Archaeological evidence of coca (Erythroxylum coca,erythroxylaceae) in the upper mantaro valley,Peru

Prehistoric remains of coca (Erythroxylum spp.) are rarely uncovered by archaeologists or positively identified by botanists because of their fragile nature and the lack of rigorous archaeological collection techniques. This absence of plant evidence has made evolutionary studies of diffusion and use of coca difficult. From special depositional conditions in the Mantaro area of central Peru, one coca leaf and two endocarps have been uncovered and identified as Erythroxylum coca var. coca. These three specimens came from elite-status contexts dating to the Late Intermediate and the Late Horizon-Early Colonial Periods. These remains provide the first highland evidence for access to coca-producing, ceja de montaña farms, which lie more than 50 km away on the eastern slope of the Andes.     

Inter- and intra-specific variation among five Erythroxylum taxa assessed by AFLP

BACKGROUND: and Aims The four cultivated Erythroxylum taxa (E. coca var. coca, E. novogranatense var. novogranatense, E. coca var. ipadu and E. novogranatense var. truxillense) are indigenous to the Andean region of South America and have been cultivated for folk-medicine and, within the last century, for illicit cocaine production. The objective of this research was to assess the structure of genetic diversity within and among the four cultivated alkaloid-bearing taxa of Erythroxylum in the living collection at Beltsville Agricultural Research Center. METHODS: Amplified fragment length polymorphism (AFLP) fingerprinting was performed in 86 Erythroxylum accessions using a capillary genotyping system. Cluster analysis, multidimensional scaling (MDS) and analysis of molecular variance (AMOVA) were used to assess the pattern and level of genetic variation among and within the taxa. KEY RESULTS: A clear distinction was revealed between E. coca and E. novogranatense. At the intra-specific level, significant differentiation was observed between E. c. var. coca and E. c. var. ipadu, but the differentiation between E. n. var. novogranatense and E. n. var. truxillense was negligible. Erythroxylum c. var. ipadu had a significantly lower amount of diversity than the E. c. var. coca and is genetically different from the E. c. var. ipadu currently under cultivation in Colombia, South America. CONCLUSIONS: There is a heterogeneous genetic structure among the cultivated Erythroxylum taxa where E. coca and E. novogranatense are two independent species. Erythroxylum coca var. coca is most likely the ancestral taxon of E. c. var. ipadu and a founder effect may have occurred as E. c. var. ipadu moved from the eastern Andes in Peru and Bolivia into the lowland Amazonian basin. There is an indication of artificial hybridization in coca grown in Colombia.     

Flavonoids as chemotaxonomic markers for cultivated Amazonian coca

Purported ‘Amazonian coca,’ Erythroxylum coca var. ipadu Plowman (E. coca var. ipadu) was harvested from cultivated fields in Colombia, South America to determine: (a) its identity; (b) if its leaf flavonoids were complimentary to those present in leaf tissue of E. c. var. ipadu in our collection derived from Colombia; and (c) if complimentary, indicative of kinship to E. coca var. ipadu obtained from Colombia, or a related Erythroxylum taxon. Leaf extracts from Amazonian field-grown coca afforded eight O-conjugated flavonoids: two O-conjugates of taxifolin, one O-conjugate of quercetin, two O-conjugates of eriodictyol and three O-conjugates of kaempferol. Present also in leaf tissue of Amazonian field-grown coca, but lacking in leaf tissue from our collection of E. c. var. ipadu was an O-ethyl ester typically found in E. coca var. coca, kaempferols and a 7-O-rutinoside commonly encountered in the E. novogranatense taxons. Flavonoids found in our collection of E. coca var. ipadu were five O-conjugated derivatives of taxifolin and an O-conjugated quercetin. Leaf flavonoids of currently cultivated Amazonian coca are a mixture of those present in E. coca var. coca, E. coca var. ipadu and E. novogranatense var. truxillense, whereas those present in our authenticated living collection are derivatives of E. coca var. coca. Our data suggest that the Amazonian coca under cultivation in Colombia is a genetic hybrid cross between E. coca var. coca and E. novogranatense var. truxillense, occurring after 1972.     

Identification of Erythroxylum taxa by AFLP DNA analysis

Erythroxylum coca, indigenous to the Andean region of South America, is grown historically as a source of homeopathic medicine. However, in the last century, cultivation of E. coca and several closely-related species for the production of illicit cocaine has become a major global problem. Two subspecies, E. coca var. coca and E. coca var. ipadu, are almost indistinguishable phenotypically; a related cocaine-bearing species also has two subspecies (E. novogranatense var. novogranatense and E. novogranatense var. truxillense) that are phenotypically similar, but morphologically distinguishable. The purpose of this research was to discover unique AFLP DNA patterns ("genetic fingerprinting") that characterize the four taxa and then, if successful, to evaluate this approach for positive identification of the various species of coca. Of seven different AFLP primer pairs tested, a combination of five proved optimal in differentiating the four taxa as well as a non-cocaine-bearing species, E. aerolatum. This method of DNA fragment separation was selective, and faster, for coca identification, compared with analyses based on flavonoid chemotaxonomy. Using the 5-primer AFLP approach, 132 known and unknown coca leaf accessions were evaluated. Of these, 38 were collected in 1997-2001 from illicit coca fields in Colombia, and all were genetically differentiated from coca originating in Peru and Bolivia. Based on the DNA profiling, we believe that the Colombian coca now represents a hybridization of E. coca var. ipadu. Geographical profiling within Colombia also seems feasible as new coca production areas are developed or new types of coca are introduced within traditional growing areas.     

Storage effects on genomic DNA in rolled and mature coca leaves

Rolled and mature leaf tissue was harvested from Erythroxylum coca var. coca Lam. (coca) to determine a method for storage that would maintain DNA with high quality and content up to 50 days. Harvesting coca leaf tissue under Andean field conditions often requires storage from 3 to 10 days before extraction where tissue integrity is lost. All samples of rolled and mature coca leaf tissue were harvested and separately stored fresh in RNAlater for 50 days at 4 degrees, -20 degrees, and 23 degrees C, while similar samples were air-dried for 72 h at 23 degrees C or oven-dried for 72 h at 40 degrees C after storage, before extraction. Triplicate samples of each tissue type were extracted for DNA at 10-day intervals and showed that DNA integrity and content were preserved in leaf tissue stored at 4 degrees and -20 degrees C for 50 days. Rolled and mature leaf tissue stored at 4 degrees, -20 degrees, and 23 degrees C showed insignificant degradation of DNA after 10 days, and by day 50, only leaf tissue stored at 4 degrees and -20 degrees C had not significantly degraded. All air- and oven-dried leaf tissue extracts showed degradation upon drying (day 0) and continuous degradation up to day 50, despite storage conditions. Amplified fragment length polymorphism analysis of DNA from rolled and mature leaf tissue of coca stored at 4 degrees and -20 degrees C for 0, 10, and 50 days showed that DNA integrity and content were preserved. We recommend that freshly harvested rolled or mature coca leaf tissue be stored at 4 degrees, -20 degrees, and 23 degrees C for 10 days after harvest, and if a longer storage is required, then store at 4 degrees or -20 degrees C.     

Heterostyly in Erythroxylum coca (Erythroxylaceae)

Erythroxylum coca is a distylous species with a strong self-incompatibility system linked with the floral dimorphism. The two sets of stamens in the flowers are usually unequal in length, but between individuals of both morphs there is considerable variation in the relative qengths of the two sets of stamens, which is unrelated to the incompatibility system. Pin flowers produce more pollen grains than thrum flowers, but thrum pollen is larger than pin pollen. Within each morph the two sets of stamens produce pollen grains of slightly different diameter. Erythroxylum novogranatewe is also distylous. Pin flowers of E. novogranatense var. novogranatense are partially self-compatible, while thrum flowers of E. novogranatense var. truxillense are self-incompatible. Reports of tristyly and of four different morphs in species of Erythroxylum are probably misinterpretations, resulting from limited sampling, of the continuous variation in the relative lengths of the two sets of stamens.     

Calystegines in wild and cultivated Erythroxylum species

Calystegines were identified in the genus Erythroxylum for the first time. Erythroxylum novogranatense var. novogranatense, a species cultivated for cocaine production, contained 0.2% total calystegines in dry leaves. Forty six Erythroxylum herbarium species consisting mostly of leaf tissue were analysed for calystegines, and 38 were found positive. Calystegines were compared qualitatively and quantitatively between individual Erythroxylum species. Calystegines A(3) and B(2) were the major calystegines in most species. Total calystegine content reached up to 0.32% dry mass. The simultaneous occurrence of calystegines, cocaine, other alkaloids of a 3alpha-hydroxy- or 3beta-hydroxytropane structure together with nicotine supports the concept of common biosynthetic steps of these alkaloids in Erythroxylum. The present results are the basis for further investigations of the phylogenetic origin of tropane alkaloid biosynthesis in the taxonomically remote families Solanaceae and Erythroxylaceae.     

Tropane alkaloids from Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae)

Six tropane alkaloids were isolated from the Sri Lankan endemic plant Erythroxylum zeylanicum O.E. Schulz (Erythroxylaceae) and structurally elucidated by NMR and MS measurements. Three of them, erythrozeylanines A [1R,3R,5S,6R-6-acetoxy-3-(3',4',5'-trimethoxybenzoyloxy)tropane], B [cis-3 beta-(cinnamoyloxy)tropane], and C [cis-6 beta-acetoxy-3 alpha-(cinnamoyloxy)tropane] are new, whereas the others have already been found in other Erythroxylum species. For the first time, the absolute configuration of a tropane alkaloid (erythrozeylanine A) has been determined by quantum chemical CD calculations.     

The use of multiple alien chromosome addition aneuploids facilitates genetic linkage mapping of the Gossypium G genome.

Primary germplasm pools represent the most accessible source of new alleles for crop improvement, but not all effective alleles are available in the primary germplasm pool, and breeders must sometimes confront the difficulties of introgressing genes from the secondary and tertiary germplasm pools in cotton by using synthetic polyploids as introgression bridges. Two parental Gossypium nelsonii x Gossypium australe AFLP genetic linkage maps were used to identify G genome chromosome-specific molecular markers, which in turn were used to track the fidelity and frequency of G. australe chromosome transmission in a Gossypium hirsutum x G. australe hexaploid bridging family. Conversely, when homoeologous recombination is low, first generation aneuploids are useful adjuncts to genetic linkage mapping. Although locus ordering was not possible, the distribution of AFLP markers among 18 multiple chromosome addition aneuploids identified mapping errors among the G. australe and G. nelsonii linkage groups and assigned non-segregating G. australe AFLPs to linkage groups. Four putatively recombined G. australe chromosomes were identified in 5 of the 18 aneuploids. The G. australe and G. nelsonii genetic linkage maps presented here represent the first AFLP genetic linkage maps for the Gossypium G genome.     

Variation of Alkaloid Content in Erythroxylum coca Leaves from Leaf Bud to Leaf Drop

A sequential study describing the content (%) of alkaloids inleaves of Erythroxylum coca var. coca Lam. from leaf bud developmentto leaf drop has not previously been conducted. The length oftime the leaf resides on the plant and its concurrent metabolicactivity also has not been defined. In the present study, cocaine,methyl ecgonine, hygrine, tropinone, trans -cinnamoylcocaine,cis-cinnamoylcocaine, tropacocaine and cuscohygrine were monitoredto determine: (1) their content and patterns of accumulationfrom leaf bud to leaf drop; (2) the time leaves resided on theplant; and (3) whether leaves were metabolically active untilleaf drop. E. coca plants were grown in a controlled environmentfor 37 months. Leaves similar in chronological age were extractedand analysed for alkaloid content by gas chromatography (GC)and gas chromatography/mass spectrometry (GC/MS). Cocaine washighest in 7 d old rolled leaves (0·75%) and declinedto 0·39% at leaf drop. Cocaine, methyl ecgonine, hygrine,tropinone, trans -cinnamoylcocaine, cis-cinnamoylcocaine, cuscohygrineand tropacocaine content in 35 d old (mature) leaves was 0·61,0·59, 0·68, 0·08, 0·31, 0·55,0·52, and 0·05%. respectively. Cocaine, methylecgonine, hygrine, cis -cinnamoylcocaine, and cuscohygrine displayeda gradual decline from week 2 to week 36 of leaf duration. Tropinoneand tropacocaine were the least abundant of the alkaloids monitored.Cis-cinnamoylcocaine content exceeded cocaine at week 12, 16,and weeks 19 to 23 of leaf duration. Trans -cinnamoylcocainewas highest in rolled leaves (week 1) and in expanded leavesafter week 30. The monitored alkaloids appeared to be part ofthe metabolically active pool of the leaf. Leaves remained onthe E. coca plants for 36 weeks.Copyright 1994, 1999 AcademicPress Cocaine, methyl ecgonine, hygrine, tropinone, trans-cinnamoylcocaine, cis-cinnamoylcocaine, cusco-hygrine, tropacocaine, leaf bud, rolled leaves, expanded leaves, alkaloids, patterns, fluctuation, Erythroxylum coca var. coca, E, coca     

An Alginate Prill Formulation of Fusarium oxysporum Schlechtend: Fr. f. sp. erythroxyli for Biocontrol of Erythroxylum coca var. coca

A rice alginate prill formulation of isolate EN - 4 of Fusarium oxysporum f . sp . erythroxyli, pathogenic to Erythroxylum coca var . coca (coca) , was evaluated in greenhouse and field studies for its ability to enhance pathogen populations in the soil and cause disease in coca . The formulation was applied to four different soil types in the greenhouse at 33 . 6 kg ha 1 . It enhanced the population of EN - 4 in each soil and most ( > 90%) of the fungal population remained in the upper 5 cm of soil during the 49 - day experiment . When applied in field experiments , the formulation enhanced the population of EN - 4 in the soil . Isolate EN - 4 was present in the upper 7 . 6 cm of soil 28 days after application at populations similar to those in the greenhouse studies (1 103 to 1 104 colony - forming units (CFUs) / g of soil) . Elevated populations of the pathogen (1 102 CFUs / g of soil) were still present in treated soils 229 days after application of the formulation . The areas used for field studies were already infested with the pathogen and typically developed high levels of fusarium wilt within 2 years of planting with coca . The formulated F. oxysporum began having a significant effect on plant death 100 - 200 days after application based on repeated measures analysis . These data suggest that a formulation of F. oxysporum f . sp . erythroxyli which enhances the incidence of fusarium wilt in coca fields can be produced using established techniques .     

Content and De Novo Synthesis of Cocaine in Embryos and Endosperms from Fruit of Erythroxylum coca Lam

There is controversy as to whether the mature fruit of Erythroxylumcoca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine).In the present study, cocaine was monitored to determine ifit was present in embryos and endosperms of mature fruit ofE. coca var. coca Lam., and if present, the time required forde novo synthesis in imbibing seed. Seeds from mature fruitof E. coca were dissected to separate the embryos from the endosperms.The separated embryos and endosperms were analysed for cocaine.Subsequently, endosperms and embryos from seed imbibed. undera light and dark treatment were separated on days 3, 6, 9, 12and 15 and analysed for cocaine. Cocaine was present in embryos(0.005% of d. wt) and endosperms (0–001% of d. wt) ofmature fruit of E. coca. De novo synthesis of cocaine occurredonly in embryos of seed imbibed under light after day 9 of imbibition. Erythroxylum coca, alkaloid, benzoylmethylecgonine, cocaine, embryo, endosperm, seed imbibition     

Content and Distribution of Erythroxylum coca Leaf Alkaloids

Fully expanded leaves of Erythroxylum coca var. coca Lam. (Erythroxylaceae)35-70-d-old were harvested from 21-month-old plants grown undergreenhouse conditions. Each harvested leaf was placed on a plexiglasssilhouette according to its dimensions and divided into fourprimary sections (petiole, base, mid and anterior) and threesubsections (lamina periphery, false mid-rib, and true mid-rib)to determine the distribution and content of hygrine, cuscohygrine,trans-cinnamoylcocaine, cis -cinnamoylcocaine, tropacocaine,tropinone, methyl ecgonine and cocaine. The leaf sub-sectionsand petiole were extracted and analysed for alkaloid content(%) by HPLC (cocaine alkaloid only) gas chromatography and GC/MS.Cocaine, methyl ecgonine and hygrine were highest in the laminaperiphery with a content of 0·48, 0·46 and 0·32%,respectively. Trans-cinnamoylcocaine was pre-eminent of thecinnamoylcocaines and was most abundant in the petiole at acontent of 0·24%. Cis-cinnamoylcocaine, tropinone, cuscohygrineand tropacocaine were ubiquitously distributed throughout theleaf where the average contents were 0·14, 0·004,0·16 and 0·04%, respectively.Copyright 1995, 1999Academic Press Alkaloids, Erythroxylum coca var. coca, E. coca, leaf alkaloids, petiole alkaloids, cocaine, cuscohygrine, methyl ecgonine, hygrine, tropinone, tropacocaine, trans-cinnamoylcocaine, cis-cinnamoylcocaine     

Experimental design in supercritical fluid extraction of cocaine from coca leaves

An optimisation procedure for the supercritical fluid extraction (SFE) of cocaine from the leaves of Erythroxylum coca var. coca was investigated by means of experimental design. After preliminary experiments where the SFE rate-controlling mechanism was determined, a central composite design was applied to evaluate interactions between selected SFE factors such as pressure, temperature, nature and percentage of the polar modifier, as well as to optimise these factors. Predicted and experimental contents of cocaine were compared and robustness of the extraction method estimated by drawing response surfaces. The analysis of cocaine in crude extracts was carried out by capillary GC equipped with a flame ionisation detector (GC-FID), as well as by capillary GC coupled with a mass spectrometer (GC-MS) for peak identification.     

Cocaine and Cinnamoylcocaine Content of Erythroxylum Species

The dried leaves of 31 species and two varieties of the genusErythroxylum were quantitatively analysed for cocaine and bothcis- and trans-cinnamoylocaine, using a stable-isotope dilutionmethod and gas chromatography-mass spectrometry selected-ionmonitoring. Both cocaine and cinnamoylcocaine were detectedin all four varieties of cultivated coca (E. coca var. coca,E. coca var. ipadu, E. novogranatense var. novogranatense andE. novogranatense var.truxillense). Cinnamoylcocaines (cis andtrans) were found in much higher concentrations in both varietiesof E. novogranatense than in either variety of E. coca. Amazoniancoca (E. coca var. ipadu) contained consistently lower cocainelevels than the montane variety (E. coca var. coca). Twenty-ninewild species of Erythroxylum, selected to represent morphological,ecological and taxonomic diversity, were analysed for the samealkaloids; cocaine was detected in 13 neotropical species, representingfive sections of the genus. No cocaine was detected in the OldWorld species. Two wild species from Venezuela, E. recurrensand E. steyermarkii, contained cocaine levels comparable tothose found in the commercially cultivated species. Erythroxylaceae, Erythroxylum, coca, cocaine, cinnamoylcocaine, alkaloids, chemotaxonomy     

Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli

Flavonols are plant polyphenolic compounds that belong to the class of molecules collectively known as flavonoids. Because of their demonstrated health benefits towards a wide array of human pathological conditions, a great interest has emerged for their biosynthesis from well-characterized microbial hosts. We present the functional expression in Escherichia coli of a plant P450 flavonoid 3', 5'-hydroxylase (F3'5'H) as a fusion protein with a P450 reductase. This expression allowed metabolic engineering of E. coli to produce the flavonol kaempferol and the 3', 4' B-ring hydroxylated flavonol quercetin from the p-coumaric acid precursor by simultaneously co-expressing the fusion protein with 4-coumaroyl:CoA-ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3beta-hydroxylase (FHT) and flavonol synthase (FLS). Biosynthesis of the B-ring tri-hydroxylated flavonol myricetin from the engineered strains was accomplished when flavanones rather than phenylpropanoid acids were used as precursor molecules. Cultivation of the recombinant strains in rich medium increased the synthesis of all flavonoids with the exception of myricetin. The present work opens the possibility of the future production of several other hydroxylated flavonoid molecules in E. coli.     

A RAD Tag Derived Marker Based Eggplant Linkage Map and the Location of QTLs Determining Anthocyanin Pigmentation

Both inter- and intra-specific maps have been developed in eggplant (Solanum melongena L.). The former benefit from an enhanced frequency of marker polymorphism, but their relevance to marker-assisted crop breeding is limited. Combining the restriction-site associated DNA strategy with high throughput sequencing has facilitated the discovery of a large number of functional single nucleotide polymorphism (SNP) markers discriminating between the two eggplant mapping population parental lines '305E40' and '67/3'. A set of 347 de novo SNPs, together with 84 anchoring markers, were applied to the F(2) mapping population bred from the cross '305E40' x '67/3' to construct a linkage map. In all, 415 of the 431 markers were assembled into twelve major and one minor linkage group, spanning 1,390 cM, and the inclusion of established markers allowed each linkage group to be assigned to one of the 12 eggplant chromosomes. The map was then used to discover the genetic basis of seven traits associated with anthocyanin content. Each of the traits proved to be controlled by between one and six quantitative trait loci (QTL), of which at least one was a major QTL. Exploitation of syntenic relationships between the eggplant and tomato genomes facilitated the identification of potential candidate genes for the eggplant QTLs related to anthocyanin accumulation. The intra-specific linkage map should have utility for elucidating the genetic basis of other phenotypic traits in eggplant.     

Geographical variation in the surface flavonoids of Pulicaria dysenterica

Four chemical races were detected in Pulicaria dysenterica, when sampled within Europe, on the basis of the surface flavonoids present. One race uniquely contained quercetagetin 3,7-dimethyl ether and another 6-hydroxykaempferol 3,4'-dimethyl ether. A third race was based on plants having 6-hydroxykaempferol 3,7-dimethyl ether together with quercetagetin 3,7,3'-trimethyl ether. The fourth race contained the above two compounds, as well as quercetagetin 3,7,3',4'-tetramethyl ether and 6-hydroxykaempferol 3,7,4'-trimethyl ether. These lipophilic constituents were variously present on the surfaces of leaf, ray floret, disc floret and fruit. By contrast, the vacuolar flavonoid of all tissues and all races was uniformly quercetin 3-glucuronide. The kaempferol 3-glucoside previously reported in flowers was not detected. Of the lipophilic flavonoids newly reported from this plant, one 6-hydroxykaempferol 3,7,4'-trimethyl ether is new to nature.     

Alteration of location of dimer linkage sequence in retroviral RNA: little effect on replication or homologous recombination.

Retrovirus particles contain a dimer of retroviral genomic RNA. A defined region of the retrovirus genome has previously been shown to be important for both dimerization and encapsidation. To study the importance of the position of this encapsidation and dimerization signal for retroviral replication and homologous recombination, we used a previously described spleen necrosis virus-based helper cell system. This system allows retroviral vectors with multiple genetic markers to be studied after a single cycle of retroviral replication. The sequence responsible for dimerization, the encapsidation/dimer linkage sequence (E/DLS), was moved from its normal location near the 5' end of the retroviral genome to a location near the 3' end of the genome. We characterized four pairs of retroviral vectors: (i) with both E/DLSs at the 5' ends of the genomes, (ii) with both E/DLSs at the 3' ends of the genomes, and (iii) two with one E/DLS at the 5' end of the genome and one at the 3' end of the genome. We found that moving the E/DLS to the 3' end of the genome resulted in at most an approximately factor of 5 reduction in virus titer in a single cycle of retroviral replication. Furthermore, we found no changes that were attributable to the alteration of the position of the E/DLS in the minus-strand DNA primer transfers or the plus-strand DNA primer transfers, the rate of homologous recombination, or the number of internal template switches in recombinant proviruses. These results indicate that any alignment or conformation necessary for retroviral replication or recombination is not the result of the position of the E/DLS.