Isolated Mesophyll Protoplasts Research Articles

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.

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.

Synthetic Promoter Screening Using Poplar Mesophyll Protoplast Transformation.

Plant protoplasts are useful to study both transcriptional regulation and protein subcellular localization in rapid screens. Protoplast transformation can be used in automated platforms for design-build-test cycles of plant promoters, including synthetic promoters. A notable application of protoplasts comes from recent successes in dissecting synthetic promoter activity with poplar mesophyll protoplasts. For this purpose, we constructed plasmids with TurboGFP driven by a synthetic promoter together with TurboRFP constitutively controlled by a 35S promoter, to monitor transformation efficiency, allowing versatile screening of high numbers of cells by monitoring green fluorescent protein expression in transformed protoplasts. Herein, we introduce a protocol for poplar mesophyll protoplast isolation followed by protoplast transformation and image analysis for the selection of valuable synthetic promoters. Graphical overview.

Isolation, purification and PEG-mediated transient expression of mesophyll protoplasts in Camellia oleifera

BackgroundCamellia oleifera (C. oleifera) is a woody edible oil crop of great economic importance. Because of the lack of modern biotechnology research, C. oleifera faces huge challenges in both breeding and basic research. The protoplast and transient transformation system plays an important role in biological breeding, plant regeneration and somatic cell fusion. The objective of this present study was to develop a highly efficient protocol for isolating and purifying mesophyll protoplasts and transient transformation of C. oleifera. Several critical factors for mesophyll protoplast isolation from C. oleifera, including starting material (leaf age), pretreatment, enzymatic treatment (type of enzyme, concentration and digestion time), osmotic pressure and purification were optimized. Then the factors affecting the transient transformation rate of mesophyll protoplasts such as PEG molecular weights, PEG4000 concentration, plasmid concentration and incubation time were explored.ResultsThe in vitro grown seedlings of C. oleifera ‘Huashuo’ were treated in the dark for 24 h, then the 1st to 2nd true leaves were picked and vacuumed at − 0.07 MPa for 20 min. The maximum yield (3.5 × 107/g·FW) and viability (90.9%) of protoplast were reached when the 1st to 2nd true leaves were digested in the enzymatic solution containing1.5% (w/v) Cellulase R-10, 0.5% (w/v) Macerozyme R-10 and 0.25% (w/v) Snailase and 0.4 M mannitol for 10 h. Moreover, the protoplast isolation method was also applicable to the other two cultivars, the protoplast yield for ‘TXP14’ and ‘DP47’ was 1.1 × 107/g·FW and 2.6 × 107/g·FW, the protoplast viability for ‘TXP14’ and ‘DP47’ was 90.0% and 88.2%. The purification effect was the best when using W buffer as a cleaning agent by centrifugal precipitation. The maximum transfection efficiency (70.6%) was obtained with the incubation of the protoplasts with 15 µg plasmid and 40% PEG4000 for 20 min.ConclusionIn summary, a simple and efficient system for isolation and transient transformation of C. oleifera mesophyll protoplast is proposed, which is of great significance in various aspects of C. oleifera research, including the study of somatic cell fusion, genome editing, protein function, signal transduction, transcriptional regulation and multi-omics analyses.

Efficient leaf mesophyll protoplast isolation and transient gene expression in bermudagrasses (Cynodon spp.)

Efficient leaf mesophyll protoplast isolation and transient gene expression in bermudagrasses (Cynodon spp.)

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.

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.

Optimizing Protocols for Arabidopsis Shoot and Root Protoplast Cultivation.

Procedures for the direct regeneration of entire plants from a shoot and root protoplasts of Arabidopsis thaliana have been optimized. The culture media for protoplast donor-plant cultivation and protoplast culture have been adjusted for optimal plant growth, plating efficiency, and promotion of shoot regeneration. Protocols have been established for the detection of all three steps in plant regeneration: (i) chromatin relaxation and activation of auxin biosynthesis, (ii) cell cycle progression, and (iii) conversion of cell-cycle active cells to totipotent ones. The competence for cell division was detected by DNA replication events and required high cell density and high concentrations of the auxinic compound 2,4-D. Cell cycle activity and globular structure formation, with subsequent shoot induction, were detected microscopically and by labeling with fluorescent dye Rhodamine123. The qPCR results demonstrated significantly upregulated expression of the genes responsible for nuclear reorganization, auxin responses, and auxin biosynthesis during the early stage of cell reprogramming. We further optimized cell reprogramming with this protocol by applying glutathione (GSH), which increases the sensitivity of isolated mesophyll protoplasts to cell cycle activation by auxin. The developed protocol allows us to investigate the molecular mechanism of the de-differentiation of somatic plant cells.

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.

Isolation of mesophyll protoplasts from tea (<i>Camellia sinensis</i>) and localization analysis of enzymes involved in the biosynthesis of specialized metabolites

Specialized metabolites in tea (<i>Camellia sinensis</i>) are fundamental quality factors. It is important to characterize gene function <i>in vivo</i> to identify key enzymes and reactions involved in the biosynthesis of such metabolites. Here we report a transient expression method to analyze gene function in isolated tea mesophyll protoplasts. This method is an alternative approach to traditional genetic transformation for studies on gene function <i>in vivo</i>. We screened several tea cultivars and different digestion conditions to optimize protoplast isolation. Digestion of newly emerged leaves of <i>C. sinensis</i> ‘Zhongbai 4’ with 3% cellulase R-10 and 0.3% macerozyme R-10 for about 12 h yielded approximately 10⁷ mesophyll protoplasts. Genes encoding enzymes involved in secondary metabolite synthesis were transiently expressed in the protoplasts, and their subcellular locations were determined. With further improvements in the transfection efficiency, this transient expression system will contribute greatly to the analyses of <i>in vivo</i> gene function in tea.

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.

Electrofusion of mesophyll protoplasts from two varieties of sugar beet, (Beta vulgaris L.)

Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.

Isolation and Purification of Mesophyll Protoplasts from &lt;i&gt;Ginkgo biloba&lt;/i&gt; L.

Isolation and Purification of Mesophyll Protoplasts from &lt;i&gt;Ginkgo biloba&lt;/i&gt; L.

Mesophyll protoplast isolation technique and flow cytometry analysis of ancient Platycladus orientalis (Cupressaceae)

Platycladus orientalis (Cupressaceae) is a native tree species used widely for landscaping and afforestation in arid and semiarid areas in Asia. Ancient P. orientalis trees not only have an important humanistic and historical value but also have a significant scientific value in aging mechanism research. Mesophyll protoplasts are an important material for studies of plant regeneration, transient gene expression, and senescence. Although an easy and effective technique of mesophyll protoplast isolation from mature trees is urgently needed, the isolation parameters have great material specificity. The young tissue of plants is an ideal material for protoplast preparation. In this study, we employed an orthogonal experiment and several single-variable experiments to determine the main factors influencing the successful isolation of mesophyll protoplasts and established an efficient technique for isolating mesophyll protoplasts from the young scale leaves of ancient P. orientalis. The optimal parameters for mesophyll protoplast isolation are as follows: mechanical homogenization of the leaf tissue, 1.5% Cellulase R-10, 0.4% Macerozyme R-10, 0.4% Pectolyase Y-23, 1.0% ligninase, 0.7 M mannitol (pH 5.8), and 16 h of incubation. Two centrifugations (100 × g for 3 min and 500 × g for 5 min) were repeated 2 times to obtain purified protoplast suspension. The yield and viability of protoplasts under optimal conditions were 4.8 × 106 g FW-1 (per gram fresh weight) and 82.5%, respectively. The results of flow cytometry analysis showed that the isolated protoplasts had ideal viability to meet the demands of further protoplast-based research.

Somatic Embryogenesis in the Medicago truncatula Model: Cellular and Molecular Mechanisms.

Medicago truncatula is now widely regarded as a legume model where there is an increasing range of genomic resources. Highly regenerable lines have been developed from the wild-type Jemalong cultivar, most likely due to epigenetic changes. These lines with high rates of somatic embryogenesis (SE) can be compared with wild-type where SE is rare. Much of the research has been with the high SE genotype Jemalong 2HA (2HA). SE can be induced from leaf tissue explants or isolated mesophyll protoplasts. In 2HA, the exogenous phytohormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) are central to SE. However, there are interactions with ethylene, abscisic acid (ABA), and gibberellic acid (GA) which produce maximum SE. In the main, somatic embryos are derived from dedifferentiated cells, undergo organellar changes, and produce stem-like cells. There is evidence that the SE is induced as a result of a stress and hormone interaction and this is discussed. In M. truncatula, there are connections between stress and specific up-regulated genes and specific hormones and up-regulated genes during the SE induction phase. Some of the transcription factors have been knocked down using RNAi to show they are critical for SE induction (MtWUSCHEL, MtSERF1). SE research in M. truncatula has utilized high throughput transcriptomic and proteomic studies and the more detailed investigation of some individual genes. In this review, these studies are integrated to suggest a framework and timeline for some of the key events of SE induction in M. truncatula.

Efficient mesophyll protoplast isolation and development of a transient expression system for castor-oil plant (Ricinus communis L.)

IntroductionWe investigated the main factors affecting the efficacy of protoplast isolation, including leaf-obtaining period, cutting shapes of leaf material, enzyme concentration, enzymolysis time, and centrifugal speed.MethodsProtoplast isolation was optimal on the condition of 20 days of leaf materials, 2-mm filament of leaves, 1.6% RS and 0.8% R-10, 80 min of enzymolysis, and 700 rpm of centrifugation, resulting in the best yield (1.19 X 106 protoplasts/g FW) and vitality (80.34%) of mesophyll protoplasts. The transient expression vector pGFPl with green fluorescent protein was transfected into the obtained protoplasts from castor by polyethylene glycol-mediated method with a transformation efficiency of 12.37%.ResultsMoreover, the applicability of the system for studying the subcellular localization of Re FATA (an acyl-ACP thioesterase) was validated via the protoplast isolation and transient expression protocol in this study.DiscussionCollectively, the efficient mesophyll protoplast isolation and protoplast transient expression system facilitate to analyze the function of specific gene in castor (Ricinus communis L).

An efficient mesophyll protoplast isolation, purification and PEG-mediated transient gene expression for subcellular localization in Chinese kale

Abstract Transient gene expression serves as a valuable tool for gene functional study in plants. Here we reported protoplast isolation and purification from the plantlet young leaves of Chinese kale (Brassica oleracae var. alboglabra Bailey), and their transient gene expression with polyethylene glycol (PEG)-mediated transformation and subcellular localization of phytoene desaturase (BaPDS1). The procedures of isolation andtransformation ofmesophyll protoplasts derived from Chinese kale were optimized, and the influencing factors were analyzed. The results showed that the optimal protocol of protoplast isolation and purification was initialized by digestion in enzyme solution (2.0% cellulase, 0.1% pectolase, and 0.6 M mannitol) for 9 h. After filtered through 400 mesh and centrifuged at 179 ×g for purification, the total yield of protoplast reached as high as 6.04 × 107 protoplasts g−1 fresh weight (FW) and the viability of the protoplasts was up to 95%. A maximum transformation efficiency of approximately 30% measured by using green fluorescent protein (GFP) as a detecting gene was obtained when PEG4000 was at a final concentration of 40% and transformation time was set to 15 min. In addition, the subcellular localization of BaPDS1 in Chinese kale was targeted to the chloroplast, confirming the efficiency and reliability of this transient transformation system. Taken together, an efficient protoplast isolation, purification and transformation system in Chinese kale was established in this study, laying a foundation for future research in molecular biology and gene function in Chinese kale and other Brassica vegetables.

Optimized conditions for the isolation of mesophyll protoplasts along the growing season from Arbutus unedo and their use in single cell gel electrophoresis

Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation.

An efficient protocol for perennial ryegrass mesophyll protoplast isolation and transformation, and its application on interaction study between LpNOL and LpNYC1

BackgroundPerennial ryegrass (Lolium perenne L.) is an important temperate grass used for turf and forage purposes. With the increasing accumulation of genomic and transcriptomic data of perennial ryegrass, an efficient protoplast and transient gene expression protocol is highly desirable for in vivo gene functional studies in its homologous system.ResultsIn this report, a highly efficient protoplast isolation (5.6 × 107 protoplasts per gram of leaf material) and transient expression (plasmid transformation efficiency at 55.2%) was developed and the detailed protocol presented. Using this protocol, the subcellular locations of two ryegrass proteins were visualized in chloroplasts and nuclei, respectively, and protein–protein interaction between two chlorophyll catabolic enzymes (LpNOL and LpNYC1) was recorded in its homologous system for the first time.ConclusionThis efficient protoplast isolation and transformation protocol is sufficient for studies on protein subcellular localization and protein–protein interaction, and shall be suitable for many other molecular biology applications where the mesophyll protoplast system is desirable in perennial ryegrass.

Highly efficient mesophyll protoplast isolation and PEG-mediated transient gene expression for rapid and large-scale gene characterization in cassava (Manihot esculenta Crantz)

BackgroundCassava (Manihot esculenta Crantz) is a major crop extensively cultivated in the tropics as both an important source of calories and a promising source for biofuel production. Although stable gene expression have been used for transgenic breeding and gene function study, a quick, easy and large-scale transformation platform has been in urgent need for gene functional characterization, especially after the cassava full genome was sequenced.MethodsFully expanded leaves from in vitro plantlets of Manihot esculenta were used to optimize the concentrations of cellulase R-10 and macerozyme R-10 for obtaining protoplasts with the highest yield and viability. Then, the optimum conditions (PEG4000 concentration and transfection time) were determined for cassava protoplast transient gene expression. In addition, the reliability of the established protocol was confirmed for subcellular protein localization.ResultsIn this work we optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and PEG-mediated transient gene expression in cassava. The suitable enzyme digestion system was established with the combination of 1.6% cellulase R-10 and 0.8% macerozyme R-10 for 16 h of digestion in the dark at 25 °C, resulting in the high yield (4.4 × 107 protoplasts/g FW) and vitality (92.6%) of mesophyll protoplasts. The maximum transfection efficiency (70.8%) was obtained with the incubation of the protoplasts/vector DNA mixture with 25% PEG4000 for 10 min. We validated the applicability of the system for studying the subcellular localization of MeSTP7 (an H+/monosaccharide cotransporter) with our transient expression protocol and a heterologous Arabidopsis transient gene expression system.ConclusionWe optimized the main influencing factors and developed an efficient mesophyll protoplast isolation and transient gene expression in cassava, which will facilitate large-scale characterization of genes and pathways in cassava.