Strategies and Recent Achievements in Selection of Medicinal and Aromatic

J. Bernáth SZI University, Department of Medicinal and Aromatic Plants, Budapest, Hungary, P.O. Box 53, H-1502

Keywords: active agents, chemical taxa, breeding, crossing, polyploids, mutation, genetic engineering

Abstract In harmony with requirements of safety, efficacy, and stability of medicinal and aromatic drugs and their products, the need for high quality raw is increasing. This phenomenon is proven by number of breeding results and registration procedures, concerning medicinal and aromatic plant cultivars, recently reported from different countries. The worldwide progress achieved in breeding is obvious, in spite of the fact, that different strategies and methods are used country by country. In the present work breeding strategies used for changing the accumulation level and composition of active agents are overviewed. The utilization of the chemical diversity of the plant kingdom is one of main direction of investigations. Search for inter-, or intraspecific distribution of well- defined active compounds is one of the most important goal of analysis, resulting in large numbers of cultivars of practical importance. The majority of plant materials under cultivation have been created by conventional selection methods, even today, including individual- mass-, or special selection methods. More recently outstanding results achieved by hybridization, polyploidization and mutation are reported. The application of transformed cell cultures and genetic-engineering based on the over expressing or suppressing of genes responsible for biosynthesis of a special active agents may offer new possibilities and might became a novel tool in the breeding of medicinal and aromatic plants in the future.

INTRODUCTION In harmony with recent regulations (WHO, ESCOP) the quality of medicinal and aromatic plants and their products have to fulfill requirements of safety, efficacy and stability (Franz, 2000). To meet these demands, either the plant comes from wild or cultivation, more detailed knowledge on chemical and production-biological properties of species are required. However, the reliable standard production of raw, supposed to be achieved by using GAP system of cultivation, requires cultivars of high productivity. Large numbers of breeding and registration of medicinal and aromatic plant cultivars are being reported from different countries (Sarma et al., 1996, Bernáth, 2000a, Pank, 1998, Paun et al. 1996, Zheljazkov et al., 1996). The goal of the breeding varies in the great deal. In some cases the improvement of the morphological and production biological characters of plants has to be achieved. Modification of the accumulation level of the active agents can be chosen for the goal of the selection either the increase or decreases of the biologically active compounds are necessitated. Especially from point of view the cultivation practice the life form of the species has to be modified. As a result of selection, annual forms of Salvia sclarea, Carum carvi, Verbascum officinalis were registrated. In some cases, the success of cultivation depends on resistance of species. In this respect cultivars of high pathogen resistance (Mentha piperita, Hypericum perforatum) and cold tolerance (Rosmarinus officinalis) were selected and registrated. In the present work the overview is restricted to the breeding strategies used for changing the accumulation level and compositional characters of active agents.

Proc. Int. Conf. on MAP Eds. J. Bernáth et al. 115 Acta Hort. 576, ISHS 2002 EVALUATION OF STRATEGIES AND RESULTS CONCERNING GENETICAL IMPROVEMENT OF MEDICINAL AND AROMATIC PLANTS

Exploitation of Natural Biodiversity The chemical diversity of the plant kingdom is a well-known phenomenon (Swain, 1976, Tétényi, 1970) and has been utilized for improving medicinal and aromatic plant production, since centuries (Van Harten, 1984). As it was justified by the data, both interspecific and intraspecific chemical diversity may occur. The accumulated diversity of on both family (genus) and intraspecific level is an excellent example of this phenomenon (Bernáth, 1996a). As it is obvious from the data of Table 1, 61 taxa of six family based on their chemical similarity can be ranged into the same practical (interspecific) chemical group and described as origanum, commercially (Lawrence and Reynolds, 1984). Contrary, inside the genus Origanum there are species (intraspecific chemical taxa) having no "" character at all (Fig. 1). Motivated by the results of the former chemotaxonomical findings exploitation of the chemical diversity of natural flora had been intensified and widened, recently. The main directions of investigations are as follows: a) Search for inter-, or intraspecific distribution of well-defined active compounds is one of the most important goal of analysis, even today. As an example, the well known chamazulene and bisabolol-types of Matricaria recutita were found, checking 29 local populations in Bulgaria (Stanev et al., 1996), two new chemotypes of the species accumulating jaceidin and chrysosplanitin were identified in Slovakia (Repcak et al., 1999), while in contrast no special chemotypes of practical importance were found in Greece (Wogiatzi et al., 2000). In Argentina, the presence of , thymol- carvacrol, and pulegone types of Minthostachys verticillata were detected in different regions (Zygaldo et al., 1996). High chemical biodiversity of many other species, including Peumus boldus (Vogel et al., 1999), Cinnamomum verum ( Joy et al., 1998), Borago officinalis ( De Haro et al., 2000), and Achillea crithmifolia (Németh et al., 1993) were found and utilized. b) The identification and selection of plants (chemotypes) with absence of compounds of adverse effects have became an important subject, too. In this respect works of Rode et al. (1996) for detecting asaron content of Acorus calamus and finding the distribution of pyrrolizidin alkaloids in Petasites hybridus are good examples (Chizzola, 2000). c) Correlation analysis between the chromosome number and production ability of the natural populations is a new line of investigation. In this respect works concerning Marrubium vulgare (Letchamo and Mukhopadhyay, 1997), Acorus calamus (Rode et al., 1996) and 31 other species by Murin (1997) are worth to mention. d) Number of natural populations and their active agents were checked for special biological activity. Origanum species collected from Euboea and Crete were characterized according to their antimicrobial and cytotoxic activities (Sivropoulou et al., 1996). Ocimum species from India were evaluated analyzing their efficacy against Aspergillus niger, Rhizopus stolonifer, Escherichia coli, Bacillus subtilis and B. megaterium. The essential oil of O. basilicum var. purpurescens was the most potent according to the results (Thoppil et al., 1998). In the case of Lippia alba and Cymbopogon species large chemotype dependencies in analgesic, antiinflammatory and antibacterial activities of essential oils were found (Viana et al., 1998, Pattnaik et al., 1996). e) Introduction of current biochemical and analytical methods for identification and description of chemical biodiversity of medicinal and aromatic plants are in progress. In Finland, 20 local Tanacetum vulgare genotypes (Keskitalo et al., 1998), while in Japan five species and subspecies of Atracytylodes genus were characterised by DNA content (Kohjyouma et al., 1997). In both cases, the effectiveness of the methods in chemotype distinction was proven. By the help of DNA analysis the distinction of various samples of Melissa officinalis subspecies was effectual, too (Wolf et al.,

116 1999). Similarly, on the examples of Foeniculum vulgare, Angelica archangelica, Levisticum officinale, Anethum graveolens, Coriandrum sativum the isoelectric focusing can considered to be an effective method for identification of medicinal and aromatic plant taxa (Stefanovits-Bányai et al., 1999). As a new direction of chemo the analysis and characterization of enantiomers were introduced. This technique makes possible the distinguishing of closely related types of medicinal and aromatic plants, including populations of Rosmarinus officinalis, Salvia officinalis, and Salvia fruticosa (Ravid et al., 2000).

Selection The majority of plant materials under cultivation have been created using conventional selection method. Even in the countries involved in the large scale cultivation of medicinal and aromatic plants, like Hungary, 60-75 per cent of cultivars are developed by simple selection methods from local, or introduced populations. The high efficacy of selection is based on the variability, which exists either registrated cultivars or wild populations are used as a starting material. As it was demonstrated by the example of Achillea crithmifolia (Németh et al., 2000), the large chemical diversity occurring under natural habitats can be explained by inhomogenity of genes responsible for accumulation of active agents (Table 2.). The segregation of self-pollinated chemotypes in I1 reveals that each of the investigated mother plant processed a heterozygotic structure for genes regulating the level of the main compounds. 1. Individual Selection. Based on the above mentioned genetic diversity - has been applied in many cases with high efficacy. Cinchona and Eucalyptus cultures were established by this method (Bernáth, 1996b). Even today in Australia, the natural populations serve as a starting material for improving productivity of Eucalyptus and Melaleuca alternifolia stands. Plantations of Melaleuca have been established from clonal stock and have proved to be vigorously growing and frost resistant, and produce oil of supernal amount and chemical composition (Williams, 1995, Taylor, 1996). In the family the clone and line selection are applied very frequently. Special “high carvone content” type plants have been produced by clonal selection from Mentha spicata (Shikimara et al., 1993), citral rich populations from Ocimum americanum (Sarin et al., 1992) and methyl cinnamate population from O. basilicum (Simon et al., 1990). In the case of genus Salvia the selection proved to be a rather effective method. The first Salvia sclarea cultivars were developed using continuous individual selection (Bernáth, 2000b), and this method were applied in the case of Salvia officinalis, with high efficacy. By the results of Bezzi (1966), the accumulation level of essential oil can be increased. As a result of clonal selection special chemotype of low thujone content (2% of beta-thujone, or less) were developed, proving that the chemical diversity of species can be utilized and stabilized by continuous selection. (Fig. 2.). Selection under environmental pressure is a new challenge in the field of medicinal and aromatic plant breeding. This method is supported by the results of the physio-ecological investigations (Bernáth, 1986) and by the practical experiences of the selectors. From the practical point of view, the modification of chemical character of the original population affected by their simple introduction into the experimental field, or planting them to the different (Paneva et al., 1989) or extreme locations (Franz et al., 1983) call our attention to the possibilities of this method. More recently NaCl tolerant Cymbopogon martinii lines were selected by using callus cultures grown under increasing concentration stress of NaCl (Patnaik and Debata, 1997). In the case of Papaver somniferum using special test conditions in the phytotron, the narcotine, thebaine and codeine content of the original populations were shifted in the direction of continuously higher accumulation levels (Bernáth, 1998). The stabilization of high accumulation level of alkaloid components made possible the official registration of cultivars 'Kék Gemona' (with high narcotine content), 'Monaco' (accumulating codeine), as well as 'Tebona' (showing thebain accumulation character). 2. Mass selection. It is a well-known method for improving plant materials. As an

117 example Buplerum falcatum variety was developed by mass selection recently with about 1.05-7.43 % crude saponin content (Liu et al., 1989). The essential oil accumulation can be improved by this method too, based on the experiments of other authors. Its efficacy has been proved in the case of Artemisia pallens (Farooqi et al., 1990), Cymbopogon martinii var. motia (Kulkarni, 1990) and many other cases.

Crossing 1. Interspecific Crossing (or Natural Hybrid). This method has many practical application forms in the case of medicinal and aromatic plants. Mentha piperita is one of the main examples which is supposed to be a natural poly-hybrid by the literature data (Heeger, 1956). There are other crops, which are the results of the purposive interspecific crossing. The poppy cultivar 'Kék Duna', which is cultivated on a large scale in Hungary is the result of the crossing of Papaver orientale and P. somniferum, followed by individual selection. The interspecific crossing may have an advantage in the case of the woody plants, which are propagated vegetatively. There are efforts to select new Eucalyptus hybrids, of special essential oil composition (Menut et al., 1992). The crossing of Eucalyptus urophylla and E. grandis resulted intermediate hybrids, which are rich in both 1-8-cineole and p-cymene - alpha-pinene. Crossing the diploid and induced tetraploid forms of Lavandula angustifolia and L. latifolia resulted special hybrids including sesquidiploid ones (Rabotyagov and Akimov, 1990). Marked heterosis for linalool synthesis and borneol synthesis was found. However there are remarkable efforts to clear up the genetic regularity (heritability) of the secondary compounds in the course of interspecific hybridization. Seemingly the Mentha (Maffei, 1990) and Salvia species are good plant-organisms for it (Putievsky et al., 1990). The comparison of the essential oil content of the parents and hybrids of Salvia species shows a special picture (Table 3.). In the essential oil of S. fruticosa and S. officinalis hybrids there is a medium level of 1,8-cineole, α- and β-thujone, camphor, β- caryophyllene, α- and β-pinene. However, the oil of the hybrid between S. tomentosa and S. officinalis is very similar to that of S. officinalis and there is no increase of β-thujone because of the expected influence of S. tomentosa. In spite of the contradictory results it was proposed by the authors (Putievsky et al., 1990), that the large unexploded gene pool of S. officinalis group could be utilized in the future to develop new sage cultivars suited to specific agronomic conditions, and containing desired combinations of essential oil components. 2. Intraspecific Crossing. This method is frequently used to improve the genetic background of the medicinal and aromatic plant cultures. Different types of methods are used including simple crosses (such as: selfing, crossing, polycrossing, and backcrossing) and complex hybridization. In Table 4, some results concerning to the active agents of the plants are summarized, which have been published recently. In order to achieve successful breeding and cultivar development and by realizing lack of knowledge in this context, studies on the inheritance of the most important plant traits were done by several authors. As an example chemotypes of Salvia officinalis were crossed by Franz (1994). It is obvious from the data presented in Table 5, that crossing of cineol type (P132) and α-thujone type (P191) plants resulted in both α-thujone and β- thujone type individuals, as well. Also, it is a very interesting phenomenon, that while both parents contain relatively large amounts of α-thujone (29.66-34.66 %), its amount decreased to below 1 % in one group of hybrids. The same unexpected trend of changes can be observed in the case of the majority of the other compounds. Based on the results of the above experiment, it was admitted by the author, that additive or epistatic gene effects should be present in the regulation of formation of essential oil compounds. However there are observations that the correct evaluation of the progenies after crossing may require well defined ecological conditions. By the experiments of Sacco et al. (1992) in the F1 hybrids of Mentha viridis var. lavanduliodorata linalyl acetate variation appears to be more influenced by environmental changes than genotype, thereby

118 demonstrating a high degree of phenotypic plasticity. In the Mentha species it can be explained by the action of dominant ‘E’ gene which causes etherification of alcohol to yield acetates. The same gene activity may cause consequent decrease of menthol in peppermint under special ecological condition. Similar type of changes were registrated in F1 and F2 progenies of Papaver somniferum cultivars, showing characteristically high (‘Australian’ population) and low (Reading cultivar) alkaloid formation ability (Bernáth, 1993). It does mean that the actual ecological condition may have an influence on the phenotypic appearance of chemism helping, or hindering our evaluation process.

Natural Polyploids - Poliploydisation The natural polyploids can be utilized for improving the raw materials of medicinal and aromatic plants. However, the advantage of ploydization on accumulation of active agents seems to be uncertain. Comparing Valeriana exaltata, V. collina and V. procurrens populations large chemical differences were proved in relation of the chromosome number (Noller, 1989). The higher chromosome number resulted higher accumulation in valepotriates and valerenic acid. In relation of the valerenic acid the connection seems to be a very strict one. While the valerenic acid content in the root of the diploid and tetraploid forms was as low as 5-32 mg%, in the individuals of octoploid plants 138.9-264.7 mg% accumulation level was found. Similarly, the essential oil content of Lavandula angustifolia (Raev et al., 1996) and Salvia sclarea (Mekhraz et al., 1988) had been increased. Polyploids of O. gratissimum (rich in ) x O. viride (rich in thymol) were induced (Khosla et al., 1989, 1990) too. The polyploids (2n=80) exhibited gigantism, showing heterosis for both vegetative and floral characters accumulating both eugenol (50-55 %) and thymol (7-10 %). However the chemical composition of Matricaria recutita cultivars shows no correlation with the ploidy level (Franz, 1995).

Mutation Both the spontaneous and induced mutants are frequently used for improving genetic background of medicinal and aromatic plants. Among others applying gamma-irradiation dwarf mutants of Jasminum grandiflorum (Srivastava and Karmakar,1988) and Solanum laciniatum (Bernáth and Tétényi, 1973) were developed. The irradiation was successfully applied to promote alkaloid production of Claviceps purpurea (the level of alkaloid accumulation increased from 2 % to 10 %) and at the same time strains containing exclusively ergo-tamine, ergo- kristine, ergo-kornyne or ergo-kryptine were developed (Németh, 1994). Mentha arvensis of unique odour (containing 58 % of carvone) was developed by Indian (Anonym, 1993) and M.piperita population of increased menthol content by Bulgarian scientists (Zheljazkov et al., 1996). The irradiation of hyssop (Mekhraz et al., 1989) resulted similar changes; the original aroma of the plants were modified. The chemical mutagens were applied successfully too. The essential oil accumulation pattern of Cymbopogon flexuosus was modified by ethyl methanesulfonate (Kulkarni et al., 1992). Three types of plants were separated: plant possessing high oil content (1.30-1.53 %) and rich in geraniol (66-73%), plants of extremely low oil content (0-traces), plants with equalized distribution of different essential oil components (beta- ocimen, gamma-terpinene, linalool, neral, and geraniol). More recently the alkaloid composition of poppy (Papaver somniferum) was modified by mutagenic treatments in both Australia an India. Based on the results of Satpute (2000) applying different mutagenes (gamma- irradiation - 15 kR and ethyl methane sulfonate - 0.4 %) codeine-rich and thebaine-rich chemotypes were developed from the local 'Sanchita' cultivar.

Genetic Engineering There are enormous amount of publications dealing with production of active agents in callus and suspension cultures. Because of the complexity of these processes the

119 majority of the attempts has failed (Charlwood and Pletsch, 2000). There are only a few results of practical importance. The application of transformed cell cultures is a new line in this respect. As an example, Tagetes laxa root culture (transformed with Sclerotiana sclerotiorum) produced more thiophene, by about 90 % (Rodriguez and Giulietti, 1995). Based on the result of the protoplasm fusion of Tanacetum vulgare and T. cinerariifolium Keskitalo (2000) states that this form of genetic engineering may have many advantages and will became a novel tool in the future. This method requires not too much knowledge of specific biochemical steps. However, genetic-engineering based on the overpexpressing or suppressing of genes responsible for biosynthesis of a special compounds may offer much more possibilities. By the results of Nessler (1998), the genetic engineering on formation of poppy (Papaver somniferum) alkaloids can be realized in the very near future. Based on the isolation and identification of the new members of TyDC/DODC gene family, responsible for the activity of tyrosine/dopa decarboxylase, the intervention into the one of the most important starting step of alkaloid biosynthesis seems to be realistic (Fig. 3). If sufficient suppression of that gene family is achieved it may even be possible to recover plants which produce very low alkaloid levels or no alkaloids at all.

ACKNOWLEDGEMENTS The present work is completed by the help of OTKA subventions number 32393 and 34289 and 30658.

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Tables

Table 1. Species used in the world commercially as oregano, based on their chemical similarity

Family Genus and species Lamiaceae Calamintha potosina Schaf., amboinicus Lour., C. aromaticus Benth., Hedeoma floribunda Standl., H. incona Torr., H. patens Jones, Hyptis albida H.B.K., H. americana (Aubl.)Urb, H.capitata Jacq., H. pectinata Poit., H.suaveolens (L.) Poit., Monarda austromontana Epling, Ocimum basilicum L., Origanum compactum Benth., O. dictamnus L., O. elongatum (Bonnet)Emberger et Maire), O. floribundum Munby, O. grosii Pau et Font Quer ex Ietswaart, O. majorana L., O. microphyllum (Benth) Vogel, O. onites L., O. scabrum Boiss et Heldr., O. syriacum L. var. syriacum,O. vulgare L. subsp. gracile (Koch) Ietswaart, O. vulgare subsp. Hirtum (Link) Ietswaart, O. vulgare subsp. virens (Hoffmanns et Link) Ietswaart, O. vulgare subsp. viride (Boiss.) Hayek, O. vulgare L. subsp. vulgare, O. vulgare L., Poliomintha longiflora Gray, Salvia spp., Satureja thymbra L., Thymus capitatus (L.) Hoffmanns et Link Verbenaceae Lantana citrosa (Small) Modenke, L. glandulosissima Hayek, L. hirsuta Mart. et Gall., L.involucrata L., L. purpurea (Jacq.) Benth.&Hook., L. trifolia L., L. velutina Mart. & Gal., Lippia myriocephala Schlecht.& Cham., L. affinis Schau., L. alba (Mill) N.E. Br., L. Berlandieri Schau., L. cardiostegia Benth., L. formosa T.S.Brandeg., L. geisseana (R.A.Phil.)Soler., L. graveolens H.B.K., L. helleri Britton, L. micromera Schau., L. micromera var.Helleri (Britton)Moldenke, L. origanoides H.B.K., L. palmeri var. spicata Rose, L. palmeri Wats., L. umbellata Cav., L. velutina Mart. et Galeotti Rubinaceae Borreria spp. Scrophulari- Limnophila stolonifera (Blanco) Merr. aceae Apiaceae Eryngium foetidum L. Asteraceae Coleosanthus veronicaefolius H.B.K. Eupatorium macrophyllum L.

Table 2. Examples for segregation of self pollinated Achillea crithmifolia chemotypes in the first I1 progenies (Németh et al. 2000)

Original chemotypes Proportion of chemotypes in the I1 progeny Camphoraceous Cineolic Mixed Camphoraceous 1 0 6 Camphoraceous 6 2 3 Camphoraceous 1 0 0 Cineolic 0 7 2 Mixed 7 2 2 Mixed 1 0 5 Camphoraceous: ≥45% camphor, ≤15% 1,8-cineol, ~10% borneol Cineolic: 20- 30% camphor, ≥30% 1,8-cineol, ≥5% borneol Mixed: ≥45% camphor, 15-30% 1,8-cineol, 5-10% borneol

125

Table 3. Cross ability and oil composition of three Salvia species and their hybrids (Putievsky et al., 1990)

Parents Cross- Seed Main compounds of essential oil (%) ability set (%) (%) α- β- 1,8- α- β- β- pinene pinen cineole thujone thujo Camph Caryoph e ne or yllene S. officinalis 85 65 1 1 13 55 10 2 0

S. fruticosa 92 37 6 11 48 0 0 8 8

S. tomentosa 96 40 2 2 19 0 59 1 0

S. off. x S. frut. 36 9 3 7 30 27 7 4 4 S. frut. x S. off. 34 9 3 7 24 29 7 4 2 S. tom. x S. off. 2 4 2 3 12 49 6 4 2

Table 4. Some examples of the intraspecific crossing of medicinal and aromatic plants for changing the accumulation of their active agents

Species Method Goal of selection Number of references

Carum carvi crossing different ess. oil (carvone) Toxopeus et al., 1995 forms Cymbopogon flexuosus crosses citral accumulation Kulkarni and Ramesh, 1992 C. flexuosus crosses ess. oil Kulkarni,1997 C. martinii crosses of ess. oil and geraniol Sethi et al., 1990 accessions Matricaria recutita crosses Ess. oil, apigenin Kirsh and Franke ,1993 Melissa officinalis line crossing accumulation level of Adzet et al. 1992 ess. oil Ocimum americanum multiple crossing, poor methyl chavicol Gupta and Sobti 1990 selfing and citral O. canum multiple crossing, ess. oil; linalool Gupta and Sobti, 1994 selfing accumulation Salvia officinalis crossing compositional Franz ,1994 chemotypes, segrehgation, ess. oil Solanum laciniatum crossing heritability, soalasodine Vogel et al., 1992 Valeriana officinalis crosses, reciprocal heritability, ess. oil, Noller, 1989 valerenic acid etc. Ocimum gratissimum hybridization heterosis, ess. oil Bradu et al., 1990 Thymus vulgaris hybridization- heterosis, ess. oil Rey, 1993 Vetiver zizanioides hybridization heritability, ess. oil Lal et al., 1997

126

Table 5. Compositional segregation of hybrids made by crossing of different Salvia officinalis chemotypes (Franz, 1994)

Genotype Main constituents of the essential oil (%) Cineol α-thujone β-thujone camphor α-pinene camphen 132 (P) 25.89 29.66 8.09 13.08 4.62 2.52 191 (P) 17.16 34.66 6.87 13.88 8.57 4.72

132 x 191/14 33.06 0.20 20.77 10.26 21.34 3.92 132 x 191/13 32.19 0.50 24.68 15.66 9.12 4.64

132 x 191/19 23..29 22.94 6.44 14.37 13.85 6.85 132 x 191/15 22.06 27.76 6.10 11.31 13.98 3.83 132 x 191/15 18.49 25.82 5.49 18.70 12.24 5.72

Figures

O.vulgare subsp. viride O.heracleoticum

Essential oil

%

70 60 50 40 carvacrol 30 gamma-terpinene 20 thymol 10 p-cymene 0 1 2 364 5 Sezik et al 2 Lawrence and Akgül and 1993 Reynolds 1984 Bayrak 1987 Fleisher and Sneer 1982

Fig. 1. Intraspecific chemical diversity of Origanum taxa having "oregano" or no "oregano" character based on the presence of carvacrol

127 RATIO ( %)

30

25

20

15

10

TN58 5 Fl7 TN B 0 euchaliptol camphor alpha-tujone beta-thujone

Fig. 2. Special thujone low chemotype of Salvia officinalis "TN B" (2 % of beta-thujone, or less) created by clonal selection (Bezzi,1966)

COOH + COOH H N 3 C C O H H C 2 HOOC CH 2

OH OH Arogenate 4-Hydroxyphenyl-pyruvate

+ NADP NADPH CO 2 CO c b 2

COOH H H CH C H C C H C 2 H C O 2 2 2

NH b 2 d

OH CO 2 OH OH Tyrosine Tyramine 4-Hydroxyphenyl acetaldehyd (TYRAL)

1/2 O a 2 1/2 O a 2 e COOH H CH C 2 H C NH H C HO 2 2 2 NH 2 NH b HO H

CO OH OH 2 OH OH HO

Dopa Dopamine (S)- Norcoclaurine

TyDC/DODC gene family

Fig. 3. TyDC/DODC gene family, which is responsible for the activity of tyrosine/dopa decarboxylase having outstanding importance in biosynthesis of poppy alkaloids

128