Efficient Protoplast Isolation Research Articles

Establishment of an Efficient Protoplast Isolation and Transfection Method for Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a unique high-quality natural rubber tree species and rare medicinal tree species in China. The rapid characterization of E. ulmoides gene function has been severely hampered by the limitations of genetic transformation methods and breeding cycles. The polyethylene glycol (PEG)-mediated protoplast transformation system is a multifunctional and rapid tool for the analysis of functional genes in vivo, but it has not been established in E. ulmoides. In this study, a large number of highly active protoplasts were isolated from the stems of E. ulmoides seedlings by enzymatic digestion, and green fluorescent protein expression was facilitated using a PEG-mediated method. Optimal enzymatic digestion occurred when the enzyme was digested for 10 h in an enzymatic solution containing 2.5% Cellulase R-10 (w/v), 0.6% Macerozyme R-10 (w/v), 2.5% pectinase (w/v), 0.5% hemicellulase (w/v), and 0.6 mol/L mannitol. The active protoplast yield under this condition was 1.13 × 106 protoplasts/g fresh weight, and the protoplast activity was as high as 94.84%. This study established the first protoplasm isolation and transient transformation system in hard rubber wood, which lays the foundation for subsequent functional studies of E. ulmoides genes to achieve high-throughput analysis, and provides a reference for future gene function studies of medicinal and woody plants.

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

The 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.

Preparation of leaf protoplasts from Populus (Populus × xiaohei T. S. Hwang et Liang) and establishment of transient expression system

Poplar, as a typical woody plant, is an ideal raw material for the production of lignocellulose biofuel. However, the longer life cycle is not conducive to the rapid identification of poplar genes. At present, protoplasts have been used for gene function identification and high-throughput analysis in many model plants. In this paper, a simplified and efficient protoplast isolation and transient expression system of Populus (Populus × xiaohei T. S. Hwang et Liang) is described. Firstly, we proposed an efficient enzyme hydrolysis method for isolating protoplasts from leaves of Populus × xiaohei. Secondly, we optimized the conditions of protoplast transformation mediated by PEG, and established an efficient transient expression system of protoplasts of Populus × xiaohei. Finally, the subcellular localization of three identified Dof transcription factors (PnDof19, PnDof20 and PnDof30) was also observed in the nucleus by using this scheme, which proved that the method was feasible. In general, this efficient method of protoplast isolation and transformation can be used for the study of protein subcellular localization and can be applied to other fields of molecular biology, such as protein interaction, gene activation and so on.

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.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu)

BackgroundAreca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier.ResultsHere, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0–1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%.ConclusionWe have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein–protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Efficient isolation and transformation of protoplasts in coconut endosperm and leaves for gene function studies

Efficient isolation and transformation of protoplasts in coconut endosperm and leaves for gene function studies

Establishment of a Protoplasts-Based Transient Expression System in Banana (Musa spp.)

The breeding of triploid banana cultivars with improved traits, such as yield and disease resistance, remains a major challenge for breeders. One reason is that the molecular study and functional gene analysis in bananas fall behind due to the difficulties of its genetic manipulation. The plant protoplast-based transient transformation has been documented and widely used as a versatile and convenient system for functional gene analysis in many plant species. However, an efficient high-quality protoplast isolation and transformation system is still lacking for bananas. Here, we established an efficient protoplast isolation and transformation method for bananas by selecting proper source materials, optimizing conditions for enzymatic hydrolysis and PEG-mediated transfection. We found the best source materials for banana protoplasts’ isolation are young suckers, which give a yield of protoplasts ranging from 2.5 × 106 to 10.1 × 107 g−1 fresh weight after 5 to 6 h of enzymolysis. The yield is sufficient for most assays that have been established in protoplasts-based systems, such as protein subcellular localization and protein interaction assays. Moreover, using the established transient gene expression system in banana protoplasts, we validated the subcellular localization of Arabidopsis VESICLE SORTING RECEPTOR 1 (VSR1) and the protein self-interaction of Arabidopsis CNGC20 on the cell membrane. The results indicated this system works well and could be routinely used for the functional characterization of banana genes.

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.

Efficient Protoplast Isolation and DNA Transfection for Winter Oilseed Crops, Pennycress (Thlaspi arvense ) and Camelina (Camelina sativa ).

Pennycress (Thlaspi arvense) and camelina (Camelina sativa) are nonfood winter oilseed crops that have the potential to contribute to sustainable biofuel production. However, undesired agronomic traits of pennycress and camelina currently hinder broad cultivation of these plants in the field. Recently, genome editing using the CRISPR-Cas technology has been applied to improve poor agronomic traits such as the weedy phenotype of pennycress and the oxidation susceptible lipid profile of camelina. In these works, the CRISPR reagents were introduced into the plants using the Agrobacterium-mediated floral dipping method. For accelerated domestication and value improvements of these winter oilseed crops, DNA-free genome editing platform and easy evaluation method of the CRISPR-Cas reagents are highly desirable. Cell wall-free protoplasts are great material to expand the use of gene engineering tools. In this chapter, we present a step-by-step guide to the mesophyll protoplast isolation from in vitro culture-grown pennycress and soil-grown camelina. The protocol also includes procedures for DNA transfection and protoplast viability test using fluorescein diacetate. With this protocol, we can isolate an average of 6×106 cells from pennycress and 3×106 cells from camelina per gram of fresh leaf tissues. Using a 7.3kb plasmid DNA carrying green and red fluorescent protein marker genes, we can achieve an average transfection rate of 40% validated by flow cytometry for both plants.

Protoplast Isolation and Transfection in Rice.

Rice (Oryza sativa) is an important cereal crop and a model monocot plant for biology research. The reliable system of foreign DNA transformation and expression is a valuable strategy for basic research and molecular breeding application in rice. The Agrobacterium tumefaciens-mediated foreign DNA transformation system was a powerful tool for genetic research. However, it needs a long period to obtain the stable transformants for further analysis and the transformation rate limits in some organism. Protoplasts are plant cells without a cell wall, and it is much easier for foreign DNA transformation and expression. It has been widely applied in transient expression. Here, we describe a simple method for efficient protoplast isolation and transfection in rice.

Efficient isolation and purification of tissue-specific protoplasts from tea plants (Camellia sinensis (L.) O. Kuntze)

BackgroundPlant 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.ResultsThe 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 × 106 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.ConclusionsIn 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.

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.

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

BackgroundAn 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.ResultsHere 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.ConclusionThe 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.

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.

Isolation of leaf mesophyll protoplasts optimized by orthogonal design for transient gene expression in Catalpa bungei

Catalpa bungei is an important ornamental plant and timber species that originated in China. With the forthcoming C. bungei genome sequence, an efficient and convenient transformation system should be developed. The transient gene expression system using protoplasts is fast and efficient and can be applied to study the functions of genes in C. bungei. In this study, the main factors influencing the isolation of protoplasts from C. bungei were optimized by orthogonal design. The results showed that when the enzymolysis solution contained 3% Cellulase RS (w/v), 2% Macerozyme R-10 (w/v), and 0.4 M mannitol and the enzymolysis time was 8 h, the protoplast yield was 1 × 106 protoplasts/g fresh weight [FW]. The viability of protoplasts was approximately 90 % under optimal enzymolysis conditions. Furthermore, using GFP as the reporter gene, we verified the feasibility of PEG-mediated protoplast transformation of C. bungei. We established an efficient protoplast isolation procedure and feasible transient gene expression system. This methodology, when combined with genetics and omics techniques, will allow further study of the functions of the genes in C. bungei.

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.

Development of a rapid and efficient protoplast isolation and transfection method for chickpea (Cicer arietinum)

Chickpea (Cicer arietinum L.) is the second most important grain legume worldwide. Recent advances in the sequencing of the chickpea genome has provided a new and valuable resource to aid efforts in gene discovery and crop trait improvement. Technical difficulties in stable chickpea transgenics and the lack of a transient expression system for rapid analysis of gene expression and function; however, has limited the usefulness of this genomic resource. As a step toward alleviating this limitation, we report here the development of a simple and efficient transient gene expression protocol. Using leaves from chickpea seedlings, we have established a procedure that enables the generation of large quantities of vital chickpea protoplasts within only a few hours. In addition, we have optimized a PEG-calcium-mediated transfection method to efficiently deliver exogenous DNA into the chickpea protoplast. The current study is the first to present a detailed step-by-step procedures for protoplast isolation, evaluation, transfection, and application in chickpea. In addition, we optimize the transfection efficiency which has not been previously reported. Our protoplast transfection approach provides a platform that will allow rapid high-throughput screening and systematic characterization of gene expression and function. Knowledge gained through such studies will benefit current efforts to improve chickpea production and quality.•Modified enzymatic digestion solution for higher yield and viability.•Optimize transfection of chickpea protoplasts.

Efficient isolation of protoplasts from freesia callus and its application in transient expression assays

Freesia hybrida is one of the most famous cut flower variety domesticated from wild species in Freesia genus, distinguished for its colorful flower colors and sweet scents. It has potential to be a model plant to unravel the molecular basis of plant specialized metabolites biosynthesis in family Iridaceae, which are crucial to genetically modify ornamental traits such as flower color and flower scents. However, the lengthy life cycle propagated from tissue culture and extremely low transformation rate severely decelerate functional characterization of genes through transgenic method in this iridaceous plant. Using calluses induced from young flower buds from one popular cultivar of Freesia hybrida, ‘Red River®’, an efficient protoplast isolation method was established and optimized, which was further employed in the gene transient expression system. Results demonstrated that the newly explored transient system was versatile and could be applied in numerous aspects, such as protein immunoblotting, enzyme activity, protein subcellular localization, protein–protein interaction, protein–DNA interaction and regulatory gene transregulation property assays. Moreover, calluses induced from another cultivar of Freesia hybrida, ‘Ambiance’ could also be used to isolate protoplasts for gene transient transfection analysis, indicating the wide applicability of this system. Results in this study provided new methods for the elucidation of gene function in Freesia in vivo, which might also be inspirational in other species with long life cycles or hardly to be transformed, such as plants in family Iridaceae. Freesia protoplast isolation and transfection methods were established and illustrated the utilization in protein immunoblotting, enzyme activity, subcellular localization, protein-protein or protein-DNA interaction, and investigation of transactivation capacities of regulators.