Browsing by Subject "Tissue Culture."

The objectives of the study were to assess the efficiency of transient expression of sgRNA/Cas9 construct in Petunia V26, where sgRNA targeted a cytosine deaminase gene (CodA) that converts 5-fluorocytosine into the toxic compound 5-fluorouracil. Disrupting CodA by transient expression of sgRNA/Cas9 introduced a conditional negative selection system that allowed plants with mutated CodA to regenerate on media containing 5-fluorocytosine. The single transcriptional unit vector pMOH2 was designed to carry two amplified sgRNAs guiding Cas9 targeting at HinfI cutting sites. The expression vector was transformed into Petunia V26 using Agrobacterium tumefaciens (pGV2260). Successful mutations were detected on 62.5 mg/L 5-fluorocytosine. Large numbers of in vitro shoots were regenerated from the transformed leaves on a modified MS-media containing 1 mg/L zeatin. The study revealed that transient expression of the sgRNA/Cas9 construct is efficient and can be used to target other genes in Petunia V26. pMOH2 targeted its sites successfully, and proved that CodA can be used as a conditional negative selection marker to detect cells with an edited genome.