Development of an improved Agrobacterium-mediated transformation and callus-based regeneration system for sugarbeet (Beta vulgaris L.)

Authors: KHAN, ZAINUL - Oak Ridge Institute For Science And Education (ORISE); HATHAWAY, TINLEY - North Dakota State University; Chu, Chenggen; Wyatt, Nathan; Bolton, Melvin; Ramachandran, Vanitharani

Submitted to: Plant Cell Tissue and Organ Culture
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/18/2025
Publication Date: 10/7/2025
Citation: Khan, Z., Hathaway, T., Chu, C.N., Wyatt, N.A., Bolton, M.D., Ramachandran, V. 2025. Development of an improved Agrobacterium-mediated transformation and callus-based regeneration system for sugarbeet (Beta vulgaris L.). Plant Cell Tissue and Organ Culture. 163:30. https://doi.org/10.1007/s11240-025-03230-z.
DOI: https://doi.org/10.1007/s11240-025-03230-z

Interpretive Summary: Sugarbeet is cultivated for its ability to produce sucrose, a staple sugar widely used in the United States and globally. The crop is affected by several diseases caused by many different pathogens. In plants, disease-resistant traits can be generated using both traditional methods and molecular techniques. Traditional breeding for disease resistance in sugarbeet is time-consuming due to its genetic complexity. On the other hand, the application of biotechnological approaches to introduce new genes is considerably faster, but it requires a reliable transformation and regeneration system. We developed a straightforward method for efficient sugarbeet transformation using a bacterial species that can transfer genes of interest to the sugarbeet genome. In addition, a regeneration protocol was established to produce whole plants after the transformation event. The protocols developed in this study can facilitate the development of transgenic lines that are suitable for evaluating RNAi and gene editing approaches to target important sugarbeet pathogens, particularly viruses.

Technical Abstract: Sugarbeet (Beta vulgaris L.) is an important crop cultivated for its sucrose production, the only ingredient of table sugar, and is susceptible to many different pathogens. The application of recombinant DNA technologies for generating disease-resistance in crops requires a reliable transformation and regeneration system. In this study, we have developed a straightforward method for efficient Agrobacterium-mediated sugarbeet transformation followed by callus formation and regeneration of whole plants. Among the two types of explants derived from 10 to 14-day-old sugarbeet seedlings, the rate of callus induction was highest with cotyledons compared to hypocotyls. The frequency of callus induction was higher when the explants were treated for 15 minutes with 0.4 optical density Agrobacterium culture with two days cocultivation and continued incubation in the dark. A regeneration protocol was established for the hypocotyl-derived callus by optimizing several conditions from callus culturing to shoot formation and root development. The occurrence of shoot regeneration was 36% for hypocotyl-derived callus by gradually increasing the N6-benzylaminopurine (BAP) and kinetin up to 2.0 mg l-1 each, and a-napthaleneacetic acid (NAA) 0.5 mg l-1. The initiation of root formation occurred within two weeks when the root-induction medium was supplemented with 3.0 mg l-1 each of NAA and indole-3-butyric acid. RUBY, a visual reporter to track transformants, was detectable only at the callus stage. Transgene integration in the regenerated lines from hypocotyl-derived callus was confirmed by sequencing of PCR amplicons. Further, the transgene expression in the regenerated plants was confirmed by reverse transcription PCR analysis. Finally, the obtained transgenic lines were acclimatized and grown in soil under controlled environmental conditions. The transformation and callus-based regeneration protocol developed in this study can facilitate the development of non-chimeric transgenic lines compared to direct in planta regeneration methods. In this way, the first-generation transgenic lines are suitable for using RNAi and gene editing approaches to target important sugarbeet pathogens, particularly viruses.