Protocol For Isolation Of Protoplasts Research Articles

Optimized protoplast isolation and transfection with a breakpoint: accelerating Cas9/sgRNA cleavage efficiency validation in monocot and dicot

AbstractThe CRISPR-Cas genome editing tools are revolutionizing agriculture and basic biology with their simplicity and precision ability to modify target genomic loci. Software-predicted guide RNAs (gRNAs) often fail to induce efficient cleavage at target loci. Many target loci are inaccessible due to complex chromatin structure. Currently, there is no suitable tool available to predict the architecture of genomic target sites and their accessibility. Hence, significant time and resources are spent on performing editing experiments with inefficient guides. Although in vitro-cleavage assay could provide a rough assessment of gRNA efficiency, it largely excludes the interference of native genomic context. Transient in-vivo testing gives a proper assessment of the cleavage ability of editing reagents in a native genomic context. Here, we developed a modified protocol that offers highly efficient protoplast isolation from rice, Arabidopsis, and chickpea, using a sucrose gradient, transfection using PEG (polyethylene glycol), and validation of single guide RNAs (sgRNAs) cleavage efficiency of CRISPR-Cas9. We have optimized various parameters for PEG-mediated protoplast transfection and achieved high transfection efficiency using our protocol in both monocots and dicots. We introduced plasmid vectors containing Cas9 and sgRNAs targeting genes in rice, Arabidopsis, and chickpea protoplasts. Using dual sgRNAs, our CRISPR-deletion strategy offers straightforward detection of genome editing success by simple agarose gel electrophoresis. Sanger sequencing of PCR products confirmed the editing efficiency of specific sgRNAs. Notably, we demonstrated that isolated protoplasts can be stored for up to 24/48 h with little loss of viability, allowing a pause between isolation and transfection. This high-efficiency protocol for protoplast isolation and transfection enables rapid (less than 7 days) validation of sgRNA cleavage efficiency before proceeding with stable transformation. The isolation and transfection method can also be utilized for rapid validation of editing strategies, evaluating diverse editing reagents, regenerating plants from transfected protoplasts, gene expression studies, protein localization and functional analysis, and other applications.

Establishment of protoplasts isolation and transient transformation system for kiwifruit

Protoplast isolation and transient transformation have proven to be valuable tools for studying gene function in plants. However, these techniques are rarely utilized in most woody plants due to the lack of an efficient protoplast isolation system. In this study, we successfully established a protocol for isolating and purifying protoplasts from kiwifruit callus. First, loose kiwifruit callus was induced using young true leaves as explants on MS medium containing 0.5 mg·L−1 zeatin and 0.5 mg·L−1 2,4-dichlorophenoxyacetic acid (2,4-D), followed by propagation on medium supplemented with 1.0 mg·L−1 2,4-D + 0.5 mg·L−1 6-benzylaminopurine + 0.5 mg·L−1 1-naphthlcetic acid. Subsequently, the callus subcultured at 15 d was digested with an enzyme solution consisting of 2.0 % Cellulase R-10, 0.5 % Macerozyme R-10, and 0.7 M mannitol for 7 h in the dark to obtain protoplasts at a yield of approximately 2.8 × 106 protoplasts·g−1FW with viability reaching up to 95.2 %. Furthermore, we optimized the conditions for transient transformation in kiwifruit protoplasts, achieving an approximately 43.5 % transformation efficiency using a concentration of PEG4000 at 40 %. By employing this method, we successfully verified the subcellular localization of AcMYB6l-GFP fusion protein. In conclusion, we have successfully established an efficient protocol for protoplast isolation and transient transformation in kiwifruit, laying a foundation for future research on gene function and molecular breeding in Actinidia.

A highly efficient protocol for isolation of protoplast from China, Assam and Cambod types of tea plants [Camellia sinensis (L.) O. Kuntze

BackgroundTea is the most popular beverage worldwide second only to water. Its demand is tremendously rising due to increased awareness of its medicinal importance. The quality and uses of tea depend on the tea-types which are mainly three types including China, Assam and Cambod type having distinct compositions of secondary metabolites. Huge variation in secondary metabolites in different tea-types and cultivars limited the successful application of various approaches used for its trait improvement. The efficiency of a protocol for isolation of protoplast is specific to the types and cultivars of tea plants. The existing tea protoplast-isolation protocols [which were optimized for tea-types (China and Assam type) and Chinese cultivars grown in China] were found ineffective on types/cultivars grown in India due to type/cultivar variability. Therefore, optimization of protoplast-isolation protocol is essential for tea-types/cultivars grown in India, as it is the second largest producer of tea and the largest producer of black tea. Here, efforts were made to develop an efficient protoplast-isolation protocol from all major types of tea (China, Assam and Cambod types) grown in India and also from three types of tender leaves obtained from field-grown, hydroponically-grown and tissue culture-grown tea plants.ResultsDeveloped protoplast-isolation protocol was effective for different types of leaf tissue obtained from the tender leaves of field-grown, hydroponically-grown and tissue culture-grown tea plants. Moreover, optimized protocol effectively worked on all three types of tea including China, Assam and Cambod types cultivated in India. The digestion of leaves with 3% cellulase R-10, 0.6% macerozyme, 1% hemicellulase and 4% polyvinylpyrrolidone for 12 h at 28ºC yielded approximately 3.8–4.6 × 107 protoplasts per gram fresh tissue and 80–95% viability in selected tea cultivars, and tissue culture plant material was found most appropriate for protoplast isolation.ConclusionsIn conclusion, we reported an efficient protocol for isolation of protoplasts from tender tea leaves of all major tea-types (China, Assam and Cambod) grown in India. Moreover, the protocol is also effective for tender-leaf tissue of field-grown, hydroponically-grown and tissue culture-grown tea plants. The findings are expected to contribute to the genetic improvement of tea traits widely.

Improvements in Protoplast Isolation Protocol and Regeneration of Different Cabbage (Brassica oleracea var. capitata L.) Cultivars.

Protoplasts are a versatile tool in plant biotechnology since they can be used for basic biological studies as well as for breeding strategies based on genome editing. An efficient protoplast isolation protocol is essential for conducting protoplast-based studies. To optimize the protoplast isolation protocol in cabbage (Brassica oleracea var. capitata L.), different enzyme solutions were tested for the isolation of leaf mesophyll protoplasts. In our experiments, the combination of 0.5% Cellulase Onozuka RS and 0.1% Macerozyme R-10 showed the best result. The optimized protocol proved suitable for the isolation of protoplasts from five different cabbage cultivars with yields ranging from 2.38 to 4.63 × 106 protoplasts/g fresh weight (fw) and a viability of 93% or more. After three weeks in culture, protoplasts from all of the tested cultivars formed micro-calli, but further callus growth and shoot regeneration depended strongly on the genotype and regeneration protocol used. For shoot formation, 1 mg/L BAP in combination with auxin 0.2 mg/L NAA showed the best results with a regeneration of 23.5%. The results obtained will contribute to the development of different applications of cabbage protoplasts and facilitate the breeding process of this important horticultural crop.

Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene.

Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea's adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.

Establishment of an efficient cotton root protoplast isolation protocol suitable for single-cell RNA sequencing and transient gene expression analysis

BackgroundCotton has tremendous economic value worldwide; however, its allopolyploid nature and time-consuming transformation methods have hampered the development of cotton functional genomics. The protoplast system has proven to be an important and versatile tool for functional genomics, tissue-specific marker gene identification, tracking developmental trajectories, and genome editing in plants. Nevertheless, the isolation of abundant viable protoplasts suitable for single-cell RNA sequencing (scRNA-seq) and genome editing remains a challenge in cotton.ResultsWe established an efficient transient gene expression system using protoplasts isolated from cotton taproots. The system enables the isolation of large numbers of viable protoplasts and uses an optimized PEG-mediated transfection protocol. The highest yield (3.55 × 105/g) and viability (93.3%) of protoplasts were obtained from cotton roots grown in hydroponics for 72 h. The protoplasts isolated were suitable for scRNA-seq. The highest transfection efficiency (80%) was achieved when protoplasts were isolated as described above and transfected with 20 μg of plasmid for 20 min in a solution containing 200 mM Ca2+. Our protoplast-based transient expression system is suitable for various applications, including validation the efficiency of CRISPR vectors, protein subcellular localization analysis, and protein–protein interaction studies.ConclusionsThe protoplast isolation and transfection protocol developed in this study is stable, versatile, and time-saving. It will accelerate functional genomics and molecular breeding in cotton.

Evaluation and Improvement of Protocols for Ganoderma boninense Protoplast Isolation and Regeneration

Ganoderma boninense is the causal agent of basal stem rot (BSR) disease of oil palm. The BSR disease reduces oil palm yield by up to 80% of the average oil yield. Attempts to control the disease caused by this fungus in the field showed varying levels of success and cases of infection increased from year to year. Hence, the development of new efficient methods to control the spread of this fungus should be commenced promptly. To ensure a better strategy is created, more thorough research on the method deploy by this fungus to infect the host at the molecular level need to be carried out first. However, the major limitation in endeavoring into the functional analysis of virulence genes related to the pathogenicity of this fungus was hampered by the unavailability of established methods for protoplast isolation with a high regeneration rate to be used in the genetic manipulation analysis. Thus, in this paper, we report an efficient protocol for protoplast isolation and regeneration in G. boninense and successfully used the isolated protoplasts in PEG-mediated transformation analysis. A large quantity of protoplast was obtained using the protocol that utilizes the following parameters: 3 to 4-day-old mycelia, treated with 1% lysing enzyme and 0.02% Driselase, incubated at 30 °C in an osmotic medium containing 0.6 M mannitol at pH 5.8 for 2 h. The highest protoplast yield was in the range of 8.95 × 109 to 3.12 × 1010 cells/mL per 5 g of mycelia used. The regeneration rate ranged from 9.03% to 22.55%, depending on the regeneration media used. By using 5 µg of vector to transform into 1.0 × 107 protoplast/mL, around 3 – 10 mitotically stable putative transformants were successfully obtained and verified via PCR. This protocol will find useful applications in genetic studies to enhance insight into this poorly characterized and understood phytopathogen.

A versatile protoplast system and its application in Cannabis sativa L.

Cannabis sativa L. (cannabis) is one of the earliest domesticated crops cultivated for medicinal, food, industrial, and recreational use throughout the world. A growing number of jurisdictions have lifted the prohibition on medical and recreational cannabis with legislation that allows researchers to conduct important and urgently needed research, such as understanding the molecular basis of agronomically important traits. However, a dearth of suitable molecular genetic techniques for the analysis of cannabis species has hampered the elucidation of gene function. Here, we developed a robust protoplast transient transformation system by optimizing conditions of protoplast isolation and polyethylene glycol–mediated transformation using cannabis cotyledons. A protoplast isolation yield of up to 1.15 × 107 cells per gram of fresh material and 98.5% viability and a transformation efficiency of 75.4% were achieved. This optimized protocol for protoplast isolation and transformation was successfully applied to eight representative cannabis varieties, which highlight the versatility and robustness of this technique. Using this protocol, the nuclear localization of a basic helix–loop–helix transcription factor (CsMYC2) was visualized, and the transcriptional activity of CsMYC2 was assessed. These results demonstrate the effectiveness of our cannabis protoplast isolation and transient transformation method that should serve as a powerful molecular research tool in a wide range of molecular studies.

An improved protocol for protoplast production, culture, and whole plant regeneration of the commercial brown seaweed Undaria pinnatifida

Protoplasts (i.e., living plant cells devoid of cell wall) represent an important biotechnological tool with utility for the conduction of in vitro studies and crop improvement in economic cultivars. Undaria pinnatifida is an annual brown kelp commonly used in Asian cuisine and several industries because of its biological properties. It is one of the major seaweed cultivars used worldwide. This study establishes a simple and effective protocol for protoplast isolation, culture, and whole plant regeneration of U. pinnatifida . The results showed that high protoplast yields (2–4 × 10 7 protoplast g −1 of fresh weight) were obtained using the commercially available enzymes cellulase “Onozuka” RS (1 %) and alginate lyase (4 U mL −1 ) without any pre-treatment and with a short incubation time (2–4 h). The isolation protocol described herein presented with more effective results with meristematic explants and was superior to the protocols reported for U. pinnatifida and other brown algae. Additionally, the enzymatic mixture could be reused up to six times. Whole plant regeneration was accomplished after 3 months in culture at 20 °C using two culture media, and a mixture of antibiotics (penicillin G, streptomycin, and chloramphenicol) was added once at the beginning of the culture. Our regeneration protocol was less laborious and more cost-effective, exhibiting final plating efficiency higher than previously reported and demonstrating the potential to be used to produce up to 3 × 10 4 sporophytes g −1 of explant. The established protocol can be easily used for genetic manipulation, protoplast fusion, or micropropagation, thus enabling the expansion of the use of protoplasts in algae. • Single-step production of protoplasts from the edible seaweed Undaria pinnatifida using commercial enzymes • The reusability of the enzyme shows that the process is economically viable. • Antibiotics and temperature are key factors for protoplast survival, division and normal regeneration. • Regeneration protocol is simpler and more effective than previously reported.

Genome editing in the edible fungus Poria cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection.

Poria cocos is an important edible and medicinal fungus with a long history. However, the lack of adequate genetic tools has hindered molecular genetic research and the genetic modification of this species. In this study, the endogenous U6 promoters were identified by mining data from the P. cocos genome, and the promoter sequence was used to construct a sgRNA expression vector pFC332-PcU6. Then, the protoplast isolation protocol was developed, and the sgRNA-Cas9 vector was successfully transformed into the cells of P. cocos via PEG/CaCl2-mediated transformation approach. Off-target sites were genome-widely predicted and detected. As a result, the target marker gene ura3 was successfully disrupted by the CRISPR-Cas9 system. This is the first report of genome editing in P. cocos using CRISPR-Cas9 system integrating genome-wide off-target prediction and detection. These data will open up new avenues for the investigation of genetic breeding and commercial production of edible and medicinal fungus.

A highly efficient mesophyll protoplast isolation and PEG-mediated transient expression system in eggplant

Protoplasts are widely used in transient expression systems that are a simple, robust, and powerful method for gene function verification. However, such a versatile tool has not been developed using eggplant. We here established an efficient protocol for protoplast isolation from plantlet leaves of eggplant by optimizing enzymatic conditions. This isolation required an optimal D-mannitol concentration (0.5 M), enzyme concentration (1.25% (w/v) cellulose and 0.4% (w/v) macerozyme), digestion time (3 h), and temperature (26 ℃). This protocol provided the highest yield (1.2 × 107/g fresh weight (FW)) and viability (96%) of protoplasts. Furthermore, the polyethylene glycol (PEG)-mediated transient transformation of eggplant protoplasts with the marker gene yellow fluorescent protein (YFP) was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. More than 50% transfection efficiency could be obtained with the optimized system. In addition, subcellular localization of several fusion proteins was verified, and the bimolecular fluorescence complementation assay was used to detect protein–protein interaction in this system. Dual-luciferase assays in eggplant protoplasts showed that the expression of anthocyanin biosynthesis-related structural genes (SmCHS and SmDFR) was activated by the transcription factor SmMYB1. Taken together, we developed a highly efficient and stable protocol for protoplast isolation and transfection in eggplant, thereby facilitating gene function characterization in molecular breeding.

First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill.

Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.

Optimization of protocol for efficient protoplast isolation and transient gene expression in carnation

The optimal protocols for protoplast isolation and transfection in carnation cv. Scarlet were developed by optimizing the factors involved in protoplast isolation and transfection processes. The best protoplast yield and viability were achieved using 0.5 M mannitol, enzyme concentrations of 1.0% cellulase and 0.1% macerozyme, and digestion time of 8 h. In contrast, the best transfection efficiency was obtained using protoplast density of 2.5 × 105, polyethylene glycol (PEG) concentration of 20%, and transfection time of 15 min. The protocols could produce reasonable protoplast yield and transfection efficiency in other carnations. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all carnations was confirmed using PCR analysis. To our knowledge, this is the first report on transient gene expression in carnation protoplasts. This transient gene expression system could be valuable for genome editing of unwanted genes in carnations using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 technology.

Transcriptional landscape of highly lignified poplar stems at single-cell resolution

BackgroundPlant secondary growth depends on the activity of the vascular cambium, which produces xylem and phloem. Wood derived from xylem is the most abundant form of biomass globally and has played key socio-economic and subsistence roles throughout human history. However, despite intensive study of vascular development, the full diversity of cell types and the gene networks engaged are still poorly understood.ResultsHere, we have applied an optimized protoplast isolation protocol and RNA sequencing to characterize the high-resolution single-cell transcriptional landscape of highly lignified poplar stems. We identify 20 putative cell clusters with a series of novel cluster-specific marker genes and find that these cells are highly heterogeneous based on the transcriptome. Analysis of these marker genes’ expression dynamics enables reconstruction of the cell differentiation trajectories involved in phloem and xylem development. We find that different cell clusters exhibit distinct patterns of phytohormone responses and emphasize the use of our data to predict potential gene redundancy and identify candidate genes related to vascular development in trees.ConclusionsThese findings establish the transcriptional landscape of major cell types of poplar stems at single-cell resolution and provide a valuable resource for investigating basic principles of vascular cell specification and differentiation in trees.

Protoplast isolation from Dictyopteris pacifica and Scytosiphon lomentaria, using a simple commercial enzyme preparation

BackgroundProtoplasts (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. ResultsProtoplasts 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 × 106 protoplasts g−1 fresh weight, respectively. The values obtained for S. lomentaria were 2–3 orders of magnitude higher than previously reported. ConclusionsOur 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.

Development of a robust transient expression screening system in protoplasts of Cannabis

Transient expression systems in mesophyll protoplasts have been utilised in many plant species as an indispensable tool for gene function analysis and efficacious genome editing constructs. However, such a system has not been developed in Cannabis due to the recalcitrant nature of the plant to tissue culture as well as its illegal status for many years. In this study, young expanding leaves from aseptic in vitro Cannabis explants were used for protoplast isolation. Factorial designs were used to optimise variables in viable protoplast isolation and transient expression of GFP, with a range analyses performed to determine, and quantify, significantly impacting variables. Viable protoplast yields as high as 5.7 × 106 were achieved with 2.5% (w/v) Cellulase R-10, 0.3% (w/v) Macerozyme R-10 and 0.7 M mannitol, incubated for 16 h. As indicated by the transient expression of GFP, efficiency reached 23.2% with 30 μg plasmid, 50% PEG, 1 × 106 protoplasts and a transfection duration of 20 min. Application of the optimised protocol for protoplast isolation was successfully evaluated on three subsequent unrelated genotypes to highlight the robustness and broad applicability of the developed technique.

Highly Efficient Leaf Base Protoplast Isolation and Transient Expression Systems for Orchids and Other Important Monocot Crops.

Versatile protoplast platforms greatly facilitate the development of modern botany. However, efficient protoplast-based systems are still challenging for numerous horticultural plants and crops. Orchids are globally cultivated ornamental and medicinal monocot plants, but few efficient protoplast isolation and transient expression systems have been developed. In this study, we established a highly efficient orchid protoplast isolation protocol by selecting suitable source materials and optimizing the enzymatic conditions, which required optimal D-mannitol concentrations (0.4–0.6 M) combined with optimal 1.2% cellulose and 0.6% macerozyme, 5 μM of 2-mercaptoethanol and 6 h digestion. Tissue- and organ-specific protoplasts were successfully isolated from young leaves [∼3.22 × 106/g fresh weight (FW)], flower pedicels (∼5.26 × 106/g FW), and young root tips (∼7.66 × 105/g FW) of Cymbidium orchids. This protocol recommends the leaf base tissues (the tender part of young leaves attached to the stem) as better source materials. High yielding viable protoplasts were isolated from the leaf base of Cymbidium (∼2.50 × 107/g FW), Phalaenopsis (1.83 × 107/g FW), Paphiopedilum (1.10 × 107/g FW), Dendrobium (8.21 × 106/g FW), Arundina (3.78 × 106/g FW) orchids, and other economically important monocot crops including maize (Zea mays) (3.25 × 107/g FW) and rice (Oryza sativa) (4.31 × 107/g FW), which showed marked advantages over previous mesophyll protoplast isolation protocols. Leaf base protoplasts of Cymbidium orchids were used for polyethylene glycol (PEG)-mediated transfection, and a transfection efficiency of more than 80% was achieved. This leaf base protoplast system was applied successfully to analyze the CsDELLA-mediated gibberellin signaling in Cymbidium orchids. We investigated the subcellular localization of the CsDELLA-green fluorescent protein fusion and analyzed the role of CsDELLA in the regulation of gibberellin to flowering-related genes via efficient transient overexpression and gene silencing of CsDELLA in Cymbidium protoplasts. This protoplast isolation and transient expression system is the most efficient based on the documented results to date. It can be widely used for cellular and molecular studies in orchids and other economically important monocot crops, especially for those lacking an efficient genetic transformation system in vivo.

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

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.

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

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.

The plasma membrane Cation binding protein 1 affects accumulation of Potato virus Y in pepper both at the systemic level and in protoplasts

The Plasma membrane Cation binding Protein 1 (PCaP1) has been shown to be important for the intra-cellular movement of two members of the Potyvirus genus in arabidopsis and tobacco plants. In this study, the orthologous PCaP1 gene of pepper (Capsicum annuum) was examined for its role in the accumulation of Potato virus Y, type member of the Potyvirus. Downregulation of C. annuum PCaP (CaPCaP) through tobacco rattle virus-induced gene silencing, resulted in lower accumulation of potato virus Y (PVY) in pepper plants. Using an improved pepper protoplast isolation protocol, we showed that knockdown of CaPCaP negatively affected PVY accumulation at the within-cell level in pepper in contrast with the turnip mosaic virus-arabidopsis pathosystem. Conversely, following overexpression of CaPCaP, the accumulation of PVY at the systemic level was increased. The results provide further knowledge on the role of PCaP in the potyvirus infection process and reveal differences of its action among different pathosystems.