Introduction

A huge genetic diversity is available in Solanum species for various desirable traits. Wild Solanum species have been used in potato breeding but they represent only a small fraction to the total Solanum diversity (Bradshaw et al. 2006). Huge efforts are involved in successful utilization of wild species to widen the narrow genetic base of the cultivated potato. Many useful genes of wild sources cannot be transferred to common potato through conventional breeding because of sexual barriers caused by the differences in ploidy level and endosperm balance number (EBN) (Spooner and Salas 2006). However, it is now possible to overcome the sexual barriers using methods such as manipulation of ploidy and EBN, bridge crosses, mentor pollination, embryo rescue, hormone treatment, reciprocal crosses, and somatic hybridization (reviewed by Jansky 2006).

Somatic hybridization aims to strengthen the potato gene pool by introducing genes from wild species (Helgeson et al. 1993). This technique allows several advantages over conventional breeding and transgenic methods, such as: (i) produces fertile somatic hybrids with target traits of wild Solanum species, (ii) provides access to basic pre-breeding material for effective utilization in breeding, (iii) enables easy transfer of monogenic and polygenic traits in one step, (iv) results recombination of nuclear and cytoplasmic genomes, and (v) avoids biosafety regulatory issues associated with transgenics. In the past, a few researchers discussed protoplast fusion in Solanum species (Orczyk et al. 2003), plant species (Waara and Glimelius 1995; Davey et al. 2005; Eeckhaut et al. 2013), citrus (Guo et al. 2013), asymmetric fusion (Lakshmanan et al. 2013), asymmetric fusion in wheat (Liu and Xia 2014), integeneric fusion (Liu et al. 2005) and crop improvement in China (Wang et al. 2013).

During the past 40 years, hundreds of symmetric somatic hybrids were produced, while limited number of asymmetric somatic hybrids. More than a decade back, Orczyk et al. (2003) reviewed application of potato somatic hybrids in genetics and breeding, and since then several progresses have been achieved in this area. Hence, this review aims to progress in somatic hybridization research in potato and their application to genetics, breeding and genomics studies.

Production of potato somatic hybrids

Protoplast fusion and culture

Protoplast isolation was first reported by Cocking (1960) and after a decade successful regeneration of plants from tobacco protoplasts was obtained (Takebe et al. 1971). With the development of electrofusion method, reports on recovery of somatic hybrid regenerants were more prevalent (Fish et al. 1988; Bates et al. 1987) might be due to easy control of fusion parameters than the chemical method using polyethylene glycol (Wallin et al. 1974). In food crops, electrofusion was first reported in potato between Solanum tuberosum and semi-cultivated (S. phureja) (Puite et al. 1986). Leaf-mesophyll cells of in vitro-grown plants were used largely to isolate protoplasts in protoplast-digestion-medium (Binding et al. 1978), then culture of fused products in the VKM medium (Binding and Nehls 1977) followed by shoot development on the MS13K medium (Behnke 1975). Recently, selection of somatic hybrids (S. tuberosum + S. chacoense) based on callus growth tagged with green fluorescent protein has been observed (Rakosy-Tican and Aurori 2015). Over the 40 years, many fusion experiments were executed and hundreds of potato somatic hybrids were produced and characterized, as summarized in Table 1.

Table 1 Summary of production and characterization of potato somatic hybrids developed using Solanum species

Confirmation of somatic hybrids

A large number of potato somatic hybrids have been produced via protoplast fusion between the common and the wild species S. acaule, S. berthaultii, S. brevidens, S. bulbocastanum, S. cardiophyllum, S. chacoense, S. circaefolium, S. commersonii, S. etuberosum, S. × michoacanum, S. melongena, S. nigrum, S. phureja, S. pinnatisectum, S. tuberosum, S. sanctae-rosae, S. spegazzinii, S. stenotomum, S. tarnii, S. torvum, S. vernei, S. verrucosum, and S. villosum (Table 1). Following regeneration, somatic hybrids are characterized through various methods such as cytological (flow cytometry, chromosome count, guard cell count, FISH-fluorescence in situ hybridization and GISH-genomic in situ hybridization), isozyme, molecular markers (e.g. RAPD-random amplified polymorphic DNA, RFLP-restriction fragment length polymorphism, ISSR-inter simple sequence repeat, SSR-simple sequence repeat, AFLP-amplified fragment length polymorphism, and DArT-diversity array technology), phenotypes (e.g. foliage, stem, leaf, flower and tuber traits) and pollen fertility. Somatic hybrids are also analyzed for cytoplasm types (W/α, T/β, W/γ, W/δ and S/ε: Lössl et al. 1999) based on organelle (chloroplast and mitochondria) genomes-specific markers as described by Lössl et al. (2000). Finally, somatic hybrids are examined for the presence of target traits under field or controlled conditions and for many other traits (Table 1).

Application of potato somatic hybrids

Genetics

Genetics of potato somatic hybrids and their segregating progenies has been studied in nuclear genome to dissect the recombination patterns. Chromosomal segregation pattern was analyzed in hexaploid somatic hybrid (S. brevidens + S. tuberosum) and their progenies with S. tuberosum using RFLP (Williams et al. 1993) and RAPD markers (McGrath et al. 1996). Study suggests that S. brevidens ribosomal (r)DNA loci are primarily contributed to isochromosome formation in the hybrids and progenies (McGrath and Helgeson 1998). Further, a chromosome substitution line was developed in S. breviden-somatic hybrids and progenies (BC1, BC2 and BC3) with S. tuberosum. Study demonstrates that a single copy of chromosome 8 from S. brevidens replaced the same in the BC3 clone and has significant impact on transferring resistance to tuber soft rot and early blight (Tek et al. 2004). The importance of combining GISH and DNA markers was also suggested to study chromosomal behaviours in potato (Dong et al. 1999). Poor chromosomal pairing was observed in somatic hybrid (S. etuberosum + S. tuberosum) and progenies (BC1 and BC2), and further suggests that genome dosage affects tuber formation but has less effect on potato virus Y (PVY) resistance (Gavrilenko et al. 2003). Evidence of tetrasomic inheritance was investigated in a tetraploid somatic hybrid (S. commersonii + S. tuberosum) and F2 progeny (90 individuals) using RAPD and AFLP markers. Segregation pattern was investigated by RFLP markers in a progeny of hexaploid somatic hybrids (S. acaule + S. tuberosum) crossed with S. tuberosum. Study concludes that somatic hybridization allows the effective use of S. acaule genes into cultivated potato (Yamada et al. 1998). Recently, somatic hybrid (S. chacoense + S. tuberosum) exhibited tetrasomic or disomic segregation ratio using SSR markers and suggested that pentaploid hybrid exhibits tetraploid inheritance pattern (Chen et al. 2016).

Interaction between nuclear and cytoplasmic genes can affects fertility and agronomic traits of somatic hybrids and progenies (Lössl et al. 1994). Segregation and recombination patterns of organelle genomes were investigated in potato (Frei et al. 1998) and analyzed variations (Tiwari et al. 2014, 2016). Majority of somatic hybrids follow recombination of mitochondrial genome from both parents, and chloroplast pattern from only one parent (e.g. S. bulbocastanum: Iovene et al. 2007; S. pinnaticetum: Sarkar et al. 2011; S. chacoense: Chen et al. 2013) except recombination of chloroplast genome was observed only once in S. vernei-somatic hybrid (Trabelsi et al. 2005). Loss of male sterility was observed in nuclear-mitochondrial genomes re-arrangement in S. commersonii-somatic hybrids and BC1 progeny (Cardi et al. 1999). Study suggests possibility of exploitation of novel cytoplasm in potato breeding (Scotti et al. 2003), especially variation in a hot spot mitochondrial region (rpl5rps14) (Scotti et al. 2004). Recently, random and non-random segregations of organelle genomes were observed in somatic hybrid (S. × michoacanum + S. tuberosum) using DArT markers (Smyda-Dajmund et al. 2016).

In search of new genes, a few potato somatic hybrids and their progenies were exploited in linkage mapping studies. The RB gene (Rpi-blb1) originates from diploid wild species S. bulbocastanum of somatic hybrid (S. bulbocastanum + S. tuberosum) and confers durable resistance to late blight (Helgeson et al. 1998; Song et al. 2003). The RB gene was mapped to potato chromosome 8 through analysis of somatic hybrid progenies (BC1 and BC2) (Naess et al. 2000, 2001). Besides, genetic stability of in vitro plants of somatic hybrids (S. tuberosum dihaploid ‘C-13’ + S. pinnatisectum, and ‘C-13’ + S. etuberosum) was confirmed using methylation-sensitive amplified polymorphism (MSAP) and AFLP markers (Tiwari et al. 2013b, 2015d, e).

Breeding

Improvement of somatic hybrids is essential for desirable agronomic traits through breeding methods to decrease the undesirable effects of wild species. In addition, transfer of disease/pest resistance traits from somatic hybrids to progenies is also important. Assessment of genetic and phenotypic variation among somatic hybrids (Gavrilenko et al. 1999) and development of their advanced progenies such as F2, BC1, BC2 and BC3 led to the effective utilization somatic hybrids in potato breeding (Table 1). Somatic hybrids performed better in field trials in terms of tuber traits and phenotypes (Carrasco et al. 2000), and a few hybrid produced higher yield, tuber number and weight than parents (Möllers et al. 1994). Tuber yield per plant of backcrossed progenies of somatic hybrid improved considerably to that of parents (Carputo et al. 2000). Recently, potential S. pinnatisectum-somatic hybrids (P4, P8 and P10) were selected for adaptability, tuber traits, late blight resistance and keeping quality traits in the sub-tropical plains of India, where nearly 90% of potato is grown (Luthra et al. 2016). Further, hybrid progeny (BC1) was also generated that can be utilized in potato breeding (Luthra et al. 2016). Good tuber yield and quality were observed in S. tarnii-somatic hybrids derived BC1 progenies in the field trials (Thieme et al. 2008). Further, study confirmed that somatic hybrid had resistance to both PVY and late blight, of which only PVY resistance was transferred to BC1 progeny. Multiple years of field evaluations of S. etuberosum-somatic hybrids and progenies showed stable transmission and expression of PLRV and PVY resistances in three (BC1, BC2 and BC3) and two (BC1 and BC2) generations, respectively (Novy et al. 2007). Besides, resistances to PVY, potato leaf roll virus (PLRV), and Green peach aphid in BC2 progeny were also observed (Novy et al. 2002). Another study demonstrates that late blight resistance can be transferred successfully through breeding from tetraploid somatic hybrids (S. × michoacanum + S. tuberosum and autofused S. × michoacanum) to common varieties (Smyda-Dajmund et al. 2017). Bacterial wilt resistance was transferred to advanced progenies of S. commersonii-somatic hybrids, and three highly resistant clones (BC1 and BC2) were selected as breeding materials (Kim-Lee et al. 2005). The effect of genetic constitution of S. tuberosum was investigated in S. bulbocastanum-somatic hybrids and progenies (BC1 and BC2) (Rakosy-Tican et al. 2015). Field performance for foliage maturity and tuber traits (tuber yield, tuber number, tuber weight and specific gravity) was observed in somatic hybrids (S. brevidens/S. commersonii + S. tuberosum) progenies and implicated to breeding for freezing tolerance (Chen et al. 1999a, b, c). Somatic hybrids (S. commersonii + S. tuberosum) were observed more similar to cultivated potato for phenotypes in field evaluations and developed F2 progeny, and suggested to fast transfer of useful traits from S. commersonii into cultivated background (Cardi 1998; Cardi et al. 2002). Recently, SSR alleles were identified for bacterial wilt resistance breeding in S. chacoense-somatic hybrids and backcross progenies (Chen et al. 2016). To our knowledge, there are investigations up to BC3 generation and all attempts to exploit somatic hybrid material so far have not resulted in registration of a potato cultivar.

Genomics

Somatic hybrids and their parents have been exploited to a limited extent in genomics studies. Whole genome sequences of chloroplast genome of wild potato species (S. commersonii), a commonly used fusion parent, was deciphered and identified two Indel markers for application in chloroplast genotyping (Cho et al. 2016). High-throughput genotyping of somatic hybrids (S. × michoacanum + S. tuberosum) showed presence of both parental chromosomes and loss of some markers (13.9–29.6%) in the hybrids using 5358 DArT markers analysis (Smyda-Dajmund et al. 2016). In functional genomics, genes controlling potato tuberization in tuber-bearing somatic hybrids (S. tuberosum + S. etuberosum) vs. control parent (S. etuberosum-non tuberous) were identified using microarray. Findings suggest candidate genes expression in leaf tissue of somatic hybrid are implicated to tuber growth and development process such as transport, carbohydrate metabolism, phytohormones and transcription/translation/binding functions (Tiwari et al. 2015a, b). In another study, late light resistance genes were identified in somatic hybrid (S. tuberosum + S. pinnatisectum) by microarrays and study suggests a broad spectrum of candidate genes involved in late blight resistance in the hybrid (Singh et al. 2016a). Further, recent identification of eight miRNAs (miR395, 821, 1030, 1510, 2673, 3979, 5021 and 5213) in S. pinnatisectum-somatic hybrid for late blight resistance genes and their targets has led a new insight in potato biology (Singh et al. 2016b). Most of the predicted target genes are associated with different biological processes such as disease resistance proteins and transcription factors families. These miRNAs could be manipulated through RNAi technique for transgenic development (Singh et al. 2016b). Thus, with the advancement in next-generation sequencing technologies, there is a huge scope to deploy these novel tools in somatic hybrids to study potato biology and improvement.

Conclusions

Limited utilization of Solanum species and therefore narrow genetic base of the cultivated potato is the cause of concern of yield stagnation. Development of potato somatic hybrids using wild Solanum species with desirable attributes could be helpful to address this issue. Analyzing breeding potential of somatic hybrids, development of advanced progenies by hybridization with common varieties and identification of linked molecular markers are important for their successful applications in potato breeding. Further, information on nuclear-cytoplasmic interaction between cultivated and wild species could also be useful while exploitation of somatic hybrids in breeding. Novel genomics tools like whole genome sequencing, DArT markers based genotyping, microRNAs, microarrays and many others could strengthen the somatic hybridization research in potato. Moreover, access to the next-generation sequencing technologies and the potato genome sequences are essential for application of genome-enabled breeding strategies and critical investigation of complex biological processes like heat tolerance, tuberization, yield, disease-pest resistance and other traits. The sequence data could be extensively deployed for gene discovery, marker development, phylogeny, comparative genomics, breeding, and engineering of new genotypes/phenotypes. Taken together, above studies would provide a myriad of useful information available in somatic hybrids for potato genetics, breeding and genomics improvement.