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Embryogenic suspension cultures are suitable for EMS mutagenesis in grapevine, and HRM prescreening of EMS-treated somatic embryo clusters allows rapid detection of point mutations before plant regeneration. Somatic embryogenesis is an excellent system for induced mutagenesis and clonal propagation in woody plants. Our work was focused on establishing a procedure for inducing ethyl methanesulfonate (EMS) mutagenesis in grapevine. Embryogenic cell aggregates (ECAs) growing in liquid medium were treated with increasing concentrations of EMS. We found that EMS dramatically affects the viability of ECAs at concentrations above 20 mM (25.5 ± 2.9 % survival), whereas concentrations above 10 mM affect embryogenic potential (22.1 ± 1.7 % of ECAs gave rise to embryos). Embryo masses generated from EMS-treated embryogenic cell aggregates were prescreened by quantitative PCR-High Resolution Melting (qPCR-HRM) to detect single nucleotide polymorphisms (SNPs) in a 1,000-bp VvNCED1-encoding DNA fragment, which served as the target gene. Detected mutations were verified in regenerated plants by PCR and sequencing. qPCR-HRM analysis of the difference plots for the fluorescence signals allowed detection of a mutation in a sample from an embryogenic aggregate treated with 10 mM EMS. To confirm the nature of the mutation, embryos from this aggregate were recovered and germinated, and leaves were collected for PCR and sequencing analysis. The alignment of sequences from regenerated plants with the wild-type sequence revealed a transitional mutation (G/C to A/T) in the 1,000-bp VvNCED1-encoding region. To our knowledge, this is the first time that EMS mutagenesis has been performed using an embryogenic cell suspension of grapevine.
Grapevine; Ethyl methanesulfonate; Embryogenic suspension; NCED; qPCR-HRM
...The PCR reactions were performed in 20 μL volumes containing 40 ng of template DNA, 200 μM each dNTP, 0.5 μM each primer, 1.0 U Green Taq DNA Polymerase (GenScript, Piscataway, NJ, USA), 1.5 mM MgCl 2 and 2 μL of 10Χ Taq Reaction Buffer (GenScript)...
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