Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

Raikar, S. V.; Braun, R. H.; Bryant, C.; Conner, A. J.; Christey, M. C.

doi:10.1007/s11816-008-0058-3

Cited by 13 publications

(9 citation statements)

References 25 publications

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“…Protoplasts are plant cell whose cell walls are enzymatically removed using cellulose-R10 combined with macerozyme-R10 [14,34]. We noted that increase in total concentration of enzymes led to an increase in protoplast yield, while excess enzymes led to a decrease in protoplast yield and viability (data not shown), probably due to phytotoxicity of enzymes on the membrane of protoplasts [61,62]. To obtain a higher number of viable protoplasts, a suitable concentration of additional enzymes such as pectolyase Y-23 [63] or/and hemicellulose may be added [24,29].…”

Section: Discussionmentioning

confidence: 90%

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

Ren

Gao

et al. 2020

IJMS

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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.

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Section: Discussionmentioning

confidence: 90%

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

Ren

Gao

et al. 2020

IJMS

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“…Additionally, concentration gradients of cellulose-R10 combined with macerozyme-R10 were included. The increase in total enzyme concentrations resulted in increased protoplast yield and viability, whereas excess enzymes led to the phytotoxicity of enzymes on the membrane of protoplasts ( Zhu et al, 1997 ; Raikar et al, 2008 ). The optimum concentrations of cellulose-R10 combined with macerozyme-R10 were determined to be 1.2% (w/v) and 0.6% (w/v), respectively ( Table 1 ).…”

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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“…As the concentration of total enzyme in the mixture increased, protoplast yield seemed to increase but protoplast viability to decrease. High levels of cellulose R-10 and macrerozyme R-10 increased protoplast yield but decreased the viability of protoplasts, possibly due to the influence of enzymes on the integrity of membrane and the physiological activities of protoplasts [37, 38]. The highest yield (5.1 × 10 7 protoplasts/ g FW) and the lowest viability of protoplasts (83.8%) were recorded with the 2.4% cellulose and 1.2% macrerozyme, which was the most concentrated enzyme mixture in our study.…”

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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Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

Cited by 13 publications

References 25 publications

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

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

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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