Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine

Microvine 04C023V0004 (V4) is a model Vitis vinifera genotype that carries a heterozygous gibberellin insensitive 1 (Vvigail) mutation making the plants compact in stature and constantly fruiting, yet its utility is hindered by long regeneration times and modest transformation efficiency levels. To improve microvine V4 transformation, we developed a method utilizing embryogenic cell suspension (ECS) cultures and a novel protocol for Agrobacterium-mediated transformation. Friable globular (translucent or cream-colored) somatic embryos dissected from microvine embryogenic calli were used to initiate suspension cultures. ECS cultures require a modest amount of time and effort to maintain and produce abundant, rapidly growing explant material within several months. For transformation, the ECS cells were heat shocked at 45 °C and co-cultivated with Agrobacterium tumefaciens carrying a binary vector with the microvine Ubiquitin 7 (VviUbi7) promoter controlling mCherry expression allowing the use of red fluorescence as a visible marker. Development of transgenic microvine plants was improved with the addition of gibberellic acid to the shooting medium. Eighteen independent transgenic plants were characterized using droplet digital PCR (ddPCR) demonstrating that nine (50%) had one or two copies of the introduced selection marker transgene. This method produced approximately 30 transgenic plants per 100 mg fresh weight of ECS culture five months after co-cultivation. Use of microvine V4 ECS cultures and a modified transformation protocol can efficiently generate transgenic plants advancing grapevine biotechnology research. An Agrobacterium-mediated transformation protocol using embryogenic cell suspension cultures was developed for efficiently and rapidly generating microvine transgenic plants.