Nuclear behaviour of colonies and regenerated buds from protoplasts ofNicotiana plumbaginifolia Viviani haploids

Cambecèdes, Jocelyne; Boucaud, Marie‐Thérèse de; Gaultier, Jean-Maurice; Lutz, A

doi:10.1007/bf00043044

Cited by 6 publications

(5 citation statements)

References 18 publications

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“…The degree of viability is used as a criterion to determine the quality of protoplasts after enzymatic digestion. However, enzymatic structural variability, which results in anomalies in digestion during cell division and in plants regenerated from protoplasts ( Cambecedes et al., 1988 ). In this study, although high-yield (5×10 6 protoplasts/g FW) and high-vitality (> 90%) protoplasts were obtained by optimizing the enzymatic hydrolysis conditions, the cell membranes of the protoplasts perforated to different degrees post enzymolysis.…”

Section: Discussionmentioning

confidence: 99%

“…As a result, the enzymatic solution disrupts not only the physiologic function but also the cytologic at molecular mechanisms of protoplasts ( Fu and Zhuang, 2001 ). Isolation generates structural variability, thereby causing anomalies during cell division ( Tylicki et al., 2002 ), as well as in plants regenerated from protoplasts ( Cambecedes et al., 1988 ). These anomalies often hinder the introduction of new plant varieties obtained via in vitro protoplast fusion ( Handley et al., 1986 ).…”

Section: Introductionmentioning

confidence: 99%

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An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

et al. 2022

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The protoplast experimental system eis an effective tool for functional genomics and cell fusion breeding. However, the physiological and molecular mechanisms of protoplast response to enzymolysis are not clear, which has become a major obstacle to protoplast regeneration. Here, we used physiological, cytological, proteomics and gene expression analysis to compare the young leaves of sugarcane and enzymolized protoplasts. After enzymatic digestion, we obtained protoplasts with viability of > 90%. Meanwhile, the content of malondialdehyde, an oxidation product, increased in the protoplasts following enzymolysis, and the activity of antioxidant enzymes, such as peroxidase (POD), catalase (CAT), acid peroxidase (APX), and O2-, significantly decreased. Cytologic analysis results showed that, post enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed several short rod-like structures in protoplasts. In this study, a proteomics approaches was used to identify proteins of protoplasts in response to the enzymatic digestion process. GO, KEGG, and KOG enrichment analyses revealed that the abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which allow for protein biosynthesis or degradation. RT-qPCR analysis revealed that the expression of osmotic stress resistance genes, such as DREB, WRKY, MAPK4, and NAC, was upregulated, while that of key regeneration genes, such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA, and GAUT, was significantly downregulated in the protoplasts. Hierarchical clustering and identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. Our findings can facilitate the development of a standard system to produce regenerated protoplasts using molecular markers and antibody detection of enzymolysis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

et al. 2022

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“…The level of viability is usually used as a criterion to determine the quality of the protoplasts after enzymatic digestion. However, enzymatic digestion generates structural variability resulting in anomalies observed during cell division as well as in plants regenerated from protoplasts (Cambecedes et al, 1988). These anomalies often hinder the introduction of new plant varieties obtained by methods of in vitro protoplast fusion (Handley et al, 1986).…”

Section: Discussionmentioning

confidence: 99%

“…As a result, the enzymatic solution disrupts not only physiologic function but also protoplast cytology and molecule level (Fu, 2001). Isolation generates structural variability, resulting in anomalies observed during cell division (Tylicki, 2002), as well as in plants regenerated from protoplasts (Cambecedes et al, 1988). These anomalies often hinder the introduction of new plant varieties obtained by methods of in vitro protoplast fusion (Handley et al, 1986).…”

Section: Introductionmentioning

confidence: 99%

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

Zhang

Wang

Xiao

et al. 2022

Preprint

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The protoplast experimental system has been becoming a powerful tool for functional genomics and cell fusion breeding. However, the physiology and molecular mechanism during enzymolysis is not completely understood and has become a major obstacle to protoplast regeneration. Our study used physiological, cytology, iTRAQ (Isobaric Tags for Relative and Absolute Quantification) -based proteomic and RT-PCR analyses to compare the young leaves of sugarcane (ROC22) and protoplasts of more than 90% viability. We found that oxidation product MDA content increased in the protoplasts after enzymolysis and several antioxidant enzymes such as POD, CAT, APX, and O2- content significantly decreased. The cytology results showed that after enzymolysis, the cell membranes were perforated to different degrees, the nuclear activity was weakened, the nucleolus structure was not obvious, and the microtubules depolymerized and formed many short rod-like structures in protoplasts. The proteomic results showed that 1,477 differential proteins were down-regulated and 810 were up-regulated after enzymolysis of sugarcane young leaves. The GO terms, KEGG and KOG enrichment analysis revealed that differentially abundant proteins were mainly involved in bioenergetic metabolism, cellular processes, osmotic stress, and redox homeostasis of protoplasts, which would allow protein biosynthesis or / degradation. The RT-PCR analysis revealed the expression of osmotic stress resistance genes such as DREB, WRKY, MAPK4, and NAC were up-regulated. Meanwhile, the expression of key regeneration genes such as CyclinD3, CyclinA, CyclinB, Cdc2, PSK, CESA and GAUT were significantly down-regulated in the protoplasts. Hierarchical clustering, identification of redox proteins and oxidation products showed that these proteins were involved in dynamic networks in response to oxidative stress after enzymolysis. We used a variety of methods to figure out how young sugarcane leaves react to enzymes.

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“…50% of premitotic Vicia hajastana protoplasts (Simmonds 1986;Simmonds and Setterfield 1986). This indicates that the procedure of protoplast isolation generates structural variability (Korlach and Zoglauer 1995;Zhang et al 1998;Tylicki et al 2002), resulting in anomalies observed during cell division (Meijer and Simmonds 1988), as well as in plants regenerated from protoplasts (Cambecedes et al 1988). These anomalies often hinder introduction of new plant varieties obtained by methods of in vitro protoplast fusion (Handley et al 1986).…”

Section: Introductionmentioning

confidence: 93%

Changes in the organization of the tubulin cytoskeleton during the early stages of Solanum lycopersicoides Dun. protoplast culture

et al. 2003

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Changes in the tubulin cytoskeleton during protoplast culture and plant regeneration of Solanum lycopersicoides Dun. were analyzed using an immunodetection method. Directly after isolation, four groups of protoplasts were distinguished: (1) mononuclear, (2) polynuclear, (3) homogeneous, (4) anuclear. The tubulin cytoskeleton of the protoplasts underwent rearrangements, correlating to the number and structure of cell nuclei in the protoplast. All protoplast groups with the exception of mononuclear were characterized by perturbations in the organization of the tubulin cytoskeleton. Anuclear and homogeneous protoplasts did not have a tubulin cytoskeleton. Polynuclear protoplasts had cortical microtubules, but were not capable of re-forming their original arrangement and did not possess a radial or perinuclear cytoskeleton. Irregularities in microtubule arrangement of these three groups of protoplasts caused their inability to regenerate a cell wall and to divide. Anuclear, polynuclear and homogeneous protoplasts were eliminated from the culture. Mononuclear protoplasts rearranged their cortical microtubules and reestablished the radial and perinuclear tubulin cytoskeleton. Re-formation of the cell suspension and subsequent regeneration of plants occurred exclusively from mononuclear protoplasts, which were able to regenerate cell walls and to divide.

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Nuclear behaviour of colonies and regenerated buds from protoplasts ofNicotiana plumbaginifolia Viviani haploids

Cited by 6 publications

References 18 publications

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An integrated physiology, cytology, and proteomics analysis reveals a network of sugarcane protoplast responses to enzymolysis

An Integrated Physiological, Cytology and Proteomics Reveals Network of Sugarcane Protoplasts Responses to Enzymolysis

Changes in the organization of the tubulin cytoskeleton during the early stages of Solanum lycopersicoides Dun. protoplast culture

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