Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans




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Interspecific somatic hybrids Solanum villosum (+) S. tuberosum, resistant to Phytophthora infestans
Justyna Tarwacka1, Lidia Polkowska-Kowalczyk1*, Bożena Kolano2, Jadwiga Śliwka3, Bernard Wielgat1
1Department of Plant Biochemistry, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland

2Department of Plant Anatomy and Cytology, University of Silesia, Jagiellońska 28, 40-032 Katowice, Poland

3Plant Breeding and Acclimatization Institute - National Research Institute (IHAR-PIB), Młochów Research Center, Platanowa 19, 05-831 Młochów, Poland

*Corresponding author: Fax: 4822 658 46 36; Tel no. 4822 5925713

E-mail address: lidekp@ibb.waw.pl (L. Polkowska-Kowalczyk)
Abstract

The interspecific somatic hybrids, 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids), were obtained and characterized molecularly and cytogenetically.



The morphology of fusion-derived plants was intermediate in relation to the parental species. The expected ploidy level of the regenerants was 6x for the VT hybrids, but the real ploidy of the hybrids varied, with some of them being euploids, and others - aneuploids. The hybridity of the regenerants was verified by random amplified polymorphic DNA (RAPD) analysis. Despite the variation in ploidy, the RAPD patterns of the hybrids were mostly uniform, suggesting similarity of the genotypes of the VT clones. Genomic in situ hybridisation (GISH) analysis discriminated between the chromosomes of both parental genomes in VT somatic hybrids and confirmed also their hybridity. The resistance of VT somatic hybrids to Phytophthora infestans was evaluated and all the hybrids were proved to be highly resistant. In search of the mechanisms involved in resistance of the Solanum species to P. infestans, the biochemical reactions occurring early after elicitor treatment were studied. Production of reactive oxygen species (ROS), as one of the earliest reactions induced by pathogens or their elicitors, was examined in the resistant wild species S. villosum, susceptible S. tuberosum clone DG 81-68 and in the VT hybrid, resistant to P. infestans. After treatment of the leaves with elicitor, the relative increase in ROS production was higher in leaves of the susceptible potato clone than in the resistant plants of S. villosum and the somatic hybrid.
Keywords:

Phytophthora infestans, resistance, Solanum tuberosum, Solanum villosum, somatic hybrids;
Abbreviations:

Avr gene, avirulence gene; biotin-16-dUTP, biotin-16-2'-deoxy-uridine-5'-triphosphate; CF, culture filtrate; CTAB, cetyltrimethylammonium bromide; cvs, cultivars; DAPI, 4',6-diamidino-2-phenylindole; digoxigenin-11-dUTP, digoxigenin-11-2’-deoxy-uridine-5’-triphosphate; FITC, fluorescein isothiocyanate; GISH, genomic in situ hybridisation; HR, hypersensitive response; NBT, nitroblue tetrazolium; PEG, polyethylene glycol; R gene, resistance gene; RAPD, random amplified polymorphic DNA; ROS, reactive oxygen species; tbr, Solanum tuberosum; vll, Solanum villosum; VT hybrids, somatic hybrids of 4x S. villosum (+) 2x S. tuberosum clone DG 81-68;

Introduction

Phytophthora infestans (Mont.) de Bary, the pathogenic oomycete, causes late blight - the most destructive potato disease (Kamoun and Smart, 2005; Sarkar et al., 2011; etc.). The damage caused by P. infestans in agricultural crops is tremendous due to difficulties in controlling the spread of the disease. The use of chemicals targeted against P. infestans can provide some level of disease control. However, in a long term, the development of potato crops possessing durable genetic resistance, both by classical breeding and/or by genetic engineering methods, provides the best prospect for effective economical and environmentally acceptable control of late blight disease.

There are different types of resistance to P. infestans, and the resistance level varies in potato cultivars as well as in numerous wild Solanum species. It is generally accepted that the race- or cultivar-specific plant resistance requires the presence of both plant resistance (R) gene and matching pathogen avirulence (Avr) gene, whereas non-host and quantitative (partial, field) plant resistances are polygenically controlled (Keller et al., 2000; Kamoun and Smart, 2005; Kou and Wang, 2010). In the interaction between immune wild Solanum genotypes and P. infestans, the non-host type of resistance can be explained by the presence of a whole arsenal of R genes interacting with a number of Avr genes, whereas partial resistance could result from “weak” R gene - Avr gene interactions or gene-dosage effects. On the other hand, biochemical defence responses in host and non-host resistance are very similar, although the latter does not require apparent R/Avr gene combination. In Solanum/P. infestans interactions, wild Solanum species often express non-host resistance, whereas cultivated potatoes represent mostly quantitative resistance manifested by various levels of disease reduction. Moreover, in cultivated potato the presence of some unidentified R genes is not excluded (Vleeshouwers et al., 2000). Studies of Tian et al. (2006) indicated that different wild Solanum species, potato cultivars with various levels of resistance, and representative non-host species infected with P. infestans displayed a hypersensitive response (HR). Polygenic resistance to P. infestans may be of great value in potato breeding since it seems to be the only durable resistance to this disease identified so far. Some R genes can also be more durable than others. The RB gene (also known as Rpi-blb1) of S. bulbocastanum recognises a broad spectrum of P. infestans isolates and appears to be durable in the field (Helgeson et al., 1998; Song et al., 2003; Vleeshouvers et al., 2008).

The improvement of potato resistance to late blight disease can be achieved by introgression of the resistance genes from wild Solanum species by different biotechnological methods, among others, somatic hybridisation (Orczyk et al., 2003). This method allows to create potato genotypes with enhanced resistance to various biotic and abiotic stresses, including resistance to P. infestans and Globoderia pallida from S. berthaultii (Serraf et al., 1991), resistance to potato virus Y (PVY) and P. infestans from S. tarnii (Thieme et al., 2008). In the case of the latter, the obtained somatic hybrids were successfully backcrossed with cultivated potato (Thieme et al., 2008). Somatic hybridisation proves to be a valuable tool for producing pre-breeding material with increased genetic diversity and has an unique potential to transfer simultaneously both nuclear and cytoplasmic genes (Zhou et al., 2001; Tiwari et al., 2010). Because of the existence of different kinds of crossing barriers between wild and cultivated Solanum species, protoplast fusion is a method which allows to omit most of them (Szczerbakowa et al., 2010).

In our lab we have previously conducted successful fusion experiments between cultivated potato and several late blight-resistant wild Solanum species, such as S. bulbocastanum (Szczerbakowa et al., 2003a), S. nigrum (Szczerbakowa et al., 2003b), S. pinnatisectum (Szczerbakowa et al., 2005) and S.michoacanum (Szczerbakowa et al., 2010). The number of somatic hybrids obtained varied for different fusion combinations, and the hybrid level of resistance against P. infestans was not always as high as that of the wild parent, being insufficient for breeding programs (Szczerbakowa et al., 2010). Besides improvement of potato resistance against P. infestans, our research interests concern the defence mechanisms in Solanum species in response to P. infestans (Polkowska-Kowalczyk et al., 2004; 2007; 2008; 2011). Our studies showed that the same early reactions to elicitor from P. infestans occurred both in resistant and susceptible Solanum species although they differed in speed and intensity depending on the level of resistance of the species.

Studies on the mechanisms of plants’ response to pathogen attack are useful for elucidating the physiological and molecular bases of plant resistance and for plant breeding. Biochemical reports concerning polygenic resistance are limited. The diversity of potato genotypes complicates the understanding of biochemical reactions occurring in the infected plants. Moreover, phylogenic analyses suggest that oomycetes form a unique group of eukaryotic plant pathogens which may display distinct genetic and biochemical mechanisms in the interactions with plants (Kamoun et al., 1999; Kamoun and Smart, 2005). Experiments on various Solanum species expressing different type and level of resistance against P. infestans may help to understand reactions induced by pathogen or pathogen - derived elicitor.

One of the earliest observable events of plants’ defence mechanism to biotic stresses is oxidative burst, an accelerated production of reactive oxygen species (ROS) (Halliwell, 2006; Marino et al., 2012; etc.). The generated ROS are considered to play a key role in the integration of diverse strategies leading to disease resistance. ROS may act as signal transduction agents for activating defence pathways and mediators in HR.



Solanum villosum Mill. (2n = 4x = 48), a wild Solanum species, commonly known as red-fruit nighshade, is used by many African tribes because of its nutritive values. S. villosum is believed to have originated in Eurasia, and is sometimes considered to have a southern European origin. It is widespread, but absent in Central and South America, and New Guinea. Scientific reports concerning S. villosum are scarce. Ojiewo et al. (2006) have developed two genotypes from wild-type tetraploid S. villosum (2n = 4x = 48). After application of colchicine to S. villosum, octoploid plants (2n = 8x = 96) were obtained. These plants had larger stomata, fewer but larger leaves, and were late flowering as compared to the wild-type tetraploids (Ojiewo et al., 2006; Masinde et al., 2007). Increase in ploidy level resulted in enlarged vigour and more intensive growth of 8x plants (Masinde et al., 2009). S. villosum is completely resistant to P. infestans since neither spreading lesions nor sporulation were observed on it in a number of experiments; hence, this species may be used as a source of resistance for breeding potato (Colon et al., 1993; Lebecka, 2008).

In this paper we present the molecular and cytogenetic characteristics of somatic hybrids obtained via polyethylene glycol (PEG)-mediated chemical fusion performed in order to transfer resistance to P. infestans from the wild tetraploid S. villosum to a susceptible diploid clone of S. tuberosum. We present S. villosum (+) S. tuberosum (VT) somatic hybrids’ morphology, ploidy, genome composition and resistance to P. infestans. We also report generation of ROS during elicitor (culture filtrate of P. infestans, CF) treatment in both parental species and in the interspecific somatic hybrid, expressing different level of resistance to P. infestans.

The detailed characteristics of VT somatic hybrids may help in further selection of the best clones for breeding tests. Moreover, comparative studies on kinetics and intensity of ROS production may lead to a better understanding of the mechanisms involved in polygenic resistance.
Materials and Methods

Plant material

The late blight- susceptible diploid Solanum tuberosum clone DG 81-68 (2n = 2x = 24) is a potato breeding line obtained in IHAR-PIB, Młochów Research Center, Poland. Seeds of the tetraploid wild species S. villosum Mill. (2n = 4x = 48), highly resistant to P. infestans, were provided by Warsaw Botanical Garden, Poland. The species origin is Birmingham, UK, and its accession no. is 4.

Axenic shoots of the parental species (received from IHAR-PIB, Młochów Research Center, Poland) and the generated interspecific somatic hybrids expressing different level of resistance to the oomycete pathogen P. infestans, were cultured in vitro as described previously (Polkowska-Kowalczyk et al., 2004). The plants grew under controlled conditions: day/light fluorescent lamp 150 μmol m-2s-1 for 16 h, day/night temperature of 22/18 ºC.

The protoplasts isolated from leaves of the parental species, were fused in the presence of polyethylene glycol (PEG), according to Szczerbakowa et al. (2003a,b). The regenerated somatic hybrids were propagated and grown in vitro on MS/2 hormone-free medium (Murashige and Skoog, 1962), supplemented with 2 % sucrose and 0.6 % agar.


RAPD analysis


For verification of the hybridity, the random amplified polymorphic DNA (RAPD) analysis was used. The total genomic DNA was isolated from leaves of 3-week-old in vitro grown plants, following the modified cetyltrimethylammonium bromide (CTAB) protocol, as described by Szczerbakowa et al. (2003a). The molecular markers generated in the presence of decamer primer, OPH-04 (5’GGAAGTCGCC3’), were used to confirm the hybrid nature of the regenerated plants. The polymerase chain reaction (PCR) was carried out in MJ Mini-Personal Thermal Cycler (Bio-Rad) in a total volume of 25 µL of reaction mixture containing: 0.5 U Taq DNA polymerase (Perkin Elmer), 1 × buffer (10 mM Tris-HCl pH 8.3) and 1.5 mM MgCl2, 0.4 µM primer, 0.25 mM of each dNTP (Roche Diagnostic), and the DNA template (15 ng). The reaction mixture was supplemented with sterile deionised water to total volume of 25 µL. The PCR parameters were established according to Naess et al. (2001). The amplification products (12 µL) were separated using electrophoresis in 1.5 % agarose gel in 0.5 × TBE buffer for 2 h at 90 V at room temperature, stained with ethidium bromide (4 µg/ml) and evaluated under UV (254 nm). A molecular size marker, 100-bp (Fermentas), was used.

Ploidy level determination


Ploidy level was determined firstly by counting of chloroplasts in guard cells on leaf lower surface of soil-grown plants. The mean of at least 20 measurements was indicative of ploidy of plants, as shown for potato by Jakuczun et al. (1997).

Chromosome number was determined directly by chromosome counting in metaphase spreads.


Chromosome preparations


Young roots (2 cm long) were detached 7-10 days after the last passage and used for chromosome counting. The root tips were treated with 0.05 % aqueous colchicine solution for 3 h to accumulate metaphases and then fixed with Carnoy solution (ethanol : acetic acid, v : v 3 : 1) for at least 48 h at 4 ºC. The fixed tissue was washed in 10 mM citric acid-sodium citrate buffer (pH 4.8) for 15 min prior to digestion in an enzyme mixture of 1 % cellulase Onozuka R-10 (Kinki Yakult MFG CO., Japan), 1 % cellulase from Aspergillus niger (0.32 U/mg, Serva), 20 % pectolyase (0.70 U/mg, Serva) for 50 min at 37 °C. After incubation, the root tips were transferred to the citrate buffer for 15 min at 4 ºC. Meristems were dissected out from root tips, squashed in drops of 45 % acetic acid and frozen. The cover slips were removed and the preparations were dehydrated in a graded series of 70 %, 80 % and absolute ethanol and air-dried. The slides were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma) and screened under Olympus IX70 fluorescent microscope with CCD camera. Pictures were analysed using Analysis 3.0 program.
Genomic in situ hybridisation (GISH) analysis

For hybrid genome composition analysis, GISH with total genomic DNAs from S. tuberosum and S. villosum as probes was used. DNA was isolated from leaves harvested from 3-week-old in vitro grown plants according to Doyle and Doyle (1987). Genomic DNA was sheared to a length of 1-10 kb and then labelled with digoxigenin-11-2’-deoxy-uridine-5’-triphosphate (digoxigenin-11-dUTP, Amersham) or biotin-16-2'-deoxy-uridine-5'-triphosphate (biotin-16-dUTP, Roche) by nick translation.

Genomic in situ hybridisation was performed according to protocols of Schwarzacher and Heslop-Harrison (2000), with minor modifications. Briefly, the hybridisation mixture, consisting of 100 ng of labelled DNA probe, 50 % formamide, 10 % dextran sulfate and 0.1 µg / µL of sheared salmon sperm DNA, was denatured for 10 min at 85 °C, then chilled on ice and applied to the chromosome preparation. The slides and hybridisation mixture were denatured together at 72 °C for 5 min in an in situ Thermal Cycler (Thermo Hybaid, Franklin, MA, USA) and then allowed to hybridise for 48 - 72 h in a humid chamber at 37 °C. Stringent washes (10 % formamide in 0.1  SSC at 37 C) were followed by immunodetection of labelled DNA probes with primary and secondary antibodies. Digoxigenin-labelled DNA probe was detected using fluorescein-conjugated anti-digoxigenin antibodies (Roche, Switzerland) and signals were amplified with fluorescein isothiocyanate (FITC)-conjugated anti-sheep secondary antibodies (Jackson ImmunoResearch, UK). Biotin-labelled DNA probe was detected with Texas Red-conjugated anti-biotin antibodies (Jackson ImmunoResearch, UK) and signals were amplified with Texas Red-conjugated anti-mouse secondary antibodies (Jackson ImmunoResearch, UK). The preparations were mounted in Vectashield (Vector Laboratories, UK) containing 2 µg / mL DAPI. At least 30 metaphase and interphase cells were examined for each genotype. Selected spreads and interphase nuclei were photographed with CCD camera attached to Eclipse 800 Olympus epifluorescence microscope using UV excitation for DAPI visualisation, blue light excitation for FITC-conjugated antibodies and green excitation for Texas Red labelled probes. Pictures were analysed using Lucia Orca program.



Assessment of late blight resistance

The resistance of genotypes studied to oomycete pathogen P. infestans was evaluated using detached leaflet assay as described by Zarzycka (2001). One of the most aggressive and virulent isolates of P. infestans (isolate MP324 from 1997, complex race: 1.2.3.4.5.6.7.8.10.11, defined using Black’s differential set, A1 mating type and resistant to metalaxyl) received from IHAR-PIB, Młochów Research Center, Poland, was used as inoculum. Sporangia were collected in deionised water and adjusted to standard concentration of 50 sporangia mm-3. Five to ten leaves from each genotype were tested on two different dates. The leaflets were placed upside-down on a plastic tray on the wet wood wool, and a drop of inoculum was placed near the midrib on each leaflet. Inoculated leaves were incubated at 16 ºC under constant illumination of about 30 μmol m-2·s-1. After six days of incubation, the resistance was evaluated using a 9-grade scale, where 9 indicated the highest resistance. Cultivars (cvs) Tarpan (susceptible) and Bzura (mid-resistant), as well as a diploid clone DG 94-15 (resistant), were used as standards.

The virulence of inoculum (on leaflets of Black’s differentials) and its aggressiveness (on cvs Bintje and Tarpan carrying no R-genes) were examined in each test.
Pathogen elicitor

The pathogen P. infestans (isolate MP618 from 2005, of following race: 1.2.3.4.(5).6.7.10.11, A1 mating type and resistant to metalaxyl) received from IHAR-PIB, Młochów Research Center, Poland, was maintained on rye agar medium at 15 ºC in the dark. A culture filtrate (CF), which served as an elicitor, was prepared from the pathogen grown in liquid medium. After 6 weeks of growth, the medium was separated from the oomycete, dialysed against water for 48 h and lyophilised. The CF residue dissolved in distilled water was quantified as µg glucose equivalents ml-1, as described in detail by Polkowska-Kowalczyk et al. (2004).


Elicitor treatment of leaves

Leaves harvested from 4-week-old in vitro plants were placed on moist filter paper in Petri dishes. The culture filtrate was applied in droplets on the surface of each leaf at a concentration of 0.67 μg glucose equivalents·g-1 FW. An appropriate volume of the elicitor was applied with a micropipette, in small droplets onto the abaxial leaf surface. In parallel, dishes with control leaves with an equal volume of distilled water in droplets were set up. Leaves were incubated at 25 °C under continuous light 150 μmol·m-2·s-1 supplied by fluorescent tubes (Pila, Poland) and kept for 6, 18, 30 h at 25 ºC under continuous light (Polkowska-Kowalczyk et al., 2004). At given time intervals the leaves were taken for analysis.

Assay for ROS production


ROS production was evaluated by determining the reduction of nitroblue tetrazolium (NBT) in the medium by O.-2 released from leaf tissues (Doke, 1983). The leaves treated with CF and the control leaves treated with water were incubated for 1 h in a mixture containing NBT (nitroblue tetrazolium (2,2’-di-p-nitrophenyl-5,5’-diphenyl-[3,3’-dimethoxy-4,4’diphenylene]-ditetrazolium chloride, Sigma). The mixture was then heated at 85 ºC for 15 min, cooled and absorbance at 580 nm was measured (Polkowska-Kowalczyk et al., 2004).

Results

Morphology

The chemofusion experiments between the protoplasts of the diploid potato clone DG 81-68 and wild species S. villosum (4x) yielded five clones of S. villosum (+) S. tuberosum (VT) hybrids. The morphology of the individual VT clones was similar, with very subtle differences among them. Four hybrid clones (VT-2, VT-3, VT-4 and VT-5) exhibited vigorous growth and rooted easily, and only the clone VT-1 exhibited some abnormalities. None of VT clones was producing tubers. Generally, the leaf size and shape of the hybrids were quite similar to S. villosum, but their growth habit was similar to S. tuberosum. Flowers of S. villosum were white and stellate with unfused petals that bent backwards when maturing. The other parental species S. tuberosum DG 81-68 had white flowers with fivefold symmetry and fused petals. Flowers of all somatic hybrids were also white. The flowers of VT-1, VT-4 and VT-5 somatic hybrids had fivefold symmetry with partially fused petals, while VT-2 and VT-3 had fourfold symmetry with unfused petals. The morphology of parental Solanum species and the selected VT somatic hybrids is shown in Figure 1.



Verification of hybridity by RAPD analysis

The hybridity of in vitro regenerants was verified by the RAPD method on the basis of the specific parental markers shown in band patterns of all the examined hybrids. Of several decamer primer tested, only the one primer OPH-04 generated polymorphic markers specific for each of the parents. All these specific bands were present in the tested regenerants, proving their hybridity. The RAPD patterns of nuclear DNA of the parental species and the five somatic hybrids are presented in Figure 2. The RAPD products characteristic for parental species, S. tuberosum and S. villosum, appeared in all VT hybrids studied. The RAPD profiles of all the VT hybrids were mostly uniform, suggesting similar genotypes of these independent clones.



Genome size

The best vigorous plants were used to evaluate the ploidy level using two different approaches: (1) raw estimation of ploidy level by counting chloroplasts in guards cells, (2) direct method of chromosome counts in metaphase spreads. The use of two methods resulted in rather similar observations, with the first method being less accurate but faster, while the second more accurate but more time consuming. On the basis of chloroplast number, all the hybrid clones seemed to be hexaploids (6x), as expected (Table 1). However, the direct method of chromosome counting showed that only two somatic hybrids, VT-1 and VT-5, were euploids (hexaploids) and possessed full sets of chromosomes from both parents (2n = 72). The VT-2 somatic hybrid was found to be pentaploid (5x). The remaining two hybrids, VT-3 and VT-4, were aneuploids (hypohexaploids) lacking 8 chromosomes. The pictures of chromosomes of parental species and the selected somatic hybrids are shown in Figure 3.



Genome composition

Genomic in situ hybridisation (GISH) has proved to be very valuable for identifying chromosomes from different parental species in hybrids (e.g. Tang et al., 2009). It can be applied to small chromosomes of Solanum species (Wilkinson et al., 1995; Dong et al., 1999).

Two selected somatic hybrids, euploid VT-5 and aneuploid VT-4, were analysed using GISH to check their genome composition. GISH with genomic DNA of S. tuberosum and S. villosum proved the hybrid origin of VT-5 and VT-4 clones and supported the results obtained from RAPD analysis. After GISH with genomic DNA of S. tuberosum, hybridisation signals were observed only on part of the VT-5 hybrid chromosomes. The majority of the chromosomes were not labelled indicating that they originated from the other parent. In the interphase nuclei of VT-5 plant there were large and distinct sectors of green-labelled S. tuberosum chromatin visible. After GISH with genomic DNA of S. villosum to VT-4 hybrid metaphase plate the majority of the chromosomes showed hybridisation signals, although a group of chromosomes without signals was also observed. In the metaphase plate (in prophase) of the analysed VT-4 plant there were large and distinct sectors of pink-labelled S. villosum chromatin visible. Both euploid (VT-5) and aneuploid (VT-4) were somatic hybrids possessing genomes from two fusion parents (Figure 4).

Resistance to P. infestans

Resistance to P. infestans was estimated using one of the most aggressive and virulent isolates of the oomycete pathogen - MP324. Testing for resistance of somatic hybrids against P. infestans showed that wild parental species S. villosum and all somatic hybrids were very highly resistant to late blight disease (over 8 in 9-grade scale, when 9 = resistant). No disease lesions, but only HR necroses or lack of any macroscopic symptoms were observed on these leaves. Parental potato diploid clone DG 81-68 was susceptible to P. infestans (about 2.5 in 9-grade scale) (Table 1).



ROS production

Reduction of nitroblue tetrazolium (NBT) by reactive oxygen species (ROS) released from leaves was used for determination of ROS production. Leaves detached from in vitro propagated plants were treated with the elicitor, the culture filtrate (CF) of P. infestans or water (control). Changes in ROS production in leaves treated with CF were compared to mean values of control, H2O treated leaves. Since two selected hybrids, euploid VT-5 and aneuploid VT-4, were similarly resistant to P. infestans, the ROS production was estimated only in one of them - VT-5.

In the leaves of resistant S. villosum, ROS production increased significantly (170 % of control level) after 6 h of incubation with CF (Figure 5). After 18 h of treatment, ROS production reached 220 % of control level, which started to decrease slightly at 30 h of experiment (205 % of control level). In the leaves of susceptible S. tuberosum DG 81-68, ROS production increased significantly at 6 h of incubation with CF to 282 %. At 18 h of treatment, ROS level reached even 629 % of control. Afterwards, it started to decrease significantly reaching 418 % of control. In the leaves of resistant somatic hybrid, VT-5, ROS production reached 152 % of control level at 6 h after CF treatment. At 18 h we observed a further significant increase to 252 % of control. Afterwards, it started to decrease, at 30 h of incubation reaching 168 %.

We have observed that the relative increase in ROS production in response to CF treatment was higher in leaves of the susceptible S. tuberosum clone than in the wild resistant Solanum species and its somatic hybrid.



Discussion

The interspecific somatic hybrids, 4x S. villosum (+) 2x S. tuberosum clone DG 81-68 (VT hybrids), were obtained and characterized molecularly and cytogenetically. The produced S. villosum (+) S. tuberosum hybrids varied slightly in morphology and exhibited features characteristic of both parents. Higher dosage of S. villosum hexaploid genome probably resulted in a stronger expression of S. villosum features - leaf size and shape, no tubers development, although growth habit was rather similar to S. tuberosum parental species. According to Szczerbakowa et al. (2003a) dominating traits of the wild parent were also described for S. bulbocastanum (+) S. tuberosum clone H-8105 somatic hybrids.

The RAPD analysis is often used for identification and selection of somatic hybrids. According to literature data, RAPD molecular markers, have been applied successfully for hybrids’ confirmation and cytoplasmic characterization (Sarkar et al., 2011). Molecular analysis with the RAPD markers revealed the presence of products characteristic of both parental species, S. tuberosum and S. villosum, in all the VT hybrids obtained. The RAPD profiles of all the VT hybrids were rather uniform, suggesting similarity of the genotypes of the obtained VT clones (Figure 2). According to this similarity, all the VT somatic hybrids were shown to be highly resistant to P. infestans (Table 1). The lower ROS production in response to CF treatment in leaves of wild resistant S. villosum species and its somatic hybrid in comparison with the susceptible S. tuberosum parent confirmed the transfer of the effective defence mechanism from S. villosum to its potato hybrids.

Similarly to RAPD analysis, GISH analysis confirmed the presence of both parental genomes in the VT hybrid genome (Figure 4). The GISH analysis is known to be a successful approach for selection of fusion hybrids as well as for verification of origin of their chromosomes (Horsman et al., 2001). Such cytogenetic analysis as well as chromosome count enable estimation of the completeness and proportion of parental genomes in individual hybrid clones (Greplová et al., 2008). This is very worthwhile for the variable and unstable genomes of the interspecific somatic hybrids.

In our case, in fusion-derived plants of intermediate morphology in relation to the parental species, the ploidy level was expected to be 6x; however, real ploidy of VT-hybrids varied, with two of them (VT-1 and VT-5) being euploids and three (VT-2, VT-3 VT-4) - aneuploids. Hybrids VT-5 and VT-1 possessed a full set of chromosomes from both parents, while the VT-2 was pentaploid with 60 chromosomes, and VT-3 and VT-4 had 64 chromosomes (Table 1). We suppose that aneuploids possessed all the S. tuberosum chromosomes whereas a part of S. villosum chromosomes was eliminated. This supposition would be also in line with literature data. According to Gernand at al. (2006), uniparental chromosome elimination occurred in several interspecific hybrids of plants, but a clear mechanism of elimination is not yet known. Gavrilenko et al. (2002) showed that in somatic hybrids chromosome elimination and structural changes occur frequently, for example, in Solanum brevidens (+) S. tuberosum hybrids, chromosome instability with various degrees of aneuploidy have been described. The expected 6x S. brevidens (+) S. tuberosum hybrids were hypohexaploids (2n = 6x = 64-70), each of them having more frequently lost a variable number of chromosomes from S. brevidens than from the cultivated potato. Szczerbakowa et al. (2011) reported that the variability in nuclear genome size of the S. nigrum (+) S. tuberosum allopolyploids maintained in vitro probably reflected somaclonal variation occurring during protoplast regeneration through the callus dedifferentiation stage. However, the tissue-culture alterations had no substantial effect either on morphology or on resistance of somatic hybrids to P. infestans.

The produced S. villosum (+) S. tuberosum somatic hybrids were resistant to P. infestans, with mean scores over 8 on resistance scale, when 9 = the most resistant. This was presumably due to a larger input of the resistant tetraploid S. villosum genome. The opposite situation was observed for S.michoacanum (+) S. tuberosum hybrids which were all susceptible to P. infestans, probably because of the 1:1 proportion of resistant (S.michoacanum) and susceptible (S. tuberosum) parental genomes (Szczerbakowa et al., 2010). In case of 4x S. villosum (+) 2x S. tuberosum hybrids with 2:1 proportion of the resistant (S. villosum) to susceptible (S. tuberosum) parental genomes, partial elimination of S. villosum chromosomes did not affect the plant resistance, similarly as it was reported for S. nigrum (+) S. tuberosum hybrids varying in ploidy (Szczerbakowa et al., 2003b; Zimnoch-Guzowska et al., 2003).

In search of the mechanisms involved in resistance of the Solanum species to P. infestans, the biochemical reactions occurring early after elicitor treatment were studied. Production of ROS is considered to be one of the earliest reactions induced by pathogens or their elicitors (Halliwell, 2006; etc.). The present study showed for the first time a comparison of the kinetics of ROS production during CF treatment in parental species and in the interspecific somatic hybrid, expressing different levels of resistance to the P. infestans. The obtained results complement our earlier studies on oxidative processes in Solanum species representing different levels of resistance to P. infestans (Polkowska-Kowalczyk et al., 2001; 2004). We have observed that the CF-induced ROS production varied in terms of kinetics and intensity, depending on the type and level of resistance exhibited by the genotype. Now, similarly to our previous results, it has been observed that the relative increase in ROS production in response to CF treatment was much lower in leaves of the resistant S. villosum species and interspecific somatic hybrid than in the susceptible S. tuberosum clone. Differences in ROS production among these genotypes might be explained by different R genes expressed in them. We also suggest that defence mechanisms existing in resistant wild Solanum species are transferred, among others, to somatic hybrids as the result of protoplast fusion. This is confirmed by the similarly low level of ROS production in VT somatic hybrid and its resistant parental species S. villosum.

Summing up, the detailed characteristic of S. villosum (+) S. tuberosum somatic hybrids may help in further selection of the best clones for breeding tests. Until now, backcrossing was attempted with two S. tuberosum pollinators of different ploidy (2x and 4x). The berries were produced, but with few and degenerated seeds. Other pollinators will be tried in future in order to obtain the viable seeds. Moreover, further experiments will be undertaken to elucidate the mechanisms involved in polygenic resistance, the only durable resistance against late blight disease known so far.



Acknowledgements

We wish to thank Dr. Anna Szczerbakowa for helpful discussion and critical reading of the manuscript. We are grateful to Dr. Renata Lebecka (IHAR-PIB, Młochów Research Center) for providing the isolates of P. infestans and help with resistance tests. The authors are also grateful to Michał Oskiera, M. Sci., for participation in fusion experiments, to Irena Dzikowska for technical support in plant cultures and to Dr. Krzysztof Olszak for help in photography. This work was partially supported by the Ministry of Science and Higher Education Project PBZ-MNiSW-2/3/2006/33.


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Table 1

Ploidy level and late blight resistance of the parental species and their hybrids.



Clone

Chromosome counts

Number of chloroplasts

in guard cell

(mean ± SD)

Ploidy

Resistance to

P. infestans,

(mean ± SD)

S. villosum

48

10.0 ± 1.2 (28)b

4x

8.6 ± 0.5 (24)c

S. tuberosum

DG 81-68

24

6.9 ± 1.0 (57)

2x

2.5 ± 1.6 (24)

VT-1

72 (20)a

nd

6x

9.0 ± 0.2 (30)

VT-2

60 (21)

14.6 ± 1.0 (26)

5x

8.2 ± 0.4 (36)

VT-3

64 (18)

15.5 ± 1.0 (24)

6x-

8.2 ± 0.4 (60)

VT-4

64 (16)

nd

6x-

8.8 ± 0.5 (12)

VT-5

72 (25)

16.0 ± 1.0 (26)

6x

8.3 ± 0.5 (150)



SD - standard deviation, nd - not determined;

a - number of metaphase spreads analysed

b - number of guard cells checked in parentheses

c - number of tested leaves

Legends of figures


Figure 1. In vitro grown parental species A) S. villosum, B) S. tuberosum DG 81-68 and selected somatic hybrids: C) VT-3 and D) VT-5.
Figure 2. RAPD profiles of S. villosum (lane 1, vll), S. tuberosum DG 81-68 (lane 2, tbr) and their somatic hybrids (lanes 3-7, from left to right: VT-1, VT-2, VT-3, VT-4, VT-5). Amplification was done with primer OPH-04. Arrows indicate the bands specific for S. villosum and S. tuberosum DG 81-68.
Figure 3. The metaphase chromosomes in root meristem cells of A) S. villosum 2n = 4x = 48, B) S. tuberosum DG 81-68 2n = 2x = 24, and two selected hybrids C) VT-4 2n = 64 and D) VT-5 2n = 6x = 72.
Figure 4. Chromosome constitution of S. villosum (+) S. tuberosum somatic hybrids. In case of aneuploid VT-4 (2n = 64) S. villosum genomic DNA was labelled with biotin, and afterwards immunodetected with antibodies conjugated with Texas Red (A, B).

In case of euploid VT-5 (2n = 72) S. tuberosum genomic DNA was labelled with digoxigenin, and afterwards immunodetected with antibodies conjugated with FITC (C, D).


Figure 5. Changes in ROS production in leaves of Solanum species and selected somatic hybrid (VT-5) treated with culture filtrate (CF) for 6, 18 and 30 h. Control level means average values represented by control leaves treated with H2O regardless of time of treatment. At least three independent series of experiments were performed. The data presented are the mean values ±SD (n=6 at least). The significance of difference (p<0.05) between mean values was determined by the Student’s t-test. The same letters indicate significant differences (p0.05) between mean values for CF treated leaves in respective genotypes, * significant differences (p0.05) between mean values for CF treated and control leaves.

















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