The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, wa... more The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.
Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) ... more Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) in laboratory and industrial yeast strains. The plasmid, YEpS , containing the SOD2 gene was unstable in a sod2° mutant. However when Paraquat (0.5 mM) was used as a selective agent, the plasmid was maintained in the sod2° mutant but lost in the wild-type strain. When the CUP1 gene was inserted into YEpS 1 , the resulting plasmid (YEpCuS ) was 100% stable in the sod2° mutant grown in Cu -containing medium. In the absence of Cu , the proportion of plasmid-containing cells fell to 20%. YEpS was also transformed into an industrial strain, transformants could be selected in Paraquat-containing medium but showed poor stability.
The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, wa... more The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.
Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) ... more Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) in laboratory and industrial yeast strains. The plasmid, YEpS , containing the SOD2 gene was unstable in a sod2° mutant. However when Paraquat (0.5 mM) was used as a selective agent, the plasmid was maintained in the sod2° mutant but lost in the wild-type strain. When the CUP1 gene was inserted into YEpS 1 , the resulting plasmid (YEpCuS ) was 100% stable in the sod2° mutant grown in Cu -containing medium. In the absence of Cu , the proportion of plasmid-containing cells fell to 20%. YEpS was also transformed into an industrial strain, transformants could be selected in Paraquat-containing medium but showed poor stability.
Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotria... more Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotriates, which are terpene derivatives, in all of its organs. Valepotriates are the presumed sedative generic components of the pharmaceutically used species of Valeriana. The influence of various concentrations of the auxins indole-3-acetic acid, indole-3-butyric acid and α-naphthaleneacetic acid on the growth of micropropagated V. glechomifolia was investigated under conditions of transient and continuous exposure. Changes in the development of roots and shoots as well as the production of the valepotriates acevaltrate, valtrate and didrovaltrate (analyzed by high-performance liquid chromatography) were evaluated. The best performance in valepotriate production, growth and survival under ex vitro conditions following plant acclimatization was achieved in the continuous presence of 5.71 μM IAA. When cultured in medium containing IAA plants produced stable levels of valepotriates throughout the entire cultivation period.
The phenolic compound content of Hypericum ternum was investigated after micropropagation establi... more The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.
in Vitro Cellular & Developmental Biology-plant, 2006
Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all... more Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g−1 DW, valtrate (VAL) 10.2mgg−1 DW, and didrovaltrate (DID) 2.9mg g−1 DW were observed in root cultures after 7–8wk of culture.
Valtrate, DIA-valtrate, acevaltrate, 1-beta-acevaltrate and didrovaltrate have been quantitativel... more Valtrate, DIA-valtrate, acevaltrate, 1-beta-acevaltrate and didrovaltrate have been quantitatively estimated by reversed-phase HPLC in the leaves, flowers, stems and roots of Valeriana glechomifolia Meyer, V. catharinensis Graebn., V. chamaedryfolia Cham. & Schltdl., V. eichleriana (C.A.Mull.) Graebn., V. polysthachya Smith, V. scandens L., V. eupatoria Sobral, V. salicariifolia Vahl and V. tajuvensis Sobral. All plants presented valepotriates being V. glechomifolia the richest one, followed by V. eupatoria, V. eichleriana and V. tajuvensis.
Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium s... more Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC.
A system for growing in liquid medium whole plants of Valeriana glechomifolia, endemic to souther... more A system for growing in liquid medium whole plants of Valeriana glechomifolia, endemic to southern Brazil and capable of accumulating bioactive valepotriates, is described. Murashige and Skoog (MS) and Gamborg B5 (B5) media (1.0×, 0.3× and 0.1× strength) without phytohormones were evaluated after four weeks of culture in relation to growth and valepotriate yield. Plants grown in 1.0× MS displayed greatest growth and valepotriate yields and the study of the light condition showed that plants grown under light and dark had similar weight increase and maximum valepotriate yield, 27.2 mg/g DW and 25.0 mg/g DW, respectively. Valtrate was the most abundant valepotriate, followed by acevaltrate and didrovaltrate.
in Vitro Cellular & Developmental Biology-plant, 2008
Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumul... more Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumulates the bioactive terpene derivatives valepotriates in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75), or on a modified formulation (M Δ) was compared in stock cultures kept for up to 9 mo. without subculture. Changes in biomass accumulation, development of roots and shoots, and the production of the valepotriates acevaltrate, didrovaltrate, and valtrate were monthly evaluated. The highest biomass accumulation and root development was observed in plants grown on M Δ, whereas better leaf development was detected in M-Δ- and MS-medium-grown plants after 8 and 9 mo. of culture, respectively. Maximal didrovaltrate and valtrate yields were observed in M-Δ-grown plants harvested after 5 and 6 mo. of culture, respectively, whereas acevaltrate concentration was highest on M-Δ- and MS-75-grown plants after 7 mo. of culture. Plants grown for 6 mo. without subculture in M Δ were successfully propagated, showing stable growth and valepotriate yields three- to sixfold higher that those observed in field-grown plants. The results showed a positive effect of combined moderate reduction in salt concentration and increases in selected micronutrients and myo-inositol amounts on both growth and valepotriate yields of extended period stock cultures of V. glechomifolia.
Callus and cell suspension cultures were established from young leaves of Pilocarpus pennatifoliu... more Callus and cell suspension cultures were established from young leaves of Pilocarpus pennatifolius on Murashige & Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L kinetine. The pilocarpine contents of callus and cell suspension cultures were quantitatively compared by HPLC.
The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, wa... more The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.
Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) ... more Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) in laboratory and industrial yeast strains. The plasmid, YEpS , containing the SOD2 gene was unstable in a sod2° mutant. However when Paraquat (0.5 mM) was used as a selective agent, the plasmid was maintained in the sod2° mutant but lost in the wild-type strain. When the CUP1 gene was inserted into YEpS 1 , the resulting plasmid (YEpCuS ) was 100% stable in the sod2° mutant grown in Cu -containing medium. In the absence of Cu , the proportion of plasmid-containing cells fell to 20%. YEpS was also transformed into an industrial strain, transformants could be selected in Paraquat-containing medium but showed poor stability.
The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, wa... more The autonomously replicating plasmid YEpSS1, containing the S. cerevisiae SOD1 and SRB1 genes, was highly unstable in a wild-type strain. When transformed into a fragile srb1-1 mutant host, the same plasmid displayed different characteristics depending on the growth medium used. Both batch and continuous culture experiments demonstrated that the plasmid was very unstable when the transformed strain SLU15 was grown in the presence of an osmotic stabiliser (10% w/v sorbitol). However, in the absence of the osmoticum, nearly 100% of the cells retained the plasmid and produced the Sod1 protein after 80 generations of growth.
Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) ... more Saccharomyces cerevisiae SOD2and CUP1genes were used to maintain high-copy number plasmids (YEp) in laboratory and industrial yeast strains. The plasmid, YEpS , containing the SOD2 gene was unstable in a sod2° mutant. However when Paraquat (0.5 mM) was used as a selective agent, the plasmid was maintained in the sod2° mutant but lost in the wild-type strain. When the CUP1 gene was inserted into YEpS 1 , the resulting plasmid (YEpCuS ) was 100% stable in the sod2° mutant grown in Cu -containing medium. In the absence of Cu , the proportion of plasmid-containing cells fell to 20%. YEpS was also transformed into an industrial strain, transformants could be selected in Paraquat-containing medium but showed poor stability.
Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotria... more Valeriana glechomifolia is a plant species endemic to southern Brazil that accumulates valepotriates, which are terpene derivatives, in all of its organs. Valepotriates are the presumed sedative generic components of the pharmaceutically used species of Valeriana. The influence of various concentrations of the auxins indole-3-acetic acid, indole-3-butyric acid and α-naphthaleneacetic acid on the growth of micropropagated V. glechomifolia was investigated under conditions of transient and continuous exposure. Changes in the development of roots and shoots as well as the production of the valepotriates acevaltrate, valtrate and didrovaltrate (analyzed by high-performance liquid chromatography) were evaluated. The best performance in valepotriate production, growth and survival under ex vitro conditions following plant acclimatization was achieved in the continuous presence of 5.71 μM IAA. When cultured in medium containing IAA plants produced stable levels of valepotriates throughout the entire cultivation period.
The phenolic compound content of Hypericum ternum was investigated after micropropagation establi... more The phenolic compound content of Hypericum ternum was investigated after micropropagation establishment and during acclimatization over the phenological development of the plant. Plantlets cultured in vitro on full Murashige and Skoog medium without growth regulators displayed higher phenolic compound yields, were acclimatized, and field grown. Production of total phenolic compounds as well as hyperoside, chlorogenic acid, quercitrin, guaijaverin, isoquercitrin, and uliginosin B were quantified at vegetative, flowering and fructification stages, and different plant organs (roots, stems, leaves and reproductive parts) showing that reproductive parts at flowering stage and the leaves at fructification stage were the main repository site of secondary metabolites, except for uliginosin B. The stems were the least accumulative organ, while the roots accumulated only hyperoside and uliginosin B. Moreover, the accumulation of most of the flavonoids and uliginosin B in acclimatized plants surpassed the levels found in the wild plant, warranting further research with the species.
in Vitro Cellular & Developmental Biology-plant, 2006
Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all... more Valeriana glechomifolia is an endemic species of southern Brazil, capable of accumulating, in all of its organs, the terpene derivatives known as valepotriates, the presumed sedative components of the roots of pharmaceutically used species of Valeriana. In vitro cultures of the plant were established and the accumulation of acevaltrate, didrovaltrate, and valtrate in callus, cell suspension, and untransformed root cultures was studied. Leaves of in natura plants and roots of micropropagated plantlets were used as the explants for callus induction and root culture establishment, respectively, on Gamborg B5 basal medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) alone or with kinetin (KIN). Culture growth and secondary metabolite yields were enhanced with 2,4-D (4.52μM) and KIN (0.93μM). Maximum valepotriate contents, quantified by HPLC, of acevaltrate (ACE) 2.6mg g−1 DW, valtrate (VAL) 10.2mgg−1 DW, and didrovaltrate (DID) 2.9mg g−1 DW were observed in root cultures after 7–8wk of culture.
Valtrate, DIA-valtrate, acevaltrate, 1-beta-acevaltrate and didrovaltrate have been quantitativel... more Valtrate, DIA-valtrate, acevaltrate, 1-beta-acevaltrate and didrovaltrate have been quantitatively estimated by reversed-phase HPLC in the leaves, flowers, stems and roots of Valeriana glechomifolia Meyer, V. catharinensis Graebn., V. chamaedryfolia Cham. & Schltdl., V. eichleriana (C.A.Mull.) Graebn., V. polysthachya Smith, V. scandens L., V. eupatoria Sobral, V. salicariifolia Vahl and V. tajuvensis Sobral. All plants presented valepotriates being V. glechomifolia the richest one, followed by V. eupatoria, V. eichleriana and V. tajuvensis.
Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium s... more Callus and cell suspension cultures of Rauwolfia sellowii were established in Gamborg B5 medium supplemented with 1 mg l-1 2,4-dichlorophenoxyacetic acid, 0.2 mg l-1 kinetin and 30 g l-1 sucrose. The growth cycle of suspension cultures was completed in ca. 22 days and the maximum specific growth rate was 0.0098 h-1 with a doubling time of 71 h. The cultures accumulated the same major alkaloids as in the leaves of the parent plant, such as sellowiine, 19α,20α-epoxyakuammicine, vomilenine, picrinine and 12-demethoxytabernulosine. The alkaloid contents of leaves, callus and cell suspension cultures were quantitatively compared by HPLC.
A system for growing in liquid medium whole plants of Valeriana glechomifolia, endemic to souther... more A system for growing in liquid medium whole plants of Valeriana glechomifolia, endemic to southern Brazil and capable of accumulating bioactive valepotriates, is described. Murashige and Skoog (MS) and Gamborg B5 (B5) media (1.0×, 0.3× and 0.1× strength) without phytohormones were evaluated after four weeks of culture in relation to growth and valepotriate yield. Plants grown in 1.0× MS displayed greatest growth and valepotriate yields and the study of the light condition showed that plants grown under light and dark had similar weight increase and maximum valepotriate yield, 27.2 mg/g DW and 25.0 mg/g DW, respectively. Valtrate was the most abundant valepotriate, followed by acevaltrate and didrovaltrate.
in Vitro Cellular & Developmental Biology-plant, 2008
Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumul... more Valeriana glechomifolia, a southern Brazilian endemic species commonly known as Valerian, accumulates the bioactive terpene derivatives valepotriates in all of its organs. In vitro growth of V. glechomifolia on solid Murashige and Skoog (MS) without phytohormones at full, 75% (MS 75), or on a modified formulation (M Δ) was compared in stock cultures kept for up to 9 mo. without subculture. Changes in biomass accumulation, development of roots and shoots, and the production of the valepotriates acevaltrate, didrovaltrate, and valtrate were monthly evaluated. The highest biomass accumulation and root development was observed in plants grown on M Δ, whereas better leaf development was detected in M-Δ- and MS-medium-grown plants after 8 and 9 mo. of culture, respectively. Maximal didrovaltrate and valtrate yields were observed in M-Δ-grown plants harvested after 5 and 6 mo. of culture, respectively, whereas acevaltrate concentration was highest on M-Δ- and MS-75-grown plants after 7 mo. of culture. Plants grown for 6 mo. without subculture in M Δ were successfully propagated, showing stable growth and valepotriate yields three- to sixfold higher that those observed in field-grown plants. The results showed a positive effect of combined moderate reduction in salt concentration and increases in selected micronutrients and myo-inositol amounts on both growth and valepotriate yields of extended period stock cultures of V. glechomifolia.
Callus and cell suspension cultures were established from young leaves of Pilocarpus pennatifoliu... more Callus and cell suspension cultures were established from young leaves of Pilocarpus pennatifolius on Murashige & Skoog (MS) medium supplemented with 5.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg/L kinetine. The pilocarpine contents of callus and cell suspension cultures were quantitatively compared by HPLC.
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