title: Development of Agrobacterium-mediated transformation technology for mature seed-derived callus tissues of indica rice cultivar IR64.
abstract: Indica rice cultivar IR64 is most recalcitrant to regenerate, which affects the transformation efficiency especially when mature seed-derived callus tissues are used as explants. Therefore, a simple, rapid and improved genetic transformation protocol has been developed for the indica rice cultivar IR64 using Agrobacterium-mediated genetic transformation. With different hormonal combination tested, the maximum callus induction was observed on MS medium supplemented with 2.5 mg/l 2,4-D and 0.15 mg/l BAP from the scutellum explants. Three weeks old scutellum derived callus explants were immersed in Agrobacterium suspension (strain LBA4404, OD600=1.0) and co-cultured at 26±2°C in dark for 2 d. The maximum transformation efficiency (12%) was achieved with infection of callus explants for 20 min along with use of 150 μm acetosyringone. The maximum plant regeneration was observed on MS medium supplemented with 3 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA. The maximum root induction was observed on MS medium along with 10 g/l glucose and 20 g/l sucrose. The integration of the transgene in T1 transgenic plants was confirmed by polymerase chain reaction and Southern blot analyses. The copy number of transgenes has been found to vary from 1 to 2 in transgenic plants. By using this improved method we have successfully raised transgenic rice plants within 3 mo from seed inoculation to plant regeneration.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India.
authors.forename: Ranjan Kumar
authors.initials: RK
authors.lastname: Sahoo
authors.forename: Narendra
authors.initials: N
authors.lastname: Tuteja
mesh_terms: D015084:2,4-Dichlorophenoxyacetic Acid / Q000494:pharmacology; D000098:Acetophenones / Q000494:pharmacology; D060054:Agrobacterium / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D007701:Kinetin / Q000494:pharmacology; D012275:Oryza / Q000235:genetics* / Q000502:physiology; D010937:Plant Growth Regulators / Q000494:pharmacology; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000502:physiology; D012038:Regeneration / Q000187:drug effects; D012639:Seeds / Q000235:genetics* / Q000502:physiology; D013997:Time Factors; D046509:Tissue Culture Techniques; D014170:Transformation, Genetic / Q000187:drug effects / Q000235:genetics*
chemical_list: D000098:Acetophenones; D010937:Plant Growth Regulators; D015084:2,4-Dichlorophenoxyacetic Acid; C051667:acetosyringone; D007701:Kinetin