Establishment of a stable grape immature zygotic embryo-based genetic transformation system

Abstract

Grapevine, along with other woody perennials, has proven to be a recalcitrant crop to produce transgenic plants, largely restricting its potential for further breed improvement. Here we report the establishment of a stable and reproducible transformation protocol using immature zygotic embryos from 'Chardonnay' seeds. We extensively investigated parameters considered essential for a successful transformation, including antibiotic concentration, pretreatment method, Agrobacterium concentration, concentration of acetosyringone (AS), duration of immature zygotic embryo infection, and the infection method. Our results indicated that transformable immature zygotic embryos may be generated by soaking seeds in a 2.5 g·L−1 gibberellin (GA3) solution for 15 min in a 55 °C constant temperature water bath, followed by 24 h of growth at room temperature before sterilized and inoculated. Furthermore, the transformation rate increased to 12.38% when the immature zygotic embryos of split beak were immersed in the Agrobacterium tumefaciens solution containing 200 μM AS (OD600 = 0.6) for 20 min after 5 days of pre-culture. Transgene integration and expression was further confirmed by the detection of GFP and Western blot in leaves. The newly-developed method took roughly 4 months to obtain the transformation-positive plants, which marked a considerable reduction in the length of time required for the procedure. More importantly, the receptor material is easily accessible, the process unaffected by the changing of the season, and it is straightforward to implement. Our developed transformation protocol on grape immature zygotic embryos offers a tool for gene function analysis and germplasm enhancement via biotechnology.