QUANTITATIVE DETERMINATION OF CANNABINOIDS IN INDIVIDUAL GLANDULAR TRICHOMES OF CANNABIS SATIVA L. (CANNABACEAE)

Cannabinoid levels of individual mature glandular trichomes from two clones and two strains of Cannabis sativa L., which included both drug and fiber phenotypes, were investigated by gas-liquid chromatographic analyses. Capitate-stalked glands were selectively harvested from vein and nonvein areas of pistillate bracts while capitate-sessile glands were harvested from these areas of leaves. The qualitative cannabinoid profile characteristic of the strain or clone was maintained in the individual capitate-stalked glands while the quantitative cannabinoid profiles varied with each strain or clone and between vein and nonvein areas as well. Capitate-sessile glands were found to contain conspicuously lower levels of cannabinoids than capitate-stalked glands. This study emphasizes that glands of Cannabis represent a dynamic system within the cannabinoid synthesizing activities of this plant.     

GLAND DISTRIBUTION AND CANNABINOID CONTENT IN CLONES OF CANNABIS SATIVA L.

The relationship between glandular trichomes and cannabinoid content in Cannabis sativa L. was investigated. Three strains of Cannabis, which are annuals, were selected for either a drug, a non-drug, or a fiber trait and then cloned to provide genetically uniform material for analyses over several years. The distribution of the number and type of glands was determined for several organs of different ages including the bract and its subtending monoleaflet leaf and the compound leaf on pistillate plants. Quantitation of glands on these structures was integrated with gas chromatographic analyses of organ cannabinoid profiles. A negative correlation was found between cannabinoid content and gland number for each of the three clones. Isolated heads of the capitate-stalked glands also were analyzed for cannabinoid content and found to vary in relation to clone and gland age. These studies indicate that cannabinoids may occur in plant cells other than glandular trichomes. The results of these studies emphasize the need for stringent sampling procedures in micromorphological studies on trichome distribution and analytical determinations of cannabinoid content in Cannabis.     

MORPHOGENESIS OF CAPITATE GLANDULAR HAIRS OF CANNABIS SATIVA (CANNABACEAE)

The glandular secretory system in Cannabis sativa L. (marihuana) consists of three types of capitate glandular hairs (termed bulbous, capitate-sessile, and capitate-stalked) distinguishable by their morphology, development, and physiology. These gland types occur together in greatest abundance and developmental complexity on the abaxial surface of bracts which ensheath the developing ovary. Bulbous and capitate-sessile glands are initiated on very young bract primordia and attain maturity during early stages of bract growth. Capitate-stalked glands are initiated later in bract growth and undergo development and maturation on medium, to full sized bracts. Glands are epidermal in origin and derived, with one exception, from a single epidermal initial. The capitate-stalked gland is the exception and is of special interest because it possesses a multicellular stalk secondarily derived from surrounding epidermal and subepidermal cells. Glands differentiate early in development into an upper secretory portion and a subtending auxiliary portion. The secretory portion, depending on gland type, may range from a few cells to a large, flattened multicellular disc of secretory cells. The secretory portion produces a membrane-bound resinous product which caps the secretory cells. Capitate-stalked glands are considered to be of particular evolutionary significance because they may represent a gland type secondarily derived from existing capitate-sessile glands.     

MORPHOLOGY OF GLANDULAR HAIRS OF CANNABIS SATIVA FROM SCANNING ELECTRON MICROSCOPY

Three distinct types of glandular hairs of increasing morphological complexity which occur on flowering tops of Cannabis sativa L. (marihuana) are described from scanning electron microscopy. These gland types—termed bulbous, capitate-sessile, and capitate-stalked, described from pistillate plants—occur in greatest abundance on the outer surface of bracts ensheathing the ovary. Bulbous and capitate-sessile glands, which arise at an early stage in bract development, are scattered over the bract surface. Mature bulbous glands have a small swollen head on a short stalk, whereas capitate-sessile glands have a large globular head attached directly to the bract surface. Because of their numbers and large size, capitate-sessile glands are the most conspicuous gland type during the early phase of bract development. Capitate-stalked glands, which have a large globular head on a tall, multicellular stalk, differentiate during subsequent bract development. These stalked glands arise first along the bracteal veins and then over the entire bract surface. A voluminous, fluid secretory product accumulates in the glandular head of all three types. These glands are believed to be a primary site of localization of the marihuana hallucinogen, tetrahydrocannabinol.     

GLANDULAR STRUCTURES OF HOLOCARPHA AND THEIR ONTOGENY

Carlquist , Sherwin . (Rancho Santa Ana Botanic Garden, Claremont, California.) Glandular structures of Holocarpha and their ontogeny. Amer. Jour. Bot. 46(4): 300–308. Illus. 1959.—Two types of advanced glandular structures occur in the 4 species of the genus Holocarpha. Sessile disk-shaped glands occur at the tips of upper leaves and of involucral and receptacular bracts. Unlike all other glandular structures of Madinae which have been investigated, these originate from several protodermal initials rather than a single one. These glands, however, represent modifications of a glandular trichome. The other type of glandular structure, termed hollow-stalked trichome here, occurs on the outer surface of involucral and receptacular bracts. These trichomes originate from a single cell but differ from others in the formation of a hollow stalk, the wall of which is one cell in thickness. Mesophyll of the bract, often with an included vascular bundle, is present as an intrusion into the base of the hollow stalk. Corresponding to the advanced nature of the glandular structures, the leaves show specializations in the “inrolling” of margins. Upper leaves have a cylindrical organization of vascular tissue, whereas basal leaves are “normal” and leaves of the main stem are intermediate. The species of Holocarpha differ in certain details of leaf anatomy and structure of hollow-stalked trichomes. The systematic distribution of these is given. The essential unity of the various glandular structures of Madinae is discussed both in terms of mature structure and ontogeny, and the steps in the evolution of these are suggested.     

Morphology and ultrastructure of trichomes of intact and in vitro plants of stevia rebaudiana Bertoni with reference to biosynthesis and accumulation of steviol glycosides

The ultrastructure of trichomes of intact and in vitro plants of stevia were investigated under scanning and transmission electronic microscopy. Three types of trichomes were found on both the upper and lower surfaces of the leaf: viz. rounded glands and large and small trichome. The density of trichomes on the surface of upper leaves is higher than on lower leaves, while in vitro plants are characterized with lower density of trichomes than intact ones. The positive correlation between number of trichomes on leaves and content of steviol glycosides (SGs) in them was found. Three basic stages of ontogeny of gland cells were revealed which reflect the sequence of secrete accumulation.     

ULTRASTRUCTURAL DEVELOPMENT OF CAPITATE GLANDULAR HAIRS OF CANNABIS SATIVA L. (CANNABACEAE)

The capitate-sessile and capitate-stalked glands of the glandular secretory system in Cannabis, which are interpreted as lipophilic type glandular hairs, were studied from floral bracts of pistillate plants. These glands develop a flattened multicellular disc of secretory cells, which with the extruded secretory product forms the gland head and the auxiliary cells which support the gland head. The secretory product accumulates beneath a sheath derived from separation of the outer wall surface of the cellular disc. The ultrastructure of secretory cells in pre-secretory stages is characterized by a dense ground plasm, transitory lipid bodies and fibrillar material, and well developed endoplasmic reticulum. Dictyosomes and dictyosome-derived secretory vesicles are present, but never abundant. Secretory stages of gland development are characterized by abundant mitochondria and leucoplasts and by a large vacuolar system. Production of the secretory product is associated with plastids which increase in number and structural complexity. The plastids develop a paracrystalline body which nearly fills the mature plastid. Material interpreted as a secretion appears at the surface of plastids, migrates, and accumulates along the cell surface adjoining the secretory cavity. Extrusion of the material into the secretory cavity occurs directly through the plasma membrane-cell wall barrier.     

PATTERNS OF VARIATION IN FOLIAR TRICHOMES OF EASTERN NORTH AMERICAN QUERCUS

Scanning electron microscopy of leaf trichomes of the forty two native species of oaks in eastern North America indicates five patterns of variability: 1) Eight trichome types are evident among the species and each species possesses a definite complement of trichome types. Certain trichomes are restricted to particular subgenera and series. 2) An obvious seasonal loss of trichomes occurs during leaf maturation. This loss may be both quantitative in terms of trichome density and qualitative in terms of trichome type. 3) There is an obvious difference between the adaxial and abaxial surfaces. The adaxial side of most oak leaves is dark green, lustrous, and glabrous or glabrate. The abaxial surface either remains pubescent, becomes glabrate or glabrous, or maintains trichomes along the midrib or in the axils of major secondary veins. There are also initial quantitative and qualitative trichome differences between the two sides. 4) Geographical and ecological variations are due in part to non-genetic ecophenic modifications, ecotypic differentiation, and random genetic differences not necessarily correlated with environmental conditions. Trichome types are considered to be less affected by environment than is trichome density. 5) Hybridization and introgression within a subgenus leads to localized variability. Trichomes of hybrids are usually a combination of the parental types. These five patterns of variation are predictable and appear to be held within rather narrow limits. The complement of foliar trichomes, therefore, is a reliable character in the taxonomy of the oaks.     

The Ultrastructure of Glandular Trichomes ofPhillyrea latifoliaL. (Oleaceae) Leaves

The anatomy and ultrastructure of glandular trichomes at differentdevelopmental stages were investigated inPhillyrea latifoliaL.leaves by transmission electron microscopy and histochemicaltechniques. The trichome consisted of a multicellular secretoryhead, a unicellular stalk and a collecting cell surrounded byepidermal cells and spongy mesophyll cells. There were numerousplasmodesmata across the cell walls of trichome cells, and especiallybetween the stalk cell and the collecting cell. The collectingcell and stalk cell contained few chloroplasts. Mitochondria,elements of the endoplasmic reticulum and small vacuoles wereabundant in the secretory cells. Crystals were present in thesecretory cells and the collecting cell, especially at the matureand senescent stages of trichome development. As the cuticle,which covered the secretory cells, did not show pores or perforations,it is proposed that secretion occurred by accumulation of productsin subcuticular spaces followed by diffusion through the cuticle.Callose accumulation was observed between the stalk cell andthe collecting cell of senescent trichomes, especially in salt-treatedplants. Trichome ontogeny was accelerated in salt-treated plants.Copyright1998 Annals of Botany Company Cuticle;Phillyrea latifolia; secretion; transmission electron microscopy; trichome development.     

Ontogenetics of QTL: the genetic architecture of trichome density over time in Arabidopsis thaliana

Although much is known about the molecular genetic basis of trichome development in Arabidopsis thaliana, less is known about the underlying genetic basis of continuous variation in a trait known to be of adaptive importance: trichome density. The density of leaf trichomes is known to be a major determinant of herbivore damage in natural populations of A. thaliana and herbivores are a significant selective force on genetic variation for trichome density. A number of developmental changes occur during ontogeny in A. thaliana, including changes in trichome density. I used multiple interval mapping (MIM) analysis to identify QTL responsible for trichome density on both juvenile leaves and adult leaves in replicate, independent trials and asked whether those QTL changed with ontogeny. In both juvenile and adult leaves, I detected a single major QTL on chromosome 2 that explained much of the genetic variance. Although additional QTL were detected, there were no consistent differences in the genetic architecture of trichome density measured on juvenile and adult leaves. The finding of a single QTL of major effect for a trait of known adaptive importance suggests that genes of major effect may play an important role in adaptation.     

PKS activities and biosynthesis of cannabinoids and flavonoids in Cannabis sativa L. plants

Polyketide synthase (PKS) enzymatic activities were analyzed in crude protein extracts from cannabis plant tissues. Chalcone synthase (CHS, EC 2.3.1.74), stilbene synthase (STS, EC 2.3.1.95), phlorisovalerophenone synthase (VPS, EC 2.3.1.156), isobutyrophenone synthase (BUS) and olivetol synthase activities were detected during the development and growth of glandular trichomes on bracts. Cannabinoid biosynthesis and accumulation take place in these glandular trichomes. In the biosynthesis of the first precursor of cannabinoids, olivetolic acid, a PKS could be involved; however, no activity for an olivetolic acid-forming PKS was detected. Content analyses of cannabinoids and flavonoids, two secondary metabolites present in this plant, from plant tissues revealed differences in their distribution, suggesting a diverse regulatory control for these biosynthetic fluxes in the plant.     

Artificial Defoliation Induces Trichome Production in the Tropical Shrub Cnidoscolus aconitifolius (Euphorbiaceae)1

Induction of plant defenses and their spatial variability are key subjects in the field of ecology and evolution of defensive traits in plants. Nevertheless, induction has been more commonly studied under controlled environments, ignoring other factors that might influence this process in natural settings. The main goal of this study was to determine if artificial defoliation induces trichome production in three natural populations of the tropical shrub Cnidoscolus aconitifolius. First, we performed trichome counts for each population before imposing artificial defoliation to assess differences in trichome loads between populations. Trichome densities (trichomes/cm²) were quantified for leaf blades, petioles, and flower stalks. To determine if defoliation induced trichome production, three defoliation treatments (0% leaves defoliated or controls, 50% of total leaves defoliated, and 100% defoliation) were applied once at the beginning of the reproductive season. Trichome counts were performed on each structure every ～20 d during a 3‐mo period after the application of treatments. Trichome counts showed significant differences in trichome densities between populations for all three structures. In turn, artificial defoliation increased trichome density. Significant differences among treatments were found for trichome densities on leaf blades and petioles. In both these structures, the 100 percent defoliation treatment differed significantly from control plants, presenting higher trichome densities. In addition, the treatment × population interaction was not significant for leaf blades and petioles, indicating that induction is a generalized response in this species, at least at the study sites. These results indicate that trichomes in C. aconitifolius are inducible due to defoliation.     

Lima bean–lady beetle interactions: hooked trichomes affect survival of <Emphasis Type="Italic">Stethorus punctillum</Emphasis> larvae

We tested the hypothesis that Lima bean Phaseolus lunatus L. (Henderson cultivar) trichome density affects the survival of the acariphagous lady beetle Stethorus punctillum Weise. When isolated throughout larval development, 10% or less of S. punctillum larvae reared on two-spotted spider mite Tetranychus urticae Koch on small (rather than large) P. lunatus leaves survived until pupation. Although other factors might have contributed to larval mortality, we demonstrated that survival rate was related to trichome density (i.e., number of hooked trichomes per cm on the underside of leaves). Trichome density was greatest on the underside of small leaves and least on large leaves, indicating that survival rate increased as trichome density decreased. Hooked trichomes impaled S. punctillum larvae more often at the apex of the abdomen than at any other body part. Hooked trichomes also trapped S. punctillum adults, but they always managed to free themselves. This study highlights the negative effect of Lima bean hooked trichomes on a predacious lady beetle that attacks the two-spotted spider mite.     

THE LEAF OF CALYCADENIA AND ITS GLANDULAR APPENDAGES

Carlquist , Sherwin . (Rancho Santa Ana Botanic Garden, Claremont, Calif.) The leaf of Calycadenia and its glandular appendages. Amer. Jour. Bot. 46(2) : 70-80. Illus. 1959.—Large tack-shaped glands are characteristic of the leaves of Calycadenia which are associated with the inflorescence. These glands may be divided into those which are terminal on leaves and those which occur laterally on the surface of the leaf. Lateral glands show stages early in their development which are identical with those of simpler trichomes of Madinae. Terminal glands, which possess more vascularization of the stalk, show a more modified form of development. Vascularization is not derived from protoderm, but from more deeply-seated cells. These cells are included in a zone of elongation which forms the stalk. Vascular bundles may extend to the base of glands which lack vascularization in their stalks. Tack-shaped glands are considered an advanced form of trichome in which internal tissues of the leaf are involved. Within the genus Calycadenia, ontogenetic and comparative studies suggest that the following characters are advanced: reduction to a single terminal gland, “inrolling” of margins to form a cylinder of bundles, concomitant with a central core of fibers or a pectic channel. Systematic distribution of gland occurrence and of types of foliar structure are given.     

EVALUATION OF RELATIONSHIPS WITHIN ERIODICTYON (HYDROPHYLLACEAE) USING TRICHOME CHARACTERISTICS

All 13 taxa of Eriodictyon Bentham (Hydrophyllaceae) were examined to determine the range of variation in trichome characters within the genus. Four simple trichome types were found: short and straight, intermediate length and straight, long and straight, long and wavy. Glandular capitate trichomes were also found in some species. Sessile glands were also observed but not included in the study. Most taxa displayed unique combinations of trichome types on stems, leaves, inflorescence axes and flower parts that allowed those taxa to be identified using trichome types alone. Trichome data support most previous taxonomic treatments, but suggest that 1) the two varieties of E. traskiae are extremely similar, 2) the rare E. altissimum is most similar to the widespread E. californicum, 3) the rare E. capitatum is allied with E. crassifolium var. nigrescens or perhaps E. angustifolium, 4) E. crassifolium var. denudatum and E. crassifolium var. nigrescens are essentially indistinguishable using trichome characters and, coupled with variation in other characters, are best combined under the name E. crassifolium var. nigrescens. Trichome characters provide a wealth of taxonomically useful information and may prove useful in the study of related genera of Hydrophyllaceae.     

Novel reports of glands in Neotropical species of Indigofera L. (Leguminosae,Papilionoideae)

By considering controversial discussions in the literature with regard to gland denomination in Indigofera species, as well as the taxonomic value of secretory structures in Leguminosae, we aim to morphologically detail glands that had been previously observed in I. microcarpa and I. sabulicola, and to investigate the occurrence of glands in vegetative and reproductive organs of other six Neotropical species that belong to the genus. Glands analyzed through scanning electronic microscopy (SEM) in combination with anatomic analyses correspond to secretory trichomes that are classified into seven types. Main variations in relation to types occurred with regard to head shape and peduncle size. Trichome heads were multicellular, with a thin cuticle. Hollow heads with conspicuous inner space characterized only one type (type 1); the other trichome types had massive heads. Peduncles, which varied from biseriate to multiseriate, had thick, pecto-cellulosic cell walls. Trichomes were found on stems, stipules, petioles, rachis, petiolules, leaflets, bracteoles, sepals, standards and fruits, more commonly along the margins. Each of the eight Indigofera species analyzed had at least two different trichome types out of the seven types that occurred in reproductive and vegetative organs of these taxa. Various types of secretory trichomes were found in I. campestris, I. lespedezioides, I. microcarpa, I. spicata, I. suffruticosa and I. truxillensis. Stems and rachis were the vegetative organs in which a greater variety of trichomes occurred, and sepals were parts of reproductive organs with the same status. Five out of the seven secretory trichome types occurred on both vegetative and reproductive organs. Distribution and gland types differed between species and these gland distribution patterns can be used as diagnostic characters. Reports of glands in Indigofera campestris, I. hirsuta, I. lepedezioides, I. suffruticosa, I. spicata and I. truxillensis, their recognition as secretory trichomes, and the morphological variety of types found for such trichomes are novel data for Indigofera.     

6-Benzylaminopurine treatment induces increased pubescence on wheat leaves

The epidermis of wheat (Triticum aestivum L.) leaves contains trichomes that contribute to resistance to insect pests and drought tolerance. In the present study, we examined the effects of 6-benzylaminopurine (BA) and methyl jasmonate (MeJA) treatment on trichome development on the leaves of wheat cv. Norin 61 seedlings. Without phytohormone treatment, trichomes on the adaxial leaf surface were short (90 μm) and their density was low (3.6 trichomes/mm²). Both BA and MeJA treatments significantly increased the density of trichomes, and there were no significant differences between the phytohormone treatments. BA treatment increased trichome length to five times as long as that in the control, whereas MeJA treatment did not significantly affect trichome length. Since BA treatment concurrently increased the DNA content of the nuclei in trichome cells, endoreduplication of the nuclei is probably involved in trichome enlargement. These results indicate that even wheat cultivars with short trichomes retain the mechanisms for trichome enlargement and stimuli such as BA application can induce increased pubescence on wheat leaves.     

Development and Structure of Internal Glands and External Glandular Trichomes in Pogostemon cablin

Pogostemon cablin possesses two morphologically and ontogenetically different types of glandular trichomes, one type of bristle hair on the surfaces of leaves and stems and one type of internal gland inside the leaves and stems. The internal gland originates from elementary meristem and is associated with the biosynthesis of oils present inside the leaves and stems. However, there is little information on mechanism for the oil biosynthesis and secretion inside the leaves and stems. In this study, we identified three kinds of glandular trichome types and two kinds of internal gland in the Pogostemon cablin. The oil secretions from internal glands of stems and leaves contained lipids, flavones and terpenes. Our results indicated that endoplasmic reticulum and plastids and vacuoles are likely involved in the biosynthesis of oils in the internal glands and the synthesized oils are transported from endoplasmic reticulum to the cell wall via connecting endoplasmic reticulum membranes to the plasma membrane. And the comparative analysis of the development, distribution, histochemistry and ultrastructures of the internal and external glands in Pogostemon cablin leads us to propose that the internal gland may be a novel secretory structure which is different from external glands.     

Nondestructive assessment of leaf chemistry and physiology through spectral reflectance measurements may be misleading when changes in trichome density co-occur

Reflectance indices are frequently used for the nondestructive assessment of leaf chemistry, especially pigment content, in environmental or developmental studies. Since reflectance spectra are influenced by trichome density, and trichome density displays a considerable phenotypic plasticity, we asked whether this structural parameter could be a source of variation in the values of the most commonly used indices. Trichome density was manipulated in detached leaves of three species having either peltate (Olea europaea and Elaeagnus angustifolius) or tubular (Populus alba) trichomes by successive removal of hairs. After each dehairing step, trichome density was determined by light or scanning electron microscopy and reflectance spectra were obtained with a diode-array spectrometer. Although species-specific differences were evident, most of the indices were considerably affected even at low trichome densities. In general, the less-affected indices were those using wavebands within the visible spectral region. The index that could be safely used even at very high hair densities in all species was the red edge index (lambda(RE)) for chlorophyll. The results indicate that changes in reflectance indices should be interpreted cautiously when concurrent changes in trichome density are suspected. In this case, the red edge for chlorophyll content may be the index of choice.