Organogenesis and Sonication-Assisted Agrobacterium-Mediated Transformation of Poplar Roots

Giovanella, Thais Salete; Degenhardt, Juliana; Zanella, Laudiane Bruna; Filho, João Carlos Bespalhok

doi:10.21203/rs.3.rs-465228/v1

Cited by 1 publication

(2 citation statements)

References 27 publications

(42 reference statements)

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“…The most commonly used co-cultivation technique involves incubating plant explants in a liquid Agrobacteria suspension and liquid media for hours or even several days. This is often accompanied by continuous shaking, followed by several steps of rinsing and drying the explant over sterile paper before the nutrient media change [4,23,24]. However, this technique has several disadvantages, such as the complicated handling of the explants during their transfer from liquid medium to a solid, and the stressful conditions due to their extensive handling, which can lead to wounded plant tissues.…”

Section: Discussionmentioning

confidence: 99%

“…Prolonged exposure of leaf tissue to air for slicing can cause rapid drying, leading to additional challenges in the regeneration process. Other explant types, such as leaf petioles, root segments, and calli [9,24], are also widely used, each with its own advantages and disadvantages. Although internode sections of shoots are sometimes used in studies, they are not as commonly used as leaves [4,8,30].…”

Section: Discussionmentioning

confidence: 99%

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Simplified Method for Agrobacterium-Mediated Genetic Transformation of Populus x berolinensis K. Koch

Pavlichenko,

Protopopova

2024

MPs

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The rapid advancement of genetic technologies has made it possible to modify various plants through both genetic transformation and gene editing techniques. Poplar, with its rapid in vitro growth and regeneration enabling high rates of micropropagation, has emerged as a model system for the genetic transformation of woody plants. In this study, Populus × berolinensis K. Koch. (Berlin poplar) was chosen as the model organism due to its narrow leaves and spindle-shaped crown, which make it highly suitable for in vitro manipulations. Various protocols for the Agrobacterium-mediated transformation of poplar species have been developed to date. However, the genetic transformation procedures are often constrained by the complexity of the nutrient media used for plant regeneration and growth, which could potentially be simplified. Our study presents a cheaper, simplified, and relatively fast protocol for the Agrobacterium-mediated transformation of Berlin poplar. The protocol involved using internode sections without axillary buds as explants, which were co-cultivated in 10 µL droplets of bacterial suspension directly on the surface of a solid agar-based medium without rinsing and sterile paper drying after inoculation. We used only one regeneration Murashige and Skoogbased medium supplemented with BA (0.2 mg·L−1), TDZ (0.02 mg·L−1), and NAA (0.01 mg·L−1). Acetosyringone was not used as an induction agent for vir genes during the genetic transformation. Applying our protocol and using the binary plasmid pBI121 carrying the nptII selective and uidA reporter genes, we obtained the six transgenic lines of poplar. Transgenesis was confirmed through a PCR-based screening of kanamycin-selected regenerants for the presence of both mentioned genes, Sanger sequencing, and tests for detecting the maintained activity of both genes. The transformation efficiency, considering the 100 explants taken originally, was 6%.

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Section: Discussionmentioning

confidence: 99%