Poppy: In Vitro Culture Technologies

The opium poppy, like many other important crop species, has provided a number of technical challenges for the application of modern molecular approaches. These difficulties include, but are not limited to, problems in protein and nucleic acid purification, as well as the transformation and regeneration of fertile plants. Over the past decade most of these barriers have been overcome so that the opium poppy is poised to become a model system for the study of latex cell gene regulation as well as a target for metabolic engineering of important pharmaceuticals.

This section will review the features which make the opium poppy recalcitrant to standard molecular procedures and how protocols have been successfully adapted in order to work with poppies. In addition, the cloning and characterization of several poppy genes will be described. Their potential application in the metabolic engineering of transgenic poppy plants for altered alkaloid synthesis is also discussed.

Characterization Of Major Latex Proteins

Laticifers are internal secretory systems which occur in both primitive and advanced flowering plants. Although they are present in only fifteen families, latex-bearing plants may be quite valuable due to their ability to accumulate useful products such as alkaloids, hydrocarbons, or enzymes.

Opium is the air-dried cytoplasm of the opium poppy laticifer which remains metabolically active at maturity. Isolated latex is able to convert radioactive precursors into morphinane alkaloids. Enzymes of general cellular metabolism have been detected in isolated poppy latex, as have enzymes which may be involved in alkaloid biosynthesis.

Biochemical investigations of poppy alkaloid metabolism have been complicated by the presence of robust polyphenol oxidase (PPO) activity which can interfere with many enzyme assays when latex is exposed to air. Opium poppy has one of the most active PPO enzymes in the plant kingdom. Most plant PPOs have a rather low substrate affinity with a Km of about 1μM), however, the opium poppy enzyme has a Km of only 1μM.

Studies of fractionated latex have shown that polyphenol oxidase activity sediments with a 1000g vesicle pellet in centrifuged latex and is present in both aqueous and detergent-soluble forms. These two forms of PPO have different substrate affinities and are distributed in separate cytoplasmic compartments: a lighter organelle containing the soluble PPO and a heavier organelle with the bound form. In Papaver bracteatum ()polyphenol oxidase activity has been localized to largemembrane-bound inclusions within laticifer plastids which are also present in opium poppy laticifers. In view of what is now known about polyphenol oxidases in the plastids of other species, it seems likely that the membrane-bound form of polyphenol oxidase in opium poppy latex described by Roberts (1971) is the plastid form.

It is not possible to directly separate the proteins in unfractionated opium poppy latex by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) because they precipitate in the presence of morphinane alkaloids, even when boiled. Although many latex proteins remain soluble when whole latex is mixed 1:1 with a sample buffer containing a non-ionic detergent such as NP-40, the most efficient way to isolate latex proteins is to separate them from the latex alkaloids before mixing with SDS sample buffer.

Morphinane alkaloids and their precursors are stored in membrane-bound alkaloidal vesicles within the latex. Alkaloidal vesicles and their contents can be easily separated from the latex serum by gently mixing freshly collected latex with 0.05 M phosphate buffer (pH 7.0) containing 0.5M mannitol and subjecting the mixture to a 10 second spin in a microcentrifuge. The mannitol acts as an osmoticum which prevents lysis of the alkaloidal vesicle pellet leaving a clear supernatant serum fraction which can be drawn off and analysed by SDS-PAGE.

Electrophoretic analysis of poppy latex has revealed a distinct group of abundant, low molecular weight polypeptides which we have termed the major latex proteins or MLPs. Protein gel blots and immunocytochemical investigations show that MLPs are constitutively expressed in latex and thus represent good markers for laticifer development.

Two dimensional electrophoresis of poppy latex separates the MLPs into a series of discrete polypeptides with pis ranging from 6.0 to 3.5. Molecular analysis suggest that each of these MLPs is encoded by a separate member of a small gene family.

Isolation of clonable nucleic acids from opium poppy tissues is difficult because of its abundant PPO activity (see above) and the high concentration of dopamine in the latex which is quickly converted by polyphenol oxidase into highly reactive quinones in the presence of oxygen. Poppy PPO is so active that it will even use the phenol at the interface of phenol—chloroform extraction buffer as a substrate. Thus, protocols involving a phenol extraction step yield dark brown RNAs that cannot be copied by reverse transcriptase and DNA molecules that do not digest with restriction enzymes. The CTAB protocol for isolation of plant nucleic acids developed by Taylor and Powell (1982) does not use phenol and thus works well with poppy. Additionally, because dopamine and other potential polyphenol oxidase substrates are confined to the alkaloidal vesicles, it is also possible to isolate latex RNA directly from the serum by removing the vesicular fraction as outlined above.

MLPs are encoded by a family of nine genes which can be divided into two distinct subfamilies based on DNA gel blot analysis. Genomic clones and cDNAs have been obtained for four members of this family: MLP15, MLP22 and MLP146/MLP149 which are physically linked and separated by approximately 5.5kb. The organization of the MLP family is consistent with the triploidhybrid origin of the opium poppy as proposed by Kadereit (1986).

The biochemical function of the opium poppy MLPs has not been determined, however, homologous genes have been identified in several other plant species, all of which lack laticifers. It has been proposed that these proteins participate in early disease resistance responses which is consistent with the concept that laticifers are ‘pre-wounded’ cells that constitutively accumulate defence compounds that can be immediately released when the plant is injured. It will be interesting to see if the molecular regulation of laticifer-specific gene expression is controlled by signal transduction paradigms similar to those of woundinduced genes in non-laticiferous plants.

Production Of Transgenic Plants

In order to produce transgenic plants one must be able to: (1) stably integrate foreign DNA into its genome; and (2) regenerate fertile plants from transformed tissues. It is now possible to do both of these in opium poppy.

Long-term callus and suspension cultures of opium poppy have been maintained on defined media in several laboratories (reviewed elsewhere in this volume). Both roots and shoots have been regenerated from callus, however, a much simpler method for regenerating poppy suspensions through somatic embryogenesis has been developed. Opium poppy somatic embryogenesis parallels the development of zygotic embryos, with both tissues passing from globular, to heart and finally torpedo stages. One unusual feature of somatic embryos is the presence of laticifers among elements of the procambium, in contrast to the zygotic embryos which develop laticifers only after germination. Somatic embryo cultures have been shown to synthesise complex morphinane alkaloids that are not produced by normal seed or undifferentiated callus.

Large numbers of plants have been regenerated from suspension cultures via our somatic embryogenesis procedure and grown to maturity. Normal R1 plants have been also obtained by selfing these regenerated plants. The ability to induce regeneration easily from opium poppy tissue cultures through somatic embryogenesis has greatly facilitated the development of transformation/regeneration protocols for this species.

Future Prospects: Metabolic Engineering Of Alkaloid Pathways

The availability of reliable transformation/regeneration systems for opium poppy and the cloning of alkaloid pathway genes means that it should be possible to apply metabolic engineering to alter the quantity and quality of alkaloids in this species in the very near future.

Experiments are currently underway in our laboratory to overexpress tyrosine decarboxylase in transgenic poppies to determine which subclass of alkaloid might increase with a rise in the tyramine and dopamine pool sizes. Anti-sense and co-suppression approaches are also being explored to see which subclasses of alkaloids can be lowered by decreasing precursor availability. If sufficient suppression of the TyDC/DODC gene family is achieved it may even be possible to recover plants which produce very low alkaloid levels or no alkaloids at all. Such plants would undoubtedly be very useful for examining the ecophysiological roles of alkaloids in nature.

Craig L.Nessler “In Vitro Culture Technologies” (1998)