Tom XXV ROŚLINY OLEISTE 2004 OSEVA PRO Ltd., Research Institute of Oilseed Crops at Opava, Czech Republic Poppy breeding with increased content of thebaine * Hodowla maku o podwyższonej zawartości tebainy Key words: Papaver somniferum, thebaine, seed yield, commercial characteristics Thebaine is a precursor of morphine and codeine and so there may be a mutation of poppy (Papaver somniferum) containing an increased content of thebaine. Using screening methods of TLC and precise HPLC, studies were made of poppy (Papaver somniferum) genetic resources and breeding materials under development. About 10% of genotypes containing thebaine were identified but in a majority of genotypes the progenies produced unstable thebaine content. Lines with stable, although relatively low thebaine content, high seed yields and very good commercial characteristics were found. Słowa kluczowe: Papaver somniferum, tebaina, plon nasion, charakterystyka ekonomiczna W Czechach, w przeciwieństwie do innych państw, uprawa maku ma duże znaczenie gospodarcze. W Azji i Europie mak postrzegany jest jako źródło opiatów i z tego względu jego uprawa jest znacznie ograniczana, w Czechach natomiast mak uprawiany jest na 40 000 ha, co stanowi o dominującej pozycji tego kraju jako producenta maku w Europie. Ponad połowa maku w Czechach jest uprawiana z przeznaczeniem tylko na zbiór nasion wykorzystywanych w przemyśle spożywczym i jest to główny kierunek produkcji, pozostała część służy również pozyskaniu opiatów. Głównym alkaloidem pozyskiwanym z maku w Czechach jest morfina. Może ona być łatwo przekształcana w diacetylomorfinę znaną jako heroina. Przedsiębiorstwa farmaceutyczne preferują obecnie tebainę przydatną do produkcji nieaktywnych analogów trudno przekształcalnych w heroinę. Tebaina jest prekursorem morfiny i kodeiny, i z tego względu możliwa jest mutacja powodująca zahamowanie syntezy alkaloidów na etapie tebainy. Formy o zmienionym w taki sposób składzie alkaloidów uzyskano w Tasmanii przy użyciu mutacji chemicznej. W przedstawionej pracy podjęto próbę znalezienia genetycznych źródeł wysokiej zawartości tebainy w roślinach Papaver somniferum. Materiał do badań stanowiły zasoby genotypów Papaver somniferum znajdujące się w kolekcjach Instytutu Badawczego Roślin Oleistych w Opawie i Uniwersytetu Rolniczego w Pradze. Badane genotypy analizowano pod względem zawartości i składu alkaloidów przy użyciu chromatografii cienkowarstwowej TLC i dokładnych metod chromatografii cieczowej HPLC. Przebadano 565 roślin pochodzących ze 107 odmian kolekcyjnych i 1399 roślin z 241 linii hodowlanych. Tebainę w kombinacji z innymi alkaloidami, głównie morfiną, zawierało 259 roślin, a zaledwie 5 roślin zawierało samą tebainę. W pokoleniu uzyskanym z wyselekcjonowanych, izolowanych roślin o podwyższonej zawartości tebainy zaledwie 8% utrzymywało zawartość tego alkaloidu. Znaleziono linie o stabilnej, chociaż stosunkowo niewielkiej zawartości tebainy i bardzo dobrych wartościach gospodarczych. * This paper is an outcome of Project No. 521/98/0512 funded by the Grant Agency of the Czech Republic and Project No. QD1143 funded by the Ministry of Agriculture of the Czech Republic.
614 Introduction Contrary to other countries, poppy has a special position in the Czech Republic. Poppy is well-known worldwide predominantly as a source of opium, and the utilization of seeds for culinary purposes is practically unknown. This does not apply only to Asia which is a major centre of opium production. The same opinion on poppy cultivation is expressed even by northern and western European countries. This view is also inherent in restrictive measures adopted by the European Union. The result is that poppy cultivation in these countries is practically impossible. The restrictions on poppy growing imposed also in other countries of Central Europe make the Czech Republic, where poppy occupies ca 40 000 ha of cropland, the largest producer of poppy in this area today. Poppy has been recognized by the Parliament of the Czech Republic as a national specialty of Czech agriculture. Complete harvesting and threshing of poppy and subsequent approved sale of empty capsules for industrial processing are among the state-aided agricultural activities (Team of authors 2004). Poppy is a conventional crop in the Czech Republic. As it is used predominantly for the food industry, the main goal of poppy growing is seed production. Poppy straw production for the pharmaceutical industry is only an additional program, and now it is harvested from about half of the total area. Most popular are seed-type varieties. In contrast to opium types, they give high seed yields and a relatively low content of alkaloids in capsules. In spite of that, poppy is still looked upon as opium and its growing is governed strictly by the law on intoxicants. The law strictly requires compulsory registration of poppy areas and determines the process and requirements connected with juice purchase. The main alkaloid of poppy in the Czech Republic is morphine. The most widely cultivated varieties contain 0.4 0.5% of morphine in their capsules. Morphine may be easily misused for drug production because diacetylmorphine known as heroin can be easily made from it. Pharmaceutical companies show concern about thebaine suitable for the production of non-addictive analgesics which can only hardly be converted into heroin. The aim of the study was to search for suitable genetic resources with high thebaine content in Papaver somniferum and by using them to develop a line with good economic characteristics and increased thebaine content in the poppy straw. Material and Methods The source material was genetic resources of poppy collected in the Oil Crop Research Institute in Opava and breeding materials at different stages of development from locations in Opava, Keřkov and from the Czech Agricultural University in
Poppy breeding with increased content of thebaine 615 Prague. These genotypes were analyzed for thebaine and codeine contents. Selected plants were bagged before blooming and the flower on the main stem from each plant was used. From the straightening bud just before flowering, sepals and petals were removed to prevent rotting of separate flowers and the rest of the flower was isolated using a paper bag. After stamens dropped off, the bag was removed. After maturation the empty capsules were analyzed for alkaloid contents. The seeds from isolated capsules were used for potential propagation of selected plants. The seeds of plants with higher thebaine and codeine contents and with high or very low contents of morphine were propagated again in the following year. The progenies were again analyzed for alkaloid contents and the aim was to test the stability of this trait. From the lines which exhibited the desirable contents of stable alkaloids, lines with good economic characteristics and blue seeds were selected. The economic characteristics of these lines were tested in a system of pre-trials which were designed following general guidelines on experimental work. The area of the plot was 10 m 2. There were four replications and the design of plots at replications was randomized. During the tests the guidelines on official variety trials were followed. The results were statistically analyzed. Alkaloid composition and contents were determined using a screening technique based on thin-layer chromatography (TLC) and a precise method of liquid chromatography (HPLC). The description of the methods is given in Havel et al. (2001). The methods were developed in such a way that the same sample could be used both for TLC and HPLC analyses. Therefore, there was no need to prepare samples for HPLC analysis, which made the analysis cheaper and more rapid. Results Table 1 shows the results of analysis of plants from source populations. A total of 565 plants from 107 varieties of the world collection and 1399 plants from 241 breeding lines were analyzed. A total of 259 plants (13.2% plants from source genotypes) exhibited non-zero content of thebaine in combination with other alkaloids, predominantly morphine. There were only 5 plants in which only thebaine without any other alkaloids was identified. In screening tests TLC was used. Even though this method is based just on visual evaluation, it is possible to detect several levels of thebaine content. Therefore, for other tests, materials with the highest contents of thebaine were selected. They accounted for about 20% of all the detected genotypes with nonzero content of thebaine. The results are presented in Table 2.
616 Table 1 Analysis of isolated plants from source populations using a screening method (TLC) Analizy wskazanych roślin z populacji wyjściowej przy użyciu TLC Source of material Materiał wyjściowy Number of genotypes Liczba genotypów Number of plants analyzed Liczba badanych roślin Plants containing thebaine Rośliny zawierające tebainę and other alkaloids i inne alkaloidy only wyłącznie Varieties Odmiany 107 565 75 0 Breeding lines Linie 241 1399 184 5 Total Ogółem 248 1964 259 5 Table 2 Determination of thebaine contents in progenies of plants selected in the first cycle of tests using TLC Zawartość tebainy w potomstwie roślin selekcjonowanych w pierwszym cyklu przy użyciu TLC Number of lines Liczba linii Number of plants tested Liczba testowanych roślin Plants containing thebaine Rośliny zawierające tebainę and other alkaloids i inne alkaloidy only wyłącznie 67 336 26 1 The results suggested that the stability of high content of thebaine in the progenies of plants selecte in the first cycle of tests is small. Only 8% of progenies retained higher thebaine contents in the juice. From these progenies 24 lines were selected and tested for stability of thebaine content. The results are given in Table 3. Table 3 Tests for stability of thebaine contents in selected progenies using TLC Test stabilności zawartości tebainy u wyselekcjonowanych potomstw, oznaczenie przy użyciu TLC Plants from the previous cycle of tests containing Rośliny poprzedniego cyklu testowania zawierające: Number of progenies analyzed Liczba analizowanych potomstw Number of plants analyzed Liczba badanych roślin Plants containing thebaine Rośliny zawierające tebainę and other alkaloids i inne alkaloidy only wyłącznie thebaine only tylko tebainę thebaine + other alkaloids tebainę + inne alkaloidy 1 5 0 0 23 94 10 2
Poppy breeding with increased content of thebaine 617 From the test of stability of thebaine content it follows that none of the lines containing only thebaine was stable enough during the entire cycle of tests. In a group of plants containing some other alkaloids (mainly morphine) besides thebaine, two lines showed relatively good stability of higher thebaine contents in capsules. In these lines thebaine content was determined by a precise method. The results are shown in Table 4. Table 4 Determination of thebaine content in stable lines using a precise method based on HPLC Zawartość tebainy oznaczona w stabilnych liniach przy użyciu metody HPLC No. of line/no. of plant Nr linii/nr rośliny Thebaine content [%] Zawartość tebainy 1/1 0.18 1/2 0.05 1/3 0.10 1/4 0.19 1/5 0.12 2/1 0.09 2/2 too small amount of sample niewystarczająca próbka 2/3 0.12 2/4 0.10 2/5 0.08 The highest thebaine content was found in plants no. 1/1 and 1/4. Both of them had a satisfactory colour of seeds, and therefore both were used as a basis of lines put into the system of performance tests. The blue-seeded poppy variety Gerlach with a medium content of morphine (0.3 0.5%) and a high yield of seeds served as a control. The results are given in Table 5. The results suggest that the parameters of both lines are comparable to the control variety. Both varieties produced a highly significantly higher seed yield. Discussion The main objective of the growing of poppy in the Czech Republic is seed production for the food industry. The production of poppy straw for the pharmaceutical industry is only subsidiary. The highest yields of poppy seeds reach normally 1.5 t/ha, but the mean yield in the year 2003 was 0.51 t/ha. Empty capsules yield is about 80% of seed yield. The only harvesting technology which is now available is direct harvesting of a mixture of poppy seeds and juice by a harvesting thresher. This technology causes significant losses due to capsule
618 Some parameters of lines with higher thebaine content Niektóre parametry linii z wysoką zawartością tebainy Table 5 Trait Cecha Gerlach Line 1 Line 2 Colour of blossom Kolor kwiatów white biały white biały white biały Colour of petal base Kolor podstawy kwiatów Hairiness under seed pods Owłosienie pod makówką violet fioletowy non-uniform niejednolite violet fioletowy medium średnie violet fioletowy heavy mocne Seed colour Kolor nasion blue niebieski blue niebieski blue niebieski Start of blooming Początek kwitnienia Helminthosporiosis on leaves (9 1) Helminthosporiosis na liściu Helminthosporiosis on seed capsules (9 1) Helminthosporiosis na makówkach 16 June 16 czerwca 14 June 14 czerwca 16 June 16 czerwca 7.0 5.0 4.8 4.8 5.0 5.0 Stand height [cm] Wysokość 96 100 96 Seed yield [t/ha] Plon nasion 1.34 1.38** 1.45** Empty capsules yield [t/ha] Plon pustych makówek 0.62 0.80 0.82 1000-seed weight [g] Masa 1000 nasion 0.40 0.54 0.40 ** statistically significant difference at the significance level α = 0.01 różnice statystycznie istotne na poziomie α = 0,01 crushing. This means that only half of the total weight of capsules is harvested. From France comes the information about the technology of harvesting of the entire above ground parts of poppy at green stage (personal communication). This technology, however, has not been introduced in the Czech Republic yet. Komberec (2002) stated that the average costs of poppy cultivation in the year 2001 were about 14,500 CZK/ha, in the year 2003 the costs (in practice) were around 19,000 CZK/ha. If the purchase price of juice were about 10 CZK/kg, it is impossible to make the production of juice cost-effective at this price. In the present situation the best way is to develop poppy which could serve a dual purpose, i.e. poppy with good yields of high-quality seeds and a good content of desirable alkaloids in the empty poppy capsules. By using mutation breeding, Sharma et al. (1999) developed the poppy variety Sujata which has a minimum content of alkaloids in the poppy straw. In the Czech Republic the Polish variety Przemko with a low content of morphine is registered. In Poland there are also low-morphine varieties Mieszko and Michalko
Poppy breeding with increased content of thebaine 619 which also have a specific colour of flower. This colour of the flower is then used for visual identification. Growing such varieties is practically safe because their utilization for drug production is impossible. On the other hand, this will be the end of utilizing the poppy straw as a valuable source of alkaloids for the pharmaceutical industry. The pharmaceutical industry purchases the morphine-containing juice on a large scale and its purchase is well organized in the Czech Republic. The cultivated varieties of poppy contain a relatively small amount of morphine, therefore the misuse of juice for drug production is not popular here. But there are still some risks, and that is the reason why poppy cultivation and juice purchase are governed by valid laws on opiates. Another alternative is thebaine utilization. The pharmaceutical industry needs thebaine because it produces non-addictive analgesics and other medications. During the synthesis of alkaloids in poppy, thebaine creates a precursor of codeine and morphine. It is therefore probable that there is a mutation in which the conversion of thebaine into other alkaloids is inhibited. Finding such a mutation was the aim of the project. During the tests approximately 10% of genotypes containing thebaine were identified. A majority of genotypes were, however, unstable and their progenies did not produce thebaine at all. Also, the lines with stable thebaine production were identified. These lines showed very good commercial characteristics, but their thebaine content was very low. The fact that the methods applied and the trends in breeding are good and can be fruitful one day is confirmed by the first reports from Tasmania on developing poppy with a high content of alkaloids (Fist 2003). The development of the line was based on mutation breeding with subsequent selection for alkaloid contents and composition. Alkaloids were determined in the latex of leaves by rapid HPLC. A recessive mutation of poppy Papaver somniferum was found and it contained practically no morphine and codeine. Now it is grown by the Tasmanian Alkaloids Company as the variety Norman. References Fist A. 2003. Produkce máku v Tasmánii. Příležitostná studie modifikovaného výzkumu a technologie, 12. Havel J., Kolovrat O., Kameníková L., Bechyně M. 2001. The search for genetic resources of opium poppy (Papaver somniferum) with high thebaine content and the development of a screening method. Czech J. Genet. Plant Breed., 37, 3: 88-92. Kolektiv autorů Sdružení Český mák informuje. 3. makový občasník Praha únor 2004. Komberec S. 2001. Ekonomika pěstování vybraných plodin v České republice ve sklizňovém roce. Farmář, únor 2002. Sharma J.R., Lal R.K., Gupta A.P., Misra H.O., Pant V., Singh N.K., Pandey V. 1999. Development of non-narcotic (opiumless and alkaloid-free) opium poppy, Papaver somniferum. Plant Breeding, 118, 5: 449-452. Situační a výhledová zpráva Olejniny. Ministerstvo zemědělství České republiky, leden 2004.