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Delivery of tobacco mosaic virus RNA into plant protoplasts mediated by reverse-phase evaporation vesicles (Liposomes)
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Protoplasts were isolated from tobacco suspension cultures using a new cellulase preparation. Tobacco mosaic virus (TMV) RNA was encapsulated in reverse-phase evaporation vesicle (REV) liposomes of phosphatidylserine and cholesterol, and was successfully introduced into tobacco protoplasts by treatment of the REV/protoplast mixture with polyvinyl alcohol or polyethylene glycol followed by washing with high pH-high Ca buffer. Delivery of TMV-RNA was monitored by determining the number of infected protoplasts using the immunofluorescence technique. Production of TMV particles in the infected protoplasts was also confirmed by electron microscopy. Because of the high encapsulation efficiency of REV liposomes the amount of TMV-RNA necessary to cause infection in the majority of protoplasts could be reduced to 1/10 to 1/5 that required in the previous study (Fukunaga et al. 1981). The usefulness of the REV-mediated delivery of nucleic acids for genetic manipulation of plant protoplasts is discussed.
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... In experiments, the sterile suspension of the in vitro cell culture of Nicotiana tabacum, L. cv Bright Yellow 2 (BY 2) was used. Tobacco BY 2 cells were cultivated on Linsmaier and Skoog basal medium (LS) [75] in Nagata modification [76]. ...
Many studies have shown that melatonin (an indoleamine) is an important molecule in plant physiology. It is known that this indoleamine is crucial during plant stress responses, especially by counteracting secondary oxidative stress (efficient direct and indirect antioxidant) and switching on different defense plant strategies. In this report, we present exogenous melatonin’s potential to protect lipid profile modification and membrane integrity in Nicotiana tabacum L. line Bright Yellow 2 (BY-2) cell culture exposed to lead. There are some reports of the positive effect of melatonin on animal cell membranes; ours is the first to report changes in the lipid profile in plant cells. The experiments were performed in the following variants: LS: cells cultured on unmodified LS medium—control; (ii) MEL: BY-2 cells cultured on LS medium with melatonin added from the beginning of culture; (iii) Pb: BY-2 cells cultured on LS medium with Pb2+ added on the 4th day of culture; (iv) MEL+Pb: BY-2 cells cultured on LS medium with melatonin added from the start of culture and stressed with Pb2+ added on the 4th day of culture. Lipidomic analysis of BY-2 cells revealed the presence of 40 different phospholipids. Exposing cells to lead led to the overproduction of ROS, altered fatty acid composition and increased PLD activity and subsequently elevated the level of phosphatidic acid at the cost of dropping the phosphatidylcholine. In the presence of lead, double-bond index elevation, mainly by higher quantities of linoleic (C18:2) and linolenic (C18:3) acids in the log phase of growth, was observed. In contrast, cells exposed to heavy metal but primed with melatonin showed more similarities with the control. Surprisingly, the overproduction of ROS caused of lipid peroxidation only in the stationary phase of growth, although considerable changes in lipid profiles were observed in the log phase of growth—just 4 h after lead administration. Our results indicate that the pretreatment of BY-2 with exogenous melatonin protected tobacco cells against membrane dysfunctions caused by oxidative stress (lipid oxidation), but also findings on a molecular level suggest the possible role of this indoleamine in the safeguarding of the membrane lipid composition that limited lead-provoked cell death. The presented research indicates a new mechanism of the defense strategy of plant cells generated by melatonin.
... Tobacco BY-2 cells (Nicotiana tabacum L. cv. Bright Yellow 2) were cultured at 27 C in suspension using modified Linsmaire and Skoog s (mLS) medium, as described by Nagata et al. (1981). ...
Caffeine has long been known to be a specific inhibitor of plant cytokinesis. Caffeine inhibits callose deposition in the cell-plate, albeit via an unknown mechanism. We previously reported that the caffeine-sensitive processes that are required for callose deposition in the cell-plate are initiated at or before the onset of mitosis in cell-cycle-synchronized tobacco BY-2 cells. Because the content of UDP-glucose, which is a substrate for callose synthesis, reportedly increases prior to mitosis, we hypothesized that caffeine affects the generation and/or accumulation of UDP-glucose. In the present study, as a first step to verify this hypothesis, we examined whether caffeine inhibits cell-wall regeneration in protoplasts, which requires UDP-glucose. As a result, we found that caffeine inhibited the cell-wall regeneration of protoplasts in a dose-dependent manner, and that the effect was reversible. These results support the notion that the sites of action of caffeine are the generation and/or accumulation of UDP-glucose, rather than the previously proposed regulatory mechanisms mediated by Ca²⁺.
... The use of nanocarriers (NCs) for nucleic acid delivery in plant biotechnology is not new and dates back to the early 1980s. For example, liposomes were used to transport TMV RNA into tobacco BY-2 protoplasts to produce TMV particles (Nagata et al., 1981). Later, fluorescent conjugated polymer nanoparticles (CPNs) were used to deliver siRNAs targeting genes involved in the cellulose biosynthetic pathway into tobacco BY-2 protoplasts for gene silencing (Silva et al., 2010). ...
Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) via double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack. Yet, due to biosafety concerns regarding transgenics, country-specific GMO legislation has limited the practical application of desirable attributes in plants. The topical application of dsRNA/siRNA targeting essential fungal gene(s) through spray-induced gene silencing (SIGS) on host plants has opened up a transgene-free avenue for crop protection. However, several factors influence the outcome of RNAi, including but not limited to RNAi mechanism in plant/fungi, dsRNA/siRNA uptake efficiency, dsRNA/siRNA design parameters, dsRNA stability and delivery strategy, off-target effects, etc. This review emphasizes the significance of these factors and suggests appropriate measures to consider while designing in silico and in vitro experiments for successful RNAi in open-field conditions. We also highlight prospective nanoparticles as smart delivery vehicles for deploying RNAi molecules in plant systems for long-term crop protection and ecosystem compatibility. Lastly, we provide specific directions for future investigations that focus on blending nanotechnology and RNAi-based fungal control for practical applications.
... Tobacco mosaic virus (TMV) RNA has been entrapped in negative liposomes, which could be expressed in target cells. According to previous results, entrapped TMV RNA infected Vinca, Petunia, and the protoplasts of tobacco [94][95][96][97][98]. GAD et al. (1988) found that negative liposomes could adhere to watermelon pollen grains and other organs [99]. ...
Highly efficient gene delivery systems are essential for genetic engineering in plants. Traditional delivery methods have been widely used, such as Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated delivery, biolistic particle bombardment, and viral transfection. However, genotype dependence and other drawbacks of these techniques limit the application of genetic engineering, particularly genome editing in many crop plants. There is a great need to develop newer gene delivery vectors or methods. Recently, nanomaterials such as mesoporous silica particles (MSNs), AuNPs, carbon nanotubes (CNTs), and layer double hydroxides (LDHs), have emerged as promising vectors for the delivery of genome engineering tools (DNA, RNA, proteins, and RNPs) to plants in a species-independent manner with high efficiency. Some exciting results have been reported, such as the successful delivery of cargo genes into plants and the generation of genome stable transgenic cotton and maize plants, which have provided some new routines for genome engineering in plants. Thus, in this review, we summarized recent progress in the utilization of nanomaterials for plant genetic transformation and discussed the advantages and limitations of different methods. Furthermore, we emphasized the advantages and potential broad applications of nanomaterials in plant genome editing, which provides guidance for future applications of nanomaterials in plant genetic engineering and crop breeding.
The plant cytokinetic microtubule array, called the phragmoplast, exhibits higher microtubule dynamics in its center (midzone) than at the periphery (distal zone). This behavior is known as the axial asymmetry. Despite being a major characteristic of the phragmoplast, little is known about regulators of this phenomenon. Here we address the role of microtubule nucleation in axial asymmetry by characterizing MACERATOR (MACET) proteins in Arabidopsis thaliana and Nicotiana benthamiana with a combination of genetic, biochemical, and live-cell imaging assays, using photo-convertible microtubule probes, and modeling. MACET paralogs accumulate at the shrinking microtubule ends and decrease the tubulin OFF rate. Loss of MACET4 and MACET5 function abrogates axial asymmetry by suppressing microtubule dynamicity in the midzone. MACET4 also narrows the microtubule nucleation angle at the phragmoplast leading edge and functions as a microtubule tethering factor for AUGMIN COMPLEX SUBUNIT 7 (AUG7). The macet4 macet5 double mutant shows diminished clustering of AUG7 in the phragmoplast distal zone. Knockout of AUG7 does not affect MACET4 localization, axial asymmetry, or microtubule nucleation angle, but increases phragmoplast length and slows down phragmoplast expansion. The mce4-1 mce5 aug7-1 triple knockout is not viable. Experimental data and modeling demonstrate that microtubule nucleation factors regulate phragmoplast architecture and axial asymmetry directly by generating new microtubules and indirectly by modulating the abundance of free tubulin.
For almost 50 years, tobacco (Nicotiana tabacum) BY-2 cells have been widely recognized as an important cell line for plant biology. The cell line grows rapidly, can be synchronized to a high degree, and is excellent for imaging; over the years, these features have led to many high-impact discoveries. However, certain other uses of this cell line are virtually unknown. In the early days, I was involved in distributing the cells to laboratories around the world. Many of these scientists wanted to study the cell cycle; however, I also distributed the cells to scientists who were elucidating the mechanism of plant transformation by Agrobacterium tumefaciens. In fact, BY-2 cells played an essential role in the identification and analysis of Vir genes on the Ti plasmid; likewise, the cells were important for discovering the factor that induces the expression of Vir genes. Thus, BY-2 cells were crucial for the development of modern plant biotechnology. Here, I recount the story of how this came to pass and explain why the use of BY-2 cells in this work was never recognized.
Many precursors of plant arabinogalctan proteins (AGPs) contains a C-terminal glycosylphosphatidylinositol (GPI)-anchoring signal. Using NtAGP1, a classical tobacco AGP, as a model, and green fluorescent protein (GFP) and sweet potato sporamin (SPO) as tags, we analyzed the localization and modification of AGP and its mutant without GPI-anchoring signal (AGPΔC) in tobacco BY-2 cells. The NtAGP1 fusion proteins migrated as large smear on SDS-polyacrylamide gel and these proteins also localized preferentially to the plasma membrane. In contrast, fusions of AGPΔC with GFP- and SPO yielded several forms: The largest were secreted, whereas others were recovered in the endomembrane organelles including vacuoles. Comparison of the glycan structures of the microsomal SPO-AGP and the secreted SPO-AGPΔC using antibodies against the glycan epitopes of AGP indicated that the glycan structures of these proteins are different. These observations indicate that GPI-anchoring is required for the proper transport and glycosylation of the AGP precursor.
Recent metagenomic studies which focused on virus characterization in the entire plant environment have revealed a remarkable viral diversity in plants. The exponential discovery of viruses also requires the concomitant implementation of high-throughput methods to perform their functional characterization. Despite several limitations, the development of viral infectious clones remains a method of choice to understand virus biology, their role in the phytobiome, and plant resilience. Here, we review the latest approaches for efficient characterization of plant viruses and technical advances built on high-throughput sequencing and synthetic biology to streamline assembly of viral infectious clones. We then discuss the applications of plant viral vectors in fundamental and applied plant research as well as their technical and regulatory limitations, and we propose strategies for their safer field applications.
Hairy roots were induced on leaf explants excised from sugarbeet (Beta vulgaris) plants when inoculated by Agrobacterium rhizogenes R1601. Paper electrophoresis proved the presence of agropine, the unusual amino acid in these roots which demonstrated the transformation of these tissues. Transformed hairy roots were easily grown on agar-solidified Murashige and Skoog, 1962 (MS) medium containing 0.5mgl-1 2,4dichlorophenoxy acetic acid (2,4-D) or B5 medium supplemented with a mixture of 0.7 mgl-1 Benzyl Adenine (BA) and 0.7 mgl-1 (2,4-D). Callus produced from these hairy roots grown on MS medium was of friable and white in color, while brown in color on B5 medium. Calli were grown efficiently in liquid B5 medium provided with a mixture of 1.0 mgl-1 kinetine (Kin.) and 1.0 mgl-1 2,4-D. These results may contribute in transformation of sugarbeet plants. 456 اﻟﻛﺎﻟس ﺗﻛوﯾن اﻟﻣﻌدل اﻟﻣﺷﺗق ً اﺛﯾﺎ ور ً اﺛﯾﺎ ور اﻟﻣﺣوﻟﺔ اﻟﺷﻌرﯾﺔ اﻟﺟذور ﻣن اﻟﻣﺗﻛوﻧﺔ ﻋﻠﻰ أ اﻟﺳﻛري اﻟﺑﻧﺟر اق ور Beta vulgaris ﺑ ﺑﻛﺗرﯾﺎ Agrobacterium rhizogenes 1601 ﺑﻼزﻣﯾدات ﻋﻠﻰ اﻟﺣﺎوﯾﺔ Ri اﻟﻧﻌﻣﺔ ﺷﻌﯾب ﻗﺗﯾﺑﺔ اﻟﻣﻼح ﻗﺎﺳم اﺣم ﻣز اﻟ اﻟﺗﻘﻧﯾﺎت وﺣدة ﺣﯾﺎﺗﯾﺔ ، اﻟﺣﯾﺎة ﻋﻠوم ﻗﺳم ، اﻟﺗرﺑﯾﺔ ﻛﻠﯾﺔ ، اﻟﻣوﺻل ﺟﺎﻣﻌﺔ اﻟﺧﻼﺻﺔ ﻋﻠﻰ اﻟﺷﻌرﯾﺔ اﻟﺟذور ﺗﻛوﻧت اق أور اﻟﺳﻛري اﻟﺑﻧﺟر ﻧﺑﺎﺗﺎت) Beta vulgaris (، ﻣن اﻟﻣﻔﺻوﻟﺔ ﻧﺎﻣﯾﺔ ﻧﺑﺎﺗﺎت اﻟﺣﻘل ﻓﻲ ، اﻟﻣ ﻓﻲ اﻗﻊ و ﺑﺑﻛﺗرﯾﺎ اﻟﻣﻠﻘﺣﺔ Agrobacterium rhizogenes R1601. ﻧﺗﺎﺋﺞ أﻛدت و اﻟ ﺗرﺣﯾل اﻻﻛروﺑﯾن، ﻋن ﻟﻠﻛﺷف اﻟﻛﻬرﺑﺎﺋﻲ اﻟ ﺣﺎﻣض اﻷ ﻣﯾﻧﻲ ﻏﯾر اﻻ ﻋﺗﯾﺎدي، ﻧﺟﺎح ﻫذﻩ ﻓﻲ اﺛﻲ اﻟور اﻟﺗﺣول اﻷﻧﺳﺟﺔ. و ٕ ا ﻧﻣوﻩ ﺑﺳﻬوﻟﺔ اﻟﺟذور ﻫذﻩ ﻣن اﻟﻣﺗﻛون اﻟﻛﺎﻟس اﻣﺗﺎز ﻋﻠﻰ وﺳط MS ﻋﻠﻰ اﻟﺣﺎوي اﻟﺻﻠب 0.5 ﻟﺗر ﻣﻠﻐم-1 2,4-D و وﺳط ﻋﻠﻰ B5 اﻟﺻﻠب اﻟ ﺑـ ﻣﺟﻬز 0.7 ﻣﻠﻐم ﻟﺗر-1 BA و 0.7 ﻣﻠ ﻐم ﻟﺗر-1 2,4-D و أ ﺗﺻف اﻣﻪ ﺑﻘو وﻟ اﻟﻬش وﻧﻪ وﺳط ﻋﻠﻰ اﻻﺑﯾض MS وﻟوﻧﻪ اﻟﺑﻧﻲ وﺳط ﻋﻠﻰ B5. ً ﺳﻠوﻛﺎ ﻧﻣوﻩ ﻓﻲ اﻟﻛﺎﻟس وﺳﻠك ً ﻣﻣﺎﺛﻼ ﻋﻠﻰ وﺳط B5 اﻟﺳﺎﺋل ﻋﻠﻰ اﻟﺣﺎوي 1.0 ﻣﻠﻐم ﻟﺗر-1 Kin. و 1.0 ﻣﻠﻐم ﻟﺗر-1 2,4-D. ً ﺟدا ﻣﻬﻣﺔ اﻟﻧﺗﺎﺋﺞ ﻫذﻩ ﻓﻲ اﻟﺣﯾوﯾﺔ اﻟﺗﻘﺎﻧﺔ ﻣﺟﺎﻻت ﻟ ﻠﻧﺑﺎﺗﺎت ﻻن ً ﺧﻠوﯾﺎ ً ﺧطﺎ ﺗﻌد اﻟﻣﺗﻛوﻧﺔ اﻟﻛﺎﻟس ارع ﻣز ﻓﻲ ﻣﻬﻣﺎ إ ﻧﺗﺎج ﺳﻛري ﺑﻧﺟر ﻧﺑﺎﺗﺎت ً اﺛﯾﺎ ور ﻣﺣوﻟﺔ ﻣن ﻣﺑﺎﺷرة ﻫذا اﻟﻛﺎﻟس .
There are sharply rising demands for pharmaceutical proteins, however shortcomings associated with traditional protein production methods are obvious. Genetic engineering of plant cells has gained importance as a new strategy for protein production. But most current genetic manipulation techniques for plant components, such as gene gun bombardment and Agrobacterium mediated transformation are associated with irreversible tissue damage, species-range limitation, high risk of integrating foreign DNAs into the host genome, and complicated handling procedures. Thus, there is urgent expectation for innovative gene delivery strategies with higher efficiency, fewer side effect, and more practice convenience. Materials based nanovectors have established themselves as novel vehicles for gene delivery to plant cells due to their large specific surface areas, adjustable particle sizes, cationic surface potentials, and modifiability. In this review, multiple techniques employed for plant cell-based genetic engineering and the applications of nanovectors are reviewed. Moreover, different strategies associated with the fusion of nanotechnology and physical techniques are outlined, which immensely augment delivery efficiency and protein yields. Finally, approaches that may overcome the associated challenges of these strategies to optimize plant bioreactors for protein production are discussed.
A newly synthesized phospholipid, l, 2-0-Dipentadecylmethylidene-glycerol-3-phosphoryl-(N-ethylamino) -ethanolamine, having positive charge was found to exercise fusion activity on plant protoplasts. This phospholipid is not found in cells and has an unusal chemical structure with a dioxolane ring. Some properties of this compound are discussed in relation to phospholipid-induced cell fusion.
Protoplasts of Vicia hajastana Grossh. obtained from suspension-culture cells and Pisum sativum L. obtained from leaves adhered tightly to each other in concentrated solutions of high-molecular-weight polyethylene glycol (PEG). The adhesion occurred non-specifically between the free protoplasts from the same species as well as from the different species and genus. It was enhanced by enrichment of the PEG solution with calcium. Very few heteroplasmic fusions occurred during the period when the protoplasts were incubated in the PEG solution. However, many heterokaryons (up to 10%) were formed soon after the PEG solution was diluted out. The same phenomena were also observed in protoplasts from suspension-culture cells of Glycine max L. and from leaves of Hordeum vulgare L. Vicia and soybean protoplasts obtained from cultured cells regenerated cell walls and underwent sustained cell division after such treatment. Some Vicia-pea heterokaryons divided once. Over 10% of the soybean-barley hybrids divided in 7 days. Some divided 4-5 times and formed small clusters of cells in 10 days. The hybrids were recognizable because they contained chloroplasts from the leaf protoplast and exhibited morphological characters typical of the chlorophyll-less cells. None of the protoplasts from pea and barley leaves, either with or without PEG treatment, underwent cell division during the period of observation. The mechanism of adhesion and fusion of the protoplasts has been discussed.
Products of polyethylene glycol-induced fusion between pea root nodule and tobacco mesophyll protoplasts were examined by transmission electron microscopy. Protoplasts from nodule cells in early symbiotic stages formed heteroplasmic fusion bodies with tobacco mesophyll protoplasts. These fusion bodies had a continuous plasmalemma enclosing the contents of two fusion partners with no trace of boundary membrane inbetween. Bacteroids were present as groups, but individual bacteroids were surrounded by well preserved membrane. Th.e fusion bodies showed no ultrastructural feature which might suggest degenerative changes. Protoplasts from older nodule cells apparently failed to complete heteroplasmic fusion; only some bacteroids were introduced into the heterologous cytoplasm, while major portion of the disintegrated nodule protoplast remained adhering to the surface of tobacco protoplasts.
THE ability to insert messenger RNA selectively into large numbers of differentiated eukaryotic cells and thereby alter the expression of the eukaryotic genome would provide a new and direct approach to the study of the molecular mechanisms involved in protein synthesis. Several procedures have been used to facilitate the cellular incorporation of administered RNA, including microinjection of RNA into Xenopus oocytes1-6 and into fully differentiated eukaryotic cells7,8, direct co-cultivation of cells with naked RNA (refs 9-12), and encapsulation of mRNA within erythrocyte ghosts followed by Sendai virus induced fusion of the ghosts to recipient cells in culture13. We describe here the use of liposomes for the selective insertion of functional mRNA into differentiated eukaryotic cells in vitro. We provide evidence which indicates that cultured human epithelial carcinoma cells (HEp-2) treated with liposomally encapsulated rabbit globin mRNA are stimulated to produce a globin-like protein.
By subverting and/or circumvernting normal pathways of endocytosis, liposomes bypass cell surface restrictions and introduce their contents into cells. Lipoposomes offer a promising new approach for altering the genotype of cells in addition to being a versatile carrier system with numerous therapeutic applications.
Pectolyase Y23, a new maceration enzyme from Aspergillus japonicus, combined with Cellulase Onozuka R10 liberated protoplasts from tobacco mesophyll in less than 25 min. The protoplasts divided to form cell colonies. This combination of enzymes also liberated protoplasts from mesophyll of rice and oat. The enhanced macerating activity of Pectolyase Y23 was found to be due not only to the high activity of pectin lyase and polygalacturonase, but also to a factor which stimulated maceration.