ArticleLiterature Review

Plant protoplasts: Status and biotechnological perspectives

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Abstract

Plant protoplasts ("naked" cells) provide a unique single cell system to underpin several aspects of modern biotechnology. Major advances in genomics, proteomics, and metabolomics have stimulated renewed interest in these osmotically fragile wall-less cells. Reliable procedures are available to isolate and culture protoplasts from a range of plants, including both monocotyledonous and dicotyledonous crops. Several parameters, particularly the source tissue, culture medium, and environmental factors, influence the ability of protoplasts and protoplast-derived cells to express their totipotency and to develop into fertile plants. Importantly, novel approaches to maximise the efficiency of protoplast-to-plant systems include techniques already well established for animal and microbial cells, such as electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers, especially perfluorochemicals and hemoglobin. However, despite at least four decades of concerted effort and technology transfer between laboratories worldwide, many species still remain recalcitrant in culture. Nevertheless, isolated protoplasts are unique to a range of experimental procedures. In the context of plant genetic manipulation, somatic hybridisation by protoplast fusion enables nuclear and cytoplasmic genomes to be combined, fully or partially, at the interspecific and intergeneric levels to circumvent naturally occurring sexual incompatibility barriers. Uptake of isolated DNA into protoplasts provides the basis for transient and stable nuclear transformation, and also organelle transformation to generate transplastomic plants. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, synthesis of pharmaceutical products, and toxicological assessments. This review focuses upon the most recent developments in protoplast-based technologies.

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... Protoplasts represent a versatile experimental system to perform functional genetic analysis and to study a wide range of cellular processes, e.g. cellular structure, membrane function, hormonal signaling (Davey et al., 2005). Furthermore, protoplasts might also be an important biotechnological tool in breeding programs, especially in vegetatively propagated species of high heterozygosity and ploidy level, such as strawberry (Nyman and Wallin, 1988). ...
... Through protoplasts, it is possible to create gene combinations of nucleus and/or cytoplasmic organelles that are unique, being the obtainment of asymmetric hybrids and cybrids one the main applications for protoplast fusion (Geerts et al., 2009;Li et al., 1999). As the variation obtained in plants regenerated from protoplasts is much greater than that in plants coming from cultures with organized growth (Karp, 1993), protoplast culture could have an enormous value as a source of somaclonal variation, so-called protoclonal variation, expanding the existing germplasm by creating variations that may be used within breeding programs (Davey et al., 2005). ...
... Protoplast isolation is a stress-inducing process (Papadakis et al., 2001;Papadakis and Roubelakis-Angelakis, 2002), in which waste products accumulation could induce cell disruption. Sucrose and glucose are usually used in the protoplast isolation and purification media (Davey et al., 2005). In the strawberry cv. ...
Article
Protoplasts are an useful biotechnological tool for plant improvement. In strawberry, very few studies on protoplast technology have been carried out. In this investigation, a procedure for the isolation and culture of strawberry protoplasts, cv. ‘Chandler’, has been developed. The effect of several factors affecting the successful isolation of protoplasts and formation of microcalli, e.g. explant source, washing procedure, hormonal composition of the culture medium and protoplast density, were evaluated. For shoot regeneration, microcalli derived from isolated protoplasts were transferred to MS medium supplemented with 0.2 mg l⁻¹ NAA and either 5 mg l⁻¹ BA or 3 mg l⁻¹ TDZ, obtaining a similar regeneration rate, 17%, in both media. Twenty-one independent protoclones were transferred to field conditions for agronomic evaluation. Significant alterations in the growth habit, density of foliage, leaf color and leaf morphology were detected in some lines. Fruit yield was significantly reduced in 15 out of the 21 protoclones evaluated due to a reduction in fruit weight and/or the number of fruits. Ploidy level was unaffected in a sample of 6 lines selected at random; however, a study of genetic stability by using 10 EST-SSR markers showed genetic alterations in all the lines analyzed. Despite the high rate of somaclonal variation detected in the protoclones, some of the lines displayed an agronomical behavior similar to control plants, indicating that this protocol could be useful for genetic improvement in this species.
... The microalgae cell wall is constituted by rigid components embedded in a polymeric matrix constituted by 70% cellulose as well as glycoproteins, pectin, and algaenan, which enhance the stiffness of their cell walls [12]. High-pressure homogenization following acidic and thermal pre-treatment, single enzymatic digestion with cellulase, hemicellulase, and pectinase, or a mix of them as well as combinatory methods of enzymatic and mechanical treatment (sonication and microwaves) are used for microalgae protoplast formation [13][14][15]. ...
... Nowadays, protoplast fusion of different species or organisms is a common technique for genome combination and development of microorganisms with desired properties as well as in biotechnological applications [14,[16][17][18][19]. The extent of cell wall thickening, temperature and duration of the enzymatic incubation, pH, agitation, as well as the nature of osmotic solution are key factors for protoplast formation [13]. ...
... After protoplast isolation, the regeneration of the cell wall is of vital importance in order to fully exploit the genetic or biotechnological features that have been modified in the parental cells. Medium with a non-metabolizable sugar alcohol like mannitol or sorbitol, and with auxins and cytokinins, may provide protection against osmotic pressure and support mitosis and daughter cell formation [13]. Studies on Chlorophyta species after physical wounding demonstrate the recreation of a cell membrane on the surface of protoplasts 12 h after the wounding, while Golgi bodies with numerous vesicles at the peripheral region of the rebuilding cell at 24 h after the wounding began to develop [22]. ...
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Intensive research on the use of magnetic nanoparticles for biotechnological applications of microalgae biomass guided the development of proper treatment to successfully incorporate them into these single-cell microorganisms. Protoplasts, as cells lacking a cell wall, are extensively used in plant/microalgae genetic manipulation as well as various biotechnological applications. In this work, a detailed study on the formation of protoplasts from Haematococcus pluvialis with the use of enzymatic and mechanical procedures was performed. The optimization of several parameters affecting the formation of protoplasmic cells and cell recovery was investigated. In the enzymatic treatment, a solution of cellulase was studied at different time points of incubation, whereas in the mechanical treatment, glass beads vortexing was used. Mechanical treatment gave better results in comparison to the enzymatic one. Concerning the cell recovery, after the protoplast formation, it was found to be similar in both methods used; cell viability was not investigated. To enhance the protoplast cell wall reconstruction, different "recovery media" with an organic source of carbon or nitrogen were used. Cell morphology during all treatments was evaluated by electron microscopy. The optimal conditions found for protoplast formation and cell reconstruction were successfully used to produce Haematococcus pluvialis cells with magnetic properties.
... The list of plant species that can be regenerated from protoplasts is, however, limited. The common approach to design a regeneration protocol is trial and error based, an exhaustive strategy given the number of parameters that is relevant for protoplast regeneration (reviewed by Davey et al., 2005). On top of that, the genotype dependency of regeneration, e.g., in Apium graveolens or Kalanchoe blossfeldiana (Bruznican et al., 2017;Cui et al., 2019) limits the design of widely applicable protocols in many crops. ...
... On top of that, the genotype dependency of regeneration, e.g., in Apium graveolens or Kalanchoe blossfeldiana (Bruznican et al., 2017;Cui et al., 2019) limits the design of widely applicable protocols in many crops. Innovative approaches as, e.g., electrical stimulation or adding surfactants or antibiotics have significantly contributed to the regeneration of protoplasts isolated from recalcitrant genotypes or species (Davey et al., 2005;Eeckhaut et al., 2013). This has increased the application potential of protoplasts for plant breeding. ...
... It is also impossible to introgress particular DNA sequences or genes through somaclonal variation. Furthermore, when using protoplasts as source explants, it should be kept in mind that regeneration is the most common bottleneck in any protoplast related work (Davey et al., 2005;Eeckhaut et al., 2013) and as a result this strategy is confined to a limited number of species, including Chrysanthemum. Nonetheless, contemporary tools like next generation sequencing, metabolite profiling and in situ hybridization offer prospects to gain insight in the regeneration process and consequently to expand the group of regenerative species. ...
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Chrysanthemum × morifolium protoplasts were isolated and regenerated to assess possible protoclonal variation in the regenerants. After a preliminary screening of the potential of different regeneration systems for protoplast regeneration, we produced a series of cut chrysanthemum ‘Arjuna’ leaf protoplast regenerants through liquid culture. Regenerants (54) were vegetatively propagated and grown under a commercial production system in 2 different seasons. All screened regenerants were significantly affected with regard to either flower number, flower size, flower weight, leaf weight, stalk weight, or plant size. A significant plant size reduction in 43/52 and 48/49 regenerants for both seasons was the most recorded effect. Also a reduction in flowering induction time up to 10 days, altered flower types and colors were observed. Differences between growing seasons were notable. Possible molecular backgrounds including genome size variation and commercial applications in breeding of chrysanthemum are discussed.
... It takes 2-3 days, sometimes even more to form cell walls around the plasma membrane. The loss of the spherical shape of protoplasts in cultures is an indication of cell wall formation (Davey et al. 2005). The cell wall formation is again reliant on the culture conditions, nutrient supplementation, the concentration of the growth regulators, external culture cues such as light intensity (2000-5000 lx) and temperature (22-30°C), and above all the genotype of the parent plant (Davey et al. 2005). ...
... The loss of the spherical shape of protoplasts in cultures is an indication of cell wall formation (Davey et al. 2005). The cell wall formation is again reliant on the culture conditions, nutrient supplementation, the concentration of the growth regulators, external culture cues such as light intensity (2000-5000 lx) and temperature (22-30°C), and above all the genotype of the parent plant (Davey et al. 2005). Protoplast derived cells with defective cell wall do not undergo normal mitotic division. ...
... The window period of * 2-5 weeks is an appropriate time point to carry out all the single-cell targeted experiments, including heterofusions (van Ark et al. 1992). Protoplast fusion enabled cytoplasmic male sterility, expression genes for synthesis of recombinant proteins, vitamins, and natural products of therapeutic relevance as well as to incorporate resistance genes towards stress caused by salinity, drought, cold and pathogens such as viruses, fungi, and bacteria (Davey et al. 2005). ...
Article
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Metabolic pathway reconstruction and gene edits for native natural product synthesis in single plant cells are considered to be less complicated when compared to the production of non-native metabolites. Being an efficient eukaryotic system, plants encompass suitable post-translational modifications. However, slow cell division rate and heterogeneous nature is an impediment for consistent product retrieval from plant cells. Plant cell synchrony can be attained in cultures developed in vitro. Isolated plant protoplasts capable of division, can potentially enhance the unimpaired yield of target bioactives, similar to microbes and unicellular eukaryotes. Evidence from yeast experiments suggests that ‘critical cell size’ and division rates for enhancement machinery, primarily depend on culture conditions and nutrient availability. The cell size control mechanisms in Arabidopsis shoot apical meristem is analogous to yeast notably, fission yeast. If protoplasts isolated from plants are subjected to cell size studies and cell cycle progression in culture, it will answer the underlying molecular mechanisms such as, unicellular to multicellular transition states, longevity, senescence, ‘cell-size resetting’ during organogenesis, and adaptation to external cues.
... Nowadays, somatic hybridization through protoplast fusion is a well refined and routinely used method in order to create Solanum hybrids with different useful properties [126,127]. Plant protoplasts are naked somatic cells from which the cell wall has been removed by enzymatic digestion, therefore these cells can be used for gene transfer, somatic hybridization [128], and more recently for targeted mutagenesis and genome research. Protoplasts are still totipotent and they are able to regenerate new cell wall, divide to form new cell colonies, microcalluses, calluses and finally new plants. ...
... Recently, selection of SHs (S. tuberosum + S. chacoense) based on callus growth tagged with gfp has been also observed [138]. Different methods are available for protoplast fusion, but only two are generally used: electrofusion and PEG (polyethylene glycol) induced fusion [128]. Electrofusion is the most widely used method since its discovery in 1979 [139], and it consists in first instance of protoplast agglutination induced by the use of an alternating current (AC) field, the so-called dielectrophoresis [140]. ...
... In the second phase the agglutinated aligned protoplasts are induced to fuse by using direct current (DC) square wave pulses with a high intensity (2000 V cm −1 ) and very short duration (10-100 μs) [141]. PEG-induced fusion generally has a similar efficiency as electrofusion, especially after applying calcium solution washing step [128]. Immediately after fusion or after the plants have been regenerated, the obtained SHs are subject to different analysis, such as cytological (flow cytometry, chromosome counts, chloroplasts counts in guard cells, FISH -fluorescence in situ hybridization and GISH -genomic in situ hybridization), molecular: isozyme, molecular markers (e.g. ...
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The aim of this chapter is to describe in a synthetic manner the most efficient biotechnological techniques which can be applied in potato breeding with emphasis on multiple resistance traits. To this end, most important results of all biotechnological techniques will be pointed out including new biotechnological tools of genome editing. The somatic hybridization will be the core of the presentation as the only non-GMO strategy with good results in transferring multiple resistances into potato gene pool. The chapter is presenting all data in a synthesized form and made comparisons between the existing techniques and their possible adoption in breeding in different parts of the world, depending on regulations and consumer choice. Moreover, the recently discovered value of potato as a healthy food and its possible applications in cancer treatment will be also discussed with new data on both potato and some of its wild relatives.
... Protoplasts are the cells with the cell wall removed, which is accomplished with an enzymatic treatment [4]. Somatic fusion depends on many factors, e.g., the source of In contrast, research on PA activity in protoplasts is relatively rare. ...
... However, the overall frequency of cell division was lower in the tested breeding lines compared to the commercial cultivar "Sława z Gołębiewa." Such results showed the genotypic effect on cell proliferation, which was commonly observed in protoplast cultures of B. oleracea and other species [4,7,8,10,48]. Considering the aforementioned studies, the different responses might also be related to the different levels of endogenous PAs between the tested accessions [14,56], which might further affect their response after an exogenous PA application. ...
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Polyamines (PAs) are organic molecules that are found in plants and animals. In plants, they are involved in the regulation of cellular growth, apoptosis, rooting, flower development, and stress responses. The effect of exogenously applied polyamines on the development of Brassica oleracea L. var. capitata protoplast cultures was studied. Protoplasts were isolated from hypocotyls of 2-week-old seedlings of three accessions and they were cultured in liquid media supplemented with putrescine (Put), spermidine (Spd), and spermine (Spm) at concentrations of 0 (control), 10, 20, and 40 µM. In the very early culture (24 and 48 h), cellular reactive oxygen species levels (ROS) in live cells were monitored using a fluorescent probe. The Put- and Spd-treated protoplasts exhibited lower fluorescence intensities, which corresponded to lower ROS accumulation as compared to the PA-free control. The protoplast viability was affected by the type of polyamine applied rather than its concentration. Put and Spd had a beneficial effect on the mitotic activity of the cultured cells, which was observed in all tested accessions. The highest frequency of shoot organogenesis (21%) was obtained from microcalli derived from the protoplasts cultured on the medium supplemented with 10 µM Put. Analysis of the ploidy level of the regenerants showed that the vast majority were diploids. Our results demonstrated that exogenously applied PAs maintained the viability of B. oleracea L. var. capitata protoplasts by alleviating oxidative stress and stimulating mitotic activity, which further affected the plant regeneration process.
... The choice of proper culture systems is an important factor. Culture systems, such as liquid, semi-liquid, protoplast embedded in agar, filter paper overlaid on agar, and alginate culture technique, have been used in previous studies (Davey et al., 2005a;Eeckhaut et al., 2013;Eeckhaut and Van Huylenbroeck, 2011;Fiuk and Rybczyński, 2007;Kim and Lee, 1996;Meyer et al., 2009;Marchant et al., 1997;Pati et al., 2008;Zhou et al., 2005), whereas the latter culture systems are likely easier to be established and more effective to reduce phenolic accumulation and osmolarity. However, an optimal culture system that can be used for a wide range of species has not yet been developed so far. ...
... This is because isolated protoplasts do not generally fuse and tend to repel each other due to negative charges on the plasma membrane surfaces of isolated protoplasts. To overcome this issue, techniques such as electrofusion and chemical treatment of protoplasts with polyethylene glycol (PEG), calcium ions, or a combination of these treatments] remain the most commonly used techniques (Davey et al., 2005a). In Petunia, the fusion of two protoplasts from two sexually incompatible cultivars was successful; the fused protoplasts provided a genetically improved mutant (Power et al., 1980). ...
Article
The international floriculture industry is expected to become bigger in the near future, owing to the continuous increase in the demand for ornamental plants. To facilitate the demand, many efforts are being paid to produce new cultivars with superior floral traits, such as novel color patterns and shapes and improved flower longevity. Protoplasts have been used to produce new cultivars, especially commercially important ornamental plants with incompatibility barriers in sexual hybridization, using protoplast fusion and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated genome editing. However, the success of the protoplast isolation to shoot regeneration process remains a bottleneck for most ornamental plants. In this review, we highlighted the role of the factors that affect the protoplast isolation to shoot regeneration process of ornamental plants. The practical application of protoplasts in developing new genetically improved cultivars from some ornamental plants via somatic hybridization and genome editing was also described. Information in this review will contribute to the success of plant regeneration from protoplasts for ornamental plants and provide insights on new cultivar production, mainly from commercially important ornamental plants that cannot produce a new cultivar via sexual breeding.
... Enzymatic methods, such as algae cell treatment with cellulase, hemi-cellulase, and pectinase or mixtures of them, or mechanical methods, such as sonication and microwaves, as well as glass beads agitation, are the most frequently used techniques for protoplast formation [24][25][26]. Factors such as cell wall thickness, temperature, enzymes incubation time, pH, agitation, and the nature of the osmotic solution can affect protoplast formation [27]. Since protoplast formation is a stress-controlled process, re-creation of a fully structured and functional cell should be followed by treatment aiming at efficient regeneration of the cell walls [28]. ...
... Proper osmotic conditions led to enhanced protoplast generation yields and viability, as well as regeneration, protecting the fragile cell from bursting or shrinking. Usually mannitol and sorbitol, as well as sodium chloride, increased protoplasts yields [36], supporting mitosis and daughter cell formation and protecting protoplasts from osmotic shocks [27]. Studies on Chlorophyta species revealed cell membrane regeneration 12 h after wounding, supported by an extensive protein expression governed by Golgi apparatus [41], and furthermore, cell-wall-digested cells of Bryopsis plumose cells developed in 9-12 h a lipid membrane demonstrating a regeneration efficiency of 40% [42]. ...
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Microalgae are used in industrial and pharmaceutical applications. Their performance on biological applications may be improved by their immobilization. This study presents a way of cell immobilization using microalgae carrying magnetic properties. Nannochloropsis oceanica and Scenedasmus almeriensis cells were treated enzymatically (cellulase) and mechanically (glass beads), generating protoplasts as a means of incorporation of magnetic nanoparticles. Scanning electron microscopy images verified the successful cell wall destruction for both of the examined microalgae cells. Subsequently, protoplasts were transformed with magnetic nanoparticles by a continuous electroporation method and then cultured on a magnetic surface. Regeneration of transformed protoplasts was optimized using various organic carbon and amino acid supplements. Both protoplast preparation methods demonstrated similar efficiency. Casamino acids, as source of amino acids, were the most efficient compound for N. oceanica protoplasts regeneration in enzymatic and mechanical treatment, while for S. almeriensis protoplasts regeneration, fructose, as source of organic carbon, was the most effective. Protoplasts transformation efficiency values with magnetic nanoparticles after enzymatic or mechanical treatments for N. oceanica and S. almeriensis were 17.8% and 10.7%, and 18.6% and 15.7%, respectively. Finally, selected magnetic cells were immobilized and grown on a vertical magnetic surface exposed to light and without any supplement.
... In 1892, Klercher was the first to isolate protoplasts (reviewed by Cocking, 1972;Davey et al., 2005) by using a mechanical method to remove plant cell walls. In this approach, an onion bulb (Allium cepa) is sliced and placed in a plasmolysing solution to pull the membranes of epidermal cells away from their walls. ...
... Protoplasts can then be isolated by removing these intact plasmolysed cells from the remaining cell wall (Chambers and Höfler, 1931). However, this method is only feasible for storage tissues such as bulbs; meristematic cells require more extensive plasmolysis and only yield a small number of protoplasts (Cocking 1972;Davey et al., 2005). ...
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In the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas) system, protoplasts are not only useful for rapidly validating the mutagenesis efficiency of various RNA-guided endonucleases, promoters, sgRNA designs, or Cas proteins, but can also be a platform for DNA-free gene editing. To date, the latter approach has been applied to numerous crops, particularly those with complex genomes, a long juvenile period, a tendency for heterosis, and/or self-incompatibility. Protoplast regeneration is thus a key step in DNA-free gene editing. In this report, we review the history and some future prospects for protoplast technology, including protoplast transfection, transformation, fusion, regeneration, and current protoplast applications in CRISPR/Cas-based breeding.
... The cell wall can be removed either by mechanical approach or enzymatic hydrolysis. The latter method is easier to perform and widely used to achieve high protoplast yield (Davey et al., 2005). Protoplasts are suitable for DNA delivery either by polyethylene glycol (PEG), electroporation or DNA microinjection (Eeckhaut et al., 2013;Masani et al., 2014). ...
... As expected, the absence of one of these enzymes in the enzyme mixture decreased the protoplast yield. Cellulase involves removing the cell wall, while the macerozyme and pectinase aid cell separation (Cocking, 1972;Davey et al., 2005). The degradation of cellulosic contents on the plant cell wall has improved the adsorption of pectinase into the tissues, thus improving the protoplast release from source tissues. ...
Article
The absence of a cell wall on the protoplast contributes to its versatility. Its flexibility for DNA manipulation and the possibility of rapid cell-based assay is desirable in the plant biotechnology field. This study was carried out to improve protoplast isolation from oil palm in vitro leaf and mesocarp tissues. The factors affecting protoplast isolation efficiency were optimized, including the protocols and enzyme composition involved, focusing on the oil palm in vitro leaf first. Incubation of oil palm leaf sample with an enzyme mixture of cellulase R-10, macerozyme R-10, driselase, and pectolyase Y-23, for 14 h has successfully produced up to 2.5 × 106 protoplasts g-1 fresh weight (FW)-1 with 95% viability. Incubation of oil palm mesocarp tissue with the optimized enzyme mixture for 2 h at static condition has also successfully produced 3.98 × 106 protoplasts g-1 FW-1 with 85% viability. Besides, it was found that increasing the sample's surface area in contact with enzyme solution by slicing the samples into narrow strips and thin layers has improved the penetration of enzymes into the tissues and enhanced the isolation efficiency. In addition, a plasmolysis step before enzymatic treatment has also improved the protoplast viability by minimizing the damage incurred during isolation. The successful isolation of protoplast from oil palm leaf and mesocarp has enabled the study of gene function and the characterization of endogenous tissue-specific promoter being carried out in vivo.
... Totipotente Protoplasten gelten als ein sehr wichtiges Versuchsmaterial für die Gentechnik (Davey et al, 2005). Die somatische Hybridisierung durch Protoplastenfusion ermöglicht die vollständige oder teilweise Kombination von Kern-und Zytoplasma-Genomen auf interspezifischer und intergenerischer Ebene, um natürlich auftretende sexuelle Inkompatibilitätsbarrieren zu umgehen. ...
... Totipotente protoplasten worden beschouwd als een zeer belangrijk experimenteel materiaal voor genetische manipulatie (Davey et al, 2005). Somatische hybridisatie door protoplastfusie maakt het mogelijk de nucleaire en cytoplasmatische genomen geheel of gedeeltelijk te combineren op interspecifiek en intergeneriek niveau om natuurlijk voorkomende seksuele incompatibiliteitsbarrières te omzeilen. ...
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Somatische embryogenese is een van de belangrijkste technologieën voor de microvermeerdering van dadelpalmcultivars. Onlangs is aanzienlijke vooruitgang geboekt bij de ontwikkeling en optimalisering van deze techniek op basis van embryogene celsuspensieculturen. Dit boek beschrijft een procedure voor de snelle ontwikkeling van een groot aantal somatische embryo's uit embryogene celsuspensieculturen, beschrijft ook een efficiënt micropropagatieprotocol van somatische embryogenese uit celsuspensieculturen te beginnen met scheutpunt-explantaten tot acclimatisatie van de plant en vernieuwende informatie over recente vooruitgang op het gebied van suspensieculturen in de dadelpalm.
... I protoplasti totipotenti sono considerati un materiale sperimentale molto importante per l'ingegneria genetica (Davey et al, 2005). L'ibridazione somatica tramite fusione di protoplasti permette di combinare i genomi nucleari e citoplasmatici, completamente o parzialmente, a livello interspecifico e intergenerico per aggirare le barriere naturali di incompatibilità sessuale. ...
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A embriogénese somática é uma das tecnologias mais importantes para a micropropagação de cultivares de palmeiras tâmaras. Recentemente, foram feitos progressos consideráveis no desenvolvimento e optimização desta técnica a partir de culturas de suspensão de células embriogénicas. Este livro descreve um procedimento para o rápido desenvolvimento de um grande número de embriões somáticos a partir de culturas em suspensão de células embriogénicas, descreve também um protocolo eficiente de micropropagação a partir de embriogénese somática a partir de culturas em suspensão de células, começando com explantes de pontas de rebentos até à aclimatação das plantas e informação inovadora sobre o progresso recente das culturas em suspensão de palma tâmara.
... Los protoplastos totipotentes se consideran un material experimental muy importante para la ingeniería genética (Davey et al, 2005). La hibridación somática mediante la fusión de protoplastos permite combinar los genomas nuclear y citoplasmático, total o parcialmente, a nivel interespecífico e intergenérico para sortear las barreras de incompatibilidad sexual que se producen de forma natural. ...
... So, future studies must focus on examination of callus regeneration ability (Farsi et al. 2012;Cai et al. 2013). Friable callus has been proven to be crucial for the establishment of cell suspension cultures, which played an important role in successful somatic hybridization (Davey et al. 2005) and genetic transformation (Chumakov and Moiseeva 2012) in many plants. One of the most chal-lenges in walnut tissue culture is browning. ...
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Present study was conducted to achieve intergeneric hybridization in Juglandaceae for the first time. Due to embryo abortion possibility, the embryo rescue technique was conducted via callus induction from immature hybrid embryo culture. For this reason, embryos were sampled at 20, 30, 40 and 60 days after pollination and cultured on two enrichment media (WPM and DKW). The developmental stage of immature embryo, kind of medium and addition of growth regulators to the induction medium played the role in promoting better callus and plantlet formation. The best quality and quantity of callus induction were achieved in 60 days’ explant. Maximum induction was in WPM and there was no callus induction in PGRs free medium. Browning was the most obstacle to conquer in callus proliferation. Germination of remained mature hybrid embryo until fruit harvest time was induced on three media (MS, WPM, and DKW). There were no significant differences between medium and male parents effect on germination parameters. But, MS and WPM performed better for hybrid embryo germination. The first germination was 7 days in MS and WPM and about 10 days for DKW after culture. In all medium the roots were robust and more developed than the leaves. The microplant transferred to soil after hardening.
... Récemment, l'hybridation somatique a été utilisée avec succès chez les bananiers à propagation végétative (Musa spp.) (Assani, et al,. 2005 ), chez les Solanaceae pour introduire des traits de résistance aux maladies, en transférant la résistance de parents sauvages à des variétés cultivées ( Les protoplastes totipotents sont considérés comme un matériel expérimental très important pour le génie génétique (Davey et al, 2005). L'hybridation somatique par fusion de protoplastes permet de combiner les génomes nucléaires et cytoplasmiques, totalement ou partiellement, aux niveaux interspécifique et intergénérique afin de contourner les barrières d'incompatibilité sexuelle naturelles. ...
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L'embryogenèse somatique est l'une des technologies les plus importantes pour la micropropagation des cultivars de palmiers dattiers. Récemment, des progrès considérables ont été réalisés dans le développement et l'optimisation de cette technique à partir de cultures de suspension de cellules embryogènes. Ce livre décrit une procédure pour le développement rapide d'un grand nombre d'embryons somatiques à partir de cultures de suspension de cellules embryogènes. Il décrit également un protocole efficace de micropropagation à partir de l'embryogenèse somatique à partir de cultures de suspension de cellules, en commençant par des explants de bouts de pousses jusqu'à l'acclimatation de la plante, ainsi que des informations innovantes sur les progrès récents des cultures de suspension chez le palmier dattier.
... Later, plant protoplasts were successfully used for RNP delivery in a variety of plants such as tobacco, Arabidopsis, lettuce, rice, and Petunia (Woo et al., 2015;Subburaj et al., 2016;Murovec et al., 2018;Park et al., 2019). However, for most monocot species, including major crops such as maize, wheat, rice, barley, and sorghum, regeneration of plants from protoplasts remains either unattainable or inefficient (Davey et al., 2005). ...
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Despite the conspicuous and rapid development of genome editing tools, implementing this technology in plants often remains constrained by our inability to regenerate fertile genome-modified plants. Finding a viable combination of delivery method, genome editing reagents, and plant regeneration system has often been both crop-specific and crop-limited. Recent progress using morphogenic genes such as Wuschel2 (Wus2) and Babyboom (Bbm) has alleviated some of these crop-specific challenges, and along with other improvements, continues to expand the range of plants that can be edited.
... Additionally, the transcription levels for NtCes1-A in CPN-siRNA treated protoplasts after a 48 h incubation were reduced by over 76%, compared to 17% NtCes1-A mRNA reduction with CPN treatment alone. Internalisation of nucleic acids by protoplasts has traditionally been achieved by using poly(ethylene glycol) (PEG) or by electroporation, which enhances the cell membrane permeability through short, highintensity electrical pulses (Fisk and Dandekar, 2004;Davey et al., 2005). However, with use of both PEG and electroporation there was a detrimental impact on protoplast viability (Fromm et al., 1985;Fisk and Dandekar, 2004;Silva et al., 2010). ...
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Since the discovery of RNA interference (RNAi) in the nematode worm Caenorhabditis elegans in 1998 by Fire and Mello et al., strides have been made in exploiting RNAi for therapeutic applications and more recently for highly selective insect pest control. Although triggering mRNA degradation in insects through RNAi offers significant opportunities in crop protection, the application of environmental naked dsRNA is often ineffective in eliciting a RNAi response that results in pest lethality. There are many possible reasons for the failed or weak induction of RNAi, with predominant causes being the degradation of dsRNA in the formulated pesticide, in the field or in the insect once ingested, poor cuticular and oral uptake of the nucleic acid and sometimes the lack of an innate strong systemic RNAi response. Therefore, in the last 10 years significant research effort has focused on developing methods for the protection and delivery of environmental dsRNA to enable RNAi-induced insect control. This review focuses on the design and synthesis of vectors (vehicles that are capable of carrying and protecting dsRNA) that successfully enhance mRNA degradation via the RNAi machinery. The majority of solutions exploit the ability of charged polymers, both synthetic and natural, to complex with dsRNA, but alternative nanocarriers such as clay nanosheets and liposomal vesicles have also been developed. The various challenges of dsRNA delivery and the obstacles in the development of well-designed nanoparticles that act to protect the nucleic acid are highlighted. In addition, future research directions for improving the efficacy of RNA-mediated crop protection are anticipated with inspiration taken from polymeric architectures constructed for RNA-based therapeutic applications.
... Later, plant protoplasts were successfully used for RNP delivery in a variety of plants such as tobacco, Arabidopsis, lettuce, rice, and Petunia (Woo et al., 2015;Subburaj et al., 2016;Murovec et al., 2018;Park et al., 2019). However, for most monocot species, including major crops such as maize, wheat, rice, barley, and sorghum, regeneration of plants from protoplasts remains either unattainable or inefficient (Davey et al., 2005). ...
... It has been proven that embryogenic friable calliwere essential for the development of cell suspension cultures that have played an important role in successful somatic hybridization (Davey et al., 2005;Chumakov and Moiseeva, 2012). In the present study, embryogenic calli were produced using the ovule culture of C. volkameriana at different immature fruit stages on EME medium supplemented with 2,4-D, BAP, and KIN. ...
Article
The present study reports on the isolation of viable protoplast from ovule-derived embryogenic calli of Volkameriana (Citrus volkameriana L.), which is a rootstock in high demand for lemon production.Ovules of C. volkameriana isolated at 3 different immature fruit stages, comprising4, 8, and 12 weeks after anthesis (WAA),were cultured on5 different media in order to produce embryogenic callus lines as a source material for protoplast isolation. EME medium (MT basal medium + 0.5 gL–1 malt extract), with the addition of phytohormones [kinetin (KIN), 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BAP)] at different concentrations, were tested for callogenesis. According to 2-way ANOVA, significant effects were determined as a result of the immature fruit stage and type of culture media (P ≤ 0.01) on the callogenesis and embryogenic callus induction frequency. First, callus formation was recorded after 4 WAA on medium comprising EME + 2,4-D (1.0 mg L–1) + BAP (0.5 mg L–1). Callus induction frequency was the highest (90.00%) in the same culture medium when the ovules wereculturedat8 WAA. In addition, culturing the ovules isolated from 12 WAA immature fruits of C. volkameriana resulted in the highest indirect somatic embryogenesis (24%). Embryogenic callus initiation was the highest (25.56%) using EME + KIN (1.0 mg L–1) and ovules cultured at 8 WAA (14%) resulted in the highest embryogenic callus formation. Effects of different enzyme concentrations on the efficiency of protoplast isolation were calculated using the hemocytometer cell counting method. The combination of 2% cellulase and 0.2% pectinase gave the highest numbers of protoplasts, at 12.33 × 105protoplast/mL. Embryogenic callus lines obtained by culturing ovules of C. volkameriana yielded high-quality protoplasts after isolation and could be useful as a protoplast source for further somatic hybridization studies.
... Protoplasts are plant cells that have had the cell wall removed, but they retain the main cell features (Davey et al. 2005). Like eukaryotic cells, they are a versatile model system for observing various cellular events. ...
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Castor bean is an oil crop plant (Euphorbiaceae) found across the tropical, subtropical, and temperate regions. Despite its important oil properties and cultivation in a wide range of environments, the molecular mechanisms of castor’s adaptation and metabolism have not been fully clarified due to difficulties in genetic modification approaches. The protoplasts of several other plant species have been used as versatile cell-based model systems to elucidate the biological functions of genes and proteins. Here, we report an optimized protocol for protoplast isolation from the leaves and cotyledons of castor bean. The main parameters evaluated to achieve the maximum protoplast yield were the application of a cell wall-degrading enzyme solution, the osmotic pressure of the enzymolysis solution, and the enzymolysis time. Transient expression and the main influencing factors were validated by fluorescence microscopy of castor protoplasts. Our results suggest that castor protoplasts can be used as a productive cell-based system to explore the mechanisms involved in the molecular, biochemical, and functional characterization of castor bean genes.
... However, regeneration of plant from the protoplast of Brassica is genotype dependent (Hansen et al. 1999). In addition, the use of protoplast requires a longer tissue culture period, which will lead to a rising risk of contamination and undesirable somaclonal variations (Bhalla and Singh 2008;Davey et al. 2005). Thus, application of PEGmediated or electroporation-mediated protoplasts transformation method for regenerating transgenic plants is limited. ...
Article
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Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol.
... Since the molecular mechanisms involved in shoot formation in protoplast-derived calli remain largely unresolved, the capacity to induce protoplast-to-plant regeneration is a major limit to the development of T-DNA-free gene-edited potato plants. Innovative approaches were developed to enhance protoplast regeneration and included electrostimulation and exposure of protoplasts to surfactants and respiratory gas carriers (Davey et al. 2005). Besides, the BBM transcription factor activates a broad set of genes involved in the transcription of cellular signaling molecules, cell wall biosynthesis proteins, and protein turnover (Horstman et al. 2017a). ...
Article
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Main conclusion An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor. Abstract Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts’ transgene expression and protoplasts’ regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.
... Protoplast technology has focused especially on the generation of interspecific hybrids between wild and cultivated species that cannot be produced via conventional hybridization (Davey et al., 2005). In potato culture, wild Solanum species are commonly used to introduce desirable traits to commercial genotypes. ...
Chapter
The demand of horticultural crops is booming day by day due to vagaries in consumption patterns, so the need of the hour is to develop technologies that boost the production at a rapid rate. Plant Tissue culture is one such noteworthy biotechnological tool that has its application in propagation and improvement, disease eradication, herbicide resistance, salinity tolerance, amalgamation of high nutrient content, genetically improved plants and conservation of endangered plant species. In the near future usage of this technology is going to increase further manifold. It is used for production of disease-free quality planting material and development of varieties through direct regeneration, anther/ovule culture, somatic embryogenesis etc. Plant tissue culture has to pilot notable contributions in present times and today they constitute an essential tool in the improvement of horticultural sciences and modern agriculture.
... The preparation of protoplasts, first described in 1960 [1], has since that time been tailored to the plant species, the tissue under study, and the developmental stage of the plant. As a consequence, a large number of protocols for protoplast isolation have been developed [2][3][4][5][6][7][8]. In protoplast preparation, variable factors between protocols concern mainly the components of the digestion mixture (composition and concentrations) and the incubation conditions [9][10][11][12]. ...
Article
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Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single‐cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single‐cell suspensions can be obtained through digestion of the cells walls and release of the so‐called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.
... Some studies suggested that relatively high protoplast culture densities would promote cell growth and division (Chuong et al., 1985;Kiełkowska and Adamus, 2012). It could be that growing protoplasts stimulate growth and mitotic division of adjacent cells by releasing growth factors into the medium (Davey et al., 2005). In this study, we also observed that a low protoplast density would usually result in poor protoplast viability during the early stage of cultures. ...
Article
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Field cress ( Lepidium campestre ) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L ⁻¹ NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L ⁻¹ TDZ combined with either 0.05 mg L ⁻¹ NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L ⁻¹ TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.
... Protoplast fusion and somatic hybridization offers the potential to produce novel crops and overcome breeding obstacles in polyploid and apomictic warm-season grasses [7,[119][120][121]. It could provide alternative ways to produce hybrids from sexually incompatible species and offers opportunity for intergeneric hybridization [16]. ...
Article
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Warm-season grasses are C4 plants and have a high capacity for biomass productivity. These grasses are utilized in many agricultural production systems with their greatest value as feeds for livestock, bioethanol, and turf. However, many important warm-season perennial grasses multiply either by vegetative propagation or form their seeds by an asexual mode of reproduction called apomixis. Therefore, the improvement of these grasses by conventional breeding is difficult and is dependent on the availability of natural genetic variation and its manipulation through breeding and selection. Recent studies have indicated that plant tissue culture system through somatic embryogenesis complements and could further develop conventional breeding programs by micropropagation, somaclonal variation, somatic hybridization, genetic transformation, and genome editing. This review summarizes the tissue culture and somatic embryogenesis in warm-season grasses and focus on current status and above applications including the author’s progress.
... Thus, a plant cell without a cell wall or protoplast is the most suitable transformation material as the DNA could easily pass through the cell membrane. The cell wall can be removed by either mechanical approach or enzymatic hydrolysis, which the latter method is easier to perform and widely being used for high protoplast yield [12]. Protoplasts are suitable for DNA delivery by either polyethylene glycol (PEG), electroporation, or DNA microinjection [13]. ...
Chapter
The protocol outlined in this chapter describes a detailed procedure for protoplast isolation and transformation using polyethylene glycol (PEG)-mediated transfection and DNA microinjection, highlighting also the critical steps associated with the method. Briefly, we will describe the efficient isolation of protoplasts from 3-month-old suspension calli collected at 14 days after cultured. Digestion of the calli with an optimal composition of enzyme solution yielded over 2 × 106 protoplasts/mL with the viability of more than 80%. The concentrations of DNA, PEG, and magnesium chloride and application of heat shock treatment are the crucial determinants for efficient PEG-mediated transfection. Using the optimal PEG transfection conditions, a transfection efficiency of more than 20% could be obtained. At the same time, protoplasts embedded in alginate layer cultured for 3 days and injected with 100 ng/μL of total DNA solution are the optimal factors for microinjection. We successfully regenerated the injected protoplasts to calli expressing green fluorescent protein (GFP) signals when cultured in optimal medium and cultivation procedures.
... Later, plant protoplasts were successfully used for RNP delivery in a variety of plants such as tobacco, Arabidopsis, lettuce, rice, and Petunia (Woo et al., 2015;Subburaj et al., 2016;Murovec et al., 2018;Park et al., 2019). However, for most monocot species, including major crops such as maize, wheat, rice, barley, and sorghum, regeneration of plants from protoplasts remains either unattainable or inefficient (Davey et al., 2005). ...
... Transformation is the first approach with which to genetically manipulate water chestnut. Later, as cell culture technology for this hydrophyte improves, it may be possible to extend somatic cell techniques to expose somaclonal variation, and to isolate and fuse protoplasts to effect gene transfer by generating somatic hybrid and cybrid plants (Davey et al. 2005). In the likely changing environmental conditions associated with global warming, water chestnut should be given serious consideration as an alternative to cereals in some areas. ...
Article
Water chestnut (Trapa sp.), a starch-producing hydrophyte, is a plant with potential as a secure food source, especially in wasteland regions that are prone to flooding. Information is available that relates to the morphology and taxonomy of the plant, but there is an urgent need to characterize existing germplasm at the molecular level as a basis for future breeding. Effort needs to be invested in establishing internationally recognized germplasm collections and the instigation of extensive breeding programs. Somatic cell technologies could enable the mass propagation of elite plants of water chestnut and provide a basis for longer-term genetic manipulation of water chestnut using transformation and somatic hybridization approaches.
... Protoplasts are naked living plant cells that can be obtained by enzymatic digestion of the cell walls. These are widely used for studying plant genetics, for breeding, and, more recently, for genome-editing and gene silencing technologies (Davey et al. 2005;Burris et al. 2016). In seaweeds, protoplasts can be also used as seeds for largescale farming, offering the advantage of continuous supply of plantlets without a waiting period (Gupta et al. 2018). ...
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Petalonia fascia is a widespread brown alga with economic potential due to its use as raw or dried powder, and in the bio-medical field. Protoplasts are living plant cells devoid of cell wall with a wide range of applications in basic and applied research, especially in crop improvement, and as a seedstock in seaweeds. Protoplasts have been previously isolated from P. fascia, but, their regeneration ability, an important prerequisite for protoplast applications, has not been explored. In this work, we report the protoplast isolation and successful regeneration from P. fascia using the commercially available cellulase "Onozuka" RS (1%) and alginate lyase (4 U mL −1). Protoplast production was enhanced under increased osmolarity (2512 mOsm L −1 H 2 O), with chelation pre-treatment, and short incubation time (4 h). Our protocol produced more than 14 times the number of protoplasts obtained using previously reported protocols. After 4 weeks in culture, protoplasts developed into prostrate and discoid and mixed thallus, as well as cell clumps. Blades mostly emerged from prostrate thalli. Antibiotics were not crucial for improving protoplast regeneration, and temperature did not affect the development of the morphological forms and blades. Our results show that high yields of protoplasts (10 7 protoplasts g −1 FW) with good regeneration ability can be obtained from P. fascia using a simple mixture of commercial enzymes. This represents the first report of successful protoplast regeneration in P. fascia.
... Protoplast technology has focused especially on the generation of interspecific hybrids between wild and cultivated species that cannot be produced via conventional hybridization (Davey et al., 2005). In potato culture, wild Solanum species are commonly used to introduce desirable traits to commercial genotypes. ...
Chapter
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The potato (Solanum tuberosum L.) is important tuber commercial crop in the genus Solanum, originated in the Andes, Bolivia and Peru, South America. After rice and wheat, potato is the third most important food crop in the world in terms of human consumption. Vegetative propagation using whole or cut tubers is the most common method for propagation of potato crop in developing countries. The main drawback of conventional seed production system is rapid degeneration of seed tubers by the viruses and other pathogens, low seed multiplication, larger size of tubers hence high cost of seed production and time duration is more to get breeder seed. The cultivated potato (Solanum tuberosum L., 2n = 4x = 48) is highly amenable to biotechnology. Tremendous advances and refinements in the techniques of plant biotechnology have allowed the problems of crop productivity and quality seed production. This is the transition phase of shifting whole seed production system from conventional system to hi-tech seed production system which involves in-vitro micro-propagation techniques. In-vitro multiplication of virus-free microplants and microtuber followed by minituber production in net house and aeroponic techniques is most important components of high-tech seed production which overcomes drawbacks of conventional seed production system. In this book chapter the emerging trends on propagation of potato is discussed.
... Plant protoplasts, totipotent, viable cells from which the cell walls have been enzymatically or mechanically removed, are targeted for the fusion of exogenous nucleic acids and cell organelles [1,2]. Plant protoplasts provide unique, single-cell systems for investigating the aspects of genomics, proteomics, and metabolomics [3]. With the rapid development of genome editing and gene silencing techniques, protoplasts have wide utility in technologies involving clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9. Thus, the development of an efficient protoplast preparation method constitutes a profitable and worthwhile endeavor for research on the biology and physiology of the tea plant. ...
Article
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Background Plant protoplasts constitute unique single-cell systems that can be subjected to genomic, proteomic, and metabolomic analysis. An effective and sustainable method for preparing protoplasts from tea plants has yet to be established. The protoplasts were osmotically isolated, and the isolation and purification procedures were optimized. Various potential factors affecting protoplast preparation, including enzymatic composition and type, enzymatic hydrolysis duration, mannitol concentration in the enzyme solution, and iodixanol concentration, were evaluated. Results The optimal conditions were 1.5% (w/v) cellulase and 0.4–0.6% (w/v) macerozyme in a solution containing 0.4 M mannitol, enzymatic hydrolysis over 10 h, and an iodixanol concentration of 65%. The highest protoplast yield was 3.27 × 10 ⁶ protoplasts g ⁻¹ fresh weight. As determined through fluorescein diacetate staining, maximal cell viability was 92.94%. The isolated protoplasts were round and regularly shaped without agglomeration, and they were less than 20 μm in diameter. Differences in preparation, with regard to yield and viability in the tissues (roots, branches, and leaves), cultivars, and cultivation method, were also observed. Conclusions In summary, we reported on a simple, efficient method for preparing protoplasts of whole-organ tissue from tea plant. The findings are expected to contribute to the rapid development of tea plant biology.
... Some studies suggested that higher culture densities would promote the growth and division of protoplast cells (Chuong et al., 1985;Kielkowska and Adamus, 2012). The reason for this could be that cultured protoplasts stimulate growth and mitotic division of adjacent cells by releasing growth factors into the surrounding medium (Davey et al., 2005). In this study, we also found that a low density of protoplasts could result in poor cell division and thus reduced callus formation. ...
Article
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Difficulty in protoplast regeneration is a major obstacle to apply the CRISPR/Cas9 gene editing technique effectively in research and breeding of rapeseed (Brassica napus L.). The present study describes for the first time a rapid and efficient protocol for the isolation, regeneration and transfection of protoplasts of rapeseed cv. Kumily, and its application in gene editing. Protoplasts isolated from leaves of 3-4 weeks old were cultured in MI and MII liquid media for cell wall formation and cell division, followed by subculture on shoot induction medium and shoot regeneration medium for shoot production. Different basal media, types and combinations of plant growth regulators, and protoplast culture duration on each type of media were investigated in relation to protoplast regeneration. The results showed that relatively high concentrations of NAA (0.5 mg l −1) and 2,4-D (0.5 mg l −1) in the MI medium were essential for protoplasts to form cell walls and maintain cell divisions, and thereafter auxin should be reduced for callus formation and shoot induction. For shoot regeneration, relatively high concentrations of cytokinin were required, and among all the combinations tested, 2.2 mg l −1 TDZ in combination with auxin 0.5 mg l −1 NAA gave the best result with up to 45% shoot regeneration. Our results also showed the duration of protoplast culture on different media was critical, as longer culture durations would significantly reduce the shoot regeneration frequency. In addition, we have optimized the transfection protocol for rapeseed. Using this optimized protocol, we have successfully edited the BnGTR genes controlling glucosinolate transport in rapeseed with a high mutation frequency.
... Moreover, they might need specific requirements such as the addition of PSK (Phytosulfokin) to culture medium used in such protoplasts to stimulate the division of protoplasts (Grzebelus et al., 2012). Additionally, protoplasts density might be reason since the cultured density affected the cell wall regeneration and division (Davey et al., 2005). ...
... Although they are usually produced from plant cells, protoplasts can also be obtained from bacteria [1] and fungi [2]. Protoplasts represent a powerful experimental material for in vivo manipulations, offering a wide range of applications for basic and applied research, including genome-editing and gene silencing techniques, and crop improvement bypassing sexual reproduction from species of economic importance [3][4][5]. The success of this technology relies on reproducible protocols for protoplast isolation, which are mainly achieved by using commercial enzymes [6,7]. ...
Article
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Background Protoplasts (i.e., naked plant cells) can be used for in vitro manipulations and genetic improvement in cultivars with economic value. During the last decade, protoplast research in economic brown algae has been scarce, and it is usually hampered by the use of non-commercial enzymes or crude extracts for isolating protoplasts. Dictyopteris pacifica is part of a brown algal genus well known by its wide chemical diversity and biological properties. Scytosiphon lomentaria is an edible brown seaweed with antioxidant, antitumor, and antiviral properties. So far, there are no protoplast isolation protocols using commercial enzymes for these two economic brown algae. In this study, we obtained protoplasts from cultured samples of D . pacifica and S . lomentaria using commercially available enzymes. Additionally, we investigated the effects of Driselase inclusion and Ca-chelation pre-treatment on protoplast yields in order to optimize the conditions for protoplast preparations. Results Protoplasts were isolated from Dictyopteris pacifica and Scytosiphon lomentaria using the commercially available Cellulase Onozuka RS (1%) and Alginate lyase (4 U mL ⁻¹ ), and short incubation time (4 h). Driselase did not show significant effects on protoplast production in both species. Ca-chelation pre-treatment only increased the number of protoplasts in D . pacifica . Under optimal conditions, the protoplast yields from D . pacifica and S . lomentaria were 4.83 ± 2.08 and 74.64 ± 32.49 × 10 ⁶ protoplasts g ⁻¹ fresh weight, respectively. The values obtained for S . lomentaria were 2–3 orders of magnitude higher than previously reported. Conclusions Our results show that high protoplast yields can be obtained from D . pacifica and S . lomentaria using a simple mixture of commercial enzymes (Cellulase RS and Alginate lyase) and short incubation time (4 h). This work also represents the first report of protoplast isolation in D . pacifica . The method proposed here can help to expand protoplast technology in more brown algal species.
Article
Hydrogel encapsulation has been widely utilized in the study of fundamental cellular mechanisms and has been shown to provide a better representation of the complex in vivo microenvironment in natural biological conditions of mammalian cells. In this review, we provide a background into the adoption of hydrogel encapsulation methods in the study of mammalian cells, highlight some key findings that may aid with the adoption of similar methods for the study of plant cells, including the potential challenges and considerations, and discuss key findings of studies that have utilized these methods in plant sciences.
Article
Cannabis sativa L. is a valuable, up-and-coming industrial crop with a substantially growing market. However, due to an extended period of legal restriction, research with cannabis has been limited, particularly in laboratory settings. Expanding the application of biotechnological techniques to cannabis can facilitate addressing species-specific impediments to improving crop traits and further fundamental understanding of its intricacies. Here, we describe application of protoplast transformation for the study of transient gene expression in a low-THC cannabis cultivar. To produce explant tissue as a source of protoplasts, a method for hormone-free in vitro micropropagation is established. Protoplasts are isolated from young leaves of the micropropagated stocks and transiently transformed with plasmid DNA carrying a fluorescent marker gene. This is the first report of protoplast transformation in this species. A protoplast isolation yield is achieved of up to 2 × 10⁶ cells per gram of leaf material, vitality staining shows that up to 82 % of isolated protoplasts are viable, and quantification of the cells expressing a fluorescent protein indicates that up to 31 % of the cells can be successfully transformed. Additionally, protoplasts are transformed with an auxin-responsive reporter gene and the reaction to treatment with indole-3-acetic acid is quantified using flow cytometry. This work demonstrates that relatively minor modification of standard techniques can be used to study this important emerging crop.
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إن نمو فكرة تأليف هذا الكتاب، الذي يتناول احد المكونات المهمة في الخلية، تبلورت اثناء تدريسنا في السنوات العشرة الماضية مجموعة من موضوعات التقانات الحيوية الحديثة في النبات ضمن برامج الدراسات الاولية والعليا. وفضلاً عن ذلك فقد انجزت في مختبرات وحدة التقنيات الحياتية مجموعة من البحوث في هذه الموضوعات ونشرت في مجلات عالمية وعربية ومحلية متخصصة، كما اجيزت مجموعة من اطاريح الدكتوراه والماجستير في كليات التربية والعلوم والزراعة ضمن هذه الموضوعات ايضاً. وجاء اصطفاء هذا المؤلَف الذي افترضت هيكليته بصورة مناسبة للطلبة والباحثين مع عرض المصادر السابقة والتقانات المعتمدة بطريقة ضامنة للاستفادة والانتفاع منها. وان المراجع المذكورة تتيح للقارئ سهولة ايجاد الموضوعات التي يرغبها، وتمثل نخبة من المصادر (الحاكمة) السابقة التي لابد من الاشارة اليها وبالأخص في مجال علم النبات والتقنية الحيوية. وكيفية استخدام البروتوبلاست في دراسات بناء البروتين والاحماض النووية RNA، DNA، وفي استقطاب المواد الغريبة متمثلة بالبلاستيدات الخضراء والمايتوكوندريا والبكتريا والفايروسات. وايضاً تضمين الحامض النووي DNA او الحامض النووي البلازميدي P-DNA والتي تهدف الى تحسين النوع النباتي من خلال نقل بعض العناصر الوراثية الى الذخيرة الوراثية (Genome) للبروتوبلاست بواسطة تقانات التثقيب الكهربائي، الحقن المجهري الدقيق، الاندماج الخلوي، القذائف البيولوجية، الزراعة المرافقة، النواقل البكتيرية والتي جميعاً تهدف الى انتاج نباتات معدَلة وراثياً تتمتع بصفات محسنة. وقد عكف المؤلفًين على اعتماد فيض من المصادر المتآلفة مع هذه الموضوعات واخرى اشارت فقط الى المعلومات ذاتها. إن التطور الحاصل في انظمة البروتوبلاست النباتي احتل مشروعات البحوث والدراسات عبر السنوات الاخيرة. فقد اقيمت العديد من المؤتمرات العلمية وورش العمل وصدَرت مجموعة من المراجع والمقالات التي تتفاعل وتتعامل مع هذه الانظمة النباتية وبدأت تحتل ذات مكانتها كأنظمة مستقلة بعيدة عن الخلايا النباتية التي هي اساس ووحدة البناء في النبات.
Chapter
We have developed an optimized protocol for isolating protoplasts from chlorenchyma cells of the single-cell C4 species Bienertia sinuspersici. The isolated protoplasts maintained the integrity of the unique single-cell C4 intracellular compartmentation of organelles as observed in chlorenchyma cells after cell wall digestion. Approximately over 80% of isolated protoplasts expressed the fusion reporter gene following the polyethylene glycol-mediated transfection procedures. Overall, fluorescent protein fusion tagged with various intraorganellular sorting signals validated the potential use of the transient gene expression system in subcellular localization and organelle dynamics studies.
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Key message Floral thermogenesis is an important reproductive strategy for attracting pollinators. We developed essential biological tools for studying floral thermogenesis using two species of thermogenic aroids,Symplocarpus renifolius and Alocasia odora. Abstract Aroids contain many species with intense heat-producing abilities in their inflorescences. Several genes have been proposed to be involved in thermogenesis of these species, but biological tools for gene functional analyses are lacking. In this study, we aimed to develop a protoplast-based transient expression (PTE) system for the study of thermogenic aroids. Initially, we focused on skunk cabbage (Symplocarpus renifolius) because of its ability to produce intense as well as durable heat. In this plant, leaf protoplasts were isolated from potted and shoot tip-cultured plants with high efficiency (ca. 1.0 × 10⁵/g fresh weight), and more than half of these protoplasts were successfully transfected. Using this PTE system, we determined the protein localization of three mitochondrial energy-dissipating proteins, SrAOX, SrUCPA, and SrNDA1, fused to green fluorescent protein (GFP). These three GFP-fused proteins were localized in MitoTracker-stained mitochondria in leaf protoplasts, although the green fluorescent particles in protoplasts expressing SrUCPA-GFP were significantly enlarged. Finally, to assess whether the PTE system established in the leaves of S. renifolius is applicable for floral tissues of thermogenic aroids, inflorescences of S. renifolius and another thermogenic aroid (Alocasia odora) were used. Although protoplasts were successfully isolated from several tissues of the inflorescences, PTE systems worked well only for the protoplasts isolated from the female parts (slightly thermogenic or nonthermogenic) of A. odora inflorescences. Our developed system has a potential to be widely used in inflorescences as well as leaves in thermogenic aroids and therefore may be a useful biological tool for investigating floral thermogenesis.
Chapter
Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.
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In contrast to animals, plants cannot avoid unfavorable temperature conditions. Instead, plants have evolved intricate signaling pathways that enable them to perceive and respond to temperature. General acclimation processes that prepare the plant to respond to stressful heat and cold, usually occur throughout the whole plant. More specific temperature responses, however, are limited to certain tissues or cell types. While global responses are amenable to epigenomic analyses, responses which are highly localized are more problematic as the chromatin in question is not easily accessible. Here we review the current knowledge of the epigenetic regulation of FLOWERING LOCUS C and FLOWERING LOCUS T as examples of temperature-responsive flowering time regulators that are expressed broadly throughout the plants and in specific cell types, respectively. While undoubtably extremely successful, we reason that future analyses would benefit from higher spatiotemporal resolution. We conclude by reviewing methods and successful applications of tissue- and cell type-specific epigenomic analyses and provide a brief outlook into the future, single-cell epigenomics.
Chapter
Citrus is a very nutritious fruit that is consumed worldwide, making it one of the most economically important fruits. Like other crops, citrus must cope with multiple biotic and abiotic stressors in its growing environment. These stressors represent obstacles which must be overcome by horticultural improvement programs. Conventional breeding and genetic transformation are the most popular techniques for citrus improvement. Because conventional breeding has several limitations, biotechnology-mediated strategies are being increasingly used, allowing better management of genetic resources and faster development of superior genotypes. Gene transfer in citrus demands reproducibility as well as effective genetic transformation and plant regeneration protocols. Although Agrobacterium-mediated transformation is the main method of citrus genetic modification, other direct gene transfer techniques have also attracted attention recently. Together, these methods have produced promising results against abiotic and biotic stresses which affect citrus production and has also helped in enhancing yield and fruit quality. Nevertheless, novel techniques are constantly being developed for increasing the efficiency of citrus improvement efforts. Currently, CRISPR/Cas9 is the most efficient genome-editing tool with good potential for developing edited citrus plants. In this chapter, we present the tools and techniques used with citrus biotechnology. We also highlight the efforts of the scientific community toward the generation of genetically modified citrus plants.
Chapter
Carrot (Daucus carota) is a useful plant model for the study of carotenoid biosynthesis, specifically in roots which are enriched in carotenoids. Carrot genome and transcriptome sequences, complemented by optimized methods for carrot transformation, contribute to a comprehensive toolbox for exploring pathway regulation. To expand the repertoire of tools available for the study of D. carota, we present protocols for the isolation of protoplasts from D. carota cell suspension cultures and polyethylene glycol (PEG)-mediated transformation. To obtain carrot protoplasts, in vitro somatic embryogenesis from epicotyls is induced. The somatic embryogenic tissue that develops is transferred to liquid medium to obtain a suspension of cells which are homogenized and incubated with cell-wall degrading enzymes to release protoplasts. For transfection, protoplasts are incubated with a plasmid encoding a protein of interest prior to examination of protein localization by light microscopy. As an example, we demonstrate nuclear localization of a carrot transcription factor, DcAREB3.
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Embriogeneza somatyczna jest jedną z najważniejszych technologii mikrorozmnażania odmian palmy daktylowej. Ostatnio dokonano znacznego postępu w rozwoju i optymalizacji tej techniki z kultur zawiesinowych komórek embriogennych. Ta książka opisuje procedury szybkiego rozwoju dużej liczby zarodków somatycznych z embriogenicznych kultur zawiesin komórkowych, opisuje także efektywny protokół mikrorozmnażania z embriogenezy somatycznej z kultur zawiesin komórkowych począwszy od eksplantatów z końcówki pędu do aklimatyzacji roślin oraz innowacyjne informacje na temat ostatnich postępów w kulturach zawiesinowych palmy daktylowej.
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Somatic hybridisation in the carrot, as in other plant species, enables the development of novel plants with unique characteristics. This process can be induced by the application of electric current to isolated protoplasts, but such electrofusion requires an effective hybrid cell identification method. This paper describes the non-toxic fluorescent protein (FP) tagging of protoplasts which allows discrimination of fusion components and identification of hybrids in real-time during electrofusion. One of four FPs: cyan (eCFP), green (sGFP), yellow (eYFP) or the mCherry variant of red FP (RFP), with a fused mitochondrial targeting sequence, was introduced to carrot cell lines of three varieties using Agrobacterium-mediated transformation. After selection, a set of carrot callus lines with either GFP, YFP or RFP-labelled mitochondria that showed stable fluorescence served as protoplast sources. Various combinations of direct current (DC) parameters on protoplast integrity and their ability to form hybrid cells were assessed during electrofusion. The protoplast response and hybrid cell formation depended on DC voltage and pulse time, and varied among protoplast sources. Heterofusants (GFP + RFP or YFP + RFP) were identified by detection of a dual-colour fluorescence. This approach enabled, for the first time, a comprehensive assessment of the carrot protoplast response to the applied electric field conditions as well as identification of the DC parameters suitable for hybrid formation, and an estimation of the electrofusion success rate by performing real-time observations of protoplast fluorescence.
Article
Over the past few decades, additive manufacturing, commonly known as three-dimensional (3D) printing, has proven to be a powerful innovation in numerous scientific areas. Thanks to continuous and progressive advances, 3D printing has demonstrated tremendous possibilities offered for tissue engineering and regenerative medicine, where complex structures of living cells and biocompatible materials were shaped into functional tissues and organs. Unlike animal cells, the use of green plant cells (green algae and land plants cells) for 3D bioprinting is still at its very early stage. The main feature that could be explored by green plant 3D bioprinting is the totipotency of the land plant cells. This asset enables any given part of the plant to regenerate a complete individual, identical or almost identical to the original one. Moreover, due to their high growth rate, green algae appear to be a handy tool to characterize the process of printing plant cells, called “Green bioprinting”. Successful transfer of 3D bioprinting know-how to green plants could lead to a breakthrough in the understanding of the so-called in planta behavior, but also in innovation within plant-based industry as for food industry or bioproduction of active biomolecules or plant-based (bio)materials. Here we review the use of 3D bioprinting in plant science and biotechnology and we define the requirements to achieve plant cell 3D bioprinting by microextrusion. On the other hand, we discuss current and future applications, as well as the limitations that may be encountered.
Chapter
Plant secondary metabolites are biosynthetically derived from primary metabolites. Recent surveys have established that nearly 35% of molecules in the pharmaceutical industry are of plant origin. The production of plant secondary metabolites for a long period of time was relayed on natural stands of the source plant or cultivation in the field. This poses problems such as increasing pressure on naturally growing plant resources, consequent depletion of plants, the geographic variation that is causing inconsistency in the total content of metabolites, seasonal variation in metabolite content, and growth of plants. Also, a large area is to be maintained for raising resources for the industrial level extraction of metabolites. Scientists and biotechnologists have considered plant cell, tissue, and organ cultures as an alternative way to produce the corresponding secondary metabolites. Plant cell culture technology deals with the in vitro culture of plant cells for the production of specific metabolites. Different strategies, using in vitro systems, have been explored for improving the production of secondary metabolites. Alternate strategies such as in vitro mutagenesis and induction of polyploids can also be explored to achieve enhanced productivity of metabolites. It is now possible to manipulate the pathways that lead to enhanced production of secondary metabolites. Successful cloning of entire genes in a pathway to plasmid vectors and expression in microbial systems, makes simple and highly efficient metabolite production system in future.
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The Colorado potato beetle (CPB) is a major insect pest that is controlled mainly through the use of pesticides. Development of potato clones with multiple forms of host plant resistance may provide a stable alternative or supplemental form of CPB control. Tetraploid hybrids were developed by somatic fusion of diploid interspecific Solanum clones with different forms of resistance to CPB. Hybrids were created between a clone containing leptine glycoalkaloids and four clones producing glandular trichomes. One fusion produced vigorous hybrids that were analyzed for CPB resistance traits. Somaclonal variation among hybrids was detected for trichome density and resistance to feeding by adult and larval beetles. Somatic hybrids were less resistant than the parents in adult feeding preference trials, but several were more resistant than either parent in larval feeding trials. Future studies are needed to determine whether clones producing both glandular trichomes and leptines express resistance that is more stable than that of clones with only one resistance factor. The Colorado potato beetle (CPB) (Leptinotarsa decemlineata Say) is the major defoliator of potato and, in some cases, can be the limiting factor for potato production. This insect is largely controlled by chemical means, which is expensive and potentially damaging to the environment. Furthermore, some CPB populations have developed resistance to sev-eral major classes of insecticides (Mowry and Sandvol, 1995). Utilization of host plant resis-tance offers a feasible alternative or supple-ment to chemical insect control, but is difficult because modern potato cultivars have a nar-row genetic base and do not offer adequate genetic variability for selection of insect-re-sistant types (Sanford et al., 1984). However, sources of resistance have been found in some wild Solanum species.
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A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.
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In Rorippa indica, a wild crucifer, high-frequency plant regeneration through organogenesis (∼38%) was achieved from in vitro-grown leaf explants. When the same-aged in vitro-grown leaves were utilized for protoplast isolation, ∼4 × 106 protoplasts g-1 leaf tissue were obtained after digestion with an enzyme solution containing cellulase and macerozyme. A maximum of ∼66% first division, ~∼3% second division and ∼35% plating efficiency were obtained when protoplasts were plated at 5 × 104 protoplasts ml-1 plating density. All the 528 protoplast-derived calli tested for regeneration exhibited shoot organogenesis. Regenerated shoots were transferred to soil after rooting and hardening. Chromosome analysis of microspore mother cells from pot-grown protoplast regenerated plants showed that all were diploid (2n = 32).
Article
Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.
Book
Electrical Manipulation of Cells provides an authoritative and up-to-date review of the field, covering all the major techniques in a single source. The book features broad coverage that ranges from the mechanisms of action of external electrical fields on biological material to the ways in which electrical stimuli are employed to manipulate cells. Bringing together the work of leading international authorities, the book covers membrane breakdown, gene delivery, electroporation, electrostimulation, cell movement, hybridoma production, plant protoplasts, electrorotation and stimulation, and electromagnetic stimulation. For each topic, the authors discuss the relevance of the approach to the current state of the art of biotechnology. Electrical Manipulation of Cells is an unmatched source of information for anyone involved in the manipulation of cells, particularly biotechnologists, cell biology, microbiologists, biophysicists and plant scientists. For researchers, the book provides technical material that ccan be employed in their own work. Students will gain thorough appreciation of the applications of this important technique.
Chapter
During the last decade, the isolation and culture of protoplasts has been described for grain legumes of the genera Cajanus,Pisum and Vigna, while in forage legumes, protoplasts of Astragalus, Medicago, Onobrychis and Trifolium have received most attention. Reports on the transformation of legume protoplasts are limited. Somatic hybridisation has been directed towards the grain legumes Lathryus, Pisum and Vigna, with Lotus,Medicago, Onobrychis and Trifolium being targeted in forage crops. Several reports have described the use of legume protoplasts as experimental material in physiological investigations and in studies of plant-pathogen interactions.
Article
Protoplasts of Valencia sweet orange ( Citrus sinensis Osb.),irradiated by X-ray with a dose rate of 3.8 krad/min for 45 min, were electrically fused with protoplasts of Murcott tangor ( C. reticulata x C. sinensis) that were treated with 0.25 mmol/L iodoacetic acid for 15 min. It took nearly 15 months for the fusion-derived calli to develop into embryoids that were only originated in the medium of MT supplemented with 2% glycerol. The shoots were recalcitrant to rooting in the root-induction medium. In vitro grafting was employed to produce whole plants though one self-rooting plant was obtained. Cytological determination of root and shoot tips showed mainly diploid and aneuploid cells, together with few tetraploid cells in some plants. RAPD (random amplified polymorphism DNA) analysis with 10-mer primers demonstrated that bands specific to the fusion parents were detected in the regenerated plants, indicating that interspecific somatic hybrids have been obtained via protoplast asymmetric fusion in Citrus.
Article
The gaseous phytohormone ethylene regulates many aspects of plant morphogenesis. Growth and development of cells cultured in vitro are largely dependent on the presence of phytohormones, including ethylene in the culture environment. Hence, modification of phytohormone composition and interaction in the nutrient medium has been the primary strategy to manipulate morphogenesis in vitro. Such studies have shown the importance of ethylene, as well as the inhibition of its synthesis or action, in growth and organized development in, vitro, including xylogenesis, organogenesis, somatic embryo genesis, and androgenesis. More recently, mutants and transgenic plants have been used to elucidate the role of ethylene in various cellular and developmental processes. In this review, we concentrate on the more recent advances in the study of ethylene in plant morphogenesis in vitro. We also include information about the various chemical modulators of ethylene biosynthesis and action employed in plant tissue culture.
Chapter
Some cells can detect voltage gradients as low as 0.5 μV/m and current densities of as little as 5 nA/cm2. These extremely sensitive electrosensing mechanisms are used by animals for navigation and to find prey and by plants for predicting the availability of water. They probably evolved from somewhat less-sensitive mechanisms used by most cells for sensing the weak currents that flow within organisms to control their growth. The artificial application of weak electrical currents can sometimes interact with these to stimulate growth, the healing of injuries, and the regeneration of organs. Examples are given, the mechanisms of the effects discussed, and a hypothesis presented for their likely evolution.
Chapter
High-voltage, short-duration electrical pulses stimulate DNA synthesis in isolated higher plant protoplasts. They also promote the growth of protoplast-derived cells, and shoot regeneration from protoplast-derived tissues. Such effects of electrostimulation persist over many cell generations. This enhancement of growth and organogenesis has application in the multiplication of elite individuals and in maximizing the recovery of genetically engineered plants following somatic hybridization and transformation. Detailed knowledge of the precise mechanisms of action of electrical pulses on the stimulation of growth and morphogenesis in plant cells are still lacking. The possible synergistic effects of electrical and chemical parameters require further investigation.
Article
RFLP (restriction fragment length polymorphism) was employed to analyze cytoplasmic genome of diploid somatic hybrid plant, morphologically similar to rough lemon which was leaf parent, that was produced via protoplast fusion between rough lemon (Citrus jambhiri Lush) and Hamlin sweet orange (C. sinensis Osb.), the embryogenic parent. Three enzyme-mitocondrial probe combinations and one enzyme-chloroplast probe combination demonstrated that the plant had identical band profiles to Hamlin sweet orange as far as mtDNA and cpDNA were concerned.
Article
The effect of glycine, at several different concentrations, on the yield of viable protoplasts derived from hypocotyls of Cucumis melo cv. Fastoso and Green Delica and Cucumis metuliferus was investigated. A glycine concentration of 0.05 M in the enzyme solution was optimal for protoplast isolation of C. melo cv. Fastoso. However, glycine had no significant effect on protoplast isolation from C. melo cv. Green Delica. With C. metuliferus, 0.1 M glycine was found to be optimal for the isolation of protoplasts. Protoplasts were obtained in high yield from both hypocotyl and cotyledon tissue. The yield from cotyledon tissue of C. melo Green Delica as well as C. metuliferus was optimised by dark pretreatment for four days on medium containing 1.1 μM naphthalene acetic acid plus 4.6 μM kinetin. Subsequent work on plating efficiency of the protoplasts showed that only the cotyledon-derived protoplasts from C. melo cv. Green Delica and C. metuliferus were able to undergo cell division. Dividing protoplasts from C. melo cv. Green Delica progressed to form microcolonies.
Article
Asymmetric hybridization was conducted between wheat and Bromus inermis Leyss which is a distantly related intergeneric plant (belonging to different tribe) of wheat and possesses some favorable traits, such as resistant to cold, drought and disease. Protoplasts isolated from young embryo-derived calli of common wheat (Triticum aestivum L., cv. 99P, (AABBDD), 2n = 42) were fused with UV-treated protoplasts isolated from young embryo-derived calli of Bromus inermis by PEG method. Three clones (No. 1 - No.3) were regenerated from the fusion products and differentiated into albino seedlings. The clones and the seedlings were all verified as hybrids by chromosome counting, isozyme and RAPD analysis. Their isozyme and RAPD pattern contained the characteristic bands of both parents as well as new band(s) . The chromosome numbers of albino were in the range of 42-54 with small chromosomes of Bromus inermis and chromosome fragments. The above results confirmed that hybrid albinos were obtained.
Article
Plant tissue culture is recognised as a disposable tool to generate useful genetic variability for crop improvement. Nine somaclones of tetraploid potato cv. Bintje regenerated from stem, tuber or protoplast-derived callus were analysed in vitro and in the field. All of them were morphological and cytological chimeras. They had alterations in general appearance, leaf morphology and tuber characteristics. The phenotypic variations of the first vegetative progeny (SC2 plants) were in most cases different in comparison with the primary regenerants (SC1 plants). The observed phenotypic instability was in correlation with chromosome mosaic ism (chromosomal instability) as aneuoploid and polyploid cells were detected at high frequency (57-89 %) in root tips of all somaclons investigated.
Article
Comparison between second generation of functional cybrid (containing Solanum nigrum genome and Solanum tuberosum plastome) and original (S. nigrum) plants was used to study the effect of nucleus/chloroplast interaction on the physiology of the respective cybrid. Percentage of seed germination, response of shoot apices to form adventitious shoots on B5 medium supplemented with 5% liquid coconut endosperm and 2.5 M BAP, and morphological characters of cybrid were similar to that of the original plant. The cybrid shoots were light green and bleached under atrazine stress while the original plant shoots were dark green and were not affected by atrazine. Fresh and dry matter as well as carbohydrate contents of cybrid shoots were lower than those of original plant, generally, these values were reduced under atrazine stress. Protein and sodium contents remained unaffected under chloroplast substitution and atrazine stress. Potassium content of cybrid was higher than those of corresponding original plant, but with a tendency to be lowered under atrazine stress. X-ray microanalysis data showed differences in ion contents between the cybrid and original plant on atrazine free medium or under atrazine stress.
Article
Cyclophilins were first identified as intracellular targets of the immunosuppressant cyclosporin A. Using a cDNA library constructed from petunia petal protoplast culture cells, we have isolated cDNAs corresponding to three different cyclophilins and the corresponding genes have been designated PhCyP1, PhCyP2 and PhCyP3 (Petunia hybrida Cyclophilin). Here we report expression patterns of these three genes by Northern analysis and by RT-PCR. PhCyP1 and PhCyP3 transcripts were expressed in all plant tissues (root, stem, leaf, and flower) examined, although PhCyP1 showed much stronger expression than PhCyP3. Examination of expression patterns in whole plants showed that PhCyP transcripts were predominantly expressed at the early stage of floral development. The accumulation of PhCyP1 mRNA in petal protoplast cultures increased after 1 day and reached a maximum level at 4 days of culture. These findings further suggest that cyclophilins might play a functional role in actively growing cells and plant growth.
Article
Hybrid plants were obtained from protoplast fusion between Triticum aestivum L. (2n = 42) and Agropyron elongation (2n = 70) via PEG method, but they did not produce seeds. The ovaries of hybrid plants were used to induce hybrid calli again from which was followed by plant regeneration. The hybrid characteristics of the calli and plants were determined by chromosome counting and analysis of esterase isozyme. The results revealed that both of them still retained the hybrid nature. Two of these hybrid plants survived and produced seeds after they were transported into soil. The analysis of phenotype, chromosome, isozyme and RAPD of F1 plants again proved their hybridity thus indicating that fertile hybrid plants were produced. Chromosome fragments appeared in the root cells of F1 and F2 plants. The analysis of PMCs of F2 plants revealed that the range of chromosomal number were 18 II - 22 II and pairing or segregating chromosomal fragments were observed, confirming that the chromosomal fragments were minichromosomes. The hybrid F1 and F2 plants grew vigorously, the stalks were strong, the ears and grains were bigger than parent wheat ("Jinan 177"). Now a lot of F2 spike lines are growing and further analysis will be underway.
Article
Yields of 106-107 apricot mesophyll protoplasts g fw-1 were obtained depending on factors such as plasmolysing pretreatment, digesting enzymes and digestion time. Onozuka R-10 (1%) in combination with Pectolyase Y-23 (0.1%) and Hemicellulase (1%) was found best for protoplast isolation among several enzyme combinations tested. Viability was 83% with this enzyme combination. Plasmolysis of leaves for 90 min in a 13% sorbitol solution greatly increased the number of protoplasts obtained. Optimum incubation time of 13-16 h produced the best combination of yield and viability. During the purification of protoplasts a critical step was the centrifugation of the sucrose gradient at 75 g with a dramatic decrease in the recovery of protoplasts at lower and higher centrifugation speeds. Protoplasts were cultured under different media composition and growth regulator combinations. Limited growth and division of protoplasts embedded in agarose drops were obtained.
Article
A procedure using a fungal pectinase was developed to rapidly release mesophyll cells from tobacco leaves. Fifty to ninety per cent of the released cells were morphologically intact and were converted into spherical protoplasts by cellulase treatment. Cells isolated from tobacco mosaic virus-inoculated leaves supported multiplication of the virus during subsequent incubation.
Chapter
Plant protoplasts, when released from the confines of their cell walls by enzymatic digestion, will readily fuse following chemical and/or electrical treatment. The fusion of protoplasts from somatic cells has resulted in the generation of novel somatic hybrid plants, circumventing sexual incompatibility, particularly in inter-generic and inter-specific combinations. Thus, both nuclear and organellar genomes can be combined to generate novel nuclear-organellar genomic combinations in fusion products (heterokaryons), heterokaryon-derived tissues and regenerated plants. Theoretically, the plants regenerated from the fusion of two diploid somatic (cell) protoplasts should be tetraploid. In practice, somatic hybrid plants may have the chromosome complement of one parent with the addition of a few chromosomes from the other fusion partner, or complements which exceed the summation of those of the two parents (Ward et al.,1994). In general, it is comparatively rare to find chromosome complements which are a simple summation of those of the two parents. At the organellar level, the chloroplasts of one partner usually dominate; plastids of the other partner are normally eliminated. However, on rare occassions, a mixed population of plastids may persist in heterokaryon-derived cells and regenerated plants, or plastid DNA recombination may give rise to “new” organelles (Medgyesy et al., 1985). At the mitochondrial level, DNA recombination is common. The transfer of organelles with associated traits, such as mitochondrial DNA encoded cytoplasmic male sterility or chloroplast-encoded herbicide (e.g., atrazine) or antibiotic (e.g., streptomycin) resistance, can be promoted by irradiation of one of the fusion partners to fragment its nuclear genome. Thus, the irradiated partner acts, effectively, as a donor of organellar genomes, resulting in the production of cytoplasmic hybrids (cybrid) cells from which plants can be regenerated. In both somatic hybridization and cybridization, protoplast fusion constitutes the initial process, followed by selection of heterokaryon-derived cells and tissues which are induced to regenerate intact plants. Several selection systems have been developed to recover hybrid and cybrid tissues. These include simple heterokaryotic effects and media to encourage the growth of heterokaryons and hybrid cells (Davey and Kumar, 1983), the use of biochemical mutants sometimes in combination with expression of genetic markers (e.g., antibiotic resistance), micro-manipulation (Gilmour et al., 1987) and high technology flow cytometry (Hammatt et al., 1990).
Article
No presently available rootstock combines all the available rootstock attributes necessary for efficient long-term citriculture (production and harvesting) of Mexican limes and other commercially important scions. In the present study, somatic hybridization techniques were used to combine the widely adapted Amblycarpa mandarin (also known as Nasnaran mandarin) with six different trifoliate/trifoliate hybrid selections: Benton, Carrizo, and C-35 citranges; Flying Dragon and Rubidoux trifoliate oranges; and a somatic hybrid of sour orange + Flying Dragon. The ultimate goal of this research is to generate polyploid somatic hybrids that express the complementary horticultural and disease resistance attributes of the corresponding parents, and have direct potential as improved tree-size controlling rootstocks. Somatic hybrids from all six parental combinations were confirmed by a combination of leaf morphology, flow cytometry, and randomly amplified polymorphic DNA (RAPD) (for nuclear hybridity) and cleaved amplified polymorphic sequence (CAPS) analyses (for mtDNA and cpDNA). This is the first report of citrus somatic hybridization using Amblycarpa mandarin. Unexpected hexaploid somatic hybrid plants were recovered from the fusion of Amblycarpa mandarin + C-35 citrange. Hexaploid hybrids should be very dwarfing and may have potential for producing potted ornamental citrus. Resulting somatic hybrid plants from all six combinations have been propagated by tissue culture and/or rooted cuttings and are being prepared for commercial field evaluation for their potential as improved rootstocks for Mexican lime and other important scions.
Article
Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.
Article
We previously showed that during protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197–205). We have now further tested if the expression of totipotency in protoplasts is related to the activity of cellular antioxidant machinery during protoplast culture. Totipotent (T) tobacco protoplasts had 2-fold lower contents of intracellular O2.− and H2O2 and 7-fold lower levels of O2.− and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2.−, resulted in isolation of tobacco protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine protoplasts during culturing, although higher contents of O2.− and H2O2 were still found in NT- compared with T-tobacco protoplasts, after 8 d in culture. In T-tobacco protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine protoplasts, the oxidized forms predominated. In addition, T-tobacco protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in protoplasts is correlated with reduced activity of the cellular antioxidant machinery.
Article
Although nonsymbiotic hemoglobins (Hbs) are found in different tissues of dicots and monocots, very little is known about hb genes in monocots and the function of Hbs in nonsymbiotic tissues. We report the cloning and analysis of two rice (Oryza sativa L.) hb genes, hb1 and hb2, that code for plant Hbs. Rice hb1 and hb2 genes contain four exons and three introns, as with all of the known plant hb genes. At least three copies of the hb gene were detected in rice DNA, and analysis of gene expression shows that hb1 and hb2 are expressed in leaves but only hb1 is expressed in roots. A cDNA for rice Hb1 was expressed in Escherichia coli, and the recombinant Hb (rHb1) shows an unusually high affinity for O2 because of a very low dissociation constant. The absorbance spectra of the ferric and deoxyferrous rHb1 indicate that, in contrast to symbiotic Hbs, a distal ligand is coordinated to the ligand-binding site. Mutation of the distal His demonstrates that this residue coordinates the heme Fe of ferric and deoxyferrous rHb1 and stabilizes O2 in oxy-rHb1. The biochemical properties of rice rHb1 suggest that this protein probably does not function to facilitate the diffusion of O2.
Article
Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l-1 myo-inositol, 4.4 M BA, and 1.4 M 2,4-{D}) at a density of 5×104 protoplasts ml-1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l-1 myo-inositol was replaced with the same osmolarity of 90 g l-1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l-1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.
Article
When Vicia hajastana Grossh. cells or protoplasts were cultured at a high population density (ca. 5000 cells or protoplasts/ml), they were able to grow in a mineral-salt solution supplemented with sucrose (or glucose), a few vitamins, and 2,4-dichlorophenoxyacetic acid. They were not able to survive when cultured at a low population density unless the medium was supplemented with zeatin, naphthalene-1-acetic acid, nucleic-acid bases, amino acids, other sugars, sugar alcohols, and organic acids. Vicia cells were able to grow at an initial population density of 25-50 cells/ml in this defined medium. The population density could be lowered to 1-2 cells/ml with good growth when the mineral-salt medium was enriched with organic acids, sugars, sugar alcohols, coconut water, and casamino acids. The protoplasts also grew best in a medium enriched with these supplements. Three individual protoplasts were isolated and each one was cultured in a separate dish containing 4 ml of this medium. Within 30-40 days, each one had grown indefinitely and formed a mass of cells (ca. 10(7)).
Article
Nucellar callus lines were established from two orange cultivars ('Nucellar Shamouti', 'Shamouti Landau'), three mandarin cultivars ('Murcott', 'Dancy', 'Ponkan') one grapefruit cultivar ('Duncan') and sour orange (Citrus aurantium). These callus lines were initiated from in vitro cultured ovules of young fruits and maintained an embryogenic capacity. The plating efficiencies of protoplasts derived from these calli, as well as those of protoplasts from lemon (cv. 'Villafranca') nucellar callus were differentially affected by the maceration enzymes and by the sugars used as osmotic stabilizers. Plants with normal morphological features were regenerated from cultured protoplasts derived from each of the nucellar callus lines. The establishment of eight new protoplast systems in Citrus paves the way for cell genetics studies and for novel breeding approaches in these economically important orchard trees.