Establishment of efficient callus genetic transformation system for Pyrus armeniacaefolia

Abstract

The purpose of the present study was to establish an efficient and stable callus genetic transformation system for Pyrus armeniacaefolia. Fruits 15 days after flowering were used for callus inducing. Major factors that influence growth state of callus (concentrations of plant growth regulators, subculture cycle) and transformation (Agrobacterium tumefaciens concentration, infection time, co-culture time, AS and kanamycin concentrations) were examined. The results showed that the optimum mediums for callus induction was MS supplemented with 3.0 mgL−1 2,4-Dichlorophenoxyacetic acid (2, 4-D) and 0.5 mgL−1 6-Benzylaminopurine (6-BA). The appropriate proliferation medium was MS containing 2.5 mgL−1 2,4-D and 0.5 mgL−1 6-BA with 21 d subculture cycle. Transformation was successfully achieved using the following protocol: callus were soaked in A. tumefaciens strain EHA105 (OD600 = 0.8) harboring pBI121 plasmid for 15 min. After 3 d co-culture on proliferation medium supplemented with 20 mgL−1 AS, the callus were then screened on the selection medium (proliferation medium containing 25 mgL−1 kanamycin and 200 mgL−1 cefotaxime) to select the resistant callus. The GUS-positive rate was 96%. And the GUS-positive callus were further identification at DNA and transcription levels. We believe that the present study provides technical support for functional researches on fruit quality related genes.