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

Authors: SHIVANI - Oak Ridge Institute For Science And Education (ORISE); Mohr, Toni; POLITO, JOSH - Oak Ridge Institute For Science And Education (ORISE); Thilmony, Roger

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/7/2026
Publication Date: 5/8/2026
Citation: Shivani, Mohr, T.J., Polito, J.T., Thilmony, R.L. 2026. Harnessing embryogenic cell suspension culture for Agrobacterium-mediated transformation of microvine. Plant Cell Tissue and Organ Culture. 165.Article 50. https://doi.org/10.1007/s11240-026-03453-8.
DOI: https://doi.org/10.1007/s11240-026-03453-8

Interpretive Summary: Grapes are an important high-value fruit crop that could benefit from genetic improvements made using modern biotechnology. To utilize biotechnology for improving grapes, effective techniques are required to enable their efficient manipulation. Research was performed with microvine, a model grape variety that has a desirable compact stature and can rapidly produce fruit in a single growing season. A novel approach for growing microvine cells in a controlled environment and performing genetic engineering was developed. The reported technique involves growing cells in liquid media that have an efficient ability to regenerate plants. The study provides a detailed and reliable step-by-step workflow that researchers can utilize to efficiently perform microvine biotechnology.

Technical Abstract: 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. 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 reduced the time needed to maintain the culture and produced abundant starting material with modest growth periods. A protocol was 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 (OD600 0.2) carrying a binary vector with the microvine Ubiquitin 7 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 (10 mg/L) to the shooting medium. Eighteen independent transgenic plants were characterized by using droplet digital PCR demonstrating that eight (44%) had one or two copies of the introduced transgene. This method can produce approximately 30 transgenic plants per 100 mg of ECS culture within six months from the start of Agrobacterium co-cultivation. 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.