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: Ranjan Kumar Sahoo, Narendra Tuteja
• Journal: GM crops & food (GM Crops Food) 2012 Apr-Jun Vol. 3 Issue 2 Pg. 123-8 ISSN: 2164-5701 [Electronic] United States
• PMID: 22538224 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Acetophenones
• Plant Growth Regulators
• 2,4-Dichlorophenoxyacetic Acid
• acetosyringone
• Kinetin

Topics

• 2,4-Dichlorophenoxyacetic Acid (pharmacology)
• Acetophenones (pharmacology)
• Agrobacterium (genetics)
• Dose-Response Relationship, Drug
• Kinetin (pharmacology)
• Oryza (genetics, physiology)
• Plant Growth Regulators (pharmacology)
• Plants, Genetically Modified (genetics, physiology)
• Regeneration (drug effects)
• Seeds (genetics, physiology)
• Time Factors
• Tissue Culture Techniques
• Transformation, Genetic (drug effects, genetics)