The opium poppy's ability to produce various alkaloids is both useful and problematic. Breeding of new varieties with varying alkaloid content is therefore an important task. In this paper, the breeding technology of new low morphine poppy genotypes, based on a combination of a TILLING approach and single-molecule real-time NGS sequencing, is presented. Verification of the mutants in the TILLING population was obtained using RT-PCR and HPLC methods. Only three of the single-copy genes of the morphine pathway among the eleven genes were used for the identification of mutant genotypes. Point mutations were obtained only in one gene (CNMT) while an insertion was obtained in the other (SalAT). Only a few expected transition SNPs from G:C to A:T were obtained. In the low morphine mutant genotype, the production of morphine was decreased to 0.1% from 1.4% in the original variety. A comprehensive description of the breeding process, a basic characterization of the main alkaloid content, and a gene expression profile for the main alkaloid-producing genes is provided. Difficulties with the TILLING approach are also described and discussed.
Keywords: TILLING; chemical mutagenesis; expression profiles; morphine content; new breeding methods; opium poppy.