Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts

Yasuda, Kaori; Watanabe, Yukio; Watanabe, Masami

doi:10.5511/plantbiotechnology.24.361

Cited by 18 publications

(10 citation statements)

References 43 publications

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“…Abundant calcium oxalate crystals accumulated in the ruptured protoplasts, which could puncture other protoplasts ( Pindel, 2007 ; Lin et al, 2018b ), and were observed in the protoplast solution ( Supplementary Figure 1 ). Peroxides released by the ruptured protoplasts could also damage the protoplasts ( de Marco and Roubelakis-Angelakis, 1996 ; Yasuda et al, 2007 ). To address these problems and protect protoplasts from rupturing, a suitable concentration of the reducing agent 2-mercaptoethanol was added to the enzyme solution.…”

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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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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“…In non-regenerating protoplasts, with a lower capacity of antioxidant machinery, intracellular ROS induced by the stress of isolation can accumulate in a compartment-specific manner (De Marco and RoubelakisAngelakis 1996). Accumulated ROS are associated with increased levels of lipid peroxidation (Papadakis et al 2001) and participate in the initiation of apoptosis-like cell death of cultured protoplasts (Yasuda and Watanabe 2007). In a similar way to protoplast regeneration, the capacity of callus regeneration might be under the control of ROS production systems induced by growth regulators and controlled by antioxidant cellular systems, as previously demonstrated in wheat (Szechynska-Hebda et al 2007).…”

Section: Introductionmentioning

confidence: 90%

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

Petřivalský

Vaníčková

Ryzí

et al. 2011

Plant Cell Tiss Organ Cult

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The present study investigated changes in the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in isolated mesophyll protoplasts and cell cultures of the cucumber Cucumis sativus cv. Marketer. Although only a minor increase in the level of nitrogen oxide (NO) was observed during the first 7 days of culture following protoplast isolation, a substantial accumulation of ROS was detected. Compounds known to modulate endogenous ROS and RNS levels were employed to study their role in cucumber protoplast regeneration and growth. Supplementing the culture medium with the NO donors S-nitrosoglutathione and sodium nitroprusside and the ROS scavenger ascorbate significantly increased protoplast viability and cell density. In contrast, cell density was significantly decreased following the addition of catalase to the medium. Scavenging of ROS and RNS induced the formation of cucumber microcalli, thus suggesting a differential role of NO in the maintenance of cell viability and in the control of cell division. Our findings confirm the crucial role of controlled ROS and RNS production in both protoplast regeneration and cellular growth and differentiation.

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“…The isolation procedure can also induce further chromatin condensation and the apoptotic pathway [72]. This condensation is accompanied by a reduction in the scavenging capacity of reactive oxygen species (ROS) [73], which leads to an increased ROS accumulation [74,75]. Recently, the presence of chromatin condensation has also been demonstrated during protoplast isolation followed by subsequent cultivation in a buffer without PGRs [76].…”

Section: The Protoplast Isolation Step As the Key For Reprogrammingmentioning

confidence: 99%

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming

Pasternak

Lystvan

Betekhtin

et al. 2020

IJMS

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Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.

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Generation of intracellular reactive oxygen species during the isolation of Brassica napus leaf protoplasts

Cited by 18 publications

References 43 publications

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

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

The effects of reactive nitrogen and oxygen species on the regeneration and growth of cucumber cells from isolated protoplasts

From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming

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