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)
1
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-
2
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
3
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).
4
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.
5
Solanum villosum Mill. (2n = 4x = 48), a wild Solanum species, commonly known as redfruit 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.
6
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 MiniPersonal 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
7
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
8
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
9
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,
10
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).
11
Results
Morphology
The chemofusion experiments between the protoplasts of the diploid potato clone DG 8168 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.
12
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
13
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 9grade 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,
14
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
15
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
16
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,
17
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.
18
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Table 1
Ploidy level and late blight resistance of the parental species and their hybrids.
Clone
S. villosum
S. tuberosum
DG 81-68
VT-1
VT-2
VT-3
VT-4
VT-5
48
Number of chloroplasts
in guard cell
(mean ± SD)
10.0 ± 1.2 (28)b
24
72 (20)a
60 (21)
64 (18)
64 (16)
72 (25)
Chromosome
counts
4x
Resistance to
P. infestans,
(mean ± SD)
8.6 ± 0.5 (24)c
6.9 ± 1.0 (57)
2x
2.5 ± 1.6 (24)
nd
14.6 ± 1.0 (26)
15.5 ± 1.0 (24)
nd
16.0 ± 1.0 (26)
6x
5x
6x6x6x
9.0 ± 0.2 (30)
8.2 ± 0.4 (36)
8.2 ± 0.4 (60)
8.8 ± 0.5 (12)
8.3 ± 0.5 (150)
Ploidy
SD - standard deviation, nd - not determined;
ab
number of metaphase spreads analysed
- number of guard cells checked in parentheses
c-
number of tested leaves
25
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
26
(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.
27
28
29
30
31
32