Voi 511996): 449^60. Electrofusion of protoplasts of anther-derived dihaploid lines of commercial potato cultivars Veli-Matti Rokka Agricultural Research Centre ofFinland, Institute ofCrop and Soil Science, Plant Breeding Section, FIN-31600 Jokioinen, Finland Yong-Sheng Xu Department ofPlant Production, P.O. Box 27, FIN-00014 University ofHelsinki, Finland Pirjo Tanhuanpää Agricultural Research Centre of Finland, Institute of Crop and Soil Science, FIN-31600 Jokioinen, Finland Leena Pietilä Boreal Plant Breeding, Myllytie 10, FIN-31600 Jokioinen, Finland Eija Pehu Department ofPlant Production, P.O. Box 27, FIN-00014 University ofHelsinki, Finland Somatic hybrids of anther-derived dihaploid (2x) potato (Solanum tuberosum L.) lines were pro- duced by electrofusion of protoplasts. Using RAPD (randomly amplified polymorphic DNA) mark- ers, six new combinations of dihaploid parental lines from cultivars Matilda, Nicola, Pito, Stina, Van Gogh and White Lady were identified. RAPD marker identification of the putative hybrids was most- ly done using two distinct parental line specific primers. 43% of the 76 regenerated calli from the six combinations produced hybrid shoots. Most of the somatic hybrids were tetraploid (4x), but in four fusion combinations plants of hexaploid (6x), octoploid (8x) or mixoploid level were also identified by chromosome counts or flow cytometric nuclear DNA analysis. The mean nuclear DNA content (2C value) of the tetraploid and hexaploid somatic hybrids was lower than the expected DNA content (i.e. the 2C values of the original tetraploid cultivars or the sum of the 2C values of the dihaploid fusion parents). Some somatic hybrids having the expected nuclear DNA content were also found. Key words: flow cytometry, ploidy level, RAPD, Solanum tuberosum , somatic hybridization ntroduction ticularly successful in species from the Solanaceae and Brassicaceae (Glimelius 1988). In potato breeding, protoplast technology has been applied extensively. Recent results have shown that es- pecially intraspecific fusion of dihaploid potato Protoplast fusion has become an important method for crop improvement, and has been par- © Agricultural and Food Science in Finland Manuscript received June 1996 449 AGRICULTURAL AND FOOD SCIENCE IN FINLAND https://www.c-info.fi/en/info/?token=3weSFkGIOfUGrBez.cWCGJJfCxYEpTCFEqzQtzw.Fjn-DZfrnj5zby-JKXi9eH25Fnqwr925M1hwYAUrW5G00h66M81gZ9r_QbUllhdDHCra7Mjl4_tvjcGuQG0lum__gXJjtzieOp6kcUehijbh6u902sC60skj8uVulQduT7NiH7csAZwZHeReqG41LMa7K0hshkkJmlLZdc17uVtZz-uoG3zBg3H7NmJsM7WwvDMTNjlOyPxmSbz99Hr-kMPvZUuFpqZ3Go08ciieyTc8G6RsQXjOxATuE6OEhYs0pksju-Kq9QLpSa6krme3GC4Y7MGK4QY1F1wqlMjr-yQTuH0CkuvPgNvOhqONQOdutFDiBs7sqUspP9_7o3KyO_CfuuWn8PJQksfucwK2pIVj 2 Rokka, V.-M. et al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato lines cannot be considered only as a sophisticated research method, but rather a technique which can now be applied successfully in potato breed- ing (Möllers and Wenzel 1992, Schweis and Munzert 1993, Möllers et al. 1994). Cultivated potato, Solanum tuberosum L. ssp. tuberosum, is tetraploid (2n=4x=4B) and highly heterozygous, and breeding programmes based on crossing seldom produce progenies superior to the parental lines (Ross 1986). Therefore, re- duction in ploidy level has been attempted, ei- ther through chromosome elimination effected by pollination with Solanum phureja Juz. etBuk. (Hougas and Peloquin 1957) or anther culture (Dunwell and Sunderland 1973). Resulting di- haploids (2n=2x=24) expressing the desired phe- notype can be screened and the tetraploid con- dition reconstituted by fusion of protoplasts of two different dihaploid lines (Wenzel et al. 1979). Dihaploid-dihaploid fusion programmes have been initiated by several research groups (e.g. Austin et al. 1985, Debnath and Wenzel 1987,Waara et al. 1989, Baird et al. 1992, Schweis and Munzert 1993), but only Waara et al. (1989, 1991, 1992) have published the use of anther- derived dihaploids in their intraspecific somatic hybridization programme. Somatic hybrids must be distinguished from unfused material or fusion products resulting from homokaryon fusions. Various selection methods have been developed. Hybrid fusion products can be selected using a micromanipu- lator (Waara et al. 1991), flow sorter (Puite et al. 1988), hybrid vigour (Debnath and Wenzel 1987), intermediate morphology (Gleddie et al. 1986), mutant lines (White and Vasil 1979) or with selectable markers (Masson et al. 1989). Identification of hybridity based on molecular (Pehu et al. 1989, 1990, Baird et al. 1992) and biochemical analysis (Waara et al. 1989, Coop- er-Bland et al. 1994) has also been applied. It is essential that the identification for hybridity is simple and quick due to the high number of fu- sion products to be screened. The genetic composition of the intraspecific somatic hybrids is expected to be balanced (the hybrids are euploid tetraploids), but aneuploidy and different ploidy levels are common among hybrid regenerants (Waara et al. 1992, Rasmus- sen and Rasmussen 1995). In this study, anther- derived dihaploid potato lines derived from cvs. Matilda, Nicola, Pito, Stina, Van Gogh and White Lady were electrofused in various combinations. The somatic hybrids were characterized by chro- mosome counts and nuclear DNA content deter- minationby flow cytometry. The objective of the study was to produce and characterize new in- traspecific somatic hybrids for potato breeding purposes. The aim of the study was also to get more information on potato breeding at the dip- loid level in order to move anther cultures and protoplast fusions as part of the practical potato breeding. Material and methods Plant material Dihaploid potato lines ‘Nicola 2.dh.2.1.1.’, ‘Pito 35.dh.7.4.1.’, ‘Pito 30.dh. 16.1.1.’, ‘Pito 12.dh.57.3.1.’, ‘Van Gogh 13.dh.l 1.3.1.’, ‘Van Gogh 7.dh.12.2.1.’, ‘Van Gogh 19.dh.37.1.1.’ and ‘White Lady 4.dh.2.3.2.’ were produced by anther culture (Tiainen 1992, Rokka et al. 1996). Dihaploid lines ‘Matilda 1.dh.536.6’ and ‘Stina 4.dh.161.15’ were provided from The Swedish University of Agricultural Sciences. All of the genotypes were aseptically cultured in vitro on MS2O medium (Murashige and Skoog 1962) containing 20 g1 1 sucrose, 100 mg 1 1 caseinhy- drolysate, 0.05 mg 1 1 NAA (oc-naphthaleneace- tic acid) and 2 mg f STS (silver thiosulphate). The cultures were maintained in a photoperiod of 16 h per day (63 pE nv 2 s ') at a temperature of 24°C. Protoplast isolation Leaf material of 4 to 6-week-old plants was cut into small sections and placed in 10-20 ml of preplasmolysis solution (0.5 M mannitol) for I h. 450 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 449^460. The material was then transferred into 10 ml of enzyme solution (Rokka et al. 1994). After 16-18 h enzyme treatment in the dark at 24°C, the protoplast suspension was filtered through a 48 pm nylon sieve. The filtrate was centrifuged at 80 g for 5 min. The protoplast pellet was resuspended in wash solutioncontain- ing the major salts of CPW medium with man- nitol (Jones et al. 1989). Viable protoplasts were separated from dead protoplasts by centrifuga- tion at 120-160 g for 5 min on 30% (v/v) Per- coll (Pharmacia Fine Chem. AB). The layer of viable protoplasts on the surface of the Percoll solution was collectedand washed with the wash solution followed by two further washes in the fusion solution (0.5 M mannitol, 0.2 mM CaCl2). Electrofusion and culture of the protoplasts For the fusion experiments the protoplasts of the fusion parents were mixed in a 1:1 ratio. The protoplast mixture, adjusted to a density of 2 x 105 ml 1 with the fusion solution, was transferred into a lamellar chamber. The protoplasts were aligned and fused according to Rokka et al. (1994). Following fusion, the protoplasts in man- nitol solution (500 pi) were pipetted into 3.5 cm diameter Petri dishes to which double strength V-KM culture medium (Bokelmann and Roest 1983) was added in a ratio of 1:1. The proto- plasts were embedded adding 0.9% (w/v) low- gelling-temperature agarose (Type VII, Sigma) and cultured in the dark at 24°C. After 10-14 days the cultures were resuspended with 3-7 ml per plate normal strength V-KM medium and transferred to dim light. When colonies devel- oped (after 3-5 weeks), the cultures were con- tinued as described by Rokka et al. (1994), ex- cept that STS (2 mg I 1) was added to the media D and SP (Creissen and Karp 1985). Analysis of hybridit/ by RAPD patterns DNA extraction was carried out according to Rokka et al. (1994). The RAPD primers were synthesized either on an Applied Biosystems 372 DNA/RNA Synthesizer or purchased from Oper- on Technologies (Alameda, USA). Different 10- or 11-mer primer sequences were used to estab- lish polymorphisms between the dihaploid lines. Primers producing unique amplification products in both parental lines, or preferably, sequential- ly used primers producing genotype specific bands, were used in identification of somatic hybrids. The PCR (polymerase chain reaction) was carried out as described by Rokka et al. (1995). Chromosome counts and nuclear DNA content determination Chromosome numbers were counted from root tip cells of in vitro (MS2O + 0.05 mg 1 1 NAA) cultured plants according to Tiainen (1992). Nuclear DNA content (2C values) was measured from the dihaploid parental lines, the tetraploid original cultivars and the somatic hybrids using flow cytometry as described by Rokka et al. (1995). 1000-4000 nuclei were analysed in each sample. Results Protoplast isolation and culture The protoplast yields varied considerably be- tween dihaploids, and in many cases one of the parental lines of the fusion combination had more burst and collapsed protoplasts than the other (data not shown). Embedding in 0.9% aga- rose decreased the burst of the protoplasts dur- ing the first days of culture. Compared to cul- turing of the protoplasts in liquid medium, more divisions and colonies occured in embedded medium. The resuspension of cultures by liquid V-KM medium after 11-14 days enhanced strongly the rate of divisions, and the dilutions also prevented browning of the growing colo- 451 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka, V.-M. el al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato Table 1.Nucleotide sequences of the RAPD primers, which were used in identification of hybridity. Fusion combination Primer code* Sequence (s'to 3') Bands specific to Pito 35.dh.7.4.1.(+) Matilda 1.dh.536.6 107 GAC TGC AGA C Pito and Matilda Pito 35.dh.7.4.1.(+) Stina 4.dh. 161.15 102 TGATCGACTCG Pito and Stina Pito 35.dh.7.4.1.(+) Van Gogh 7.dh. 12.2.1. OPB-08 GTC CAC ACG G Pito OPK-08 GAA CAC TGG G Van Gogh Pito 30.dh.16.1.1.(+) Van Gogh 13.dh.l 1.3.1. OPB-09 TGGGGGACTC Pito OPK-08 GGA CAC TGG G Van Gogh Pito 12.dh.57.3.1.(+) Nicola 2.dh.2.1.1. OPB-09 TGGGGGACTC Pito OPB-05 TGC GCC CTT C Nicola Van Gogh 19.dh.37.1.1. (+) White Lady 4.dh.2.3.2. OPK-02 GTCTCCGCAA Van Gogh OPB-10 CTG CTG GGA C White Lady * primers assigned with numbers were synthesized on a DNA synthesizer, primers with the prefix OP were purchased from Operon Technologies Table 2. Frequency of regeneration and hybridity of the dihaploid-dihaploid fusion products. Fusion combination no. ofcalli no. of hybrid calli (%) no. of regenerated hybrid from all hybrid into shoots calli regenerated shoots Pito 35.dh.7.4.1.(+) Matilda 1.dh.536.6 22 3 14% 21 Pito 35.dh.7.4.1. (+) Stina 4.dh. 161.15 5 1 20% 32 Pito 35.dh.7.4.1.(+) Van Gogh 7.dh. 12.2.1. 4 4 100% 7 Pito 30.dh.16.1.1.(+) Van Gogh 13.dh.l 1.3.1. 21 6 29% 47 Pito 12.dh.57.3.1.(+) Nicola 2.dh.2.1.1. 12 II 92% 30 Van Gogh 19.dh.37.1.1. (+) White Lady 4.dh.2.3.2. 12 8 67% 78 TOTAL 76 33 43% 215 nies. The time required from protoplast isolation and fusion to shoot formation ranged from 5 to 8 months depending on the fusion combination. Identification of somatic hybrids using RAPDs Polymorphisms between parental dihaploids were tested with several primers. In the dihap- loid combination 'Pito 35.dh.7.4.1. (+) Stina 4.dh. 161.15’ six primers were tested, three of which generated significantly different banding patterns. In the combination ‘Pito 35.dh.7.4.1. (+) Matilda 1.dh.536.6’ two primers out of 14 primers produced different patterns for the pa- rental lines. In these two combinations, a single primer allowed successful identification of hy- bridity (Table 1). The somatic hybrids contained the combined pattern of the parental lines, where- as unfused material and regenerants derived from homokaryon fusions had the pattern of only one of the parental dihaploid lines. Two distinct parental-line-specific primers were used in the identification of the hybrids in the other four fusion combinations (‘Pito 35.dh.7.4.1. (+) Van Gogh 7.dh. 12.2.1.’, ‘Pito 30.dh. 16,1.1. (+) Van Gogh 13.dh. 1 1.3.1.’, ‘Pito 12.dh.57.3.1. (+) Nicola 2.dh.2.1.1.’ and ‘Van Gogh 19.dh.37.1.!.(+)White Lady 4.dh.2.3.2.’) (Table 1).Thus, 90% (44/49) of the primers gen- erated polymorphism specific to one of the pa- rental lines. Using two such parental-line- spe- 452 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 449^)60. cific primers required twice as much resources, but were more reliable in the hybridity verifica- tion than the use of a single primer. An example of the identificationof somatic hybrids is shown in Figure 1. The frequency of somatic hybrids was estimated for six different fusion combina- tions (Table 2). In total, 215 hybrid shoots were recovered from a total of 33 calli (Table 2). Chromosome counts and flow cytometric determination of nuclear DNA content Chromosomes of 24 shoots regenerated from one callus of a fusion combination ‘Pito 35.dh.7.4.1. (+) Stina 4.dh. 161.15’ were counted. All of the hybrids were tetraploid, chromosome numbers ranging from 45 to 50. Ploidies of all of the so- matic hybrids derived from seven different calli of the fusion combination ‘Pito 35.dh.57.3.1. (+) Nicola 2.dh.2.1.1.’ were also tetraploid. The oth- er fusion combinations produced both tetraploid, hexaploid, octoploid and mixoploid hybrids (Ta- ble 3). The total number of dihaploid-dihaploid fusion calli, which produced somatic hybrid re- generants of different ploidy levels is shown in Table 4. Yet, the tetraploid level was the most common. The mean DNA content (2C value) deter- mined from leafnuclei in dihaploid parental lines of the three fusion combinations (Table 5) was Fig. 1. Regenerants from fusion combination of dihap- loid potato lines (‘Pito 30.dh.16.1.1. (+) Van Gogh 13-dh.l 1.3.1.’) identified by RAPDs. The amplification was made by primer 809 (OPB-09) (lanes 1-10) and primer KOB (OPK-08) (lanes 11-20). Primer 809 produced a ‘Pito 30.dh.16.1.1.‘ specific band of 1600 bp (lane 1), which was visible both in the mixed DNA of the dihaploids (lane 3) and in all protoplast fusion regenerants (lanes 4- 10). Primer KOB amplified a ‘VanGogh 13.dh.l I.3.l.‘spe- cific band of 1900 bp (lane 12), which was visible in the mixed DNA of the dihaploids (lane 13) and in two protoplast fusion regenerants: 1302(lane 14) and 1903 (lane 20) i.e. those regenerants were somat- ic hybrids. A 100bp DNA lad- der (Gibco BRL) was used as a molecular weight marker, (Photo: Veli-Matti Rokka). 453 AGRICULTURAL AND FOOD SCIENCE IN FINLAND 3 Rokka , V.-M. et al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato Table 3. Ploidy level of the dihaploid-dihaploid somatic hybrids of potato derived from six fusion combinations. The ploidy levels were determined with chromosome counts and/or flow cytometric nuclear DNA analysis. Fusion combination callus No. of regenerants/ no. ploidy level Pito 35.dh.7.4.1. (+) Matilda 1 .dh.536.6 01 5/6x 02 14/8x 10 2/4x, l/8x Pito 35.dh.7.4.1.(+) Stina 4.dh.161.15 01 24/4x Pito 35.dh.7.4.1.(+) Van Gogh 7.dh. 12.2.1. 01 2/8x 02 l/4x, l/6x-Bx* 03 l/4x-6x*, 2/8x Pito 30.dh.16.1.1.(+) Van Gogh 13.dh.l 1.3.1. 08 5/6x 10 4/6x 11 7/4x 12 6/4x 19 4/6x Pito 12.dh.57.3.1. (+) Nicola 2.dh.2.1.1. 01 2/4x 02 2/4x 03 l/4x 04 2/4x 06 l/4x 11 l/4x 18 l/4x Van Gogh 19.dh.37.1.1. (+) White Lady 4.dh.2.3.2. 04 2/8x 06 7/4x 08 13/6x 09 9/4x 10 6/6x 12 5/6x 13 4/6x * mixoploid shoots at 6x and 8x (6x-8x) levels or 4x and 6x (4x-6x) levels Table 4. Number ofdihaploid-dihaploid fusion calli, which produced somatic hybrid regenerants of different ploidy levels. Ploidy No. ofcalli (%) 4x 12 46.2 6x 8 30.8 8x 3 11.5 4x/6x/Bx* 3 11.5 TOTAL 26 100.0 * shoots derived from the same callus, but having different ploidy levels between 1.65 and 1.73 pg. The mean 2C values of the original tetraploid cultivars were general- ly two times higher (3.32 pg in cv. Pito, 3.44 pg in cv. White Lady, 3.47 pg in cv. Nicola and 3.48 pg in cv. Van Gogh) compared to the correspond- ing dihaploid lines. The mean 2C values of all of the tetraploid somatic hybrids (3.18-3.29 pg) derived from three fusion combinations, were lower than the expected 2C values (i.e. the 2C values of the original tetraploid cultivars or the sum of the 2C values of the dihaploid parents) (Table 5). Also in hexaploid somatic hybrids the 454 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 449^460. Table 5. DNAcontent (2C values) in leaf nuclei of dihaploid parental lines and the corresponding somatic hybrids in three fusion combinations. Plant ploidy 2C value (pg)* mean s.d. Fusion combination 'Pito 12.dh.57.3.1. (+) Nicola 2.dh.2.1.1.' Pito 12.dh.57.3.1. 2x 1.65 0,03 Nicola 2.dh.2. 1.1. 2x 1.73 0,01 expected 2C values 2x + 2x 3.38 Pito 4x 3.32 0,05 Nicola 4x 3.47 0,05 somatic hybrids 4x 3.24 0,07 Fusion combination 'Pito 30.dh. 16.1.1. (+) Van Gogh 1 3.dh. 11.3.1.' Pito 30.dh. 16.1.1. 2x 1.66 0,05 Van Gogh 12.dh.l 1.3.1. 2x 1.65 0,00 expected 2C values 2x + 2x 3.31 Pito 4x 3.32 0,05 Van Gogh 4x 3.48 0,03 somatic hybrids 4x 3.18 0,13 expected 2C values 2x + 2x + 2x 4.97 somatic hybrids 6x 4.67 0,26 Fusion combination 'Van Gogh 19.dh.37.1.1. (+) White Lady 4.dh.2.3.2.' Van Gogh 19.dh.37.1.1. 2x 1.73 0,02 White Lady 4.dh.2. 3.2. 2x 1.72 0,03 expected 2x + 2x 3.45 Van Gogh 4x 3.48 0,03 White Lady 4x 3.44 0,02 somatic hybrids 4x 3.29 0,33 expected 2C values 2x + 2x + 2x 5.18 somatic hybrids 6x 4.77 0,41 * mean of three flow cytometric measurements and standard deviation mean 2C values were lower (4.56-4.77 pg) than the expected 2C values (4.97-5.18 pg) (Table 5). However, in each fusion combination there were a number ofregenerants having the same or high- er 2C values than the original cultivars. The hy- brids having higher 2C values were probably hypertetraploids or hyperhexaploids. Examples of the flow cytometric DNA content determina- tion of the potato material are shown in Figure 2. Discussion In this study, six new dihaploid potato line com- binations were produced by electrofusion ofpro- toplasts. The genetic material of the anther-de- rived dihaploid parents originated from Scandi- navian (cvs. Matilda, Pito and Stina), Dutch (cv. Van Gogh) and Hungarian (cv. WhiteLady) po- tato cultivars. Furthermore, the results of this study demonstrate the applicability of RAPD analysis in identificationof intraspecific somat- 455 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka, V.-M. et al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato Fig. 2. Flow cytometry of leaf nuclei of a dihaploid potato ‘Nicola 2.dh.2.1.1.’ (Fig. a.), a tetraploid culti- var Nicola (Fig. b.), a tetraploid somatic hybrid 0406 between two dihaploid lines (‘Pito 12.dh.57.3.1. (+) Nicola 2.dh.2.1.1.’) (Fig. c.) and a hexaploid somatic hybrid 0402 between two dihaploids (‘Van Gogh 19.dh.37.1.1. (+) White Lady 4,dh.2.3.2.’) (Fig. d.). The histograms were generated by propidium-iodide stained leaf nuclei and chicken red blood cell (CRBC) controls using linear scale of fluorescence intensity (FL2-H). CRBC were added as an internal standard to the plant nuclei samples. The signal threshold was adjusted to eliminate most debris from analysis. The nuclear DNA content (2C value) was calculated by direct comparison of the modal position of the plant peaks to the modal position of the CRBC peak (DNA content = 2.33 pg). 2C is defined as the DNA contentof the plant in the Gl phase of the cell cycle and 4C in G 2 phase (C is the DNA content of a haploid cell). 456 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Vol. 5 (1996): 449^460. ic hybrids of potato, and flow cytometric nucle- ar DNA content analysis of the hybrids. In previous research works, anther-derived dihaploids have seldom been used in intraspe- cific somatic hybridizations, because anther cul- ture of S. tuberosum has been considered inef- fective in the production of dihaploids. Our re- cent results, however, in androgenesis of agro- nomically important potato cultivars, have been promising (Rokka et al. 1996). The advantage of producing dihaploid lines through anthercul- ture is that the anther-derived dihaploids do not contain any other genetic material than that of the anther culture source plant. Dihaploids pro- duced by S. phureja pollinations may contain S. phureja DNA or variable chromosome num- bers (Clulow et al. 1993). The application of electrofusion rather than chemical fusion in- creases also the final number of somatic hybrids (Tempelaar and Jones 1985). The embedding of fused protoplasts with agarose followed by di- lution steps, enhanced first divisions of the cul- tured protoplasts. However, the whole culturing process of 5. tuberosum protoplasts is still quite limiting, if a wide range of genotypes is to be included in a protoplast fusion programme. Generally, the methods for identification and selection of hybrid plants have also been a bot- tle-neck in protoplast fusion. However, RAPDs offer an opportunity to confirm the hybridity during very early stages of cultures. Because simple DNA extraction method can be applied for RAPD analysis, it is possible to screen a large number of regenerated plants in a short time (Rokka et al. 1994). Compared with isozyme (Waara et al. 1989, Möllers and Wenzel 1992) and RFLP (restriction fragment length polymor- phism) analysis (Pehu et al. 1989), the RAPD method is fast. Isozyme analysis and RAPDs have given similar results in verification of so- matic hybridity (Rasmussen and Rasmussen 1995). In the case that the hybrids should con- tain the combination of the bands of the both parental lines (using a single primer), competi- tion for amplification sites in the target DNA may result in the absence of line-specific bands in the true somatic hybrids. Thus, the reproduc- ibility ofparental-specific bands would be more reliable using two primers, when each produce parental-specific bands rather than a single prim- er. However, one parental band may also be miss- ing as noted by Rasmussen and Rasmussen (1995), who suggested this to be due to the lack of specific chromosomes carrying the corre- sponding primer sequence in the hybrids. In the present work, all the strongly diagnostic bands gave consistent results in the identification of hybridity. Primers can also amplify sequences of mitochondrial and chloroplast origin (Lorenz et al. 1994). Complete chloroplast segregation is normal in intraspecific somatic hybrids (Lössi et al. 1994), but mitochondria can produce rear- rangements (Xu et al. 1993, Lössi et al. 1994). In rare cases nuclear hybrids may be identified as non-hybrids, if the primer amplifies chloro- plast DNA of the other parent. In this study, two fusion combinations pro- duced only tetraploid somatic hybrids, but in four combinations either tetraploid, hexaploid, octo- ploid or mixoploid hybrids were regenerated. In some cases the same callus regenerated into shoots which differed in ploidy levels from each other, which may be due to genetic rearrange- ments during the callus stage and shootregener- ation or the calli were derived from aggregated protoplasts or cell colonies (Waara et al. 1992). Rasmussen and Rasmussen (1995) noticed that in one fusion combination only few of the hy- brids were tetraploid. Other than the expected tetraploid levels can also be explained by fusion of more than two individual protoplasts. Chime- ras are also possible to occur after compaction of protoplasts or because of the grafted groups of cells as described by Binding et al. (1988). The mean nuclear DNA content of the intraspe- cific somatic hybrids was lower than the expect- ed DNA content. Valkonen et al. (1994) found a high correlationbetween 2C values and chromo- some numbers in diploid, tetraploid and hexa- ploid Solanum species. The low 2C value of most of the somatic hybrids produced in the present experiment could be due to aneuploidy. One to three individual chromosomes may be missed in many regenerants. There were, however, also 457 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka , V.-M. et al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato some individual hybrids that had the expected or higher DNA content than the original culti- vars. Also Rasmussen and Rasmussen (1995) noticed that hypoploidy was more general than hyperploidy among somatic hybrids. However, the association between the number of chromo- somes and the phenotype of the plant is unclear. Lössi et al. (1994) have found no association, but Karp et al. (1989) have noticed that some aneuploids expressed phenotypic differences. This paper reports successful production of somatic hybrids of anther-derived dihaploid po- tato lines. Further experiments are underway to characterize the disease resistance traits of the hybrids and to fuse new dihaploid lines having superior agronomic traits. Acknowledgements. The authors wish to thank Ms. Leena Lohermaa and Ms. Kirsti Salmi for excellent technical as- sistance. Prof M. Umaerus, who kindly provided the di- haploid lines from The Swedish University ofAgricultural Sciences, is also acknowledged. The authors want to thank Ann Fenwick M.S. forcorrections inEnglish language. 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Theoreti- cal and Applied Genetics 85: 1017-1022. 459 AGRICULTURAL AND FOOD SCIENCE IN FINLAND Rokka, V.-M. et al. Electrofusion ofprotoplasts ofdihaploid lines ofpotato SELOSTUS Perunalajikkeiden ponsiviljelyllä tuotettujen dihaploidien protoplastien sähköfuusio Veli-Matti Rokka, Yong-Sheng Xu, Pirjo Tanhuanpää, Leena Pietilä ja Eija Pehu Maatalouden tutkimuskeskus, Helsingin yliopisto ja Boreal Suomen Kasvinjalostus Protoplasteilla tarkoitetaan soluja, joiden solunseinä on poistettu entsymaattisesti. Tällaisia soluja voidaan fuusioida yhteen, jolloin kahden eri perunalinjan yh- distelmästä muodostuu solujen kasvatuksen jälkeen somaattisia hybridejä. Somaattiset hybridit ovat pe- runan jalostukselle tärkeitä, koska tavanomainen ja- lostus, joka tapahtuu tetraploidien (4x) kasvien su- vullisin risteytyksin, tarvitsee runsaasti risteytyksiä ja suuren määrän jälkeläisiä. Käyttämällä hyväksi di- haploideja (2x) perunoita ja fuusioimalla linjoja yh- teen (2x + 2x), voidaan perunan jalostusta sekä no- peuttaa että tehostaa. Tässä työssä ponsiviljelyllä tuotetuista dihaploi- deista perunalinjoista (peräisin lajikkeista Matilda, Nicola, Pito, Stina, Van Gogh ja White Lady) eris- tettiin protoplasteja, joita fuusioitiin sähköisesti. Kah- den eri dihaploidin protoplasteja fuusioimalla saatiin tuotettua somaattisia hybridejä. Saadut kuusi uutta fuusioyhdistelmää analysoitiin käyttämällä RAPD- merkkejä. Oletetut hybridit määritettiin useimmiten kahdella alukkeella, jotka kumpikin tuottivat dihap- loideille vanhempaislinjoille spesifisen merkin. Useimmat tuotetut somaattiset hybridit olivat tet- raploideja (4x), mutta neljästä fuusioidusta yhdistel- mästä muodostui myös kasveja, joilla oli joko hek- saploidinen (6x), oktoploidinen (8x) tai miksoploidi- nen genomi. Tämä tutkittiin laskemalla hybridien kromosomit tai analysoimalla kasvit virtaussytomet- rillä. Somaattisten hybridien DNA-pitoisuuksien ole- tettiin olevan yhtä suuria kuin tetraploidien perunoi- den DNA-pitoisuudet tai dihaploidien fuusiovanhem- pien DNA-pitoisuuksien summat. Kuitenkin useiden tetra- ja heksaploidien hybridien DNA-pitoisuuksien keskiarvot olivat pienempiä kuin oletetut DNA-pitoi- suudet. 460 AGRICULTURAL AND FOOD SCIENCE IN FINLAND