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Anther culture provides a tool to produce haploid lines from cultivated potato (Solanum tuberosum L.), which has a tetraploid (2n = 4x = 48) genome constitution. Shoot regeneration via direct embryogenesis in anther culture procedure is preferred to produce dihaploid (2n = 2x = 24) potato lines, which can be applied in breeding of potato varieties. The anther culture protocol described in the present chapter can be conducted not only in cultivated potato (S. tuberosum) but also in other genetically related potato species.

European hazelnut (Corylus avellana L.) is a strictly cross-pollinated diploid tree species, which has its northernmost populations in Fennoscandia, and it was one of the first species to recolonize northern Europe after the last ice age. Hazelnut produces edible nuts in Finland but nowadays they are underutilized as food, and currently no breeding programmes exist. In the present study, 300 hazelnut specimens were collected from 20 different locations (= populations) in Finland, and they were genetically analyzed using nine simple sequence repeat (SSR) markers. Most of the genetic diversity existed within populations (83%). According to different genetic analyses (STRUCTURE, principal coordinates analysis, and clustering), a general lack of structure was observed, suggesting extensive gene flow among hazelnuts between 17 investigated populations. However, genetic structuring was clearly observed in three populations: Hakavuori, Mustiala, and Pähkinämäki, which might have become isolated due to geographical barriers that kept them separate, diminishing gene flow from other populations. Studying the diversity of European hazelnut is of great interest for understanding population genetics of a species distributed in its marginal areas in the north, and the results are also valuable for further uses in plant conservation, selection, and possible future breeding actions in Finland.

Anther culture provides a tool to produce haploid lines from cultivated potato (Solanum tuberosum L.), which has a tetraploid (2n = 4x = 48) genome constitution. Shoot regeneration via direct embryogenesis in anther culture procedure is preferred to produce dihaploid (2n = 2x = 24) potato lines, which can be applied in breeding of potato varieties. The anther culture protocol described in the present chapter can be conducted not only in cultivated potato (S. tuberosum) but also in other genetically related potato species.

European hazelnut (

Streptomyces spp. are a highly diverse group of bacteria most of which are soil-inhabiting saprophytes. A few are plant pathogens that produce a family of phytotoxins called thaxtomins and cause significant economic losses, e.g., by reducing the marketability of potato tubers (Solanum tuberosum). In northern Europe, S. scabies, S. turgidiscabies and S. europaeiscabiei are the most common plant pathogenic species. In this study, a Streptomyces strain isolated from a netted scab lesion on a tuber of potato cv. Bintje in northern Sweden was identified as S. turgidiscabies but was found to differ in the genomic region carrying genes required for thaxtomin biosynthesis. Our results showed that the strain did not produce thaxtomin but rather phytotoxin fridamycin E, which is an anthraquinone novel to plant pathogenic Streptomyces spp. Fridamycin E was shown to reduce or inhibit sprouting of potato microtubers in vitro. While fridamycin E is known to have antibiotic activity against Gram-positive bacteria, the inhibitory activity of fridamycin E on plant growth is a novel finding.

Potato (Solanum tuberosum L. ssp. tuberosum) microtubers were produced in vitro with Liquid Lab™ Rocker system. A thin-layer liquid culture was applied together with a regular pitch in autoclavable simple plastic vessels. All cultures were carried out at room temperature without contamination problems. Each cultivar tested (Asterix, Timo, Van Gogh, Velox) formed microtubers in the Liquid Lab system. The mean number of microtubers per vessel (50 explants) varied between 30 (cv. Asterix in 8 weeks tuber induction) and 75 microtubers (cv. Velox in 11 weeks tuber induction). Majority (63%) of the microtubers was sufficient by size and weight (above 200 mg) for further storage at dormancy (4°C). The cv. Velox yielded the highest number of microtubers with cultivation capacity. As a result of prolonged microtuber induction of 2–3 weeks, more microtubers with competence for cultivation were obtained per cultivar, except for cv. Van Gogh. Still, the mean weight of Van Gogh microtubers was significantly higher after prolonged microtuber induction (0.67 g) than after short induction (0.51 g). In conclusion, Liquid Lab™ Rocker system is a novel, efficient and rapid system for mass propagation of potato.

Protoplast fusion has been developed as a method for plant breeding. Especially promising results in somatic genome manipulation through protoplast technology have been obtained with the plant families Brassicaceae, Rutaceae, and Solanaceae. The fusion of protoplasts derived from diverse origins generates a hybrid cell, where the protoplasm, organelles, and genetic material from both of the partner cells are combined. This fusion product is called a heterokaryon or a heterokaryocyte. The fusion of isolated protoplasts from somatic cells and regeneration of hybrid plants from the fusion products (somatic hybrids) allow combining of complete genomes of two desirable parents, irrespective of their taxonomic relationship. Usually, protoplast fusion can be classified as symmetric and asymmetric hybridization. Symmetric fusion incorporates the whole genomes of both parents, particularly nuclear ones, whereas asymmetric fusion allows partial genome transfer from a donor cell to a recipient. In asymmetric fusion, the donor cells are usually subjected to irradiation or ultraviolet treatment before fusion. In comparison to other techniques of chromosomal and genetic engineering, somatic genome hybridization has a unique potential to transfer simultaneously both nuclear and cytoplasmic genes. It overcomes gene segregation and enables the transfer of both mono- and polygenic traits among sexually incompatible species. The technique of cell fusion has already been applied to produce a large number of intergeneric, intertribal, or interfamily symmetric and asymmetric somatic hybrids by several research teams and private companies. Procedures for protoplast fusion of potato to produce symmetric somatic hybrids are included, along with detailed lists of materials and descriptions of techniques.

Solanum acaule Bitt., a wild potato species, is closely related to cultivated potato (Solanum. tuberosum L.). Incorporation of desirable traits from allotetraploid [2n=4x=48, 2 endosperm balance number (EBN)] S. acaule (acl) into autotetraploid (2n=4x=48, 4EBN) S. tuberosum (tbr) is difficult due to incongruity boundaries. In this study, three hybrid combinations, each with a specific genome constitution, were produced through protoplast fusion: (1) hexaploid 2x acl (+) 4x tbr, (2) tetraploid 2x acl (+) 2x tbr, and (3) hexaploid 4x acl (+) 2x tbr hybrids. In terms of glycoalkaloid aglycones, the hybrids produced demissidine, tomatidine and solanidine, similarly to the S. acaule parental species, but S. tuberosum synthesised only solanidine. Inoculations with Clavibacter michiganensis ssp. sepedonicus (Cms), which is the causal agent of bacterial ring rot in potato, yielded significantly lower total glycoalkaloid aglycone accumulation both in S. acaule plants and in interspecific hybrids in comparison with the corresponding mock-inoculated plants. However, in S. tuberosum the aglycone levels were either higher or unchanged as a result of infection by Cms. To incorporate the desirable traits of the interspecific somatic hybrids into 4EBN S. tuberosum, sexual backcrosses were carried out. The hexaploid 4x acl (+) 2x tbr hybrids with the hypothetical 4EBN showed the greatest capacity to undergo backcrosses with S. tuberosum.

SUMMARY Suppression subtractive hybridization was used to isolate the genes which are specifically up-regulated in the biotrophic phase of the incompatible interaction between a potato genotype, 1512 c(16), containing the resistance gene R2, and a Phytophthora infestans isolate containing the avirulence gene Avr2. Eight cDNAs were up-regulated in the biotrophic phase of the incompatible interaction. Seven of these were also up-regulated in the compatible interaction, but not until late in the necrotrophic phase. Amongst the sequences to be isolated were genes encoding the cysteine protease cathepsin B, StCathB, and an oxysterol binding protein, StOBP1; equivalent genes are involved in programmed cell death (PCD) processes in animals, but have yet to be implicated in such processes in plants. Whereas StOBP1 was up-regulated early in potato plants containing either R gene-mediated or moderate to high levels of field resistance, the highest levels of up-regulation of StCathB were observed early in R gene-mediated resistance but gradually increased from the early to late stages of field resistance, revealing these genes to be components of independent defence pathways and providing a means of distinguishing between these forms of resistance. StOBP1 was up-regulated by oligogalacturonides (plant cell wall breakdown products generated by pectinase activities), indicating that it is also a component of a general, non-specific defence pathway and is unlikely to play a role in PCD. In contrast, the expression of StCathB was unaffected by oligogalacturonide treatment, further associating its up-regulation specifically with the gene-for-gene interaction.

The development of potato (Solanum tuberosum) cultivars immune (completely free of infective agents) to bacterial ring rot disease, caused by Clavibacter michiganensis ssp. sepedonicus, would be a significant step toward eradication of the disease. Earlier results suggested that the wild potato species Solanum acaule, acc. PI472655 (genotype 7-8), was immune to C. michiganensis ssp. sepedonicus. The goal of the present study was to investigate the possibility of transferring this desirable trait to cultivated potato through interspecific somatic hybridization. Eight different somatic hybrids between S. acaule and S. tuberosum, with three different genome ratios, were tested for susceptibility to infection in the glasshouse using two C. michiganensis ssp. sepedonicus strains. All the somatic hybrids expressed symptoms of ring rot and were susceptible to infection as determined by IFAS (indirect immunofluorescent antibody staining) tests. The genome compositions of the hybrids influenced bacterial titres. Most of the hybrids with a higher proportion of the S. acaule genome contained lower numbers of C. michiganensis ssp. sepedonicus cells than hybrids with lower proportions of the S. acaule genome. In growth chamber tests, temperature was found to be a determining factor in the expression of immunity in the tetraploid S. acaule 7-8 line. At 21°C, S. acaule 7-8 was immune to infection by C. michiganensis ssp. sepedonicus, but at 15°C, plants supported large populations of the pathogen. However, none of the S. acaule plants expressed disease symptoms. Thus, S. acaule exhibits temperature-dependent immunity to infection by C. michiganensis ssp. sepedonicus and should be considered tolerant rather than immune.

Protoplasts were fused to produce somatic hybrids between a triploid (2n=3x=32-34) interspecific somatohaploid betweenSolanum brevidens Phil. andS. tuberosum L., and a dihaploid (2n=2x=24) anther-derived line ofS. tuberosum cv. Van Gogh. A total of 265 plants were regenerated from protoplast fusion derived calli and their hybridity was verified using fusion partner specific RAPD markers. These “second generation” somatic hybrids were aneuploid pentaploids (2n=5x=51-65) with a 2C DNA content ranging from 3.36 to 4.43 pg, which corresponded to the sum of the 2C values of each of the fusion partners (somatohaploid: 2.22 pg; and the dihaploid line of cv. Van Gogh: 1.87 pg). Most of the “second generation” somatic hybrids were vigorous, but variable in morphology. They were extremely resistant to PLRV and they had tolerance to PVY infection derived from the somatohaploid fusion partner. Even though most of the “second generation” hybrids tuberized, the tuber morphology was variable and most were poorly shaped. InErwinia soft rot resistance tests, the tubers showed higher level of resistance than the tetraploidS. tuberosum cultivars, the dihaploidS. tuberosum fusion partners and the hexaploid somatic hybrids betweenS. brevidens andS. tuberosum. The “second generation” somatic hybrids were all male sterile and failed to produce berries or seeds.

Nine dwarf (di)haploid lines (2n224) were obtained from the tetraploid (2n 4 48), long day-adapted potato cultivar ‘Pito’ (Solanum tuberosum subsp. tuberosum) through anther culture. They grew slowly, had very short internodes, compact and ball-shaped appearance, and dark green leaves. Dwarfism was due to a recessive gene, designated pito. Endogenous gibberellin contents were measured in the leaves of dwarf and wild-type lines by gas chromatography linked to mass spectrometry (GC-MS). High amounts of GA19 ,G A 20 ,G A 29 ,G A 1, and GA8 were detected in the wild-type plants, which indicated that the early 13-hydroxylation pathway was predominantly used for GA biosynthesis in S. t. subsp. tuberosum. Also GA53 ,G A 15 and GA9 were detected but not quantified. Very low endogenous amounts of all analysed GAs were detected in the pito mutants, indicating a block at an early part of the GA biosynthesis pathway. The dwarf lines strongly and quickly responded to the exogenous application of low amounts (79 nM) of bioactive GA (GA3), which restored normal growth and confirmed that the pito dwarfs were synthesis mutants and not GA response mutants. © 1999 Elsevier Science Ireland Ltd. All rights reserved.

Laser flow cytometry was used to analyze nuclear DNA contents (2C values) of five genera (Severinia Ten., Atalantia Corrèa, Fortunella Swing., Poncirus Raf., and Citrus L.) taxonomically grouped in subtribe Citrinae (citrus fruit trees) of the Rutaceae. The genotypes analyzed had 2C values ranging from 0.67 pg for diploid Severinia buxifolia (Poir.) Ten. to 1.27 pg for tetraploid Hongkong Fortunella hindsii Swing. There was no significant difference in the 2C values within the sexually compatible diploid species of 11 “true citrus fruit trees” [Citrus aurantium L., C. grandis (L.) Osbeck, C. limon (L.) Burm. f., C. limonia Osbeck, C. paradisi Macf., C. reshni Hort. ex Tanaka, C. sinensis (L.) Osbeck, C. volkameriana Ten. & Pasq., Poncirus trifoliata (L.) Raf., and the intergeneric hybrid C. sinensis × P. trifoliata]. The species Atalantia ceylanica (Arn.) Oliv. (a “near-citrus fruit tree”), sexually incompatible with Citrus spp., had a 2C value significantly different from those of the true citrus fruit tree species. The 2C value of Severinia buxifolia (a “primitive citrus fruit tree”), another species sexually incompatible with the Citrus spp., also differed from those of some of the true citrus fruit tree species. The data largely corresponds with taxonomical differences between a) the genera Citrus and Poncirus and b) the genera Severinia and Atalantia, all assigned to subtribe Citrinae.

Solanum acaule Bitt. is a disomic tetraploid (4x) wild potato species which is resistant to several potato diseases. Introgression of disease resistance and abiotic stress tolerance to the tetrasomic tetraploid (4x) cultivated potato (S. tuberosum L.) gene pool via crossing has been limited due to the difference in the endosperm balance number. In the present study, protoplast fusion was applied to produce hexaploid (6x) somatic hybrids between the parental lines, tetraploid (4x) S. acaule and two anther-derived dihaploid (2x) lines of S. tuberosum cv. White Lady. One callus (0.4%) of a total of 229 calli obtained regenerated into shoots in the fusion combination S. acaule (+) White Lady 15.dh.8.2.2. All the regenerated shoots were confirmed to be interspecific somatic hybrids using species-specific RAPD markers. In another fusion combination, S. acaule (+) White Lady 7.dh.23.1.1, fifteen calli (5%) regenerated into a total of sixteen shoots from 289 calli. All the analysed somatic hybrids between S. acaule and S. tuberosum were hexaploid. The mean DNA content (2C value) of the combination S. acaule (+) White Lady 15.dh.8.2.2 somatic hybrids (4.55 pg), was approximately the sum (4.69 pg) of the DNA contents of the parental lines, S. acaule (2.95 pg) and S. tuberosum (1.74 pg). In the greenhouse, the two somatic hybrids analysed were normal in their morphological characteristics and more vigorous than their parental lines. Most of the morphological characteristics were closer to the tetraploid S. acaule than to the dihaploid S. tuberosum. The interspecific somatic hybrids are currently being tested for frost tolerance and glycoalkaloid composition.

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species.

The chromosomal distribution, copy numbers, and nucleotide sequences were determined for four repetitive DNA clones, pSB1 and pSB7 of Solanum brevidens and pST3 and pST10 of Solanum tuberosum. Using fluorescence in situ hybridization (FISH), pSB1 and pSB7 were localized near the telomeres and in some centromeric and interstitial sites of S. brevidens chromosomes, but not in S. tuberosum chromosomes, after high stringency washes. The clone pST3 showed signals in the telomeric areas of a few chromosomes in S. tuberosum, but signals were not detected in S. brevidens. All three repeated sequences (pSB1, pSB7, and pST3) were detected in chromosomal areas that are typically known to contain tandemly repeated sequences. The S. tuberosum clone pST10 did not show signals in either species even at low stringency conditions. The estimated copy numbers of the four clones were 1500, 6750, 300, and 400 for pSB1, pSB7, pST3, and pST10, respectively, in the corresponding haploid genomes (S. brevidens and S. tuberosum). The inserts of the four clones pSB1, pSB7, pST3, and pST10 were 322, 167, 845, and 121 bp, respectively. After sequencing, no significant sequence homologies were found among the four clones. A homology search in sequence data bases showed that pSB7 has variable homology (78-100%) with another repetitive sequence of S. brevidens Sb4/2 depending on its subrepeat. It also showed some homology with one repeat of tomato (pLEG15) and one repeat of Solanum circaeifolium (pSC15).Key words: chromosome, copy number, fluorescence in situ hybridization, FISH, nucleotide sequence, potato.

Solanum tuberosum cv. Pentland Dell expresses single gene-mediated hypersensitive resistance (HR) to potato virus Y (strain group PVY O ), A (PVA 1 ) and X (PVX 3 ) and is resistant to cucumber mosaic virus (Fny-CMV). S. brevidens is resistant to these viruses, too, but resistance is functional through slow virus cell-to-cell movement. These two virus resistance mechanisms were combined by symmetric somatic hybridization. Six hybrid plants were regenerated from a single fusion callus and tested for virus resistance by graft-inoculation. Titers of PVY and PVA in the hybrids were higher than in S. brevidens . Only limited necrosis developed following infection with PVY and chlorotic lesions developed following infection with PVA in the hybrids, whereas top necrosis and necrotic lesions, respectively, developed in P. Dell. No virus or symptoms were observed in one hybrid inoculated with PVX and three hybrids inoculated with CMV. Other hybrids developed lethal necrosis following infection with PVX, and stunting, necrosis and yellow mosaic symptoms following infection with CMV, which was similar to P. Dell. S. brevidens contained moderate titers of PVX and CMV. It developed no symptoms following infection with PVX and only mild mosaic symptoms following infection with CMV. Altered resistance expression in the hybrids may be attributable to simultaneous expression of the two resistance mechanisms, interference in the expression of either resistance mechanism caused by the genes of the heterologous genome, and/or somaclonal variation.

Two Solanum brevidens specific repetitive DNA clones (pSB1 and pSB7) were used simultaneously as probes in fluorescence in situ hybridization (FISH) for cytological studies of somatohaploids and their somatic hybrid donors. pSB1 was labelled with digoxigenin-11-dUTP and pSB7 was labelled with biotin-14-dATP and they were detected with reporter molecules conjugated to fluorescent dyes using digital imaging. The tandemly repeated sequences hybridized mostly near the telomeres of the chromosomes of S. brevidens. Using these two probes, it was possible to identify chromosomes containing repetitive DNA of S. brevidens both in the somatic hybrids between S. brevidens and S. tuberosum, and somatohaploids derived from the somatic hybrids. These cytological analyses showed that for the largest part genomes of the hexaploid somatic hybrids and their anther-derived triploid somatohaploids were composed of the genome of S. brevidens., Somaattisia hybridejä, jotka on tuotettu villin perunalajin (Solanum brevidens) ja viljellyn perunan (S. tuberosum) soluja fuusioimalla, on käytetty ponsiviljelyssä somatohaploidien tuottamiseksi. Somatohaploidit ovat mielenkiintoisia kasvinjalostustutkimuksessa, koska niiden avulla voidaan tutkia hybridien ominaisuuksien periytymistä meioosin jälkeen. Lisäksi somatohaploideja voidaan hyödyntää perunan lajikejalostuksessa risteyttämällä niitä haploidien perunalinjojen kanssa protoplastifuusioiden avulla. Tällöin on kyseessä suvullisen takaisinristeytyksen vaihtoehtoinen jalostusmenetelmä, ja villien perunalajien haluttuja ominaisuuksia voidaan siirtää todennäköisesti tehokkaammin agronomisilta ominaisuuksiltaan hyviin perunalinjoihin. Tässä työssä tutkittiin kahden S. brevidens -lajilta eristetyn toistuvajaksoisen DNA-koettimen (pSB1 ja pSB7) avulla somaattisten hybridien ja niistä tuotettujen somatohaploidien kromosomistoja. Tutkimuksessa käytettiin fluoresenssi in situ -hybridisaaliota, jonka avulla yksijuosteinen toistuvajaksoinen DNA voitiin paikallistaa denaturoituihin kromosomeihin, jotka sisälsivät S. brevidens -lajin vastaavaa komplementaarista DNA:ta. Fluoresenssi in situ -hybridisaatiota varten pSBI-koetin leimattiin digoksigeniini-11-dUTP:IIa ja pSB7-koetin biotiini-14-dATP:lIa. Kromosomeihin hybridisoituneet leimatut DNA-koettimet paikallistettiin niihin konjugoituneiden fluoresenssiväriaineiden avulla. Molemmat koettimet olivat peräkkäisjaksoista DNA:ta ja hybridisoituivat S. brevidens -lajin kromosomien telomeerisiin päihin. Kyseisten koettimien avulla voitiin erottaa somaattisten hybridien ja niiden somatohaploidien ne kromosomit, jotka sisälsivät S. brevidens -lajin toistuvajaksoista DNA:ta. Fluoresenssi in situ -hybridisaation avulla todettiin, että heksaploidit somaattiset hybridit ja niistä tuotetut triploidit somatohaploidit sisälsivät enemmän S. brevidens -lajin genomia kuin S. tuberosum -lajilta peräisin olevaa genomia.

Somatic hybrids of anther-derived dihaploid (2x) potato (Solanum tuberosum L.) lines were produced by electrofusion of protoplasts. Using RAPD (randomly amplified polymorphic DNA) markers, 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 mostly 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., Protoplasteilla tarkoitetaan soluja, joiden solunseinä on poistettu entsymaattisesti. Tällaisia soluja voidaan fuusioida yhteen, jolloin kahden eri perunalinjan yhdistelmästä muodostuu solujen kasvatuksen jälkeen somaattisia hybridejä. Somaattiset hybridit ovat perunan jalostukselle tärkeitä, koska tavanomainen jalostus, joka tapahtuu tetraploidien (4x) kasvien suvullisin risteytyksin, tarvitsee runsaasti risteytyksiä ja suuren määrän jälkeläisiä. Käyttämällä hyväksi dihaploideja (2x) perunoita ja fuusioimalla linjoja yhteen (2x + 2x), voidaan perunan jalostusta sekä nopeuttaa että tehostaa. Tässä työssä ponsiviljelyllä tuotetuista dihaploideista perunalinjoista (peräisin lajikkeista Matilda, Nicola, Pito, Stina, Van Gogh ja White Lady) eristettiin protoplasteja, joita fuusioitiin sähköisesti. Kahden 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 dihaploideille vanhempaislinjoille spesifisen merkin. Useimmat tuotetut somaattiset hybridit olivat tetraploideja (4x), mutta neljästä fuusioidusta yhdistelmästä muodostui myös kasveja, joilla oli joko heksaploidinen (6x), oktoploidinen (8x) tai miksoploidinen genomi. Tämä tutkittiin laskemalla hybridien kromosomit tai analysoimalla kasvit virtaussytometrillä. Somaattisten hybridien DNA-pitoisuuksien oletettiin olevan yhtä suuria kuin tetraploidien perunoiden DNA-pitoisuudet tai dihaploidien fuusiovanhempien DNA-pitoisuuksien summat. Kuitenkin useiden tetra- ja heksaploidien hybridien DNA-pitoisuuksien keskiarvot olivat pienempiä kuin oletetut DNA-pitoisuudet.

A total of 1000 anther-derived plants was regenerated from tetraploid potato (Solanum tuberosum L.) genotypes. Capacity to undergo androgenesis was analysed in 41 potato cultivars and 7 clones grown either in the greenhouse or in the field. Of the 48 genotypes, 33 produced embryos and 23 regenerated shoots from embryos. Anther-derived plantlet production was determined in genotypes 86110, Agria, Calgary, Escort, Helios, Idole, JO 0982 JO 1432, Kainuun Musta, Kardal, KE48, Matilda, Nicola, Petra, Pito, Rustica, Stirling, Torridon, Ute, Van Gogh, Vebeca, Vento and White Lady. The highest number of shoots (24 shoots/100 anthers) was obtained from cv. Calgary, when anthers were isolated from field-grown donor plants. Incubating anthers at 28 C, rather than at 20 C or 24 C, enhanced embryo production in four genotypes tested. However, shoot production was improved only in cv. Pito cultured at 28 C. When anthers of cv. Petra were cultured at 28 C for four weeks, followed by reduction of culture temperature to 24 C, a high rate of shoot production was recorded (14 shoots/100 anthers). The ratio between dihaploids and tetraploids varied among the anther-derived plants of the different genotypes. The number of dihaploids was highest in potato clone JO1432 (100%) and in cv. Calgary (93%) and lowest in cvs. Pito (21%) and Torridon (6%).

Somatic hybrids between dihaploid (2 n =2 x =24) cultivated potato Solanum tuberosum L.) and diploid (2 n =2 x =24), virus resistant wild potato species S. brevidens Phil., were tested for their feasibility for anther culture. A total of 17 shoots regenerated by embryogenesis from anthers of the hybrids. Five regenerants survived and were grown in vitro and in the greenhouse. The haploids were morphologically intermediate between S. brevidens and S. tuberosum . They were analysed for S. brevidens specific and S. tuberosum specific RAPD markers using four primers, and were found to contain the S. brevidens specific DNA markers and most of the S. tuberosum specific markers. The chromosome numbers of the haploids ranged from 34 to 36. According to flow cytometric analysis, the nuclear DNA contents of the haploids (2 C =2.15–2.30pg) were approximately half of the hexaploid somatic hybrids (2 C =4.47–4.53pg). The somatic hybrids were identified as chimeric (hypohexaploids), whereas the haploids derived from them using anther culture were all triploids according to flow cytometry. The haploids were extremely resistant to potato leaf roll virus.

Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.

Necrotic lesions and vein necrosis characteristic of the hypersensitive response (HR) controlled by the dominant resistance gene Ny develop in potato cv. Pito after infection with potato virus Y ordinary strain (PVY masculine) at a low temperature (16/18 degrees C night/day). In contrast, at high temperatures (19/24 degrees C night/day), large coalesced lesions develop in the lower infected leaves, which wither and remain hanging from stems forming the leaf-drop symptom; mosaic symptoms with no necrosis also develop in the top leaves. The genetic basis of the leaf-drop symptom and its dependence on temperature were examined using a novel approach involving 58 haploids (2n = 24) derived from 'Pito' (2n = 48) through anther culture. These haploids and 'Pito' were graft-inoculated with PVY(O) at 19/24 to 25 degrees C (night/day). Necrotic symptoms were expressed in 28 haploids, of which 18 haploids (phenotype class N) developed top necrosis, vein necrosis, or both and necrotic lesions that are characteristic of HR. Ten haploids showed leaf drop similar to 'Pito' (phenotype class LD). Thirty haploids were susceptible and showed only mosaic symptoms (phenotype class S). These data indicated that necrosis was induced by a single dominant gene, Ny, in the simplex condition. However, the three distinct phenotypic classes (N, LD, and S) among the haploids grown under the same environmental conditions showed that another locus (gene) was involved in modifying the HR triggered by Ny. Data suggested that this locus contains a dominant temperature-dependent modifier (Tdm) gene that alters the expression of PVY-induced HR at higher temperatures, resulting in leaf drop.

The present anther culture procedure has been utilized successfully for a genetically diverse range of potato material including commercial varieties of cultivated potato (Solanum tuberosum ssp. tuberosum), its related wild species and various hybrids having wild Solanum spp. germplasm. Previously, cultivated potato has been described as a recalcitrant crop plant with regard to androgenesis, and progress in haploid production through direct embryogenesis using anther culture has been considerably limited. Therefore parthenogenesis through pollination with selected clones of S. phureja is still widely utilized for haploid production in potato. However, wild characteristics derived from the S. phureja pollinator and aneusomatic genomic constitutions in the resultant haploid lines are common when parthenogenesis (gynogenic pathway) is carried out for practical haploid production in potato. Isolated microspore culture in potato has yet to provide results for plant production. Therefore, in vitro androgenesis through direct embryogenesis in anther culture, the method described in the current article, is suggested for haploid production in potato and its relatives. The present anther culture protocol has been conducted on a genetically diverse range of Solanum spp. material. The method was originally introduced for dihaploid (2n=2x=24) production from tetraploid (2n=4x=48) varieties of cultivated potato (Rokka et al., 1996), but since then it has also been utilized for: tetraploid breeding material with wild potato germplasm; dihaploid lines of cultivated potato; S. acaule ssp. acaule wild potato species (Rokka et al., 1998); interspecific hybrids between A genome potato species (S. tuberosum and S. acaule); tetraploid and hexaploid interspecific hybrids between A and E genome species [S. tuberosum x S. brevidens hybrids, Rokka et al. (1995); S. tuberosum x S. etuberosum hybrids, V-M. Rokka, R. Thieme and T. Gavrilenko, unpublished results]; and intergeneric tomato (Lycopersicon esculentum + Solanum etuberosum) somatic hybrids (Gavrilenko et al., 2001).

Interspecific somatic hybrids produced by protoplast fusion between two wild Solanum species (S. acaule, acl; S. brevidens, brd) and cultivated potato Solanum tuberosum (tbr) were analyzed in terms of the starch nanometer-range structure and glycoalkaloid (GA) contents. The crystallinity of starch granules, the average size of starch crystallites, and the lamellar distances were obtained from tuber samples using wide-angle and small-angle X-ray scattering methods. These measurements showed that incorporation of wild genomes from either nontuberous (brd) or tuberous (acl) Solanum species caused no significant modifications of the nanostructure of potato starch. In contrast, the GA profiles of the hybrids, which were analyzed by LC-ESI-MS in both tuber and foliage samples, differed considerably from those of cultivated potato. Regardless of the low total tuber GA concentrations (approximately 9 mg/100 g of fresh weight), the somatic hybrids contained GAs not detected in the parental species. A high proportion of spirotype GAs consisting of 5,6-dihydrogenated aglycons, for example, alpha-tomatine and tomatidine bound with solatriose, and chacotriose were found in the hybrids. In conclusion, the foliage of interspecific hybrids contained a higher variation in the structures of GAs than did the tubers.

Potato is world’s fourth most important food crop after wheat, maize and rice (Hawkes, 1990). However, it is generally felt that the nutrition and breeding potential of potato have not yet been fully exploited. The nutritional constituents of potato tubers are well-balanced for human requirements. In developing countries, where the immediate demand for food is often crucial, potato outcompetes rice and wheat in production of edible energy per hectare and is nearly as efficient as groundnut, lentil, cabbage, rice, and wheat in production of edible protein (Horton, 1986). In most parts of the world, except the natural habitats of potato species in Latin America, a single potato species (Solanum tuberosum L.) is cultivated and just a minor part of existing potato germplasm (ca. 230 species) has been used in potato breeding and production (Ross, 1986). Many useful traits that are yet to be transferred to cultivated potato lines have been identified in wild potato germplasm; still, many traits and useful genotypes await identification (Hawkes, 1990). The unexploited traits identified in the non-cultivated tuber-bearing species include resistance to various stresses, such as plant pathogens, insect pests and frost, and also tolerance to heat under tropical climates (Hawkes, 1990).

Potato is one of the most important crops worldwide. Genetic resources of potato (Solanum tuberosum L. ssp. tuberosum) and related cultivated species are conserved through storage of tubers, in vitro plants and in cryopreservation. Cryopreservation, storage in or above liquid nitrogen, is the best option to maintain vegetatively propagated plants in the long term. The present review gives comprehensive information about various cryopreservation techniques for potato published from 1977 until the present. It discusses factors that affect the process and success of cryopreservation, such as donor culture conditions, preculture, cooling, warming and post-culture treatments. Studies are presented that analyse the histological and ultrastructural changes after different cryopreservation steps and the morphological pathways during regeneration of plants after rewarming. The maintenance of genetic stability in potato after cryopreservation has also been demonstrated by various phenotypic and molecular methods. The first thermal analyses on potato shoot tips are presented using differential scanning calorimetry to analyse the state of water during cooling and warming. Biochemical analyses of different compounds, such as soluble sugars and proteins, have been performed to understand and improve existing cryogenic methods. Potato is an example where successful virus elimination has been obtained via cryopreservation of shoot tips (cryotherapy). There are already cryopreserved collections of potato shoot tips in Germany, Peru, Czech Republic, South Korea and USA, but additional experiments on fundamental aspects of potato cryopreservation will help to improve understanding of the different cryopreservation methods, start new collections in other countries and also build up existing cryocollections of potato.