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

Background 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. While these traits make V4 desirable for research, genetic engineering is challenging because of long regeneration times and modest transformation efficiency levels. Results To improve microvine V4 transformation, we established a method utilizing embryogenic cell suspension (ECS) cultures and a novel protocol for Agrobacterium- mediated transformation. Friable translucent or cream-colored globular somatic embryos 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. A protocol was also developed that utilized these embryogenic suspension cells for Agrobacterium -mediated transformation. The ECS cells were heat shocked at 45°C for 5 minutes and then combined with a co-cultivation medium containing 400 mM acetosyringone, Agrobacterium tumefaciens AGL1 (OD ₆₀₀ 0.2) carrying a binary vector with the microvine Ubiquitin 7 ( VviUbi7 ) promoter controlling mCherry expression allowing the use of red fluorescence as a visible marker. After co-cultivation, washing the ECS cells with cefotaxime (400 mg/L) medium successfully inhibited bacterial growth. Development of healthy, actively growing transgenic microvine plants was achieved with the addition of gibberellic acid (GA ₃ ) (10 mg/L) to the shooting medium. Eighteen independent transgenic plants were characterized using droplet digital PCR (ddPCR) demonstrating that eight (44%) had one or two copies of the introduced transgene. This method produced approximately 30 transgenic plants per 100 mg of ECS culture within five months from the start of Agrobacterium co-cultivation. Conclusion Use of microvine V4 ECS cultures and a modified transformation protocol can efficiently generate transgenic plants advancing grapevine biotechnology research. In the future, this protocol can potentially be adapted for other grapevine genotypes.