Adventitious shoot regeneration and Agrobacterium tumefaciens-mediated transformation of leaf explants of sweet cherry (Prunus avium L.)

Abstract

ABSTRACT Sweet cherry (Prunus avium L.) remains recalcitrant for genetic transformation due to the lack of efficient plant regeneration systems via organogenesis or somatic embryogenesis. In this study, in vitro shoot cultures were derived from a single mature embryo (open pollinated) of ‘Selah’ sweet cherry. Leaf explants were cultured on Woody Plant Medium supplemented with different plant growth regulators to induce shoot regeneration. The optimal regeneration at a frequency of 32.5% and an average of 1.1 shoots per explant occurred on the medium containing 4.54 µM thidiazuron (TDZ) and 2.95 µM indole-3-butyric acid (IBA). Transient transformation showed an efficient delivery of the β-glucuronidase (GUS) reporter gene (gusA) using Agrobacterium tumefaciens strain EHA105. Under the optimal gene delivery conditions, stable transformations were conducted using pGA643 and pBI-VcFT containing a blueberry FLOWERING LOCUS T (VcFT). A total of 500 leaf explants, 250 for each construct, were used for transformation. After 10-week selection, three leaf explants transformed with the pGA643 produced four kanamycin-resistant shoots, in which stable integration and expression of the nptII were confirmed by Southern blot and RT-PCR analysis, respectively. This study demonstrated that it was possible to produce stable transgenic sweet cherry using Agrobacterium tumefaciens-mediated transformation of leaf explants.