Showing papers on "Protoplast published in 2020"

Establishment of a PEG-mediated protoplast transformation system based on DNA and CRISPR/Cas9 ribonucleoprotein complexes for banana.

Abstract: To date, CRISPR/Cas9 RNP editing tools have not been applied to the genetic modification of banana. Here, the establishment of a PEG-mediated banana protoplast transformation system makes it possible to build an efficient DNA-free method for a site-directed mutagenesis system. Protoplasts constitute a versatile platform for transient expression in plant science. In this study, we established a PEG-mediated banana protoplast transformation system. This system was further optimized for successfully delivering CRISPR/Cas9 and CRISPR/Cas12a plasmids and CRISPR/Cas9 ribonucleoproteins (RNPs) for targeted delivery of the PDS gene into banana protoplasts. Specific bands were observed in PCR-Restriction Enzyme Digestion (PCR-RE) assays, and Sanger sequencing of single clones further confirmed the occurrence of indels at target sites. Deep amplicon sequencing results showed that the editing efficiency of the CRISPR/Cas9 system was higher than that of the other two systems. The PEG-mediated banana protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in banana. The application of the CRISPR/Cas9 RNP system enables the generation of banana plants engineered by DNA-free gene editing.

Highly Efficient Protoplast Isolation and Transient Expression System for Functional Characterization of Flowering Related Genes in Cymbidium Orchids.

Abstract: Protoplast systems have been proven powerful tools in modern plant biology. However, successful preparation of abundant viable protoplasts remains a challenge for Cymbidium orchids. Herein, we established an efficient protoplast isolation protocol from orchid petals through optimization of enzymatic conditions. It requires optimal D-mannitol concentration (0.5 M), enzyme concentration (1.2 % (w/v) cellulose and 0.6 % (w/v) macerozyme) and digestion time (6 h). With this protocol, the highest yield (3.50 × 107/g fresh weight of orchid tissue) and viability (94.21%) of protoplasts were obtained from flower petals of Cymbidium. In addition, we achieved high transfection efficiency (80%) through the optimization of factors affecting polyethylene glycol (PEG)-mediated protoplast transfection including incubation time, final PEG4000 concentration and plasmid DNA amount. This highly efficient protoplast-based transient expression system (PTES) was further used for protein subcellular localization, bimolecular fluorescence complementation (BiFC) assay and gene regulation studies of flowering related genes in Cymbidium orchids. Taken together, our protoplast isolation and transfection protocol is highly efficient, stable and time-saving. It can be used for gene function and molecular analyses in orchids and other economically important monocot crops.

Protoplast isolation and shoot regeneration from protoplast-derived calli of Chrysanthemum cv. White ND

Abstract: In this study, we sought to optimize the isolation of protoplasts from chrysanthemums by manipulating the mannitol and cellulase levels, the incubation period, and the purification method, followed by the conversion of the protoplasts into calli and shoots. A high protoplast yield was achieved using 0.5 M mannitol, 1.5% cellulase, and a 4 h incubation period. Cell wall regeneration was observed after 3 days, with the first cell division occurring approximately 4–5 days after culturing. The addition of sucrose to the culture media was more beneficial than glucose; in sucrose media the protoplasts grew more rapidly and successfully reached the colony and microcalli stage. The addition of activated charcoal to the culture improved colony and microcalli formation. Greater proliferation of microcalli was also achieved using solid Murashige & Skoog (MS) media supplemented with 1 mg l−1 6-Benzylaminopurine (BA) and 2 mg l−1 Naphthaleneacetic acid (NAA). The calli produced shoots THE on media supplemented with 2 mg l− 1 BA and 0.5 mg l−1 NAA. These findings could facilitate further chrysanthemum protoplast-based research. The regeneration of chrysanthemum protoplasts into whole plants is difficult due to the recalcitrant nature of these plants and because of their genotype-dependent response. This study is appropriate for protoplast isolation and callus formation of Chrysanthemum cv. White ND, and it also achieved regenerating chrysanthemum protoplasts-derived calli into whole plants.

Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection

Abstract: Nowadays, genome editing in plants has become much easier thanks to the recently developed clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (CRISPR–Cas9) nuclease system. However, to combine protoplast technology with the CRISPR–Cas9 system in plants, a stable and an efficient foreign DNA delivery system is essential for gene editing. In the present study, we developed an electro-transfection system for CRISPR–Cas9 ribonucleoprotein (RNP) delivery to cabbage protoplasts. Under 1000 V treatment, the frequency of initial cell division and total number of cell colonies formed were 47.7 ± 2.5% and 52 ± 7.5%, respectively. The total number of cell colonies formed following 1000 V treatment was 1.4 times higher than that following polyethylene glycol (PEG) treatment. However, the frequency of initial cell division and total number of cell colonies formed from protoplasts decreased with increasing voltage. Cy3–Cas9 protein delivery into the nucleus was confirmed through both electro-transfection and PEG-mediated transfection using confocal laser scanning microscopy. The frequency of insertions and deletions in the synthesized guide RNA of phytoene desaturase 1 was the highest at 3.4% following electro-transfection at 1000 V with a pulse width of 20 ms and only 1.8% following PEG-mediated transfection. These results indicate that electro-transfection is more efficient in RNP delivery to protoplast than PEG-mediated transfection in cabbage for PDS1 sgRNA delivery. Therefore, the electro-transfection system developed in the present study presents the possibility it could be used for DNA-free genome editing of other crops.

Optimization of isolation and transfection conditions of maize endosperm protoplasts.

Abstract: Endosperm-trait related genes are associated with grain yield or quality in maize. There are vast numbers of these genes whose functions and regulations are still unknown. The biolistic system, which is often used for transient gene expression, is expensive and involves complex protocol. Besides, it cannot be used for simultaneous analysis of multiple genes. Moreover, the biolistic system has little physiological relevance when compared to cell-specific based system. Plant protoplasts are efficient cell-based systems which allow quick and simultaneous transient analysis of multiple genes. Typically, PEG-calcium mediated transfection of protoplast is simple and cost-effective. Notably, starch granules in cereal endosperm may diminish protoplast yield and integrity, if the isolation and transfection conditions are not accurately measured. Prior to this study, no PEG-calcium mediated endosperm protoplast system has been reported for cereal crop, perhaps, because endosperm cells accumulate starch grains. Here, we showed the uniqueness of maize endosperm-protoplast system (EPS) in conducting endosperm cell-based experiments. By using response surface designs, we established optimized conditions for the isolation and PEG-calcium mediated transfection of maize endosperm protoplasts. The optimized conditions of 1% cellulase, 0.75% macerozyme and 0.4 M mannitol enzymolysis solution for 6 h showed that more than 80% protoplasts remained viable after re-suspension in 1 ml MMG. The EPS was used to express GFP protein, analyze the subcellular location of ZmBT1, characterize the interaction of O2 and PBF1 by bimolecular fluorescent complementation (BiFC), and simultaneously analyze the regulation of ZmBt1 expression by ZmMYB14. The described optimized conditions proved efficient for reasonable yield of viable protoplasts from maize endosperm, and utility of the protoplast in rapid analysis of endosperm-trait related genes. The development of the optimized protoplast isolation and transfection conditions, allow the exploitation of the functional advantages of protoplast system over biolistic system in conducting endosperm-based studies (particularly, in transient analysis of genes and gene regulation networks, associated with the accumulation of endosperm storage products). Such analyses will be invaluable in characterizing endosperm-trait related genes whose functions have not been identified. Thus, the EPS will benefit the research of cereal grain yield and quality improvement.

Effective callus induction and plant regeneration in callus and protoplast cultures of Nigella damascena L.

Abstract: In this study we report the development of effective in vitro systems for a medicinal plant Nigella damascena L. comprising: (1) callus induction, (2) somatic embryogenesis in callus cultures with subsequent plant regeneration, and (3) isolation and regeneration of callus-derived protoplasts. Callus development was achieved on 83–100% of hypocotyl and cotyledon explants, whereby Murashige and Skoog medium (MS) supplemented with 3 mg L−1 6-benzylaminopurine and 0.5 mg L−1 α-naphthaleneacetic acid (NAA; BN medium) was more advantageous than MS with kinetin and NAA (KN medium). Histological observations of calli revealed the presence of embryogenic zones from which somatic embryos developed on the hormone-free medium. Plant regeneration was observed on 76–95% of calli. A high capacity to form somatic embryos and regeneration was maintained in long-lasting cultures, i.e. even in 2 year old callus. The obtained callus was also a good source tissue for protoplast isolation. By applying a mixture of cellulase and pectolyase, the acceptable yield of viable protoplasts was achieved, especially from hypocotyl-derived callus maintained on BN medium. Protoplasts embedded in an alginate matrix and cultured in modified Kao and Michayluk media re-constructed their cell wall and re-entered mitotic divisions. About 30% of small cell aggregates formed microcalli, which, after the release from alginate, proliferated continuously on KN and BN media, irrespective of the tissue variant used as the protoplast source. Somatic embryo formation and plant regeneration were successful on hormone-free media. An effective plant regeneration system of N. damascena protoplast cultures has been developed and is being reported for the first time. The main objective of this study was to develop in vitro systems utilizing N. damascena seedlings, as an easily accessible explant source, for efficient callus induction and proliferation, and plant regeneration via somatic embryogenesis. Moreover, we attempted to validate the usefulness of the obtained callus as a source of protoplasts and their capability to develop into plants.

Improvement of Ethanol Tolerance by Inactive Protoplast Fusion in Saccharomyces cerevisiae.

Abstract: Saccharomyces cerevisiae is a typical fermentation yeast in beer production. Improving ethanol tolerance of S. cerevisiae will increase fermentation efficiency, thereby reducing capital costs. Here, we found that S. cerevisiae strain L exhibited a higher ethanol tolerance (14%, v/v) than the fermentative strain Q (10%, v/v). In order to enhance the strain Q ethanol tolerance but preserve its fermentation property, protoplast fusion was performed with haploids from strain Q and L. The fusant Q/L-f2 with 14% ethanol tolerance was obtained. Meanwhile, the fermentation properties (flocculability, SO2 production, α-N assimilation rate, GSH production, etc.) of Q/L-f2 were similar to those of strain Q. Therefore, our works established a series of high ethanol-tolerant strains in beer production. Moreover, this demonstration of inactivated protoplast fusion in industrial S. cerevisiae strain opens many doors for yeast-based biotechnological applications.

Optimization of the protoplast transient expression system for gene functional studies in strawberry (Fragaria vesca)

Abstract: Polyethylene glycol (PEG)-mediated transient expression system in plant protoplasts has been widely used in a variety of plants for gene function characterization. However, such a system has not been developed for strawberry. In this study, we report a method for obtaining high quality and high yield protoplasts from strawberry leaves. In addition, we developed an efficient transient gene expression system based on the PEG-mediated method, and the system has been successfully applied to studies of protein expression, protein subcellular localization and protein–protein interaction in strawberry. Furthermore, the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) technology was used to enable targeted mutagenesis of the FvTCP17 gene in strawberry protoplasts. Taken together, our results demonstrate the potential of this highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9-mediated genome editing in strawberry. We report a highly efficient mesophyll protoplast system for transient gene expression and induction of CRISPR/Cas9‐mediated genome editing in strawberry.

Development of an efficient protoplast isolation and transfection system for castor bean (Ricinus communis L.)

Abstract: 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.

Protoplast Isolation and Shoot Regeneration from Protoplast-Derived Callus of Petunia hybrida Cv. Mirage Rose.

Abstract: Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.

Efficient isolation of protoplasts from rice calli with pause points and its application in transient gene expression and genome editing assays

Abstract: An efficient in vivo transient transfection system using protoplasts is an important tool to study gene expression, metabolic pathways, and multiple mutagenesis parameters in plants. Although rice protoplasts can be isolated from germinated seedlings or cell suspension culture, preparation of those donor tissues can be inefficient, time-consuming, and laborious. Additionally, the lengthy process of protoplast isolation and transfection needs to be completed in a single day. Here we report a protocol for the isolation of protoplasts directly from rice calli, without using seedlings or suspension culture. The method is developed to employ discretionary pause points during protoplast isolation and before transfection. Protoplasts maintained within a sucrose cushion partway through isolation, for completion on a subsequent day, per the first pause point, are referred to as S protoplasts. Fully isolated protoplasts maintained in MMG solution for transfection on a subsequent day, per the second pause point, are referred to as M protoplasts. Both S and M protoplasts, 1 day after initiation of protoplast isolation, had minimal loss of viability and transfection efficiency compared to protoplasts 0 days after isolation. S protoplast viability decreases at a lower rate over time than that of M protoplasts and can be used with added flexibility for transient transfection assays and time-course experiments. The protoplasts produced by this method are competent for transfection of both plasmids and ribonucleoproteins (RNPs). Cas9 RNPs were used to demonstrate the utility of these protoplasts to assay genome editing in vivo. The current study describes a highly effective and accessible method to isolate protoplasts from callus tissue induced from rice seeds. This method utilizes donor materials that are resource-efficient and easy to propagate, permits convenience via pause points, and allows for flexible transfection days after protoplast isolation. It provides an advantageous and useful platform for a variety of in vivo transient transfection studies in rice.

Somaclonal Variation in Chrysanthemum × morifolium Protoplast Regenerants.

Abstract: 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.

Optimization of protoplast isolation from the gametophytes of brown alga Undaria pinnatifida using response surface methodology

Abstract: The microscopic gametophytic phase of the commercial brown alga, Undaria pinnatifida, can be used for several applications, including the production of bioactive compounds, aquaculture and as germplasm bank. Therefore, gametophytes are good candidates for cellular biotechnology techniques, many of which rely on protoplasts (“naked” living plant cells). This study reports on the optimization of protoplast yield from male and female gametophytes of U. pinnatifida using different mixtures of commercial enzymes and chelation pre-treatment. Key conditions for achieving the highest protoplast yield, such as enzyme combinations, chelation pre-treatment, growth, temperature, incubation time, pH and osmolarity, were investigated. Protoplast isolation conditions were modelled by using response surface methodology (RSM) via Box-Behnken design (BBD) and subsequently experimentally verified in its predictability of protoplast production. The enzyme composition with 1% cellulase RS, 2 U mL−1 alginate lyase and 1% driselase with chelation pre-treatment, at 2481–2591 mOsm L−1 H2O and adjusted to pH 5.8–6.1, produced the highest protoplast yields of 3.12 ± 0.51 × 106 protoplasts g−1 fresh weight for male gametophyte and 2.11 ± 0.08 × 106 for female gametophyte when incubated at 20 °C for 4–7 h using cultures at mid or early exponential phase, respectively. These conditions also gave high amounts of protoplasts from other strains of Korea. Our results show the effectiveness of commercial enzymes combined with chelation pre-treatment in protoplast isolation and RSM with BBD is a useful method for rapidly producing the higher yields of protoplasts from brown alga.

Ethanol production from cassava starch by protoplast fusants of Wickerhamomyces anomalus and Galactomyces candidum

Abstract: The potential of protoplast fusants of Wickerhamomyces anomalus and Galactomyces candidum isolates for ethanol production from cassava starch was evaluated. The protoplasts were obtained from 18 h ...

Interspecific hybridization between cultivated morels Morchella importuna and Morchella sextelata by PEG-induced double inactivated protoplast fusion

Abstract: The commercial production of Morchella mushrooms calls for urgent breeding of excellent varieties or strains with appropriate tools, such as protoplast fusion. However, the protoplast fusion in morels has not been studied. In this paper, interspecific hybridization between cultivated morels of M. importuna and M. sextelata by PEG-induced protoplast fusion was conducted. Apart from functional complementation of double inactivated protoplasts, the fusants were characterized by cultural and cultivated characters and molecular markers of random amplified polymorphic DNA (RAPD). The results suggested that the hybrids and their parents showed significant difference in their inoculum recovery time, mycelial growth rate, yield of cultivation and total amino acid content of ascocarps. Moreover, positive barrage reactions were observed between parental strains as well as between each parent and a hybrid line. A dendrogram created on the basis of RAPD fingerprints exhibited three major clusters, in which morel hybrids showed intra-cluster variations, M. sextelata #6 formed an out group, while M. importuna #4 was phylogenetically closer to morel hybrids. All the results demonstrated that real fusants were obtained in our study. Protoplast fusion may provide an ideal alternative for new strain selection, and thus will promote the healthy development of morel industry.

An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9

Abstract: Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.

Enhancement of protoplast preparation and regeneration of Hirsutella sinensis based on process optimization

Abstract: To explore the optimal methods for the protoplast preparation and regeneration of Hirsutella sinensis by optimizing the limiting factors. During the treatment of enzymatic protoplast preparation, mycelium cultured for 7 days was the optimal start material. The maximum protoplast preparation rate of 4.3 × 107 protoplasts/g fresh weight (FW) was obtained after 0.5 h treatment of 1 mg/ml mixed lytic enzymes in KH2PO4-K2HPO4 buffer (pH 5.5) with 0.6 M KCl at 18 °C. As for the protoplast regeneration, the maximum protoplast regeneration rate reached 12.32% through 5 × 103 protoplasts mL−1 cultivated for 20 days in the regeneration medium with 0.6 M mannitol and 1.5% agar. The preparation and regeneration of H. sinensis protoplasts was firstly established based on process optimization and it provided a foundation for the study of H. sinensis mutagenesis.

Incorporation of Magnetic Nanoparticles into Protoplasts of Microalgae Haematococcus pluvialis: A Tool for Biotechnological Applications.

Abstract: 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.

ACTIN7 Is Required for Perinuclear Clustering of Chloroplasts during Arabidopsis Protoplast Culture.

Abstract: In Arabidopsis, the actin gene family comprises eight expressed and two non-expressed ACTIN (ACT) genes. Of the eight expressed isoforms, ACT2, ACT7, and ACT8 are differentially expressed in vegetative tissues and may perform specific roles in development. Using tobacco mesophyll protoplasts, we previously demonstrated that actin-dependent clustering of chloroplasts around the nucleus prior to cell division ensures unbiased chloroplast inheritance. Here, we report that actin-dependent chloroplast clustering in Arabidopsis mesophyll protoplasts is defective in act7 mutants, but not act2-1 or act8-2. ACT7 expression was upregulated during protoplast culture whereas ACT2 and ACT8 expression did not substantially change. In act2-1, ACT7 expression increased in response to loss of ACT2, whereas in act7-1, neither ACT2 nor ACT8 expression changed appreciably in response to the absence of ACT7. Semi-quantitative immunoblotting revealed increased actin concentrations during culture, although total actin in act7-1 was only two-thirds that of wild-type or act2-1 after 96 h culture. Over-expression of ACT2 and ACT8 under control of ACT7 regulatory sequences restored normal levels of chloroplast clustering. These results are consistent with a requirement for ACT7 in actin-dependent chloroplast clustering due to reduced levels of actin protein and gene induction in act7 mutants, rather than strong functional specialization of the ACT7 isoform.

Stay in Touch-The Cortical ER of Moss Protonemata in Osmotic Stress Situations.

Abstract: Plasmolysis is usually introduced to cell biology students as a tool to illustrate the plasma membrane: hypertonic solutions cause the living protoplast to shrink by osmotic water loss; hence, it detaches from the surrounding cell wall. What happens, however, with the subcellular structures in the cell cortex during this process of turgor loss? Here, we investigated the cortical endoplasmic reticulum (ER) in moss protonema cells of Physcomitrella patens in a cell line carrying a transgenic ER marker (GFP-HDEL). The plasma membrane was labelled simultaneously with the fluorescent dye FM4-64 to achieve structural separation. By placing the protonemata in a hypertonic mannitol solution (0.8 M), we were able to follow the behaviour of the cortical ER and the protoplast during plasmolysis by confocal laser scanning microscopy (CLSM). The protoplast shape and structural changes of the ER were further examined after depolymerisation of actin microfilaments with latrunculin B (1 µM). In its natural state, the cortical ER is a dynamic network of fine tubes and cisternae underneath the plasma membrane. Under acute and long-term plasmolysis (up to 45 min), changes in the protoplast form and the cortical ER, as well as the formation of Hechtian strands and Hechtian reticula, were observed. The processing of the high-resolution z-scans allowed the creation of 3D models and gave detailed insight into the ER of living protonema cells before, during and after plasmolysis.

Exogenous Nitric Oxide Delays Plant Regeneration from Protoplast and Protonema Development in Physcomitrella patens

Abstract: Nitric oxide (NO) has been recognized as a major player in the regulation of plant physiology and development. NO regulates cell cycle progression and cell elongation in flowering plants and green algae, although the information about NO function in non-vascular plants is scarce. Here, we analyze the effect of exogenous NO on Physcomitrella patens protonema growth. We find that increasing concentrations of the NO donor sodium nitroprusside (SNP) inhibit protonema relative growth rate and cell length. To further comprehend the effect of NO on moss development, we analyze the effect of SNP 5 and 10 µM on protoplast regeneration and, furthermore, protonema formation compared with untreated plants (control). Isolated protoplasts were left to regenerate for 24 h before starting the SNP treatments that lasted five days. The results show that SNP restrains the protoplast regeneration process and the formation of new protonema cells. When SNP treatments started five days after protoplast isolation, a decrease in cell number per protonema filament was observed, indicating an inhibition of cell cycle progression. Our results show that in non-vascular plants, NO negatively regulates plant regeneration, cell cycle and cell elongation.

Improvement of vincamine production of endophytic fungus through inactivated protoplast fusion

Abstract: Improvement of the production of vincamine in endophytic fungus VINI-7 was performed by using the inactivated protoplast fusion method. The preparation conditions of protoplasts were optimized by systematic trials with various parameters, and inactivated protoplast fusion was subsequently performed. The mycelium in logarithmic growth phase was treated with 1500 U/mL lywallzyme, 1500 U/mL lysozyme, 2000 U/mL cellulase, and 1000 U/mL snailase solution for 3 h at 30 °C and had the best conditions, in which the concentration of the protoplast was 3.17 × 107 cells/mL. Protoplasts were inactivated by heat, ultraviolet, microwave, sodium nitrite, and diethyl sulfate, respectively. Subsequently, protoplasts inactivated by different methods were subjected to respective protoplast fusion. The results showed that the yield of vincamine in fusants inactivated by mutagens was generally higher than that of fusants inactivated by heat. The highest yield of vincamine in two fusants (U-U1 and N-N1) was 31.6 and 38.7 mg, which increased to 162.24 and 221.16%, respectively, as compared to the parent strain (12.05 mg). LC-MS/MS analysis showed that U-U1 and N-N1 fusants could produce vincamine. Furthermore, the results of genetic stability experiments indicated that U-U1 and N-N1 were genetically stable.

Application of the protoplast co-culture method for evaluation of allelopathic activities of volatile compounds, safranal and tulipalin A

Abstract: The effects of two plant volatile compounds (VOCs), safranal and tulipalin A were investigated using the protoplast method with digital image analysis, which was developed to investigate allelopathic activities of plants at the cellular level in a 50 μL liquid medium. Both VOCs showed inhibition on lettuce protoplasts growth at all three stages, i.e., the cell wall formation stage, the cell division stage, and the yellow colour accumulation stage. Among the three stages of protoplast growth, differences of inhibition patterns were observed. Inhibitions at the cell wall formation stage were stronger than those at the latter two stages in both VOCs. Tulipalin A showed higher inhibitory activity (total inhibition at 100 μM) than that of safranal (44% inhibition at 1 mM) at cell division stage. They were also compared with the direct exposure method in an enclosed vial using germinated lettuce seedlings, which was developed to investigate allelopathic activities of VOCs at plant level. Tulipalin A showed less inhibitory activity on radicle growth of lettuce, than that of safranal reported. Inhibition on hypocotyl growth was stronger than that on radicle in both VOCs. The causes of the differences between two VOCs and two bioassay methods, and the application of the protoplast method, were discussed for clarifying the contribution of VOCs as allelochemicals at cellular level.

Establishment of Pluripotent Cell Cultures to Explore Allelopathic Activity of Coffee Cells by Protoplast Co-Culture Bioassay Method.

Abstract: We focused on the demonstration of a new pluripotent coffee cell culture system to control the growth and metabolic functions. Somatic cells in the epidermal layer of in vitro somatic embryos (SEs) of Coffea canephora expressed higher pluripotency to produce secondary SEs than primary or secondary meristematic tissue. SEs were ideal explants to selectively induce functionally-differentiated cell lines, both non-embryogenic callus (nEC) and embryogenic callus (EC). The protoplast co-culture bioassay method was used to explore allelopathic activity of these cultured coffee cells. Cell wall formation of lettuce protoplasts varied after five days of co-culture. A strong stimulative reaction was observed at lower nEC protoplast densities, whereas growth was inhibited at higher densities. The reaction of lettuce protoplasts after 12 days of co-culture was recognized as an inhibitory reaction of colony formation.

Genome rearrangements and megaplasmid loss in the filamentous bacterium Kitasatospora viridifaciens are associated with protoplast formation and regeneration.

Abstract: Filamentous Actinobacteria are multicellular bacteria with linear replicons. Kitasatospora viridifaciens DSM 40239 contains a linear 7.8 Mb chromosome and an autonomously replicating plasmid KVP1 of 1.7 Mb. Here we show that lysozyme-induced protoplast formation of the multinucleated mycelium of K. viridifaciens drives morphological diversity. Characterisation and sequencing of an individual revertant colony that had lost the ability to differentiate revealed that the strain had not only lost most of KVP1 but also carried deletions in the right arm of the chromosome. Strikingly, the deletion sites were preceded by insertion sequence elements, suggesting that the rearrangements may have been caused by replicative transposition and homologous recombination between both replicons. These data indicate that protoplast formation is a stressful process that can lead to profound genetic changes.