Callus induction and Agrobacterium tumefaciens– mediated genetic transformation in redbud (Cercis griffithii Boiss.)

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   Redbud (Cercis spp.), has a great importance in urban forestry and landscape architect, due to its ornamental values. But its use is hampered by some unfavorable traits that can be improved by genetic engineering. In vitro grown of cotyledons and stem explants of redbud were cultured on MS basal medium supplemented with different concentrations of growth regulators for callus induction. EHA105 and AGL1 strains of Agrobacterium tumefaciens were transformed with pCAMBIA2301 binary vector, harboring nptII and gus genes conferring kanamycin (Kan) resistance and reporter enzymatic protein of β-glucuronidase activity, respectively, using calcium chloride (CaCl2) and Tris. Calli and zygotic embryo cells were infected with different densities of Agrobacterium strains under vacuum and normal conditions. Addition of 22.4 and 9.1 μM of 2,4-Dicholophenoxy acetic acid (2,4-D) to medium resulted in the highest 92 and 97% callus induction in cotyledon and stem explants, respectively. Optimum concentration of Kan for selecting of transformed shoot and callus was also detected. The results showed that the highest transformation level can be obtained by OD600 = 0.8 of AGL1 strain in vacuum condition. Histochemical analysis of reporter gene, and polymerase chain reaction (PCR) analysis confirmed transfering of the transgene from agrobacterium to embryo-derived plantlets and callus induced from regenerated shoots. The Agrobacterium tumefaciens-transformation procedure described here for the first time may pave an efficient way for generating transgenic C. griffithii trees using zygotic embryos as explants.

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