Alkaloid Spectrum of Different Germplasm Lines in Opium Poppy (Papaver somniferum L.)

The opium latex of 1470 individual plant samples of 98 germplasm lines were screened for five major economic alkaloids. The alkaloids profile showed that morphine content ranged from 9.20–20.86% with an average of 15.00 ± 0.267. Codeine ranged from 1.69–6.48% with arithmetic mean of 3.35 ± 0.091, thebaine from 0.52–7.95% with an average of 2.27 ± 0.116. Narcotine ranged from 8.79–17.97% with an average 8.79 ± 0.266 and papaverine content ranged from 0.00–6.07% with an average 1.18 ± 0.167 in over all the germplasm lines. The content of different alkaloids categorized into class interval which exhibited that maximum number of plants and accessions for morphine content fall in group of 10–15% followed by 15–20%, for codeine content in group of 2–4% followed by 4–6%, for thebaine content in 1–2% followed by 2–4%, for narcotine in 5–10% followed by 10–15% and for papaverine content 0–2%. 24 germplasm lines had morphine content above 16.0%. Similarly, 37 germplasm lines for codeine and 13 for thebaine showed consistently high content, i.e., above 3%. 33 germplasm lines had narcotine content above 9% and 20 lines exhibited papaverine content above 2%. Line BR 316 was found unique having thebaine content 6.27–9.33%. The probable reasons of increase or decrease of different alkaloids based on their biosynthetic pathway has been discussed in detail.     

Inter-relation between descriptors and morphine yield in Asian germplasm of opium poppy Papaver somniferum

A set of 208 Indian and two Thai germplasm accessions of opium poppy Papaver somniferum were assessed for variation in 17 morphological characters, seed yield and content and yield of morphine from capsules and peduncles. The germplasm was found to be highly variable for all the characters evaluated. In the harvested peduncles and capsules, 13% was peduncle straw, 61% seeds and the rest capsule husk. The peduncle and capsule straw yields ranged between 0.6–2.2 and 1.4–5.3 g plant^-1, respectively. Morphine content in the peduncle varied between about 0.001–0.24% and that in the capsule from 0.02 to 1.05%. On average basis morphine content in the capsule husk was more than 9-fold higher than the peduncle straw. The plant morphine yields from peduncles and capsules ranged between 1.2 and 28.6 mg plant^-1. Four accessions yielded more than 20 mg of morphine plant^-1. Among these, in one of the accessions about 13% of the morphine was contributed by the peduncle. The plants of high morphine yielding accessions were generally small in height, and bore white flowers and large sized ungrooved capsules with a small number of seeds, on a large peduncle.     

High pressure liquid chromatographic determination of the five major alkaloids in Papaver somniferum L. and thebaine in Papaver bracteatum Lindl. capsular tissue.

A high pressure liquid chromatographic isocratic procedure is described for determining and quantitating the 5 major alkaloids narcotine, papaverine, thebaine, codeine, and morphine in Papaver somniferum L. and thebaine in Papaver bracteatum Lindl. Other papaveraceous alkaloids, including salutaridine, oripavine, laudanosine, isothebaine, cryptopine, alpinigenine, narceine, protopine, and gnoscopine, were also quantitated. The values for morphine, codeine, and thebaine in P. somniferum were in agreement within 5--9% with values obtained by the United Nations Narcotics Laboratory by other methods. In contrast to previously reported procedures, the advantage of this method is that no precolumn or other purification other than solvent extraction of the capsular tissue is necessary. Isocratic chromatography alone on a single column resolved the 5 major alkaloids.     

Application of reverse phase ion-pair partition chromatography to drugs of forensic interest.

A method is described for determining a wide range of abused drugs by using 1 column, a single isocratic system, and a fixed wavelength (254 nm) ultraviolet detector. Paired-ion chromatography is performed on a reverse phase muBondapak C18 column. Acidic, basic, and neutral drugs, including their corresponding salts, can be determined without prior cleanup. A counter ion, 1-heptane sulfonate, is dissolved in the aqueous organic mobile phase to give a final pH of approximately 3.5. This technique is applicable to ergot alkaloids, phenylethylamines, opium alkaloids, local anesthetics, barbiturates, and other drugs of forensic interest. Five major opium alkaloids in gum opium, namely, morphine, codeine, thebaine, narcotine, and papaverine, can be separated in approximately 20 min.     

Pattern of diversity for morphological and alkaloid yield related traits among the periwinkle Catharanthus roseus accessions collected from in and around Indian subcontinent

Thirty two accessions of periwinkle Catharanthus roseus collected from different semitemperate to tropical geographical areas of Indian subcontinent, Madagascar, Singapore and Malaysia were characterized under field conditions for 53 growth, development, morphogenesis and alkaloid yield related characters over a few seasons at Lucknow, India. Large differences were observed among the accessions for each of the characters examined. The differences among the accessions ranged 3, 80 and 15 fold for the alkaloid yield related traits – leaf dry matter yield and leaf vincristine and vinblastine concentration, respectively. Strong correlations were observed between leaf area and leaf yield with leaf alkaloid, root and root alkaloid yields, contents of dimeric alkaloid in leaves with yields of respective alkaloids, leaf number and root alkaloid content with leaf vinblastine yield and leaf vinblastine content with leaf vincristine and vinblastine yields. Multi-variate analyses allowed classification of the accessions into 5 to 7 morphologically and presumably genetically distinct groups. Generally, the accessions coming from tropical agro-climates tended to get separated from those having origins in subtropical to semi-temperate environments. Three clusters were observed to bear complementary characters for possibly cross-parenting high alkaloid yielding transgressive segregants. One of the accessions of C. roseus in which the leaf vincristine content was 5 fold and vinblastine content one and a half fold of the respective all accession means appeared suitable for the domesticated cultivation. It was proposed that accumulation of high amounts of total alkaloids and one or more dimeric alkaloids in leaves of certain accessions may be a reflection of reproductive fitness achieved under the biotic and abiotic stresses imposed on their parent populations, sometime in their evolutionary pathway.     

Simultaneous assay for six alkaloids in opium, using high-performance liquid chromatography.

A method is described for the quantitative determination of morphine, codeine, cryptopine, thebaine, papaverine, and narcotine in opium by high-performance liquid chromatography. The alkaloids are isolated from a dilute acid extract by adsorption on an Amberlite XAD-2 resin column and eluted first with methanol and then with chloroform-methanol (3+1). After solvent removal by reduced pressure evaporation, the alkaloids are redissolved in chloroform-methanol (3+1). The sample solution, plus brucine as an internal standard, is injected onto a Corasil II column and eluted with hexane that is gradient programmed with a solution of chloroform-methanol-diethylamine (100+300+1). The absorbances of the separated alkaloids are continuously monitored at 254 nm, using a flow-through ultraviolet double-beam photometer.     

Variation in the seed and oil yields and oil quality in the Indian germplasm of opium poppy Papaver somniferum

The variation in the seed shape, colour and yield, and content, yield and fatty acid composition of seed oil of 109 accessions of opium poppy Papaver somniferum, (majority of them Indian land races), was investigated. The seeds were white, pale yellow or light brown in colour, reniform or round in shape and varied in size up to three fold. The oil content, seed and the oil yield varied between 26 to 52%, 1.0 to 7.4 g/plant and 0.4 to 2.7 g/plant, respectively. The % content of palmitic, oleic and linoleic acid in the seed oil ranged between 9.3 to 40.0%, 7.5 to 58.4% and 0.7 to 72.7%, respectively. On average basis, the levels of major fatty acids in the seed oil were: oleic (37.1%) > palmitic (27.3%) > linoleic acid (17.2%). The palmitoleic, stearic and linolenic acids were present in the oils of only some of the accessions. Two of the accessions yielded linoleic acid rich seed oil of about the same quality as soybean and maize oils, and in four accessions, the proportion of palmitic, oleic and linoleic acids was roughly equal. The palmitic acid was relatively less and linoleic acid more in the seed oil from accessions rich in oil content. The oil that contained higher amount of oleic acid also contained higher amount of palmitic acid and relatively lower amount of linoleic acid. The correlation analyses revealed a strong positive relationship between seed yield and oil yield (r = +0.81), oil yield and oil content (r = +0.54) and oleic acid and palmitic acid content in the seed oil (r = +0.49), and a weak positive relationship between oil content and linoleic acid content of oil (r = +0.24), and a negative correlation was observed between oil content and palmitic acid content (r = –0.32), palmitic acid and linoleic acid (r = –0.55) and oleic acid and linoleic acid contents of oil (r = –0.68). The observations have permitted selection of accessions that are high seed and oil yielding and/or rich in linoleic, palmitic and oleic acids or containing palmitic, oleic and linoleic acids in about equal amounts.     

Optimized LC/MS/MS analysis of morphine and codeine in poppy seed and evaluation of their fate during food processing as a basis for risk analysis

The opiate alkaloids present in poppy seed intended for use in food recently have raised major concerns. An efficient method for routine analysis of morphine and codeine using liquid chromatography in combination with tandem mass spectrometry on a triple quadrupole instrument (LC/MS/MS) was therefore developed. The optimal sample preparation was found to be cold extraction of 10 g of unground poppy seed with 30 mL of methanol containing 0.1% acetic acid for 60 min shaken at 250 rpm. The fate of morphine during food processing was also studied. All experiments led to a significant reduction of morphine and codeine. For poppy cake only 16-50% of the morphine was recovered, and in poppy buns at the highest temperature (220 degrees C) only 3% of the original morphine content was found. Ground poppy seed showed significantly lower recoveries than untreated seed. Morphine elimination during food processing has to be taken into account in the current discussion about its maximum limits in poppy seed.     

Genetic diversity analysis in Boerhavia diffusa L. of different geographic locations in India using RAPD markers

Boerhavia diffusa is extensively used in herbal medicines as well as in the Ayurvedic system, because it contains a set of clinically important compounds. In the present study, the genetic variability in Boerhavia diffusa between accessions of different geographical origin within the Indian territory is assessed through random amplified polymorphic DNA (RAPD) markers. Twenty-eight accessions of Boerhavia were screened with eighteen primers of which nine were found to be the most informative. The degree of polymorphism was found to be high in accessions collected from different places of Uttar Pradesh (Set II) in comparison to other states of India (Set I). A relatively lower level of polymorphism was recorded in accessions collected from diverse locations around Lucknow (Set III). Accessions from neighbouring geographical regions exhibited more similarity than those from distant regions (as revealed by the set I analysis). Certain diagnostic markers may be correlated with morphological character(s) such as plant type. BDL appeared most distinct and divergent from the rest of the accessions and the BDJ plant in set II also showed least similarity estimate. Fragments of 5.62 Kb and 4.47 Kb with primer GN59 was found to be unique for BDP and BD2 having ovate leaf character, whereas ovoid leaf genotype exhibited 0.79 Kb (GN34 primer) fragment. Similarly a unique band type (0.35 Kb) with primer GN83 was present in BDL and BD1 that share light pink flower. Jaccard's, and Nei and Li similarity coefficient values amongst the accessions were in the range of 0.22 to 0.89 and 0.33 to 0.93, respectively. Association of RAPD markers with the leaf characteristics, flower colour as well as with geographical locations has been made. This shows that RAPD markers are also useful for the study of genetic structure of Boerhavia populations.     

Diversity among landraces of Indian snapmelon (<Emphasis Type="Italic">Cucumis melo</Emphasis> var. <Emphasis Type="Italic">momordica</Emphasis>)

Diversity among 36 snapmelon landraces, collected from 2 agro-ecological regions of India (9 agro-climatic sub-regions), was assayed using RAPD primers, morphological traits of plant habit and fruit, 2 yield-associated traits, pest and disease resistance and biochemical composition (TSS, ascorbic acid, titrable acidity). Typical differences among accessions were observed in plant and fruit characteristics and snapmelon germplasm with high titrable acidity and possessing resistance to downy mildew, Cucumber mosaic virus, Zucchini yellow mosaic virus, Papaya ringspot virus, Aphis gossypii and Meloidogyne incognita was noticed in the collection. RAPD based grouping analysis revealed that Indian snapmelon was rich in genetic variation and region and sub-region approach should be followed across India for acquisition of additional melon landraces. Accessions of var. agrestis and var. momordica clustered together and there was a separate cluster of the accessions of var. reticulatus. Comparative analysis of the genetic variability among Indian snapmelons and an array of previously characterized reference accessions of melon from Spain, Israel, Korea, Japan, Maldives, Iraq, Pakistan and India using SSRs showed that Indian snapmelon germplasm contained a high degree of unique genetic variability which was needed to be preserved to broaden the genetic base of melon germplasm available with the scientific community. N. P. S. Dhillon and Ranjana contributed equally to this work and are considered the first authors.     

Application of random amplified polymorphic DNA to study genetic diversity in Paspalum scrobiculatum L. (Kodo millet, Poaceae)

Summary Genetic diversity and patterns of geographic variation among collections of Paspalum scrobiculatum (kodo millet) and P. polystachyum were studied using molecular markers generated through the random amplified polymorphic DNA (RAPD) method. A high level of polymorphism in RAPD markers was observed among the individual accessions, demonstrating the high genetic diversity of the crop. The markers obtained from the RAPD method were analyzed with the cluster analysis, principal coordinates and minimum spanning tree methods. Three major groups were resolved, one representing the African accessions, and two for the Indian accessions. The accessions of the north African kodo millet and P. polystachyum (considered conspecific with P. scrobiculatum) were quite distinct. The Australian kodo millet showed higher affinity to the African types. The study demonstrated that the RAPD technique can be applied to resolving degrees and patterns of genetic variation at the population and species levels, identifying cultivars, and defining gene pools of this crop.     

Analysis of genetic diversity in Indian taro [Colocasia esculenta (L.) Schott] using random amplified polymorphic DNA (RAPD) markers

Taro [Colocasia esculenta (L.) Schott] germplasm accessions collected from different parts of India were subjected to RAPD (Random Amplified Polymorphic DNA) analysis to assess the genetic diversity prevalent and also to test the genetic basis of morphotypic classification. Thirteen random decamer primers out of the 22 tested were used to analyse 32 taro accessions belonging to 28 morphotypes. Three out of these thirteen primers analysed showed 100 per cent polymorphism. Per cent polymorphism varied from 60 to 100 among the polymorphic primers. High genetic diversity was revealed as the similarity coefficient values ranged from 0.50 to 0.98. No two accessions analysed in the present study showed a similarity coefficient value of one thereby indicating their distinctness and presence of high genetic diversity in Indian taro germplasm. Dendrogram obtained from UPGMA analysis grouped 32 accessions in four clusters and three accessions were placed as outliers. Clustering pattern did not show any strict relationship with geographical distribution, morphotype classification and genotypic diversity. Further, accessions classified, as belonging to the same morphotypic group did not always cluster together. Presence of a very close genepool of the wild, weedy and cultivated forms with extreme levels of phenotypic and genotypic variation is suggested as the reason for high genetic diversity reported. Usefulness of DNA markers such as RAPD in characterising and assessing the genetic diversity in Indian taro germplasm is hereby demonstrated.     

Evaluation of emmer wheat and other Triticeae for resistance to Russian wheat aphid

Summary During the winters of 1990/91 and 1991/92, 181 accessions of Triticum dicoccon Schrank from the CIMMYT gene-bank were screened in the field for resistance to Russian wheat aphid, Diuraphis noxia (Kurdjumov). Accessions were sown in hill plots of 10 seeds and artificially infested with D. noxia at the two-leaf growth stage. Hills were visually assessed for damage at tillering, booting and heading. Entries differed significantly in their reaction to D. noxia, and severity of symptoms increased with time. Twenty four of the entries were highly resistant to the aphid. In winter 1991/92, 807 accessions of wild and cultivated wheats (26 species) and synthetic hexaploids were screened similarly for resistance to D. noxia. A large number of A-genome species were resistant, while few D-genome species were identified as resistant. These newly discovered sources of resistance can be used to expand the genetic base of resistance to D. noxia in both bread (T. aestivum L.) and durum wheats (T. turgidum L. convar. durum (Desf.) Mackey).     

Realizing value of genetic resources of <Emphasis Type="Italic">Allium</Emphasis> in India

The genetic resources of Allium in India are potential source of genes for widening the crop genetic base. Despite their high economic value, limited number of germplasm accessions of wild species have been collected and conserved mainly due to difficult access to areas of occurrence. The present work mainly highlights information on species diversity distribution and utilization of wild Allium species to assess the value of Indian germplasm and prioritization for future collection and conservation programmes.     

Low levels of genetic diversity detected by RAPD analysis in geographically distinct accessions of Bacopa monnieri

Brahmi (Bacopa monnieri) is atraditional Indian medicinal plant known for its natural nootropicaction of saponins present in large amount in its shoots. Acollection of 24 B.monnieri accessions from differentagro-climatic zones of India and an introduction from Malaysiamaintained in the field genebank at CIMAP was analysed for RAPDvariation. Among the 40 random primers tested, 29 primers generatedone or more polymorphic bands. The number of polymorphic bandsgenerated was primer dependent, ranging from 2 to maximum of 8.Similarity matrices were generated from the RAPD data on the basis ofNei's estimates of similarity indices and dendrograms wereconstructed based on UPGMA clustering. All the accessions were foundto be in the range of 0.8–1.0 of similarity, which isindicative of a narrow genetic base among the various accessions witha medium level of polymorphism. It was possible to differentiateindividual accessions, showing differences in morphological andgrowth properties at DNA level. The observed low levels of geneticvariation were attributed to interplay of sexual and vegetative modesof reproduction and similarity of local environments in habitats ofB. monnieri.     

Genetic diversity and structure of Ethiopian,Yemen and Brazilian C<Emphasis Type="Italic">offea arabica</Emphasis> L. accessions using microsatellites markers

Genetic diversity among 115 coffee accessions from the Coffea Germplasm Collection of IAC was assessed using SSR markers. The germplasm represents 73 accessions of Coffea arabica derived from spontaneous and subspontaneous plants in Ethiopia and Eritrea, species center of origin and diversity, 13 commercial cultivars of C. arabica developed by the Breeding Program of IAC, 1 accession of C. arabica cv. ‘Geisha’, 13 accessions of C. arabica from Yemen, 5 accessions of C. eugenioides, 4 accessions of C. racemosa and 6 accessions of C. canephora. Genetic analysis was performed using average number of alleles per locus (A), proportion of polymorphic loci (P), Shannon’s genetic index (H′ and G′_ST) and clustering analysis. All evaluated species were distinguished by a cluster analysis based on Jaccard’s coefficient. Differentiation between the cultivated plants of C. arabica and accessions derived from spontaneous and subspontaneous plants was observed. Spontaneous and subspontaneous accessions from Ethiopia were separated according to the geographical origin: east and west of the Great Rift Valley. Cultivated plants showed a low genetic diversity with a division in two groups: accessions from Yemen (H′=0,028) and Brazilian commercial cultivars (H′=0,030). The results agreed with previously reported narrow genetic basis of cultivated plants of C. arabica and supported the hypotheses about domestication of the species. This study also showed a significant genetic diversity among accessions from Ethiopia and Eritrea present in the Germplasm Collection of IAC. This diversity is specially observed in accessions from Sidamo (H′=0,143), Kaffa (H′=0,142) and Illubabor (H′=0,147) indicating their importance as source of genetic variability for coffee breeding programs.     

Genetic Diversity of the Vigna Germplasm from Thailand and Neighboring Regions Revealed by AFLP Analysis

Thailand is a center of diversity for section Angulares of the Asian Vigna (genus Vigna subgenus Ceratotropis) and 4 Vigna species are cultivated in Thailand. Using newly collected wild and cultivated germplasm of Vigna from Thailand and outgroup accessions AFLP analysis was conducted to clarify genetic diversity and relationships. The results suggest that cultivated V. umbellata and V. mungo evolved from wild relatives in a single domestication event. Vigna umbellata is poorly differentiated from its wild and weedy relatives compared to V. mungo. Results suggest northern Thailand and the neighboring Shan state, Myanmar, is the probable center of domestication for V. umbellata as wild accessions from this area and cultivated rice bean from a wide area in Asia are not greatly diverged. Vigna minima, V. tenuicaulis and V. exilis accessions in Thailand are well differentiated with considerable intra-specific variation. Vigna hirtella consists of two well differentiated subgroups, suggesting taxonomic revision may be necessary. Close genetic relationships between V. radiata and V. grandiflora, and between V. mungo and V. trinervia are confirmed. Naturally growing V. mungo populations in northern Thailand appear to be true wild species as they are well differentiated from Indian wild and Thai cultivated populations. The origin of naturally growing cowpea in Thailand needs to be further studied using a more comprehensive set of materials. This study clarifies inter and intra-specific genetic diversity and inter species relationships of Thai Vigna species.     

Evaluating landraces of bread wheat <Emphasis Type="Italic">Triticum aestivum</Emphasis> L. for tolerance to aluminium under low pH conditions

Bread wheat (Triticum aestivum L.) landraces held within ex situ collections offer a valuable and largely unexplored genetic resource for wheat improvement programs. To maximise full utilisation of such collections the evaluation of landrace accessions for traits of interest is required. In this study, 250 accessions from 21 countries were screened sequentially for tolerance to aluminium (Al) using haematoxylin staining of root tips and by root regrowth measurement. The staining test indicated tolerance in 35 accessions, with an intermediate response to Al exhibited in a further 21 accessions. Of the 35 accessions classified as tolerant, 33 also exhibited increased root length following exposure to Al. The tolerant genotypes originated from Bulgaria, Croatia, India, Italy, Nepal, Spain, Tunisia, and Turkey. AFLP analysis of the 35 tolerant accessions indicated that these represent diverse genetic backgrounds. These accessions form a valuable set of germplasm for the study of Al tolerance and may be of benefit to breeding programs for expanding the diversity of the gene pool from which tolerant cultivars are developed.     

Genetic identity and relationships of Iranian apple (Malus?×?domestica Borkh.) cultivars and landraces, wild Malus species and representative old apple cultivars based on simple sequence repeat (SSR) marker analysis

In order to shed light on the role of Iran in apple evolution and domestication, we chose to investigate the relationships of a collection of 159 accessions of wild and domesticated apples including Iranian indigenous apple cultivars and landraces, selected wild species, and old apple scion and rootstock cultivars from different parts of the world. The majority of the wild species belonged to M. sieversii, which is widely believed to be the main maternal wild ancestor of domestic apples, from Kazakhstan and M. orientalis, which is one of the probable minor ancestors of domestic apples, from Turkey and Russia located on the east and west of Iran, respectively. The accessions were assigned into six arbitrary populations for the purpose of generating information on genetic parameters. Nine simple sequence repeat (SSR) loci selected from previous studies in apple were screened over DNA extracted from all the accessions. Results showed that all SSR loci displayed a very high degree of polymorphism with 11–25 alleles per locus. In total, there were 153 alleles across all loci with an average of 17 alleles per locus. The SSR allelic data were then used for estimation of population genetic parameters, including genetic variation statistics, F-statistics, gene flow, genetic identity, genetic distance and then cluster analysis using POPGENE 1.32 software. The F-statistics and gene flow in particular, showed that there was more intra-population than between population variation. The genetic identity and genetic distance estimates, and the dendrogram generated from the un-weighted pair group arithmetic average (UPGMA) method of cluster analysis showed that the Iranian cultivars and landraces were more closely related to M. sieversii from Central Asia (east of Iran) and M. orientalis native to Turkey and Russia than to other accessions of Malus species. Also, the old apple cultivars from different parts of the world have a closer genetic relationship to M. sieversii, M. orientalis and the Iranian apples, than to other wild species. Based on these results, we suggest that the Iranian apples may occupy an intermediate position between the domesticated varieties and wild species. We propose that Iran could be one of the major players in apples’ domestication and transfer from Central Asia to the western countries.