Construction of high efficiency regeneration and transformation systems of <Emphasis Type="Italic">Pyrus ussuriensis</Emphasis> Maxim

The purpose of the present study was to establish a simple and efficient protocol for pear regeneration and transformation. Major factors that influence multiplication, rooting, regeneration, and transformation (concentrations of plant growth regulators, pre-culture or co-culture times, and kanamycin concentration) were examined in wild Pyrus ussuriensis Maxim ‘Shanli’. The results showed that the best multiplication index (5.1) was obtained on Murashige and Skoog (MS) medium containing 1.5 mg L^?1 N6-benzyladenine (BA) and 0.2 mg L^?1 indole-3-butyric acid (IBA) with healthy and strong growth. On 1/2 MS medium supplemented with 1.0 mg L^?1 IBA or 0.2 mg L^?1 indole-3-acetic acid (IAA), the rooting percentage was up to 94.4%. The highest regeneration rate (100%) and number of adventitious shoots per explant (6.30) were obtained on Nitsch and Nitsch 1969 (NN69) medium containing 2.0 or 3.0 mg L^?1 thidiazuron (TDZ) and 0.2 mg L^?1 IAA with leaves as explants. Transformation was successfully achieved using the following protocol: cut-wounding leaves pre-cultured for 5 days were co-cultivated with the Agrobacterium tumefaciens strain EHA105 harboring 35S::DHN3 plasmid for 2 days, then were transferred to regeneration medium containing 15 mg L^?1 kanamycin to select the resistant plants, and followed by being cultured on rooting medium supplemented with 5 mg L^?1 kanamycin. PCR analysis revealed that 27 independent transgenic lines were obtained, with the transformation rate up to 11.72%. We postulate that the regeneration and transformation system in this study is an alternative method for pear breeding.