CMS LINES FOR EVALUATION OF POLLEN FLOW IN SUNFLOWER RELEVANCE FOR TRANSGENE FLOW MITIGATION

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1 Articles A&EB CMS LINES FOR EVALUATION OF POLLEN FLOW IN SUNFLOWER RELEVANCE FOR TRANSGENE FLOW MITIGATION T. Hvarleva 1, M. Hristova 2, A. Bakalova 1, M. Hristov 3, I. Atanassov 1, A. Atanassov 1 AgroBioInstitute, Sofia, Bulgaria 1 University of Forestry, Faculty of Agronomy, Department of Genetic of Agricultural Culture, Sofia, Bulgaria 2 Dobroudja Agricultural Institute, General Toshevo, Bulgaria 3 Correspondence to: Tzvetanka Hvarleva hvarleva@abi.bg Abstract Sunflower pollen flow over long distance was evaluated. Plants from three CMS lines were cultivated at distances 10 m, 1.0 km and 1.5 km from the male fertile line used as pollen donor. The assessment of the seed set of the CMS plants showed a decrease in the pollination with the distance from the pollen donor plants. The microsatellite analysis of bulk samples from the collected seeds demonstrated pollen dispersal from the pollen donor plants over all three tested distances. Further analysis of individual seeds suggested that a small portion of the collected seeds may have resulted from selfpollination of studied CMS lines and crosspollination between them. The observed restoration of fertility in the CMS lines was most probably caused by the excessive rainfall and lower average temperatures during the period of the flowering of the CMS lines. The stability of a wider range of CMS lines carrying cytoplasms from H. petiolaris, H. argophylis, H. rigidus and H. praoxes species were evaluated within three years ( ) in two different locations in Bulgaria. Sixteen CMS lines, studied in 2006, were sterile in tested environmental conditions. Six of the CMS lines were further assessed in the period after two periods of sowing and parallel cultivation in greenhouse. The investigated CMS lines differed in regard to the stability of cytoplasmic male sterility. Four of them were fully sterile across all nine different environmental conditions in the period Two CMS lines showed unstable sterility that varied under the different environmental conditions. The restoration of fertility in these CMS lines was partial and affected only few individual plants. Taken together these data suggest that the climatic condition (temperature, humidity and daily light intensity) may affect the stability of sterility of the sunflower CMS lines depending of their genotype. The results from the present study indicated that long distance pollen dispersal is a major factor limiting the cultivation of transgenic sunflower plants. The prevention of transgenic pollen spread through cultivation of transgenic CMS plants has to be well evaluated in direction to the stability of the used CMS lines for each specific location, multiple periods of cultivation and various climatic conditions. Keywords: sunflower, pollen flow, CMS lines, cytoplasmic male sterility trait, microsatellite analysis, transgene flow mitigation Introduction The main concern related to the transgenic crops cultivation is the ecological impact of transgene escape. Pollen mediated transfer of a transgene to the wild relatives and related cultivars has been considered a major environmental risk. Gene flow is a naturally occurring process and for many taxa is often sufficiently high to homogenize even neutral alleles (15). Gene flow from crops to wild relatives was studied for a number of crops (for review see 8 and 9). If the transgenic trait (disease/insect resistance, tolerance to herbicides or to stress environmental conditions such as drought or salinity) provides fitness advantages to wild relatives, the corresponding favorable allele may spread quickly and be maintained for a long term period in a wild population under the selective pressure. This may lead to the creation of invasive weeds and hampering of weed control. During the last two decades a number of studies were devoted to the assessment and modeling of pollen flow in different crops in order to evaluate the possibility for coexistence between transgenic and nontransgenic plants (sunflower (1); trees (6); maize (3); oilseed rape (4, 5, 23). Sunflower (Helianthus annuus) is a crop of major economic importance, being among the five most important oilseed crops in the world. Sunflower is an outcrossing crop that is pollinated by insects, mainly honey bees, so the risk of undesired long distance spread of pollen that carries a transgene is much greater than in crops pollinated by wind. This places the sunflower in a high risk category in terms of the likelihood of transgene escape. Presently no genetically modified sunflower is commercially grown worldwide, but a number of transgenic traits such as tolerance to herbicides, resistance to insects and improved quality, have been introduced in sunflower by means of biotechnology and are under field experiments (2). If transgenic sunflower will be considered for commercial growth in the future, methods for preventing transgene escape have to be developed to allow the co-existence of genetically modified (GM) and non-gm plants on the farms. The assessment of gene flow in sunflower is hampered by several factors referring to the type of pollination: the necessity of a large cultivation area in order that a large scale trials are performed, 1309

2 giving consideration to the behavior of honeybees and the occurrence of bumblebees and other insect, the assessment of the competition between sunflower and the surrounding plant species for bee foraging, the impact of landscape specificity, etc. The studies on gene flow in sunflower are mainly focused on the dispersal of pollen between H. annuus species and between cultivated sunflower and its wild relatives. The interspecies and intraspecies crossing in H. annuus and its closed relatives were determined (7, 21, 22). Chromosomal structure differences between species were suggested as significant barriers to gene flow between sunflower species (19, 20). A number of studies documented the introgression and persistence of cultivar alleles in wild relatives (1, 8, 9, 14, 17). Several factors assist exchange of genes between wild and cultivated sunflowers: overlapping of flowering time, shared pollinators, self-incompatibility of wild sunflower, self compatibility of domesticated sunflower, and high rate of outcrossing. Pollen flow from imidazolinone (IMI)-resistant domesticated sunflower to IMI-susceptible common sunflower and prairie sunflower was assessed in a small scale experiment. Gene flow at frequency of 2-5% was measured at 30 m from the pollen source (14). Gene flow from cultivated to wild sunflower was found to decrease with the distance (1). It was highest (27%) within 3 m, but occurred at a low frequency up to a distance of 1000 m from the source of pollination. The role of climatic conditions on the vitality of pollen, and correspondingly on the rate of pollination over long distances were suggested. Both biotic and abiotic factors were pointed to influence pollen dispersal and hybridization (humidity, temperature, cultivar genotype, bees). The alleles introgressed in wild populations can be further spread by both pollen and seed dispersal, providing an additional source for secondary gene flow. The F1 hybrids between cultivated and wild sunflower are fertile and may hybridize more easily with wild relatives than cultivated sunflower (24). The long time persistence up to five generations (29) and 40 years (12) of cultivar alleles in wild populations was obtained. These studies demonstrate that sunflower pollen can be spread at long distances and the crop alleles can be introgressed and persist for a long time in the wild relatives. Methods, such as physical distances and crop based barriers, used with wind pollinated crops to prevent transgene flow, are not effective enough for sunflower. Arias and Reisenberg (1) considered that the isolation distance for sunflower must be greater than 1000 m and this barrier cannot be considered impermeable. For commercial sunflower seed production the isolation distances between hybrid seed production fields and wild sunflower and/ or other cultivated sunflower are required to be km. The use of cytoplasmic male sterility could serve as an alternative route to prevent transgene escape in sunflower. Cytoplasmic male sterility is a maternally inherited trait resulting in pollen abortion, while the female fertility and the normal vegetative development are not interfered with. In most cases the CMS phenotype is associated with mutation in mitochondrial genome that is controlled by dominant nuclear restorer (Rf) genes. The inbred lines that harbor the Rf alleles, restorer (R) lines, are used for pollination of CMS lines in hybrid seed production. The cross between a CMS line and a restorer line results in a progeny that is heterozygous for the Rf gene and correspondingly fertile. Currently CMS inbred lines, used for the production of hybrid seeds in sunflower, are developed on the basis of a single CMS source, CMS-PET1 cytoplasm. This CMS was developed by Leclercq (13) in an interspecific cross between the wild species H. petiolaris Nutt and H. annuus. From then until 2005 seventy two CMS new sources were identified in sunflower and described worldwide, but are still not used in agricultural practice (24). Most of them were derived from crosses between wild annual species and cultivated sunflower or were induced by mutagenesis. CMS may also occur in wild H. annuus populations as spontaneous mutations or naturally occurring interspecies cross. In this study CMS lines were used with aim to evaluate: 1. pollen flow in sunflower over a long distance; 2. reliability of cytoplasmic male sterility in sunflower as a tool for preventing pollen flow from transgenic sunflower. Materials and Methods Plant material Eighteen CMS lines, which differ in respect to the origin of cytoplasm (H. petiolaris, H. argophylis, H. rigidus and H. praoxes) as well as restorer line 7015R, were obtained from the sunflower collection of Dobrudja Agricultural Institute. The set of investigated CMS lines included 14 Bulgarian CMS lines, developed in the same institute, (10, 11), two originating from the USA and two-from Ukraine (Table 1). The locally selected CMS lines are well adapted to the climatic conditions in Bulgaria and harbor a number of agronomical important characteristics including disease resistance. Field trials and analysis of seed progeny 1. Assessment of pollen flow over long distance The field trial accomplished in the summer of 2005 was situated in village Opitsvet, Sofia district, Bulgaria. Three CMS lines, 6134A, 6148A, 6149A, sown in three lots at distances 10 m, 1.0 km and 1.5 km from the male restorer line, were used as pollen acceptors (Fig. 1). In each lot each CMS line was sown in two rows, consisting of 25 plants. The area between the donor plants sown in lot 1 and target plants in lot 2 was planted with maize, while between lot 1 and lot 3 there was a meadow. No other sunflowers were sown within 3 km. All CMS plants were left without bags for free pollination and after seeds set, were covered with bags to protect them from the birds. The mature heads were harvested and air dried. The number of seeds in each sunflower head was counted. The rate of pollination per each combination CMS line/distance 1310

3 List of investigated CMS lines, origin of cytoplasm and stability of the CMS trait in the period under stud TABLE 1 CMS lines/origin Origin of Sofia Sofia cytoplasm Sofia Sofia GT GT* GT 1sd 2nd gh 1sd 2sd gh A (Bulgaria) Petiolaris unstable stable unstable stable stable unstable unstable stable stable A (Bulgaria) Petiolaris stable stable stable stable stable stable stable stable stable 3.197A (Bulgaria) Petiolaris stable stable A (Bulgaria) Petiolaris stable stable A (Bulgaria) Petiolaris unstable stable stable unstable unstable stable A (Bulgaria) Petiolaris unstable unstable stable A (Bulgaria) Petiolaris unstable stable stable stable stable stable stable stable stable stable A (Bulgaria) Petiolaris stable stable A (Bulgaria) Petiolaris stable 10. U14A (Ukraine) Petiolaris stable stable 11. U66A (Ukraine) Petiolaris stable stable 12. HA89 (USA) Petiolaris stable stable stable stable stable stable stable stable stable 13. HA402 (USA) Petiolaris stable stable A (Bulgaria) Petiolaris stable stable stable stable stable stable stable stable A (Bulgaria) Praoxes stable stable A (Bulgaria) Argophillus stable stable A (Bulgaria) Argophillus stable stable A (Bulgaria) Rigidus stable stable The highlighted cells represent the period under study and stability of the cytoplasmic male sterility trait of the corresponding CMS line; GT- General Toshevo; gh-greenhouse; 1st and 2sd-first and second sowing date; *due to the severe drought and high temperatures in 2007 and the lack of possibility for watering, the field trial set in General Toshevo was destroyed; was calculated as a percentage of filled (fertile) seeds over all seeds found in 25 heads per line/distance. Fig. 1. Field plan of the sunflower experiment 2005 Ten seeds from each head were selected randomly. The selected seeds per combination CMS line/distance were mixed. Ten seeds from this mix were used for bulk analysis and 25 seeds for microsatellite analysis of individual seeds. Seeds were potted and were germinated in a growth chamber. Leaves from the seedlings were sampled for DNA extraction. 2. Assessment of the stability of cytoplasmic male sterility During the period identical field trials were performed in two locations in Bulgaria with different climatic conditions, soil type and altitude: Dobrudja Agricultural Institute, near General Toshevo city in Northeast Bulgaria and Research Station Kubratovo in Midwest Bulgaria, near Sofia city, both with moderate continental climate. The climate of General Toshevo is characterized by less rainfalls and higher daily temperature than that in Sofia. Leaf samples from all plants were collected and frozen for verification of the genetic authenticity of plants. At the beginning of florescence, sunflower heads were covered with bags to prevent the pollination with foreign pollen. The assessment of the release of pollen in the bags was carried out during the flowering period. The mature sunflower heads were collected and assessed for the presence of filled seeds. The number of plants with filled seeds and the number of filled seeds in each fertile plant was assessed. The source of pollen contributing to the seed set was determined by microsatellite analysis. 1311

4 Eighteen sunflower CMS lines were tested for stability of sterility in 2006 in both locations, with the exception of two CMS lines, 6023A and 6027A, which were sown only in General Toshevo (Table 1). For each line 30 plants were sown in each location. Ten of them were isolated with bags at the beginning of flowering for testing of sterility, ten plants were left for free pollination and ten plants were pollinated with pollen from restorer line HA89R to test their ability for pollination. Six out of 18 CMS lines (HA89, 6023A, 6134A, 6149A, 6116A and 2607A in H. petiolaris cytoplasm) were selected for further evaluation in 2007 and five (HA89, 6023A, 6149A, 6116A and 2607A) - in In order to increase the variability of climatic conditions, the CMS lines in research station Kubratovo were sown at two sowing dates in a field, about one month apart, and at one sowing date in a greenhouse (Table 1). Thus, 50 plants per CMS line and per location (1 st sowing date), 40 plants per 2 nd sowing date and 30 plants per greenhouse were tested. Each combination of sowing date/ location was considered different environment. Meteorological data, temperatures, rainfall and daily light intensity were provided by the Sofia Meteorological Service. DNA extraction DNA was extracted from 100 mg leaf tissue following the procedure described by Murray and Thompson (16). Leaves were sampled from the plats in the field or from seedlings grown in a climatic chamber. PCR and microsatellite analysis The investigated CMS lines were characterized through 18 microsatellite markers uniformly distributed in the genome of sunflower: ORS 16, ORS 316, ORS 857, ORS613, ORS 230, ORS 1079, ORS 309, ORS 307, ORS 925, ORS 894, ORS 885, ORS 949, ORS 610, ORS 878, ORS 7, ORS 1065, ORS 761, ORS 561 (26, 27). PCR reaction was performed according to the protocol of Tang et al. (27) in a volume of 20 μl in a GeneAmp PCR System 9700 (Applied Biosystem). The PCR mixture contained 30 ng genomic DNA, 1.6x PCR reaction buffer with 2.5 mm MgCl 2 (Fermentas), 250 nm of each dntp, 4-14 pmole of each primer depending on the primer and 1U Taq DNA-polymerase (Fermentas). The forward primer of each pair was labelled with fluorescent Cy-5 dye. The touchdown PCR were used according to Tang et al. (27) for the amplification of all investigated loci: 95ºC for 2min, 1 cycle of 94ºC for 30 sec, 63ºC for 30 sec, 72ºC for 45 sec, followed by 5 cycles with decreasing of temperature with 1ºC per cycle, followed by 33 cycles of 94ºC for 30 sec, 57ºC for 30 sec, 72ºC for 45 sec. Final extension was 20 min at 72ºC. Internal standards were produced by amplification of puc19 fragments with sizes 50, 100, 150, 200, 250, 300, 350, 400, 450 and 500 bp. PCR products were separated by electrophoresis on a sequencing gel in ALF Express II sequencer (GE Healthcare). The alleles were automatically sized with software AlleleLocator 1.03 (GE Healthcare). The software Identity (28) was used for calculation of genetic diversity among investigated CMS lines at 18 microsatellite loci. Results and Discussion Genotyping of sunflower lines Eighteen CMS lines as well as restorer line 7015R were genotyped with 18 microsatellite markers uniformly distributed through the genome of sunflower. A unique microsatellite profile was determined for each line. A low level of polymorphism (genetic diversity, GD=0.51) was observed among the investigated lines. This was probably due to the use of genetically similar initial lines used for the development of the studied CMS lines. The number of alleles per microsatellite locus ranged from 2 to 5. A set of 5 SSR markers (ORS 16, ORS613, ORS 230, ORS 307 and ORS 925) was composed, which allowed for discrimination of the investigated CMS lines as well as the male fertile line. The allelic profiles of 18 investigated CMS lines at these five loci are presented in Table 2. The set was used for verification of genetic authenticity of CMS and male fertile plants in the field experiment and for determination of pollen source that contributed to the seed set at CMS plants. TABLE 2 Genotype of 18 CMS lines at SSR loci ORS16, ORS613, ORS230, ORS307, ORS925 CMS line/ SSR locus ORS16 ORS613 ORS230 ORS307 ORS A A A A A A A A A U 14A U 66A HA 89A / HA A 138/ / A A A A Evaluation of pollen flow over long distance Pollen flow from male fertile (restorer) 7015R line was assessed in specially designed experiment, in which lots of CMS plants were sown at distances 10 m, 1.0 km and 1.5 km from the male fertile plants The CMS lines 6134A, 6148A, 6149A were used 1312

5 as pollen acceptors. The rate of pollination at each distance was evaluated by measuring the seed set in 25 heads per CMS line. Table 2 presents the average value of percentage of filled seeds over all seeds found per combination CMS line/distance. The data showed that the rate of pollination decreased with the distance (Table 3). At distance 10 m the percentage of seed set varied between 50% and 80% among the CMS lines. At distance 1.0 km the seed set ranged from 20% to 44%, while at distance 1.5 km - from 4% to 11%. The observed differences in the pollination rate among the three CMS lines at each distance could be associated with the differences in the flowering time of the CMS lines as well as with bees preferences for forage. TABLE 3 Percentage of fertile seeds obtained per CMS line at distance 10m, 1.0 km and 1.5 km Percentage of fertile seeds CMS line 10m 1.0km 1.5km 6134A A A To determine the source of pollen that contributed to the seed set parentage analysis of the seed progeny was performed with the selected set of five SSRs. The bulk samples of 10 seeds from analyzed CMS line/distance combinations were subject of microsatellite analysis. The presence of two alleles, one transferred from the CMS mother line and a second from the 7015 R pollinator line were observed in all tested samples. Occasionally additional SSR alleles were observed for some of the samples. The origin of these SSR alleles was clarified through the genotyping of 25 individual seeds per combination CMS line/distance. The SSR analysis demonstrated that most of the seeds were a result of pollination of the given CMS line with pollen from the male fertile line 7015R. These seeds were heterozygous for each SSR loci, possessing alleles of male fertile line and the respective CMS line. A small portion of seeds was homozygous containing alleles identical with the alleles of the mother CMS line, suggesting that they most possibly resulted from self-pollination. The percentage of these seeds calculated per CMS line over the three distances (75 seeds) varied among CMS lines: 2.7% for line 6149, 5.3% for line 6134 and 6.7% for line The seeds of the third group were found to be heterozygous containing the combination of alleles of two mother CMS lines. These seeds, which represent 4% of all tested seeds, were considered a result of cross-pollination between two CMS lines. The data obtained from the field trial indicated that the pollen of male fertile line 7015R can be spread at a distance of up to 1.5 km. The evidence for long distance pollen flow was obtained in other studies. Arias and Rieseberg (1) found that pollen from cultivated sunflower can be spread over a distance of 1 km and to pollinate the wild sunflowers. Pollen flow at long distances was also observed in canola (Brassica napus), which is pollinated by wind and insects. A large-scale study on pollen movement from herbicide resistant canola to the commercial canola fields demonstrated low level of cross pollination occurred at 3 km distance (18). The seed set obtained at the three distances demonstrated the gradual decrease of pollination rate of tested CMS lines with the distance from the male fertile line. The restorer line was determined as a major pollen donor contributing to the seed set at the CMS lines at the tree distances evaluated. However, a small number of seeds resulting from self- ( %) and cross-pollination (4%) between the CMS lines also contributed to the overall seed set. This suggested the existence of certain rate of reversal to fertility in the investigated CMS lines. Since no male fertile restoration was reported for the CMS lines used in the study, the observed partial male fertility restoration could be caused by the excessive rate of rainfall and moderate temperatures during the summer of Such low frequency restoration of male fertility could present a potential risk of transgenic pollen spread if transgenic CMS lines are planted as part of measures of preventing of pollen flow from transgenic plants. To evaluate the reliability of CMS for preventing transgene pollen flow, the CMS stability has to be assessed for each genotype for the specific environmental conditions over longer period of multiple cultivations. Stability of cytoplasmic male sterility of sunflower CMS lines under different climatic conditions To evaluate the stability of the male sterility trait in sunflower, eighteen sunflower CMS lines carrying cytoplasms from H. petiolaris, H. argophylis, H. rigidus and H. praoxes species, were assessed in two locations in Bulgaria in 2006 (Table 1). Ten plants per each CMS line were covered with bags at the beginning of the flowering period and surveyed for release of pollen and for seed set. Among the sixteen CMS lines grown in Sofia district, nine CMS lines were found to be fully sterile, while individual plants (10 plants) from the remaining 7 CMS lines were obtained to release pollen during the flowering period and to set seeds. The number of seeds obtained at these plants varied from 3 to 200. Taking into consideration the possible presence of impurities, the genetic authenticity of the fertile plants was verified. Ten out of 12 fertile plants were found to be hybrids that shared one allele with the corresponding CMS line. The obtained segregation of alleles in the seed progeny of these plants showed that the seeds were result of self-pollination. These plants were considered contamination of fertile hybrids, presented in the corresponding lines. The remaining two fertile plants had a microsatellite profile, identical to the corresponding CMS lines 2607A and 6148A. Both plants formed few seeds, 18 for the 2607A plant and 10 for the 6148A plant. Microsatellite analysis of these seeds proved that they originated from self-pollination. Both plants did not flower at the beginning of the flowering period. The flowers appeared in the middle of the sunflower heads after rainfall and abrupt decrease in temperature and daily light intensity. Together these data suggests that the restoration of 1313

6 fertility in these two plants was partial and was most probably caused by the change in climatic conditions. The lack of seed set was found for all 18 CMS lines in General Toshevo, where the temperature and humidity during the flowering period were relatively stable in comparison to these conditions in Sofia district. Six of the CMS lines assessed in 2006: HA89A, 2607A, 6023A, 6116A, 6134A, 6149A, were further evaluated in the period in two locations, Sofia and General Toshevo. These CMS lines were selected, because they harbour a number of valuable agronomic characteristics including resistance to Orobanche, race E (CMS lines 6116A and 6134A) and Plasmopara helianthi (line 2607A). To increase the variability of the climatic conditions, in Sofia district the cultivation of the CMS lines was performed, with two sowing dates in the field and one sowing date in the greenhouse. Thus, in total, five out of the sixth CMS lines were assessed in three consecutive years ( ) in 9 climatic conditions. Line 6134A was studied in two consecutive years in 5 climatic conditions (Table 1). The three vegetative periods ( ) were characterized by high temperatures and low values of rainfall. Due to the severe drought and high temperatures in 2007 and the lack of possibility for watering, the field trial set in General Toshevo was destroyed. The investigated CMS lines were found to differ in regard to the stability of cytoplasmic male sterility. Four of them, HA89A, 6023A, 6116A, 6149A, were fully sterile across all 9 climatic conditions in the period Two CMS lines 2607A and 6134A showed unstable male sterility that varied over different climatic conditions. CMS line 6134A was found unstable in 2 out of five climatic conditions. The percentage of the obtained fertile plants was 4% and 8%, respectively. The number of seeds set at these plants varied from 5 to 70. More affected by the restoration of fertility was line 2607A, which was found to be unstable in 5 out of 9 climatic conditions. The percentage of the obtained fertile plants varied over the five climatic conditions between 2% and 6% with a number seeds varying between 50 and 200. These data indicated that the restoration of fertility observed in both CMS lines was partial and affect individual plants. At the same time the lines were sterile in two years of assessment (2006 and 2008) in General Toshevo where the climate is characterized with higher temperatures and less rainfall than in Sofia. Both lines were also sterile in greenhouse conditions in Sofia in This was probably due to the high daily temperatures ( C) that affected the lifetime of pollen. The data obtained in the period suggest that the climatic condition (temperature, humidity and daily light intensity) may have an effect on the stability of sterility of the investigated CMS lines and that this effect is genotype dependent. Conclusions The obtained results in this study provided evidence for pollen spread in sunflower up to 1.5 km, suggesting that the long distance pollen dispersal is a major factor limiting the cultivation of transgenic sunflower. Thus, the physical distances alone could not ensure prevention of the transgene spread in the environment. Isolation by time of flowering seems to be suitable for small-scale research trials, but its application in conventional farming requires the development of corresponding management strategies. Cytoplasmic male sterility could serve as a reliable technique for preventing pollen mediated transgene dispersal. Our results on the stability of the CMS trait showed that partial reversal to fertility affecting individual plants in a particular CMS lines could occur under the influence of climatic conditions. Hence, the use of transgenic CMS lines has to follow a precise evaluation of the stability of sterility of particular CMS line over various climatic conditions and multiple periods of cultivation. The research effort should also be placed for development of suitable strategies that include genetic isolation by male sterility. Acknowledgements This study is financially supported by the Integrated project Co-extra (contract no ) within the 6 th Framework Program Priority 5 : Food Quality and Safety. References 1. Arias D., Rieseberg L. (1994) Theor. Appl. Genet., 89, Cantamutto M. and Poverene M. (2007) Field crop research, 100, Devos Y., Ruheul D., De Schrijver A. (2005) Environ. Biosafety Res., 4, Devaux C., Klein E.K., Lavigne C., Sausse C., Messean A. 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