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Evaluation of alkaloid profiles in hybrid generations of different poppy (Papaver somniferum L.) genotypes
Industrial Crops and Products 33 (2011) 690–696
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Evaluation of alkaloid profiles in hybrid generations of different poppy (Papaver somniferum L.) genotypes Éva Németh-Zámbori ∗ , Csilla Jászberényi, Péter Rajhárt, Jeno˝ Bernáth Corvinus University of Budapest, Department of Medicinal and Aromatic Plants, 1118 Budapest, Villányi str. 35-43, Hungary
a r t i c l e
i n f o
Article history: Received 27 July 2010 Received in revised form 7 January 2011 Accepted 10 January 2011 Available online 4 February 2011 Keywords: Inheritance Breeding Morphine Thebaine Codeine Narcotine
a b s t r a c t Five varieties (‘Minoan’, ‘Medea’, ‘Korona’, ‘Przemko’, ‘Kozmosz’) of poppy representing different chemotypes were combined and the alkaloid profiles of F1–F3 progenies were studied during 2006–2009. In the crosses of high alkaloid containing varieties the content of total alkaloids and that of morphine and thebaine showed increased levels in the hybrid generations which persisted till F3. Narcotine (noscapine), however accumulated at lower level than the midparent values and showed a decreasing tendency over generations. A higher number of homogenous strains starts to appear in F3 progenies. The majority of codeine containing individuals concentrates to certain strains. A growing number of high thebaine containing individuals was observed in several combinations. In the crosses with low alkaloid containing parent (‘Przemko’) the F1 exhibits considerable heterosis for total alkaloid content. Low alkaloid containing recessive individuals segregate in F2 and stabilise in F3. Results of our crossing experiments reflected well the effects of genetic regulation at three levels of enzymatic processes during the biosynthesis of poppy alkaloids. Data support the recessive determination of transformations at TyDC (tyrosine decarboxylase) and BBE (berberine bridge enzyme) levels while more complex (polygenic) effects are supposed in controlling the quantity of narcotine (noscapine) and morphinanes. Selection for fixing very low alkaloid content and narcotine may be effective in early F2 generations, however a selection for morphinanes is not worthy before F3. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Poppy, (Papaver somniferum L.) is one of the most important medicinal plant species worldwide. While the seeds are utilised in culinary, the capsules provide a valuable raw for the pharmaceutical industry. Total demand for opiate raw materials has increased continuously over the last decades and has been anticipated to rise further in the next years even till 450 tonnes of morphine equivalent (Anon., 2009). Poppy has been utilised for thousands of years. The main alkaloid of the latex, morphine is one of the most effective analgesics, codeine, and narcotine (noscapine) are antitussive agents, however, the last one was proved to possess potential anticancer effects, too (Facchini et al., 2007). Thebaine is the source of other important analgesics and sanguinarine, accumulating in the roots, has antimicrobial properties.
∗ Corresponding author at: Corvinus University of Budapest, Department of Medicinal and Aromatic Plants, H-1518 Budapest, P.O. Box 53, Hungary. Tel.: +36 1 482 6252; fax: +36 1 482 6330. E-mail address: [email protected] (É. Németh-Zámbori). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.01.013
The large ecological flexibility of poppy enables its cultivation in the most diverse areas of the word. Among medicinal plant species, the genetic resources of poppy are very rich. Almost 50 cultivars are officially registered worldwide and there might exist a pool of other genotypes patented and cultivated in controlled production systems (Bernáth and Németh, 2009). Breeding methods include selection, crossing and mutation breeding, while in the last decade genetic engineering has became a potentially new tool in manipulating metabolic productivity. In spite of the considerable number of studies, information on inheritance of alkaloids is still contradictious. Based on crossing, back-crossing and diallele trials, polygenic determination was assumed for the accumulation of main alkaloids. The majority of authors describe heterosis for the most important alkaloids (Kálmán-Pál et al., 1987; Singh and Khanna, 1991; Singh et al., 1999), however, several other publications mention intermediate levels or mixed findings (Dános, 1965; Morice and Louarn, 1971; Krenn et al., 1998). The dominance variance proved to be outstanding, although in some cases (e.g. content of thebaine and narcotine) considerable additive variances were also registered. Heritability of morphine content is in most cases low or at least moderate (8–36%), however, high h2 is also reported (Singh et al., 1999). In crosses with top1 mutant Millgate
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et al. (2004) concluded single gene regulation of thebaine content. In the last years, molecular genetical studies have been involved to explain the genetical background of poppy alkaloids. The key enzymes catalysing the biosynthetic processes in formation of these alkaloids have already been isolated and the correspondent cDNAs cloned. Especially the basic pathway leading to (S)-reticuline seems to be cleared up. Gene-transformations and cDNA transcript studies showed, that the regulation may be a complex, metabolon type one, where yet unknown transcription factors, enzymatic feed back and feed forward reactions, regulon systems are assumed (Allen et al., 2004; Ziegler et al., 2009). Till now, the joint interpretation of experimental results by classical genetic and molecular aspects has hardly happened. Besides, the majority of studies focuses only on some selected alkaloid compounds and reports the analysis of the first or at least the second – inbred, hybrid, transformant or mutant – generations. Recently, we carried out a comprehensive evaluation of F1–F3 progenies where the parent varieties represented different chemotypes of poppy. Aim of these investigations was the study of the alkaloid accumulation and profile in reflection of the recent molecular genetic informations at the same time with conclusions for the practical breeding. 2. Material and methods 2.1. Plant material The following parental varieties of Papaver somniferum L. were used for crossings: ‘Minoan’: industrial cultivar with high accumulation level of morphine (18–20 mg/g) in dry capsules. ‘Medea’: industrial cultivar accumulating high levels of morphine (16–18 mg/g) and thebaine (4–6 mg/g). ‘Korona’: industrial cultivar showing especially high levels (18–20 mg/g) of narcotine (noscapine) and ‘Przemko’: culinary cultivar of restricted alkaloid accumulation (below 1 mg/g) in the capsules. Each of the mentioned varieties had been crossed by the cultivar ‘Kozmosz’ having medium (6 mg/g) total alkaloid content with main compound morphine in the capsules. This is an old variety of high yields and cold tolerance, used mainly for food.
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2.3. Chemical analysis Individual capsules were used for the phytochemical analysis. For the extraction 0.2 g of pulverized capsule and 20–25 ml solvent chloroform–methanol (4:1) was used in Soxhlet apparatus (Sigma–Aldrich Kft. Budapest, Hungary). After vacuum distillation and repeated solution of samples in 1 ml solvent, the separation procedure was carried out in horizontal chambers (H-Trennkammer Desaga Nr. 120150, Wiesloch, Germany). The procedure consisted of toluol-ethyl-acetate-diethyl-amin (7:2:1) forming the mobil phase on Silica gel Plates 60 F254 (Merck Kft. Budapest, Hungary). Evaluation was carried out by densitometric analysis (CHR-SCAN TR-541 equipment with a LabChromTM Chromatographic Data processing System Version 5.2; Chemotron, Budapest, Hungary). The densitometric scanning profiles of four alkaloids (morphine, codeine, thebaine and narcotine = noscapine) were calibrated against the corresponding standards. The accuracy of the measurements is the level of 0.0001%, CV of parallel measurements is 3.19%. In the following, alkaloid data always refer to the content of ripe capsules in % dry mass. 2.4. Statistical analysis Biometrical analysis was carried out by Statistica 8 software. Descriptive statistical data (mean, standard deviation, maximum and minimum values) and coefficient of variation were calculated for each strain and each combination in every generations. Linear regression was determined in each combination between the alkaloid values of mother plants in F1–F2 and means of their progeny in F2–F3, respectively. Correlation matrices were calculated among the main alkaloid compounds in each generation of each combination. For determination of the differences among strains belonging to the same combination in respect of the total alkaloid content, morphine, codeine, thebaine and narcotine, Anova was performed for each variable. Evaluation of the differences among strains of different combinations was carried out by pincipal component analysis using all of the mentioned compounds as variables. 3. Results
2.2. Field experiments
3.1. Alkaloid profiles of the progenies in cross ‘Minoán’ × ‘Kozmosz’
The open field experiments took place at the Research Station of the Corvinus University, in Soroksár, Budapest. Crossing of the parental varieties was carried out in 2006 by castration of the mother plants at bud stage, and pollination by the pollen mixture of the other variety. After fertilisation the flowers were isolated by paper bags, which remained on the plants for about a week. In 2007 the F1 generation was studied, using the progenies (strains) of 50–75 mother plants/combination. The F2 generation was formed by selfings of the F1 plants and evaluated in the following year. Similarly, the F3 generation was developed by selfings of the F2 individuals and studied in 2009 under the same experimental circumstances. In each year, parental varieties were investigated as controls for comparison with the progenies. Seeds of each population were sown into small plots (10 m2 ) by hand at the beginning of March each year. Row and plant distances were 0.5 m and 0.05 m, respectively. Plants were harvested in July, at the stage of full ripening. The three generations were checked for their alkaloid contents, based on 15–20 individual measurements pro strain. Reciprocal progenies of the same crosses were evaluated together because evaluation of maternal effects was beyond the scope of this report.
Total alkaloid contents of the progenies each of the F1, F2 and F3 exceeded the mean values of the two parents (Table 1). Standard deviation was similar in F1 and F2 (CV = 51–55%), and decreased in F3 (CV = 33%). Among the strains of this combination, significant differences appeared in F2 (p = 0.09). However, separation of the strains did not show connection with the alkaloid level of the corresponding mother plants. In the progeny plants, – similarly to both parental varieties – morphine is the main alkaloid. Its value in the hybrid populations is higher than parental mean. Standard deviation is slightly decreasing in F3. Morphine is present in each sample but the special chemotype of the ‘Minoan’ parent (accumulating almost exclusively morphine) appeared only in F3 in 20% of the individuals. Codeine can be measured in both parents only in relative low level (below 1 mg/g). In F2 and F3 the content of the hybrid progenies exceeds that of the parental varieties. The proportion of individuals accumulating codeine in higher than trace levels is growing through the generations. In F1 it is 14%, in F2 34% and in F3 it is 55%. The majority of codeine containing individuals concentrates to certain strains and the homogeneity from this respect is growing. In F3 already 41% of the strains is homogenous: in 18%
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Table 1 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies ‘Minoán’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (188)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 2.0
20.0 2.0 14.0 0.0 26.0
9.4 0.2 2.2 0.0 11.7
4.34 0.45 2.73 0.00 5.99
F2 (289)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 0.0
18.0 7.0 15.0 9.0 35.0
7.6 0.7 2.4 0.8 11.5
4.12 1.28 2.94 1.59 6.34
F3 (284)
Morphine Codeine Thebaine Narcotine Total
6.0 0.0 0.0 0.0 6.0
26.0 4.0 14.0 4.0 42.0
11.5 0.6 3.3 0.3 16.0
4.03 0.76 2.65 0.86 5.94
‘Minoan’ ‘Kozmosz’
Morphine:16.0 Morphine: 3.7
Codeine: 0.5 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 16.5 Total: 5.8
of them each individual accumulates codeine, in 23% of the strains no one does. In the parents, thebaine accumulates only in Kozmosz (about 1 mg/g). The average content of thebaine in each hybrid generation exceeds considerably the mid parent values. Besides, the number of high thebaine containing individuals is growing. Higher levels than that of the ‘Kozmosz’ parent were found in 47% of the F1 progeny plants, in 49% of the F2 and in 78% of the F3 ones. Both in F2 and F3 generations, even very high values (above 6 mg/g) were measured in 11.0 and 10.7% of the individuals, respectively. Thebaine appeared in 51% of the F2 progeny plants even in strains, where it was not detected in the mother plant. Narcotine (noscapin) can be found only in the ‘Kozmosz’ parent, although even there, in relatively low abundance (0.9 mg/g). It was not detected in the F1 generation of this combination but it was measured in F2 in 25% of the individuals and in F3 in 14% of them. F3 progeny plants of narcotine containing F2 mother plants always inherit this feature. However, the accumulation level is low, its average values do not exceeds that of the ‘Kozmosz’ parent.
levels below the mid parent values (Table 2). However, the proportion of individuals containing high (above 6 mg/g) values is increasing and – similarly to the ‘Minoan’ × ‘Kozmosz’ combination –, in F2–F3 reaches 9–13% of individuals. On the other side, the number of individuals free of thebaine is decreasing. Strains are going to be more uniform through the generations also in respect of thebaine, in F3 already characteristic strains including high thebaine or low thebaine accumulating individuals can be distinguished. The accumulation of narcotine is not significant in this combination, in the parental varieties it can be found only in ‘Kozmosz’ in low quantities (0.9 mg/g). It could not be detected in F1 but appears in F2 (in 13% of the individuals) and is present also in F3 (in 36% of the individuals), but the average level is low, below that of ‘Kozmosz’. The standard deviation is, however high. Similarly, to the morphinane alkaloids, numerous strains are getting uniform in F3.
3.2. Alkaloid profiles of the progenies in cross ‘Medea’ × ‘Kozmosz’
In contrary to the formerly mentioned combinations, in this cross the average total alkaloid content of the hybrid progenies is lower than midparent level (Table 3). It is mainly due to the major component narcotine. Standard deviation is relatively high in each generation (CV = 39–53%). Significant differences among strains (progenies of individual crossings) of this combination are detected from F2 generation (p = 0.04). In this case, morphine contents of both parents are similar and the mean values of the F1 and F2 hybrids reflect also this level, while it is somewhat higher in F3. Individual maximum values are increasing thorough the generations, similarly to the formerly mentioned combinations. Codeine accumulates in this cross only at minimum levels. Among the parents, only ‘Kozmosz’ contains it in higher quantities than traces. This level (1 mg/g or below) appears in each generations, except some F2 plants. The proportion of individuals which accumulate codeine at all, is also low, around 8–10% in each generations and they concentrate to some distinct strains. Similarly to the morphine-type combinations, an increase of the mean accumulation level of thebaine can be observed during the generations. The standard deviaton of F2 and F3 is relatively high. The maximum thebaine values exceed those of the parents in each generation (in F2 up to 12 mg/g). As in case of codeine, the individuals containing thebaine concentrate to certain strains.
The alkaloid profiles of the hybrid generations show much similarities to those of the cross ‘Minoan’ × ‘Kozmosz’ (Table 2). The average content of total alkaloids of the F1–F3 generations exceeds the midparent values. Standard deviation is highest in F2 (CV = 60%) and decreases in F3 (CV = 30%). There are significant differences among the progeny strains in F2 (p = 0.03), but this difference was not in connection with the alkaloid level of the corresponding mother plants. In this combination both parents accumulate morphine as main alkaloid but besides, ‘Medea’ contains a significant amount of thebaine (6–8 mg/g). In the hybrid progenies morphine appears as main component. It is present in each individuals, but the level is variable (0.5–3.0 mg/g). Standard deviation is similar in each of the three generations. In F3 already more strains became homogenous and the variance is decreasing (CV below 15%). Codeine is a minor compound here, in many cases it could not be detected at all. The proportion of the individuals accumulating codeine in more than trace levels is however, increasing: in F1 generation 24%, in F2 it is 25% and in F3 it appears in 42% of the individuals. The level is, however under 1 mg/g, except 2 individuals in F2 where it reaches 1%. The high thebaine content of the parent ‘Medea’ does not appear in the progenies, in each of the hybrid generations we find medium
3.3. Alkaloid profiles of the progenies in cross ‘Korona’ × ‘Kozmosz’
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Table 2 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Medea’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (185)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 3.0
20.0 5.0 14.0 0.0 32.0
10.0 0.4 4.0 0.0 14.6
4.47 0.94 3.29 0.00 6.88
F2 (294)
Morphine Codeine Thebaine Narcotine Total
0.5 0.0 0.0 0.0 0.0
30.0 10.5 15.0 6.0 42.0
9.4 0.5 3.2 0.3 13.6
5.42 1.71 3.31 1.11 8.29
F3 (283)
Morphine Codeine Thebaine Narcotine Total
6.0 0.0 0.0 0.0 8.5
30.0 3.0 16.0 8.0 41.0
12.5 0.3 3.3 1.0 19.7
4.83 0.55 3.25 1.67 6.63
‘Medea’ ‘Kozmosz’
Morphine: 13.5 Morphine: 3.7
Codeine: 0.5 Codeine: 0.1
Thebaine: 8.7 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 22.7 Total: 5.8
Narcotine is the main alkaloid of the parent ‘Korona’, where it reaches 78% of the total alkaloid accumulation. In the hybrid progenies however, the mean concentration is below the midparent level and both this accumulation level and the maximal individual values are decreasing through the generations. As narcotine is present in both parental varieties, it can be detected already in F1 progenies, in contrary to the formerly mentioned crosses. In F1 only 2% in F2 and F3 8–8% of the individuals are narcotine free. An exceeding level like the ‘Korona’ parent can be found only in about one quarter of the progenies (F1: 28%, F2: 27%, F3: 18%). The standard deviation is considerably high in each generation (CV = 49–63%). 3.4. Alkaloid profiles of the progenies in cross ‘Przemko’ × ‘Kozmosz’ In the first progeny of the cross of ‘Kozmosz’ and a partner of very low (less than 1 mg/g) total alkaloid content, the mean values show strong heterosis effect (Table 4). 98% of the F1 individuals has higher contents than those of ‘Kozmosz’ and there is not a single individual of very low alkaloid content. In F2 a segregation was observed: a group of low alkaloid containing individuals is present, but on the other side, the maximum values also increase. The mean value is dropping both in F2 and F3. In F1 there is no significant difference among the strains of this combination (p = 0.138). This is not the case even in F2 gen-
eration because in each strain a big segregation is present and the variance is high (p = 0.737). In F3 there is already a statistically approved difference among the progenies of different mother plants (p = 0.000). In this combination, morphine is the main alkaloid, but the level of thebaine is also significant. Similarly to and in connection with the total alkaloid contents, the level of morphine exhibits a heterosis effect. Later, in F3 it is less than the level of ‘Kozmosz’ (Table 4). Standard deviation is considerable, especially in the segregating generations. The accumulation level of codeine in F1 exceeded that of the hybrid progenies of the high alkaloid containing varieties. In the further generations the mean values – similarly to the total alkaloid content – are decreasing, but remain at a level higher than the ‘Kozmosz’ parental variety. Marginal values are found between zero and 4 mg/g. Like the former combinations, an increasing homogeneity of strains (codeine containing ones or lacking of codeine) can be observed. In F3 one third of the strains contains codeine in each individual. The content of thebaine is similar to the codeine. There was a consdiderable heterosis found in F1 and the level decreases afterwards – that is a deviation from the combinations of high alkaloid containing parents. Similarly to codeine, in F3 already a clear distinction can be seen among high thebaine and low thebaine containing strains.
Table 3 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Korona’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (271)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 4.0
12.0 2.0 4.0 20.0 30.0
4.9 0.1 0.3 7.9 13.2
2.33 0.54 0.70 3.96 5.75
F2 (280)
Morphine Codeine Thebaine Narcotine Total
0.5 0.0 0.0 0.0 0.5
15.0 5.0 13.0 12.0 32.0
5.3 0.2 0.9 6.0 12.5
2.84 0.81 2.21 3.85 6.52
F3 (284)
Morphine Codeine Thebaine Narcotine Total
2.0 0.0 0.0 0.0 5.0
20.0 2.0 8.0 16.0 30.1
8.2 0.2 1.8 5.2 15.3
3.88 0.47 2.14 3.97 5.89
‘Korona’ ‘Kozmosz’
Morphine: 5.3 Morphine: 3.7
Codeine: 0.0 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 19.6 Narcotine: 0.9
Total: 24.9 Total: 5.8
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Table 4 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Przemko’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (158)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 2.0 0.0 4.5
8.0 4.0 7.0 0.0 16.0
5.6 1.9 4.1 0.0 11.6
1.66 1.25 1.32 0.00 2.60
F2 (298)
Morphine Codeine Thebaine Narcotine Total
0.0 0.0 0.0 0.0 0.0
13.0 7.0 12.0 10.0 22.0
3.4 0.9 1.8 0.8 6.9
2.70 1.32 2.49 0.90 5.49
F3 (248)
Morphine Codeine Thebaine Narcotine Total
0.1 0.0 0.0 0.0 0.1
9.0 4.0 4.5 2.0 15.0
3.2 0.8 1.1 0.3 5.4
2.84 0.89 1.27 0.56 4.56
‘Przemko’ ‘Kozmosz’
Morphine: 0.2 Morphine: 3.7
Codeine: 0.0 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 0.2 Total: 5.8
Narcotine content is very low in this combination, present only in ‘Kozmosz’ parent. It was not measured in F1 but appears in F2 and F3 (in 44 and 62% of the individuals, respectively). Highest values were found in F2 generation, however, this mean value is only 1.2 mg/g. 3.5. Differences among combinations In spite of several general observations and many similarities of the inheritance of alkaloid accumulation in the combinations of high alkaloid containing varieties, their progenies differ significantly (p = 0.000) from each other based on multivariate statistics. The different progeny strains of the common partner, ‘Kozmosz’ are separated from each other, based on morphine, codeine, thebaine and narcotinee contents both in F2 (Fig. 1) and in F3. 4. Discussion Results of our crossing experiments showed a practical reflexion of three characteristic levels of biosynthetic regulation of poppy alkaloids (Fig. 2).
3,5 3,0
1
2,5
4
2,0
44 4
Factor 2: 32,04%
1,5
1
1,0
2
4
0,5
4
1
0,0
1
2
-0,5
2
-1,0
1 2
2
3
23 1
3
3
33 33
-1,5 -2,0 -2,5 -3,0
-5
-4
-3
-2
-1
0
1
2
3
4
Factor 1: 43,17% Fig. 1. Separation of the F2 progenies of different crosses in the multivarate coordinate system. (Combinations: (1) ‘Minoán’ × ‘Kozmosz’; (2) ‘Medea’ × ‘Kozmosz’; (3) ‘Korona’ × ‘Kozmosz’; (4) ‘Przemko’ × ‘Kozmosz’. Factor 1: morphine, thebaine; Factor 2: codeine, narcotine.)
The combination where ‘Przemko’ (low alkaloid containing variety) is one of the parents shows the most definite differences from other crosses. In the hybrid generations of high alkaloid containing varieties zero-variants (with trace level of alkaloids) are present in very low proportions (2.8–3.6%), and this low content does not stabilise in the following progenies. In the cross of ‘Przemko’ and ‘Kozmosz’, zero variants like ‘Przemko’ parent appear in F2 and they do not segregate further, thus may be recessive genotypes. It means that a selection for fixing low alkaloid content may be effective in early F2 generations. Lack or appearance of alkaloids in poppy is be connected to the TyDC/DoDC gene family, while the quantitaive and qualitative characteristics are determined in later biosynthetic steps (Psenak, 1998). In varieties like ‘Przemko’ the inhibition at the first steps leading dopamine is masking the genetic background and potential of the further transformations. In our experiment, the broad phenotypic variability (4.5–16 mg/g of total alkaloids) in F1 reflects the heterozygotic structure of ‘Przemko’ at the downstream levels of genetic regulation. In progenies of a low alkaloid containing variety, the individuals carrying dominant alleles in TyDC assure the potential for exploiting the genetical potential of downstream levels and breeding for special alkaloid profile. Based on this, also the “alkaloid free” varieties have the potential to provide high accumulation rates in their progenies even in short term (2–3 generations) if there is a dominant allele in the locus determining the initial steps. At the same time, phenotype of the individuals which carry dominant alleles in the mentioned locus may also manifest itself in very low alkaloid accumulation and this trait be fixed by selection of genes acting in this direction. The next main step determining the formation of major poppy alkaloids is the branching point after (S)-reticuline leading to different alkaloid types, among them the most important morphinanes and phthalideisoquinolines. Among the latter ones, the compound narcotine seems to have the largest significance. Nyman (1979) concluded that its appearance seems to depend on the presence of recessive alleles in the regulation. This biosynthetic pathway which begins with BBE (berberine bridge enzyme) is less understood even today compared to that of the morphinanes (Facchini et al., 2007). In our experimental varieties both ‘Korona’ and ‘Kozmosz’ enable the synthesis, while the route is blocked in ‘Minoan’ and ‘Medea’. Based on the data of the hybrid progenies, it can be assumed, that also ‘Przemko’ may carry dominant alleles at this regulation level. Narcotine containing individuals apper in F2 generations of
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695
Fig. 2. The sheme of biosynthesis of main alkaloids in poppy.
each tested combinations. In their F3 progeny plants the narcotine still remains, thus screening and selection for this trait may be started already in the second generation. A heterozygotic structure of the narcotine free individuals in F2 is supposed because narcotine containing plants segregates also in their progenies. However, as the proportion of narcotine containing individuals do not increase in all of the F3 and the F2 segregation ratio is not always identical with 1:3 the regular, monogenic determination may be excluded. When after (S)-reticuline the synthesis into the direction of phthalideisoquinolines is assured, the actual level of narcotine may be determined by polygenic effects through the following biosynthetic steps including at least five, less known transformations (Facchini et al., 2007). Based on the above mentioned appearance of narcotine in each combination of our study, it seems that the appropriate alleles are present even in the parents like ‘Minoan’ where they cannot manifest phenotypically because of the block at the level of BBE. Abundance of these alleles acting into the direction of a high narcotine accumulation level may, however be rare in varieties ‘Minoán’, ‘Medea’ and ‘Przemko’, thus, the probablility of really high potential hybrids in this respect is low. In the progenies of high narcotine containing variety ‘Korona’ a fraction of individuals possessing the transgessive high level, can be well distinguished (Table 5). This offer a well established background for the positive selection as has been the case during breeding of ‘Korona’ itself (Németh et al., 2010). The main biosynthetic route of morphinanes results in thebaine, codeine and morphine. The enzymes and encoding genes are almost
fully known (Ziegler et al., 2009). These alkaloids were present in each of our parental varieties, their variability in the hybrid accessions is significant. The progenies of the parents having medium or high values of morphine, show similar segregation patterns. This happened both in F2 and F3 generations, thus, a selection into the direction of increased morphine content does not seem worthy very early, in F2. In earlier publications the majority of authors describe heterosis for the most important alkaloids (Kálmán-Pál et al., 1987; Singh et al., 1999), however, several other publications mention opposite results (Dános, 1965; Morice and Louarn, 1971). Neither of them followed the progenies till the F3 generations.
Table 5 Segregation of F2 hybrids in combination ‘Korona’ × ‘Kozmosz’ for narcotine content in the capsules. Upper boundary 0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000 16.000 18.000 20.000
Frequency 5 5 65 55 65 20 35 15 10 0 5
Cumulative frequency
Percent
Cumulative percent
5 10 75 130 195 215 250 265 275 275 280
1.78571 1.78571 23.21429 19.64286 23.21429 7.14286 12.50000 5.35714 3.57143 0.00000 1.78571
1.7857 3.5714 26.7857 46.4286 69.6429 76.7857 89.2857 94.6429 98.2143 98.2143 100.0000
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Table 6 Changes in proportion of individuals (% of the total population) accumulating thebaine in different combinations during the hybrid generations. Combination
F1
F2
F3
Kozmosz × Minoan Kozmosz × Medea Kozmosz × Korona Kozmosz × Przemko
47 90 18 100
69 76 28 53
78 91 56 65
Thebaine appeared in 30–75% of the F2 progeny plants even in strains, where it was not detected in the mother plant. This refers to the possible recessive determination of thebaine accumulation. However, a monogenic simply recessive determination can be excluded according to the further segregation of thebaine containing F2 plants. Already in 1965 Böhm mentioned that transformation of thebaine into morphine is more dominant than the blocking of the synthesis. Based on the results of crosses between morphine and thebaine chemotypes Millgate et al. (2004) supposed recessive or incomplete dominance regulation of thebaine accumulation through blocking of the following demethylation steps. Recently, studies on transgenic poppy genotypes resulted in the conclusion, that the genetic regulation of the morphinane pathway includes complex, metabolon like processes (Facchini et al., 2007; Ziegler et al., 2009). For practical breeding it seems to be of importance, that an increase of individuals accumulating thebaine was observed during the hybrid generations. Especially significant is this tendency for the crosses, where thebaine is not characteristic for both parents (Table 6). A similar tendency was registered for codeine. However, in the early generations, the appearance of these compounds in the progenies does not show a significant correlation with the phenotype of the mother plants (Table 7), therefore this characteristics can not be fixed easily. Even in F3, when the presence of codeine and thebaine may start to stabilise, the level shows a large variance. It was observed that the accumulation levels of thebaine and codeine are in siginficant positive correlation with each other (correlation coefficient in ‘Minoán’ × ‘Kozmosz’ F2: r = 0.54; F3: r = 0.57; in ‘Medea’ × ‘Kozmosz’ F2: r = 0.40; F3: r = 0.39). In the majority of cases, high thebaine and codeine containing individuals have also an enhanced morphine content. Formerly, Kálmán-Pál et al. (1987) described this feature as a temperature dependent accumulation of morphinanes due to an increased precursor supply under favourable ecological cirsumstances. Based on molecular genetical data, the reaction is highly dependent on the activity of codeineone
Table 7 Correlation coefficients between parent individuals and mean values of their progenies concerning the studied alkaloid compounds (Bold: significant at p = 5%). Combination
Alkaloid
F1–F2
F2–F3
Kozmosz × Minoan
Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine
−0.22 X −0.26 X 0.11 X 0.23 X −0.26 X −0.05 0.29 0.20 0.79 0.02 x
0.16 x 0.15 x 0.27 x 0.35 x 0.31 x 0.23 0.35 0.78 0.18 0.94 0.77
Kozmosz × Medea
Kozmosz × Korona
Kozmosz × Przemko
X: no calculation because there are many zero values among the parental data.
reductase enzyme encoded by a multigene family (Larkin et al., 2007). At the same time it seems to be obvious, that the activity of this gene (Cor) alone may not be responsible for the accumulated quantities of morphinanes (Allen et al., 2004). Our results let to conclude the importance of additive gene actions in determination of biosynthesis of morphinanes. The practical results of many new cultivars developed by individual selection contribute to this assumption (Bernáth and Németh, 2009). However, in the hybrid generations of crosses with low alkaloid containing variety (‘Przemko’) we detected heterosis effect indicating dominance gene actions. It is similar to our previous findings during development of the culinary poppy cultivar ‘Ametiszt’ (Németh et al., 2002). In some publications, dominance and additive effects apper also together (Németh, 2002). It seems, that the former statements on the mode of inheritance of morphinanes may be only partially right, because of the complexity of this regulation. Additionally, in practical breeding even unexpected, genotype specific reactions may be anticipated. Acknowledgement The work was supported by the OTKA Research Found Project No. K62732. References Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A.C., Fist, A.G., Gerlach, W.A., Larkin, P.J., 2004. RNAi mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 1559– 1566. Anon., 2009. Report of International Narcotics Control Board for 2007. United Nations Publications, Vienna, Sales No. E 08.XI.1. Bernáth, J., Németh, É., 2009. Breeding of poppy. In: Vollmannn, J., Rajcan, I. (Eds.), Oil crops, Series: Handbook of Plant Breeding. Springer Science + Business Media, Dordrecht, pp. 449–468. Böhm, H., 1965. Über Papaver bracteatum Lindl II. Mitteilung: Die Alkaloide des reifen Bastards aus der reziproken Kreuzung dieser Art mit Papaver somniferum L. Planta Med. 2, 234–240. Dános, B., 1965. Wirkung der generativen Hybridisierung auf die Gestaltung des Alkaloidgehalts des Mohns. Pharmazie 20, 727–730. Facchini, P.J., Hagel, J.M., Liscombe, D.K., Loukanina, N., MacLeod, B.P., Samanani, N., Zulak, K.G., 2007. Opium poppy: blueprint for an alkaloid factory. Phytochem. Rev. 6, 97–124. Kálmán-Pál, Á., Bernáth, J., Tétényi, P., 1987. Phenotypic variability in the production and alkaloid spectrum of the Papaver somniferum L. hybrid. Herba Hung. 26, 75–82. Krenn, L., Dobos, G., Gabriel, E., 1998. Alkaloidgehalt und -spektrum verschiedener Mohn-Genotypen. Z. Arzn. Gew. Pfl. 6, 118–124. Larkin, P.J., Miller, J.A.C., Allen, R.S., Chitty, J.A., Gerlach, W.L., Frick, S., Kutchan, T.M., Fist, A.J., 2007. Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum. Plant Biotechnol. J. 5, 26–37. Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach, W.I., Fist, A.J., Larkin, P.I., 2004. Morphine-pathway block in top1 poppies. Nature 431, 413. Morice, J., Louarn, J., 1971. Study of morphine content in the oil poppy (P. somniferum L.). Ann. Amelior. Pl. 21, 465–485. Németh, É., 2002. World tendencies aims and results of poppy (Papaver somniferum L.) breeding. In: Govil, J.N., Kumar, A.P., Singh, V.K. (Eds.), Series Recent Progress in Medicinal Plants Biotechnology and Genetic Engineering, 4. SCI TECH Pub, Houston, USA, pp. 129–141. ˝ F., 2002. New results of poppy (Papaver Németh, É., Bernáth, J., Sztefanov, A., Petheo, somniferum L.) breeding for low alkaloid content in Hungary. Acta Hortic. 576, 151–158. Németh, É., Bernáth, J., Jászberényi, Cs., 2010. Studies on the inheritance of poppy (Papaver somniferum) alkaloids and the new cultivar ‘Korona’ accumulating high concentrations of narcotine. Acta Hortic. 860, 153–160. Nyman, U., 1979. Papaver somniferum L.—Breeding for a Modified Morphinane Alkaloid Pattern. University of Uppsala, Dissertation. Psenak, M., 1998. Biosynthesis of morphinane alkaloids. In: Bernáth, J. (Ed.), PoppyGenus Papaver. Harwood Academic Press, Amsterdam, pp. 159–188. Singh, S.P., Khanna, K.R., 1991. Heterosis in opium poppy. Ind. J. Agric. Sci. 61, 259–263. Singh, S.P., Tiwari, R.K., Dubey, T., 1999. Heterosis ind inbreeding depression in opium poppy (Papaver somniferum). J. Med. Arom. Plants 21, 23–25. Ziegler, J., Facchini, P.J., Geissler, R., Schmidt, J., Ammer, Ch., Kramell, R., Voigtlander, S., Gesell, A., Pienkny, S., Brandt, W., 2009. Evolution of morphine biosynthesis in opium poppy. Phytochemistry 70, 1696–1707.
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Evaluation of alkaloid profiles in hybrid generations of different poppy (Papaver somniferum L.) genotypes Éva Németh-Zámbori ∗ , Csilla Jászberényi, Péter Rajhárt, Jeno˝ Bernáth Corvinus University of Budapest, Department of Medicinal and Aromatic Plants, 1118 Budapest, Villányi str. 35-43, Hungary
a r t i c l e
i n f o
Article history: Received 27 July 2010 Received in revised form 7 January 2011 Accepted 10 January 2011 Available online 4 February 2011 Keywords: Inheritance Breeding Morphine Thebaine Codeine Narcotine
a b s t r a c t Five varieties (‘Minoan’, ‘Medea’, ‘Korona’, ‘Przemko’, ‘Kozmosz’) of poppy representing different chemotypes were combined and the alkaloid profiles of F1–F3 progenies were studied during 2006–2009. In the crosses of high alkaloid containing varieties the content of total alkaloids and that of morphine and thebaine showed increased levels in the hybrid generations which persisted till F3. Narcotine (noscapine), however accumulated at lower level than the midparent values and showed a decreasing tendency over generations. A higher number of homogenous strains starts to appear in F3 progenies. The majority of codeine containing individuals concentrates to certain strains. A growing number of high thebaine containing individuals was observed in several combinations. In the crosses with low alkaloid containing parent (‘Przemko’) the F1 exhibits considerable heterosis for total alkaloid content. Low alkaloid containing recessive individuals segregate in F2 and stabilise in F3. Results of our crossing experiments reflected well the effects of genetic regulation at three levels of enzymatic processes during the biosynthesis of poppy alkaloids. Data support the recessive determination of transformations at TyDC (tyrosine decarboxylase) and BBE (berberine bridge enzyme) levels while more complex (polygenic) effects are supposed in controlling the quantity of narcotine (noscapine) and morphinanes. Selection for fixing very low alkaloid content and narcotine may be effective in early F2 generations, however a selection for morphinanes is not worthy before F3. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Poppy, (Papaver somniferum L.) is one of the most important medicinal plant species worldwide. While the seeds are utilised in culinary, the capsules provide a valuable raw for the pharmaceutical industry. Total demand for opiate raw materials has increased continuously over the last decades and has been anticipated to rise further in the next years even till 450 tonnes of morphine equivalent (Anon., 2009). Poppy has been utilised for thousands of years. The main alkaloid of the latex, morphine is one of the most effective analgesics, codeine, and narcotine (noscapine) are antitussive agents, however, the last one was proved to possess potential anticancer effects, too (Facchini et al., 2007). Thebaine is the source of other important analgesics and sanguinarine, accumulating in the roots, has antimicrobial properties.
∗ Corresponding author at: Corvinus University of Budapest, Department of Medicinal and Aromatic Plants, H-1518 Budapest, P.O. Box 53, Hungary. Tel.: +36 1 482 6252; fax: +36 1 482 6330. E-mail address: [email protected] (É. Németh-Zámbori). 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.01.013
The large ecological flexibility of poppy enables its cultivation in the most diverse areas of the word. Among medicinal plant species, the genetic resources of poppy are very rich. Almost 50 cultivars are officially registered worldwide and there might exist a pool of other genotypes patented and cultivated in controlled production systems (Bernáth and Németh, 2009). Breeding methods include selection, crossing and mutation breeding, while in the last decade genetic engineering has became a potentially new tool in manipulating metabolic productivity. In spite of the considerable number of studies, information on inheritance of alkaloids is still contradictious. Based on crossing, back-crossing and diallele trials, polygenic determination was assumed for the accumulation of main alkaloids. The majority of authors describe heterosis for the most important alkaloids (Kálmán-Pál et al., 1987; Singh and Khanna, 1991; Singh et al., 1999), however, several other publications mention intermediate levels or mixed findings (Dános, 1965; Morice and Louarn, 1971; Krenn et al., 1998). The dominance variance proved to be outstanding, although in some cases (e.g. content of thebaine and narcotine) considerable additive variances were also registered. Heritability of morphine content is in most cases low or at least moderate (8–36%), however, high h2 is also reported (Singh et al., 1999). In crosses with top1 mutant Millgate
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et al. (2004) concluded single gene regulation of thebaine content. In the last years, molecular genetical studies have been involved to explain the genetical background of poppy alkaloids. The key enzymes catalysing the biosynthetic processes in formation of these alkaloids have already been isolated and the correspondent cDNAs cloned. Especially the basic pathway leading to (S)-reticuline seems to be cleared up. Gene-transformations and cDNA transcript studies showed, that the regulation may be a complex, metabolon type one, where yet unknown transcription factors, enzymatic feed back and feed forward reactions, regulon systems are assumed (Allen et al., 2004; Ziegler et al., 2009). Till now, the joint interpretation of experimental results by classical genetic and molecular aspects has hardly happened. Besides, the majority of studies focuses only on some selected alkaloid compounds and reports the analysis of the first or at least the second – inbred, hybrid, transformant or mutant – generations. Recently, we carried out a comprehensive evaluation of F1–F3 progenies where the parent varieties represented different chemotypes of poppy. Aim of these investigations was the study of the alkaloid accumulation and profile in reflection of the recent molecular genetic informations at the same time with conclusions for the practical breeding. 2. Material and methods 2.1. Plant material The following parental varieties of Papaver somniferum L. were used for crossings: ‘Minoan’: industrial cultivar with high accumulation level of morphine (18–20 mg/g) in dry capsules. ‘Medea’: industrial cultivar accumulating high levels of morphine (16–18 mg/g) and thebaine (4–6 mg/g). ‘Korona’: industrial cultivar showing especially high levels (18–20 mg/g) of narcotine (noscapine) and ‘Przemko’: culinary cultivar of restricted alkaloid accumulation (below 1 mg/g) in the capsules. Each of the mentioned varieties had been crossed by the cultivar ‘Kozmosz’ having medium (6 mg/g) total alkaloid content with main compound morphine in the capsules. This is an old variety of high yields and cold tolerance, used mainly for food.
691
2.3. Chemical analysis Individual capsules were used for the phytochemical analysis. For the extraction 0.2 g of pulverized capsule and 20–25 ml solvent chloroform–methanol (4:1) was used in Soxhlet apparatus (Sigma–Aldrich Kft. Budapest, Hungary). After vacuum distillation and repeated solution of samples in 1 ml solvent, the separation procedure was carried out in horizontal chambers (H-Trennkammer Desaga Nr. 120150, Wiesloch, Germany). The procedure consisted of toluol-ethyl-acetate-diethyl-amin (7:2:1) forming the mobil phase on Silica gel Plates 60 F254 (Merck Kft. Budapest, Hungary). Evaluation was carried out by densitometric analysis (CHR-SCAN TR-541 equipment with a LabChromTM Chromatographic Data processing System Version 5.2; Chemotron, Budapest, Hungary). The densitometric scanning profiles of four alkaloids (morphine, codeine, thebaine and narcotine = noscapine) were calibrated against the corresponding standards. The accuracy of the measurements is the level of 0.0001%, CV of parallel measurements is 3.19%. In the following, alkaloid data always refer to the content of ripe capsules in % dry mass. 2.4. Statistical analysis Biometrical analysis was carried out by Statistica 8 software. Descriptive statistical data (mean, standard deviation, maximum and minimum values) and coefficient of variation were calculated for each strain and each combination in every generations. Linear regression was determined in each combination between the alkaloid values of mother plants in F1–F2 and means of their progeny in F2–F3, respectively. Correlation matrices were calculated among the main alkaloid compounds in each generation of each combination. For determination of the differences among strains belonging to the same combination in respect of the total alkaloid content, morphine, codeine, thebaine and narcotine, Anova was performed for each variable. Evaluation of the differences among strains of different combinations was carried out by pincipal component analysis using all of the mentioned compounds as variables. 3. Results
2.2. Field experiments
3.1. Alkaloid profiles of the progenies in cross ‘Minoán’ × ‘Kozmosz’
The open field experiments took place at the Research Station of the Corvinus University, in Soroksár, Budapest. Crossing of the parental varieties was carried out in 2006 by castration of the mother plants at bud stage, and pollination by the pollen mixture of the other variety. After fertilisation the flowers were isolated by paper bags, which remained on the plants for about a week. In 2007 the F1 generation was studied, using the progenies (strains) of 50–75 mother plants/combination. The F2 generation was formed by selfings of the F1 plants and evaluated in the following year. Similarly, the F3 generation was developed by selfings of the F2 individuals and studied in 2009 under the same experimental circumstances. In each year, parental varieties were investigated as controls for comparison with the progenies. Seeds of each population were sown into small plots (10 m2 ) by hand at the beginning of March each year. Row and plant distances were 0.5 m and 0.05 m, respectively. Plants were harvested in July, at the stage of full ripening. The three generations were checked for their alkaloid contents, based on 15–20 individual measurements pro strain. Reciprocal progenies of the same crosses were evaluated together because evaluation of maternal effects was beyond the scope of this report.
Total alkaloid contents of the progenies each of the F1, F2 and F3 exceeded the mean values of the two parents (Table 1). Standard deviation was similar in F1 and F2 (CV = 51–55%), and decreased in F3 (CV = 33%). Among the strains of this combination, significant differences appeared in F2 (p = 0.09). However, separation of the strains did not show connection with the alkaloid level of the corresponding mother plants. In the progeny plants, – similarly to both parental varieties – morphine is the main alkaloid. Its value in the hybrid populations is higher than parental mean. Standard deviation is slightly decreasing in F3. Morphine is present in each sample but the special chemotype of the ‘Minoan’ parent (accumulating almost exclusively morphine) appeared only in F3 in 20% of the individuals. Codeine can be measured in both parents only in relative low level (below 1 mg/g). In F2 and F3 the content of the hybrid progenies exceeds that of the parental varieties. The proportion of individuals accumulating codeine in higher than trace levels is growing through the generations. In F1 it is 14%, in F2 34% and in F3 it is 55%. The majority of codeine containing individuals concentrates to certain strains and the homogeneity from this respect is growing. In F3 already 41% of the strains is homogenous: in 18%
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Table 1 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies ‘Minoán’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (188)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 2.0
20.0 2.0 14.0 0.0 26.0
9.4 0.2 2.2 0.0 11.7
4.34 0.45 2.73 0.00 5.99
F2 (289)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 0.0
18.0 7.0 15.0 9.0 35.0
7.6 0.7 2.4 0.8 11.5
4.12 1.28 2.94 1.59 6.34
F3 (284)
Morphine Codeine Thebaine Narcotine Total
6.0 0.0 0.0 0.0 6.0
26.0 4.0 14.0 4.0 42.0
11.5 0.6 3.3 0.3 16.0
4.03 0.76 2.65 0.86 5.94
‘Minoan’ ‘Kozmosz’
Morphine:16.0 Morphine: 3.7
Codeine: 0.5 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 16.5 Total: 5.8
of them each individual accumulates codeine, in 23% of the strains no one does. In the parents, thebaine accumulates only in Kozmosz (about 1 mg/g). The average content of thebaine in each hybrid generation exceeds considerably the mid parent values. Besides, the number of high thebaine containing individuals is growing. Higher levels than that of the ‘Kozmosz’ parent were found in 47% of the F1 progeny plants, in 49% of the F2 and in 78% of the F3 ones. Both in F2 and F3 generations, even very high values (above 6 mg/g) were measured in 11.0 and 10.7% of the individuals, respectively. Thebaine appeared in 51% of the F2 progeny plants even in strains, where it was not detected in the mother plant. Narcotine (noscapin) can be found only in the ‘Kozmosz’ parent, although even there, in relatively low abundance (0.9 mg/g). It was not detected in the F1 generation of this combination but it was measured in F2 in 25% of the individuals and in F3 in 14% of them. F3 progeny plants of narcotine containing F2 mother plants always inherit this feature. However, the accumulation level is low, its average values do not exceeds that of the ‘Kozmosz’ parent.
levels below the mid parent values (Table 2). However, the proportion of individuals containing high (above 6 mg/g) values is increasing and – similarly to the ‘Minoan’ × ‘Kozmosz’ combination –, in F2–F3 reaches 9–13% of individuals. On the other side, the number of individuals free of thebaine is decreasing. Strains are going to be more uniform through the generations also in respect of thebaine, in F3 already characteristic strains including high thebaine or low thebaine accumulating individuals can be distinguished. The accumulation of narcotine is not significant in this combination, in the parental varieties it can be found only in ‘Kozmosz’ in low quantities (0.9 mg/g). It could not be detected in F1 but appears in F2 (in 13% of the individuals) and is present also in F3 (in 36% of the individuals), but the average level is low, below that of ‘Kozmosz’. The standard deviation is, however high. Similarly, to the morphinane alkaloids, numerous strains are getting uniform in F3.
3.2. Alkaloid profiles of the progenies in cross ‘Medea’ × ‘Kozmosz’
In contrary to the formerly mentioned combinations, in this cross the average total alkaloid content of the hybrid progenies is lower than midparent level (Table 3). It is mainly due to the major component narcotine. Standard deviation is relatively high in each generation (CV = 39–53%). Significant differences among strains (progenies of individual crossings) of this combination are detected from F2 generation (p = 0.04). In this case, morphine contents of both parents are similar and the mean values of the F1 and F2 hybrids reflect also this level, while it is somewhat higher in F3. Individual maximum values are increasing thorough the generations, similarly to the formerly mentioned combinations. Codeine accumulates in this cross only at minimum levels. Among the parents, only ‘Kozmosz’ contains it in higher quantities than traces. This level (1 mg/g or below) appears in each generations, except some F2 plants. The proportion of individuals which accumulate codeine at all, is also low, around 8–10% in each generations and they concentrate to some distinct strains. Similarly to the morphine-type combinations, an increase of the mean accumulation level of thebaine can be observed during the generations. The standard deviaton of F2 and F3 is relatively high. The maximum thebaine values exceed those of the parents in each generation (in F2 up to 12 mg/g). As in case of codeine, the individuals containing thebaine concentrate to certain strains.
The alkaloid profiles of the hybrid generations show much similarities to those of the cross ‘Minoan’ × ‘Kozmosz’ (Table 2). The average content of total alkaloids of the F1–F3 generations exceeds the midparent values. Standard deviation is highest in F2 (CV = 60%) and decreases in F3 (CV = 30%). There are significant differences among the progeny strains in F2 (p = 0.03), but this difference was not in connection with the alkaloid level of the corresponding mother plants. In this combination both parents accumulate morphine as main alkaloid but besides, ‘Medea’ contains a significant amount of thebaine (6–8 mg/g). In the hybrid progenies morphine appears as main component. It is present in each individuals, but the level is variable (0.5–3.0 mg/g). Standard deviation is similar in each of the three generations. In F3 already more strains became homogenous and the variance is decreasing (CV below 15%). Codeine is a minor compound here, in many cases it could not be detected at all. The proportion of the individuals accumulating codeine in more than trace levels is however, increasing: in F1 generation 24%, in F2 it is 25% and in F3 it appears in 42% of the individuals. The level is, however under 1 mg/g, except 2 individuals in F2 where it reaches 1%. The high thebaine content of the parent ‘Medea’ does not appear in the progenies, in each of the hybrid generations we find medium
3.3. Alkaloid profiles of the progenies in cross ‘Korona’ × ‘Kozmosz’
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Table 2 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Medea’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (185)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 3.0
20.0 5.0 14.0 0.0 32.0
10.0 0.4 4.0 0.0 14.6
4.47 0.94 3.29 0.00 6.88
F2 (294)
Morphine Codeine Thebaine Narcotine Total
0.5 0.0 0.0 0.0 0.0
30.0 10.5 15.0 6.0 42.0
9.4 0.5 3.2 0.3 13.6
5.42 1.71 3.31 1.11 8.29
F3 (283)
Morphine Codeine Thebaine Narcotine Total
6.0 0.0 0.0 0.0 8.5
30.0 3.0 16.0 8.0 41.0
12.5 0.3 3.3 1.0 19.7
4.83 0.55 3.25 1.67 6.63
‘Medea’ ‘Kozmosz’
Morphine: 13.5 Morphine: 3.7
Codeine: 0.5 Codeine: 0.1
Thebaine: 8.7 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 22.7 Total: 5.8
Narcotine is the main alkaloid of the parent ‘Korona’, where it reaches 78% of the total alkaloid accumulation. In the hybrid progenies however, the mean concentration is below the midparent level and both this accumulation level and the maximal individual values are decreasing through the generations. As narcotine is present in both parental varieties, it can be detected already in F1 progenies, in contrary to the formerly mentioned crosses. In F1 only 2% in F2 and F3 8–8% of the individuals are narcotine free. An exceeding level like the ‘Korona’ parent can be found only in about one quarter of the progenies (F1: 28%, F2: 27%, F3: 18%). The standard deviation is considerably high in each generation (CV = 49–63%). 3.4. Alkaloid profiles of the progenies in cross ‘Przemko’ × ‘Kozmosz’ In the first progeny of the cross of ‘Kozmosz’ and a partner of very low (less than 1 mg/g) total alkaloid content, the mean values show strong heterosis effect (Table 4). 98% of the F1 individuals has higher contents than those of ‘Kozmosz’ and there is not a single individual of very low alkaloid content. In F2 a segregation was observed: a group of low alkaloid containing individuals is present, but on the other side, the maximum values also increase. The mean value is dropping both in F2 and F3. In F1 there is no significant difference among the strains of this combination (p = 0.138). This is not the case even in F2 gen-
eration because in each strain a big segregation is present and the variance is high (p = 0.737). In F3 there is already a statistically approved difference among the progenies of different mother plants (p = 0.000). In this combination, morphine is the main alkaloid, but the level of thebaine is also significant. Similarly to and in connection with the total alkaloid contents, the level of morphine exhibits a heterosis effect. Later, in F3 it is less than the level of ‘Kozmosz’ (Table 4). Standard deviation is considerable, especially in the segregating generations. The accumulation level of codeine in F1 exceeded that of the hybrid progenies of the high alkaloid containing varieties. In the further generations the mean values – similarly to the total alkaloid content – are decreasing, but remain at a level higher than the ‘Kozmosz’ parental variety. Marginal values are found between zero and 4 mg/g. Like the former combinations, an increasing homogeneity of strains (codeine containing ones or lacking of codeine) can be observed. In F3 one third of the strains contains codeine in each individual. The content of thebaine is similar to the codeine. There was a consdiderable heterosis found in F1 and the level decreases afterwards – that is a deviation from the combinations of high alkaloid containing parents. Similarly to codeine, in F3 already a clear distinction can be seen among high thebaine and low thebaine containing strains.
Table 3 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Korona’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (271)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 0.0 0.0 4.0
12.0 2.0 4.0 20.0 30.0
4.9 0.1 0.3 7.9 13.2
2.33 0.54 0.70 3.96 5.75
F2 (280)
Morphine Codeine Thebaine Narcotine Total
0.5 0.0 0.0 0.0 0.5
15.0 5.0 13.0 12.0 32.0
5.3 0.2 0.9 6.0 12.5
2.84 0.81 2.21 3.85 6.52
F3 (284)
Morphine Codeine Thebaine Narcotine Total
2.0 0.0 0.0 0.0 5.0
20.0 2.0 8.0 16.0 30.1
8.2 0.2 1.8 5.2 15.3
3.88 0.47 2.14 3.97 5.89
‘Korona’ ‘Kozmosz’
Morphine: 5.3 Morphine: 3.7
Codeine: 0.0 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 19.6 Narcotine: 0.9
Total: 24.9 Total: 5.8
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Table 4 Basic statistics of alkaloid values (mg/g in the capsules) of the progenies. ‘Przemko’ × ‘Kozmosz’ (in brackets: nr. of individuals in each progeny). Generation
Alkaloid
Minimum
Maximum
Mean
Standard deviation
F1 (158)
Morphine Codeine Thebaine Narcotine Total
1.0 0.0 2.0 0.0 4.5
8.0 4.0 7.0 0.0 16.0
5.6 1.9 4.1 0.0 11.6
1.66 1.25 1.32 0.00 2.60
F2 (298)
Morphine Codeine Thebaine Narcotine Total
0.0 0.0 0.0 0.0 0.0
13.0 7.0 12.0 10.0 22.0
3.4 0.9 1.8 0.8 6.9
2.70 1.32 2.49 0.90 5.49
F3 (248)
Morphine Codeine Thebaine Narcotine Total
0.1 0.0 0.0 0.0 0.1
9.0 4.0 4.5 2.0 15.0
3.2 0.8 1.1 0.3 5.4
2.84 0.89 1.27 0.56 4.56
‘Przemko’ ‘Kozmosz’
Morphine: 0.2 Morphine: 3.7
Codeine: 0.0 Codeine: 0.1
Thebaine: 0.0 Thebaine: 1.1
Narcotine: 0.0 Narcotine: 0.9
Total: 0.2 Total: 5.8
Narcotine content is very low in this combination, present only in ‘Kozmosz’ parent. It was not measured in F1 but appears in F2 and F3 (in 44 and 62% of the individuals, respectively). Highest values were found in F2 generation, however, this mean value is only 1.2 mg/g. 3.5. Differences among combinations In spite of several general observations and many similarities of the inheritance of alkaloid accumulation in the combinations of high alkaloid containing varieties, their progenies differ significantly (p = 0.000) from each other based on multivariate statistics. The different progeny strains of the common partner, ‘Kozmosz’ are separated from each other, based on morphine, codeine, thebaine and narcotinee contents both in F2 (Fig. 1) and in F3. 4. Discussion Results of our crossing experiments showed a practical reflexion of three characteristic levels of biosynthetic regulation of poppy alkaloids (Fig. 2).
3,5 3,0
1
2,5
4
2,0
44 4
Factor 2: 32,04%
1,5
1
1,0
2
4
0,5
4
1
0,0
1
2
-0,5
2
-1,0
1 2
2
3
23 1
3
3
33 33
-1,5 -2,0 -2,5 -3,0
-5
-4
-3
-2
-1
0
1
2
3
4
Factor 1: 43,17% Fig. 1. Separation of the F2 progenies of different crosses in the multivarate coordinate system. (Combinations: (1) ‘Minoán’ × ‘Kozmosz’; (2) ‘Medea’ × ‘Kozmosz’; (3) ‘Korona’ × ‘Kozmosz’; (4) ‘Przemko’ × ‘Kozmosz’. Factor 1: morphine, thebaine; Factor 2: codeine, narcotine.)
The combination where ‘Przemko’ (low alkaloid containing variety) is one of the parents shows the most definite differences from other crosses. In the hybrid generations of high alkaloid containing varieties zero-variants (with trace level of alkaloids) are present in very low proportions (2.8–3.6%), and this low content does not stabilise in the following progenies. In the cross of ‘Przemko’ and ‘Kozmosz’, zero variants like ‘Przemko’ parent appear in F2 and they do not segregate further, thus may be recessive genotypes. It means that a selection for fixing low alkaloid content may be effective in early F2 generations. Lack or appearance of alkaloids in poppy is be connected to the TyDC/DoDC gene family, while the quantitaive and qualitative characteristics are determined in later biosynthetic steps (Psenak, 1998). In varieties like ‘Przemko’ the inhibition at the first steps leading dopamine is masking the genetic background and potential of the further transformations. In our experiment, the broad phenotypic variability (4.5–16 mg/g of total alkaloids) in F1 reflects the heterozygotic structure of ‘Przemko’ at the downstream levels of genetic regulation. In progenies of a low alkaloid containing variety, the individuals carrying dominant alleles in TyDC assure the potential for exploiting the genetical potential of downstream levels and breeding for special alkaloid profile. Based on this, also the “alkaloid free” varieties have the potential to provide high accumulation rates in their progenies even in short term (2–3 generations) if there is a dominant allele in the locus determining the initial steps. At the same time, phenotype of the individuals which carry dominant alleles in the mentioned locus may also manifest itself in very low alkaloid accumulation and this trait be fixed by selection of genes acting in this direction. The next main step determining the formation of major poppy alkaloids is the branching point after (S)-reticuline leading to different alkaloid types, among them the most important morphinanes and phthalideisoquinolines. Among the latter ones, the compound narcotine seems to have the largest significance. Nyman (1979) concluded that its appearance seems to depend on the presence of recessive alleles in the regulation. This biosynthetic pathway which begins with BBE (berberine bridge enzyme) is less understood even today compared to that of the morphinanes (Facchini et al., 2007). In our experimental varieties both ‘Korona’ and ‘Kozmosz’ enable the synthesis, while the route is blocked in ‘Minoan’ and ‘Medea’. Based on the data of the hybrid progenies, it can be assumed, that also ‘Przemko’ may carry dominant alleles at this regulation level. Narcotine containing individuals apper in F2 generations of
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695
Fig. 2. The sheme of biosynthesis of main alkaloids in poppy.
each tested combinations. In their F3 progeny plants the narcotine still remains, thus screening and selection for this trait may be started already in the second generation. A heterozygotic structure of the narcotine free individuals in F2 is supposed because narcotine containing plants segregates also in their progenies. However, as the proportion of narcotine containing individuals do not increase in all of the F3 and the F2 segregation ratio is not always identical with 1:3 the regular, monogenic determination may be excluded. When after (S)-reticuline the synthesis into the direction of phthalideisoquinolines is assured, the actual level of narcotine may be determined by polygenic effects through the following biosynthetic steps including at least five, less known transformations (Facchini et al., 2007). Based on the above mentioned appearance of narcotine in each combination of our study, it seems that the appropriate alleles are present even in the parents like ‘Minoan’ where they cannot manifest phenotypically because of the block at the level of BBE. Abundance of these alleles acting into the direction of a high narcotine accumulation level may, however be rare in varieties ‘Minoán’, ‘Medea’ and ‘Przemko’, thus, the probablility of really high potential hybrids in this respect is low. In the progenies of high narcotine containing variety ‘Korona’ a fraction of individuals possessing the transgessive high level, can be well distinguished (Table 5). This offer a well established background for the positive selection as has been the case during breeding of ‘Korona’ itself (Németh et al., 2010). The main biosynthetic route of morphinanes results in thebaine, codeine and morphine. The enzymes and encoding genes are almost
fully known (Ziegler et al., 2009). These alkaloids were present in each of our parental varieties, their variability in the hybrid accessions is significant. The progenies of the parents having medium or high values of morphine, show similar segregation patterns. This happened both in F2 and F3 generations, thus, a selection into the direction of increased morphine content does not seem worthy very early, in F2. In earlier publications the majority of authors describe heterosis for the most important alkaloids (Kálmán-Pál et al., 1987; Singh et al., 1999), however, several other publications mention opposite results (Dános, 1965; Morice and Louarn, 1971). Neither of them followed the progenies till the F3 generations.
Table 5 Segregation of F2 hybrids in combination ‘Korona’ × ‘Kozmosz’ for narcotine content in the capsules. Upper boundary 0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000 16.000 18.000 20.000
Frequency 5 5 65 55 65 20 35 15 10 0 5
Cumulative frequency
Percent
Cumulative percent
5 10 75 130 195 215 250 265 275 275 280
1.78571 1.78571 23.21429 19.64286 23.21429 7.14286 12.50000 5.35714 3.57143 0.00000 1.78571
1.7857 3.5714 26.7857 46.4286 69.6429 76.7857 89.2857 94.6429 98.2143 98.2143 100.0000
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Table 6 Changes in proportion of individuals (% of the total population) accumulating thebaine in different combinations during the hybrid generations. Combination
F1
F2
F3
Kozmosz × Minoan Kozmosz × Medea Kozmosz × Korona Kozmosz × Przemko
47 90 18 100
69 76 28 53
78 91 56 65
Thebaine appeared in 30–75% of the F2 progeny plants even in strains, where it was not detected in the mother plant. This refers to the possible recessive determination of thebaine accumulation. However, a monogenic simply recessive determination can be excluded according to the further segregation of thebaine containing F2 plants. Already in 1965 Böhm mentioned that transformation of thebaine into morphine is more dominant than the blocking of the synthesis. Based on the results of crosses between morphine and thebaine chemotypes Millgate et al. (2004) supposed recessive or incomplete dominance regulation of thebaine accumulation through blocking of the following demethylation steps. Recently, studies on transgenic poppy genotypes resulted in the conclusion, that the genetic regulation of the morphinane pathway includes complex, metabolon like processes (Facchini et al., 2007; Ziegler et al., 2009). For practical breeding it seems to be of importance, that an increase of individuals accumulating thebaine was observed during the hybrid generations. Especially significant is this tendency for the crosses, where thebaine is not characteristic for both parents (Table 6). A similar tendency was registered for codeine. However, in the early generations, the appearance of these compounds in the progenies does not show a significant correlation with the phenotype of the mother plants (Table 7), therefore this characteristics can not be fixed easily. Even in F3, when the presence of codeine and thebaine may start to stabilise, the level shows a large variance. It was observed that the accumulation levels of thebaine and codeine are in siginficant positive correlation with each other (correlation coefficient in ‘Minoán’ × ‘Kozmosz’ F2: r = 0.54; F3: r = 0.57; in ‘Medea’ × ‘Kozmosz’ F2: r = 0.40; F3: r = 0.39). In the majority of cases, high thebaine and codeine containing individuals have also an enhanced morphine content. Formerly, Kálmán-Pál et al. (1987) described this feature as a temperature dependent accumulation of morphinanes due to an increased precursor supply under favourable ecological cirsumstances. Based on molecular genetical data, the reaction is highly dependent on the activity of codeineone
Table 7 Correlation coefficients between parent individuals and mean values of their progenies concerning the studied alkaloid compounds (Bold: significant at p = 5%). Combination
Alkaloid
F1–F2
F2–F3
Kozmosz × Minoan
Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine Morphine Codeine Thebaine Narcotine
−0.22 X −0.26 X 0.11 X 0.23 X −0.26 X −0.05 0.29 0.20 0.79 0.02 x
0.16 x 0.15 x 0.27 x 0.35 x 0.31 x 0.23 0.35 0.78 0.18 0.94 0.77
Kozmosz × Medea
Kozmosz × Korona
Kozmosz × Przemko
X: no calculation because there are many zero values among the parental data.
reductase enzyme encoded by a multigene family (Larkin et al., 2007). At the same time it seems to be obvious, that the activity of this gene (Cor) alone may not be responsible for the accumulated quantities of morphinanes (Allen et al., 2004). Our results let to conclude the importance of additive gene actions in determination of biosynthesis of morphinanes. The practical results of many new cultivars developed by individual selection contribute to this assumption (Bernáth and Németh, 2009). However, in the hybrid generations of crosses with low alkaloid containing variety (‘Przemko’) we detected heterosis effect indicating dominance gene actions. It is similar to our previous findings during development of the culinary poppy cultivar ‘Ametiszt’ (Németh et al., 2002). In some publications, dominance and additive effects apper also together (Németh, 2002). It seems, that the former statements on the mode of inheritance of morphinanes may be only partially right, because of the complexity of this regulation. Additionally, in practical breeding even unexpected, genotype specific reactions may be anticipated. Acknowledgement The work was supported by the OTKA Research Found Project No. K62732. References Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A.C., Fist, A.G., Gerlach, W.A., Larkin, P.J., 2004. RNAi mediated replacement of morphine with the nonnarcotic alkaloid reticuline in opium poppy. Nat. Biotechnol. 22, 1559– 1566. Anon., 2009. Report of International Narcotics Control Board for 2007. United Nations Publications, Vienna, Sales No. E 08.XI.1. Bernáth, J., Németh, É., 2009. Breeding of poppy. In: Vollmannn, J., Rajcan, I. (Eds.), Oil crops, Series: Handbook of Plant Breeding. Springer Science + Business Media, Dordrecht, pp. 449–468. Böhm, H., 1965. Über Papaver bracteatum Lindl II. Mitteilung: Die Alkaloide des reifen Bastards aus der reziproken Kreuzung dieser Art mit Papaver somniferum L. Planta Med. 2, 234–240. Dános, B., 1965. Wirkung der generativen Hybridisierung auf die Gestaltung des Alkaloidgehalts des Mohns. Pharmazie 20, 727–730. Facchini, P.J., Hagel, J.M., Liscombe, D.K., Loukanina, N., MacLeod, B.P., Samanani, N., Zulak, K.G., 2007. Opium poppy: blueprint for an alkaloid factory. Phytochem. Rev. 6, 97–124. Kálmán-Pál, Á., Bernáth, J., Tétényi, P., 1987. Phenotypic variability in the production and alkaloid spectrum of the Papaver somniferum L. hybrid. Herba Hung. 26, 75–82. Krenn, L., Dobos, G., Gabriel, E., 1998. Alkaloidgehalt und -spektrum verschiedener Mohn-Genotypen. Z. Arzn. Gew. Pfl. 6, 118–124. Larkin, P.J., Miller, J.A.C., Allen, R.S., Chitty, J.A., Gerlach, W.L., Frick, S., Kutchan, T.M., Fist, A.J., 2007. Increasing morphinan alkaloid production by over-expressing codeinone reductase in transgenic Papaver somniferum. Plant Biotechnol. J. 5, 26–37. Millgate, A.G., Pogson, B.J., Wilson, I.W., Kutchan, T.M., Zenk, M.H., Gerlach, W.I., Fist, A.J., Larkin, P.I., 2004. Morphine-pathway block in top1 poppies. Nature 431, 413. Morice, J., Louarn, J., 1971. Study of morphine content in the oil poppy (P. somniferum L.). Ann. Amelior. Pl. 21, 465–485. Németh, É., 2002. World tendencies aims and results of poppy (Papaver somniferum L.) breeding. In: Govil, J.N., Kumar, A.P., Singh, V.K. (Eds.), Series Recent Progress in Medicinal Plants Biotechnology and Genetic Engineering, 4. SCI TECH Pub, Houston, USA, pp. 129–141. ˝ F., 2002. New results of poppy (Papaver Németh, É., Bernáth, J., Sztefanov, A., Petheo, somniferum L.) breeding for low alkaloid content in Hungary. Acta Hortic. 576, 151–158. Németh, É., Bernáth, J., Jászberényi, Cs., 2010. Studies on the inheritance of poppy (Papaver somniferum) alkaloids and the new cultivar ‘Korona’ accumulating high concentrations of narcotine. Acta Hortic. 860, 153–160. Nyman, U., 1979. Papaver somniferum L.—Breeding for a Modified Morphinane Alkaloid Pattern. University of Uppsala, Dissertation. Psenak, M., 1998. Biosynthesis of morphinane alkaloids. In: Bernáth, J. (Ed.), PoppyGenus Papaver. Harwood Academic Press, Amsterdam, pp. 159–188. Singh, S.P., Khanna, K.R., 1991. Heterosis in opium poppy. Ind. J. Agric. Sci. 61, 259–263. Singh, S.P., Tiwari, R.K., Dubey, T., 1999. Heterosis ind inbreeding depression in opium poppy (Papaver somniferum). J. Med. Arom. Plants 21, 23–25. Ziegler, J., Facchini, P.J., Geissler, R., Schmidt, J., Ammer, Ch., Kramell, R., Voigtlander, S., Gesell, A., Pienkny, S., Brandt, W., 2009. Evolution of morphine biosynthesis in opium poppy. Phytochemistry 70, 1696–1707.