements impact on bone tissue regeneration. This work opens new avenues on the development of novel strategies, using in situ bioprinting, for the building of tissues, from the ground up.
Article
Full-text available
Treating a myocardial infarction (MI), the most frequent cause of death worldwide, remains one of the most exciting medical challenges in the 21st century. Cardiac tissue engineering, a novel emerging treatment, involves the use of therapeutic cells supported by a scaffold for regenerating the infarcted area. It is essential to select the appropriate scaffold material; the ideal one should provide a suitable cellular microenvironment, mimic the native myocardium, and allow mechanical and electrical coupling with host tissues. Among available scaffold materials, natural scaffolds are preferable for achieving these purposes because they possess myocardial extracellular matrix properties and structures. Here, we review several natural scaffolds for applications in MI management, with a focus on pre-clinical studies and clinical trials performed to date. We also evaluate scaffolds combined with different cell types and proteins for their ability to promote improved heart function, contractility and neovascularization, and attenuate adverse ventricular remodeling. Although further refinement is necessary in the coming years, promising results indicate that natural scaffolds may be a valuable translational therapeutic option with clinical impact in MI repair.
Article
Full-text available
The global extent and distribution of forest trees is central to our understanding of the terrestrial biosphere. We provide the first spatially continuous map of forest tree density at a global scale. This map reveals that the global number of trees is approximately 3.04 trillion, an order of magnitude higher than the previous estimate. Of these trees, approximately 1.39 trillion exist in tropical and subtropical forests, with 0.74 trillion in boreal regions and 0.61 trillion in temperate regions. Biome-level trends in tree density demonstrate the importance of climate and topography in controlling local tree densities at finer scales, as well as the overwhelming effect of humans across most of the world. Based on our projected tree densities, we estimate that over 15 billion trees are cut down each year, and the global number of trees has fallen by approximately 46% since the start of human civilization.
Article
Full-text available
Endosperm transfer cells in maize have extensive cell wall ingrowths that play a key role in kernel development. Although the incorporation of lignin would support this process, its presence in these structures has not been reported in previous studies. We used potassium permanganate staining combined with transmission electron microscopy – energy dispersive X-ray spectrometry as well as acriflavine staining combined with confocal laser scanning microscopy to determine whether the most basal endosperm transfer cells (MBETCs) contain lignified cell walls, using starchy endosperm cells for comparison. We investigated the lignin content of ultrathin sections of MBETCs treated with hydrogen peroxide. The lignin content of transfer and starchy cell walls was also determined by the acetyl bromide method. Finally, the relationship between cell wall lignification and MBETC growth/flange ingrowth orientation was evaluated. MBETC walls and ingrowths contained lignin throughout the period of cell growth we monitored. The same was true of the starchy cells, but those underwent an even more extensive growth period than the transfer cells. Both the reticulate and flange ingrowths were also lignified early in development. The significance of the lignification of maize endosperm cell walls is discussed in terms of its impact on cell growth and flange ingrowth orientation.
Article
Full-text available
The development of second-generation biofuels--those that do not rely on grain crops as inputs--will require a diverse set of feedstocks that can be grown sustainably and processed cost-effectively. Here we review the outlook and challenges for meeting hoped-for production targets for such biofuels in the United States.
Article
Full-text available
TREES ARE MAJOR COMPONENTS OF THE BIOSPHERE, AND THEIR WOOD HAS A MAJOR ROLE AS A SUSTAINABLE AND RENEWABLE ECOMATERIAL Wood is the most important natural and endlessly renewable source of energy and therefore has a major future role as an environmentally cost-effective alternative to burning fossils fuels. The major role of wood is not only the provision of energy but also the provision of energy-sufficient material for our buildings and many other products. In addition, developing wood cells represent one of the most important sinks for excess atmospheric CO2 ,t hereby reducing one of the major contributors to global warming.
Article
Full-text available
Hybrid aspen (Populus tremula x tremuloides) cell cultures were grown for 7, 14 and 21 days. The cell cultures formed primary cell walls but no secondary cell wall according to carbohydrate analysis and microscopic characterization. The primary walls were lignified, increasingly with age, according to Klason lignin analysis. Presence of lignin in the primary walls, with a higher content in 21-day old cells than in 7-day old cells, was further supported by phloroglucinol/HCl reagent test and confocal microscopy after both immunolocalization and staining with acriflavin. Both laccase and peroxidase activity were found in the cultures and the activity increased during lignin formation. The lignin from the cell culture material was compared to lignin from mature aspen wood, where most of the lignin originates in the secondary cell wall, and which served as our secondary cell wall control. Lignin from the cell walls was isolated and characterized by thioacidolysis followed by gas chromatography and mass spectrometry. The lignin in the cell cultures differed from lignin of mature aspen wood in that it consisted exclusively of guaiacyl units, and had a more condensed structure. Five lignin structures were identified by mass spectrometry in the cell suspension cultures. The results indicate that the hybrid aspen cell culture used in this investigation may be a convenient experimental system for studies of primary cell wall lignin.
Article
3D food printing allows creation of foods by depositing food material according to computer aided designs. However, the number of printable materials for food is still low which limits the possibilities of creating specific structures and textures. A novel approach is tested of using food printing materials incorporating plant cells in order to print foods that resemble plant tissues in various ways. A 3D printing method was developed based on the extrusion of bio-inks composed of a low-methoxylated pectin gel and embedded lettuce leaf cells. Bovine serum albumin was added in order to increase the air fraction in the printed gel matrix. Objects containing up to 5 × 10⁶ cells/mL were successfully 3D printed. The mechanical strength increased by the pectin concentration and decreased with the increase of air fraction and concentration of encapsulated cells. The viability of the encapsulated plant cells depended on the pectin concentration and varied from 50 to 60%.
Article
Recent advances applying mammalian tissue engineering to in vitro plant cell culture have successfully cultured single plant cells in a 3D microstructure, leading to the discovery of plant cell behaviours that were previously not envisaged. Animal and plant cells share a number of properties that rely on a hierarchical microenvironment for creating complex tissues. Both mammalian tissue engineering and 3D plant culture employ tailored scaffolds that alter a cell's behaviour from the initial culture used for seeding. For humans, these techniques are revolutionizing healthcare strategies, particularly in regenerative medicine and cancer studies. For plants, we predict applications both in fundamental research to study morphogenesis and for synthetic biology in the agri-biotech sector.
Article
The Zinnia elegans mesophyll cell culture is a useful system for xylogenesis studies. The system is associated with highly synchronous tracheary element (TE) differentiation, making it more suitable for molecular studies requiring larger amounts of molecular isolates, such as mRNA and proteins and for studying cellulose synthesis. There is, however, the problem of non-uniformity and significant variations in the yields of TEs (%TE). One possible cause for this variability in the %TE could be the lack of a standardized experimental protocol in various research laboratories for establishing the Zinnia culture. Mesophyll cells isolated from the first true leaves of Z. elegans var Envy seedlings of approximately 14 days old were cultured in vitro and differentiated into TEs. The xylogenic culture medium was supplied with 1mg/l each of benzylaminopurine (BA) and alpha-naphthalene acetic acid (NAA). Application of this improved culture method resulted in stable and reproducible amounts of TE as high as 76% in the Zinnia culture. The increase was mainly due to conditioning of the mesophyll cell culture and adjustments of the phytohormonal balance in the cultures. Also, certain biochemical and cytological methods have been shown to reliably monitor progress of TE differentiation. We conclude that, with the adoption of current improvement in the xylogenic Z. elegans culture, higher amounts of tracheary elements can be produced. This successful outcome raises the potential of the Zinnia system as a suitable model for cellulose and xylogenesis research.
Article
In vitro models of normal mammary epithelium have correlated increased extracellular matrix (ECM) stiffness with malignant phenotypes. However, the role of increased stiffness in this transformation remains unclear because of difficulties in controlling ECM stiffness, composition and architecture independently. Here we demonstrate that interpenetrating networks of reconstituted basement membrane matrix and alginate can be used to modulate ECM stiffness independently of composition and architecture. We find that, in normal mammary epithelial cells, increasing ECM stiffness alone induces malignant phenotypes but that the effect is completely abrogated when accompanied by an increase in basement-membrane ligands. We also find that the combination of stiffness and composition is sensed through β4 integrin, Rac1, and the PI3K pathway, and suggest a mechanism in which an increase in ECM stiffness, without an increase in basement membrane ligands, prevents normal α6β4 integrin clustering into hemidesmosomes.
Article
Wood is a complex cellular structure with different properties in the radial and tangential direction. Many researchers have measured dynamic properties in the longitudinal direction and a few in the radial direction but very little data can be found in the literature on dynamic mechanical properties in the tangential direction. The purpose of the work presented in this paper was to investigate the dynamic mechanical behaviour in the radial and tangential directions of wood (Pinus sylvestris). Testing was done in tension at 1 Hz with a Dynamic Mechanical Thermal Analyser. Properties in radial and tangential direction were different. The radial direction showed a higher elastic modulus and lower loss factor levels at temperatures between –120C and 80C. The tangential direction had on average a higher peak temperature than the radial direction for a loss factor peak around –80C. It is the opposite of synthetic composites where the stiffer direction has a higher peak temperature. A loss factor peak at around 0C was seen, most significantly in the tangential direction. This peak has scarcely been reported in the literature before. The distance between annual rings did not significantly affect the dynamic behaviour in the tangential direction.
Article
Cellulose is generally found in the context of complex plant cell wall materials and mostly in association with other glycans. Cellulose-directed carbohydrate-binding modules (CBMs) can be readily adapted to a range of methods for the in situ imaging of cellulose structures within plant cell walls or other cellulose-based materials. Protocols for the preparation and selection of plant materials, their fixation and processing for preparation of sections for CBM labeling, and fluorescence imaging procedures are described. Approaches to direct methods in which CBMs are directly coupled to fluorophores and indirect methods in which staged incubations with secondary reagents are used for the fluorescence imaging of CBM binding to materials are discussed and presented.
Article
The past three decades have seen the emergence of an endeavor called tissue engineering and regenerative medicine in which scientists, engineers, and physicians apply tools from a variety of fields to construct biological substitutes that can mimic tissues for diagnostic and research purposes and can replace (or help regenerate) diseased and injured tissues. A significant portion of this effort has been translated to actual therapies, especially in the areas of skin replacement and, to a lesser extent, cartilage repair. A good amount of thoughtful work has also yielded prototypes of other tissue substitutes such as nerve conduits, blood vessels, liver, and even heart. Forward movement to clinical product, however, has been slow. Another offshoot of these efforts has been the incorporation of some new exciting technologies (e.g., microfabrication, 3D printing) that may enable future breakthroughs. In this review we highlight the modest beginnings of the field and then describe three application examples that are in various stages of development, ranging from relatively mature (skin) to ongoing proof-of-concept (cartilage) to early stage (liver). We then discuss some of the major issues that limit the development of complex tissues, some of which are fundamentals-based, whereas others stem from the needs of the end users.
Article
Goldsworthy, A. and Rathore, K. S. 1985. The electrical control of growth in plant tissue cultures: The polar transport of auxin.—J. exp. Bot. 36: 1134–1141. The reasons for a many-fold stimulation of shoot formation and a 60–70% stimulation of growth in tobacco callus caused by passing a very weak electric current (1 or 2 μA) between the callus and the culture medium have been investigated. The stimulation of callus growth occurred only when the callus was made negative to the medium and then only when IAA was added. It was abolished, even in the presence of IAA, by the addition of TIBA which is an inhibitor of polar auxin transport, and also when the IAA was substituted by either IAN or the synthetic auxin 2,4-D, neither of which show significant polar transport. This suggests that the electrical treatment may have aligned the physiological polarities of the callus cells so as to promote the polar transport of IAA into the tissue when the callus was negative to the medium. If so, the enhanced shoot formation may have been due to the parallel orientation of the growth axes of individual cells so as to make the production of organforming meristems more likely. The mechanism of the effect and its relationship to the natural forces controlling differentiation is discussed.
Article
Single cells were isolated mechanically from the mesophyll of adult plants and of seedlings of Zinnia elegans L. cv. Canary bird. When single cells isolated from the first leaves of seedlings were cultured in a liquid medium in the dark with rotation, they differentiated to tracheary elements with a reasonable degree of synchrony in the 24-hour period between days 2 and 3 after culture. The proportion of tracheary elements as a percentage of total cells reached nearly 30% 3 days after culture. Factors favoring cytodifferentiation were certain optimum levels of both alpha-naphthalene-acetic acid (0.1 milligram per liter) and benzyladenine (1 milligram per liter), a low concentration of ammonium chloride (0 to 1 millimolar), and an initial cell population density in the range 0.4 to 3.8 x 10(5) cells/ml. It was possible to follow analytically the sequence of cytodifferentiation in individual cells in this system.
Biotechnology in agriculture and forestry. 17. high-tech and micropropagation I, The Indian Journal of Genetics and Plant Breeding