Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Yao, Liping; Liao, Xiudong; Gan, Zhizhe; Xiang, Peng; Wang, Peng; Li, Shaojuan; Li, Tianhong

doi:10.1016/j.scienta.2016.06.003

Cited by 34 publications

(27 citation statements)

References 48 publications

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“…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]. The efficiency of protoplast isolation from orchids was greatly increased from 10 6 to 10 7 /g FW (almost ten folds) with this protocol.…”

Section: Discussionmentioning

confidence: 95%

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: 95%

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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“…Source material is the most critical factor affecting protoplast release ( Jeon et al, 2007 ; Pindel, 2007 ; Yoo et al, 2007 ; Zhang et al, 2016b ). Various tissues of in vitro grown plantlets, including leaf tissues, flower pedicel, and column ( Pindel, 2007 ), and ex vitro grown leaves and flower petals ( Nanjareddy et al, 2016 ) as well as suspension cultured cells ( Mazarei et al, 2008 ; Wang et al, 2015 ; Yao et al, 2016 ) have been used, and protoplasts have been isolated from various plant species ( Table 1 ). In the present study, protoplasts were successfully isolated from various ex vitro tissues of orchids via the selection of suitable source materials and the optimization of enzymatic conditions.…”

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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“…In P. persica , different tissues were successfully used as explants for biolistic-mediated β-glucuronidase gene expression 203 in the search for a stable transformation procedure. In addition, protoplast procedures in P. avium 204 and P. avium hybrids 205 can be proposed as optimal tools for gene editing capability for in vivo evaluation. This technique could help to overcome chimerism, a significant issue in woody plant genetic transformation.…”

Section: Development Of Biotechnology Tools: Genetic Transformation Amentioning

confidence: 99%

Prunus genetics and applications after de novo genome sequencing: achievements and prospects

et al. 2019

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Prior to the availability of whole-genome sequences, our understanding of the structural and functional aspects of Prunus tree genomes was limited mostly to molecular genetic mapping of important traits and development of EST resources. With public release of the peach genome and others that followed, significant advances in our knowledge of Prunus genomes and the genetic underpinnings of important traits ensued. In this review, we highlight key achievements in Prunus genetics and breeding driven by the availability of these whole-genome sequences. Within the structural and evolutionary contexts, we summarize: (1) the current status of Prunus whole-genome sequences; (2) preliminary and ongoing work on the sequence structure and diversity of the genomes; (3) the analyses of Prunus genome evolution driven by natural and man-made selection; and (4) provide insight into haploblocking genomes as a means to define genome-scale patterns of evolution that can be leveraged for trait selection in pedigree-based Prunus tree breeding programs worldwide. Functionally, we summarize recent and ongoing work that leverages whole-genome sequences to identify and characterize genes controlling 22 agronomically important Prunus traits. These include phenology, fruit quality, allergens, disease resistance, tree architecture, and self-incompatibility. Translationally, we explore the application of sequence-based marker-assisted breeding technologies and other sequence-guided biotechnological approaches for Prunus crop improvement. Finally, we present the current status of publically available Prunus genomics and genetics data housed mainly in the Genome Database for Rosaceae (GDR) and its updated functionalities for future bioinformatics-based Prunus genetics and genomics inquiry.

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Protoplast isolation and development of a transient expression system for sweet cherry (Prunus avium L.)

Cited by 34 publications

References 48 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

Prunus genetics and applications after de novo genome sequencing: achievements and prospects

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