Transformation of opium poppy (Papaver somniferum L.) with antisense berberine bridge enzyme gene (anti-bbe) via somatic embryogenesis results in an altered ratio of alkaloids in latex but not in roots

• Published in: Transgenic research 2004-12, Vol.13 (6), p.607-613
• Main Authors: Frick, S, Chitty, J.A, Kramell, R, Schmidt, J, Allen, R.S, Larkin, P.J, Kutchan, T.M
• Format: Article
• Language: English
• Subjects: Agrobacterium tumefaciens - genetics; alkaloids; Alkaloids - analysis; Alkaloids - chemistry; antisense DNA; bbe geme; berberine bridge enzyme; Biological and medical sciences; biosynthesis; Biotechnology; Cells, Cultured; Chromatography, High Pressure Liquid; complementary DNA; enzymes; Fundamental and applied biological sciences. Psychology; Gene Dosage; Genetic engineering; Genetic technics; genetic transformation; Genome, Plant; latex; Latex - chemistry; Methods. Procedures. Technologies; Papaver - chemistry; Papaver - enzymology; Papaver - genetics; Papaver somniferum; Plant Roots - chemistry; Plant Roots - genetics; Plants, Genetically Modified; Polymerase Chain Reaction; roots; Seedlings - genetics; somatic embryogenesis; Transformation, Genetic; Transgenes; Transgenic animals and transgenic plants; transgenic plants
• ISSN: 0962-8819; 1573-9368
• DOI: 10.1007/s11248-004-2892-6

The berberine bridge enzyme cDNA bbe from Papaver somniferum L. was transformed in antisense orientation into seedling explants of the industrial elite line C048-6-14-64. In this way, 84 phenotypically normal To plants derived from embryogenic callus cultures were produced. The selfed progeny of these 84 plants yielded several T1 plants with an altered alkaloid profile. One of these plants T1-47, and its siblings T2-1.2 and T2-1.5 are the subject of the present work. The transformation of these plants was evaluated by PCR, and northern and Southern hybridisation. The transgenic plants contained one additional copy of the transgene. The alkaloid content in latex and roots was determined with HPLC and LC-MS. We observed an increased concentration of several pathway intermediates from all biosynthetic branches, e.g., reticuline, laudanine, laudanosine, dehydroreticuline, salutaridine and (S)-scoulerine. The transformation altered the ratio of morphinan and tetrahydrobenzylisoquinoline alkaloids in latex but not the benzophenanthridine alkaloids in roots. The altered alkaloid profile is heritable at least to the T2 generation. These results are the first example of metabolic engineering of the alkaloid pathways in opium poppy and, to our knowledge, the first time that an alkaloid biosynthetic gene has been transformed into the native species, followed by regeneration into a mature plant to enable analyses of the effect of the transgene on metabolism over several generations.