| This thesis presents a study on plant regeneration, genetic transformation, and metabolic engineering of the Papaveraceae. For metabolic engineering in the Papaveraceae an optimized protocol for plant regeneration and transformation was developed as follows: (1) somatic embryogenesis and plant regeneration from seed-derived embryogenic callus and cell suspension cultures of California poppy (Eschscholzia californica Chain.) was established; (2) Agrobacterium-mediated stable genetic transformation of E. californica via somatic embryogenesis was achieved; (3) an Agrobacterium-mediated protocol for the stable genetic transformation of intact opium poppy, Papaver somniferum L., plants via shoot organogenesis was developed; and (4) transgenic opium poppy and California poppy root cultures were established using Agrobacterium rhizogenes.; California poppy cell and hairy root cultures produce several benzophenanthridine alkaloids with potent pharmacological activity. Antisense constructs of genes encoding two enzymes involved in benzophenanthridine alkaloid biosynthesis, the berberine bridge enzyme (BBE) and N-methylcoclaurine 3′-hydroxylase (CYP80B1), were introduced separately into California poppy cell cultures. Transformed cell lines expressing antisense- BBE or antisense-CYP80B1 constructs and displaying low levels of BBE or CYP80B1 mRNAs, respectively, showed reduced accumulation of benzophenanthridine alkaloids compared to control cultures transformed with a β-glucuronidase gene.; Sense and antisense constructs of genes encoding the BBE were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with the GUS gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense- BBE also reduced the growth rate of the root cultures. These data provide new insight into the metabolic engineering of benzophenanthridine alkaloid pathways. |
