Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC
Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among...
Ausführliche Beschreibung
Autor*in: |
Jogam, Phanikanth [verfasserIn] |
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Format: |
E-Artikel |
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Sprache: |
Englisch |
Erschienen: |
2022 |
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Schlagwörter: |
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Anmerkung: |
© King Abdulaziz City for Science and Technology 2022 |
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Übergeordnetes Werk: |
Enthalten in: 3 Biotech - Berlin : Springer, 2011, 12(2022), 9 vom: 28. Juli |
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Übergeordnetes Werk: |
volume:12 ; year:2022 ; number:9 ; day:28 ; month:07 |
Links: |
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DOI / URN: |
10.1007/s13205-022-03251-x |
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Katalog-ID: |
SPR047708131 |
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100 | 1 | |a Jogam, Phanikanth |e verfasserin |4 aut | |
245 | 1 | 0 | |a Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
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520 | |a Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. | ||
650 | 4 | |a Candidate genes |7 (dpeaa)DE-He213 | |
650 | 4 | |a GUS |7 (dpeaa)DE-He213 | |
650 | 4 | |a Stable transformation |7 (dpeaa)DE-He213 | |
650 | 4 | |a Suspension cells |7 (dpeaa)DE-He213 | |
700 | 1 | |a Sandhya, Dulam |4 aut | |
700 | 1 | |a Alok, Anshu |4 aut | |
700 | 1 | |a Shekhawat, Mahipal S. |4 aut | |
700 | 1 | |a Peddaboina, Venkataiah |4 aut | |
700 | 1 | |a Singh, Kashmir |4 aut | |
700 | 1 | |a Allini, Venkateswar Rao |0 (orcid)0000-0002-2514-2828 |4 aut | |
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10.1007/s13205-022-03251-x doi (DE-627)SPR047708131 (SPR)s13205-022-03251-x-e DE-627 ger DE-627 rakwb eng Jogam, Phanikanth verfasserin aut Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © King Abdulaziz City for Science and Technology 2022 Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 Sandhya, Dulam aut Alok, Anshu aut Shekhawat, Mahipal S. aut Peddaboina, Venkataiah aut Singh, Kashmir aut Allini, Venkateswar Rao (orcid)0000-0002-2514-2828 aut Enthalten in 3 Biotech Berlin : Springer, 2011 12(2022), 9 vom: 28. Juli (DE-627)655133887 (DE-600)2600522-0 2190-5738 nnns volume:12 year:2022 number:9 day:28 month:07 https://dx.doi.org/10.1007/s13205-022-03251-x lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_266 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2022 9 28 07 |
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10.1007/s13205-022-03251-x doi (DE-627)SPR047708131 (SPR)s13205-022-03251-x-e DE-627 ger DE-627 rakwb eng Jogam, Phanikanth verfasserin aut Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © King Abdulaziz City for Science and Technology 2022 Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 Sandhya, Dulam aut Alok, Anshu aut Shekhawat, Mahipal S. aut Peddaboina, Venkataiah aut Singh, Kashmir aut Allini, Venkateswar Rao (orcid)0000-0002-2514-2828 aut Enthalten in 3 Biotech Berlin : Springer, 2011 12(2022), 9 vom: 28. Juli (DE-627)655133887 (DE-600)2600522-0 2190-5738 nnns volume:12 year:2022 number:9 day:28 month:07 https://dx.doi.org/10.1007/s13205-022-03251-x lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_266 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2022 9 28 07 |
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10.1007/s13205-022-03251-x doi (DE-627)SPR047708131 (SPR)s13205-022-03251-x-e DE-627 ger DE-627 rakwb eng Jogam, Phanikanth verfasserin aut Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © King Abdulaziz City for Science and Technology 2022 Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 Sandhya, Dulam aut Alok, Anshu aut Shekhawat, Mahipal S. aut Peddaboina, Venkataiah aut Singh, Kashmir aut Allini, Venkateswar Rao (orcid)0000-0002-2514-2828 aut Enthalten in 3 Biotech Berlin : Springer, 2011 12(2022), 9 vom: 28. Juli (DE-627)655133887 (DE-600)2600522-0 2190-5738 nnns volume:12 year:2022 number:9 day:28 month:07 https://dx.doi.org/10.1007/s13205-022-03251-x lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_266 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2022 9 28 07 |
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10.1007/s13205-022-03251-x doi (DE-627)SPR047708131 (SPR)s13205-022-03251-x-e DE-627 ger DE-627 rakwb eng Jogam, Phanikanth verfasserin aut Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © King Abdulaziz City for Science and Technology 2022 Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 Sandhya, Dulam aut Alok, Anshu aut Shekhawat, Mahipal S. aut Peddaboina, Venkataiah aut Singh, Kashmir aut Allini, Venkateswar Rao (orcid)0000-0002-2514-2828 aut Enthalten in 3 Biotech Berlin : Springer, 2011 12(2022), 9 vom: 28. Juli (DE-627)655133887 (DE-600)2600522-0 2190-5738 nnns volume:12 year:2022 number:9 day:28 month:07 https://dx.doi.org/10.1007/s13205-022-03251-x lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_266 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2022 9 28 07 |
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10.1007/s13205-022-03251-x doi (DE-627)SPR047708131 (SPR)s13205-022-03251-x-e DE-627 ger DE-627 rakwb eng Jogam, Phanikanth verfasserin aut Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC 2022 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier © King Abdulaziz City for Science and Technology 2022 Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 Sandhya, Dulam aut Alok, Anshu aut Shekhawat, Mahipal S. aut Peddaboina, Venkataiah aut Singh, Kashmir aut Allini, Venkateswar Rao (orcid)0000-0002-2514-2828 aut Enthalten in 3 Biotech Berlin : Springer, 2011 12(2022), 9 vom: 28. Juli (DE-627)655133887 (DE-600)2600522-0 2190-5738 nnns volume:12 year:2022 number:9 day:28 month:07 https://dx.doi.org/10.1007/s13205-022-03251-x lizenzpflichtig Volltext GBV_USEFLAG_A SYSFLAG_A GBV_SPRINGER SSG-OLC-PHA GBV_ILN_11 GBV_ILN_20 GBV_ILN_22 GBV_ILN_23 GBV_ILN_24 GBV_ILN_31 GBV_ILN_32 GBV_ILN_39 GBV_ILN_40 GBV_ILN_60 GBV_ILN_62 GBV_ILN_63 GBV_ILN_65 GBV_ILN_69 GBV_ILN_70 GBV_ILN_73 GBV_ILN_74 GBV_ILN_90 GBV_ILN_95 GBV_ILN_100 GBV_ILN_101 GBV_ILN_105 GBV_ILN_110 GBV_ILN_120 GBV_ILN_138 GBV_ILN_150 GBV_ILN_151 GBV_ILN_152 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_187 GBV_ILN_206 GBV_ILN_213 GBV_ILN_224 GBV_ILN_230 GBV_ILN_250 GBV_ILN_266 GBV_ILN_281 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_636 GBV_ILN_702 GBV_ILN_2001 GBV_ILN_2003 GBV_ILN_2004 GBV_ILN_2005 GBV_ILN_2006 GBV_ILN_2007 GBV_ILN_2008 GBV_ILN_2009 GBV_ILN_2010 GBV_ILN_2011 GBV_ILN_2014 GBV_ILN_2015 GBV_ILN_2020 GBV_ILN_2021 GBV_ILN_2025 GBV_ILN_2026 GBV_ILN_2027 GBV_ILN_2031 GBV_ILN_2034 GBV_ILN_2037 GBV_ILN_2038 GBV_ILN_2039 GBV_ILN_2044 GBV_ILN_2048 GBV_ILN_2049 GBV_ILN_2050 GBV_ILN_2055 GBV_ILN_2056 GBV_ILN_2057 GBV_ILN_2059 GBV_ILN_2061 GBV_ILN_2064 GBV_ILN_2065 GBV_ILN_2068 GBV_ILN_2088 GBV_ILN_2093 GBV_ILN_2106 GBV_ILN_2107 GBV_ILN_2108 GBV_ILN_2110 GBV_ILN_2111 GBV_ILN_2112 GBV_ILN_2113 GBV_ILN_2118 GBV_ILN_2122 GBV_ILN_2129 GBV_ILN_2143 GBV_ILN_2144 GBV_ILN_2147 GBV_ILN_2148 GBV_ILN_2152 GBV_ILN_2153 GBV_ILN_2188 GBV_ILN_2190 GBV_ILN_2232 GBV_ILN_2336 GBV_ILN_2446 GBV_ILN_2470 GBV_ILN_2472 GBV_ILN_2507 GBV_ILN_2522 GBV_ILN_2548 GBV_ILN_4012 GBV_ILN_4035 GBV_ILN_4037 GBV_ILN_4046 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4242 GBV_ILN_4246 GBV_ILN_4249 GBV_ILN_4251 GBV_ILN_4305 GBV_ILN_4306 GBV_ILN_4307 GBV_ILN_4313 GBV_ILN_4322 GBV_ILN_4323 GBV_ILN_4324 GBV_ILN_4325 GBV_ILN_4326 GBV_ILN_4328 GBV_ILN_4333 GBV_ILN_4334 GBV_ILN_4335 GBV_ILN_4336 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4393 GBV_ILN_4700 AR 12 2022 9 28 07 |
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English |
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Enthalten in 3 Biotech 12(2022), 9 vom: 28. Juli volume:12 year:2022 number:9 day:28 month:07 |
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Enthalten in 3 Biotech 12(2022), 9 vom: 28. Juli volume:12 year:2022 number:9 day:28 month:07 |
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Candidate genes GUS Stable transformation Suspension cells |
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3 Biotech |
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Jogam, Phanikanth @@aut@@ Sandhya, Dulam @@aut@@ Alok, Anshu @@aut@@ Shekhawat, Mahipal S. @@aut@@ Peddaboina, Venkataiah @@aut@@ Singh, Kashmir @@aut@@ Allini, Venkateswar Rao @@aut@@ |
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2022-07-28T00:00:00Z |
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Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. 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|
author |
Jogam, Phanikanth |
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Jogam, Phanikanth misc Candidate genes misc GUS misc Stable transformation misc Suspension cells Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
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Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC Candidate genes (dpeaa)DE-He213 GUS (dpeaa)DE-He213 Stable transformation (dpeaa)DE-He213 Suspension cells (dpeaa)DE-He213 |
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misc Candidate genes misc GUS misc Stable transformation misc Suspension cells |
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Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
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Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
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Jogam, Phanikanth |
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3 Biotech |
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Jogam, Phanikanth Sandhya, Dulam Alok, Anshu Shekhawat, Mahipal S. Peddaboina, Venkataiah Singh, Kashmir Allini, Venkateswar Rao |
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agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of artemisia pallens wall. ex dc |
title_auth |
Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
abstract |
Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. © King Abdulaziz City for Science and Technology 2022 |
abstractGer |
Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. © King Abdulaziz City for Science and Technology 2022 |
abstract_unstemmed |
Abstract A reliable and stable Agrobacterium-mediated genetic transformation system for Artemisia pallens has been developed using cell suspension cultures derived from cotyledon explants. Cotyledon, attached cotyledon, and compound leaves were found to be suitable for the induction of callus among five different types of explants tested. The yellow friable callus derived from attached cotyledon was used to initiate suspension cultures in Suspension Culture Medium (SCM) which was supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) at 2.0 mg $ L^{−1} $ and in combination with different concentrations of Zeatin (ZEA) at 0.25 mg $ L^{−1} $. Two different shock treatments, cold shock (at 4 ℃) for 20 min and heat shock (at 45 ℃) treatment for 5 min, heat shock treatment increased the transformation efficiency. The supplementation of Pluronic F-68 (0.05%) significantly enhanced the transformation efficiency of suspension cultures, whereas Silwet L-77 (0.05%) leads to more browning of the cells and reduced the transformation efficiency. The maximum GUS intensity was recorded with an optimal intensity of blue spots in the transformed cells. The highest GUS fluorometric activity measured was 879.4 ± 113.7 nmol 4MU/mg/min in transformed cell suspension cultures. The hygromycin-resistant calli showed intense blue color in GUS histochemical assay. The transgene integration into the plant genome was confirmed by polymerase chain reaction (PCR) using uidA specific primers in six hygromycin-resistant cell lines. The partial coding sequence of three candidate reference genes, i.e., ADP-ribosylation factor (Arf), β-actin (Act), and ubiquitin (Ubi), and carotenoid biosynthesis pathway gene, i.e., Phytoene desaturase (Pds) were cloned, sequenced, and submitted to NCBI for the first time. The quantitative mRNA expression of the transgene (uidA) and internal ApPds gene were evaluated in transgenic callus lines. The present Agrobacterium-mediated genetic transformation protocol could help in better understanding of the metabolic pathways of this medicinally important plant and its genetic improvement. © King Abdulaziz City for Science and Technology 2022 |
collection_details |
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container_issue |
9 |
title_short |
Agrobacterium-mediated genetic transformation and cloning of candidate reference genes in suspension cells of Artemisia pallens Wall. ex DC |
url |
https://dx.doi.org/10.1007/s13205-022-03251-x |
remote_bool |
true |
author2 |
Sandhya, Dulam Alok, Anshu Shekhawat, Mahipal S. Peddaboina, Venkataiah Singh, Kashmir Allini, Venkateswar Rao |
author2Str |
Sandhya, Dulam Alok, Anshu Shekhawat, Mahipal S. Peddaboina, Venkataiah Singh, Kashmir Allini, Venkateswar Rao |
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doi_str |
10.1007/s13205-022-03251-x |
up_date |
2024-07-03T14:28:47.035Z |
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|
score |
7.399579 |