Paul G. Mahlberg

A chemotaxonomic analysis of cannabinoid variation in Cannabis (Cannabaceae)

Cannabinoids are important chemotaxonomic markers unique to Cannabis. Previous studies show that a plants dry-weight ratio of Δ(9)-tetrahydrocannabinol (THC) to cannabidiol (CBD) can be assigned to one of three chemotypes and that alleles B(D) and B(T) encode alloenzymes that catalyze the conversion of cannabigerol to CBD and THC, respectively. In the present study, the frequencies of B(D) and B(T) in sample populations of 157 Cannabis accessions were determined from CBD and THC banding patterns, visualized by starch gel electrophoresis. Gas chromatography was used to quantify cannabinoid levels in 96 of the same accessions. The data were interpreted with respect to previous analyses of genetic and morphological variation in the same germplasm collection. Two biotypes (infraspecific taxa of unassigned rank) of C. sativa and four biotypes of C. indica were recognized. Mean THC levels and the frequency of B(T) were significantly higher in C. indica than C. sativa. The proportion of high THC/CBD chemotype plants in most accessions assigned to C. sativa was <25% and in most accessions assigned to C. indica was >25%. Plants with relatively high levels of tetrahydrocannabivarin (THCV) and/or cannabidivarin (CBDV) were common only in C. indica. This study supports a two-species concept of Cannabis.

Laticifers: An Historical Perspective

This review describes the development of the laticifer concept, with emphasis upon the nonarticulated type, from early observations of plant exudates and “juices” to the presentation of laticifers by Esau (1953). Classical writers and herbalists described practical applications of these substances. With the advent of the microscope early investigators believed that these substances occurred in structures present in most, if not all, plants and, wrongly, equated these structures to the circulatory system in animals. Introduction of the term, latex, into botany derived from its early use as a term for a blood component by physicians, and not for analogy to milk. However, the origin of the terms, laticifer and laticiferous, remains uncertain. Initial studies of laticifers were marked by the controversy of whether they represented intercellular spaces or elongated cells. Confirmation of their cellular character led to the designation of nonarticulated and articulated laticifers. Nonarticulated laticifers were shown to arise during early embryogeny in some plants. The ontogenetic origin of the articulated laticifer was unclear to early workers, but new laticifers were detected to be formed by cambium activity. Nonarticulated laticifers were described to develop by intrusive growth whereby tips of the cell penetrated between adjacent cells. The coenocytic condition of the nonarticulated laticifer resulted from nuclear divisions along the cell positioned in the growth region of the shoot and the subsequent distribution of the daughter nuclei along the length of the cell.ZusammenfassungDie vorliegende Übersicht beschreibt die Entwicklung des Milchröhrenkonzeptes, beginnend mit den frühen Beobachtungen an Pflanzenausscheidungen und “Pflanzensäften” bis hin zu ihrer Darstellung bei Esau (1953). Dabei stehen ungegliederte Milchröhren im Vordergrund. Die klassischen Schriftsteller sowie die Verfasser der Kräuterbücher haben die Nutzanwendungen dieser Stoffe geschildert. Mit der Erfindung des Mikroskops wurden frühe Forscher zu der Annahme verleitet, daß derartige Stoffe in Strukturen vorkämen, die den meisten, wenn nicht allen Pflanzen gemeinsam seien. Diese Strukturen wurden dann, fälschlicherweise, mit dem Kreislauf der Tiere homoligisiert. Die Einführung des Begriffs “Latex” in die botanische Terminologie beruht auf der frühen ärztlichen Verwendung dieses Begriffs für einen Blutbestandteil, und nicht auf einer Analogie zu Milch. Die genaue Herkunft der Bezeichnungen “Milchröhre” und “Milchsaft führend” bleibt jedoch im Dunkeln. Erste Untersuchungen an Milchröhren waren von der Kontroverse geprägt, ob es sich um sehr stark gestreckte Zellen oder um Hohlräume zwischen Zellen handelt. Mit der Bestätigung der zellulären Natur der Milchröhren fand ihre Einteilung in gegliederte und ungegliederte Milchröhren statt. Es konnte gezeigt werden, daß ungegliederte Milchröhren schon in den frühen Embryonalstadien milchsaftführender Pflanzen angelegt werden. Die ontogenetische Herkunft gegliederte Milchröhren konnte von den frühen Bearbeitern nicht geklärt werden; sie stellten jedoch fest, daß neue Milchröhrenzellen durch kambiale Aktivität abgegliedert werden. Es wurde auch beschrieben, daß ungegliederte Milchröhren während des Wachstums in bereits vorhandene Gewebe eindringen, wobei ihre Zellspitzen sich zwischen die Zellwände zweier benachbarter Zellen drängen. Die coenocytische Natur ungegliederter Milchröhren kommt durch Kernteilungen an der Spitze der Milchröhre und damit des Sprosses zustande, wobei sich die Tochterkerne nach ihrer Abgliederung entlang der gesamten Zelle verteilen.

Morphinane alkaloids in cultured tissues and redifferentiated organs of Papaver somniferum

Abstract The capacity of alkaloid synthesis was examined in cultured tissues of Papaver somniferum . Callus, derived meristemoids, redifferentiated roots and

Immunochemical localization of tetrahydrocannabinol (THC) in cryofixed glandular trichomes of Cannabis (Cannabaceae).

Delta 9-tetrahydrocannabinol (THC) localization in glandular trichomes and bracteal tissues of Cannabis, prepared by high pressure cryofixation-cryosubstitution, was examined with a monoclonal antibody-colloidal gold probe by electron microscopy (EM). The antibody detected THC in the outer wall of disc cells during the presecretory cavity phase of gland development. Upon formation of the secretory cavity, the immunolabel detected THC in the disc cell wall facing the cavity as well as the subcuticular wall and cuticle throughout development of the secretory cavity. THC was detected in the fibrillar matrix associated with the disc cell and with this matrix in the secretory cavity. The antibody identified THC on the surface of secretory vesicles, but not in the secretory vesicles. Gold label also was localized in the anticlinal walls between adjacent disc cells and in the wall of dermal and mesophyll cells of the bract. Grains were absent or detected only occasionally in the cytoplasm of disc or other cells of the bract. No THC was detected in controls. These results indicate THC to be a natural product secreted particularly from disc cells and accumulated in the cell wall, the fibrillar matrix and surface feature of vesicles in the secretory cavity, the subcuticular wall, and the cuticle of glandular trichomes. THC, among other chemicals, accumulated in the cuticle may serve as a plant recognition signal to other organisms in the environment.

Accumulation of Cannabinoids in Glandular Trichomes of Cannabis (Cannabaceae)

Abstract Sessile- and capitate-stalked secretory glands are sites of cannabinoid accumulation in Cannabis (Cannabaceae). Analyses show cannabinoids to be abundant in glands isolated from bracts or leaves of pistillate plants. Cannabinoids are concentrated in the secretory cavity formed as an intrawall cavity in the outer wall of the disc cells. Specialized plastids, lipoplasts, in the disc cells synthesize lipophilic substances, such as terpenes, that migrate through the plasma membrane and into the cell wall adjacent to the secretory cavity. These substances enter the cavity as secretory vesicles. An antibody probe for THC shows it to be most abundant along the surface of vesicles, associated with fibrillar material in the cavity, in the cell wall and in the cuticle; little THC was detected in the cytoplasm of disc or other cells. The phenol, phloroglucinol, is abundant in both gland types. A working hypothesis for the site of cannabinoid synthesis is proposed, and must be examined further. Knowledge of the mechanism of cannabinoid synthesis and localization can contribute to efforts to further reduce the THC content in hemp strains for potential agricultural use in the United States and elsewhere.

QUANTITATIVE ANALYSIS OF CANNABINOIDS IN THE SECRETORY PRODUCT FROM CAPITATE-STALKED GLANDS OF CANNABIS SATIVA L. (CANNABACEAE)

Capitate-stalked glandular trichomes from pistillate bracts of Cannabis sativa L. were analyzed to determine the cannabinoid composition of secretory products within the secretory sac. Analyses were performed on cloned materials of a strain characteristically high in cannabidiol. The secretory product is accumulated in a single large secretory sac, which develops above a multicellular disk of secretory cells during gland ontogeny. The content of the secretory sac was removed with micropipets, using a micromanipulator without damaging or removing disk cells or their contents, and analyzed by gas-liquid chromatography. The cannabinoid content of the secretory sac was compared with previously quantitated cannabinoids of whole capitate-stalked glands. Results indicated that nearly all of the cannabinoid content of capitate-stalked glands was present in the secretory sac.

Accumulation of non-utilizable starch in laticifers of Euphorbia heterophylla and E. myrsinites.

Starch biosynthesis and degradation was studied in seedlings and mature plants of Euphorbia heterophylla L. and E. myrsinites L. Mature embryos, which lack starch grains in the non-articulated laticifers, develop into seedlings that accumulate starch rapidly when grown either in the light or the dark. Starch accumulation in laticifers of dark-grown seedlings was ca. 47 and 43% of total starch in light-grown controls in E. heterophylla and E. myrsinites, respectively. In light-grown seedlings, starch was present in laticifers as well as parenchyma of stems and leaves, whereas in dark-grown seedlings starch synthesis was almost exclusively limited to laticifers. In 7-month-old plants placed into total darkness, the starch in chyma was depleted within 6 d, whereas starch in laticifers was not mobilized. The starch content of latex in plants during development of floral primordia, flowering, and subsequent fruit formation remained rather constant. The results indicate that laticifers in seedlings divert embryonal storage reserves to synthesize starch even under stress conditions (darkness) in contrast to other cells, and that starch accumulated in laticifers does not serve as a metabolic reserve. The laticifer in Euphorbia functions in the accumulation and storage of secondary metabolites yet retains the capacity to produce, but not utilize starch, a primary metabolite.

Cell Wall Perforation in Laticifers of Papaver somniferum L.

Cell wall perforation in the articulated anastomosing laticifers of the opium poppy, Papaver somniferum L., was examined by electron microscopy. Perforation resulted from a gradual thinning of the walls on both sides of the middle lamella. Thinning occurred uniformly over transverse walls during articulation and at localized sites along lateral walls during anastomosis. Electron-dense globular deposits were seen at perforation sites; their function in the perforation process was not apparent. The progressive and simultaneous thinning of the cell wall on either side of the middle lamella at the site of a developing perforation suggests that wall-degrading enzymes (cellulases), rather than pressure, may be involved in the wall-perforation process.

Sterols and triterpenols in latex and cultured tissues of Euphorbia pulcherrima

Abstract The sterol and triterpenol constituents of Euphorbia pulcherrima latex and cultured callus tissues were examined by GLC and mass spectrometry. Latex extracts from different varieties contained sitosterol, β-amyrin, germanicol, cycloartenol, β-amyrin acetate, and germanicol acetate. Capillary GC profiles of these varieties indicated that the triterpene content was essentially identical for examined latices. Cultured tissues derived from petioles and stem internodes synthesized only sitosterol in significant quantities, although trace amounts of several sterols that occur in latex were also detected in cultured tissues. This study supports the interpretation that the pattern of triterpene synthesis in the laticifer of the normal plant is a highly controlled and stable phenomenon among varieties of this species.

Mitotic Waves in Laticifers of Euphorbia marginata

A successive pattern of nuclear divisions that result in mitotic waves has been observed within the coenocytic nonarticulated laticifers of embryos of Euphorbia marginata Pursh. These waves originate independently in the cotyledonary or hypocotyl portion of the laticifer and exhibit uni-or bidirectional movement at variable velocities. Individual nuclei or groups of neighoring nuclei in a laticifer were observed in a sequence of mitotic stages ranging from prophase to telophase; division activity varied with individual laticifers in an embryo. Two mitotic patterns were apparent in the embryo: a random pattern associated with various cells in the meristematic area, and a successive pattern restricted to the laticifer. A substance, synthesized by and restricted to the laticifer, may be associated with this mitotic pattern.