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Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition
Division of Tsukuba, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation, 1-2 Hachimandai, Tsukuba, Ibaraki 305-0843, Japan
Faculty of Pharmacy, Keio University, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
* Author to whom correspondence should be addressed.
Received: 29 November 2011; in revised form: 15 January 2012 / Accepted: 17 January 2012 / Published: 2 February 2012
Abstract: The in vitro shoot culture of a T-DNA insertional mutant of Papaver somniferum L. established by the infection of Agrobacterium rhizogenes MAFF03-01724 accumulated thebaine instead of morphine as a major opium alkaloid. To develop a non-narcotic opium poppy and to gain insight into its genetic background, we have transplanted this mutant to soil, and analyzed its alkaloid content along with the manner of inheritance of T-DNA insertion loci among its selfed progenies. In the transplanted T0 primary mutant, the opium (latex) was found to be rich in thebaine (16.3% of dried opium) by HPLC analysis. The analyses on T-DNA insertion loci by inverse PCR, adaptor-ligation PCR, and quantitative real-time PCR revealed that as many as 18 copies of T-DNAs were integrated into a poppy genome in a highly complicated manner. The number of copies of T-DNAs was decreased to seven in the selected T3 progenies, in which the average thebaine content was 2.4-fold that of the wild type plant. This may indicate that the high thebaine phenotype was increasingly stabilized as the number of T-DNA copies was decreased. In addition, by reverse transcription PCR analysis on selected morphine biosynthetic genes, the expression of codeine 6-O-demethylase was clearly shown to be diminished in the T0 in vitro shoot culture, which can be considered as one of the key factors of altered alkaloid composition.
Keywords: Papaver somniferum L.; opium alkaloid; Agrobacterium rhizogenes; T-DNA insertional mutant
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Kawano, N.; Kiuchi, F.; Kawahara, N.; Yoshimatsu, K. Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition. Pharmaceuticals 2012, 5, 133-154.
Kawano N, Kiuchi F, Kawahara N, Yoshimatsu K. Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition. Pharmaceuticals. 2012; 5(2):133-154.
Kawano, Noriaki; Kiuchi, Fumiyuki; Kawahara, Nobuo; Yoshimatsu, Kayo. 2012. "Genetic and Phenotypic Analyses of a Papaver somniferum T-DNA Insertional Mutant with Altered Alkaloid Composition." Pharmaceuticals 5, no. 2: 133-154.