Euphytica 85 : 2 1 7 -233,1995 . 217 © 1995 Kluwer Academic Publishers. Printed in the Netherlands .

The potential of somatic hybridization in crop breeding

Sylvia Waara & Kristina Glimelius Uppsala Genetic Center, Department of , Swedish University of Agricultural Sciences, Box 7003, S-750 07 Uppsala, Sweden

Key words : crop improvement, alien transfer, progeny analysis, somatic hybridization

Summary

In recent years, the rapid development of genetics has made possible the transfer of alien over wide taxonomic distances by somatic hybridization . In this review, the potential of somatic hybridization in the breeding of crops within the Brassicaceae, Fabaceae, Poaceae and Solanaceae is discussed . It is evident from these studies that many hybrids, either symmetric or asymmetric, which are fertile have the potential to be used as a bridge between the alien species and the crop . Progeny analysis of some combinations also reveals intergenomic translocations which may lead to the introgression of the alien genes . Furthermore, fusion techniques enable the resynthesis of allopolyploid crops to increase their genetic variability and to restore level and heterozygosity after breeding at reduced ploidy level in polyploid crops.

Introduction been identified, and somatic hybridization might then be the method of choice . Besides being of value for Genetic variability within the species has been effi- the transfer of unidentified genes, somatic hybridiza- ciently utilized by breeders in their efforts to improve tion is a tool for the modification and improvement crops. However, the existing variability in a breed- of polygenic traits . Furthermore, the modification of ing population may not be sufficient for modem plant organellar genetic material is possible via somatic breeding purposes, and thus great efforts have been hybridization since a mixture of the two fusion part- made to broaden the existing gene pool of crops . Intro- ners is obtained in the hybrid cell . In this presentation, duction of new traits has been based mainly on sexual an overview will be given regarding the utilization crosses between different genotypes within or between of somatic hybridization as a method of transferring closely related species . However, due to the presence alien genes to crop species. The potential of somat- of various reproductive barriers, gene transfer has been ic hybridization for restoring ploidy level in polyploid restricted to sexually-compatible species, thus limit- species after breeding at reduced ploidy level, as well as ing the possibilities of modifying and improving crop the challenge of resynthesising allopolyploid species, plants. Many desirable and agronomically-interesting will also be discussed . We will focus on work with traits may only be found in distantly related species crops from the Brassicaceae, Fabaceae, Poaceae and or even in unrelated organisms. Since they constitute Solanaceae, and discuss the methodologies and the fate a genetic resource potential, considerable effort has of the transferred alien DNA in the specific hybrids and been allocated to identify and isolate these genes and their progeny. transfer them into crops . Through the rapid develop- ment of somatic cell genetics, methods now exist for transferring genes across sexual borders and over wide taxonomic distances . Where interesting genes have been identified and isolated, they can be transferred by transformation, but, for most traits the genes have not 218

Methodology trofusion and it might have several advantages over chemical fusion ; fusion conditions can be more easi- technology ly controlled, single pairs of may be fused and very fragile protoplasts that do not survive chem- Two criteria have to be fulfilled to exploit the use of ical fusion, may survive the electrofusion conditions protoplast fusion technology ; protoplasts must be iso- (Bates et al., 1987b). lated in large quantities and the isolated protoplasts must be totipotent, i .e. they should have the ability to Selection of hybrids proliferate and regenerate into new plants . The first report on enzymatic release of protoplasts (Cocking, Unfortunately, the desired fusion products usually rep- 1960) was followed 11 years later by the first report on resent less than 10% of the total fusion mixture . Much the of plants from protoplast of tobacco time and effort have therefore been devoted to develop (Nagata & Takebe, 1971 ; Takebe et al ., 1971) . Since methods for selection of hybrid cells, and this is still then, regeneration of plants from protoplasts has been a bottle-neck in several systems . Initially, hybrid cells reported for more than 320 higher plant species rep- were selected by employing complementation selec- resenting 146 genera and 49 plant families (reviewed tion procedures using auxotrophic, resistant or chloro- by Roest & Gilissen, 1989, 1993) . The most amenable phyll - deficient mutants or non-regenerating, wild- plants for tissue culture can generally be found within type lines (Schieder, 1982) . The general application of the Solanaceae and Brassicaceae but success has also these selection strategies is, however, limited . Mutants recently been reported for more recalcitrant species for agronomically-important genotypes are scarce and including many members of the Poaceae (Vasil & Vasil, the production of mutants is both difficult and may 1992), the Fabaceae (Puite, 1992), berries such as have a negative influence on the genetic fidelity of strawberry (Nyman & Wallin, 1988, 1992) and sever- the plant material. The same condition also applies to al woody plant species, including temperate fruit trees more recent selection strategies employed where drug and Citrus species (see reviews by Puite, 1992 ; Grosser resistance genes have been introduced into one or both & Gmitter, 1990) . fusion partners by transformation, although somatic hybrids can be easily identified by selecting for drug Production of somatic hybrids resistance (Bates et al ., 1987a; Masson et al ., 1989 ; Toriyama et al ., 1987a; Wijbrandi et al., 1990d). Most somatic hybrid plants obtained so far have been Various methods with more general applicability produced after chemical treatment with polyethylene have therefore been developed . These include methods glycol, PEG (Kao & Michayluk, 1974; Wallin et al., based on visual identification after fusion of morpho- 1974). The PEG treatment induces agglutination of the logically different cell types or differentially stained protoplasts and fusion will occur after dilution of the protoplasts. The hybrid cells may then be mechani- PEG with a solution containing a high concentration of cally isolated (Kao, 1977 ; Hein et al ., 1983; Puite et calcium ions at high pH . The resulting fusion frequen- al., 1986; Sundberg & Glimelius, 1986; Waara et al ., cy can vary roughly from 1 to 20% depending upon 1991) or enriched by a flow cytometer and cell sorter cell types and fusion conditions employed . Methods (Afonso et al ., 1985 ; Glimelius et al ., 1986; Puite et al ., for electrofusion have also been developed and reports 1988 ; Hammatt et al., 1990) . Alternatively, one or both of somatic hybrid plants recovered from such experi- parents can be inactivated with chemical compounds ments are also prevalent (Bates & Hasenkampf, 1985 ; such as iodoacetamide and rhodamine 6-G (Menczel Koop & Schweiger, 1985; Puite et al., 1985 ; Puite et et al ., 1982 ; Barsby et al ., 1987 ; Terada et al ., 1987b; al. 1986; Fish et al ., 1988) . The electrofusion process Bottcher et al ., 1989) . is initiated by resuspending protoplasts in a medium of Hybrid cells can also be identified at later stages low conductivity in a chamber separated by two elec- during culture . The expression of hybrid vigour at the trodes and applying a high alternating electric field . callus stage (Debnath et al ., 1987 ; Deimling et al ., The protoplasts will move in the electric field by dielec- 1988; Waara et al ., 1989 ; Polgar et al ., 1993) as well trophoresis and will thereby become attached to each as differences in parental and hybrid tissue morphol- other like chains of pearls . A short pulse of direct cur- ogy (Gleddie et al., 1986; Handley et al ., 1986 ; Fish rent is then applied to induce fusion . Very high fusion et al ., 1987 ; Klimaszewska & Keller, 1988) have been frequencies have sometimes been reported after elec- used. One alternative method which avoids all selection 2 1 9

methods is the fusion of individual pairs of protoplasts chloroplasts of the mesophyll fusion partner in favour in a microculture system (Koop & Schweiger, 1985) . of the suspension fusion partner have been observed However, the applicability of this method for produc- in Solanum/Lycopersicon somatic hybrids (Levi et al ., tion of somatic hybrids for breeding purposes is limited 1988; Li & Sink, 1992) . Until now, mixed popula- due to cumbersome manipulations. tions of chloroplasts (Fluhr et al., 1984; Thomzik & Hain, 1988) and recombined chloroplasts (Medgyesy Identification and characterization of somatic hybrid et al., 1985) have only rarely been detected . In con- plants trast to what has been observed with the chloroplast , recombination and/or rearrangements of the Regardless of the selection strategy used, the regen- mitochondrial genome is common (Belliard et al ., erated plants have to be analysed to verify their 1979; Kemble et al ., 1986 ; Landgren & Glimelius, hybrid nature. Preliminary evidence of hybridity can 1990). The sorting out of chloroplasts and mitochon- be obtained by scoring the morphology which is often dria is generally random and independent of the nuclear intermediate between the parents. More direct proof fusion process ; thus somatic hybrids as well as cybrids can be obtained by biochemical analysis using the may be produced. The transfer of organelles to create isoenzyme pattern (Scandalios & Sorenson, 1977 ; novel organellar combinations without the fusion of Gleba & Sytnik, 1984) or various types of molec- nuclei has therefore been utilized to transfer organelle- ular probes such as species-specific repetitive DNA encoded traits by, for example, the donor-recipient sequences (Saul & Potrykus, 1984; Imamura et al., fusion method (Zelcer et al ., 1978). By this method, 1987; Schweizer et al ., 1988), rDNA (Gleba et al ., traits encoded by the chloroplast genome such as tri- 1988) and other RFLP markers (Fish et al ., 1988; azine resistance (Barsby et al ., 1987) and traits encod- Melzer & O'Connell, 1990) . Recently, the use of PCR ed by the mitochondrial genome such as cytoplasmic technology has also been utilized for hybrid identifi- male sterility (Zelcer et al ., 1978 ; Kumashiro & Kubo, cation (Baird et al ., 1992; Xu et al ., 1993b). A cyto- 1986; Perl et al ., 1990) have been transferred to a novel logical analysis must also be performed since geneti- nuclear background. cal changes may arise during the tissue culture phase, and multiple fusion events may give rise to polyploid Symmetric vs asymmetric hybrids plants. Interspecific somatic hybrids are mostly polyploid and Organellar composition often contain many unwanted traits derived from the wild species. Therefore, several backcrosses with the In the majority of angiosperms, maternal inheritance cultivated species are necessary to remove the unde- of organelles precludes the independent assortment of sired wild characteristics and to establish the optimum organellar in most crop plants . Conversely, ploidy level for crop production . Fertile hybrids are protoplast fusion of somatic cells produces heteroplas- thus of utmost importance in order to obtain proge- mic cells. Subsequent sorting out of the organelles ny and to eliminate undesired characters of the wild during and organogenesis, often results species. in novel combinations of genomes in the regenerat- In many cases where two complete genomes from ed plants. The plastid types generally rapidly sort out phylogenetically-distant species have been combined, bi- or unidirectionally to homogeneity for one or the the resulting hybrid has been found to be sterile . Only other parent, which may depend upon the phylogenet- occasionally may the somatic hybrid be used directly ic distance between the species (Thanh et al., 1988 ; e.g. the resynthesis of the allopolyploid species Bras- Bonnett & Glimelius, 1990 ; Sundberg & Glimelius, sica napus (Schenk & Robbelen, 1982; Sundberg & 1991b; Derks et al ., 1992) and the presence of selec- Glimelius, 1986; Taguchi & Kameya, 1986, and see tion pressure for organellar type (Medgyesy et al ., below), the production of heterozygous tetraploid pota- 1980; Glimelius et al., 1981 ; Menczel et al ., 1982 ; to cultivars after fusion of dihaploid clones (Wenzel et Cseplo et al ., 1984; Menczel et al., 1986 ; Malone al ., 1979), and the use of interspecific fruit tree somatic et al., 1992 ; Jansen, 1993). The cell type had no hybrids as root stocks (Ochatt et al ., 1989; Grosser & apparent influence on the segregation of chloroplas- Gmitter, 1990) . ts in several Brassicaceae fusion combinations (Sund- Methods limiting the amount of genetic informa- berg et al ., 1991) . In contrast, biased transmission of tion passed on to the somatic hybrids from the wild 220 species have therefore been of interest, creating asym- also recently been presented (Dixelius & Glimelius, metric hybrids . This can be achieved by spontaneous 1994). elimination (Pijnackeret al ., 1987; Fahle- son et al, 1988 ; Pijnacker et al ., 1989; Sundberg et al ., 1991b; Babiychuk et al., 1992) or by irradiation prior Somatic hybridization within the Brassicaceae to fusion (Dudits, 1980 ; Gupta et al., 1984 ; Bates et al ., 1987a; Imamura et al ., 1987) . Alternative methods lim- Resynthesis of rapeseed iting the amount of DNA transferred, such as micronu- cleation (Ramulu et al ., 1992) and UV irradiation (Hall The Brassicaceae is almost a model family regarding et al., 1992), are currently also being investigated. To somatic hybridization. Protoplasts of a large number date, asymmetric hybridization has met with mixed of crop species, including oilseed rape, turnip rape, results. In general, irradiation reduced the amount of cabbage and radish, can be cultured and regenerated transferred donor DNA but the fraction may vary from to plants (Vamling & Glimelius, 1990). This has made a few traits (Dudits et al., 1987), one or a few chromo- possible the production of a large number of somatic somes (Gupta et al., 1984 ; Bates et al ., 1987a; Melzer hybrids between species of varying relatedness within & O'Connell, 1992) to numerous (Gle- the family (for a review see Glimelius et al ., 1991) . ba et al., 1988 ; Famelaer et al ., 1989; Sacristan et Hybridizations have been performed between species al., 1989; Sjodin & Glimelius, 1989b ; Yamashita et within the genus Brassica, combining both diploid and al., 1989 ; Wijbrandi et al., 1990a, b, c, Wolters et allopolyploidBrassica species into hybrid plants . The al., 1991 ; Derks et al ., 1992) the latter result being resynthesis of Brassica napus (rapeseed), combining obtained in the majority of cases . Furthermore, chro- B. campestris with B. oleracea, has been regarded as an mosome elimination is random and it is not possible interesting and important possibility for increasing the to predict which of the chromosomes will be lost . One genetic diversity of that species . Most of the breeding way to circumvent this is to tag the chromosomes with material of rapeseed originates from a small portion selection markers which enables the use of selection of the total genetic variation inherent in the parental pressures for retaining the specific chromosomes in the species (Crisp, 1976 ; Tsunoda et al ., 1980), thus, a asymmetric hybrids (Bates et al., 1987a; Sacristan et widening of the gene pool for further evaluations and al., 1989 ; McCabe et al ., 1993) . Successful use of the breeding has been a desired breeding aim (Schenk & selection strategy requires knowledge of the chromo- Robbelen, 1982; Sundberg et al., 1987; Terada et al ., somal location of agronomically-important genes, and 1987b; Rosen et al ., 1988) . Besides working with the these genes must be located on the same or on a few progenitors for oilseed rape, somatic hybrids have been chromosomes . Alternatively, selection pressure for the produced between heading type vegetables ofB. oler- desired trait may be applied . In the work of Sjodin acae (e.g. cabbage) and B. campestris (e.g. chinese & Glimelius (1989b), addition of a fungal toxin to the cabbage) also with the purpose of widening the genet- culture medium was shown to increase the frequency to ic diversity (Taguchi & Kameya, 1986) . Even though recovered disease-resistant hybrids when transferring a variation in chromosome number of the resynthe- Phoma lingam resistance into Brassica napus . sized B. napus was obtained, several of the hybrids In the previous section, the methodology of somat- contained the sum of the chromosome numbers of ic hybridization was presented . We will now turn to the the two parental species. Most of the hybrids were more direct use of somatic hybridization in the breed- fertile even though a low seedset was recorded after ing of different crop plants within the Brassicaceae, self-fertilization . Fertility was slightly higher, howev- Fabaceae, Poaceae and Solanaceae. We will main- er, when the hybrids were backcrossed to the parents ly discuss the potential of symmetric and asymmet- or to ordinary rapeseed varieties, which has enabled ric hybrids and will not directly elaborate on the suc- utilization of the material in rapeseed breeding pro- cessful use of cybridization for transfer of organelle- grammes. encoded traits; this subject has been reviewed in more Breeding goals of great importance for rapeseed detail elsewhere (Kumar & Cocking, 1987 ; Pelletier are increased resistance to blackleg, Phoma lingam, et al ., 1988 ; Glimelius et al ., 1991) . A detailed exam- and clubroot, Plasmodiophora brassicae. Somatic ination on the potential use of somatic hybridization hybridization has been used to enhance those traits for transfer of traits conferring disease resistance has (Sacristan et al, 1989 ; Sjodin & Glimelius, 1989a, b) and, as regards resistance towards blackleg, it has 22 1 also been very successful . Resistance to blackleg was showed reduced fertility and could not be backcrossed found in B. nigra, B . juncea and B. carinata (Sjodin to B. napus, preventing the transfer of the BCN resis- & Glimelius, 1988) and after production of symmet- tance to the B. napus gene pool (Lelivelt & Krens, ric as well as asymmetric somatic hybrids between 1992). The same results were reported from experi- these gene-donors and rapeseed, resistant hybrids were ments where resistance to BCN was transferred to rape- obtained (Sjodin & Glimelius, 1989a, b) . By utilizing seed from Sinapis alba (Lelivelt, 1993) ; again, the pri- a toxin, sirodesmin PL, isolated from the culture fil- mary hybrids expressed a high level of resistance, but trate of the fungus, a selection pressure exerted by the the somatic hybrids were mitotically unstable and ster- toxin could be used to select for asymmetric, resistant ile, which hampered further breeding. In contrast, the hybrids (Sjodin & Glimelius, 1989b) . Stable inher- somatic hybrids produced between B. napus and Eru- itance and possible introgression of the gene(s) for ca sativa by Fahleson et al. (1988) were fertile, which resistance toPhoma lingamhave been recorded in lines has enabled back-crossing to rapeseed . After one back- derived from a back-crossing programme of the origi- cross to rapeseed, followed by selfing of this progeny, nal hybrid with rapeseed (Dixelius, pers . comm.). the material has been divided into two groups. In one of these groups, high concentrations of erucic acid were Intergeneric somatic hybrids used as an indicator that genetic material from E. sati- va was still present in the progeny ; as the rapeseed Several intergeneric hybrids have also been produced parent in the somatic hybridization, a variety with low via protoplast fusions. In the tribe Brassiceae, species concentrations of erucic acid was used . Besides eru- from the genera Eruca (Fahleson et al ., 1988; Sikdar et cic acid, three isoenzymes and two E. sativa-specific, al., 1990), Sinapis (Toriyama et al ., 1987a; Primard et repetitive DNA sequences were used to confirm pres- al., 1988), Raphanus (Kameya et al ., 1989 ; Sundberg ence of DNA from E. sativa (Fahleson et al., 1993) . & Glimelius, 1991a), Moricandia (Toriyama et al ., This hybrid material has been introduced into breed- 1987b) and Diplotaxis (Klimazewska & Keller, 1988 ; ing programmes of rapeseed in Sweden and investi- Chatterjee et al., 1988 ; McLellan et al ., 1988) have gations are in progress to screen for traits of interest . been hybridized with different Brassica species, main- The first check, however, for presence of insect toler- ly B. napus. Compared with the intrageneric somat- ance in some of the lines, was not successful (Ahman, ic hybrids, the intergeneric hybrids showed a larg- 1993). er variation in chromosome number ; more aneuploid hybrids that had lost one or several chromosomes, as Intertribal somatic hybrids well as polyploid hybrids that had gained one or more chromosome sets, were found (Sundberg & Glimelius, Intertribal hybrids can only be produced via somat- 1991 b). Of the intergeneric hybrids produced, attempts ic hybridization . Arabidopsis thaliana from the tribe were made to introduce tolerance to Alternaria bras- Sisymbriae has been hybridized with B. campestris siceae and the beet cyst nematode (BCN), Heterodera (Gleba & Hoffman, 1979, 1980), and with B. napus schachtii from Sinapsis alba (B . hirta) (Primard et al ., (Bauer-Weston et al., 1993 ; Forsberg et al ., 1994) . Fur- 1988; Lelivelt et al ., 1993), tolerance to drought and thermore, Thlaspi perfoliatum from the tribe Lepidiae aphids from Eruca sativa (Fahleson et al ., 1988), resis- (Fahleson et al ., 1994b), Barbarea vulgaris, from the tance to BCN from Raphanus sativus L. (Lelivelt & tribe Arabideae (Fahleson et al ., 1994a; Oikarinen & Krens, 1992), a low C02-compensation point from Ryoppy, 1992) and Lesquerella fendleri from the tribe Moricandia arvensis (Toriyama et al ., 1987b) as well Drabeae (Skarzynskaya et al ., 1994) have been com- as cytoplasmic male sterility from Diplotaxis harra bined with B. napus and, in the case of Barbarea vul- (Klimaszewska & Keller, 1988), and Brassica tourne- garis, also with B. campestris. In all these hybridiza- fortii (Liu & Glimelius, 1994) . tions plants have been obtained which, according to Concerning Alternaria tolerance, the somatic isoenzyme and RFLP analysis, were hybrids. Howev- hybrids obtained between B. napus and S. alba were er, evaluation of the chromosome number and hybrid not as tolerant to Alternaria brassicae as the gene markers, including species-specific markers, showed donor (Primard et al., 1988) . In the work where that more asymmetric hybrids were found among attempts were made to introduce resistance to BCN the intertribal hybrids than among the intrageneric fromRaphanus sativus, resistance was expressed in the and intergeneric hybrids . Interestingly, though, when primary hybrids at a high level . However, these hybrids comparing the intertribal hybrids with those obtained 222 from hybridization experiments between more closely- ture and somatic hybridization have been developed related species, no differences in the frequency of (reviewed by Kumar & Davey, 1991) . Regeneration hybrid fusions or hybrid shoot regeneration were of plants from protoplasts of this family was initial- recorded . It was only after attempts to culture and ly troublesome but has recently been achieved from establish the hybrid material in the greenhouse that several genera including Glycine, Lotus, Medicago, a clear difference was noted . The intertribal combi- Pisum and Trifolium (reviewed by Webb, 1988 ; Puite, nations were, in general, more difficult to culture to 1992; Roest & Gilissen, 1993) . It is mainly in the mature plants outside in vitro conditions . This was genusMedicago that somatic hybridization as a method especially pronounced in the fusion between B. napus for crop improvement has been investigated . Unfortu- and Barbarea vulgaris which, in spite of the fact nately, in the majority of fusion experiments, hybrid that hybrid plants could be cultured in vitro, never cell lines but no hybrid plants have been recovered resulted in plants that could be established and cul- (Gilmour et al ., 1987; Damiani et al ., 1988; Deak et tured under ordinary growth conditions in the green- al., 1988; Walton & Brown, 1988 ; Gilmour et al ., house (Fahleson et al ., 1994a). According to Oikari- 1989; Niizeki & Saito, 1989 ; Pupilli et al ., 1991 ; nen & Ryoppy (1992), putative fertile hybrids between Kihara et al., 1992) . However, a few encouraging B. campestris and Barbarea vulgaris that could be results demonstrate that fusion technology may be established in the greenhouse were obtained. Clear of agronomic value in this family. Flowering somat- evidence that these plants indeed were hybrids has ic hybrids have been obtained between the sexually- not been reported, however. Nevertheless, intertribal compatible species Medicago sativa (alfalfa) and M. somatic hybrids have been produced that differenti- falcata (Teoule, 1983; Mendis et al ., 1991), as well as ate and develop into normal hybrid plants that could between alfalfa and M. coerulea (Pupilli et al ., 1992) even be successfully selfed, as for example some of and alfafa and M. intertexta (Thomas et al ., 1990). the 'Arabido-Brassica' hybrids (Forsberg et al ., 1994) . Asymmetric intra-generic Medicago hybrids have been Even though most of the hybrids produced displayed regenerated but not analysed in detail (Kuchuk et al ., some problems when flowering and did not produce 1990). Interspecific somatic hybrids within the genus pollen, several were female-fertile and could set seeds Lotus have also been produced (Wright et al ., 1987; when backcrossed with pollen from rapeseed . In the Aziz et al ., 1990) and these hybrids cannot be pro- case of the `Thlaspo-Brassica' hybrids it was even duced by sexual hybridization . Furthermore, plantlets possible to detect the presence of reasonably high con- but no mature plants were recovered after fusion of centrations of the Thlaspi-specific fatty acid, neuronic Glycine max (soybean) with other Glycine spp. (Ham- acid, in some progeny obtained from the initial hybrid matt et al., 1992) . The most interesting somatic hybrids plants backcrossed to rapeseed (Fahleson et al ., 1994b). obtained so far are probably those obtained after asym- Thus, since very low concentrations of this fatty acid metric intergeneric fusion of the sexually-incompatible are present in rapeseed, this clearly indicates that genet- species alfalfa and Onobrychis viciifolia, sainfoin (Li ic material coding for the synthesis of neuronic acid et al., 1993) . The purpose of this study was to transfer has been transferred via the hybridization . With regard the trait for the production of foliar condensed tan- to the somatic hybrids produced between Lesquerella nins from sainfoin to alfalfa . These condensed tan- fendleri and rapeseed, a large number of hybrids have nins are thought to play an important role in prevent- been obtained (Skarzhynskaya & Glimelius, 1994) . ing bloat in grazing animals by preventing gas escape Investigations have started by evaluating the presence in the rumen. A large number of highly asymmetric of lesquerolic acid, a Lesquerella-specific hydroxy- hybrids were recovered after fusion of irradiated sain- unsaturated fatty acid, in the progeny obtained when foin protoplasts and iodoacetamide-inactivated alfalfa backcrossing the hybrids with rapeseed . protoplasts. Pollen germination tests revealed that the majority of the hybrids were male-fertile . However, so far no tannin-positive regenerated plant has been Somatic hybridization in the Fabaceae detected among 43 plants analysed (Li et al ., 1993) . It is speculated that this is because only a small amount Several members of the Fabaceae are important crop of the large genome of sainfoin has been transferred plants and a broadening of the existing gene pool to the hybrids, and probably about 200 hybrids should has therefore attracted considerable interest (Kumar be screened in order to recover a highly asymmetric, & Davey, 1991) . Various methods of protoplast cul- tannin-positive hybrid . 223

Somatic hybridization within the Poaceae folium (Kameya et al., 1990), S. khasianum (Sihachakr et al., 1988), S. nigrum (Guri & Sink, 1988a), S . sisym- Successful regeneration of plants from protoplast cul- brifolium (Gleddie et al., 1986) and S . torvum (Guri tures of members of the grass family was initially diffi- & Sink, 1988b; Sihachakr et al ., 1989) have been cult and the grasses have been regarded as some of the produced. Preliminary evaluation of the agronomic most recalcitrant species for use in tissue culture tech- traits exhibited by these somatic hybrids reveals that niques . However, during the last ten years an increas- the desired properties have been incorporated but their ing number of reports on regeneration of plants from high sterility limits the potential value in future breed- embryogenic cell suspension cultures of both forage ing programmes . Only the somatic hybrids between grasses and cereals have been presented (reviewed by species with the closest phylogenetic relationship, i .e. Vasil & Vasil, 1992 ; Roest & Gilissen, 1993) . Inter- the somatic hybrids obtained after fusion of eggplant generic hybrids have been produced in several combi- and S. aethiopicum orS. integrifolium, are fertile. Such nations, e .g. Panicum maximum (+) Pennisetum amer- hybrids can also be produced by sexual hybridiza- icanum (Ozias-Akins et al., 1986), Saccharum offici- tion although they have very low fertility and different narum (+) P. americanum (Tabaeizadeh et al ., 1986), organellar composition (Daunay et al ., 1993) . Oryza sativa (+) Eichinochloa oryzicola (Terada et al., 1987a) Triticum monococcum (+) P. americanum (Vasil et al., 1988), Festuca arundinacea (+) Lolium multiflorum (Takamizo et al.,1991) . However, regener- As potato (Solanum tuberosum) is an autotetraploid, ation of mature plants was only possible in the last com- potato breeding requires extensive selection in a large bination ; this hybrid can also be produced by sexual population to obtain the desired agronomic traits com- hybridization. Nevertheless, interesting mature asym- bined in one genotype . Therefore, breeding at diploid metric hybrids between F arundinacea and L. multi- level followed by somatic hybridization (to restore the florum (Spangenberg et al ., 1994) have been recov- ploidy level) has been suggested as an alternative for ered. Attempts to overcome conventional breeding the production of superior cultivars (Wenzel et al., barriers by interspecific fusion of rice with four dif- 1979). In recent years, a large number of intraspecific ferent wild species including Oryza brachyantha, O. fusion combinations have been conducted (Austin et eichingeri, O . officinalis, and O. perrieri (Hayashi et al ., 1985a ; Debnath & Wenzel, 1987 ; Deimling et al ., al., 1988) were more successful. Mature plants with 1988; Waara et al ., 1989; Chaput et al ., 1990; Waara viable pollen could be obtained in all but the first et al., 1991 ; Mollers & Wenzel, 1992 ; Thach et al ., Oryza combinations . In addition, the production of fer- 1993). In a similar approach, dihaploid potato clones tile, inter-specific, diploid rice hybrid plants (Toriya- have been fused with diploid S. phureja or diploid S. ma & Hinata, 1988) as well as rice cybrids (Aka- tuberosum x S. phureja clones (Puite et al ., 1986 ; gi et al ., 1989; Kyozuka et al., 1989; Yang et al ., Puite et al ., 1988 ; Mattheij & Puite, 1992) . Analy- 1989) has been reported . These studies now demon- sis of the hybrid plants has demonstrated the potential strate that the fusion technology can also be extended value of this breeding method as it is possible to pre- to graminaceous crops using donor species possessing dict many characters displayed by the hybrids from agronomically-interesting traits. the characters of the dihaploid parents. Dominantly- inherited disease or pest resistance genes, such as the resistance gene Ro 1 against the nematode Globodera Somatic hybridization within the Solanaceae rostochiensis, have been transferred from one of the dihaploid parents to the hybrids (Mollers & Wenzel, Eggplant 1992) . Likewise, the major genes for PVX and PVY resistance are also expressed in somatic hybrids (Thach In the case of eggplant (Solanum melongena), sexual et al., 1993). Phenotypic characterization has shown crossing barriers have limited the possibility of trans- that most hybrid combinations show a general vege- ferring agronomically-important traits such as resis- tative hybrid vigour (Deimling et al ., 1988 ; Chaput et tance to insect attack, or resistance to various diseases al., 1990 ; Waara et al ., 1991 ; Mattheij & Puite, 1992 ; to eggplant from related wild species (Daunay et al ., Mollers et al ., 1992 ; Waara et al., 1992) . Furthermore, 1991).. Symmetric somatic hybrids between eggplant field trials reveal that several hybrid combinations give and S. aethiopicum (Daunay et al., 1993), S. integri- tuber yields as high as or higher than standard cultivars 224

(Mattheij & Puite, 1992 ; Mollers et al ., 1994) and it ed (Feher et al., 1992; Puite & Schaart, 1993 ; Xu et appears that the yield of the hybrid is highly correlated al., 1993a). Traits conferring tolerance of environmen- with the yield of the parental clones (Mattheij & Puite, tal stresses such as cold have been transferred from 1992; Mollers et al ., 1994) . The yield increase seems to cold-tolerant species such as S. brevidens and S. com- be mainly due to an increased tuber weight whereas in mersonii to potato via somatic hybrids . The hybrids several combinations, the number of tubers per plant showed intermediate tolerance compared with the par- is intermediate between the two parents (Mattheij & ents (Preiszner et al ., 1991 ; Cardi et al ., 1993). In Puite, 1992; Waara et al ., 1992; Mollers et al ., 1994) . addition, fertile hybrids exhibiting resistance to both Tuber characters such as red skin colour, yellow tuber Phytophthora infestans and Globodera pallida have flesh and round form also appear to be inherited dom- also been produced by fusion of potato and its wild inantly (Mollers & Wenzel, 1992 ; Waara et al ., 1992 ; relative S. circaeifolium (Mattheij et al ., 1992) . Mollers et al ., 1994) . The possibility of alien gene transfer through inter- Genetic improvement of the potato by interspe- generic hybridization has also been attempted ; somatic cific somatic hybridization has also been accom- hybrid plants have been recovered from fusion exper- plished. Somatic hybrid plants have been produced iments between potato and Lycopersicon esculentum between potato and several partly or completely (Melchers et al ., 1978 ; Shepard et al., 1983) and sexually-incompatible wild species including S. brevi- L. pimpinellifolium (Okamura, 1988) . Fertile hybrids dens (Barsby et al ., 1984; Austin et al ., 1985b; Austin were obtained in both cases, even though production et al., 1986; Fish et al., 1987, 1988; Preiszner et al., of seeds from the potato- combination seems to 1991) S. bulbocastanum (Austin et al ., 1993), S. cha- be very rare (Jacobsen et al ., 1994) . coense (Butenko & Kuchko, 1980), S . circaeifolium From the data presented, it is clear that potato (Mattheij et al ., 1992), S. commersonii (Cardi et al ., is a species where fusion technology has been very 1993), S. nigrum (Binding et al ., 1982), S. pinnati- successful . It is therefore encouraging that somatic sectum (Sidorov et al ., 1987) and S. torvum (Jadari et hybridization programmes are now included in com- al., 1992) . Furthermore, somatic hybrids between the mercial potato breeding schemes in the The Nether- sexually-compatible species S. berthaultii and potato lands (Mattheij & Puite, 1992) and in Germany have been produced (Serraf et al ., 1991). The most (Mollers, pers . comm .). widely-used wild species in somatic hybridization experiments is the diploid non-tuberous wild species S . Tobacco brevidens. This species carries resistance genes to sev- eral viral diseases including PLRV (Jones, 1979), PVX Tobacco has long been one of the model species for (Gibson et al., 1990) and PVY (Gibson et al., 1988) cell and tissue culture studies and the first somatic and the transfer of these traits to somatic hybrids is hybrid plant was produced between Nicotiana glau- well documented (Helgeson et al ., 1986 ; Austin et al ., ca and Nicotiana langsdorffii (Carlson et al ., 1972). 1988; Gibson et al ., 1988; Pehu et al., 1990b). Some The majority of the hybrid-enrichment methodologies of these hybrid combinations were fertile and could be and hybrid analysis systems were primarily developed backcrossed to potato (Ehlenfeldt & Helgeson, 1987 ; for various tobacco somatic hybrids which, by them- Williams et al., 1990) . These sexual progeny showed selves, did not have any agronomic importance, but substantial variation but plants with improved agricul- they paved the way for further somatic hybridization tural traits combined with retained disease resistance experiments in tobacco as well as in other crops (see have been identified (Helgeson et al ., 1993). Anoth- review by Gleba & Sytnik, 1984 ; Kubo, 1988 ; Gle- er interesting feature of these hybrids was that they ba & Schlumukov, 1990). Somatic hybrids of poten- also showed resistance to Erwinia soft rot, a tuber dis- tial economic importance were recovered from fusion ease. This trait was not inherited from the tuberizing S . experiments between N. tabacum and N. rustica (Dou- tuberosum parent which was susceptible to the disease glas et al ., 1981). The somatic hybrids expressed an (Austin et al ., 1988) . Thus, the resistance trait might elevated nicotine content (Pandeya et al ., 1986) as well have been transferred from the nontuberizing parent S . as resistance to black root rot (Thielaviopsis basicola) brevidens to the tuber-forming somatic hybrids . Asym- and blue mould (Peronospora tabacina) . Curiously, metric somatic hybrids between S. brevidens and pota- resistance against the latter disease was not found in to have also been produced but the fertility and dis- any of the parents and might have arisen due to inter- ease resistance of these has not yet been investigat- action between the different nuclear and/or organellar 225 genomes (Pandeya et al ., 1986) . The transfer of TMV fertile, intergeneric tomato x S. etuberosum somat- resistance into fertile tobacco somatic hybrids from ic hybrids (Gavrilenko et al ., 1992) are also valu- N. nesophila or N. stocktonii has also been achieved able as this combination cannot be obtained by sexual (Evans et al., 1981) . The above-mentioned hybrids hybridization. can be produced by sexual crossings although special The possibilities of partial gene transfer by asym- techniques such as ovule culture in vitro are necessary . metric somatic hybridization have also been investi- Attempts to transfer disease resistance genes into N. gated in tomato although the transfer of specific agro- tabacum from the sexually-incompatible species of N. nomic traits from the donor has not been determined repanda were also successful but the resulting somatic (O'Connell & Hanson, 1987 ; Melzer & O'Connell, hybrids were sterile (Nagao, 1982) . To overcome the 1990; Wijbrandi et al., 1990a; Ratushnyak et al ., 1991 ; sterility of both sexual and somatic hybrids, asymmet- Wolters et al. 1991 ; Derks et al ., 1992; Melzer & ric hybridization was attempted (Bates, 1990) . From O'Connell, 1992; McCabe et al ., 1993) . In the major- this work two asymmetric somatic hybrids were recov- ity of these studies, the extent of chromosome elimi- ered which showed TMV resistance and female fertility nation is rather limited and irradiation has only deter- which enabled them to be backcrossed to tobacco . mined the direction of the chromosome elimination (reviewed by Lefrancois et al ., 1993) . However, in one Tomato study where the recipient was treated with the metabol- ic inhibitor iodoacetatamide prior to fusion, a clear Tomato is the only crop plant within the small genus correlation with irradiation dose and asymmetry could Lycopersicon but several of the wild species have be established (Melzer & O'Connell, 1992) . Some of served as important sources of agronomic traits via the asymmetric hybrids between tomato and irradiated sexual crosses (Rick, 1982; Daunay et al ., 1991) . L. pennellii are also fertile, and sexual progeny can be However, many sexual crosses between tomato and the obtained after self fertilization (Melzer & O'Connell, wild species are cumbersome to perform due to bilat- 1992). This observation is especially intriguing as the eral and unilateral incompatibility, and several of the corresponding symmetric hybrids are sterile . resulting F l hybrids are sterile (reviewed by Lefrangois et al., 1993) . Therefore, the potential of somatic hybridization in tomato breeding has been investigat- Conclusions ed (see reviews by Hille et al ., 1989; Lefrancois et al ., 1993) . Interspecific symmetric hybrid plants have From this overview of somatic hybridization it is obvi- been produced between tomato (L. esculentum) and L. ous that the method can be used for combining species chilense (Bonnema & O'Connell, 1992), L. pennel- with different degrees of genetic divergence into func- lii (O'Connell & Hanson, 1987) and L. peruvianum tional hybrids, and that even species from different (Kinsara et al ., 1986; San et al., 1990; Wijbrandi tribes can be hybridized . Successful hybridization has et al., 1990d). Intergeneric somatic hybridization has also been reported in previously recalcitrant species also been possible between tomato and several species such as those within the Fabaceae and the Poaceae . including Nicotiana tabacum (Turpin, 1986), Solanum Low fertility of somatic hybrids has sometimes been etuberosum (Gavrilenko et al., 1992), S. etuberosum x reported and this could, of course, severely restrict S. brevidens (Gavrilenko et al., 1992), S. lycopersi- their utilization in breeding programmes . However, coides (Handley et al ., 1986; Tan, 1987 ; Levi et al ., in many cases only pollen fertility has been analysed 1988), S. muricatum (Sakamoto & Taguchi, 1991), S. and attempts have been restricted to self-fertilization nigrum (Guri et al., 1988), S . rickii (O'Connell & Han- of the hybrids. In general, somatic hybrids have a son, 1986) and S. tuberosum (Melchers et al ., 1978 ; low pollen fertility. Nevertheless, hybrids have been Shepard et al ., 1983). The morphology and organel- used as female parents (even though the female fer- lar composition of these hybrids have been extensively tility might be low) in backcrosses with the cultivated studied but no report on the transfer of agronomic traits species (Ehlenfeldt & Helgeson, 1987; Mattheij et al ., to the somatic hybrid is available. The somatic hybrids 1992; Cardi et al., 1993; Helgeson et al ., 1993 ; Pupilli produced between tomato and L. peruvianum are of et al., 1992; Fahleson et al ., 1993 ; 1994a, 1994b ; Dix- particular interest since this combination is one of the elius, unpublished) . Fertility has also in some cases few that is fertile, while the corresponding diploid sex- been higher in asymmetric hybrids than in the corre- ual hybrid is sterile (Lefrancois et al ., 1993). The sponding symmetric hybrid (Bates, 1990; Melzer & 226

O'Connell, 1992) . Thus, several of the hybrids, either Vincente et al ., 1992; Fahleson et al., 1994a, 1994b ; symmetric or asymmetric, have the potential to be used Forsberg et al ., 1994). Furthermore, by utilizing in as a bridge between the alien species and the crop situ DNA hybridization to chromosome spreads, a plant. detailed characterization of the hybrids regarding pres- Provided hybrids contain the alien genes of interest, ence of chromosomes from the parental species can a back-cross programme to the crop plant and analy- be obtained . For example, Itoh et al . (1991) showed sis of the progeny to determine the presence of alien that somatic hybrids between B. oleracea and X-ray- genes can be carried out . From investigations made treated B. campestris were asymmetric, while Piastuch of progeny obtained after repeated backcrossings of & Bates (1990) were able to demonstrate the presence the Brassicaceae hybrids with pollen from rapeseed, it of translocations between chromosomes of Nicotiana has been possible to follow inheritance of certain traits tabacum and N. plumbaginifoliain asymmetric somat- from the gene donors . Resistance to Phoma lingam, for ic hybrids. example, which was transferred via protoplast fusion Thus, since somatic hybridization enables trans- from B. nigra to B. napus was present even after twelve fer of genetic material between distantly-related and back-crosses (Dixelius, unpublished) . In the case of the sexually-incompatible species, it is a method with great `Thlaspo-Brassica' hybrids it was possible to obtain potential for improvement of our crops . However, to transfer of the gene(s) coding for the Thlaspi- specific be of practical value, introgression and stable inher- fatty acid, nervonic acid, to some progeny obtained itance of the alien DNA carrying the desired genes from the initial hybrid plants backcrossed to rapeseed has to take place. An important factor to investigate (Fahleson et al ., 1994b). Helgeson et al . (1993) have in the future is the mechanism involved in the incor- also shown that hexaploid somatic hybrids produced poration of donor DNA into recipient chromosomes . between cultivated potato and S. brevidens can be of Parokonny et al. (1992) have started to investigate potential value for further breeding, since progeny intergenomic translocations and the possible mecha- with disease resistance and with improved yield were nisms involved by utilizing genomic in situ hybridiza- obtained . tion (GISH) . From their studies of somatic hybrids pro- With the development of molecular techniques, bet- duced between Nicotiana plumbaginifolia and Nico- ter methods to detect and follow the introgression of tiana sylvestris, it could be revealed that intergenomic alien DNA in the genome have been obtained translocations containing chromosome segments from (see Lydiate et al ., this volume) . The existence of high- both species had been obtained . Another possible way density RFLP maps in tomato (Bernatzky & Tanksley, to investigate this is to analyse somatic hybrid progeny 1986) and potato (Bonierbale et al ., 1988 ; Gebhardt et by RFLP. Differences in the linkage groups obtained al ., 1989) has, for example, made it possible to deter- will reveal whether recombination and translocations mine the extent and direction of elimination in sev- between the genomes have occurred. eral asymmetric hybrids (Melzer & O'Connell, 1990 ; Wijbrandi et al ., 1990c; Wolters et al ., 1991 ; Melzer & O'Connell, 1992) . The presence of species-specific Acknowledgements repetitive DNA sequences in plant genomes is also valuable . Repetitive DNA is present in most species The authors gratefully acknowledge financial support and because of the tolerance of such sequences to from the Swedish Council for Forestry and Agricultural mutations, species-specific sequences are common, as Research . has been shown e.g. for species in the Brassicaceae (Simoens et al ., 1988 ; Iwabuchi et al ., 1991 ; Gupta et al ., 1992; Fahleson et al ., 1994a, 1994b), Solanaceae References (Schweizer et al ., 1988 ; Pehu et al ., 1990a; Piastuch & Bates, 1990) and Poaceae (Metzlaff et al ., 1986; Afonso, C .L., K .R . Harkins, M.A . Thomas-Compton, A .E. Krejci & D .W. Galbraith, 1985 . Selection of somatic hybrid plants in Nico- ., 1992). Iso- Zhang & Dvorak, 1989 ; Cordesse et al tiana through fluorescence-activated cell sorting of protoplasts . lated species-specific, repetitive DNA sequences have Bio/Technology 3 : 811-816 . proved to be excellent markers to verify the presence Akagi, H ., M . Sakamoto, T. Negishi & T. Fujimura, 1989. Construc- of alien DNA in somatic hybrids and their proge- tion of rice cybrid plants. Mol . Gen. Genet . 215 : 501-506. Austin, S., E . Lojkowska, M .K. Ehlenfeldt, A . Kelman & J.P. Helge- ny (Saul & Potrykus, 1984; Imamura et al ., 1987; son, 1988 . Fertile interspecific somatic hybrids of Solanum : A Piastuch & Bates, 1990 ; Itoh et al ., 1991 ; Perez- 227

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