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Factors affecting Agrobacterium-mediated transformation in Hybanthus enneaspermus (L.) F. Muell
Ganeshan Sivanandhan,Chinnathambi Arunachalam,Venkatachalam Vasudevan,Gnanajothi Kapildev,Ali Alharbi Sulaiman,Natesan Selvaraj,Andy Ganapathi,임용표 한국식물생명공학회 2016 Plant biotechnology reports Vol.10 No.2
Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day precultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD600, infection time of 6 min, AS concentration of 150 lM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.