Susan J. Murch

Thidiazuron: A potent regulator ofin vitro plant morphogenesis

SummaryTDZ (N-phenyl-N’-1,2,3-thidiazol-5-ylurea) is a substituted phenylurea compound which was developed for mechanized harvesting of cotton bolls and has now emerged as a highly efficacious bioregulant of morphogenesis in the tissue culture of many plant species. Application of TDZ induces a diverse array of cultural responses ranging from induction of callus to formation of somatic embryos. TDZ exhibits the unique property of mimicking both auxin and cytokinin effects on growth and differentiation of cultured explants, although structurally it is different from either auxins or purine-based cytokinins. A number of physiological and biochemical events in cells are likely to be influenced by TDZ, but these may or may not be directly related to the induction of morphogenic responses, and hence, the mode of action of TDZ is unknown. However, the recent approaches applied to study the morphogenic events initiated by TDZ are clearly beginning to reveal the details of a variety of underlying mechanisms. Various reports indicate that TDZ may act through modulation of the endogenous plant growth regulators, either directly or as a result of induced stress. The other possibilities include the modification in cell membranes, energy levels, nutrient uptake, or nutrient assimilation. In this review, several of these possiblities are presented and discussed in light of recently published studies on characterization of TDZ-induced morphogenic effects.

Tryptophan is a precursor for melatonin and serotonin biosynthesis in in vitro regenerated St. John's wort (Hypericum perforatum L. cv. Anthos) plants

Hypericum perforatum cv. Anthos) is presented. Isotope tracer experiments were performed on plantlets regenerated from thidiazuron-induced stem explants and grown on MS basal medium for 2 months. Radiolabel from 14C-tryptophan was recovered as 14C-indoleacetic acid, 14C-tryptamine, 14C-5-hydroxytryptophan, 14C-serotonin and 14C-melatonin in the treated St. Johns wort plantlets. Chromatographic peak identity was confirmed by high performance liquid chromatography-mass spectrometry-mass spectrometry and quantification of melatonin by radioimmunoassay. Significantly more radiolabel was recovered in serotonin relative to melatonin under low light conditions with this ratio being reversed under increased lighting, indicating that the rate of flow through this biosynthetic pathway is regulated, at least in part, by light.

Melatonin in feverfew and other medicinal plants

1598 Vol 350 • November 29, 1997 35% relative to the first trimester, in contrast to the 16% average decrease in the years 1993–96. During the third trimester of 1997, the number of cases of diarrhoea decreased, yet were still 25% above the figures seen in previous years for the same trimester. In Peru and neighbouring countries affected by El Nino— Chile, Ecuador, and Colombia—we anticipate an outbreak of diarrhoea and dehydration in the coming summer months when the high seasonal temperatures will be exacerbated by the presence of El Nino. This outbreak will burden local health services treating infections and/or diseases for which temperature may be an important determinant. Should we prepare for another cholera outbreak? Over the past 2 years, few cases of cholera have been seen in Peru, mainly, we think, because there was no susceptible population left after the huge 1991 and 1992 outbreaks. Children born after 1991 and 1992 who were neither infected nor exposed to Vibrio cholerae are the most susceptible population now. V cholerae can survive in a dormant stage in cool, brackish water, in association with plankton. When the water temperature rises, plankton blooms, and the population of V cholerae increases. R Colwell has suggested that remote satellite sensing of plankton beds might be used as an early warning system to help predict outbreaks of cholera. Analysts have examined the agricultural and economic implications of El Nino. Our data lead us to believe that it is equally imperative that we begin to examine El Nino’s implications on health and health care.

THE ROLE OF SEROTONIN AND MELATONIN IN PLANT MORPHOGENESIS: REGULATION OF AUXIN-INDUCED ROOT ORGANOGENESIS IN IN VITRO-CULTURED EXPLANTS OF ST. JOHN'S WORT (HYPERICUM PERFORATUM L.)

SummarySt. Johns wort (Hypericum perforatum cv. Anthos) is a medicinal plant with historical and anecdotal evidence of efficacy as an anti-depressant. Recent research has demonstrated an active biosynthetic pathway leading to the production of the mammalian neurohormone melatonin in St. Johns wort plantlets. The objective of the current study was to assess the physiological role of melatonin and related indoleamines in plant morphogenesis. In the initial experiments, two of the indoleamines; serotonin and melatonin, were supplemented to the culture medium. In subsequent research, six inhibitors of auxin and indoleamine action or transport, 2,3,5-triiodobenzoic acid, p-chlorophenoxyisobutyric acid, p-chlorophenyl-alanine, d-amphetamine, fluoxetine (ProzacTM), and methylphenidate (RitalinTM), were included in a culture medium in the presence or absence of the auxin, indoleacetic acid (IAA). The rate of de novo root and shoot organogenesis and the endogenous concentrations of auxin and indoleamines were determined in cultured explants. Significant reductions in de novo root regeneration were found to correspond with decreases in the pool of both IAA and melatonin. An increase in the endogenous concentration of melatonin was correlated with an increase in de novo root formation, and increased serotonin levels corresponded to increased shoot formation on the explants. Our findings provide the first evidence that a balance of the endogenous concentration of serotonin and melatonin may modulate plant morphogenesis in vitro.

Molecular fate of thidiazuron and its effects on auxin transport in hypocotyls tissues of Pelargonium × hortorum Bailey

Thidiazuron, a synthetic phenylurea-type cytokinin, has previously beenfound to induce somatic embryogenesis and organogenesis in a wide range ofplantspecies and to modulate the metabolism of endogenous auxins and cytokinins. Inspite of these findings, the precise mode of action of TDZ remainsundetermined.The current studies were undertaken to determine the fate of the TDZ moleculeand the effects of TDZ exposure on auxin transport in plants. The fate of tworadiolabelled versions of thidiazuron, [14C-5-thidiazol]-TDZ and[14C-U-phenyl]-TDZ, was investigated in sterile hypocotyl culturesofgeranium (Pelargonium × hortorumBailey). Radiolabelled TDZ was recovered from the tissue explants inethanol-insoluble, ethanol-soluble and chloroform fractions as well as inacidic, basic and neutral eluants from Dowex resins. Hypocotyl sections thathadbeen exposed to TDZ were found to accumulate more 14C-IAA from theculture medium and to translocate the auxin over a greater distance within thetissues. These data provide the first evidence that the TDZ molecule remainsintact in both a free and conjugated form within the plant tissues and providesome indication that TDZ-exposure enhances the accumulation and translocationofauxin within the tissues.

Nickel contamination affects growth and secondary metabolite composition of St. John's wort (Hypericum perforatum L.)

Abstract St. Johns wort is a medicinal plant used in the treatment of neurological disorders and recently identified as a possible treatment for cancer tumors. The principal medicinally important secondary metabolites present in St. Johns wort are thought to be hypericin, hyperforin and pseudohypericin. The current study was undertaken to determine the effects of environmental contamination with a common inorganic pollutant, nickel, on the synthesis and accumulation of bioactive molecules in St. Johns wort. Seedlings grown in a sterile, controlled environment supplemented with 25 or 50 mM nickel lost completely the capacity to produce or accumulate hyperforin and demonstrated a 15–20-fold decrease in the concentration of pseudohypericin and hypericin. These results provide the first indication that metal contamination can change the chemical composition of St. Johns wort, thereby, seriously impacting the quality, safety and efficacy of natural plant products produced by medicinal species.

Morphoregulatory role of thidiazuron: Evidence of the involvement of endogenous auxin in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium × hortorum Bailey)

Summary Thidiazuron-induced somatic embryogenesis in hypocotyl cultures of geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) consists of an induction phase on 10 or 20 (µmol·L −1 thidiazuron (TDZ) followed by an expression phase on a basal medium lacking growth regulators. The induction and development of somatic embryos were found to be closely related to levels of endogenous and exogenous auxin. Inclusion of the auxin-action inhibitor, PCIB, and the auxin-transport inhibitor, TIBA, in a TDZ-induction medium decreased embryogenic response of the cultures by different mechanisms. The decrease of the embryogenic response in the presence of PCIB was accompanied by a corresponding decrease in endogenous levels of auxins, cytokinins, and ABA. These changes in the profiles of endogenous plant growth regulators were not evident when TIBA was used. High concentrations of exogenous LAA in the expression medium suppressed the development of somatic embryos. Although the precise mode of action of TDZ is unknown, the results of this study provide evidence that TDZ modulates endogenous auxin metabolism during somatic embryo development in geranium hypocotyl cultures.

Melatonin: A potential regulator of plant growth and development?

SummaryRecent research has reported the presence of melatonin (N-acetyl-5-methoxytryptamine), a mammalian indoleamine neurohormone, in higher plants, indicating that melatonin may be an important metabolic regulator that has been highly conserved across biological kingdoms. Melatonin is synthesized from tryptophan in the mammalian pineal gland and a similar biosynthetic pathway was recently described in St. Johns wort shoot tissues, wherein radiolabel from tryptophan was recovered in serotonin and melatonin as well as indoleacetic acid. There is growing information describing melatonin control of physiological processes in mammals, yeast, and bacteria, including diurnal responses, detoxification of free radicals, and environmental adaptations. However, at the current time, there is no known specific role for melatonin in plant physiology. Alterations in melatonin concentrations in plant tissues have been shown to affect root development, mitosis, and mitotic spindle formation. The recent advancements in melatonin research in plants and some directions for important areas of future research are reviewed in this article.

Thidiazuron-induced de novo shoot organogenesis on seedlings, etiolated hypocotyls and stem segments of Huang-qin

Development of an efficient in vitro propagation system for Huang-qin (Scutellaria baicalensis), a traditional Chinese medicinal plant used in the treatment of a wide range of human ailments, is described. Thidiazuron [TDZ: N-phenyl-N′- (1,2,3-thidiazol-5-ylurea)] effectively induced regeneration on cultured intact seedlings, etiolated hypocotyl explants and sterile stem segments of Huang-qin. Histological examinations of excised hypocotyl or nodal explants revealed that adventitious shoots formed through an intermediate callus. Comparison of TDZ-induced regeneration in the three tissue types indicated that isolation of explants was not essential for optimal regenerative efficiency. Significantly more regenerants formed along hypocotyls of intact seedlings (20 shoots/explant) than were observed on excised hypocotyls (9.7 shoots/explant) indicating that endogenous metabolites produced in adjacent tissues provided resources for the shoot initiation. More than 95% of de novo regenerants formed roots and then intact plantlets under either sterile culture or greenhouse conditions. Regeneration protocols developed in this study may provide the basis for improvement of this crop through the identification of medicinally active constituents and eventual development optimized pharmaceutical products.

In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture

SummaryAn in vitro propagation system was developed for Echinacea purpurea L. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Echinacea seeds were found to be contaminated with systemic fungi and therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was incorporated in the seed germination medium to recover sterile seedlings. Regeneration was induced on petiole explants from 2-month-old sterile seedlings cultured on medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) in combination with indoleacetic acid (IAA). Two distinct forms of regeneration were identified in cultured petiole explants with histological and morphological observations, viz. the direct formation of somatic embryos on the epidermis and the de novo development of shoots from callus tissues formed in subepidermal cell layers. the results of this study have established a micropropagation system for E. purpurea that will provide sterile plant material for further investigations into medicinally active biochemicals and may facilitate mass production of high-quality E. purpurea plants for the commercial market.