Art18_Nemeth.indd Journal of Applied Botany and Food Quality 85, 116 - 119 (2012) 1Department of Medicinal and Aromatic Plants, Corvinus University of Budapest, Hungary Connection of frost tolerance and alkaloid accumulation potential in poppy (Papaver somniferum L.) C. Jászberényi1, É. Németh1 (Received December 22, 2011) Summary In the Central-European region, a growing interest for winter poppy varieties both for industrial and culinary purposes can be observed. In connection with breeding, we studied if winter hardiness and al- kaloid accumulation capacity are linked in some way. Experimental plots sown in autumn and thinned (selected) due to winter frosts were compared with spring sown ones of the same genotypes during 2009-2010. We found, that the overwintered populations exhibited signifi cantly lower total alkaloid contents (1.065%) than the ones from spring sowing (1.479%). This tendency was proved for each of the individual alkaloid compounds morphine, codeine, thebaine and narcotine, too. At the same time, maximum values in some in- dividual samples of the overwintered plants reached similar or higher contents than the spring sown ones. Therefore it is supposed that even if gene frequency in direction for enhanced alkaloid syn- thesis might be larger in frost sensitive ecotypes, the potential for breeding high alkaloid accumulating cultivars exists also among frost tolerant genotypes. Introduction In Hungary, poppy (Papaver somniferum L.) is an important indus- trial crop which is cultivated on large scale thorough the country, on more than ten thousand hectares. Besides, poppy is an essential culinary crop producing delicious, fatty oil containing seeds both for food industry and the households. In the Central-European region, especially in Hungary and Austria, poppy cultivation may take place either as winter or a spring crop, similarly to cereals. Winter poppy ecotypes possess enhanced frost tolerance in leaf rosette stage, while spring poppy is generally killed or severely damaged by frosts un- der average winter conditions. Cultivation of winter poppy in our region obtained enhanced signifi cance in the last decade, because of the higher and more stable yields among the changing and varying climatic conditions. Until recently, breeding has not been focused on this aspect. In the Hungarian list of varieties, only fi ve cultivars belong to the winter poppy group while 14 ones are registered as spring poppy cultivars. Breeding has now started to establish new, frost tolerant genotypes which possess also high capsule and seed yields and can be used either as industrial varieties of high alkaloid contents or as culinary ones with very low alkaloid contents in coincidence with the 162/ 2003. (X. 16.) Government Decree of Hungary. Unfortunately, there is practically no scientifi c background infor- mation on the genetical determination of frost tolerance in poppy. Not even the physiological or morphological protection mechanisms against low temperatures are fully ascertained. Our recent results suggested, that proline and soluble sugars (glucose, fructose, suc- rose) might contribute to the osmotic protection mechanism against cold stress, however, presumably it is not the only and main factor in it (JÁSZBERÉNYI and NÉMETH, 2011a). DOBOS (2010) observed that winter poppy is a morphologically adapted genotype with greyish coloured and horizontally laying leaf rosette, violet coloured petals and shorter vegetation cycle compared with spring types. However, these traits may only marginally stay in real connection with winter hardiness. Information on the inheritance of opiate alkaloids is much more abundant (BERNÁTH and NÉMETH, 2009; NÉMETH et al., 2011). However, it has been proved, that accumulation of individual al- kaloids is regulated in a complex, metabolon like system including multigene effects, enzymatic interactions and compartmentation. Therefore, till now practical results in breeding have mainly been achieved by individual selection. In the practice it seems to be accepted that winter tolerant genotypes are not able to accumulate high levels of alkaloids. However, as no scientifi c information and experimental results exist on the linkage of the winter hardiness and alkaloid content, it can be presumed as a statement based on limited information and experience. Therefore, in our work we studied the question if there is any well established connection between alkaloid accumulation and the overwintering potential of poppy genotypes. Using several breeding strains – be- cause of the varieties of original spring ecotype usually killed by frost – and sowing them both in autumn and spring, we examined if the selection by winter frosts resulted in any shift in alkaloid accu- mulation level and/or pattern of the capsules of the same genotypes. Materials and methods Our examination was carried out at the research fi eld of the Department of Medicinal and Aromatic Plants of Corvinus Uni- versity of Budapest, in 2009-2010. The experimental area is located in the south-eastern part of Budapest, at a geographical elevation of 100-150m. The soil is light sand, the organic matter content is low (0.2-0.4%), humus content is 1.5%, nitrogen and phosphorous levels medium, potassium levels show good supply. Fig. 1 and 2. illustrate the precipitation and the temperature values during the examination period. Different crossed F2 and F3 breeding strains were used for this experiment. Its reason was that based on the earlier experiences the varieties belonged to spring ecotype are often damaged or killed by frost in winter, contrary to the winter poppy varieties which are able to overwinter. The experimental population propagated by selfi ngs afterwards (JÁSZBERÉNYI and NÉMETH, 2011b). The parental combi- nations were as follows: ‘Medea’ x ‘No.67’, ‘Minoán’ x ‘Kozmosz’, ‘Medea’ x ‘Kozmosz’, ‘Korona’ x ‘No.67’, ‘Korona’ x ‘Kozmosz’, ‘Ametiszt’ x ‘Leila’, ‘No.1/172’ x ‘Leila’. The names in italics show registered varieties, while the ones indicated by numbers were se- lected and stabilized breeding strains. They included both high and low alkaloid accumulating genotypes. Reciprocal crossings were handled jointly. Each progeny represented the full genotypic consti- tution without previous selection into any direction. Sowing of each seed plot was carried out at the end of September 2009 and at the end of March 2010 by hand into open fi eld. The plots were 10m2 in 3 replications. During the vegetation period, the usual poppy agrotechnics had been applied. To avoid the effects of metaxenia (BERNÁTH et al., 2003), isolation of individual fl owers was carried out before anthesis with removing the isolation after Ecotypes of poppy 117 fertilisation. The capsules were harvested on 7th July (autumn sown plots) and the 13th of July (spring sown plots). Analysis of alkaloids was carried out by TLC method in 12 repli- cations per population (NÉMETH et al., 2011). After extraction of pulverized capsules in chloroform- methanol (4:1) solvent in Soxhlet apparatus, for separation horizontal TLC chambers (H- Trennkammer Desaga Nr. 120150) were used. Evaluation was car- ried out by densitometric analysis (CHR-SCAN TR-541 equipment with a LabChromTM ried out by densitometric analysis (CHR-SCAN TR-541 equipment TM ried out by densitometric analysis (CHR-SCAN TR-541 equipment Chromatographic Data processing System Version 5.2). The densitometric scanning profi les of four alkaloids (morphine, codeine, thebaine and narcotine) were calibrated against the corresponding standards. The accuracy of the measurements is the level of 0.0001%, coeffi cient of variation of parallel measure- ments is 3.19%. In the following, alkaloid data refer to the content of ripe capsules in % dry mass. Biometric evaluation of data (descriptive statistics, distributions fi t- ting, t-test) was carried out by PASW Statistics 18 software. Results Based on fi eld evaluation at the end of November and beginning of April, density of the overwintered F2 and F3 populations decreased by 30-50%. It proves the intended natural selection by winter frosts and shows in general a medium level frost tolerance. Alkaloid content of the experimental populations Because of the different levels, the evaluation of the total alkaloid content of the experimental population were at fi rst carried out in two groups: combinations of low alkaloid containing varieties and that of high alkaloid containing cultivars. In both categories, samples from autumn sown plots are inferior (0.37% and 1.27%, respectively) compared to the spring sown ones (0.57% and 1.76%, respectively). Standard deviations of data in autumn sown and spring sown plants are practically the same (0.66; 0.68) in the high alkaloid group, while it is somewhat different in the low alkaloid group (0.26; 0.48). In Fig. 3. distributions of the studied samples are illustrated together with the fi tted normal equations whose peaks show the mean values of the populations. The negative shift in the alkaloid content of the overwintered plots is obvious. Evaluation has been carried out also according hybrid strains, sepa- rately. Comparing the total alkaloid values of the same genotypes originating from autumn or spring sowing, it could be established that in the majority of cases there are signifi cant deviations between them (Tab. 1). The presence of this difference seems to be independ- ent of the genetic combination or of the fact if it is an F2 or F3 generation. Not only means but also the marginal values show the difference among the experimental populations. Except the combinations ‘Korona’ x ‘Kozmosz’, ‘Ametiszt’ x ‘Leila’, ‘No.1/172’ x ‘Leila’, the minimum values of the strains are lower in autumn sown plots than in the spring sown ones in each case. The lowest individual value in the populations is 0.10% both in case of autumn hybrids or the population not being selected by frost (Tab. 1.). The maximum values are even more interesting. Existence of some single plants in the frost selected population reaching similar values than the ones in the non-selected ones may indicate the potential of winter hardy genotypes for high alkaloid accumulation. In the recent study both similar (‘Medea’ x No.67 F2, ‘Medea’ x ‘Kozmosz’ F3) and higher (‘Minoán’ x ‘Kozmosz’ F2, ‘Korona’ x No. 67 F3) indi- vidual values had been found in the autumn sown plots compared 0,0 2,0 4,0 6,0 8,0 10,0 12,0 09 .09 .3r d 10 .09 .1s t 10 .09 .2n d 10 .09 .3r d 11 .09 .1s t 11 .09 .2n d 11 .09 .3n d 12 .09 .1n d 12 .09 .2n d 12 .09 .3n d 01 .10 .1n d 01 .10 .2n d 01 .10 .3n d 02 .10 .1s t 02 .10 .2n d 02 .10 .3r d 03 .10 .1s t 03 .10 .2n d 03 .10 .3r d 04 .10 .1s t 04 .10 .2n d 04 .10 .3r d 05 .10 .1s t 05 .10 .2n d 05 .10 .3r d 06 .10 .1s t 06 .10 .2n d 06 .10 .3r d 07 .10 .1s t 07 .10 .2n d mm Precipitation -15,0 -10,0 -5,0 0,0 5,0 10,0 15,0 20,0 25,0 30,0 35,0 09 .09 .3r d 10 .09 .1s t 10 .09 .2n d 10 .09 .3r d 11 .09 .1s t 11 .09 .2n d 11 .09 .3n d 12 .09 .1n d 12 .09 .2n d 12 .09 .3n d 01 .10 .1n d 01 .10 .2n d 01 .10 .3n d 02 .10 .1s t 02 .10 .2n d 02 .10 .3r d 03 .10 .1s t 03 .10 .2n d 03 .10 .3r d 04 .10 .1s t 04 .10 .2n d 04 .10 .3r d 05 .10 .1s t 05 .10 .2n d 05 .10 .3r d 06 .10 .1s t 06 .10 .2n d 06 .10 .3r d 07 .10 .1s t 07 .10 .2n d °C Mean Min Max Fig. 2: The temperature values at the experimental fi eld in 2009/2010 from the time of sowing to harvesting by decadal (oC) Fig. 1: Precipitation amount at the experimental fi eld in 2009/2010 from the time of sowing to harvesting by decadal (mm) 118 C. Jászberényi, É. Németh with the spring sown ones. Nevertheless, in majority of cases, the populations’ maximum values were higher in the non-frost selected, spring sown plots (1.00%-4.20%) (Tab. 1.). The total mean standard deviation of the tested combinations in autumn and spring sowing plots are 0.70 and 0.82 respectively. It indicates similar order of magnitude and no obvious connection with the experimental treatments. Alkaloid spectrum of the experimental combinations Fig. 4 shows the levels of four measured alkaloid compounds: mor- phine, codeine, thebaine and narcotine, separately. (As any other alkaloids are present only in traces, the total values are identical with the mean values of the total alkaloid levels in Tab. 1.). It seems that in the majority of strains each alkaloid compound was reduced in the samples from overwintered plants, although a signifi cant difference could be proved only for thebaine. Opposite tendencies were found only in two cases: for codeine content in F2 population of the cross ‘Medea’x No.67 and for narcotine content in F3 population of the cross ‘Korona’ x No. 67. Mean values for morphine, codeine, thebaine and narcotine in samples of overwintered plants are 0.68, 0.06, 0.16 and 0.16%, re- spectively. The same compounds in samples of spring sown plants were 0.84, 0.10, 0.34 and 0.21%, respectively. Maximum values of alkaloids in the overwintered populations were 1.12, 0.29, 0.50 and 0.53 for morphine, codeine, thebaine and narco- tine, respectively, while they reached 1.26, 0.49, 0.93 and 0.60% for these four compounds, respectively. Discussion The study focused on the question if alkaloid accumulation may be limited to winter hardy genotypes of poppy as frequently anticipated in the practice. Fig. 3: Distributions of experimental data from autumn sown (dark) and spring sown (light) plots. Left: high alkaloid containing group; Right: low alkaloid containing group of hybrids. Tab. 1: Total alkaloid content values of the experimental populations in autumn and spring sown (selected by frosts) plots Total alkaloid content (%) Autumn sowing Spring sowing Min. Max. Mean Std. Dev. Min. Max. Mean Std. Dev. F2 ’Medea’ x No. 67 0.10 2.90 1.54 A 0.94 0.60 2.90 1.90 A 0.63 ’Minoán’ x ’Kozmosz’ 0.15 2.20 1.24 A 0.48 0.70 2.05 1.39 A 0.47 ’Medea’ x ’Kozmosz’ 0.40 2.30 1.43 A 0.61 1.15 2.60 1.91 B 0.46 ’Korona’ x No. 67 0.40 2.20 1.28 A 0.54 1.10 4.20 2.18 B 0.90 ’Korona’ x ’Kozmosz’ 0.30 1.70 0.88 A 0.44 0.30 2.50 1.59 B 0.80 ’Ametiszt’ x ’Leila’ 0.05 1.05 0.48 A 0.30 0.00 1.20 0.51 A 0.41 ’No. 1/172’ x ’Leila’ 0.00 0.85 0.36 A 0.22 0.00 2.30 0.58 A 0.72 Mean 0.27 2.26 1.04 A 0.69 0.77 2.85 1.43 B 0.88 F3 ’Medea’ x No. 67 1.05 2.40 1.73 A 0.43 1.70 3.20 2.37 B 0.44 ’Minoán’ x ’Kozmosz’ 0.20 2.30 1.26 A 0.64 0.85 2.95 1.84 B 0.64 ’Medea’ x ’Kozmosz’ 0.00 2.50 1.23 A 0.60 0.60 2.60 1.56 A 0.54 ’Korona’ x No. 67 0.25 3.10 1.33 A 0.90 0.55 2.10 1.36 A 0.47 ’Korona’ x ’Kozmosz’ 0.10 1.65 0.78 A 0.38 0.50 2.70 1.52 B 0.74 ’Ametiszt’ x ’Leila’ 0.00 0.75 0.28 A 0.23 0.25 1.00 0.61 B 0.25 Mean 0.32 2.39 1.10 A 0.72 0.84 2.71 1.54 B 0.74 Total mean 1.07 A 0.70 1.48 B 0.82 Ecotypes of poppy 119 The experimental plots thinned due to winter frosts exhibited sig- nifi cantly lower total alkaloid contents than the samples from spring sown plots of the same populations. Although in open fi eld, weather conditions – especially summer rain – may contribute to alkaloid accumulation and eventual losses (BERNÁTH and TÉTÉNYI, 1981), our fi ndings ascertained the old practical observations. The lower level in overwintered plants was proved for each of the individual al- kaloid compounds morphine, codeine, thebaine and narcotine, too. The genes which might be in linkage with those regulating frost tolerance seem to be more likely present at the primarly level of alkaloid biosynthesis. These regulate the synthesis of general pre- cursors and not the characteristic opiate alkaloid compounds of the poppy capsule formed at the fi nal stages of biosynthesis (ZIEGLER et al., 2009). Clarifi cation of this question needs further research. However, at the same time, maximum values in some individual samples of the overwintered plants reached similar or higher con- tents than the spring sown ones. Therefore it is supposed that even if gene frequency in direction for high alkaloid level might be larger in frost sensitive ecotype, the potential for breeding high alkaloid ac- cumulating cultivars exists also among frost tolerant genotypes. Acknowledgement The work was supported by OTKA grant no. K 62732 and TÁMOP- 4.2.1/B-09/1/KMR-2010-0005 grant. References BERNÁTH, J., TÉTÉNYI, P., 1981: The effect of environmental factors on growth, development and alkaloid production of poppy (Papaver som- niferum L.) II. Interaction of Light and Temperature. Biochem. Physiol. Pfl . 176, 599-605. BERNÁTH, J., NÉMETH, É., 2009: Breeding of poppy. In: Vollmann, J., Rajcan, I. (eds), Oil crops, 449-468. Springer Series „Handbook of Plant Breeding” Springer Sciences + Business Media, Dordrecht. BERNÁTH, J., NÉMETH, É., PETHEŐ, F., 2003: Alkaloid accumulation in capsules of the selfed and cross-pollinated poppy. Plant Breeding 122, 263-267. DOBOS, G., 2010: Personal communication Government Regulation 162/ 2003. (X. 16.) on the order of growing, traffi cking and using plants suitable for producing drugs. JÁSZBERÉNYI, C., NÉMETH, É., 2011a: Frost tolerance of spring and winter ecotypes of poppy (Papaver somniferum L.). International Symposium on Papaver, Book of Abstracts, 7-11, February, Lucknow, India. JÁSZBERÉNYI, C., NÉMETH, É., 2011b: Observation on the inheritance of some morphological characteristics of poppy (Papaver somniferum L.). Kertgazdaság 43, 53-62. NÉMETH-ZÁMBORI, É., JÁSZBERÉNYI, Cs., RAJHÁRT, P., BERNÁTH, J., 2011: Evaluation of alkaloid profi les in hybrid generations of different poppy (Papaver somniferum L.) genotypes. Industrial Crop. Prod. 33, 690-696. ZIEGLER, J., FACCHINI, P.J., GEISSLER, R., SCHMIDT, J., AMMER, C., KRAMELL, R., VOIGTLANDER, S., GESELL, A., PIENKNY, S., BRANDT, W., 2009: Evolution of morphine biosynthesis in opium poppy. Phytochem. 70, 1696-1707. Address of the authors: Csilla Jászberényi (corresponding author) and Éva Németh, Department of Medicinal and Aromatic Plants, Corvinus University of Budapest, Villányi str. 35-43., H-1118 Budapest, Hungary Fig. 4: Alkaloid spectrum of the experimental populations in autumn (’a’) and spring (’s’) sown (selected by frosts) plots 0,0 0,5 1,0 1,5 2,0 2,5 F2 _M ex 67 _a F2 _M ex 67 _s F2 _M i x Ko _a F2 _M i x Ko _s F2 _M ex Ko _a F2 _M ex Ko _s F2 _K or x6 7_ a F2 _K or x6 7_ s F2 _K or xK o_ a F2 _K or xK o_ s F2 _A m xL e_ a F2 _A m xL e_ s F2 _1 /17 2 x Le _a F2 _1 /17 2 x Le _s F3 _M ex 67 _a F3 _M ex 67 _s F3 _M i x Ko _a F3 _M i x Ko _s F3 _M ex Ko _a F3 _M ex Ko _s F3 _K or x6 7_ a F3 _K or x6 7_ s F3 _K or xK o_ a F3 _K or xK o_ s F3 _A m xL e_ a F3 _A m xL e_ s al ka lo id co nt en t( % ) morphine codeine thebaine narcotine