Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata
Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector p...
Ausführliche Beschreibung
Gespeichert in:
| Autor*in: |
Yadav, Sushil Kumar [verfasserIn] Katikala, Sweety [verfasserIn] Yellisetty, Varalaxmi [verfasserIn] Kannepalle, Annapurna [verfasserIn] Narayana, Jyothi Lakshmi [verfasserIn] Maddi, Vanaja [verfasserIn] Mandapaka, Maheswari [verfasserIn] Shanker, Arun Kumar [verfasserIn] Bandi, Venkateswarlu [verfasserIn] Bharadwaja, Kirti Pulugurtha [verfasserIn] |
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E-Artikel |
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| Sprache: |
Englisch |
| Erschienen: |
2012 |
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| Übergeordnetes Werk: |
Enthalten in: SpringerPlus - London : Biomed Central, 2012, 1(2012), 1 vom: 10. Dez. |
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| Übergeordnetes Werk: |
volume:1 ; year:2012 ; number:1 ; day:10 ; month:12 |
| Links: |
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| DOI / URN: |
10.1186/2193-1801-1-59 |
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| Katalog-ID: |
SPR032747012 |
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| 520 | |a Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. | ||
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| 700 | 1 | |a Katikala, Sweety |e verfasserin |4 aut | |
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| 700 | 1 | |a Kannepalle, Annapurna |e verfasserin |4 aut | |
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| 700 | 1 | |a Shanker, Arun Kumar |e verfasserin |4 aut | |
| 700 | 1 | |a Bandi, Venkateswarlu |e verfasserin |4 aut | |
| 700 | 1 | |a Bharadwaja, Kirti Pulugurtha |e verfasserin |4 aut | |
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10.1186/2193-1801-1-59 doi (DE-627)SPR032747012 (SPR)2193-1801-1-59-e DE-627 ger DE-627 rakwb eng 600 ASE Yadav, Sushil Kumar verfasserin aut Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. mediated transformation (dpeaa)DE-He213 Annexin (dpeaa)DE-He213 Double cotyledonary node (dpeaa)DE-He213 Katikala, Sweety verfasserin aut Yellisetty, Varalaxmi verfasserin aut Kannepalle, Annapurna verfasserin aut Narayana, Jyothi Lakshmi verfasserin aut Maddi, Vanaja verfasserin aut Mandapaka, Maheswari verfasserin aut Shanker, Arun Kumar verfasserin aut Bandi, Venkateswarlu verfasserin aut Bharadwaja, Kirti Pulugurtha verfasserin aut Enthalten in SpringerPlus London : Biomed Central, 2012 1(2012), 1 vom: 10. Dez. (DE-627)718615298 (DE-600)2661116-8 2193-1801 nnns volume:1 year:2012 number:1 day:10 month:12 https://dx.doi.org/10.1186/2193-1801-1-59 kostenfrei 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_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_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 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_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 1 2012 1 10 12 |
| spelling |
10.1186/2193-1801-1-59 doi (DE-627)SPR032747012 (SPR)2193-1801-1-59-e DE-627 ger DE-627 rakwb eng 600 ASE Yadav, Sushil Kumar verfasserin aut Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. mediated transformation (dpeaa)DE-He213 Annexin (dpeaa)DE-He213 Double cotyledonary node (dpeaa)DE-He213 Katikala, Sweety verfasserin aut Yellisetty, Varalaxmi verfasserin aut Kannepalle, Annapurna verfasserin aut Narayana, Jyothi Lakshmi verfasserin aut Maddi, Vanaja verfasserin aut Mandapaka, Maheswari verfasserin aut Shanker, Arun Kumar verfasserin aut Bandi, Venkateswarlu verfasserin aut Bharadwaja, Kirti Pulugurtha verfasserin aut Enthalten in SpringerPlus London : Biomed Central, 2012 1(2012), 1 vom: 10. Dez. (DE-627)718615298 (DE-600)2661116-8 2193-1801 nnns volume:1 year:2012 number:1 day:10 month:12 https://dx.doi.org/10.1186/2193-1801-1-59 kostenfrei 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_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_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 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_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 1 2012 1 10 12 |
| allfields_unstemmed |
10.1186/2193-1801-1-59 doi (DE-627)SPR032747012 (SPR)2193-1801-1-59-e DE-627 ger DE-627 rakwb eng 600 ASE Yadav, Sushil Kumar verfasserin aut Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. mediated transformation (dpeaa)DE-He213 Annexin (dpeaa)DE-He213 Double cotyledonary node (dpeaa)DE-He213 Katikala, Sweety verfasserin aut Yellisetty, Varalaxmi verfasserin aut Kannepalle, Annapurna verfasserin aut Narayana, Jyothi Lakshmi verfasserin aut Maddi, Vanaja verfasserin aut Mandapaka, Maheswari verfasserin aut Shanker, Arun Kumar verfasserin aut Bandi, Venkateswarlu verfasserin aut Bharadwaja, Kirti Pulugurtha verfasserin aut Enthalten in SpringerPlus London : Biomed Central, 2012 1(2012), 1 vom: 10. Dez. (DE-627)718615298 (DE-600)2661116-8 2193-1801 nnns volume:1 year:2012 number:1 day:10 month:12 https://dx.doi.org/10.1186/2193-1801-1-59 kostenfrei 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_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_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 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_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 1 2012 1 10 12 |
| allfieldsGer |
10.1186/2193-1801-1-59 doi (DE-627)SPR032747012 (SPR)2193-1801-1-59-e DE-627 ger DE-627 rakwb eng 600 ASE Yadav, Sushil Kumar verfasserin aut Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. mediated transformation (dpeaa)DE-He213 Annexin (dpeaa)DE-He213 Double cotyledonary node (dpeaa)DE-He213 Katikala, Sweety verfasserin aut Yellisetty, Varalaxmi verfasserin aut Kannepalle, Annapurna verfasserin aut Narayana, Jyothi Lakshmi verfasserin aut Maddi, Vanaja verfasserin aut Mandapaka, Maheswari verfasserin aut Shanker, Arun Kumar verfasserin aut Bandi, Venkateswarlu verfasserin aut Bharadwaja, Kirti Pulugurtha verfasserin aut Enthalten in SpringerPlus London : Biomed Central, 2012 1(2012), 1 vom: 10. Dez. (DE-627)718615298 (DE-600)2661116-8 2193-1801 nnns volume:1 year:2012 number:1 day:10 month:12 https://dx.doi.org/10.1186/2193-1801-1-59 kostenfrei 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_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_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 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_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 1 2012 1 10 12 |
| allfieldsSound |
10.1186/2193-1801-1-59 doi (DE-627)SPR032747012 (SPR)2193-1801-1-59-e DE-627 ger DE-627 rakwb eng 600 ASE Yadav, Sushil Kumar verfasserin aut Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata 2012 Text txt rdacontent Computermedien c rdamedia Online-Ressource cr rdacarrier Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. mediated transformation (dpeaa)DE-He213 Annexin (dpeaa)DE-He213 Double cotyledonary node (dpeaa)DE-He213 Katikala, Sweety verfasserin aut Yellisetty, Varalaxmi verfasserin aut Kannepalle, Annapurna verfasserin aut Narayana, Jyothi Lakshmi verfasserin aut Maddi, Vanaja verfasserin aut Mandapaka, Maheswari verfasserin aut Shanker, Arun Kumar verfasserin aut Bandi, Venkateswarlu verfasserin aut Bharadwaja, Kirti Pulugurtha verfasserin aut Enthalten in SpringerPlus London : Biomed Central, 2012 1(2012), 1 vom: 10. Dez. (DE-627)718615298 (DE-600)2661116-8 2193-1801 nnns volume:1 year:2012 number:1 day:10 month:12 https://dx.doi.org/10.1186/2193-1801-1-59 kostenfrei 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_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_95 GBV_ILN_105 GBV_ILN_110 GBV_ILN_151 GBV_ILN_161 GBV_ILN_170 GBV_ILN_171 GBV_ILN_213 GBV_ILN_230 GBV_ILN_285 GBV_ILN_293 GBV_ILN_370 GBV_ILN_602 GBV_ILN_2014 GBV_ILN_4012 GBV_ILN_4037 GBV_ILN_4112 GBV_ILN_4125 GBV_ILN_4126 GBV_ILN_4249 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_4335 GBV_ILN_4338 GBV_ILN_4367 GBV_ILN_4700 AR 1 2012 1 10 12 |
| language |
English |
| source |
Enthalten in SpringerPlus 1(2012), 1 vom: 10. Dez. volume:1 year:2012 number:1 day:10 month:12 |
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Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata |
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Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. |
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Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. |
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Abstract A reproducible and highly efficient protocol for genetic transformation mediated by Agrobacterium has been established for greengram (Vigna radiata L. Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. Integration of annexin gene into the greengram genome was confirmed by Southern blotting. |
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Wilczek). Double cotyledonary node (DCN) explants were inoculated with Agrobacterium tumefaciens strain LBA 4404 harboring a binary vector pCAMBIA 2301 containing neomycin phosphotransferase (npt II) gene as selectable marker, β-glucuronidase (GUS) as a reporter (uidA) gene and annexin 1 bj gene. Important parameters like optical density of Agrobacterium culture, culture quantity, infection medium, infection and co-cultivation time and acetosyringone concentration were standardized to optimize the transformation frequency. Kanamycin at a concentration of 100 mg/l was used to select transformed cells. Transient and stable GUS expressions were studied in transformed explants and regenerated putative plants, respectively. Transformed shoot were produced on regeneration medium containing 100 mg/l kanamycin and 250 mg/l cefotaxime and rooted on ½ MS medium. Transient and constitutive GUS expression was observed in DCN explants and different tissues of $ T_{0} $ and $ T_{1} $ plants. Rooted $ T_{0} $ and $ T_{1} $ shoots confirming Polymerase Chain Reaction (PCR) positive for npt II and annexin 1bj genes were taken to maturity to collect the seeds. 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