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.
Highlights
Many attempts have been made to use breeding or molecular biological methods to modify the ability to produce secondary metabolites in medicinal plants
The first report was on the introduction of a gene encoding berberine bridge enzyme (BBE) to P. somniferum in antisense orientation [1]
Several reports on metabolic engineering of P. somniferum have appeared, such as RNAi-mediated gene silencing of codeinone reductase (COR) [2], overexpression of COR [3], overexpression and antisense co-suppression of (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B3) [4], overexpression and RNAi-mediated gene silencing of salutaridinol-7-O-acetyltransferase (SalAT) [5], and RNAi-mediated gene silencing of SalAT [6]
Summary
Many attempts have been made to use breeding or molecular biological methods to modify the ability to produce secondary metabolites in medicinal plants. Among the challenges being addressed, manipulations of the morphine biosynthesis in the opium poppy (Papaver somniferum L.), the conversion of narcotic morphine to codeine, which is of high importance as an antitussive and a synthetic source of dihydrocodeine, or to thebaine, which is an important starting material for the semi-synthesis of the analgesic oxycodone, will contribute to the control of narcotics, and to the supply of useful alkaloids for the production of pharmaceuticals. Several reports on metabolic engineering of P. somniferum have appeared, such as RNAi-mediated gene silencing of codeinone reductase (COR) [2], overexpression of COR [3], overexpression and antisense co-suppression of (S)-N-methylcoclaurine-3'-hydroxylase (CYP80B3) [4], overexpression and RNAi-mediated gene silencing of salutaridinol-7-O-acetyltransferase (SalAT) [5], and RNAi-mediated gene silencing of SalAT [6]. Mutant poppy top1 [7] which accumulates thebaine and oripavine as major alkaloids instead of morphine was established by the treatment of mutagen (ethyl methanesulphonate) and screening of progeny plants
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