Identification of mitotic chromosomes of tuberous and non-tuberous solanum species (Solanum tuberosum and Solanum brevidens) by GISH in their interspecific hybrids.

GISH (genomic in situ hybridization) was applied for the analysis of mitotic chromosome constitutions of somatic hybrids and their derivatives between dihaploid clones of cultivated potato (Solanum tuberosum L.) (2n = 2x = 24, AA genome) and the diploid, non-tuberous, wild species Solanum brevidens Phil. (2n = 2x = 24, EE genome). Of the primary somatic hybrids, both tetraploid (2n = 4x) and hexaploid (2n = 6x) plants were found with the genomic constitutions of AAEE and AAEEEE, respectively. Androgenic haploids (somatohaploids) derived from the tetraploid somatic hybrids had the genomic constitutions of AE (2n = 2x = 24) and haploids originating from the hexaploid hybrids were triploid AEE (2n = 3x = 33 and 2n = 3x = 36). As a result of subsequent somatic hybridization from a fusion between dihaploid S. tuberosum (2n = 2x = 24, genome AA) and a triploid somatohaploid (2n = 3x = 33, genome AEE), second-generation somatic hybrids were obtained. These somatic hybrids were pentaploids (2n = 5x, genome AAAEE), but had variable chromosome numbers. GISH analysis revealed that both primary and second-generation somatic hybrids had lost more chromosomes of S. brevidens than of S. tuberosum.

Transgenic resistance to PVY(O) associated with post-transcriptional silencing of P1 transgene is overcome by PVY(N) strains that carry highly homologous P1 sequences and recover transgene expression at infection.

Resistance to Potato virus Y (PVY) has been obtained in our previous studies through expression of the PVY P1 gene in sense or antisense orientation in potato cv. Pito. In the present study, the mechanism and strain specificity of the resistance were analyzed. Several features including low steady-state P1 mRNA expression in the resistant P1 plants indicated that resistance was based on post-transcriptional gene silencing (PTGS). Resistance was specific to PVY(O) isolates, the PVY strain group from which the P1 transgene was derived. However, according to group analyses, there was no distinguishing characteristic between the PVY(O) and PVY(N) strains P1 gene sequences. Therefore, the ability of the PVY(N) strains to overcome resistance could not be explained solely based on their P1 gene sequences. Infection with PVY(N) of the PVY(O)-resistant transgenic lines led to a recovery of expression of the P1 transgene. These data suggested that factors other than sequence homology are required in determination of the resistance specificity.

Comparison of commercial solid-phase extraction sorbents for the sample preparation of potato glycoalkaloids.

In this study five different commercial sorbents C18, SCX, CN, Certify and Oasis HLB were compared for the solid-phase extraction of potato glycoalkaloids. The recoveries were determined using alpha-solanine, alpha-chaconine and alpha-tomatine, which contained dehydrotomatine as an impurity, as standard compounds. The samples were analysed by reversed-phase liquid chromatography under gradient elution conditions using a Zorbax Rx-C18 column and acetonitrile-25 mM triethylammonium phosphate buffer (pH 3.0) as the mobile phase. The highest recovery (approximately 100%) was achieved with Oasis HLB (60 mg) cartridges. An acetic acid extract of wild Solanum brevidens leaf material was used for the testing of a clean-up procedure. The SCX proved to be the most selective and efficient for removing the undesired components from the leaf extract.

Reversed-phase liquid chromatographic separation and simultaneous profiling of steroidal glycoalkaloids and their aglycones.

Improved and simplified reversed-phase liquid chromatographic conditions for the separation and simultaneous profiling of both steroidal glycoalkaloids and their aglycones, having solanidane- or spirosolane-type structures, are described. The most reproducible retention behavior for these ionizable compounds on C18 columns was achieved under isocratic and gradient elution conditions using acetonitrile in combination with triethylammonium phosphate buffer at pH 3.0, when basic functional groups of solutes and silanol groups on the silica are fully protonated minimizing ionic interactions. Gradient elution was the only feasible approach for the simultaneous separation of steroidal glycoalkaloids and their aglycones. A Zorbax SB C18 column, specially designed for low-pH separations, showed good performance in critical separations. The impurities of the commercial tomatine and tomatidine standards were studied and confirmed using mass spectrometric, liquid chromatographic and thin-layer chromatographic methods.

Localization of the P1 protein of potato Y potyvirus in association with cytoplasmic inclusion bodies and in the cytoplasm of infected cells.

The N-terminal P1 proteinase of potato virus Y (ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the expressed protein and used to detect the viral proteins in infected tobacco leaf tissue by Western blotting and by electron microscopy with immunogold labelling. In the immunogold localization studies P1 protein was detected in association with the cytoplasmic inclusion bodies characteristic of PVY infections and in the cytoplasm of the infected plant cells. No significant P1 antibody binding with other plant cell organelles, or with the cell wall and plasmodesmata, was detected by immunogold labelling.

Cytological and molecular characterization of repetitive DNA sequences of Solanum brevidens and Solanum tuberosum.

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).