Protoplast Yield Research Articles

Establishment of transient and stable gene transformation systems in medicinal woody plant Acanthopanax senticosus

BackgroundTransient and stable gene transformation systems play a crucial role in elucidating gene functions and driving genetic improvement in plants. However, their application in medicinal woody plants has been hampered by inefficient procedures for isolating protoplasts and regenerating plants in vitro.ResultsEmbryogenic callus protoplast isolation and transient transformation system were successfully established. The highest yield of protoplasts was approximately 1.88 × 106 cells per gram with a viability of 90% under the combination of 1.5% cellulase and 0.2% macerozyme, with enzymatic digestion for 6 h in darkness followed by centrifugation at 400×g for 5 min. The transient transfection rate of protoplast reached 45.56% at a PEG 4000 concentration of 40%, a transfection time of 40 min, 16 h of dark incubation, a plasmid concentration of 1.5 ng μL−1, and 25 min heat shock at 45 °C. In addition, 15 Agrobacterium tumefaciens-mediated GUS-positive seedlings were obtained through the somatic embryogenetic pathway under the optimized conditions.ConclusionThis study successfully established both transient and stable genetic transformation systems, paving the way for future molecular biology research on A. senticosus.Graphical

Isolation of Haustorium Protoplasts Optimized by Orthogonal Design for Transient Gene Expression in Phelipanche aegyptiaca.

The efficient protoplast transient transformation system in plants is an important tool to study gene expression, metabolic pathways, and various mutagenic parameters, but it has not been established in Phelipanche aegyptiaca. As a root parasitic weed that endangers the growth of 29 species of plants in 12 families around the world, there is still no good control method for P. aegyptiaca. Even the parasitic mechanisms of P. aegyptiaca and the related genes regulating parasitism are not yet understood. In this study, by comparing the factors related to protoplast isolation and transfection, we developed the optimal protocol for protoplast isolation and transfection in Phelipanche aegyptiaca haustorium. The optimal protoplast yield and activity were 6.2 × 106 protoplasts/g fresh weight [FW] and 87.85%, respectively, by using 0.5 mol/L mannitol, enzyme concentrations of 2.5% cellulase R-10 and 0.8% Macerozyme R-10 at 24 °C for 4 h. At the same time, transfection efficiency of protoplasts was up to 78.49% when using 30 μg plasmid, 40% polyethylene glycol (PEG) concentration, 24 °C incubation temperature, and 20 min transfection time. This is the first efficient protoplasts' isolation and transient transformation system of Phelipanche aegyptiaca haustorium, laying a foundation for future studies on the gene function and mechanisms of haustorium formation in parasitic plants.

An efficient and universal protoplast-based transient gene expression system for genome editing in Brassica crops

Protoplast-based transient gene expression system has been widely used in plant genome editing because of its simple operation and less time-consuming. In order to establish a universal protoplast-based transient transfection system for verifying activities of genome editing vectors containing targets in Brassica, we systematically optimized factors affecting protoplast isolation and transient gene expression. We established an efficient protoplast-based transient gene expression system (PTGE) in Chinese cabbage, achieving high protoplast yield of 4.9 × 105 · g−1 FW, viability over 95%, and transfection efficiency of 76%. We showed for the first time that pretreatment of protoplasts with a hypotonic MMG could significantly enhance the transfection efficiency. Furthermore, protoplasts incubated at 37 °C for 6 min improved the transfection efficiency to 86%. We also demonstrated that PTGE worked well (more than 50% transfection efficiency) in multiple Brassica species including cabbage, Pak Choi, Chinese kale, and turnip. Finally, PTGE was used for validating the activities of CRISPR/Cas9 vectors containing targets in Chinese cabbage, cabbage, and pak choi, demonstrating the broad applicability of the established PTGE for genome editing in Brassica crops.

Correction to: Influences of factors affecting the induction of high protoplast yield and callus induction in two different carnations

Correction to: Influences of factors affecting the induction of high protoplast yield and callus induction in two different carnations

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.

Influences of factors affecting the induction of high protoplast yield and callus induction in two different carnations

Influences of factors affecting the induction of high protoplast yield and callus induction in two different carnations

Optimization of protoplast isolation and subsequent regeneration from the economically important brown alga Ecklonia cava using response surface methodology

Ecklonia cava is an edible kelp species distributed in Korea and Japan with diverse uses in ecological restoration, traditional medicine, and the pharmaceutical and abalone industries. These features make E. cava a good candidate for cellular biotechnology research, which is often dependent on protoplasts. To date, however, there have been no studies that have focused on the isolation of protoplast from E. cava. In this study, we sought to optimize the yield of protoplasts from cultured young sporophytes of E. cava using commercial enzymes, with a particular focus on identifying the key conditions for achieving high protoplast yield, including enzyme concentration, chelation pre-treatment, growth, temperature, incubation time, pH, and osmolarity. Protoplast production conditions were modeled using response surface methodology (RSM) via Box–Behnken design (BBD), and the practical utility of these conditions was experimentally verified. We found that an enzyme composition of 1.67 % cellulase RS and 4 U mL−1 alginate lyase without chelation pre-treatment, at 1570 mOsm L−1 H2O and pH 6 produced the highest protoplast yield of 4.45 ± 2.16 × 107 protoplasts g−1 fresh weight when incubated at 20 °C for 4 h using young (2-week-old) cultures. Our data revealed a strong positive correlation between growth rate and protoplast yield, thereby indicating that growth rate could serve as an index for determining the optimal timing of protoplast isolation from cultured material. The optimized protocol resulted in protoplasts capable of cell wall formation, division, and regeneration. These findings indicate the efficacy of commercial enzymes in protoplast isolation and that BBD-mediated RSM is a useful approach for the production of viable protoplasts from kelp sporophytes.

Optimization of preparation conditions for Salsola laricifolia protoplasts using response surface methodology and artificial neural network modeling

BackgroundSalsola laricifolia is a typical C3–C4 typical desert plant, belonging to the family Amaranthaceae. An efficient single-cell system is crucial to study the gene function of this plant. In this study, we optimized the experimental conditions by using Box-Behnken experimental design and Response Surface Methodology (RSM)-Artificial Neural Network (ANN) model based on the previous studies.ResultsAmong the 17 experiment groups designed by Box-Behnken experimental design, the maximum yield (1.566 × 106/100 mg) and the maximum number of viable cells (1.367 × 106/100 mg) were obtained in group 12, and the maximum viability (90.81%) was obtained in group 5. Based on these results, both the RSM and ANN models were employed for evaluating the impact of experimental factors. By RSM model, cellulase R-10 content was the most influential factor on protoplast yield, followed by macerozyme R-10 content and mannitol concentration. For protoplast viability, the macerozyme R-10 content had the highest influence, followed by cellulase R-10 content and mannitol concentration. The RSM model performed better than the ANN model in predicting yield and viability. However, the ANN model showed significant improvement in predicting the number of viable cells. After comprehensive evaluation of the protoplast yield, the viability and number of viable cells, the optimal results was predicted by ANN yield model and tested. The amount of protoplast yield was 1.550 × 106/100 mg, with viability of 90.65% and the number of viable cells of 1.405 × 106/100 mg. The corresponding conditions were 1.98% cellulase R-10, 1.00% macerozyme R-10, and 0.50 mol L−1 mannitol. Using the obtained protoplasts, the reference genes (18SrRNA, β-actin and EF1-α) were screened for expression, and transformed with PEG-mediated pBI121-SaNADP-ME2-GFP plasmid vector. There was no significant difference in the expression of β-actin and EF1-α before and after treatment, suggesting that they can be used as internal reference genes in protoplast experiments. And SaNADP-ME2 localized in chloroplasts.ConclusionThe current study validated and evaluated the effectiveness and results of RSM and ANN in optimizing the conditions for protoplast preparation using S. laricifolia as materials. These two methods can be used independently of experimental materials, making them suitable for isolating protoplasts from other plant materials. The selection of the number of viable cells as an evaluation index for protoplast experiments is based on its ability to consider both protoplast yield and viability. The findings of this study provide an efficient single-cell system for future genetic experiments in S. laricifolia and can serve as a reference method for preparing protoplasts from other materials.

Advances in Quercus ilex L. breeding: the CRISPR/Cas9 technology via ribonucleoproteins.

The CRISPR/Cas9 ribonucleoprotein (RNP)-mediated technology represents a fascinating tool for modifying gene expression or mutagenesis as this system allows for obtaining transgene-free plants, avoiding exogenous DNA integration. Holm oak (Quercus ilex) has an important social, economic, and ecological role in the Mediterranean climate zones of Western Europe and North Africa and is severely affected by oak decline syndrome. Here we report the first example of the application of the CRISPR/Cas9-RNP technology in holm oak. Firstly, we evaluated the protoplast isolation from both in vitro leaves and proembryogenic masses. Proembryogenic masses represented the best material to get high protoplast yield (11 x 106 protoplasts/ml) and viability. Secondly, the protoplast transfection ability was evaluated through a vector expressing green fluorescence protein as marker gene of transfection, reaching a transfection percentage of 62% after 24 hours. CRISPR/Cas9 RNPs were successfully delivered into protoplasts resulting in 5.6% ± 0.5% editing efficiency at phytoene desaturase (pds) target genomic region. Protoplasts were then cultured in semisolid media and, after 45 days in culture, developed embryogenic calli were observed in a Murashige and Skoog media with half concentration of NH4NO3 and KNO3 supplemented with 0.1 mg/L benzylaminopurine and 0.1 mg/L 2,4-dichlorophenoxyacetic acid.

Isolation, culture of Platycodon grandiflorus protoplasts: factors affecting protoplast yield, cell division, and micro-callus formation

Isolation, culture of Platycodon grandiflorus protoplasts: factors affecting protoplast yield, cell division, and micro-callus formation

Enhancing Protoplast Isolation and Early Cell Division from Cannabis sativa Callus Cultures via Phenylpropanoid Inhibition.

De novo regeneration of Cannabis sativa L. (cannabis) using tissue culture techniques remains unreliable and infrequent. Conventional methods for the regeneration and transformation of cannabis have not achieved the reliability and replicability that need to be integrated into research and breeding programs. Protoplast systems are effective for gene expression studies and transformation and genome-editing technologies and open the possibility of somatic hybridization to create interspecific hybrids. To date, leaf-derived protoplasts have been isolated for transient gene expression studies, but protoplast-to-plant regeneration has not been reported. The present study aims to evaluate the efficacy of using a callus culture system as an abundant tissue source for protoplast isolation and lays the groundwork for a protoplast-to-plant regeneration system. Using hypocotyl-derived callus cultures, which are known to have relatively greater regenerative potential, the efficacy of protoplast isolation and initial cell division were assessed. In this study, the effect of 2-aminoindane-2-phosphonic acid (AIP), a competitive inhibitor of phenylalanine ammonia lyase (PAL), in callus culture media and the effect of subculture frequency on protoplast yield were assessed. This study found that inclusion of AIP at 1 mM resulted in a 334% increase in protoplast yield compared with AIP-free medium, representing the first known use of AIP in cannabis tissue culture. Inclusion of AIP led to a 28% decrease in total soluble phenolics and 52% decrease in tissue browning compared with the control medium. Lastly, a two-phase culture system for protoplast regeneration was tested. At a concentration of 2.0 × 105 protoplasts per mL, cell wall reconstitution and cell division were observed, providing one of the first know reports of cell division from cannabis protoplasts and setting the stage for the future development of a protoplast-to-plant regeneration system.

Establishment of a genetic transformation system for cordycipitoid fungus Cordyceps chanhua.

Cordyceps chanhua is a well-known edible and medicinal mushroom with a long history of use in China, and it contains a variety of secondary metabolites with interesting bioactive ingredients. However, recent researches have mainly focused on cultivation conditions, secondary metabolite compositions and pharmacological activities of C. chanhua, the lack of an efficient and stable genetic transformation system has largely limited further research on the relationship between secondary metabolites and biosynthetic gene clusters in C. chanhua. In this study, single-factor experiments were used to compare the effects of different osmotic stabilizers, enzyme concentrations and enzyme digestion times on protoplast yield, and we found that the highest yield of 5.53 × 108 protoplasts/mL was obtained with 0.7 M mannitol, 6 mg/mL snail enzyme and 4 h of enzyme digestion time, and the regeneration rate of protoplasts was up to approximately 30% using 0.7 M mannitol as an osmotic stabilizer. On this basis, a PEG-mediated genetic transformation system of C. chanhua was successfully established for the first time, which lays the foundation for further genetic transformation of C. chanhua.

Polyethylene glycol–mediated genetic transformation and fluorescent labelling of Colletotrichum sublineola for studying sorghum anthracnose pathogenicity

AbstractAnthracnose, a disease primarily caused by the fungus Colletotrichum sublineola, affects sorghum production in China. However, the infection mechanisms and complex host interactions of C. sublineola remain to be elucidated. Thus, this study aimed to establish and optimize a protoplast transformation system of C. sublineola so as to lay a foundation for further research on the infection mechanisms and complex host interactions of C. sublineola. A genetic transformation system was established and optimized for C. sublineola strain HX‐5‐1 and used to introduce fluorescent reporter genes, namely, green fluorescent protein (GFP) and mCherry. Results showed that the growth of HX‐5‐1 was completely inhibited on a regenerating medium containing 300 μg mL−1 hygromycin B, indicating that hygromycin B acted as a transformant‐selective marker. HX‐5‐1 produced the highest yield of protoplasts when the mycelia were cultured for 60 h and dissolved in a 0.1% lysozyme solution (0.7 M NaCl) for 3 h. Subsequently, a 106 mL−1 protoplast solution transformed using 5 μg of plasmid DNA exhibited the highest transformation efficiency. Laser confocal microscopy showed that GFP and mCherry fluorescent signals were successfully expressed in the mycelia and spores of HX‐5‐1, indicating that they were stably inherited in the transformants and did not affect colony morphology, growth rate, or pathogenicity. In conclusion, we successfully established a genetic transformation system for C. sublineola and employed it to obtain stable fluorescent protein expression. This study may serve as a foundation for understanding the infection process of C. sublineola.

First Report on Mesophyll Protoplast Isolation and Regeneration System for the Duboisia Species.

The Duboisia species, a group of plants native to Australia, have been historically valued for their pharmacological properties and have played a significant role in traditional medicine and pharmaceutical research. Persistent efforts are underway to enhance the efficacy of the active ingredient scopolamine, employing both conventional breeding methods and advanced biotechnology tools. The primary objective of this research was to establish a highly efficient method for isolating mesophyll protoplasts and facilitating their regeneration, thereby laying a robust foundation for the application of various advanced plant biotechnology tools in the pursuit of genetic enhancement. The mesophyll protoplast isolation process was developed for hybrid D. myoporoides × D. hopwoodii with careful optimisation of the following parameters: leaf strip size; incubation conditions; physical treatment; and enzyme concentration. The optimal parameters were combined in each individual step; the best enzyme concentration was determined to be 2% (w/v) cellulysin and 0.5% (w/v) macerase. Protoplast yield was found to be greatly affected by the enzyme concentrations. The isolated protoplasts were cultured at a density of 0.5 × 105 to best sustain the highest cell division (33.2%) and a microcalli induction frequency of 17.9%. After 40 days of culture in a modified KM8P medium at 25 °C in darkness, visible microcalli were transferred to a solidified Murashige and Skoog (MS) medium with 1 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction under a 16 h photoperiod. After 30 days of culture, compact organogenic calli were transferred into a solid MS medium with 6-benzylaminopurine (BA) alone or thidiazuron (TDZ) alone or in combination with BA or naphthalene acetic acid (NAA) for shoot regeneration. The maximum shoot regeneration frequency (63.3%) was observed in the medium with 1.5 mg L-1 TDZ alone. For the first time, a reliable protoplast isolation and regeneration system from mesophyll cells was established for Duboisia with high protoplast viability, successful microcalli formation, and intact plant regeneration. This innovation will significantly contribute towards the genetic enhancement of the Duboisia species.

Establishing Gene Expression and Knockout Methods in Esteya vermicola CBS115803.

Pine wilt disease, which is caused by the nematode Bursaphelenchus xylophilus, is one of the most destructive forest diseases worldwide. Esteya vermicola, a nematophagous fungus, has emerged as a promising biological control agent. However, the limited availability of gene function analysis techniques hinders further genetic modification of this fungus. In this study, we employed a combination of enzymes (driselase, snailase, and cellulase) to enzymatically degrade the cell wall of the fungus, resulting in a high yield of protoplasts. Furthermore, by utilizing 0.6M sucrose as an osmotic pressure stabilizer, we achieved a significant protoplast regeneration rate of approximately 31%. Subsequently, we employed the polyethylene glycol-mediated protoplast transformation method to successfully establish a genetic transformation technique for E. vermicola CBS115803. Additionally, through our investigation, we identified the Olic promoter from Aspergillus nidulans, which effectively enhanced the expression of the DsRed gene encoding a red fluorescent protein in E. vermicola CBS115803. Moreover, we successfully implemented a split-marker strategy to delete the EvIPMD gene in E. vermicola CBS115803. In summary, our findings present valuable experimental methodologies for gene function analysis in E. vermicola CBS115803.

A streamlined guide RNA screening system for genome editing in Sorghum bicolor

BackgroundGenome editing tools derived from clustered regularly interspaced short palindromic repeats (CRISPR) systems have been developed for generating targeted mutations in plants. Although these tools hold promise for rapid crop improvement, target-specific guide RNAs exhibit variable activity. To improve genome editing, a rapid and precise method for evaluating their efficiency is necessary.ResultsHere we report an efficient system for screening single guide RNAs (sgRNAs) for genome editing in sorghum using a transient protoplast transfection assay. Protoplasts were isolated from leaves from sorghum plants cultivated under three different conditions. Cultivation for three days of continuous darkness following seven days with a 16-h light and 8-h dark photoperiod resulted in the highest yield of viable protoplasts and the highest protoplast transfection efficiency. We tested both plasmid-mediated and ribonucleoprotein-based delivery to protoplasts, via polyethylene glycol-mediated transfection, of CRISPR components targeting the sorghum genome. The frequencies of small insertions and deletions induced by a set of sgRNAs targeting four endogenous sorghum genes were analyzed via targeted deep sequencing. Our screening system induced indels in sorghum protoplasts at frequencies of up to 77.8% (plasmid) and 18.5% (RNP). The entire screening system was completed within 16 days.ConclusionsThe screening system optimized in this study for predicting sgRNA activity for genome editing in sorghum is efficient and straightforward. This system will reduce the time and effort needed for sorghum genome editing.

Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens

Protoplast culture and transformation technology offer a novel method for developing new plant varieties. Nonetheless, the effective preparation of protoplasts and transformation technology specific to celery has yet to be achieved. This study utilized celery seedling leaves as the primary materials to examine the key factors influencing protoplast isolation. The aim was to prepare leaf protoplasts with a high yield and of high quality and subsequently conduct transient gene transformation and expression. The findings indicated that the most effective procedure for isolating and purifying protoplasts was enzymatic digestion using an enzyme solution consisting of 2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol for a duration of 8 h. Subsequently, the protoplasts were filtered through a 400-mesh sieve and purified through centrifugation at 200× g. Within this system, the overall protoplast yield was exceptionally high, reaching a viability rate of up to 95%. The transient transformation system yielded a maximum transformation efficiency of approximately 53%, as evaluated using the green fluorescent protein (GFP) as a reporter gene. The parameters of the transient transformation system were as follows: a protoplast concentration of 5 × 105 cells·mL−1, exogenous DNA concentration of 500 μg·mL−1, final concentration of PEG4000 at 40%, and transformation duration of 15 min. The transient transformation system was also utilized to further analyze the protein localization characteristics of the celery transcription factor AgMYB80. The findings indicated that AgMYB80 predominantly localizes in the nucleus, thereby confirming the reliability and effectiveness of the transient transformation system. This study successfully established an efficient system for isolating, purifying, and transforming celery protoplasts, and will serve as a basis for future studies on molecular biology and gene function.

Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia Oleifera

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.

Efficient and rapid system of plant regeneration via protoplast cultures of Fagopyrum esculentum Moench

In the present study, a high yield of isolated protoplasts from the agronomically important crop Fagopyrum esculentum was obtained by applying a mixture of cellulase, pectolyase, and driselase. We demonstrated that the yield of morphogenic callus-derived protoplasts was 1 × 106 protoplasts per g of fresh tissue. For hypocotyls used as the protoplast source, the number of released cells was twice lower. The protoplasts, embedded in an agarose matrix and cultured in a modified Kao and Michayluk media supplemented with phytosulfokine, re-enter the cell cycle and start to develop, forming microcalli. The plating efficiency was about 20% in the case of hypocotyl- and morphogenic callus-derived protoplasts. For plant regeneration, the medium was supplemented with different combinations of cytokinin. Somatic embryogenesis and organogenesis occur during the cultivation of the protoplast-derived tissues, depending on the applied protoplast source. For the first time, an effective protoplast-to-plant system for F. esculentum has been developed.

Optimization of Isolation and Transformation of Protoplasts from Uncaria rhynchophylla and Its Application to Transient Gene Expression Analysis

Protoplast-based engineering has become an important tool for basic plant molecular biology research and developing genome-edited crops. Uncaria rhynchophylla is a traditional Chinese medicinal plant with a variety of pharmaceutically important indole alkaloids. In this study, an optimized protocol for U. rhynchophylla protoplast isolation, purification, and transient gene expression was developed. The best protoplast separation protocol was found to be 0.8 M D-mannitol, 1.25% Cellulase R-10, and 0.6% Macerozyme R-10 enzymolysis for 5 h at 26 °C in the dark with constant oscillation at 40 rpm/min. The protoplast yield was as high as 1.5 × 107 protoplasts/g fresh weight, and the survival rate of protoplasts was greater than 90%. Furthermore, polyethylene glycol (PEG)-mediated transient transformation of U. rhynchophylla protoplasts was investigated by optimizing different crucial factors affecting transfection efficiency, including plasmid DNA amount, PEG concentration, and transfection duration. The U. rhynchophylla protoplast transfection rate was highest (71%) when protoplasts were transfected overnight at 24 °C with the 40 µg of plasmid DNA for 40 min in a solution containing 40% PEG. This highly efficient protoplast-based transient expression system was used for subcellular localization of transcription factor UrWRKY37. Finally, a dual-luciferase assay was used to detect a transcription factor promoter interaction by co-expressing UrWRKY37 with a UrTDC-promoter reporter plasmid. Taken together, our optimized protocols provide a foundation for future molecular studies of gene function and expression in U. rhynchophylla.