Introduction

Blackspot bruising is an undesired formation of discolorations under the potato tuber skin initiated by mechanical impact. When cells are damaged, polyphenol oxidase (PPO) and phenols, normally separated by cellular compartmentation, come into contact. A series of oxidative reactions of phenols, primarily tyrosine, result in the formation of melanins (Cobb 1999). According to studies 42 % of internal bruising is formed after harvesting, and that value increases to 54 % after grading (Peters 1996). Internal discolorations drastically reduce commercial value of tubers, lead to rejection of the crop by consumers and processing industry and considerable economic losses (Storey 2007).

Blackspot bruising is a complex trait and depends on multiple factors: genotype, environment, anatomical, physical and physiological properties of tuber tissues such as starch content (Zgórska 2000; Urbany et al. 2011), tuber age, turgor pressure (Lærke et al. 2000, 2002a). According to many authors, blackspot bruise resistance depends essentially on the genetic factors which was indicated by the large share of this component in the analysis of variance (Zgórska 2000; Komorowska-Jędrys et al. 2002; Domkářová and Vokál 2005; Domański et al. 2007). Pavek et al. (1993) have showed very high heritability in narrow sense (Hn = 0.85) of blackspot bruise susceptibility. Results of Komorowska-Jędrys et al. (2002), Domański et al. (2007) and Urbany et al. (2011) studies have indicated mid- high to high heritability in broad sense.

The utilization of wild potato species could be potential approach in prevention of bruising in the breeding programs. So far, one of wild species, Solanum hjertingii, has been identified as potential source of resistance to blackening (Woodwards and Jackson 1985). S. hjertingii has been an object of studies on PPO role in discoloration formation by several authors (Sim et al. 1997; Brown et al. 1999; Culley et al. 2002).

The level of tyrosine content and activity of PPO are considered to be the main factors involved in blackspot bruise formation (McGarry et al. 1996). The importance of PPO in discoloration process was confirmed by results in many studies (Coetzer et al. 2001; Arican and Gozukirmizi 2003; Urbany et al. 2011, 2012). Tyrosine is an essential substrate for discoloration reaction. The results of some earlier studies have indicated that tyrosine level is well correlated with enzymatic discoloration potential after tissue homogenization and after abrasive peeling (Dean et al. 1993). On the other hand, no correlation between bruising and tyrosine level has been found in studies using impact method (Mondy and Munshi 1993; Stevens and Davelaar 1997; Strehmel et al. 2010). Mondy and Munshi (1993) studied two potato cultivars, one resistant and one susceptible to bruising, have found that tyrosine level was not a limiting factor for bruising determination, since in the susceptible cultivar they observed a lower level of tyrosine. Stevens and Davelaar (1997) have demonstrated positive correlation between potential to discoloration formation and tyrosine content, but at the same time they have not found correlation between blackspot bruise susceptibility and level of tyrosine. Strehmel and coworkers (2010) have proved that the level of tyrosine did not change significantly after mechanical impact in two tested cultivars. Before impact, the content of tyrosine was higher in the cultivar resistant to blackspot bruise. Other potential monophenolic substrates present in tubers, like caffeic and chlorogenic acid were not correlated with the blackspot formation (Laerke et al. 2002b).

Until now complex diploid Solanum hybrids obtained in IHAR-PIB Młochów, broadly tested and used for many purposes, were not evaluated for blackspot bruise resistance. They originated from a long- term program aimed at improving resistances to pathogens and quality traits by exploitation of new variation present in wild and primitively cultivated Solanum species (Zimnoch-Guzowska et al. 1998). The goal of the present study was to define variation in susceptibility to blackspot bruising among interspecific diploid hybrids. We would like to demonstrate usefulness of selected Solanum hybrids in potato breeding as sources of high resistance to blackspot bruise.

Material and Methods

Plant Material

From crosses between interspecific hybrids, 31 diploid clones were selected for this study (Table 1). They were resulted from long-term recombinant breeding process. In their pedigree there are following Solanum species: Solanum acaule, S. chacoense, S. demissum, S. goniocalyx, S. gourlayi, S. microdontum, S. phureja, S. stenotomum, S. verrucosum, S. yungasense and dihaploids of S. tuberosum. A detailed composition of the material is shown in Table 1. Resistances to PLRV, PVX, PVY, PVM, Synchytrium endobioticum, Phytophthora infestans and Pectobacterium carotovorum spp. carotovorum were identified in these clones along selection process (Table 2). Diploid clones, used in the experiment, possessed different leading traits: table usefulness, usefulness for chip processing, high starch content, resistance to soft rot and resistance to late blight. Non- bruising table cultivar (cv.) Vitara and bruising starch cv. Hinga were used as positive and negative standards.

Table 1 Pedigree of tested diploid interspecific hybrids. Diploid clones are ordered according to increasing susceptibility to bruising
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Table 2 Characteristics of diploid clones and standard cultivars: blackspot bruising, starch content and quality traits, resistance to pathogens, ability to produce 2n gametes and leading traits. Diploid clones are ordered according to increasing susceptibility to bruising
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Growing and Storage Conditions

All tested diploid clones and cultivars were planted in the end of April and harvested in the end of September in 2008 and 2009. They were propagated in experimental fields of IHAR-PIB Młochów. They were fertilized with 90, 90, 170 kg h−1 of N, P and K in 2008 and 120, 90, 180 kg h−1 of N, P and K in 2009, treated with pesticide whenever necessary. The total amounts of rainfall during the entire growing period were 187.7 mm and 348.2 mm in 2008 and 2009, respectively. The average daily temperatures during 5 months of growing season (from May to September) were similar in both years, 13.5 °C, 18.7 °C, 19.2 °C, 19.0 °C, 14.6 °C and 13.5 °C, 16.2 °C, 20.0 °C, 18.5 °C, 14.3 °C in 2008 and 2009, respectively. Diploid clones were hand-harvested in September and stored up to a bruising test at 5 to 10 °C and 95 % relative humidity.

Assessment of Blackspot Bruise Resistance

Experiment was conducted in mid - February in two consecutive years 2009 and 2010. To assess the susceptibility to blackspot bruising 20 undamaged, non-greening tubers with standardized weight, were selected for tests. Sample tubers were stored for 5 months at 5 to 10 °C. Blackspot bruise susceptibility was evaluated according to Domański et al. (2007). Just before test tubers were incubated for 12 h at 11 °C. After that tubers were placed in hexagon playwood drum and rotated ten times to produce simulated bruising (Douches et al. 2003). Subsequently, samples of tubers were stored for 72 h at 20 °C. Two-person team estimated visually a degree of bruising. Mean surface of blackspot bruises was evaluated for each tuber separately in scale 1 to 9, where 1 = 80.1 to 100 % of tuber surface covered by bruises, 2 = 60.1 to 80 %, 3 = 40. 1 to 60 %, 4 = 25.1 to 40 %, 5 = 15.1 to 25 %, 6 = 10.1 to 15 %, 7 =5.1 to 10 %, 8 = 0.1 to 5 %, 9 - lack of bruises. Evaluations were performed in two replications, in each 10 tubers were tested.

Assessment of Starch Content

Starch content was determined by underwater weight method. Starch content (%) was estimated from ratio of tuber weight in air (g)/tuber weight in water (g) according Lunden (1956).

Assessment of L-Tyrosine Content

L-tyrosine content was evaluated in tuber tissue extracts from 26 diploid hybrids collected from field in 2010.

Sample Preparation

For analysis, five undamaged tubers from each diploid clone were taken. After skin removal, a cylindrical sample of tissue (ca 10 mm) from the stolon and bud ends was taken using cork borer. Samples were immediately frozen in liquid nitrogen, freeze-dried and grounded to powder. 0.5 g of sample was homogenized for 2 min at maximum speed in Ultra-Turrax homogenizer with 8 mL of mixture containing methanol and 0.5 % orthophosphoric acid (1 : 1) with BHT (butylated hydroxytoluene; 1 mg mL−1). The extracts were centrifuged at 4,500 × g for 25 min at 20 °C and 6 ml of supernatant was filtered and evaporated to dryness in a vacuum evaporator. Samples were diluted in 1. 5 mL mixture of methanol and 0.5 % orthophosphoric acid (in ratio 1 : 1), filtered through a 25 mm, 0.2 μm syringe filter and 20 μL was injected for HPLC analysis. Each extract was injected in triplicate.

HPLC Analysis

RP-HPLC was performed with LC Prominence HPLC system (Shimadzu, Japan) comprising a pump LC - 20 AD, a vacuum degasser (DGU - 20 A3), UV - VIS detector (SPD-20 A) and using autosampler (SIL 20 ACHT). L - tyrosine standard was HPLC - grade purity from Roth (Switzerland). Methanol, acetonitrile, water and orthophosphoric acid were of HPLC-grade purity (POCh, Poland). Separation was performed using Vertex C-18 column (4.6 × 250 mm, 5 μm particle, Knauer) at 40 °C. The mobile phase consisted of a binary solvent - 0.5 % orthophosphoric acid (solvent A) and acetonirile (solvent B) and gradient elution (0 to 2 min, 5 % B; 2 to 5 min, 5 to10 % B; 5 to 7 min, 10 % B; 7 to 10 min, 10 to 15 % B; 10 to 13 min, 15 to 20 % B; 13 to 23 min, 20 % B; 23 to 25 min, 20 to 30 % B;25 to 28 min, 30 % B; 28 to 30 min, 30 to 50 % B; 30 to 32 min, 50 % B; 32 to 33 min, 50 to 5 % B; 33 to 35 min, 5 % B) were used. Detection was at 275 nm. The flow rate was set to 1 mL min−1. The external standard method of calibration was used with curve prepared from 5 different concentrations of the standard solution. The standard stock solution of L - tyrosine 1 mg mL−1 was prepared in methanol. L - tyrosine peak was identified by a direct comparison of its retention time with the standard.

Statistical Analyses

Analysis of variance (ANOVA) was performed to assess the effects of genotype, year and their interaction on the results of the blackspot bruise resistance. Broad-sense heritability for diploid hybrids was estimated according following formula: Hb = δ 2 g/δ 2 g + δ 2 ge + δ 2 e (where, δ 2 g = M1 − M2/L;  δ 2 ge = M2 − δ 2 e; M1 = mean square of genotype; M2 = mean square of interaction year × genotype; L = number of year) (Domański et al. 2007). Homogenous groups of genotypes according to mean blackspot bruise resistance were distinguished by the Tukey’s test. The reproducibility of the tests between years and the relationships of blackspot bruise resistance with starch content and L - tyrosine content were evaluated by calculating linear Pearson’s correlation coefficients. All calculations were performed using computer program STATISTCA for Windows (Soft, Inc., Tulsa, OK, U.S.A.).

Results

Among tested diploid interspecific hybrids originated from wild Solanum species and dihaploids of S. tuberosum, 11 forms were highly resistant to bruising, scored above 8 (Table 2). S. tuberosum, S. phureja and S. chacoense were predominant in pedigrees of majority of the tested clones in comparison to remaining species. Additionally, in two clones, high contribution of S. goniocalyx in pedigree was observed (Table 1). Prevailing theoretical contribution of S. tuberosum (range 46.8 – 74.1 %) was noted both in susceptible and resistant forms. The associations between blackspot bruise resistance and content of any of predominant species in assessed 31 diploid clones were not proved. Most clones resistant to bruising were also resistant to potato viruses, some were resistant to S. endobioticum, P. infestans or P. carotovorum (Table 2). All these resistant genotypes produced big pollen grains which effectively worked in 4x × 2x crosses in other studies.

Majority of clones classified as table stocks were highly resistant to bruising (Table 2). In other groups of usefulness, only single forms were resistant to blackspot bruise. All clones with high starch content were very susceptible to bruising. Starch content of all diploid hybrids varied from very low (8.8 %) to very high (26.4 %). Starch content of highly resistant clones ranged from 8.8 to 22.9 %, while starch content of diploids susceptible to bruising varied between 15.3 and 26.4 %. Starch content of cv. Vitara was 12.8 % and cv. Hinga 20.8 %. Correlation coefficient between blackspot bruise resistance and starch content for all diploid genotypes was significant and negative r = −0.67 (p < 0.05). All tested diploid clones produced good yield. Average tuber weight of tested clones was ca. 30 g and the average yield was about 500 g/hill.

Two-year mean values of blackspot bruise resistance of diploid clones ranged from 1.8 to 9 and covered the whole scale. Significant variability in blackspot bruising among tested genotypes was confirmed by Tuckey’s test at p < 0.05 (Fig. 1, Table 3). Cv. Vitara was estimated as highly resistant to bruising (grade 8.9) and cv. Hinga as moderately susceptible (grade 5.0). The mean blackspot bruise resistance of diploid hybrids was significantly correlated between 2 years and the correlation coefficient at p < 0.05 was r = 0.73. Generally, standard deviations of two-year mean values of bruise resistance (Table 2, Fig. 1) were lower for highly resistant and highly susceptible clones than for clones classified as intermediate.

Analysis of variance of diploid genotypes showed significant effect of genotype and year on blackspot bruising susceptibility (Table 4). Genotype had the largest influence on blackspot bruise formation, explaining 85.1 % of variance. The effect of year was weak, but significant and explained 5.0 % of variance. Interaction between genotype and year was significant and explained next 13.4 % of variance. Broad-sense heritability of blackspot bruise for diploid genotypes was estimated as moderately high Hb = 0.73.

The endogenous amounts of tyrosine for 26 diploid clones varied between 0.23 and 0.77 mg per 1 g dry weight. In tubers of resistant and susceptible clones both high and low content of substrate was observed. For tested 26 genotypes correlation coefficient between blackspot bruise resistance and tyrosine content was not significant (r = 0.33, p > 0.05).

Fig. 1
figure 1

Variability in blackspot bruise resistance among tested diploid potato clones with different leading traits and standard cultivars. Two-year mean values in 1 – 9 scale, where 9 = lack of bruises

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Table 3 Mean blackspot bruise susceptibility of 31 diploid genotypes and their classification according to Tukey’s test at p < 0.05
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Table 4 Analysis of variance for two-year mean values of blackspot bruise resistance for 31 diploid genotypes
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Discussion

Presented studies were the first effort to evaluate the blackspot bruise resistance variation of diploid interspecific hybrids obtained in IHAR-PIB Młochów. Diploid clones used in our study have in their pedigree various Solanum species, like S. acaule, S. chacoense, S. demissum, S. goniocalyx, S. gourlayi, S. microdontum, S. phureja, S. verrucosum, S. yungasense and dihaploids of S. tuberosum. None of these species was documented before as a source of resistance to blackspot bruise or enzymatic blackening, a trait significantly correlated with bruising. 11 of tested clones were highly resistant to brusing and scored above 8, in 1–9 scale. Analyzing theoretical composition of species in pedigree of majority of tested clones S. tuberosum, S. phureja and S. chacoense were found as predominant species (Table 1). In highly resistant hybrids higher contribution of S. phureja in origin was observed in comparison to the most susceptible to blackspot bruise clones. Nonetheless, we cannot indicate which of species are exactly donors of resistance to blackspot bruise, because no relationship between blackspot bruise susceptibility and contribution of any predominant species was found (Table 1). Thus, we rather suspect that combination of specific alleles of genes determines decreasing discoloration formation after the impact. One of wild tetraploid 2 EBN species, S. hjertingii, has been recognized as a source of low enzymatic blackening (Woodwards and Jackson 1985). In this species the level of PPO was lower than in cultivated lines (Sim et al. 1997; Brown et al. 1999). There have been some attempts to transfer this trait to tetraploid level (Culley et al. 2002), however the use of sexual crosses of wild species directly to S. tuberosum, without prebreeding, means a laborious elimination of negative traits associated to the wild species. This option might be easier for selection when gene cloning and genetic engineering will be applied in breeding wider.

Tested diploid clones were obtained in long-term recombinant breeding process among above presented Solanum species (Table 1). In the years of selection most of wild features were eliminated. Previously these diploids were selected for quality and resistance traits (Jakuczun and Wasilewicz - Flis 2004). Effectiveness of function of 2n pollen in 4x × 2x crosses was also checked, and this character was frequent in tested set of diploids. In prior studies among tested diploid clones resistance to PLRV, PVX, PVY, PVM, S. endobioticum, P. infestans and P. carotovorum have been found (Table 2). High contribution of S. tuberosum in origin of tested hybrids determine good level of agronomic traits. Yield and average tuber weight of tested hybrids were on a good level, typical for interspecific hybrids selected for complex quality traits in IHAR - PIB Młochów.

Most of diploid clones highly resistant to bruising were useful for table use, and two of them were cold chippers (Table 2). It should be mentioned that some of presented clones have been successfully used as a source of “cold chipping” in 4x × 2x crosses (Domański et al. 2006a, b, 2010). Authors have selected tetraploid progeny meeting desired agronomic and chipping quality traits on a better level than tetraploids obtained in traditional 4x × 4x crosses.

The results of our studies indicated that blackspot bruise resistance was mainly influenced by genotype (85.1 %). Broad-sense heritability of bruising potential was estimated as moderately high (Hb = 0.73). These results and conclusions from other studies (Pavek et al. 1993; Zgórska 2000; Komorowska-Jędrys et al. 2002; Domkářová and Vokál 2005; Domański et al. 2007; Urbany et al. 2011) have showed that the breeding for resistance to blackspot bruising could be very effective. Domański and coworkers (2007) from among 41 tested cultivars have identified several ones resistant to blackspot bruising. Authors have recommended them as source of this trait for potato breeding. Genetic pool of breeding potato needs new alleles of genes for broadening still narrowed variability. Thus, besides of cultivars the presented diploids could be the good donors of resistance to bruising. Diploid potatoes obtained in IHAR-PIB Młochów have been verified as good sources of other traits in 4x × 2x program, like resistance to P. infestans (Śliwka et al. 2010), resistance to Pectobacterium spp. (Lebecka et al. 2004), cold chipping (Domański et al. 2006a, b), starch content and table traits (Domański et al. 2000, 2010).

It was found that a low level of tyrosine, the main substrate of discoloration reaction, is not related to resistance found in our diploid hybrids. The observed lack of correlation between low tyrosine content and resistance to bruising indicated that substrate level does not seem to be preliminary factor playing a main role in mechanism of bruising formation. This conclusion is in agreement with results of other studies (Mondy and Munshi 1993; Stevens and Davelaar 1997; Strehmel et al. 2010).

In this study we presented a chosen group of diploid potato hybrids and their value as potential donors of resistance to blackspot bruising. Although this trait is not the main one in selection processing, but taking into consideration fact, that bruising is responsible for significant decreasing of tuber yield, it is worth to intensify introducing of this trait to breeding material. Diploid potato hybrids are excellent materials for clarification of genetic background of resistance to bruising, which has not been still explained.