ACTA BIOLOGICA SLOVENICA LJUBLJANA 2004 Vol. 47, Št. 2: 45-56 Sprejeto (accepted): 2004-09-17 Tissue culture of pyrethrum (Tanacetum cinerariifolium (Trevir.) Schultz Bip.) Tkivna kultura bolhača (Tanacetum cinerariifolium (Trevir.) Schultz Bip.) Mateja GSPANI, Margareta VRTAČNIKI , Jana AMBROŽIČ DOLINŠEK2*, Maja KOVAČ3, Marjana CAMLOH3, Jana ŽEL3 I Faculty of Science and Engineering, University of Ljubljana, Vegova 4, P.O.B. 18 / 1, 1000 Ljubljana, Slovenia; tel: +386(0)1 5613552, e-mail: mateja.gspan@lek.si, metka.vrtacnik@guest.ames.si 2 Department ofBiology, University ofMaribor, Koroška 160, 2000 Maribor, Slovenia; fax:+386 62 28180, e-mail:jana.ambrozic@uni-mb.si ('corresponding author) 3 National Institute of Biology, Večna pot 111, 1000 Ljubljana, Slovenia; fax:+ 386(0) 14233388, e-mail: maja.kovac@nib.si, jana.zel@nib.si, marjana.camloh@nib.si Abstract. Pyrethrum (Tanacetum cinerariifolium), is plant species with the highest amount of natural insecticides - pyrethrins. An in-house information sys- tem for the development of different plant tissue cultures and marketing of their products was designed. The application of the relational information system in set- ting up a research hypothesis of high probability is discussed. By processing the information system, plant tissue cultures' parameters were identified, selected and modified. They were tested on the plant tissue culture of Tanacetum cinerariifoli- um. The influence of jasmonic acid on axillary shoot differentiation was studied. An inhibitory effect of jasmonic acid on shoot tissue culture differentiation was proven (100 µM, 10 µM), and a prediction method for determination of the variable optimal concentration interval was presented (between 0,5 and 4,5 µM). In addition a HPLC method was introduced for pyrethrins determination. Key words: Tanacetum cinerariifolium, pyrethrum, plant tissue culture, shoot tissue culture, axillary shoot, natural insecticides, pyrethrins, jasmonic acid, infor- mation system Izvleček. Bolhač (Tanacetum cinerariifolium) je rastlinska vrsta z največjo vsebnostjo naravnih insekticidov - piretrinov. Postavili smo informacijski sistem za razvoj različnih sistemov tkivnih kultur in trženje njihovih proizvodov. 46 Acta Biologica S!ovenica, 47 (2), 2004 Preučevali smo uporabo relacijskega informacijskega sistema za vpeljavo znanstvene hipoteze z visoko verjetnostjo. Z njegovo uporabo smo prepoznali, izbrali in priredili parametre sistema tkivnih kultur. Kot poskusni sistem smo uporabili tkivno kulturo bolhača in ugotavljali vpliv jasmonske kisline (JA) na tvorbo zalistnih poganjkov v tkivni kulturi. Dokazan je bil zaviralni učinek JA na diferencijo kulture poganjkov (100 µM, 10 µM) in predstavljena metoda za napoved intervala spre- minjanja optimalne koncentracije JA (med 0,5 in 4,5 µM) . Uvedli smo metodo HPLC za ugotavljan- je vsebnosti piretrinov. Ključne besede: Tanacetum cinerariifolium, bolhač, rastlinska tkivna kultura, tkivna kultura poganjkov, zalistni poganjki, naravni insekticidi, piretrini, jasmonska kislina, informacijski sistem Abbreviations: MS = Murashige and Skoog growth medium; JA = jasmonic acid; HPLC = high pressure liquid chromatography; IAA = indole-3-acetic acid; 2,4 D = 2,4-diclorophenoxy acetic acid; NAA = naphthalene acetic acid; BAP = 6-benzylamino purine; SD = standard deviation; R = corre- lation index; EPA = Environmental Protection Agency Introduction About 10 000 million insect species cause foodstuff and wood damage. Some species are even fatal, especially for people (ELLIOT 1995). Butan extensive usage of synthetic persistent insecticides in the last fifty years has led to their dangerous accumulation in the environment and rapid insect resistance development (C0SHRAN 1995). One possible way to minimize the effect of insecticides on biological diversity, and at the same tirne to ensure food quality, is the application of biodegradable insecticides with low toxicity for Mammals. One of the most promising group of such insecticides are pyrethrins (CASIDA & QUISTAD 1995). Pyrethrins are natura! stereoisomer mixtures of six monoterpenes esters (Fig. l). The basic compo- nents of pyrethrins are rethrolone (pyrethrolone, cinerolone and jasmolone) alcohols esterified with chrysanthemic monocarboxylic (pyrethrins I) or dicarboxylic/pyrethric (pyrethrins II) acid (TOMLIN 1994, CROMBIE 1995). Pyrethrins precursor chrysanthemic acid is a volatile monoterpene formed from mevalonic acid of isoprenoid metabolism. Chrysanthemyl alcohol with its typical cyclopropane derives from two molecules of isopentenyl pyrophosphate (IPP). Oxidation of chrysanthemyl alcohol yields chrysanthemic acid. The both have been identified from explant derived callus of pyrethrum. Chrysanthemic acid is found not only as the precursor of chrysanthemic dicarboxylic/pyrethric acid and pyrethrins I/11 but also the important precursor in squalene synthesis (KEsKITALO 1999). R ~ coo o Figure 1: Monoterpenes esters of pyrethrins. Slika 1: Monoterpenski estri piretrinov. R1 pyrethrin I jasmolin I cinerin I pyrethrin II jasmolin n cinerin II R CH3, Rl CH=CH2 R CH3, Rl CH2CH3 R CH3, Rl CH3 R C02CH3, Rl CH=CH2 R C02CH3, Rl CH2CH3 R C02CH3, Rl CH3 M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of ... 47 The detailed synthesis of rethrolones stili remains unclear. However the rethrolones, charecter- ized by the cyclopentene, are derived most properly from fatty acid metabolism. The composition of the crude extracts of pyrethrum flowers is as follows: one third is the active constituents pyrethrins and two thirds are linoleic and linolenic acids (40 %), alkanes and terpenoides (CR0MBIE 1995, KESKITALO 1999). The origin of jasmolone presumably derives from linoleic acid via 12-oxophyto- dienoic acid (12--oxo PDA). Experimentally is firmly established that 12 oxo-PDA undergoes hydro- genations and 8--oxidations to form jasmonic acid (CR0MBIE 1995). A natura] means for controlling a wide range of insects and rapid insecticidal action by pyrethrins is found in the composition of esters mixture. It was also found that a moderate use might cause a slow insect resistance development (Coshran 1995). Pyrethrins were found in some species, e.g. T. coccineum, but more potent is pyrethrum (T cinerariifolium). From the middle of 19th cen- tury to World War I, Dalmatia dominated in the pyrethrum production and trade. After the war the domination was replaced by Japan, and after World War II by Kenya, Tanzania and Australia (WAINAINA 1995, GULLICKSON 1995). There were always constraints on ensuring a sufficient supply because of the laborious work, demand for low labour costs, and weather dependency of plant pro- duction. In recent years, the efficacy of pyrethrins and the unstable supply, which can no longer meet the world demand (JOVETIC & DE G0OIJER 1995, ROYAL SOCIETY OF CHEMISTRY 1988, 1996), stimu- lated interest for their biotechnological production (HITMI & al. 2000). Analysis of patents concem- ing pyrethrins in the last thirty-five years has shown a distinctive increasing research interest during the last decade (Fig. 2). 30 -~ 25 ""' 41 20 -.CI 5 = = 15 "' .... = 41 .... ~ 10 CI. 5 O+------------,-----------------< 1960 1965 1970 1975 1980 1985 1990 1995 2000 2005 Figure 2: Oscillating pyrethrins research trend in course of tirne and increasing interest in the last decade (CAplus, march 2000). Slika 2: Nihanje zanimanja za raziskovanje piretrinov v preteklih letih in naraščanje zanimanja za piretrine v zadnjem desetletju (CAplus, marec 2000). An alternative production of pyrethrins could be in vitro production by plant tissue cultures, the main advantage of which is flexible control of biotechnological processes. Therefore plant tissue cul- 48 Acta Biologica Slovenica, 47 (1), 2004 tm:es are becoming a tool for producing many important products: e.g. quality food, wood, phy- totherapy compounds, biopesticides etc (SASSON 1992, WALTON & al. 1999, RAVNIKAR & ŽEL 1992). The application of the tissue culture technique for the pyrethrum plant has developed also from the self-incompatibility of the plant (PAL & DHAR 1985). For the successful commercialisation of a biotechnological process the most important criterion must be fulfilled - less expensive in vitro in relation to in vivo pyrethrins production (JovETic & DE GooIJER 1995). The basic research problem which we tried to solve was, how to optimise tissue culture cultiva- tion of a commercially potent plant to achieve quality products, e.g. pyrethrins. The problem was structured into severa! subproblems: (ELLIOT 1995) careful selection of a substance and its source - plant, (COSHRAN 1995) selection of economic cultivating conditions, and (CASIDA & QUISTAD 1995) target-oriented development of cost-effective products. Fragmented information on the defined sub problems is dispersed among severa! bibliographic and factual sources, while the technological parameters are most often considered proprietary information and are therefore publicly available (HUMPRIES & al. 1991, GULLICKSON 1995). To increase the information content of the accessible and relevant data and information, a relational information system for plant tissue cultures development was designed. The system was tested on the experiment where the effect of JA on shoot production of pyrethrum was studied. Methods Tissue culture Plants of Tanacetum cinerariifolium were obtained from the island of Cres, Croatia. Their seeds germinated in vivo and two months old vegetative plantlets of 8 cm height were used in spring for establishment of the shoot tissue culture on MS medium (MURASHIGE & SKOOG 1962) supplement- ed with 3 % sucrose, 1 % agar, 3 µM IAA and 2 µM BAP The pH was adjusted to 5.7 and the medi- um was sterilized at 121 °C for 20 min. The induced axillary buds had been regulary subcultures, every 4 to 5 weeks, for a year. The tissue cultures were grown in a growth chamber at 23 +/- 1 °C under 100 - 110 µM m·2s-1 daylight illumination with a 16 h photoperiod. Axillary buds were separated and cultivated in 50 ml test-tubes on modified MS medium with different concentrations of JA and control medium without JA. In two experiments three JA concen- trations were tested: 1 µM, 10 µM and 100 µM JA. Growth and differentiation measurement Two and four weeks after subculture (first experiment) the height of the central shoot and shoots number in fifteen test-tubes were determined, and the results were statistically evaluated by Student's t-test: * P < 0,05, ** P < 0,01, *** P < 0,001. In the second experiment the procedure was repeated after three and five weeks. Differentiation prediction To fulfil the remaining information gap a prediction method was introduced which wili have to be tested in further research. On the basis of ali and the selected average shoots number (data inside +/- 2 SD), measured after five weeks of cultivation, the curve with the highest correlation index (R) was determined: at concentration intervals between O to 1 O µM JA and between O to 1 µM JA. M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of... 49 Isolation and quantification of pyrethrins Approximately 1 g of in vitro grown shoots was crushed in a mortar with a silicic sand and ¼ anhydrous sodium sulfate to obtain a homogenous powder. Pyrethrins were extracted two to three times with 5 ml/g FW petroleum ether (40-60°C). The clear separated extract was evaporated to dry- ness with rotated evaporator, redissolved in 3 ml CH3CN, filtered through 0,22 µm mesh filter and analysed. Pyrethrins were analyzed by Waters HPLC system with a diode array (PDA) detector. Separations were performed on Nova Pack C18 column (Waters, 150 x 3.9 mm) using a gradi- ent of CH3CN (solvent A) and Milli Q HzO (solvent B): 40 % solvent A gradually increased to 80 % during 25 min, followed by a further 10 min at 80 %. Flow rate was 1.4 ml/min. Absorbance was monitored at 225 nm. Pyrethrins in the sample were identified on the basis of retention times and characteristic absorption spectra. Standards used were cinerin I and II, pyrethrin I and II andjasmolin I and II (Pyrethrins technical mixture, PESTANAL, Riedel-de-Haen). Results and discussion Relational information system The in-house information system supports problem solving through establishing relationships among five main modules: (a) bibliography, (b) substances (properties, structures, producers, prod- ucts), (c) world market, (d) plants and (e) photos (Fig. 3). Processing the relational information sys- tem for identifying the most effective parameters for biotechnological production of the biosynthet- ic high-yielding pyrethrum tissue culture done gave the following result (Table 1): Table 1: lnformation densities of breeding parameters' values of pyrethrum plant tissue cultures. Preglednica 1: Informacijska gostota parametrov vzgoje bolhača v rastlinskih tkivnih kulturah. Plant tissue culture cultivating parameters medium modification inoculum basic medium temperature irradiation mediumpH plant no. photoperiod light quality inoculum modification storage name storage temperature lnformation densities (number of defined values of specific parameter in documents) 38 14 14 14 13 10 9 9 4 1 1 1 WUIW) MA:lKET PLANTS Figure 3: The structure of five main modules (oval boxes) of the relational information system on plant tissue culture development. Slika 3: Struktura petih glavnih modulov (ovalna polja) relacijskega informacijskega sistema za razvoj rastlinskih tkivnih kultur. GOVERNMENT PUBLICATIONS PA'll!NTS LABORATORY REPORTS RF..SEARCH ORGANISA TIONS OTIIER DOCUMENTS-. STA'll! 1 STATE2 STATE3. NATURAL SUBSTANCES SYNTHETIC ANALOGUES INPOR1"/EXPORT m 1997 INPORT/EXPORT m 1998 INPORT/EXPORT in 1999 PYRETHRINS OTHERS .. AU.ETIIRINS RESMETHRINS PROPERTIES after 2 weeeks PROPERTJES after 3 weeh ' PROPERTIES after 4 weeks CLASSIFICA TION PHYSICAL/ CHEMICAL PROPERTIES TOXICOLOGY/ ECOTOXICOLOGY PRODUCERS PRODUCER 1 ACTIV~ INGRADIENTS FORMULA TION USAGE SALE PRlCE M. Gspan, M. Vrtačnik, J. Ambrožič Dolinšek, M. Kovač, M. Camloh, J. Žel: Tissue culture of... 51 Based on the information density (Table 1), the basic parameter which promotes plant produc- tion is medium modification. This result led to setting up a high probability hypothesis which assumed that the target-oriented medium modification would have the strongest impact on the growth, differentiation and biosynthetic activity of the pyrethrum tissue culture. The information gap on medium modification possibilities was bridged by additional processing of the Bibliographic module of the Relational information system, which gave examples of medium modifications with pyrethrin biosynthesis stimulation effect (Table 2). In the literature the following pyrethrum tissue culture's stimulation medium modifications were recognised (Table 2): auxins 2,4 D, NAA; cytokinins BA, BAP, kinetin; vitamin ascorbic acid, sucrose, diluted basic medium MS and nutrient stress. It is important to stress that, regarding the bib- liographic search in Chemical Abstracts, jasmonic acid (JA) was not reported as a pyrethrum plant growth regulator although it is quite widespread among plants. It is gradually synthesised from !inoleic acid, as is suggested for pyrethrins rethrolones (CROMBIE 1995). Table 2: Medium modifications with pyrethrins biosynthesis stimulative effect obtained from bibliograph- ic module of the information system. Preglednica 2: Gojišča, ki pospešijo biosintezo piretrinov. Podatki iz bibliografskega modula informaci- jskega sistema. Reference HITMI&al., 1998 HITMI & al., 1997 DHAR & PAL, 1993 RAJASEKARAN & al., 1991 RAVISHANKAR&al., 1989 RAJASEKARAN & al., 1990 Medium modification 2 µM BAP; 21,5 µM NAA 2 µM BAP; 21,5 µM NAA; ½ MS;l,8 g/1 sncrose 2,3 µM 2,4 D; 2,2 µM BA 0,9 µM 2,4 D; 23,2 µM kinetin; MS without agar and nitrates 0,57 mM ascorbic acid; 0,9 µM 2,4 D; 2,3 µM kinetin 0,9 µM 2,4 D; 23,2 µM kinetin Statistically proven inhibitory effect of jasmonic acid After two and four weeks an inhibitory effect of 100 µM JA on pyrethrum tissue culture growth and differentiation was observed and statistically proven (Figs. 4, 5). JA clearly inhibited number of 30 ~--------------·-- 25 .. 20 .. ..C: e 15 = = ** e 10 = .C: 5 "' o OJA 1 JA 10 JA IOOJA JA concentrations ( µM) Figure 4: The effect of JA on number of shoots after two (light bar) and four (darker bar) weeks in tissue culture (first experiment). Average shoot number (n> 10), standard deviation (SD), and statistical significant differences (t-test) between control media without JA and with different concentrations of JA are shown. Slika 4: Vpliv JA na število poganjkov po dveh (svetel stolpec) in štirih (temen stolpec) tednih tkivne kul- ture (prvi poskus). Prikazana so povprečna števila poganjkov (n>lO), standardne deviacije (SD) in sta- tistično značilne razlike (t-test) med kontrolnim gojiščem brez JA in z različnimi koncentracijami JA. 52 Acta Biolooica Slovenica, 47 (2), 2004 shoots, especially on the highest JA concentration (Fig. 4), and the same trend was observed in sec- ond experiment (data not shown). JA also inhibited the early lengthening of shoots. After two weeks of culture were shoots on the highest JA concentration significantly smaller then shoots on control media without JA (Fig. 5). Differences were not observed after four weeks of culture. I 4,0 3,5 ;; 3,0 j 2,5 g 2,0 ~ 1,5 "E 1,0 ~ 0,5 e o 0JA 1 JA I0JA IOO JA JA concentrations ( pM) Figure 5: The effect of JA on central shoot length after two (light bar) and four (darker bar) weeks in tissue culture (first experiment). Average shoot number (n>lO), standard deviation (SD), and statistical significant differences (t-test) between control media without JA and with different concentrations of JA are shown. Slika 5: Vpliv JA na velikost poganjkov po dveh (svetel stolpec) in štirih (temen stolpec) tednih tkivne kul- ture (prvi poskus). Prikazana so povprečna števila poganjkov (n>lO), standardne deviacije (SD) in statistično značilne razlike (t-test) med kontrolnim gojiščem brez JA in z različnimi koncentracijami JA. Prediction of Jasmonic acid stimulative effect Based on the results (Figs. 4, 5) it was assumed that a stimulatory JA concentration on pyrethrum tis- sue culture differentiation may lie at the interval between O to 10 µM. At intervals between O to 10 µM JA and O to 1 µM JA the curve that mostly fit both the experimental raw data measured after five weeks of cultivation (second experiment) as well as selected data (+/- 2SD) was found (Figs. 6 and 7). According to the curves' peaks, which indicate the most copious pyrethrum tissue culture differentiation, the inter- 7 30 ~-.--~-~--- -...---i--+----1---'--4----i o 9 JO 11 JA concentrations (pM) Figure 6: Curves of average values from all ( •) and selected ( • )