Dalmatian pyrethrum Tanacetum cinerariifolium (Trevir.) Sch. Bip. (Asteraceae) is a perennial herb endemic to the eastern coast of the Adriatic Sea. The species is widely cultivated in many countries ...for its bioactive compounds pyrethrins, which are used as natural insecticides. Plants derived from seeds vary greatly in pyrethrin content; therefore, the vegetative propagation of high-quality individuals is very important for the establishment of agricultural pyrethrum crops. The present study deals with rapid in vitro multiplication of pyrethrum, ex vitro adaptation of selected clones and creation of an ex situ collection, as a first step towards introducing the species into agriculture in Bulgaria. Seeds from a private ex situ collection in Bulgaria and from a natural Croatian population were used as initial material for in vitro cultures initiation. Basal MS medium (Murashige and Skoog 1962) or MS supplemented with different concentrations of kinetin and indole-3-butyric acid were used for seed germination and multiplication of one-seed derived clones by consecutive subcultivations. The propagation effectiveness was evaluated as a number of new plants obtained per initial shoot. Considerable losses were noticed due to both endophytic contaminations and necrosis, especially on media supplemented with plant growth regulators. These problems were overcome by medium optimization: adding an antibiotic and modifying the medium to increase the calcium concentration using CaCOsub.3. In the best medium variant (basal MS + 200 mg/L Medaxone + 75 mg/L Ca) no more infected plants were observed, and the percentage of necrotic plants decreased threefold, which resulted in formation of 38.06±10.11 new plants per initial shoot for a period of 7 months. Three hundred and sixty plants were ex vitro adapted in a phytotron (88% surviving rate), then 16 plants from 4 selected clones were transferred to the ex situ collection and bloomed twice from the very first growing season (June and September). The number of the flower heads increased in the second year of field cultivation and an average of 328±138 capitula per plant were counted for the best clone. The first trials to establish a pilot plantation of pyrethrum are promising.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Glandular trichomes are currently known only to store mono- and sesquiterpene compounds in the subcuticular cavity just above the apical cells of trichomes or emit them into the headspace. We ...demonstrate that basipetal secretions can also occur, by addressing the organization of the biosynthesis and storage of pyrethrins in pyrethrum (Tanacetum cinerariifolium) flowers. Pyrethrum produces a diverse array of pyrethrins and sesquiterpene lactones for plant defense. The highest concentrations accumulate in the flower achenes, which are densely covered by glandular trichomes. The trichomes of mature achenes contain sesquiterpene lactones and other secondary metabolites, but no pyrethrins. However, during achene maturation, the key pyrethrin biosynthetic pathway enzyme chrysanthemyl diphosphate synthase is expressed only in glandular trichomes. We show evidence that chrysanthemic acid is translocated from trichomes to pericarp, where it is esterified into pyrethrins that accumulate in intercellular spaces. During seed maturation, pyrethrins are then absorbed by the embryo, and during seed germination, the embryo-stored pyrethrins are recruited by seedling tissues, which, for lack of trichomes, cannot produce pyrethrins themselves. The findings demonstrate that plant glandular trichomes can selectively secrete in a basipetal direction monoterpenoids, which can reach distant tissues, participate in chemical conversions, and immunize seedlings against insects and fungi.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Plants emit specific blends of volatile organic compounds (VOCs) in response to mechanical wounding. Such induced VOCs have been shown to mediate in plant and interplant communication, yet little is ...known about the time- and dose-response relationships in VOC-mediated communications. Here, we employed young seedlings of Chrysanthemum cinerariaefolium to examine the effects of volatiles emitted by artificially damaged seedlings on the biosynthesis of the natural insecticides pyrethrins in intact conspecific plants. Wounded leaves emitted (Z)-3-hexenal, (E)-2-hexenal, (Z)-3-hexen-1-ol, (Z)-3-hexen-1-yl acetate and (E)-β-farnesene as dominant wound-induced VOCs. Exposing intact seedlings to a mixture of these VOCs at concentrations mimicking those emitted from wounded seedlings, as well as placing the intact seedlings next to the wounded seedlings, resulted in enhanced pyrethrin contents in the intact seedlings. Thus we quantified mRNA transcripts of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), chrysanthemyl diphosphate synthase (CPPase), 13-lipoxygenase (13-LOX) and allene oxide synthase (AOS) genes in intact seedlings exposed to the VOC mixture to show that DXS and 13-LOX gene expression reached a maximum at 3 h, whereas CPPase and AOS reached it at 6 h. Interestingly, both increasing and decreasing the VOC mixture concentrations from those observed on injury reduced the expression of DXS, CPPase and AOS genes to the control level. Also, separating the VOC mixture into individual components eliminated the ability to enhance the expression of all the biosynthetic genes examined. This is the first study showing that the wound-induced VOCs function as a blend to control the biosynthesis of second metabolites at specific concentrations.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Although natural insecticides pyrethrins produced by Tanacetum cinerariifolium are used worldwide to control insect pest species, little information is known of their biosynthesis. From the buds of ...T. cinerariifolium, we have purified a protein that is able to transfer the chrysanthemoyl group from the coenzyme A (CoA) thioester to pyrethrolone to produce pyrethrin I and have isolated cDNAs that encode the enzyme. To our surprise, the active principle was not a member of a known acyltransferase family but a member of the GDSL lipase family. The recombinant enzyme (TcGLIP) was expressed in Escherichia coli and displayed the acyltransferase reaction with high substrate specificity, recognized the absolute configurations of three asymmetric carbons and also showed esterase activity. A S40A mutation in the Block I domain reduced both acyltransferase and esterase activities, which suggested an important role of this serine residue in these two activities. The signal peptide directed the localization of TcGLIP::enhanced green fluorescent protein (EGFP) fusion, as well as EGFP, to the extracellular space. High TcGLIP gene expression was observed in the leaves of mature plants and seedlings as well as in buds and flowers, a finding that was consistent with the pyrethrin I content in these parts. Expression was enhanced in response to wounding, which suggested that the enzyme plays a key role in the defense mechanism of T. cinerariifolium.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
•Mass selection raised pyrethrin content from 1.37% to 1.69% in progeny population.•Pyrethrin content of progeny population improved even more to 1.87% by polycross.•Family selection did not improve ...pyrethrin content.•Mass selection and polycross increased the frequency of desirable individuals in populations.•Polycross is more effective for the rapid improvement of pyrethrum populations.
Pyrethrum Tanacetum cinerariifolium (Trevir.) Sch.Bip. is a well-known plant widely used for the production of natural pyrethrin insecticides. One of the main breeding goals is to increase pyrethrin content. Comparative analysis of three breeding methods for the improvement of pyrethrin content by mass selection, family selection and polycross was carried out based on an unselected heterogeneous population. Twenty-three elite plants were identified in the base population, and one round of mass selection raised the average pyrethrin content from 1.37% to 1.69% in the progeny population. Polycross was conducted using 23 clones derived from the above same 23 elite plants derived from the base population, and the average pyrethrin content of that population improved even more to 1.87%. As a third method 12 out of 23 families were selected and hybridized within each family. This method did not improve pyrethrin content. Both mass selection and polycross increased the frequency of desirable individuals in the improved populations, and the variation coefficient of pyrethrin content was reduced from 29% in the base population to 18% and 16% after mass selection and polycross, respectively. These results show that polycross is more effective than mass selection for the rapid improvement of pyrethrum populations, and that family selection is not suitable.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
•Extract yield using methanol (9.10%) was about 10 times higher than hexane (0.85%).•Total pyrethrin content in the hexane extract was about 10% more as compared to methanolic extract.•The maximum ...pyrethrin content was obtained using acetonitrile as the extracting solvent using soxhlet method.•Energy assisted extraction techniques like agitation and sonication enhanced the extractive yield by 20–50%.•Enrichment of the pyrethrins using solid matrix technique and acetonitrile as enhanced the total pyrethrin content up to 60.37%.
This paper reports comparative extraction efficiencies and enrichment methods for natural insecticidal compounds pyrethrins from the dried flowers of Chrysanthemum cinerariaefolium using different techniques and solvent systems. Four different extraction techniques viz. percolation, agitation with heat, sonication and soxhlet using five different solvent systems hexane, ethyl acetate, acetone, acetonitrile and methanol were evaluated. In these studies, it was observed that maximum extract yield was obtained using methanol as extracting solvent by soxhlet technique, although the content of total pyrethrins was higher in the hexane extract than in methanol. The successive and maximum enrichment of the total pyrethrins was achieved by solid-matrix partitioning of the extract using acetonitrile as extracting solvent followed by treatment with activated charcoal, subsequent chilling and filtration by which enrichment of total pyrethrins up to 60.37% content was obtained. The quantitative analysis of the extraction and enrichment development protocol was validated by a simple, accurate, reproducible RP-HPLC-UV method using a binary gradient elution comprising of acetonitrile and water. The separation was achieved on a Waters-symmetry C18 column (150mm×3.9mm i.d., 5μm) that achieved a greater degree of linearity within an overall concentration range of 5–500μgmL−1 and higher degree of correlation (0.9842≤r2≤0.9987) for all the pyrethrin esters.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
The effects of growth regulators on culture response of different pyrethrum (Chrysanthemum cinerariaefolium Vis.) genotypes were investigated. In the genotype Sb/66/107, the presence of ...2,4-dichorophenoxyacetic acid (2,4-D) at 2 mg l super(-1) promoted growth of callus, whereas benzyladenine and 1-naphthaleneacetic acid had no effect. Callus growth was also affected by the 2,4-D concentration and genotypes. The optimal callus growth for the nine pyrethrum genotypes varied widely in response to 2,4-D, ranging from 0.5 mg l super(-1) for genotype Marwanga to 3.0 mg l super(-1) for Ks/75/336. Among the genotypes, shoots were regenerated from calluses of Sb/66/107, 4331, Marwanga, and MA/70/1013.
The lipid precursor alcohols of pyrethrins—jasmolone, pyrethrolone and cinerolone—have been proposed as sharing parts of the oxylipin pathway with jasmonic acid. This implies that one of the first ...committed steps of pyrethrin biosynthesis is catalyzed by a lipoxygenase, catalyzing the hydroperoxidation of linolenic acid at position 13. Previously, we showed that the expression and activity of chrysanthemyl diphosphate synthase (TcCDS), the enzyme catalyzing the first committed step in the biosynthesis of the acid moiety of pyrethrins, is trichome-specific and developmentally regulated in flowers. In the present study we characterized the expression pattern of 25 lipoxygenase EST contigs, and subsequently carried out the molecular cloning of two pyrethrum lipoxygenases,
TcLOX1
and
TcLOX2
, that have a similar pattern to
TcCDS
. Only recombinant TcLOX1 catalyzed the peroxidation of the linolenic acid substrate. Just as
TcCDS
,
TcLOX1
, are exclusively expressed in trichomes. Phylogenetic analysis showed that the enzyme shared the highest homology with chloroplast-localized 13-type-lipoxygenases that are involved in maintaining basal levels of jasmonate.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Pyrethrins and flower yield of pyrethrum (Chrysanthemum cinerariaefolium Viz.) plants were determined after application of ethrel, chlormequat chloride and paclobutrazol. Ethrel at 50, 100, 250 and ...500 mg l-l produced a significant positive effect on pyrethrins level, decreased plant height, while 50 and 100 mg l-l significantly increased fresh and dry flower yield. Chlormequat chloride at 1000 and 2000 mg l-l and paclobutrazol (80 and 160 mg l-l) increased pyrethrins level, single flower weight and decreased plant height and flower yield. ¹⁴C-acetate incorporation studies further substantiated positive effect of growth retardants on pyrethrins biosynthesis. The effect of growth retardants on pyrethrins seems to be mediated through its effect on biosynthesis.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Extraction efficiency of insecticidal active compounds from dried Dalmatian pyrethrum flowers (Chrysanthemum cinerariaefolium /Trevir/Vis) was tested using different techniques and solvents. The ...research included six treatments which are the combination of two techniques (soxtec and ultrasound) and three different solvents (hexane, ethanol and petroleum ether). Dalmatian pyrethrum is a perennial herb native to Croatia. Its powder prepared from dried flower heads has been used as natural insecticide for centuries in traditional Croatian farming systems. It has no toxicity to man and animals but possesses ecological benefits that have led to increasing worldwide production of this natural insecticide. Nowadays, it is cultivated mainly at higher altitudes in tropical countries such as Kenya, Tanzania and Rwanda. The present investigation was directed in identifying a simple and reliable extraction treatment using solvents with lower cost and toxicity and an adequate method for the identification and separation of active compounds (pyrethrins) with possible application in enterprises or industry. Best developed method was used for determination of pyrethrin content in three different natural populations of Chrysanthemum. The results revealed high content of total pyrethrins in populations grown in Croatia. Developed method and good quality product give a possibility for this culture to become again an exporting and economically valid product for Croatia.
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