Impact of Promising Genotypes of Papaver somniferum L. Developed for Beneficial Uses

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1 Impact of Promising Genotypes of Papaver somniferum L. Developed for Beneficial Uses M. Singh a, N. Chaturvedi b, A.K. Shasany and A.K. Shukla c CSIR-Central Institute of Medicinal and Aromatic Plants P.O. CIMAP Lucknow , Uttar Pradesh India Keywords: alkaloids, latex-less, narcotic, opium poppy Abstract Papaver somniferum (opium poppy) is a treasure medicinal plant known to produce therapeutically useful alkaloids like morphine, codeine, thebaine, papaverine, etc. Its seeds have high nutritional value (oil is rich in unsaturated fatty acids). Morphine, which is used as an indispensable analgesic, is in high demand. However, the narcotic nature of morphine and its easy illicit conversion into heroin has curtailed the free agriculture of P. somniferum and restricted the benefits that may accrue to the poppy farmers. Since the poppy alkaloids cannot be chemically synthesized in an economically viable manner, the plant remains the sole source of the biologically active alkaloids. Most of the licit morphine is chemically converted to codeine, which has a high pharmaceutical demand. In recent years, the trend is shifting towards thebaine and oripavine (which are precursors in the in planta morphine biosynthetic pathway) derived drugs as it is becoming increasingly difficult to control the narcotic usage of morphine. This has led to a global demand for poppy cultivars that are either (i) low in morphine but produce substantial amounts of thebaine, oripavine and codeine or (ii) are latex-less so that upon lancing the capsule do not exude latex (that contains morphine). Opium poppy crop is plagued by many devastating diseases (e.g. downy mildew). Therefore, there is a need to develop/identify disease-resistant genotypes. Significant efforts have been made globally to achieve these objectives and several speciality genotypes/cultivars of opium poppy are presently available. This paper discusses various aspects of the opium poppy crop including the present status and bottlenecks. It also highlights the future course of action to be taken for research on opium poppy. INTRODUCTION Opium poppy (Papaver somniferum) belonging to the family Papaveraceae, is one of the oldest medicinal plants known to mankind. It is well known for its economically important therapeutic alkaloids papaverine, thebaine, codeine and morphine. Papaverine has use as a vasodilator, thebaine is the starting source for medically useful derivatives (including the analgesics oxycodone and buprenorphine), codeine is an antitussive and morphine is one of the most potent analgesics known. Chemical synthesis of most of the poppy alkaloids is possible, but not commercially viable and the alternative of bioreactor production is not possible in all cases, which makes the plant more important as the source of the therapeutic alkaloids. Morphine is responsible for the addictively narcotic effect of opium. Besides, it can be easily converted into heroin (diacetylmorphine), which is more narcotic than opium. However, despite being a drug of abuse and source of other narcotics, morphine has an evergreen demand as an analgesic that warrants its licit production (Clark et al., 2010). Recent global trends indicate that it is likely that there will also be a major demand for thebaine-derived drugs. a Equal contribution as first author b Equal contribution as first author c ashupov@yahoo.com Proc. IS on Papaver Ed.: Á. Máthé Acta Hort. 1036, ISHS

2 India is the only country where Gum Harvest (GH) cultivars of P. somniferum are cultivated for producing licit opium (dried latex) for medicinal usage (Sharma et al., 2002a). In other poppy growing countries, opium collection is not prevalent and Concentrate of Poppy Straw (CPS) cultivars are grown for direct extraction of alkaloids (primarily morphine) from the capsule hulls (straw) after removal of seeds. The CPS cultivars also possess opium but they are not lanced for opium collection. Apart from the licit cultivation of GH and CPS cultivars of P. somniferum, illicit cultivation of GH cultivars is also prevalent in some regions of the world. The ideal situation would be to have poppy genotypes that are either deficient in latex morphine (designer plants blocked to produce only morphine precursors) or have latex-less cultivars that could not be lanced. P. somniferum also suffers from several bacterial, fungal, and viral diseases, among which downy mildew (DM) disease caused by the fungus Peronospora arborescens (Berk) de Bary is the most destructive and widespread. It attacks the plants at seedling stage with both systemic as well as topical symptoms. Majority of the systemically infected plants die prematurely, whereas the topically infected ones survive with reduced growth, seed, and latex yield as compared to healthy plants. Although, DM can be controlled by means of fungicides, such treatments are undesirable due to their high cost and, more significantly, their failure to achieve sustainable agricultural productivity. Hence identification of stable resistance sources and development of DMresistant genotypes appears to be the best eco-compatible solution to control DM. GENOTYPIC IMPROVEMENT IN POPPY Since varietal development is an essential prerequisite for achieving success in the commercial cultivation of any medicinal plant species, over the years, many cultivars and genotypes of P. somniferum have also been developed for different beneficial purposes. Some of these are mentioned in Table 1. A major research effort has been made at CSIR- CIMAP, Lucknow, in this direction and many cultivars of P. somniferum have been developed. Various breeding approaches like mass selection ( Shweta, Shyama, Sampada, Shubhra ), half-sib selection ( Sanchita ), induced mutation breeding ( Vivek, Sujata ) and selection in intra-specific hybrids for diverse resistance ( Rakshit ) have been used for this (Patra and Kumar, 2005). Very recently, morphine-rich CPS cultivar CIMAP Ajay has been released that was developed through recombination breeding approach (Lal et al., 2011). Apart from these cultivars, the National Gene Bank for Medicinal and Aromatic Plants at CSIR-CIMAP, Lucknow, has a rich collection of opium poppy lines and genotypes, some of which have been characterized for their morphinan alkaloid content and genetic diversity. Although it was possible to detect DNA polymorphism (through AFLP) in P. somniferum genotypes differing in straw morphinan alkaloid content, the genetic base of the opium poppy germplasm in the gene bank was found to be narrow (Dubey et al., 2010c). Earlier also, the AFLP-derived DNA polymorphisms in poppy have been found to be frequent enough to be useful for the characterization of genetic diversity in even closely related breeding lines (Saunders et al., 2001). A small number of distinct sources of resistance to DM have been identified (Singh et al., 2003; Dubey, 2008; Dubey et al., 2009a). The genotype Pps-1 is a spontaneous true breeding homeotic gene mutant having distinct partially petaloid sepals and is highly resistant to DM (Dhawan et al., 2007). Comparative analysis of the DMresistant ( Pps-1, dark green leaves with deep fringed margins) and -susceptible ( H-9, an inbred line having a yellowish green variant chlorophyll color in its broad margin leaves and developed from the same progenitor, SPS-49, as Pps-1 ) poppy genotypes has revealed genetic variation in the chloroplast-encoded RNA polymerase β subunit (rpoci) gene and amplification refractory mutation system (ARMS) has been employed to validate the detection of the nucleotide variations present in the rpoc1 gene of Pps-1 in comparison to H-9 (Dubey et al., 2009b). Resistance to downy mildew in the plant has also been studied using DNA (nuclear and chloroplast) (Dubey et al., 2010a) and 30

3 biochemical (Dubey et al., 2010b) markers. The AFLP-derived DNA markers could be used in future genetic studies for analysis of linkage to the DM-resistance trait. Since global focus has shifted to development of morphinan alkaloid-rich CPS cultivars, many such cultivars have already been developed, like Cosmos (Hungary), Lazur and Modry (Poland), Sanchita (India), etc. (Sharma et al., 1999b). Low alkaloid-containing CPS cultivars like Soma (Sweden), Przemko (Poland) and Riesenmohn (Germany) are also significant for breeding purpose. In India also, Bajpai et al. (1996) identified an accession with only 0.05% straw-morphine. Prajapati et al. (2002) evaluated various Indian land races of opium poppy for straw alkaloid content and found considerable variability for the straw alkaloid yields, which indicated that during evolution, opium poppy must have undergone selection for local adaptability and alkaloid accumulation traits. Over a period of time, CSIR-NBRI, Lucknow and Jawaharlal Nehru Krishi Vishwa Vidyalaya (JNKVV), Mandsaur have also developed a series of cultivars of P. somniferum. Poppy cultivars like Opal and Lazur have been found to perform better against herbicides as compared to cultivars, Mieszko and Michalko (Wojtowicz and Wojtowicz, 2009). Unlike P. bracteatum, P. somniferum has thebaine as a minor alkaloid but selection for high thebaine/low morphine content (cpv. Morph:The) has been carried out in it (Nyman, 1978). Quantitative 1 H NMR-based metabolite profiling has been able to distinguish a low-alkaloid poppy cultivar from a high thebaine, lowmorphine cultivar as well as pharmaceutical grade poppy from condiment poppy (Hagel et al., 2008). P. somniferum (2n=22) has cross compatibility with the wild species P. setigerum (2n=44). It has been observed that the interspecific triploid (F 1 ) hybrid (2n=33) (generated from the cross P. somniferum P. setigerum) had a considerable degree of pollen fertility and in the later generations (F 2 and onwards) it reverted back to stable diploidy through elimination of the unpaired (non-homologous) chromosomes but continued to express the dominant traits of the tetraploid parent, such as capsule dehiscence (undesirable) and novel red petal color (Patra et al., 2006). P. somniferum usually exhibits self pollination but sometimes shows often-cross pollination behaviour depending on the climatic factors etc. and estimates for the gene frequency-based outcrossing parameter (α) have been found to vary with year, cross and marker locus used (α range: %) (Kumar and Patra, 2010a). In opium poppy, biparental mating followed by recurrent selection involving desired recombinants may be utilized for improving the component traits (Kumar and Patra, 2010b). Studies on inheritance of quantitative traits in opium poppy indicated that heterotic breeding approaches should be followed for exploiting the over-dominance effects, while recurrent selection is utilized for overall population improvement (Kumar et al., 2010). Study of genetic architecture of opium poppy has also been carried out in relevance to yield improvement (Kumar, 2007). A new and distinct cultivar of Papaver ( Heartbeat ) has been developed as an interspecific hybrid, originating from a cross pollination of Papaver atlanticum, Papaver californicum, Papaver rupifragum, Papaver somniferum, and Papaver orientale (DeWelt, 2009). Upon crossing poppy cultivars of high chemical diversity, the manifestation of chemical metaxenia has been observed, whereby a change in the alkaloid pattern of the female plants was effected by foreign pollination (Bernáth et al., 2003). It has been suggested that poppy genotypes that are lacking or accumulate very little amount of alkaloids, have negligible or suppressed tyrosine decarboxylase (TYDC) activity. Codeine is used for manufacturing active pharmaceutical ingredients (APIs) such as Codeine Phosphate, Codeine Sulphate, Codeine Hydrochloride and Codeine Base that are in turn used to manufacture high-volume, over-the-counter (OTC), dosage forms for use as analgesics and antitussives. Although codeine, a minor alkaloid, can be extracted from opium and poppy straw, its demand is much higher than the natural supply due to which, 85-90% of the manufactured codeine is derived from morphine through its O- methylation. If it could be sourced from morphine-less poppies, growth of morphine poppies (which have a potential for diversion and abuse) could be drastically cut down. In 31

4 this direction, speciality genotypes of opium poppy have been developed that preferentially accumulate codeine ( 40% by weight) with little or no oripavine, morphine or thebaine (Fist et al., 2010). In a similar fashion, high thebaine ( 95% by weight) opium poppy genotypes have also been developed (Fist, 2009). Besides, there are cultivars of opium poppy that are high in thebaine and oripavine ( 50% by weight) (Fist et al., 2000, 2002, 2004). DUAL USAGE POPPY GENOTYPES Poppy genotypes are sometimes broadly classified as industrial (grown for alkaloid extraction; capsule), culinary (grown for edible seed and oil; seed) or dual purpose (grown for both alkaloid as well as seed; capsule and seed). There is difference among the operational procedures adopted by poppy growing areas across the world. In the areas like Western Europe and Australia that practice highly developed industrial production of poppy mainly for alkaloids, patented genotypes are used without cultivar registration by varietal authority. Due to industrial interest, details of such genotypes are not commonly available as they are derived from homogeneous material developed by various breeding procedures and selections are based on particular industrially relevant production characters. On the contrary, in regions like Central Europe, cultivars are registered by appropriate cultivar offices although promising ones may be patented too. The registered poppy cultivars in the European list maintained by the Community Plant Variety Office (CPVO) are differentiated based on their usage industrial ( A1, Alfa, Botond, Evelin, Buddha, Csiki kek, Kek Gemona, Medea, Minoan, Monaco, Nigra, and Tebona from Hungary; Extaz from Romania; Lazur from Poland; and Riesenmohn from Germany), culinary ( Aristo, Florian, Josef, Zeno, Zeno 2002, and Zeta from Austria; Albakomp, Ametiszt, and Kozmosz from Hungary; Albin from Slovakia; Agat, Michalko, Mieszko, and Przemko from Poland) or dual ( Bergam, Gerlach, Major, Malsar, Marathon, and Opal from Slovakia; Edel- Weiss from Austria; Kek Duna from Hungary; Marianne and Rosemarie from Netherland; Sokol from Czech Republic; Parmo from Denmark; and Rubin from Poland) (Bernáth and Németh, 2010). Figure 1 provides a Venn diagram representation of the same. The low morphine cultivar, Marianne, displayed a shifted pattern of alkaloid accumulation and was found to be rich in narcotoline and noscapine (Frick et al., 2005). In Slovakia, opium poppy is cultivated at the breeding station in Maly Saris, from where several cultivars have been released. A third dimension of utility of the opium poppy plant is added by its ornamental usage in some countries. THE PRESENT STATUS, BOTTLENECKS AND OPPORTUNITIES The present day need is to produce morphine in a controlled environment (to prevent illicit usage), like a designer plant (producing only precursors that can be converted to morphine semi-synthetically or having morphine biosynthesis displaced from the capsule latex to another plant part from where it is not-so-easy to extract) or a heterologous system (Shukla et al., 2008). In both cases, detailed knowledge of alkaloid biosynthesis (formation, trafficking, storage, regulation) is needed. A fast method for obtaining such information in non-model species is deep transcriptome sequencing as shown recently for Artemisia annua that produces the antimalarial phytomolecule artemisinin (Wang et al., 2009; Graham et al., 2010). For the Papaver species also an effort has been made in this direction, at the Donald Danforth Plant Science Center, St. Louis, MO, USA (Prof. Toni Kutchan s research group) under the One Thousand Plant Transcriptome Project (funded by government of Alberta) (One KP Project, 2011) as well at some other institutes. The Papaver species taken up for deep transcriptome sequencing include P. somniferum, P. setigerum, P. rhoeas and P. bracteatum. The complete potential of functional genomics for gene discovery/prospection in opium poppy has not been utilized till date due to limitations imposed by the relative inefficiency of genetic transformation protocols (Desgagne-Penix et al., 2009). This has also curtailed the use of insertional mutagenesis in poppy (like generation of T-DNA 32

5 and/or transposon-tagged lines). However, induced mutagenesis with chemicals like ethyl methane sulfonate (EMS) and ionizing radiations (like fast neutron bombardment) can be applied to poppy and form the basis for application of both forward (phenotype to genotype) and reverse (genotype to phenotype) genetic approaches. In case, forward genetics approaches are to be attempted, the limitation is that due to non-availability of genetic and physical maps for P. somniferum chromosome-walking methodologies cannot be used and the only alternative available is that of transcriptome/est analysis. The Top1 (thebaine oripavine poppy or NORMAN ) mutant is an outcome of such research, whereby it was suggested that the mutation was caused by lack of a functional enzyme causing oxidative demethylation of thebaine and oripavine but microarray analysis of the mutant and its parent could not provide any candidate gene to substantiate the hypothesis (Millgate et al., 2004). On the other hand, if reverse genetics approaches are to be attempted on a mutant poppy population then the screening method would depend on the type of mutagen used. With ionizing radiations causing genomic deletions, delete-a-gene method can be applied, whereas in case of alkylating mutagens like EMS that cause point mutations, TILLING (Targeting Induced Local Lesions IN Genomes) can be used to isolate mutant alleles of target genes (like for example, alkaloid biosynthesis-related genes) that have the potential to be used in metabolic engineering of poppy. Once a suitable target gene has been identified, it has to be functionally characterised through enzyme activity assay or silencing the gene through approaches like RNA interference (RNAi) and virus induced gene silencing (VIGS). Both the approaches for gene silencing, RNAi (Allen et al., 2004, 2008; Kempe et al., 2009) and VIGS (Hileman et al., 2005), have been used successfully in P. somniferum. THE PATH AHEAD GAPS TO BE FILLED Deep Transcriptome Sequencing Since P. somniferum is a model non-model species, where the genome is yet to be sequenced completely, the best possibility lies in a deep transcriptome sequencing effort targeted to prospect for the novel genes that have not been characterized till date. (S)-Reticuline the Gateway This central biosynthetic intermediate gives rise to several different alkaloidal structures (like for example protoberberine and morphinan) in P. somniferum as well as related species (Facchini and De Luca, 2008). It must be investigated that how a single and simple structure gives rise to so many complex alkaloids. The Papaverine and Narcotine Pathways The pathways for papaverine and narcotine (or noscapine) biosynthesis are poorly understood and are yet to be completely elucidated. These branches of the alkaloid biosynthesis in poppy have not been targeted as vigorously as the morphinan branch. Papaverine pathway elucidation will be facilitated by availability of papaverine-rich mutants like Pap-1 available with NBRI, Lucknow. Metabolic Blocks Bioprospecting for metabolic blocks (for specific alkaloids) in the available germplasm must be carried out. Plant breeding and selection should be continued for specific alkaloids (like thebaine, oripavine, codeine, etc.) accumulation. Novel Regulatory Genes Unlike the case of Catharanthus roseus, another model non-model alkaloidproducing plant, where transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis has been studied in some detail and the function of octadecanoid-responsive Catharanthus AP2-domain proteins (ORCAs) elucidated, in P. somniferum, the 33

6 regulatory aspects of alkaloid biosynthesis remains largely unexplored and very little effort has been made in this direction. Biotic/Abiotic Factors The effect of various biotic as well as abiotic factors on alkaloid biosynthesis and accumulation in the poppy plant needs to be elucidated. The biotic factors could be endophytes, pathogens (like fungi, virus, etc.), microbes in the rhizosphere, etc. whereas the abiotic factors could be seasonal effects, temperature variations, etc. Besides, the spatial, temporal and developmental age related variations in alkaloid content in the plant must also be defined comprehensively (although some studies have already been done in this direction) for optimal alkaloid biosynthesis and harvest. Heterologous Systems Initial attempts to produce benzylisoquinoline alkaloids in microbial systems have already been made (Hawkins and Smolke, 2008; Minami et al., 2008). These attempts must be further strengthened to produce some of the significant poppy alkaloids in heterologous systems in a cost effective manner and also to produce non-natural alkaloids. This might also help to provide some labelled intermediates for pathway elucidation of the yet uncharacterized alkaloid biosynthetic branches. Ultimately, there may be no need to grow the plant to produce the alkaloids but even then the plant would retain its importance for its nutritious seeds. Biotransformation and Production of Novel Alkaloids The P. somniferum cells could be used for biotransformation of various phytomolecules for value addition. Apart from this, the alkaloid pathway in poppy could be reprogrammed to yield alkaloids with novel structures and activities. Such an approach is being adopted on the TIA model in C. roseus by the research group of Dr. Sarah E. O Connor (earlier at the Massachusetts Institute of Technology, USA and now at the John Innes Centre, UK). Isolation of Standards of Alkaloid Biosynthetic Pathway Intermediates A chemistry effort is required to produce the standards as well as analytical methods to analyze the alkaloid intermediates. A crucial consideration for better understanding of the alkaloid biosynthetic pathway both at the regulatory as well as structural levels in future research schemes has to be the analysis of a wide range of intermediates and secondary metabolites rather than the final products alone, as it will provide information about the distribution of metabolic flux around key branchpoints like (S)-reticuline. Although major interest lies in the post reticuline pathway leading towards morphine via thebaine, measuring the level of the post-reticuline metabolic flux into all the branching pathways is required if alkaloid biosynthesis has to be studied in totality. Sequestration of Pathway Intermediates Since the alkaloid biosynthetic pathway in poppy passes through different cell types, studying the sequestration of intermediates and their ultimate storage will be an interesting aspect for studying the pathway in totality. Simultaneously, an elaborate proteomics effort may help to identify novel proteins. CONCLUSIONS P. somniferum has a great future as a model non-model species for studying alkaloid biosynthesis using both conventional as well as non-conventional approaches. Till date several speciality genotypes of the plant species have been developed in different countries. These genotypes are either oriented towards pharmaceutical usage or culinary usage and in some cases towards both. Genotype designing of P. somniferum for future 34

7 should be directed towards curtailing the abuse of morphine but at the same time allowing its beneficial therapeutic use. Although it may sound highly ambitious now, a day may come in future when morphinan alkaloids may be exclusively produced in heterologous systems and growing the plant may not be required at all for pharmaceutical purpose. Even in such a scenario, the morphologically-marked high seed and low/nil alkaloid genotypes of P. somniferum will keep its utility intact as a food crop. ACKNOWLEDGEMENTS The authors are grateful to Director, CSIR-CIMAP, Lucknow for his encouragement. They are also thankful to Drs. Raj K. Lal, Om P. Dhawan, Birendra Kumar and Anil K. Gupta, for their valuable suggestions during preparation of this article. Literature Cited Allen, R.S., Miller, J.A., Chitty, J.A., Fist, A.J., Gerlach, W.L. and Larkin, P.J Metabolic engineering of morphinan alkaloids by over-expression and RNAi suppression of salutaridinol 7-O-acetyltransferase in opium poppy. Plant Biotechnol. J. 6: Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A., Fist, A.J., Gerlach, W.L. and Larkin, P.J RNAi-mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22: Bajpai, S., Gupta, M.M. and Kumar, S A low morphine-containing accession of Papaver somniferum suitable for seed production. Plant Breed. 115: Bernáth, J. and Németh, É Poppy. p In: J. Vollmann and I. Rajcan (eds.), Oil Crops, Handbook of Plant Breeding, Vol. 4. Springer Science + Business Media, LLC, New York. DOI: / _15. Bernáth, J., Németh, É. and Petheõ, F Alkaloid accumulation in capsules of the selfed and cross-pollinated poppy. Plant Breed. 122: CIMAP Release of crop varieties: Shweta and Shyama of opium poppy. CIMAP Annual Report , CIMAP, Lucknow p CIMAP Development of improved agrotechnologies for Papaver somniferum L. CIMAP Annual Report , CIMAP, Lucknow p CIMAP Development of dual purpose mutants and interspecific hybrids in opium poppy. CIMAP Annual Report , CIMAP, Lucknow p CIMAP Development of dual purpose variety in opium poppy. CIMAP Annual Report , CIMAP, Lucknow, p Clark, P.A., Sillup, G.P. and Capo, J.A Afghanistan, poppies, and the global pain crisis. Med. Sci. Monit. 16:RA Desgagne-Penix, I., Hagel, J.M. and Facchini, P.J Mutagenesis as a functional genomics platform for pharmaceutical alkaloid biosynthetic gene discovery in opium poppy. p In: Q.Y. Shu (ed.), Induced Plant Mutations in the Genomics Era. Food and Agriculture Organization of the United Nations, Rome. DeWelt, J.P Papaver super poppy hybrid plant Heartbeat. US Patent PP20,102. Dhawan, O.P., Dubey, M.K. and Khanuja, S.P.S Detection of a true breeding homeotic gene mutant Pps-1 with partially petaloid sepals in opium poppy (Papaver somniferum L.) and its genetic behaviour. J. Hered. 98: Dhawan, O.P., Shahabuddin, S., Trivedi, M., Sattar, A., Alam, M., Samad, A., Zaim, M., Dwivedi, S., Singh, S.P., Singh, H.P., Khanuja, S.P.S., Darokar, M.P., Shasany, A.K., Gupta, M.M., Luthra, R., Sharma, J.R., Lal, R.K., Misra, H.O., Kalra, A. and Kumar, S Method of producing a poppy plant. US Patent 6,534,696. Dhawan, O.P., Shahabuddin, S., Trivedi, M., Sattar, A., Alam, M., Zaim, M., Dwivedi, S., Singh, S.P., Singh, H.P., Khanuja, S.P.S., Darokar, M.P., Shasany, A.K., Gupta, M.M., Luthra, R., Sharma, J.R., Lal, R.K., Misra, H.O., Kalra, A. and Kumar, S Registration of a new variety Rakshit of opium poppy Papaver somniferum. J. Med. Arom. Plant Sci. 23:

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9 Kempe, K., Higashi, Y., Frick, S., Sabarna, K. and Kutchan, T.M RNAi suppression of the morphine biosynthetic gene salat and evidence of association of pathway enzymes. Phytochemistry 70: Kumar, B Study on genetic architecture of opium poppy (Papaver somniferum L.) in relevance to yield improvement. PhD Thesis, University of Lucknow, Lucknow, India. Kumar, B. and Patra, N.K. 2010a. Gene frequency-based estimation of natural outcrossing in opium poppy (Papaver somniferum L.). Mol. Breeding 26: Kumar, B. and Patra, N.K. 2010b. Genetic analysis of capsule and its associated economic traits in opium poppy (Papaver somniferum L.). J. Hered. 101: Kumar, B., Singh H.P. and Patra, N.K Inheritance of quantitative traits in opium poppy (Papaver somniferum L.). Comm. Biometry Crop Sci. 5: Lal, R.K., Chandra, R., Misra, H.O., Shanker, K., Singh, A.K., Dhawan, O.P., Gupta, A,K., Kumar, B., Gupta, M.M., Verma, R.K., Alam, M., Singh, S.K., Lal, C., Kalra, A., Singh, H.N., Chauhan, H.S., Singh, H.P., Singh, S., Krishna, A., Zaim M. and Shankar H High yielding variety CIMAP Ajay morphine rich in poppy concentrate straw of opium poppy (Papaver somniferum L.). CIMAP Variety Release Document (dated 7 February, 2011). Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach, W.L., Fist, A.J. and Larkin, P.J Analgesia: morphine-pathway block in top1 poppies. Nature 431: Minami, H., Kim, J.S., Ikezawa, N., Takemura, T., Katayama, T., Kumagai, H. and Sato, F Microbial production of plant benzylisoquinoline alkaloids. Proc. Natl. Acad. Sci. USA 105: NBPGR NBPGR-Perspective Plan Vision-2025, NBPGR, New Delhi, p. 17. Németh, E., Bernáth, J. and Jászberényi, C.S Studies on the inheritance of poppy (Papaver somniferum L.) alkaloids and the new cultivar Korona accumulating high concentrations of narcotine. Acta Hort. 860: NRCMAP NRCMAP-Perspective Plan Vision-2025, NRCMAP, Anand, p. 11. Nyman, U Selection for high thebaine/low morphine content (cpv. Morph:The) in Papaver somniferum L. Hereditas 89: One Thousand Plant Transcriptome Project Patra, N.K. and Kumar, B Improved varieties and genetic research in medicinal and aromatic plants (MAPs). Proc. Second National Interactive Meet on Medicinal and Aromatic Plants. CIMAP, Lucknow, India October, p Patra, N.K., Kandpal, A. And Kumar, B Cytogenetical assessment of Papaver somniferum-p. setigerum hybrids for estimated linkage and recombination. The Nucleus 49: Prajapati, S., Bajpai, S., Singh, D., Luthra, R., Gupta, M.M. and Kumar, S Alkaloid profiles of the Indian land races of the opium poppy Papaver somniferum L. Genet. Resour. Crop Evol. 49: Saunders, J.A., Pedroni, M.J., Penrose, L.D.J. and Fist, A.J AFLP analysis of opium poppy. Crop Sci. 41: Sharma, J.R., Lal, R.K., Gupta, A.P., Misra, H.O., Pant, V., Chandra, R. and Rashid, M Opiumless and alkaloid-free non-narcotic opium poppy (Papaver somniferum) variety Sujata. US Patent 6,730,838. Sharma, J.R., Lal, R.K., Gupta, A.P., Misra, H.O., Pant, V., Singh, N.K. and Pandey, V. 1999a. Development of non-narcotic (opium less and alkaloid-free) opium poppy, Papaver somniferum. Plant Breed. 118: Sharma, J.R., Lal, R.K., Gupta, M.M., Verma, R.K. and Misra, H.O. 2002a. A superior gum variety Sampada of opium poppy (Papaver somniferum). J. Med. Arom. Plant Sci. 24: Sharma, J.R., Lal, R.K., Gupta, M.M., Verma, R.K. and Misra, H.O. 2002b. A novel nonnarcotic seed variety Sujata of opium poppy (Papaver somniferum). J. Med. Arom. Plant Sci. 24:

10 Sharma, J.R., Lal, R.K., Misra, H.O., Naqvi, A.A. and Patra, D.D. 1999b. Combating opium-linked global abuses and supplementing the production of edible seed and seed oil: A novel nonnarcotic var. Sujata of opium poppy (Papaver somniferum L.). Curr. Sci. 77: Shukla, A.K., Birtharey, V., Chaturvedi, N., Lal, R.K., Shasany, A.K. and Khanuja, S.P.S Molecular tagging of specific chemotypes of Papaver somniferum using functional genomics approach towards development of non-narcotic poppy. Presented at the Bangalore Bio 2008 Walkway of Discovery, Bangalore, India, April. poster 48. Shukla, S., Singh, S.P. and Khanna, K.R Opium poppy BROP 1. Indian Horticulture 39:7-8. Shukla, S., Singh, S.P., Singh, H.B., Pushpangadan, P High yielding multiple disease resistant/tolerant stable variety Madakini of opium poppy. US Patent 7,442,854. Singh, H.B., Singh, A., Singh, P., Srivastava, S., Shukla, S. and Singh, S.P Evaluation of germplasm of opium poppy (Papaver somniferum) for resistance to downy mildew (Peronospora arborescens). Indian J. Agric. Sci. 73: The Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute Wang, W., Wang, Y., Zhang, Q., Qi, Y., Guo, D Global characterization of Artemisia annua glandular trichome transcriptome using 454 pyrosequencing. BMC Genomics 10:465. Wojtowicz, M. and Wojtowicz, A Effectiveness of chemical protection against weeds applied to poppy (Papaver somniferum L.). J. Plant Protection Res. 49: Tables Table 1. Some prominent Papaver somniferum cultivars/genotypes. S. no. Name of the genotype/cultivar Country Characteristics/remarks Reference 1 Talia India It is sown early and has a shiny (waxy) capsule Farooqi and Sreeramu, Ranghatak India Medium-tall cultivar, capsules slightly flattened on the top, Farooqi and Sreeramu, 2004 yields opium of thin consistency that turns dark brown upon exposure 3 Dhola Chota Gotia India Dwarf cultivar (85-90 cm) Farooqi and Sreeramu, Shyama India GH cultivar; CIMAP, Lucknow CIMAP Annual (1983) 5 Shweta India GH cultivar, slightly superior to Shyama in content of main alkaloids; CIMAP, Lucknow (1983) Report, CIMAP Annual Report,

11 Table 1. Continued. S. no. Name of the genotype/cultivar Country Characteristics/remarks Reference 6 Sampada India High gum yielding superior GH Sharma et al., 2002a cultivar, remains green for longer period during lancing; CIMAP, Lucknow 7 Sujata India Latex-less cultivar; CIMAP, Lucknow Sharma et al., 1999a, 2002b, Sanchita India High seed and poppy straw yielding cultivar; CIMAP, Lucknow CIMAP Annual Report, Shubhra India Early maturing, dwarf cultivar having superior straw and seed yield and capsule number; CIMAP, Lucknow 10 Vivek India Big capsule straw cultivar; CIMAP, Lucknow 11 Rakshit India Disease-resistant (highly resistant to DM and moderately resistant to both damping off and collar rot) and high seed and straw yielding (CPS) cultivar; CIMAP, Lucknow 12 CIMAP Ajay India High straw and seed yielding CPS cultivar with high percentage of alkaloids (morphine-rich with more than 1% content in straw concentrate); CIMAP, Lucknow 13 BROP-1 (Botanical Research Opium Poppy-1) India Synthetic cultivar developed by crossing selections from Kali Dandi, Suyapankhi and Safaid Dandi, highly adaptable to different agroclimatic conditions, moderately resistant to diseases; NBRI, Lucknow 14 Madakini India Multiple disease resistant and high opium yielding cultivar; NBRI, Lucknow 15 Pps-1 India Homeotic gene mutant, highly resistant to DM; CIMAP, Lucknow 16 Jawahar Aphim- 16 India Pureline selection of local land races, early maturing, oval capsules, moderately resistant to DM; JNKVV, Mandsaur (AICRP on MAP, 1984) CIMAP Annual Report, CIMAP Annual Report, Dhawan et al., 2001; Dhawan et al., 2003 Lal et al., 2011 Farooqi and Sreeramu, 2004; Shukla et al., 1995 Shukla S. et al., 2008 Dhawan et al., 2007 Farooqi and Sreeramu, 2004; JNKVV website,

12 Table 1. Continued. S. no. Name of the genotype/cultivar 17 Jawahar Opium Poppy Jawahar Opium Poppy-540 Country Characteristics/remarks Reference India India Early maturing, spherical capsules; JNKVV, Mandsaur (AICRP on MAP, 1997) Early maturing, cylindrical capsules; JNKVV, Mandsaur (AICRP on MAP, 1998) 19 Trishna (IC-42) India Large bumble-shaped capsules, resistant to frost and root rot and tolerant to DM; NBPGR, New Delhi 20 Kirtiman India Selection from local races, moderately resistant to DM; ND University of Agriculture and Technology, Faizabad (AICRP on MAP, 1990) 21 Chetak Aphim India Moderately resistant to diseases; Rajasthan Agricultural University, Udaipur (AICRP on MAP, 1994) 22 Kék Gemona Hungary High narcotine; SZI University, Budapest 23 Tebona Hungary Reasonably high amount of morphinan alkaloids (morphine, codeine, thebaine); SZI University, Budapest 24 Top1 or NORMAN Australi a Thebaine oripavine poppy; Tasmanian Alkaloids Pty. Ltd., Westbury Tasmania 25 Opal Slovakia High morphine; Breeding Station in Maly Saris 26 Mieszko and Poland Michalko Low morphine with phenotypic markers; pink smooth petals in Mieszko and white lacinated petals with violet eyes in Michalko JNKVV website, 2011 JNKVV website, 2011 Farooqi and Sreeramu, 2004; NBPGR-Perspective Plan: Vision-2025, 2007 Farooqi and Sreeramu, 2004; NRCMAP- Perspective Plan: Vision-2025, 2007 Farooqi and Sreeramu, 2004; NRCMAP- Perspective Plan: Vision-2025, 2007 Bernáth et al., 2003 Bernáth et al., 2003 Fist et al., 2000; Millgate et al., 2004 Wojtowicz and Wojtowicz, 2009 The Plant Breeding and Acclimatization Institute (IHAR) - National Research Institute website, Kheops France High morphine Bernáth et al., A1 Hungary High morphine Bernáth et al., Lazur Poland High morphine Wojtowicz and Wojtowicz, Korona Hungary High narcotine Nemeth et al., Przemko Poland Low morphine Bernáth et al.,

13 Figures Fig. 1. Venn diagram representation of usage-based classification of registered European Papaver somniferum cultivars (based on the data provided by Bernáth and Németh, 2010). 41

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