Optimization of Agrobacterium mediated genetic transformation of cotyledonary node explants of Vigna radiata
Autor: | Annapurna Kannepalle, Kirti Pulugurtha Bharadwaja, Vanaja Maddi, S. K. Yadav, Sweety Katikala, Maheswari Mandapaka, Arun K. Shanker, Jyothi Lakshmi Narayana, Varalaxmi Yellisetty, Venkateswarlu Bandi |
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Rok vydání: | 2012 |
Předmět: |
Acetosyringone
Multidisciplinary biology Agrobacterium Research fungi food and beverages Kanamycin Annexin Agrobacterium tumefaciens biology.organism_classification Agrobacterium mediated transformation Molecular biology Double cotyledonary node Transformation (genetics) chemistry.chemical_compound Murashige and Skoog medium chemistry Botany medicine Vigna radiata Selectable marker medicine.drug Explant culture |
Zdroj: | SpringerPlus |
ISSN: | 2193-1801 |
Popis: | 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. |
Databáze: | OpenAIRE |
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