In Vitro Regeneration of Persian Poppy (Papaver Bracteatum)

Biologia 65/4: 647—652, 2010 Section Cellular and Molecular Biology DOI: 10.2478/s11756-010-0079-6

In vitro regeneration of Persian poppy (Papaver bracteatum)

Sara Rostampour1,2,HalehHashemi Sohi1*&AliDehestani3

1Department of Plant Biotechnology, National Institute of Genetic Engineering and Biotechnology, P.O. Box 14155–6343 Tehran, Iran; e-mail: [email protected] 2Department of Agronomy and Plant Breeding, University of Zabol, Zabol, Iran 3Department of Plant Breeding, Faculty of Agronomic Sciences, Sari Agricultural Sciences and Natural Resources Univer- sity, Sari, Iran

Abstract: Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25 ◦C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro wasachievedonľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum. Key words: Papaver bracteatum; callus induction; phytohormone; ascorbic acid. Abbreviations: BA, benzylamine purine; 2,4-D, 2,4-dichlorophenoxyacetic acid; DMR, Duncan’s multiple range; HPLC, high-performance liquid chromatography; MS, Murashige and Skoog; NAA, α-naphthalene acetic acid; SD, standard deviation.

Introduction there is still considerable demand. World requirements and the limited availability of codeine obtained directly Medicinal plants have been the subjects of man’s cu- from the poppy plant have made codeine production riosity since time immemorial (Constable 1990). In- through stable-cell cultures of the genus Papaver an ob- terest in phytomedicine has exploded in the last few vious target for exploitation. Papaver species produce years, and about 500 different plant species are used a wide range of isoquinolines, sometimes with very high as key ingredients, and many are still being collected yields, and within individual species there is consider- from the wild (Mendelsohn & Balick 1994). In vitro cell able intraspecific variation in alkaloid content (Phillip- and tissue culture methodology is envisaged as a mean son 1983). The major producers of the morphinans are for germplasm conservation to ensure the survival of Papaver somniferum L. and Papaver bracteatum Lindl., endangered plant species, rapid mass propagation for but this group of alkaloids has also been obtained in large-scale re-vegetation, and for genetic manipulation low yields from P. fugax L., P. setigerum D.C., P. ori- studies (Dev 1997). entale L., and P. rhoeas L. (Phillipson 1983), and from Opium is the dried cytoplasm of a specialized in- herbarium material of P. acrochaetum Borm., P. cau- ternal secretory system, the laticifer. When the unripe casicum Bieb., P. cylindricum Cullen., P. gracile Boiss., capsule is cut, cream-coloured latex oozes to the sur- and P. persicum Lind. (Wieczorek et al. 1986). Indus- face, where it dries to form a dark brown sticky mate- trial production of opiates from tissue culture is de- rial. Raw opium alkaloids have been identified in Pa- pendent on the large accumulation of alkaloids in a paver (Santacy 1970; Bentley 1971), at least 25 of which cell culture medium. While there has been great suc- occur in the latex (Osol & Pratt 1973). However, from a cess in plant-cell culture in terms of cells with high medicinal viewpoint the benzylisoquinolines, papaver- yields of isoquinolines, from commercial and pharma- ine and noscapine, and the phenanthrenenes, codeine ceutical points of view, the morphinans have proven dif- and morphine are of prime importance. The opiates ficult to produce in plant-cell cultures. Most cultured are industrial commodities of plant origin for which Papaver cells, in the form of calluses or cell suspen-

* Corresponding author

c 2010 Institute of Molecular Biology, Slovak Academy of Sciences 648 S. Rostampour et al. sion, readily produce sanguinarine, dihydrosanguinar- induction (appearance of calli) were recorded 2–12 weeks af- ine, norsanguinarine, and oxysanguinarine (Ikuta et al. ter culture. 1974; Kutchan et al. 1985). Numerous reports of the productions of the morphinans (thebaine, codeine, and Plant regeneration morphine) from cell cultures of P. somniferum and P. Preliminary experiments for testing the potential of regen- bracteatum can be found in the literature, although eration of calli were performed using different combinations yields are low compared with the high yields of the and concentrations of the above-mentioned cytokinins and auxins. Based on the test, calli were transferred into the plants. Researchers suggest that culture conditions can MS medium containing various combinations and concen- be manipulated to promote morphinan alkaloid produc- trations (0.0, 0.1, 0.5, 1.0 and 2.0 mg/L) of BA and NAA tion (Constable 1990). for regeneration. All the cultures were then incubated at We previously reported the useful protocol to in- 25 ◦C in a growth chamber with lighting of approximately troduce foreign genes into transgenic Persian poppy 1,000 lux (16 hour/day). hairy root cultures using Agrobacterium rhizogenes strain R15834 (Rostampour et al. 2009). Callus of Media and culture conditions transgenic roots were induced by 1.0 mg/L α-naphthal- The basal medium consisted of B5 or MS salts and vitamins ene acetic acid (NAA). The aim of this study was to supplemented with 3% sucrose (w/v) and solidified with 1% provide an efficient protocol for optimization of tissue (w/v) agar. The media were adjusted to pH 5.8 with 1.0 M culture in P. bracteatum as influenced by phytohor- KOH before adding agar, and then sterilized by autoclaving at 121 ◦C for 20 min. mones, ascorbic acid and temperature with different media and hormone doses. The findings of this study Statistical analysis should help to enable the production of alkaloids of bi- In this study, two cultures were raised for each treatment ological origin under controlled conditions. and each treatment was repeated thrice. Test of significance was carried out by ANOVA and the data mean±SD (stan- Material and methods dard deviation) were analyzed by Duncan’s multiple range (DMR) tests using the SAS program. Plant material The commonly cultivated Persian poppy (P. bracteatum High performance liquid chromatography (HPLC) analysis Lindl.) used as experimental material was obtained from The leaves of regenerated Persian poppy were frozen in liq- Guilan, northern Iran. Seeds were supplied by Dr. Hashemi uid N2, and extracted with methanol in a boiling water Sohi, Department of Plant Biotechnology, National Institute bath for 15 min. Extracts were reduced to dryness under of Genetic Engineering and Biotechnology, Tehran, Iran. vacuum, dissolved in 1.0 M sodium carbonate/bicarbonate (3:2, w/w), pH 10.0, and extracted three-times with ethyl Seed germination and cultivation of sterile seedlings acetate. Pooled ethyl acetate fractions were reduced to dry- Seeds, roots, cotyledons and hypocotyls of Persian poppy (P. ness and the residue taken up in 1 mL of methanol. Extracts bracteatum Lindl.) were surface-sterilized using 70% (v/v) were analyzed using a System Gold 126 HPLC (Beckman- ethanol for 1 min followed by three-times washing with dis- Coulter). Alkaloids were separated at a flow rate of 0.75 tilled sterile water. They were then kept in 5% (v/v) sodium mL/min on a C18 reverse phase column (4.6×250 mm, hypochlorite solution for 10 min followed by three washes Ultrasphere, Beckman-Coulter) using methanol:water (6:4, with distilled sterile water. The cotyledons and hypocotyls v/v) containing 0.1% (v/v) triethylamine. The identity of ± were cut into 5 1 mm segments from 7–day-old seedlings, peak was routinely analyzed by comparison of UV spectra and used as explants. and retention times with that of identified alkaloid. Induction and proliferation of callus The surface-sterilized seeds, roots, cotyledons and hypoco- Results and discussion tyls were placed on B5 (Gamborg et al. 1968) and Murashige and Skoog (MS) (Murashige & Skoog, 1962) media. Solu- Induction and proliferation of callus and plant regener- tions of cytokinins: kinetin, and 6-benzylaminopurine (BA), ation and auxins: NAA and 2,4-dichlorophenoxyacetic acid (2,4- Two different plant tissue culture media (MS and B5) D), were filter-sterilized and added in different combina- were used, and in addition to various combinations tions to the autoclaved medium when the medium temperature dropped to about 50 ◦C. All media were supplemented and concentrations of growth regulators, mainly aux- with 3% sucrose, various combinations and concentrations ins and cytokinins were used to find out their effects of cytokinins and auxins (0.0, 0.1, 0.5, 1.0 and 2 mg/L) on the callus induction (Komamine et al. 1992). Aux- and various concentrations of ascorbic acid (0.0, 5.0, 10.0, ins and cytokinins are the most widely used plant 15.0 and 20.0 mg/L) for the induction of callus at 20 ◦Cand growth regulators in plant tissue culture and usually ◦ 25 C in dark. Ten seeds, roots, cotyledons and hypocotyls used together (Gang et al. 2003). Subsequently plant were placed on 25 mL of agar-solidified culture medium in growth regulators were added into media to test their petri-dishes which were sealed with parafilm. Each petri- effects on callus formation from seed, root, cotyledon dish was considered an experimental unit. Each experiment and hypocotyl explants of P. bracteatum.Amongthe contained at least 30 replicates and the experiments were repeated three times. Well grown callus induced from ex- auxins, 2,4-D has been considered to be the most ef- plants were selected to transfer to media with appropriate fective in combination with cytokinins for callus in- hormones for subculture. Cultures were subcultured to the duction (Hazra et al. 1989; Michalczuk et al. 1992; De same medium every 15 days. Explants that responded to the Jong et al. 1993). This study showed that 2,4-D with In vitro regeneration of Persian poppy 649

Fig. 1. Callus induction and plant regeneration of Persian poppy (P. bracteatum). (A) Bright brown callus form seed after 8 weeks; (B) shoots regeneration from seed’s callus after 2 subcultures; (C) bright brown callus form root after 8 weeks; (D) shoots regeneration from root’s callus after 5 subcultures; (E) bright brown callus form hypocotyl after 16 weeks; (F) shoots regeneration from hypocotyl’s callus after 9 subcultures; (G) bright brown callus form cotyledon after 12 weeks; (H) shoots regeneration from cotyledon’s callus after 8 subcultures. All was induced on 3/4 MS medium containing 1.0 mg/L 2,4-D, 0.1 mg/L kinetin and 15 mg/L ascorbic acid at 25 ◦C in dark. Shoot regeneration from callus was achieved on 3/4 MS medium containing 0.5 mg/L BA and 1.0 mg/L NAA. a cytokinin, kinetin, was the most effective combina- tant role in the callus induction and different types of tion for inducing of callus in P. bracteatum (Table 1). auxins had various effects (Skoog & Armstrong 1970; Several researches revealed that auxins play an impor- Baskaran et al. 2006). Furthermore, the cytokinins fa- 650 S. Rostampour et al.

Table 1. The induction and proliferation of callus from seed, root, cotyledon and hypocotyl of Persian poppy with various combinations and concentrations of cytokinins and auxins.

MSa medium supplemented withb Induction rate (% ± SD)c No. 2,4-D NAA BA kinetin Seed Root Cotyledon Hypocotyl

10.100.500000 2 0.5 0 0.5 0 8.0 ± 1.7000 3 1.0 0 0.5 0 11.0 ± 2.1000 42.000.500000 50.101.000000 60.501.000000 7 1.0 0 1.0 0 12.0 ± 1.3000 82.001.000000 90.1000.10000 10 0.5 0 0 0.1 25.0 ± 3.9 22.8 ± 4.1 17.0 ± 2.7 19.0 ± 3.4 11 1.0 0 0 0.1 74.6 ± 1.2 55.0 ± 3.4 41.2 ± 2.1 43.2 ± 1.3 12 2.0 0 0 0.1 6.5 ± 2.3000 130.1000.50000 14 0.5 0 0 0.5 13.4 ± 1.3 7.4 ± 2.2 4.0 ± 1.5 4.5 ± 1.2 15 1.0 0 0 0.5 28.0 ± 2.8 16.5 ± 2.5 13.7 ± 1.8 10.0 ± 1.3 16 2.0 0 0 0.5 4.3 ± 1.4000 1700.10.500000 1800.50.500000 1901.00.500000 2000.12.000000 2100.52.000000 2201.02.000000 2300.100.10000 2400.500.10000 25 0 1.0 0 0.1 0 32.0 ± 2.4 0 0 2600.100.50000 27 0 0.5 0 0.5 0 0 h 0 0

a MS medium comprised of MS mineral salts and vitamins, 3% (w/v) sucrose, and 1% (w/v) agar. b MS medium supplemented with respective combinations of growth regulators at the indicated concentrations. c Treatment means ± S.D were separated by DMR test at 5% level.

Table 2. Eﬀect of ascorbic acid on callus induction.

Induction rate (% ± SD)c MSa medium supplemented withb Seed Root Cotyledon Hypocotyl

0.5 mg/L 2,4-D + 0.1 mg/L kinetin + 5.0 mg/L ascorbic acid 23.2 ± 1.3000 0.5 mg/L 2,4-D + 0.1 mg/L kinetin + 10 mg/L ascorbic acid 25.0 ± 2.2000 0.5 mg/L 2,4-D + 0.1 mg/L kinetin + 15 mg/L ascorbic acid 27.5 ± 1.8000 0.5 mg/L 2,4-D + 0.1 mg/L kinetin + 20 mg/L ascorbic acid 25.1 ± 1.3000 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 5.0 mg/L ascorbic acid 68.0 ± 2.3 49.2 ± 2.1 38.0 ± 2.1 39.5 ± 2.5 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 10 mg/L ascorbic acid 76.6 ± 1.2 55.3 ± 1.3 41.4 ± 1.7 44.7 ± 1.2 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 15 mg/L ascorbic acid 82.0 ± 2.4 58.3 ± 2.1 46.0 ± 1.8 49.5 ± 2.0 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 20 mg/L ascorbic acid 55.8 ± 1.4 32.5 ± 1.7 29.5 h± 1.6 30.4 ± 2.1

Table 3. Eﬀect of temperature on callus induction.

Induction rate (% ± SD)c Temperature MSa medium supplemented withb Growing state ( ◦C) Seed Root Cotyledon Hypocotyl

20 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 10 mg/L ascorbic acid 76.6 ± 1.2 55.3 ± 1.3 41.4 ± 1.7 44.7 ± 1.2 Bright brown 25 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 10 mg/L ascorbic acid 79.3 ± 4.2 58.0 ± 2.8 44.5 ± 3.7 48.0 ± 3.5 Bright brown 20 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 15 mg/L ascorbic acid 82.0 ± 2.4 58.3 ± 2.1 46.0 ± 1.8 49.5 ± 2.0 Bright brown 25 1.0 mg/L 2,4-D + 0.1 mg/L kinetin + 15 mg/L ascorbic acid 94.2 ± 6.2 77.5 ± 2.8 74.2 ± 3.0 76.7 ± 1.5 Bright brown

Fig. 2. HPLC elution profile of methanol extracts from wild-type (A) and leaves of regenerated plants (B) from Persian poppy. Peak of thebaine identified in figures exhibits UV spectra with benzylisoquinoline alkaloid signatures. cilitated the effect of auxin in callus induction (Rao et the other hand, Ilahi & Ghauri (1994) reported that al. 2006; Yang et al. 2008). Other growth-regulator com- callus was induced on P. bracteatum Lindl. seedlings binations, such as BA with 2,4-D, kinetin with NAA, inoculated on MS medium supplemented with NAA or NAA with BA, were not effective. (1.0 mg/L) and BA (0.5 mg/L). Shoots were regener- As shown in Table 1, callus was induced on MS ated in cultures grown on MS medium containing NAA medium supplemented with 3% sucrose (w/v) and 1% (1.0 mg/L), BA (0.5 mg/L) and casein hydrolyzate (2.0 (w/v) agar. The best cytokinin and auxin combina- mg/L). MS at full strength was found inhibitory for cal- tion was 1.0 mg/L 2,4-D, 0.1 mg/L kinetin and 15 lus induction and proliferation, but 1/2 MS was suit- mg/L ascorbic acid. The induction percentage initially able. Similarly, callus growth was very slow at 25 ◦C, increased then decreased with increasing 2,4-D concen- but it increased when the temperature was lowered to trations, with maximum callus observed on MS medium 20 ◦C as did bud initiation (Ilahi & Ghauri 1994). Max- with 1.0 mg/L 2,4-D. A further evaluation of 2,4-D ef- imum shoot regeneration was observed on MS medium fects showed that 2,4-D alone at either of the follow- containing 1.0 mg/L NAA and 0.5 mg/L BA (Fig. 1). ing concentrations: 0.1, 0.5, 2 and 3 mg/L, failed to Results showed that there was no significant difference induce callus. On the other hand, addition of kinetin between explants in terms of regeneration efficiency. and ascorbic acid together with 2,4-D developed callus growth (Table 2). HPLC analysis of thebaine alkaloid The callus induction initially was very slow at The benzylisoquinoline alkaloid content of Persian 20 ◦C, but increased when the temperature was in- poppy leaves was analysed by HPLC. Although the creased to 25 ◦C in dark (Table 3). However, differences only available authentic standard was thebaine, sev- based on explants were observed. As shown in Figure eral other chromatographic peaks displayed UV spec- 1, the induction of calli in cotyledons and hypocotyls tra. The identity of peak was routinely analyzed by was slower than that in seeds and roots. Results in- comparison of UV spectra and retention times with that dicated that calli in seed and root explants were in- of identified alkaloid. Analysis of alkaloid extracts from duced about 8 weeks after culture but calli in cotyle- Persian poppy tissues by HPLC revealed that thebaine dons and hypocotyl explants were induced about 12–16 accumulates in the tissues of plants (Fig. 2). weeks after culture. The maximum induction rate was Numerous research reports exist in the literature recorded as 94.2% in seeds and 77.5% in roots in MS about the effects of plant growth regulators on sec- medium with 1.0 mg/L 2,4-D, 0.1 mg/L kinetin and 15 ondary metabolites of in vitro cultures. In certain mg/L ascorbic acid at 25 ◦C in dark. The well-grown cases, they enhance alkaloid production in several plant callus were selected for subculture on MS medium sup- species, for example, berberine in Coptis japonica (Nak- plemented with 1.0 mg/L 2,4-D, 0.1 mg/L kinetin, 15 agawa et al. 1986; Ikuta & Itokawa 1988) and san- mg/L ascorbic acid, 3% sucrose (w/v) and 1% (w/v) guinarine in P. somniferum (Tyler et al. 1989). How- agar to test their growing state. After about 6 sub- ever, in this study, it seems the plant growth regulators cultures, the callus multiplied more quickly. Callus on did not have effects on the accumulation of alkaloid, MS medium containing 2,4-D, kinetin and ascorbic acid and the content of thebaine in tissues of P. bracteatum was initially bright yellow. After 12 weeks, callus was was identical with that of wild-type plants (Fig. 2). bright brown (Fig. 1). The results indicated that MS at To the best of our knowledge, this is the first report full strength was found inhibitory for callus induction, of callus induction from seeds, roots, cotyledons and while 3/4 MS was found to favour callus induction. On hypocotyls of Persian poppy (P. bracteatum Lindl.). It 652 S. Rostampour et al. can be noted that efficient callus can be initiated on Komamine A., Kawara R., Matsumoto M., Sunabori S., Toya T. 3/4 MS medium containing 1.0 mg/L 2,4-D, 0.1 mg/L & Fujimura T. 1992. Mechanisms of somatic embryogenesis in kinetin and 15 mg/L ascorbic acid at 25 ◦Cindark. cell cultures: physiology, biochemistry, and molecular biology. In Vitro Cell. Dev. Biol. 28P: 11–14. Plant regeneration of the shoots in vitro can be achieved Kutchan T.M., Ayabe S. & Coscia C.J. 1985. Cytodifferentiation on 3/4 MS medium containing 0.5 mg/L BA and 1.0 and Papaver alkaloid accumulation, pp. 281–294. In: Phillip- mg/L NAA. This established regeneration method is son J.D., Roberts M.F. & Zenk M.H. (eds), The Chemistry and Biology of Isoquinoline Alkaloids, Springer-Verlag, Berlin simple and effective to study of alkaloid content in P. and New York. bracteatum. In addition, the thebaine alkaloid profile Mendelsohn R. & Balick, M. 1994. The value of undiscovered of the Persian poppy is a well-defined model to evalu- pharmaceuticals in tropical forests. Econ. Bot. 49: 223–228. ate the potential for metabolic engineering of thebaine Michalczuk L., Cooke T.J. & Cohen J.D. 1992. Auxin levels at different stages of carrot somatic embryogenesis. Phytochem- production in P. bracteatum. istry 31: 1097–1103. Murashige T. & Skoog F. 1962. A revised medium for rapid Acknowledgements growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15: 473–497. Nakagawa K., Fukui H. & Tabata M. 1986. Hormonal regulation The authors would like to acknowledge National Institute of berberine production in cell suspension cultures of Thalic- of Genetic Engineering and Biotechnology (NIGEB), Iran, trum minus. Plant Cell. Rep. 5: 69–71. for providing financial support. Osol A. & Pratt R. 1973. Dispensatory of the United States of America. Lippincott, Philadelphia, PA, 1292 pp. Phillipson J.D. 1983. Intraspecific variation and alkaloids of Pa- References paver species. Planta Med. 48: 187–192. Rao A.Q., Hussain S.S., Shahzad M.S., Bokhari S.Y.A., Raza Baskaran P., Raja Rajeswari B. & Jayabalan. N. 2006. Develop- M.H., Rakha A., Majeed A., Shahid A.A., Saleem Z., Hus- ment of an in vitro regeneration system in sorghum [Sorghum nain T. & Riazuddin S. 2006. Somatic embryogenesis in wild bicolor (L.) Moench] using root transverse thin cell layers relatives of cotton (Gossypium spp.). J. Zhejiang Univ. Sci. (tTCLs). Turk. J. Bot. 30: 1–9. B 7: 291–298. Bentley K.W. 1971. The morphine alkaloids, pp. 3–163. In: Rostampour S., Hashemi Sohi H., Jourabchi E. & Ansari E. Manske R.H.F. (ed.), The Alkaloids; Chemistry and Physi- 2009. Influence of Agrobacterium rhizogenes on induction of ology, Vol. 13, Academic Press, New York. hairy roots and benzylisoquinoline alkaloids production in Constable F. 1990. Medicinal plant biotechnology. Planta Med. Persian poppy (Papaver bracteatum Lindl.): preliminary re- 56: 421–425. port. World J. Microbiol. Biotechnol. 25: 1807–1814. De Jong A.J., Schmidt E.D.L. & De Vriess S.C. 1993. Early events Santacy F. 1970. Papaveraceae alkaloids, pp. 333–454. In: Manske in higher plant embryogenesis. Plant Mol. Biol. 22: 367–377. R.H.F (ed.), The Alkaloids; Chemistry and Physiology, Aca- Dev S. 1997. Ethnotherapeutics and mordern drug development: demic Press, New York. the potential of Auerveda. Curr. Sci. 73: 909–928. Skoog F. & Armstrong D.J. 1970. Cytokinin annual review. Plant Gamborg O.L., Miller R.A. & Ojima K. 1968. Nutrient require- Physiol. 21: 359–384. ments of suspension cultures of soybean root cells. Exp. Cell Tyler R.T., Eilert U., Rijinders C.O.M., Roewer I.A., McNabb Res. 50: 151–158. D.K. & Kurz W.G.W. 1989. Studies on benzophenanthri- Gang Y.Y., Du G.S., Shi D.J., Wang M.Z., Li X.D. & Hua Z.L. dine alkaloid production in elicited cultured cell cultures of 2003. Establishment of in vitro regeneration system of the Papaver somniferum, pp. 200–207. In: Kurz W.G.W. (ed.), Atrichum mosses.ActaBot.Sin.45: 1475–1480. Primary and Secondary Metabolism of Plant Cell Cultures, Hazra S., Sathaye S.S. & Mascarenhas S.F. 1989. Direct somatic Spring-Verlag, Berlin. embryogenesis from peanut (Arachis hypogaea L.). BioTech- Wieczorek U., Nagakura N., Sund C., Jendrzejewski S. & Zenk, nology 7: 949–951. M.H. 1986. Radioimmunoassay determination of the six Ikuta A. & Itokawa H. 1988. Alkaloids of tissue cultures of Nan- opium alkaloids. Phytochemistry 25: 2639–2646. dina domestica. Phytochemistry 27: 2143–2145. Yang J, Gong Z.C. & Tan X. 2008. Induction of callus and ex- Ikuta A., Syono K. & Furaya T. 1974. Alkaloids of callus tis- traction of alkaloid from Yi Mu Cao (Leonurus heterophylus sues and redifferentiated plantlets in the Papaveraceae. Phy- Sw.) culture. Afr. J. Biotechnol. 7: 1157–1162. tochemistry 13: 2175–2179. Ilahi I. & Ghauri E.G. 1994. Regeneration in cultures of Papaver Received December 14, 2009 bracteatum as influenced by growth hormones and tempera- Accepted March 17, 2010 ture. Plant Cell Tissue Organ Cult. 38: 81–83.