Factors That Influence the Yield and Viability of Cucumber (Cucumis sativus L) Cotyledon Protoplasts

Kantharajah, A. S.; Dodd, W. A.

doi:10.1071/bt9900169

Cited by 6 publications

(6 citation statements)

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“…The advantage of including callus in our study was that the starting material can continuously be produced and maintained in culture. Our results reveal that the concentration of the cell wall-degrading enzymes affected protoplast yield and the source tissues for protoplast isolation influenced the release of viable protoplasts consistent with previous reports [16,67,68] The CRISPR/dCas9 system is a platform for precise genetic control of genomic targets. CRISPR/dCas9 as a re-engineering platform for regulating gene expression in plants offers several benefits including simplicity, target specificity, flexibility, and reversibility [69].…”

Section: Discussionsupporting

confidence: 91%

“…VP16 is involved in multiple interactions with components of eukaryotic basal transcriptional machinery including with the different general transcription factors and mediator complex [75] to facilitate in the assembly of the pre-initiation complex [76]. Several investigations have demonstrated that dCas9-VP64, which employs the VP16 tetramer is effective in activating gene transcription [37,68,71,72] and recent research has shown that the dCas9-VP64 may be employed in plants to activate endogenous genes [69]. In the present study, the effectiveness of targeted gene activation in plant cells was evaluated using different combinations of purified dCas9-VP64 protein and IVT-sgRNAs targeting specific regions of the UGT76G1 gene inthe stevia that make up the RNP complexes.…”

Section: Discussionmentioning

confidence: 99%

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DNA Free CRISPR/DCAS9 Based Transcriptional Activation System for UGT76G1 Gene in Stevia rebaudiana Bertoni Protoplasts

Ghose

Abdullah

Hatta

et al. 2022

Plants

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The UDP-glycosyltransferase 76G1 (UGT76G1) is responsible for the conversion of stevioside to rebaudioside A. Four single guide RNAs (sgRNAs) were designed from the UGT76G1 proximal promoter region of stevia by using the online-based tool, benchling. The dCas9 fused with VP64 as a transcriptional activation domain (TAD) was produced and purified for the formation of ribonucleoproteins (RNPs) by mixing with the in vitro transcribed sgRNAs. Protoplast yield was the highest from leaf mesophyll of in vitro grown stevia plantlets (3.16 × 106/g of FW) using ES5 (1.25% cellulase R-10 and 0.75% macerozyme R-10). The RNPs were delivered into the isolated protoplasts through the Polyethylene glycol (PEG)-mediated transfection method. The highest endogenous activation of the UGT76G1 gene was detected at 27.51-fold after 24 h of transfection with RNP30 consisting of CRISPR/dCas9-TAD with sgRNA30 and a similar activation level was obtained using RNP18, RNP33, and RNP34, produced using sgRNA18, sgRNA33, and sgRNA34, respectively. Activation of UGT76G1 by RNP18 led to a significant increase in the expression of the rate-limiting enzyme UGT85C2 by 2.37-fold and there was an increasing trend in the expression of UGT85C2 using RNP30, RNP33, and RNP34. Successful application of CRISPR/dCas9-TAD RNP in activating specific genes can avoid the negative integration effects of introduced DNA in the host genome.

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Section: Discussionsupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

DNA Free CRISPR/DCAS9 Based Transcriptional Activation System for UGT76G1 Gene in Stevia rebaudiana Bertoni Protoplasts

Ghose

Abdullah

Hatta

et al. 2022

Plants

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“…The major factors affecting protoplast preparation are osmotic pressure, enzymatic concentration, and their combination in the enzyme solution, as well as the enzymolysis time ( Kantharajah and Dodd, 1990 ; Davey et al, 2005 ). Herein, a concentration gradient of D-mannitol was used.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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“…A reliable protoplast isolation with high yield and viability is prerequisite for the success of transient gene expression. Source tissue, the type and concentration of enzymes are critical factors affecting the release of protoplasts [ 3 , 35 , 36 ]. Leaves from field or greenhouse-grown plants usually have a thick cuticle and recalcitrant cell walls, which are likely to be the major limiting factors for protoplast isolation [ 12 ].…”

Section: Discussionmentioning

confidence: 99%

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

Liu

Geng

et al. 2017

BMC Biotechnol

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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.

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Factors That Influence the Yield and Viability of Cucumber (Cucumis sativus L) Cotyledon Protoplasts

Cited by 6 publications

References 0 publications

DNA Free CRISPR/DCAS9 Based Transcriptional Activation System for UGT76G1 Gene in Stevia rebaudiana Bertoni Protoplasts

DNA Free CRISPR/DCAS9 Based Transcriptional Activation System for UGT76G1 Gene in Stevia rebaudiana Bertoni Protoplasts

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

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

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