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Kalliopi A. Roubelakis-angelakis - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Technology in Grapevine
Molecular Biology & Biotechnology of the Grapevine, 2020Co-Authors: Anastasia K. Papadakis, G. Reustle, Kalliopi A. Roubelakis-angelakisAbstract:Hastein (1880) was probably the first to use the term Protoplast, from the greek proto (πρώτο = first) and plastos (πλαστός = created), for plant cells lacking cell walls and to isolate Protoplasts from cells of Vacuheria. In 1892, Klercker was the first to isolate Protoplasts following plasmolysis of leaf tissue cells of Stratiotes aloides. Large scale isolation of Protoplasts was started in the 60’s, when cell wall hydrolytic enzymes were employed (Cocking, 1961).
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Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide
Planta, 2005Co-Authors: Anastasia K. Papadakis, Kalliopi A. Roubelakis-angelakisAbstract:Our previous results indicate that during Protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197-205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434-444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) Protoplasts reduced the accumulation of O2*- but not that of H2O2. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during Protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated Protoplasts, concomitant with H2O2 titers. A cell death program was executed in untreated Protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in Protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant Protoplast totipotency.
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Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant Protoplasts.
Plant physiology, 2001Co-Authors: Anastasia K. Papadakis, C I Siminis, Kalliopi A. Roubelakis-angelakisAbstract:We previously showed that during Protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in Protoplasts is related to the activity of cellular antioxidant machinery during Protoplast culture. Totipotent (T) tobacco Protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco Protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco Protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine Protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco Protoplasts, after 8 d in culture. In T-tobacco Protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine Protoplasts, the oxidized forms predominated. In addition, T-tobacco Protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine Protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco Protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine Protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco Protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in Protoplasts is correlated with reduced activity of the cellular antioxidant machinery.
Anastasia K. Papadakis - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Technology in Grapevine
Molecular Biology & Biotechnology of the Grapevine, 2020Co-Authors: Anastasia K. Papadakis, G. Reustle, Kalliopi A. Roubelakis-angelakisAbstract:Hastein (1880) was probably the first to use the term Protoplast, from the greek proto (πρώτο = first) and plastos (πλαστός = created), for plant cells lacking cell walls and to isolate Protoplasts from cells of Vacuheria. In 1892, Klercker was the first to isolate Protoplasts following plasmolysis of leaf tissue cells of Stratiotes aloides. Large scale isolation of Protoplasts was started in the 60’s, when cell wall hydrolytic enzymes were employed (Cocking, 1961).
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Polyamines inhibit NADPH oxidase-mediated superoxide generation and putrescine prevents programmed cell death induced by polyamine oxidase-generated hydrogen peroxide
Planta, 2005Co-Authors: Anastasia K. Papadakis, Kalliopi A. Roubelakis-angelakisAbstract:Our previous results indicate that during Protoplast isolation an oxidative burst occurs [A.K. Papadakis and KA Roubelakis-Angelakis (1999) Plant Physiol 127:197-205] and that suppression of totipotency is correlated with reduced antioxidant activity and low redox state [A.K. Papadakis et al. (2001b) Plant Physiol 126:434-444]. Polyamines are known to affect cell development and to act as antioxidants. Polyamines applied during isolation of tobacco (Nicotiana tabacum L.) Protoplasts reduced the accumulation of O2*- but not that of H2O2. This antioxidant effect is probably due to the inhibition of microsomal membrane NADPH oxidase, which occurred in a concentration-dependent manner, with spermine exerting the highest inhibitory effect. However, during Protoplast culture, polyamine oxidase activity increased severalfold in spermidine- and spermine-treated Protoplasts, concomitant with H2O2 titers. A cell death program was executed in untreated Protoplasts, as documented by membrane malfunction, induced DNase activity, DNA fragmentation and a positive TUNEL reaction. Protoplast cell death was prevented in Protoplasts treated with putrescine, but not by treatment with spermidine or spermine, which rather had the opposite effect. The data presented suggest that PAs may be implicated in the expression of plant Protoplast totipotency.
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Reduced activity of antioxidant machinery is correlated with suppression of totipotency in plant Protoplasts.
Plant physiology, 2001Co-Authors: Anastasia K. Papadakis, C I Siminis, Kalliopi A. Roubelakis-angelakisAbstract:We previously showed that during Protoplast isolation, an oxidative burst occurred and the generation of active oxygen species was differentially mediated in tobacco (Nicotiana tabacum) and grapevine (Vitis vinifera), accompanied by significant quantitative differences (A.K. Papadakis, K.A. Roubelakis-Angelakis [1999] Plant Physiol 127: 197-205). We have now further tested if the expression of totipotency in Protoplasts is related to the activity of cellular antioxidant machinery during Protoplast culture. Totipotent (T) tobacco Protoplasts had 2-fold lower contents of intracellular O2*- and H2O2 and 7-fold lower levels of O2*- and H2O2 in the culture medium, compared with non-totipotent (NT) tobacco Protoplasts. Addition of alkaline dimethylsulfoxide, known to generate O2*-, resulted in isolation of tobacco Protoplasts with reduced viability and cell division potential during subsequent culture. Active oxygen species levels decreased in tobacco and grapevine Protoplasts during culturing, although higher contents of O2*- and H2O2 were still found in NT- compared with T-tobacco Protoplasts, after 8 d in culture. In T-tobacco Protoplasts, the reduced forms of ascorbate and glutathione predominated, whereas in NT-tobacco and grapevine Protoplasts, the oxidized forms predominated. In addition, T-tobacco Protoplasts exhibited severalfold lower lipid peroxidation than NT-tobacco and grapevine Protoplasts. Furthermore, several antioxidant enzyme activities were increased in T-tobacco Protoplasts. Superoxide dismutase activity increased in tobacco, but not in grapevine Protoplasts during culturing due to the increased expression of cytoplasmic Cu/Zn-superoxide dismutase. The increase was only sustained in T-tobacco Protoplasts for d 8. Together, these results suggest that suppressed expression of totipotency in Protoplasts is correlated with reduced activity of the cellular antioxidant machinery.
Dipti Tonk - One of the best experts on this subject based on the ideXlab platform.
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Plant regeneration from Protoplasts in Indian local Coriandrum sativum L.: scanning electron microscopy and histological evidences for somatic embryogenesis
Plant Cell Tissue and Organ Culture (PCTOC), 2014Co-Authors: A. Mujib, Dipti TonkAbstract:Coriandrum sativum L. is an annual herb belonging to the family Umbelliferae. It is used as a spice plant in Indian subcontinent and it has several medicinal applications as well. In this present article, an efficient plant regeneration protocol from Protoplasts via somatic embryogenesis was established and is reported. This is the first ever Protoplast isolation study in Indian local coriander in which plant regeneration was achieved. Hypocotyl-derived embryogenic callus was used as a source of Protoplast. The embryogenic callus suspension was prepared by transferring tissues onto rotary-agitated liquid Murashige and Skoog, added with 1.0 mg l^−1 2,4-Dichlorophenoxyacetic acid (2,4-D) and 1.0 mg l^−1 KIN (6-furfurylaminopurine). The suspension was digested with enzymatic solutions and a combination of cellulase (2.0 %), pectinase (1.0 %), macerozyme (0.02 %) and driselase (0.50 %) induced maximum yield of Protoplasts (34.25 × 10^5). In 1.0 mg l^−1 2,4-D + 1.0 mg l^−1 KIN containing medium, Protoplasts divided well and formed maximum number of microcolonies (14.30/test tube). The Protoplast callus (PC) biomass grew well in solid medium. The Protoplast embryogenic callus was rich in protein, proline and sugar compared to non-embryogenic PC. The Protoplast originated callus later differentiated into somatic embryos. The somatic embryo morphology, scanning electron microscopy and histology of embryo origin and development were investigated and discussed in details in this present communication. In 1.0 mg l^−1 2,4-D + 0.5 mg l^−1 BA (6-Benzyladenine), maximum number of embryos were formed on microcallus (26.6/callus mass). The embryo matured and germinated into plantlets at a low to moderate rate, highest (31.3 %) embryo germination was observed in 1.0 mg l^−1 BA + 0.5 mg l^−1 α-Naphthalene acetic acid added medium. The entire process of regeneration took about 4–5 months’ time for recovering plantlets from Protoplasts.
Quanzi Li - One of the best experts on this subject based on the ideXlab platform.
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a simple improved throughput xylem Protoplast system for studying wood formation
Nature Protocols, 2014Co-Authors: Wei Li, Hao Chen, Quanzi Li, Jack P Wang, Hsichuan Chen, Ling ChuangAbstract:Protoplasts can be used for transient gene expression analysis. This protocol for Protoplast isolation from wood-forming tissue will be useful for genetic analysis of woody plants, particularly those that lack methods for stable transformation.
Anne-marie Rusig - One of the best experts on this subject based on the ideXlab platform.
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Production and regeneration of Protoplasts from Grateloupia turuturu Yamada (Rhodophyta)
Journal of Applied Phycology, 2011Co-Authors: Nadège Lafontaine, Isabelle Mussio, Anne-marie RusigAbstract:Protoplasts were isolated enzymatically from the carrageenophyte red alga Grateloupia turuturu (Halymeniales, Rhodophyta) that occurs along the coast of the French Channel in Normandy. Effects of the main factors on the Protoplast yield were identified to improve the isolation protocol. The optimal enzyme composition for cell wall digestion and Protoplast viability consisted of 2% cellulase Onozuka R-10, 0.5% macerozyme R-10, 2% crude extract from viscera of Haliotis tuberculata , 0.8 M mannitol, 20 mM sodium citrate, 0.3% bovine serum albumin at 25°C, and 4-h incubation period. The Protoplasts were approximately 5–15 μm in diameter, liberated mainly from the surface cell layers. Maximum yield was 1.5 × 10^7 Protoplasts g^-1 fresh tissue. The Protoplasts underwent initial division after 14 days with a high density level of 1 × 10^6 cells mL^-1 in culture medium and developed into microthalli of a line of two to six cells.
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Plant regeneration from Protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture
Journal of Applied Phycology, 2001Co-Authors: Anne-marie Rusig, Joël CossonAbstract:A continuous micropropagation was established from Protoplasts of thegreen alga Enteromorpha intestinalis . The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal Protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and Protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha Protoplasts were released (10.0 × 10^6Protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the Protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha . A promising storage process has beendeveloped which involves including Protoplasts in beads of alginic acid gel.Plants regenerated from Protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.
