Pub Date : 2023-11-13DOI: 10.1007/s11627-023-10389-7
Truyen N. Quach, Shirley J. Sato, Mark R. Behrens, Paul N. Black, Concetta C. DiRusso, Heriberto D. Cerutti, Tom Elmo Clemente
Abstract A reliable and simple Agrobacterium -mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II ( nptII ) and the phleomycin resistance ( bleI ) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens . The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 10 6 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.
{"title":"A facile Agrobacterium-mediated transformation method for the model unicellular green algae Chlamydomonas reinhardtii","authors":"Truyen N. Quach, Shirley J. Sato, Mark R. Behrens, Paul N. Black, Concetta C. DiRusso, Heriberto D. Cerutti, Tom Elmo Clemente","doi":"10.1007/s11627-023-10389-7","DOIUrl":"https://doi.org/10.1007/s11627-023-10389-7","url":null,"abstract":"Abstract A reliable and simple Agrobacterium -mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II ( nptII ) and the phleomycin resistance ( bleI ) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens . The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 10 6 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes.","PeriodicalId":13294,"journal":{"name":"In Vitro Cellular & Developmental Biology – Plant","volume":"54 8","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136282082","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-10DOI: 10.1007/s11627-023-10395-9
Manokari M., Mohammad Faisal, Abdulrahman A. Alatar, Karel Doležal, Mahipal S. Shekhawat
{"title":"Strigolactone analogue GR24 mediated somatic embryogenesis from leaf tissues of Santalum album L","authors":"Manokari M., Mohammad Faisal, Abdulrahman A. Alatar, Karel Doležal, Mahipal S. Shekhawat","doi":"10.1007/s11627-023-10395-9","DOIUrl":"https://doi.org/10.1007/s11627-023-10395-9","url":null,"abstract":"","PeriodicalId":13294,"journal":{"name":"In Vitro Cellular & Developmental Biology – Plant","volume":"112 47","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135136922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-10-25DOI: 10.1007/s11627-023-10388-8
Asmaa M. Khalifa, Mohammed A. Eid, Reda M. Gaafar, Khalil M. Saad-Allah, Dina Gad
Abstract The medicinally effective plant Achillea fragrantissima exhibits a magnitude of pharmacological activities. In this study, the effects of different ZnONP concentrations on antioxidant enzymes, bioactive secondary metabolites, redox potential, and molecular changes in A. fragrantissima callus cultures were investigated. First, the concentrations of the growth regulators 2,4-D and BA were optimized using Murashige and Skoog (MS) medium. The MS medium was then administered with 2,4-D and BA at its optimal dosage (1.0 mg.L −1 ); afterward, different ZnONP supplements (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L −1 ) were added. ZnONPs resulted in many physiological and molecular responses. ZnONPs significantly increased POD, APX, and SOD activities. While 10.0 mg.L −1 ZnONPs significantly increased POD and APX activities, 15.0 mg.L −1 ZnONPs significantly increased SOD. However, CAT activity gradually decreased with ZnONPs. Metabolically, ZnONPs increased phenolics, flavonoids, alkaloids, and saponin levels. Phenolic levels peaked at 20.0 mg.L −1 , flavonoids at 15.0 mg.L −1 , and alkaloids and saponins at 10.0 mg.L −1 . Terpenoids were more prevalent at lower levels of ZnONPs. With 15.0 and 10.0 mg.L −1 giving the maximum activity, ZnONPs enhanced the DPPH activity and TAC of the callus culture extracts, respectively. RAPD and ISSR fingerprinting were applied using 12 random and ISSR primers to evaluate the genetic stability of ZnONP-induced callus cultures. Six RAPD primers showed 83% polymorphism while the seven ISSR primers achieved 30% polymorphism. Consequently, DNA mutations may have been induced by ZnONPs and caused DNA fragments to either appear or disappear in RAPD and ISSR callus profiles. The dendrogram based on RAPD and ISSR combined data showed that by increasing ZnONP concentration the genetic differentiation among callus cultures was elevated. In conclusion, higher accumulation of secondary metabolites and redox activity were increased in A. fragrantissima callus cultures using low ZnONPs (10.0 mg.L −1 ) concentration.
{"title":"Induction of bioactive constituents and antioxidant enzyme activities in Achillea fragrantissima (Forskal) callus cultures using ZnO nanoparticles","authors":"Asmaa M. Khalifa, Mohammed A. Eid, Reda M. Gaafar, Khalil M. Saad-Allah, Dina Gad","doi":"10.1007/s11627-023-10388-8","DOIUrl":"https://doi.org/10.1007/s11627-023-10388-8","url":null,"abstract":"Abstract The medicinally effective plant Achillea fragrantissima exhibits a magnitude of pharmacological activities. In this study, the effects of different ZnONP concentrations on antioxidant enzymes, bioactive secondary metabolites, redox potential, and molecular changes in A. fragrantissima callus cultures were investigated. First, the concentrations of the growth regulators 2,4-D and BA were optimized using Murashige and Skoog (MS) medium. The MS medium was then administered with 2,4-D and BA at its optimal dosage (1.0 mg.L −1 ); afterward, different ZnONP supplements (0.0, 5.0, 10.0, 15.0, and 20.0 mg.L −1 ) were added. ZnONPs resulted in many physiological and molecular responses. ZnONPs significantly increased POD, APX, and SOD activities. While 10.0 mg.L −1 ZnONPs significantly increased POD and APX activities, 15.0 mg.L −1 ZnONPs significantly increased SOD. However, CAT activity gradually decreased with ZnONPs. Metabolically, ZnONPs increased phenolics, flavonoids, alkaloids, and saponin levels. Phenolic levels peaked at 20.0 mg.L −1 , flavonoids at 15.0 mg.L −1 , and alkaloids and saponins at 10.0 mg.L −1 . Terpenoids were more prevalent at lower levels of ZnONPs. With 15.0 and 10.0 mg.L −1 giving the maximum activity, ZnONPs enhanced the DPPH activity and TAC of the callus culture extracts, respectively. RAPD and ISSR fingerprinting were applied using 12 random and ISSR primers to evaluate the genetic stability of ZnONP-induced callus cultures. Six RAPD primers showed 83% polymorphism while the seven ISSR primers achieved 30% polymorphism. Consequently, DNA mutations may have been induced by ZnONPs and caused DNA fragments to either appear or disappear in RAPD and ISSR callus profiles. The dendrogram based on RAPD and ISSR combined data showed that by increasing ZnONP concentration the genetic differentiation among callus cultures was elevated. In conclusion, higher accumulation of secondary metabolites and redox activity were increased in A. fragrantissima callus cultures using low ZnONPs (10.0 mg.L −1 ) concentration.","PeriodicalId":13294,"journal":{"name":"In Vitro Cellular & Developmental Biology – Plant","volume":"38 4","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-10-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134973495","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-10-16DOI: 10.1007/s11627-023-10390-0
Afreen Anjum, Afaque Quraishi
{"title":"Enhanced epicurzerenone production via in vitro elicitation of microrhizomes of Curcuma caesia Roxb.","authors":"Afreen Anjum, Afaque Quraishi","doi":"10.1007/s11627-023-10390-0","DOIUrl":"https://doi.org/10.1007/s11627-023-10390-0","url":null,"abstract":"","PeriodicalId":13294,"journal":{"name":"In Vitro Cellular & Developmental Biology – Plant","volume":"76 1","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-10-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"136115406","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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