Journal of Medicinal Plants

Application of Conventional and New Biotechnological Approaches for Improving of Morphinane Alkaloids Production

1 2 3 Dehghan E (Ph.D. student) *, Hosseini B (Ph.D.) , Naghdi1 Badi H (Ph.D.) , Shahriari Ahmadi F (Ph.D.)

1- Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran 2- Department of Horticulture, Faculty of Agriculture, Urmia University, Urmia, Iran 3- Iranian Academic Center for Education, Culture & Research (ACECR), Institute of Medicinal Plants Research, Karaj, Iran *Corresponding author: Department of Biotechnology and Plant Breeding, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran Tel: +98-511- 8796818-20, Fax: +98-511- 8787430 Email: [email protected]

Receive: 4 Apr. 2010 Acceptance: 30 Aug. 2010

Abstract

More than 12,000 alkaloids are known in plants, mostly used as medicine with a world market value of about 4 billion US$. Opium poppy, Papaver somniferum, is the most important economic source of morphinane alkaloids such as morphine, codeine, thebaine, narcotine and papaverine that are exploited by the pharmaceutical industry as analgesics, antitussives and anti-spasmodics. With regard to increasing demand for these compounds, the aim of this review is presenting an outlook of classical breeding programs that successfully applied for enhancing the alkaloid content of opium poppy. The latest biotechnological approaches also are discussed to give an outlook for future trends and possibilities.

Keywords: Papaver species, Biotechnological methods, Conventional breeding, Morphinane Alkaloids, Tissue culture, Metabolic engineering Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021

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Introduction and derivatives are the most important Poppy seems to be one of the few species alkaloids produced by the opium poppy. which was utilized, even as cultivated plant, in Morphine has long occupied an eminent prehistoric times. There is no doubt about the position on the list of useful drugs. Morphine evidence that the poppy was also cultivated by and codeine are prescribed analgesics and Sumerians, Babylonians and Assyrians about cough-suppressing drugs [5]. Morphine exerts 3–6 thousand years BC [1]. The Opium poppy its effects either by hyperpolarizing or Papaver somniferum (Fig. 1) is a multipurpose inhibiting postsynaptic neurons, probably by + 2+ crop which is used as a medicinal or increasing K efflux, or by reducing Ca ornamental plant, as well as a source for seeds influx into presynaptic nerve endings and and seed oil. Opium, as dried latex of unripe thereby reducing transmitter release, including capsules of P. somniferum, contains more than acetylcholine, norepinephrine, dopamine, 80 isoquinoline alkaloids. The main alkaloids serotonin and substance P [6]. Morphine is derived in opium are morphine (4-21%), metabolized into morphine-3-glucuronide and followed by codeine, thebaine, papaverine, morphine 6-glucuronide (M6G) in the human noscapine, and narceine [2]. The Persian body; which the later one possesses analgesic genius Ebne Sina, known in the West as activity.

Avicenna (980-1037), who was revered as the Other alkaloids from poppy species have

"prince and chief of physicians" and "the various uses: noscapine has antitussive and second teacher after Aristotle," recommended antitumorogenic properties; papaverine is a opium and plants of the nightshade family as vasodilator and smooth muscle relaxant; analgesics and anaesthetics [3, 4]. The sanguinarine is antimicrobial and anti- morphinanes (5-ring benzylisoquinoline inflammatory [5, 7]. alkaloids) include morphine, codeine, thebaine

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Fig. 1- Opium poppy, P. somniferum and different parts of its vegetative and generative organs [8]

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The opiates are industrial commodities of protopines, pavines/isopavines, phthalideisoq- plant origin for which there is still uinolines, protoberberines, the true considerable demand. Globally, opium poppies benzylisoquinolines, the aporphines, the are legally cultivated on around 40,000 ha benzophenanthridines and the morphinanes annually. The major supplier of opium poppies [14]. Common to all the members of these is Tasmania, supplying around 40% of the classes are the first steps in the biosynthetic market. GlaxoSmithKline, Tasmanian pathway that lead to the central intermediate Alkaloids and Johnson-Matthey operate fully (S)-reticuline (Fig. 2). integrated supply chains for opiate production. The presence of morphinane alkaloids Levels of production are controlled by the UN (thebaine, codeine and morphine), together Single Convention on Narcotic Drugs, which with the secophthalidisoquinoline alkaloids limits production to reasonable saleable (narceine, nornarceine, narceinimide), quantities with allowance made for phthalidisoquinoline alkaloids (narcotine, contingency stocks [9]. narcotoline), benzyltetrahydroisoquinoline In the last few decades, the demand for alkaloids (reticuline, laudanosine, codamine, poppy-derived alkaloids has raised tetrahydropapaverine) and aromatic continuously, in particular the need for benzylisoquinoline alkaloids (papaverine, thebaine, which has increased by ~ 67% in the pacodine) is characteristic of Papaver last 6 years [10]. somniferum L. The carbon skeleton of benzy- With five centers of chirality, the structures lisoquinoline alkaloids is derived from two of morphinane alkaloids present a complexity molecules of tyrosine [15, 16]. The aromatic that renders commercial synthesis uneconomic amino acids, phenylalanine, tyrosine and [11] and cultivation of poppy, in spite of some tryptophan are formed via the shikimate pathway. limitation, continues to be the most effective The availability of tyrosine for alkaloid means to produce opiate analgesics [12]. biosynthetic pathways is an important Higher alkaloid content in the poppy crop determinant of the endogenous level of alkaloids. would enhance the financial return to growers The biosynthesis of benzylisoquinolines and make the industry more competitive [13]. (BIAs) starts with the condensation of two For these purposes, different efforts by tyrosine derivatives leading to the first conventional and new biotechnological tetrahydrobenzylisoquinoline norcoclaurine Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 methods have been applied to enhance alkaloid [17, 18]. Subsequent reactions include the content of P. somniferum. Thus in this methylation at position 6 of norcoclaurine by communication we will try to present an norcoclaurine 6-Omethyltransferase (6-OMT), overview of morphinanes biosynthesis in the methylation of the nitrogen by coclaurine opium poppy and different aspects of genetics, N-methyltransferase (CNMT), and the breeding, tissue culture and metabolic hydroxylation at the 3′-position by the P450 engineering of these valuable compounds. monooxygenase (S)-N-methylcoclaurine 3′-hydroxylase (Cyp80B1). One additional Benzylisoquinoline alkaloids and biosyn- methylation at the 4′position by 3′-hydroxy-N- thesis of morphinanes methylcoclaurine 4′-O-methyltransferase The benzylisoquinoline alkaloids comprise (4′-OMT) finally leads to (S)-reticuline. From a group of about 2,500 compounds, which can this central intermediate the pathway be divided into nine classes: the rhoeadines,

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Fig. 2- Benzylisoquinoline biosynthesis pathway. NCS (norcoclaurine synthase); 6-OMT (O-methyl transferase); NMT (N-methyltransferase); CPY80B3 (cytochrome P450); 4OMT (O-methyl transferase) BBE Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 (berberine bridge enzyme); STS (salutaridinol synthase); SAT (salutidarinol-7-O-acetyltransferase), COR (codeine reductase) [19]

bifurcates into the different benzylisoquinoline A series of at least 17 enzymatic steps classes and a multitude of rearrangements and contributed in the biosynthesis of morphine. modifications of the benzylisoquinoline The latter steps in the pathway that lead backbone takes place leading to the high specifically from (S)-reticuline, a central structural diversity of these alkaloids. intermediate of isoquinoline alkaloid Interestingly, whereas the biosynthesis of all biosynthesis, to morphine (Fig. 2) involve classes of benzylisoquinolines starts directly three NADPH-dependent oxidoreductases from (S)-reticuline, the conversion of [11], most probably three cytochromes P-450, stereochemistry to (R)-reticuline is a and an acetyl-CoA-dependent prerequisite for the production of morphinans. acetyltransferase [11, 19].

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Although more than 60 species or lower 100 species and is affiliated to the section taxonomic units of the genus Papaver have Mecones comprising five species, among been studied, the presence of morphine has which Papaver setigerum (2n=44) is a close been only detected in two species from the relative and probably the ancestor of the section of Mecones , i.e. P. somniferum L. and opium poppy [28]. Papaver somniferum is P. setigerum DC. The presence of morphine considered to be a predominantly self- has been also demonstrated in other plants, pollinating species with various rates of out- such as hay and lettuce and recently detected crossing depending upon variety and in mammalian tissues. Since the isolation of environmental factors; large colourful flowers morphine by Serturner in 1805, a large number with numerous stamens and large amounts of of isoquinoline alkaloids have been isolated pollen attract insects, especially bees; the from the opium poppy [21, 22]. Alkaloid transfer of pollen from one flower to another biosynthesis and accumulation is constitutive, might also be performed by wind [29]. The organ and cell type-specific processes in the divergent and long history of domestication plant. Morphine, noscapine and papaverine are and breeding of P. somniferum has resulted in generally the most abundant alkaloids in aerial the development of several different land organs, whereas sanguinarine typically races, chemotype varieties and cultivars accumulates in roots [23]. adapted to various uses and climatic Alkaloid biosynthetic enzymes and cognate conditions. Cultivation of the plant therefore transcripts have been specifically localized to covers a wide geographical area from Bombay sieve elements of the phloem and associated to Moscow in the Northern hemisphere and companion cells, respectively [24, 25]. In-situ Tasmania in the Southern hemisphere [30]. localization of alkaloid biosynthetic gene Alkaloid production is controlled by the plant transcripts indicated that seven biosynthetic genotype and by environmental factors. For enzymes (6OMT, CNMT, CYP80B, 4′OMT example cool-grown P. somniferum contains and BBE in reticuline biosynthesis, and SAT more morphine but has lower alkaloid content and COR in a morphine pathway) are localized than warm-grown. Thus secondary metabolite in sieve elements in opium poppy and accumulation generally is affected by water corresponding gene transcripts were localized availability, exposure to soil microorganisms in the supporting companion cells [24, 26]. and variations in soil pH and nutrients [31]. Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 However, another immunocytochemical Commercial cultivation of poppy have analysis clearly showed that 4′OMT and SAT been envisaged several limitation such as were localized in phloem parenchyma cells, availability of water for irrigation and need to and COR, the penultimate step in morphine fertile and free-draining soil, which is not biosynthesis, is localized in laticifers (a vessel overly acidic [13]. On the other hand several like series of long cells, which accompany diseases like powdery mildew, root rot and vascular tissues throughout the plant and leaf blight occurs in poppy. Poppy crops also contain a milky sap called latex.), which is the suffer heavily from mildew caused by site of morphinane alkaloid accumulation [27]. Peronospora arborescens and other parasitic agents. Unfortunately, at least there is no Genetics and Breeding report about occurrence of completely Papaver somniferum (2n=22) is a member resistance genetic stocks, in India [20]. Thus to of the genus Papaver, which includes some reduce the build up of diseases in

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commercially grown poppies, crop must be exhibiting heterosis for these characteristic. grown with at least a three year rotation, The implementation of these results into the before again culturing of them [12]. Regarding production of commercial high-performing of these limitations, breeding strategies, not hybrid cultivars is, however, hampered by the only must be enhancing morphinanes yield, lack of a genetic system promoting cross- but also considering the improvement of pollination. Genetic-cytoplasmic male sterility different agronomic traits. is the most appropriate and widely used The existence of substantial variations in system in several crops for the production of the available gene pool of a species is hybrid cultivars. Induced male sterile mutants necessary for any successful breeding have been obtained in plant populations of program. Several independent studies on the opium poppy, either by irradiation with evaluation of the genetic variation in the gamma rays [35], or by interspecific cultivated germplasm of P. somniferum hybridization [36], but these mutants were not reached the conclusion that only a limited characterized. In the absence of male sterility, variation prevails in Indian genetic stocks and self-incompatibility can be used for the hybrid European stocks for most agronomic and production. The use of hybrid cultivars in this chemical traits. This is related to the narrow crop is the most potent and rapid breeding genetic base of genotypes with common strategy for combining several desirable ancestry. The genetic and breeding aspects of characteristics from different parents and for opium poppy were investigated more exploiting the considerable amount of intensively in Europe in the early of 1960s and heterosis reported for morphine and seed during the past decade in India [20]. yields [34]. There are also some reports about heterosis Varietal, mass and pure line selections in P. bractatum. Thus heterotic effects can be have been applied by several breeders of used in a breeding program aimed at opium poppy for the development of improved increasing the thebaine yield. This species is cultivars [32, 33]. However, the most widely suited for the production of hybrids because of used method which has produced several the very large quantities of seeds that are commercial cultivars is the pedigree selection produced in each capsule due to its self by which, through hybridization between incompatibility nature. A significant increase Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 parents with different desirable characteristics, in the thebaine yield (more than three times) led to development of few lines. The pedigree than that of the best parent in the cross, was method has been used successfully for found in one F1 hybrid of P. bractatum [37]. increasing the yield of capsules, opium and As an interesting method, artificial seeds, the morphine content and the lodging polyploidy induction can be useful for resistance. This method, however, markedly changing morphological characteristics [38, reduces the genetic variability and contributes 39] and secondary metabolite production of to narrowing the genetic basis of the cultivated plants [40]. Polyploidization is often germplasm [34]. accompanied by increased cell size and Substantial amounts of heterosis have been conspicuous changes in secondary observed for morphine and seed yield, as well metabolism. Where vegetative plant organs are as for most of their components and a current the source of secondary metabolites, as is the goal is the development of hybrid species case with most medicinal plants, ploidy

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manipulations such as direct chromosome most widely used opiate in medicine, codeine, doubling or allopolyploidization provide a is mainly produced from the P. somniferum. rapid means to realize enhanced production of However, the plants major alkaloid, morphine, phytopharmaceuticals [40]. Triploid and and its highly addictive derivative heroin are tetraploid plants of P. somniferum, showed up also used illegally as drugs. Codeine can also to 100% increase in morphine concentration be produced from thebaine, the major alkaloid [40]. In P. bracteatum 3x and 4x plants had of P. bracteatum. The species P. bracteatum, shown higher thebaine content (respectively, Persian poppy, diploid 2n=14, is considered to 4.9% and 8.8% dry weight) than diploid (2.4% be a potential alternative to opium poppy for dry weight) counterparts, but polyploid plants codeine production due to its high content of were later in flowering and their seed setting thebaine and complete absence of morphine. was very poor, especially in triploids [41, 42]. Thebaine is a precursor of codeine and can be The polyploidy therefore seems of little use for easily converted to codeine by the breeding for high seed yield, but it might be pharmaceutical industry. In some populations considered for increasing morphinane content, of this species, the capsules and roots contain which is the primary value than the plant almost exclusively thebaine; thus the biomass component and will facilitate the extraction and purification of the raw material extraction process. is relatively easy. The addiction potential of Spontaneous and mutagen-induced thebaine or its derivatives and of the minor mutants have also been reported in alkaloids reported for P. bracteatum is P. somniferum. Such mutants can be used negligible and no cases of abuse or illicit directly as new cultivars: the ‘Soma’ variety production of thebaine have been reported was released from a spontaneous mutant in the [37]. Thus whenever the climatic conditions variety “Indra” [43]. In other cases, the are suitable for the growth of P. bracteatum it mutants have been used, frequently as parents may advantageously replace the traditional in breeding programs [44]. The occurrence of P. somniferum. biochemical mutants induced by mutagenic P. bracteatum is naturally distributed in treatments showed the good potential of this high altitudes from 1500 to 2500m. The approach in altering the alkaloid profile of species is found in three distinct areas: the plants. By the use of mutagenic agents such as Alborz Mountains north of Tehran, in the Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 gamma ray or chemical agents, some breeder Iranian Kurdistan and on the Northern slope of could produce opium poppy plants with the Caucasus [37]. A population called Arya II different characteristics of male sterility, with a thebaine content of 3.6% of dry mater opium less, high morphine yield, and high was found in Western Iran by Lalezari et al. number of capsules per plant [45]. A codeine [46]. chemotype in which the demethylation to However substantial progress, in the case morphine is blocked would be most valuable of poppy species, has been achieved, mainly both to the pharmaceutical industry and for the through genetic, during the last 30 years in prevention of the illegal use of morphine. France, where the yield of morphine has A world-wide effort was also invested increased from 4.5 kg/ha in 1961 to 10.5 kg/ha during the 1970s to domesticate and develop in 1991 [34]. But unfortunately no correlations P. bracteatum as an alternative source to the have observed between the alkaloid content opium poppy for codeine production. The and the yield of dry matter. Although

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conventional plant breeding has produced a Tissue cultures of different explants of the doubling in poppy alkaloid content over the poppy plant (i.e. seedling hypocotyls, seedling last two decades, however continued rapid roots, stalk and capsule) have been reported in improvement in morphinan yields through the literatures [48]. Callus tissues have been conventional breeding will likely be limited obtained and the presence of alkaloids has [13] and these methods did not give the been detected. However, other investigations desirable level of improvement for several have demonstrated the absence of alkaloids in reasons, including low genetic diversity, P. somniferum tissue cultures. These conflicts sterility, long generation time, perennial nature reports can be due to: the use of different and complex biosynthetic pathways involved analytical methods with varied sensibilities, [47]. Thus today we need to use of new the use of different P. somniferum cultivars biotechnological methods (in combination to and the analysis of somatic tissue classic breeding programs) to efficiently cultures at various stages of differentiation manipulate morphinanes production in poppy [49]. Industrial production of opiates from plant. tissue culture is dependent on the large accumulation of alkaloids in a cell culture Role of Biotechnology in morphinanes medium. While there has been great success in production plant-cell culture in terms of cells with high With regard to medicinal plants, yields of isoquinolines, from a commercial and biotechnology could be described as a method pharmaceutical viewpoint, the morphinans for enhancing the formation and accumulation have proved difficult to produce in plant-cell of desirable natural products, with possible cultures [50]. As an improving strategy product modification in medicinal plants. treatment with elicitor was not successful in all Micropropagation, cell and hairy root culture cases: codeine biosynthesis, for example, has as well as gene technology are all important not yet been achieved. However, compounds techniques for plant propagation, but these are sharing the same precursors and intermediates, mostly used to improve the production and such as the antimicrobial alkaloid yield of desired natural products. sanguinarine, may accumulate in quite high amounts. Using cell cultures of P. somniferum, Plant cell and tissue cultures the production of sanguinarine was shown to Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 Some compounds, such as shikonine and be elicited by preparations from fungal paclitaxel, can be produced with the mycelia [51, 52]. Likewise, Archambault et al. technology of large-scale plant cell culture. [53] were able to obtain a twofold stimulation However, other secondary metabolites, in the production of the antimicrobial particularly alkaloids, are produced at low compound, sanguinarine, through the use of a concentrations in plant cell cultures. The low chitosan elicitor in P. somniferum cell cultures. or lack of productivity of these desired However numerous studies had shown that compounds can be explained by an insufficient the production of morphinan alkaloids via in level of cell differentiation to allow a vitro cultures requires organogenesis of tissues production of secondary metabolites. In plants, in cultures. The induction of cell there is a clear correlation between cellular differentiation by the addition of exogenous differentiation and secondary metabolism. growth regulators in the culture medium

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improves alkaloid production. However, this Thebaine was present in only trace amounts in process is time-consuming and therefore it can cell cultures or absent. The self- be used only for the production of compounds incompatibility system in this species does not with a high value. The transformation of allow the creation of pure lines through medicinal plants using Agrobacterium selfing. Thus, using tissue culture as a method rhizogenes to form hairy root cultures has the for mass micropropagation in P. bracteatum potential benefits of fast growth and rates of might be useful for rapid multiplication of alkaloid production equal to or greater than superior individuals [37]. that found for the intact plant. Moreover, hairy root cultures can be scaled-up for bioreactor Production of transgenic poppy and production to allow for the large-scale metabolic engineering recovery of alkaloids or other compounds with In order to produce transgenic plants one pharmacological activities [49]. must be able to: (1) stably integrate foreign Hairy root cultures of many other DNA into its genome; and (2) regenerate medicinal plants obtained by transformation fertile plants from transformed tissues. Unlike with A. rhizogenes were examined as potential the transformation difficulty, it is now possible sources of high-value pharmaceuticals [54]. to do both of these in opium poppy. Long-term For the first time, P. somniferum hairy root callus and suspension cultures of opium poppy cultures have been established after have been maintained on certain media in transformation of hypocotyls with the several laboratories. Both roots and shoots hypervirulent A. rhizogenes strain, LBA 9402 have been regenerated from callus [58]; [55]. The total alkaloid content (morphine, however, a much simpler method for codeine and sanguinarine) was higher in hairy regenerating poppy suspensions through roots (0.46 ± 0.06 % D.W.) than in somatic embryogenesis has been developed untransformed roots (0.32 ± 0.05 % D.W.) and [59]. Hosseini [48] successfully regenerated some of the alkaloids were excreted into the transgenic poppy plants from meristemoid liquid culture medium. Rostampur et al. [56] calli of hypocotyls explants (Fig. 3). were shown that the content of different The availability of reliable transformation/ benzylisoquinoline alkaloids produced by regeneration systems for opium poppy and the Persian poppy (P. somniferum) hairy roots was cloning of alkaloid pathway genes mean that it Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 identical wild-type roots. Transformed root should be possible to apply metabolic cultures of P. somniferum and California engineering to alter the quantity and quality of poppy, Eschscholzia californica had higher alkaloids in this species. growth rate than wild roots and displayed A genetically modified opium poppy that benzilisoquinoline profile that were virtually produces the pharmaceutical precursor, identical to those of wild-type roots [57]. thebaine, instead of the narcotic alkaloids Tissue cultures of P. bracteatum have also morphine and codeine, has recently been been studied for thebaine production. described [60]. Since thebaine (in contrast to Unfortunately, little success has been achieved morphine) cannot be easily converted to heroin in producing the desirable alkaloid in (an acetyl derivative of morphine), the substantial amounts in cell or tissue culture. As genetically altered crop provides a good for P. somniferum, a different alkaloid profile solution to hamper the utilization of opium is obtained in culture, compared with plants. poppy as a source for the illicit drug market.

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a b c d

e f g h

Fig. 3- P. somniferum transgenic plants regeneration frome hypocotyl explants. Hypocotyl explant on the B5 medium (a), callus induction (b), meristemoid calli and somatic embryogenesis (c, d and e), germination and development of a somatic embryoid (f and g), root induction and regeneration of transgenic opium poppy (h) [48]

Recently, an industrial elite line of poppy concentration of (S)-scoulerine. Silencing of was genetically engineered to modify the BBE in California poppy (Eschscholzia alkaloid content of the plant by overexpression californica) resulted in increased levels of (S)- of the (S)- N -methylcoclaurine 3′ - reticuline [64]. hydroxylase [61]. The resulting transgenic Silencing of salutaridinol O-acetyltransfer- plants contained up to 450% more alkaloids in ase (SAT), an enzyme acting in the same their latex compared to untransformed plants. branch caused no accumulation of its substrate Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 Overexpression of codeinone reductase in salutaridinol but of salutaridine, the substrate poppy led to a 30% increase of morphinan of the previous enzyme [65, 66]. alkaloid content on a dry weight basis in Overexpression of SAT increased the yield of transgenic plants [62]. In a different morphine indicating a rate limiting function of experiment, the berberine bridge enzyme this enzyme [65]. The data support the (bbe), which is the first enzyme of the existence in the morphine branch of one or sanguinarine biosynthesis branch, was silenced more metabolons, i.e. multi-enzyme in poppy [63]. This resulted in an increase in complexes that allow the internal hand-over of the concentration of several pathway pathway intermediates and are disrupted by the intermediates from all biosynthetic branches of removal of one enzyme. benzylisoquinoline alkaloids in the latex of Codeine reductase is the penultimate step transgenic plants. Surprisingly, the transgenic in morphine biosynthesis. Opium poppy was plants also exhibited an increase in transformed with a chimeric cDNA hairpin

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RNA construct designed to silence codeinone transgenic source of these substances will be reductase. However, silencing of codeinone accepted by consumers. reductase resulted in the accumulation of (S)- reticuline, but not the substrate codeinone or Molecular markers other compounds on the pathway from (S)- Molecular markers, such as restriction reticuline to codeine [67]. Allen and fragment length polymorphism (RFLP) and coworkers postulated that this accumulation random-amplified polymorphic DNA (RAPD) could be due to several factors. First, appear to be good candidates for the accumulation of codeinone and morphinone identification of plant species. This could result in negative feedback on one of the methodology has become an important tool for enzymes, such as the reductase responsible for the confirmation of somatic hybrids and, more the reduction of (S)-reticuline to 1, 2- recently, reported that RAPD markers can be dehydroreticuline. Also, the biosynthetic utilized for genetic analysis of intermediates and final product may regulate micropropagated plantlets regenerated from the transcription of pathway enzymes, through somatic embryoids. This methodology has also analysis of the transcript levels of a number of been used to determine the geographic the morphine biosynthetic enzymes showed no variation of plants [69]. change in suppressed plants. Finally, It is difficult to determine P. bracteatum codeinone reductase could be a part of a compared with P. orientale L. and P. pseudo- multienzyme complex, which can't be orientale Medw, in Oxitona section, based on functioned when one of the enzymes is the morphological observation. Shoyama et al. removed. This study highlights that the [69] demonstrated that RAPD analysis can be complex metabolic networks found in plants used for a simple and rapid judgment of two are not easily or predictably redirected. The parental Papaver species in Oxitona section regulation of benzylisoquinoline alkaloid (P. bracteatum and P. pseudo-orientale) and metabolism is complex and our understanding the diagnosis of their F1 hybrids. On the other of opium poppy biochemistry at the molecular hand, this method can also be used for the level can be advanced with genetic judgment of illegally cultivated Papaver transformation and metabolic engineering species. Amplified restriction fragment length biotechnology [68]. polymorphic (AFLP) analysis, also have been Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 Results of over expression of two genes used to evaluate the genetic diversity of (sat and cor) under CaMV35s promoter breeding populations to provide information showed that transgenic plants had different on those lines with desired genetic metabolites profiles and increased content of heterogeneity [68]. some metabolites. Most transgenic lines showed significant increases in capsule Biotransformation/expression in other alkaloid content compared with non-transgenic genetically modified microorganism controls the morphine alkaloid contents on a Although many plant cell suspension dry weight basis were 14% greater than those cultures fail to produce the compounds seen in in control genotypes [48]. the plants from which they have been Since pure extracts of poppy-derived established, these cells may be used in alkaloids that are devoid of genetic material biotransformation processes where exogenous has only industrial useable, it is expected that a organic compounds are modified by living

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cells. Biotransformation studies in cell identified nor cloned. Thus, the biosynthesis of suspension cultures have been carried out with morphinan alkaloids in a microbial a view to: (i) producing new chemicals; (ii) heterologous host is not yet fully feasible. An producing known chemicals more alternative approach is microbial economically; (iii) investigating the metabolic biotransformations of morphine into valuable fate of xenobiotics; and (iv) elucidating derivatives [75]. For example, a reusable metabolic pathways [70, 71]. efficient recombinant morphine/codeine Lenz and Zenk [20] described the biotransformation system was created using biosynthetic transformation of codeine and Pseudomonas enzymes [76]. morphine from the biological precursors: codeinone and morphinone. The Systems Biology Approaches biotransformation of codeinone to codeine Recent advances in plant genomics proved to be possible in the immobilized cells. research has generated knowledge leading to a The biotransformation of thebaine via better understanding of the complex genetics codeinone and codeine to morphine also is and biochemistry involved in biosynthesis of investigated with other strains of these plant secondary metabolites. This P. somniferum cell cultures. Furuya et al. [72] genomics research also concerned described the biotransformation of codeinone identification and isolation of genes involved to codeine by the cell suspension culture and in different steps of a number of metabolic the cell-free system. This reduction required pathways. Progress has also been made in the NADH as a co-factor in the enzyme system. In development of functional genomics resources the immobilized cell system, however, this (EST databases and micro-arrays) in several reaction proceeded without NADH. Using a medicinal plant species, which offer new column bioreactor packed with the opportunities for improvement of genotypes immobilized living cells, they were also using perfect markers or genetic investigated the effects of various conditions, transformation. such as temperature and aeration, on the ESTs are generated by massive and conversion of codeinone to codeine and the random sequencing of cDNAs generated from cell viability. the mRNA of the tissue of interest. ESTs are The biosynthetic pathway to morphine in typically short (normally only partially Downloaded from jmp.ir at 10:47 +0330 on Monday September 27th 2021 the oriental poppy P. somniferum is currently represent the full-length clones) and are of being elucidated, and characterization will relatively low sequencing quality. ESTs offer a benefit the production of morphine and its quick method for cloning and examining a semi-synthetic derivatives [73, 74]. The 17- large number of genes known to be expressed step biosynthesis of morphine in this plant has in a particular cell population or tissue. Opium been almost completely elucidated, with the poppy (P. somniferum), may be the prime eventual goal being the biomimetic synthesis example for a non-model plant on the verge of of thebaine, codeine, and morphine. These becoming a model. An extensive array of cDNAs were all functionally expressed in genomics resources, including expressed insect cell culture (S. frugiperda Sf9 cells) or sequence tags (ESTs) and DNA microarrays, in E. coli. However, there are still a number of for opium poppy plants and cell cultures has biosynthetic steps in the morphinan pathway been developed, together with proteomics, for which the enzymes have neither been allowing to the development of system biology

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approaches [77]. Decker et al. [78] and (S)-norcoclaurine synthase (NCS), which Ounaroon et al. [79] used proteomic analyses catalyzes the first committed step in of P. somniferum latex (2D gel electrophoresis benzylisoquinoline alkaloid metabolism. The (microsequencing) to create proteomic maps. use of whole genome sequences and species- Beside enzymes from the primary metabolism, specific EST collections has allowed rapid a codeinone reductase was identified based on discovery of new genes involved in plant homology to known isoforms [78]. In addition, secondary metabolism. Additionally, genomic Papaver somniferum sequences coding for tools have provided the means necessary to reticuline 7- O -methyltransferase and understand intricate signaling and regulatory norcoclaurine 6- O -methyltransferase were pathways, complex phylogenic relationships, isolated based on peptide sequences and the and overall genetic architecture. respective methyl transfer enzymes of alkaloid Metabolomics is the youngest of the so- biosynthesis were characterized [79]. called “omics” methods, and ultimately Comparative macroarray analysis of opium concerns the analysis of all metabolites in an poppy and various morphine free Papaver organism. Zulak et al. [81] using Quantitative species was used to identify a P. somniferum HNMR metabolomics have drawn a high- O-methyltransferase clone [80]. In this case, resolution map of the reprogramming primary P. somniferum seedlings were used to develop and secondary metabolism in elicitor-treated a cDNA sequence library. Of the 849 opium poppy cell cultures. They have revealed sequenced elements, three were shown on a that the response of cell cultures to elicitor macroarray, differentially expressed in treatment involves the extensive P. somniferum compared to non-morphine- reprogramming of primary and secondary producing species. Whereas two of these metabolism, and associated cofactor cDNAs showed no significant homology to biosynthetic pathways. any known protein, one was found to encode a In the future, potent genomics tools will be protein identified as S-adenosyl-l-methionine combined with metabolic profiling to identify (R, S)-3′-hydroxy-N-methylcoclaurine 4′-OMT key genes that serve for engineering secondary (4′OMT). Recently, an EST sequence database product pathways. was used to obtain the P. somniferum clone of

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Journal of Medicinal Plants, Volume 9, 50 No. 35, Summer 2010