Ramona Thieme

Fertile somatic hybrids of Solanum etuberosum (+) dihaploid Solanum tuberosum and their backcrossing progenies: relationships of genome dosage with tuber development and resistance to potato virus Y

The wild non-tuberous species Solanumetuberosum is resistant to biotic andabiotic stresses, but is very difficult tocross with cultivated potato. Therefore,interspecific somatic hybrids between adihaploid clone of potato S.tuberosum (2n=2x=24, AA genome) and thediploid species S. etuberosum(2n=2x=24, EE genome) were produced byprotoplast fusion. Among the 7 fertilefusion hybrids analysed by genomic insitu hybridisation (GISH), three groups ofplants were found with the genomicconstitution of AAEE, AAEEEE and AAAAEE.Four fusion hybrids had exactly theexpected chromosome composition, while eachof the three aneuploid hybrids had lost twochromosomes of S. etuberosum. Twobackcross progenies were developed, andGISH analysis was applied to analysetransmission of the parental chromosomesinto the sexual generations. BC1hybrids derived from the crosses of thehexaploid somatic hybrids with tetraploidpotato were pentaploid with thetheoretically expected genomic compositionor with slight deviation from thisexpectation. In the three BC2 hybridsanalysed by GISH seven to 12 chromosomes ofS. etuberosum were detected in thepredominant S. tuberosum background.No recombinant chromosomes in the hybridswere detected. Genome dosage affects tuberformation in hybrids and their progenies,but has less effect on resistance to potatovirus Y (PVY) in fusion hybrids. Severalgenotypes of the fusion hybrids andBC1 progeny did not show viralinfection even in the graftingexperiments.

Cytogenetic analysis of Lycopersicon esculentum (+) Solanum etuberosum somatic hybrids and their androgenetic regenerants

Abstract  The aim of the study was to characterize genomic relationships among cultivated tomato (Lycopersicon esculentum Mill.) (2n=2x=24) and diploid (2n=2x=24) non-tuberous wild Solanum species (S.etuberosum Lindl.). Using genomic in situ hybridization (GISH) of mitotic and meiotic chromosomes, we analyzed intergeneric somatic hybrids between tomato and S.etuberosum. Of the five somatic hybrids, two plants were amphidiploids (2n=4x=48) mostly forming intragenomic bivalents in their microsporocytes, with a very low frequency of multivalents involving the chromosomes of tomato and S.etuberosum (less than 0.2 per meiocyte). Tomato chromosomes showed preferential elimination during subsequent meiotic divisions of the amphidiploids. Transmission of the parental chromosomes into microspores was also evaluated by GISH analysis of androgenic plants produced by direct embryogenesis from the amphidiploid somatic hybrids. Of the four androgenic regenerants, three were diploids (2n=2x=24 or 2n=2x+1=25) derived from reduced male gametes of the somatic hybrids, and one plant was a hypertetraploid (2n=4x+4=52). GISH revealed that each anther-derived plant had a unique chromosome composition. The prospects for introgression of desirable traits from S.etuberosum into the gene pool of cultivated tomato are discussed.

The usefulness of the gfp reporter gene for monitoring Agrobacterium-mediated transformation of potato dihaploid and tetraploid genotypes.

Potato is one of the main targets for genetic improvement by gene transfer. The aim of the present study was to establish a robust protocol for the genetic transformation of three dihaploid and four economically important cultivars of potato using Agrobacterium tumefaciens carrying the in vivo screenable reporter gene for green fluorescent protein (gfp) and the marker gene for neomycin phosphotransferase (nptII). Stem and leaf explants were used for transformation by Agrobacterium tumefaciens strain LBA4404 carrying the binary vector pHB2892. Kanamycin selection, visual screening of GFP by epifluorescent microscopy, PCR amplification of nptII and gfp genes, as well as RT-PCR and Southern blotting of gfp and Northern blotting of nptII, were used for transgenic plant selection, identification and analysis. Genetic transformation was optimized for the best performing genotypes with a mean number of shoots expressing gfp per explant of 13 and 2 (dihaploid line 178/10 and cv. ‘Baltica’, respectively). The nptII marker and gfp reporter genes permitted selection and excellent visual screening of transgenic tissues and plants. They also revealed the effects of antibiotic selection on organogenesis and transformation frequency, and the identification of escapes and chimeras in all potato genotypes. Silencing of the gfp transgene that may represent site-specific inactivation during cell differentiation, occurred in some transgenic shoots of tetraploid cultivars and in specific chimeric clones of the dihaploid line 178/10. The regeneration of escapes could be attributed to either the protection of non-transformed cells by neighbouring transgenic cells, or the persistence of Agrobacterium cells in plant tissues after co-cultivation.

The recipient potato cultivar influences the genetic makeup of the somatic hybrids between five potato cultivars and one cloned accession of sexually incompatible species Solanum bulbocastanum Dun.

Somatic hybridization is a biotechnological tool, which allows the transfer of multiple resistance genes from sexually incompatible Solanum species into cultivated potato. Here we report the effect of the recipient commercial tetraploid potato cultivar on the genetic make-up of the somatic hybrids (SHs) with an accession of the incongruent diploid species Solanum bulbocastanum Dun. The SHs were produced by mesophyll protoplast electrofusion. The analysis of ploidy by flow cytometry was first used to select hexaploid putative SH shoots but SSR and AFLP markers, DAPI staining and later flow cytometry evaluation of ploidy reveals symmetric and asymmetric SH plants regeneration in proportions that depend on the potato cultivar. The growth and fertility of the SHs support the effect of recipient cultivar. Out of five different fusion combinations, the highest number of SHs and tuberosum morphology was recorded for the combinations: blb41 (+) ‘Delikat’ 235 SHs (104 symmetric and 131 asymmetric) and blb41 (+) ‘Rasant’ 33 SH plants (22 symmetric, 11 asymmetric). There were fertile SHs of these combinations and BC1 and BC2 progenies were obtained. Less successful were the combinations: blb41 (+) ‘Quarta’ 64 SHs (57 symmetric and 7 asymmetric), blb41 (+) ‘Baltica’ 25 SHs (16 symmetric and 9 asymmetric), which were infertile and blb41 (+) ‘Agave’ with only one highly asymmetric non-viable SH plant. The production of a large number of SHs with diverse commercial cultivars is a prerequisite for further selection of useful pre-breeding material. The causes of nuclear constitution asymmetry and somatic incompatibility of the two species are also discussed.

Mismatch repair deficiency increases the transfer of antibiosis and antixenosis properties against Colorado potato beetle in somatic hybrids of Solanum tuberosum + S. chacoense

BACKGROUND Colorado potato beetle (CPB) has become the biggest enemy of cultivated potato worldwide. One of the most effective sources of resistance to CPB is Solanum chacoense, an accession with a high leptine glycoalkaloid content. The aim of our study was to assay the repellence and toxicity of S. chacoense, its somatic hybrids (SHs) and their backcross progenies (BC1 ) with potato for CPB adults and larvae. Transgenic S. chacoense, deficient in DNA mismatch repair (MMR), was also used to produce SHs, in order to increase homeologous recombination and hence introgression of wild-species DNA into the potato gene pool. RESULTS Wild-type SH was highly resistant to CPB. Resistance to CPB of BC1 progenies showed a 1:3 inheritance pattern. MMR-deficient SHs performed better in the resistance analysis. Most MMR-deficient SHs had a similar toxicity as S. chacoense and an intensely repellent effect on CPB adults. Resistance of SHs and BC1 clones may be attributed to leptine biosynthesis, which was confirmed using a RAPD marker. CONCLUSION This is the first report of SHs and their progenies exhibiting both antibiosis and antixenosis against CPB. Resistant SHs are an important step forward in combating this voracious pest of potato.

Somatic Cell Genetics and Its Application in Potato Breeding

This chapter presents a summary of published work on the development, achievements and interconnections of research on potato somatic cell genetics. To maintain genetic stability the main topics include the establishment and maintenance of in vitro cultures, micropropagation, shoot and meristem culture, somatic embryogenesis, production of micro- and mini-tubers and conservation of germplasm. In the second section, the methods presented are based on the induction and utilization of genetic variability (diversity): production of haploids, somatic hybridization via protoplast fusion, somaclonal variation and gene transfer. Another significant aspect of this review is the presentation of numerous methods used in clonal propagation, the production of healthy plants, germplasm conservation for medium-term and long-term storage, potato breeding and utilization of germplasm for the production of advanced breeding clones and potato cultivars with improved resistance to pathogens, pests and abiotic stress, and of high quality and with other specific traits for other purposes. Finally, new methods of breeding, including molecular marker development and genome editing, are briefly described to indicate the potential of somatic cell genetics for the future improvement of potato.

Homeologous chromosome pairing in distant allohaploid hybrids of the genus Solanum

The pairing of chromosomes in meiosis of unique allohaploid hybrids obtained using the methods of somatic hybridization, in vitro androgensis and chromosome engineering has been studied. Hybrids from two combinations were analyzed: (1) between cultivated potato S. tuberosum (dihaploid, 2n = 2x = 24, AA genome) and wild species S. etuberosum (2n = 2x = 24, EE genome) and (2) between cultivated tomato S. lycopersicum (2n = 2x = 24, LL) and wild species S. etuberosum (2n = 2x = 24, EE). Subsequent application of chromosome-specific BAC-clones of potato and probes of differentially labeled total DNA of the parental species allowed to identify chromosomes which were involved in pairing and to detect their genomic identity. Up to 7 intergenomic bivalents per cell were observed in allohaploids between S. tuberosum and S. etuberosum (AE); the chiasmata were distributed in the distal regions of the long arms of each chromosome and in the short arms of chromosomes 3, 6, 11, and 12. Androgenic regenerants of somatic hybrids S. lycopersicum with S. etuberosum are characterized mainly by univalent meiosis; rare bivalents (from 0 to 2 per cell) are formed by homeologs of chromosomes 4 and 6. The prospects of the proposed approach are discussed for the application of somatic hybridization, in vitro androgensis and chromosome engineering to study the potential of homeologs pairing and the strategy of introgressive hybridization of remote plant species.