In Vitro Regeneration of Persian Poppy (Papaver Bracteatum)
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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 devi- ation. 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 temper- ature 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.