Improvement of Agrobacterium-mediated transformation and rooting of black cherry

Abstract

An improved protocol for Agrobacterium-mediated transformation of an elite, mature black cherry genotype was developed. To increase transformation efficiency, vacuum infiltration, sonication, and a combination of the two treatments were applied during the cocultivation of leaf explants with Agrobacterium tumefaciens strain EHA105 harboring a PsAGAMOUS RNAi plasmid (pART27-PsAGRNAi). The effects of Agrobacterium culture density and cocultivation duration on transformation efficiency were examined using EHA105 harboring either pBI121-MDL4 or pBI121-PsTFL1. In addition, the effect of the binary vector on transformation efficiency was also studied. Fifteen-minute vacuum infiltration without sonication produced the highest transformation efficiency (21.7%) in experiments using pART27-PsAGRN Ai. OD₆₀₀ values of 1.0 and 1.5 resulted in a transformation efficiency of 5% when pBI121-PsTFL1 was used for transformation. Transformation efficiency of 5% was also obtained from 3-d cocultivation using construct pBI121-MDL4 whereas no shoots regenerated after 4-d cocultivation. The binary vectors used also impacted transformation efficiency. PCR and quantitative-PCR analyses were used to confirm the integration of transgenes and determine the copy number of the selectable marker gene, neomycin phosphotransferase II, in 18 putative transgenic lines. Rooting of transgenic black cherry shoots was achieved at a frequency of 30% using half-strength Murashige and Skoog medium supplemented with 2% sucrose, 5 µM naphthaleneacetic acid, 0.01 µM kinetin, and 0.793 mM phloroglucinol, and the resulting transgenic plants were successfully acclimatized.

Citation

Wang, Ying; Pijut, Paula M. 2014. Improvement of Agrobacterium-mediated transformation and rooting of black cherry. In Vitro Cellular and Developmental Biology - Plant. 50(3): 307-316.