Effect of photoperiod on in vitro tuberization of potato (Solanum tuberosum L.)

Single-node cuttings of potato cultivars Jemseg, Katahdin, Russet Burbank and Superior were cultured on a multiplication medium containing MS salts and no growth regulators. Cultures were exposed to 8 h (SD) and 16 h (LD) photoperiodic regimes. The subsequent plantlets were excised and single node cuttings from each photoperiodic regime were placed under SD or LD on a second medium containing growth regulators which promoted tuberization. Production of microtubers was strongly influenced by genotype and by photoperiodic treatments. Superior produced stunted plantlets and some microtubers under SD conditions in the multiplication medium. The number of microtubers formed by Jemseg was not influenced by photoperiod. However, Katahdin and Russet Burbank formed fewer microtubers under LD-LD conditions compared to LD-SD, SD-SD and SD-LD regimes. Compared with the other regimes, LD-SD photoperiod generally promoted microtuber formation with larger diameters and significantly (p<0.05) greater fresh weight. The intensity of the tuberization stimulus was affected by daylength, and this was characterized by microtubers with secondary tubers, the growth of more than one axillary microtuber, and microtubers subtended by stolons. The maturity group of the potato cultivars and photoperiodic regime in vitro strongly influenced the production of microtubers. These results can be employed to adapt light regimes for multiplication and tuberization to the specific requirements for cultivars from different maturity groups, and thus increase the efficiency of potato multiplication protocols.     

Cloning and functional analysis of a cDNA encoding a starch synthase from potato (Solanum tuberosum L.) that is predominantly expressed in leaf tissue

Three isoforms of starch synthase (SS) were shown to be present in soluble potato tuber extracts by activity staining after native gel electrophoresis. A cDNA encoding SSI from rice was used as a probe to clone a corresponding cDNA from potato. The deduced amino acid sequence identified the protein as an SS from potato with an M_r of 70.6 kDa for the immature enzyme including its transit peptide. This novel isoform was designated SSI. An analysis of the expression pattern of the gene indicated that SSI is predominantly expressed in sink and source leaves, and, to a lower extent in tubers. In several independent transgenic potato lines, where the expression of SSI was repressed using the antisense approach, the activity of a specific SS isoform was reduced to non-detectable levels as determined through activity staining after native gel electrophoresis. The reduction in the amount of this isoform of SS did not lead to any detectable changes in starch structure, probably due to the fact that this isoform only represents a minor activity in potato tubers. Received: 19 August 1998 / Accepted: 17 December 1998     

Immunocytochemical localization of patatin,the major glycoprotein in potato (Solanum tuberosum L.) tubers

Patatin is a family of glycoproteins with an apparent molecular weight of 40 kDa. The protein is synthesized as a pre-protein with a hydrophobic signal sequence of 23 amino acids. Using different immunocytochemical methods we determined the tissue-specific as well as subcellular localization of the patatin protein. Since antibodies raised against patatin showed crossreactivity with glycans of other glycoproteins, antibodies specific for the protein portion of the glycoprotein were purified. Using these antibodies for electron-microscopical immunocytochemistry, the protein was found to be localized mainly in the vacuoles of both tubers and leaves of potatoes (Solanum tuberosum L.) induced for patatin expression. Neither cell walls nor the intercellular space contained detectable levels of patatin protein. Concerning the tissue specificity, patatin was mainly found in parenchyma cells of potato tubers. The same distribution was observed for the esterase activity in potato tubers.Abbreviations PHA phytohemagglutinin - TFMS trifluoromethanesulfonic acid     

Changes in the location of polyphenol oxidase in potato (Solanum tuberosum L.) tuber during cell death in response to impact injury: comparison with wound tissue

In order to elucidate the nature of the response of potato to impact injury at the biochemical level, changes in the location of the enzyme responsible for the discoloration, polyphenol oxidase, were determined using immunogold location with an antibody specific for potato tuber polyphenol oxidase. Tissue printing revealed that the enzyme was distributed throughout the tuber. Following impact injury, both tissue printing and quantitative electron microscopy indicated that there was no increase in the level of the enzyme although there was subcellular redistribution of polyphenol oxidase. This redistribution was first apparent at 12 h after impact, as determined by the use of confocal immunolocation, and coincided with loss of membrane integrity. These changes were examined in parallel with a number of stress-related parameters in both impact and wound responses. Wounding was accompanied by active gene expression and protein synthesis, leading to metabolic activity and tissue repair. In contrast, the bruising response was characterised by a limited active response and vital-staining methods indicated that after 16 h the tissue undergoes cell death. Received: 4 June 1998 / Accepted: 18 September 1998     

Effects of activated charcoal effects on induction and development of microtubers in potato (Solanurn tuberosum L.)

The aim of this research was to compare hormone-free medium with media with regulator substances (activated charcoal, cytokinins, polyamine biosynthesis inhibitor and chlorocholine chloride) used for microtuber induction and development. Explants of cvs Monalisa, Primura and Spunta were multiplied subculturing nodal segments on plant growth regulator-free Murashige & Skoog (1962) (MS) medium. When the plantlets had 6–8 nodes, single-node stem segments were excised and transferred to eight tuberisation media, each consisting of MS basal components supplemented with sucrose (8% w/v) and various regulator substances. The control was a regulator-free medium including only sucrose. Results were expressed as the number and weight of microtubers per nodal explant.
The cultivars showed wide variations in the mean weight of microtubers, ranging from 44.6 mg (Primura) to 77.5 mg (Spunta), and nearly all plants produced tubers. Medium containing activated charcoal gave the highest rate of tuberisation and the largest microtubers. It thus played a role in optimising conditions for rapid, mass tuberisation of these cultivars, and produced large microtubers for field planting.     

Histological analysis of the maturation of native and wound periderm in potato (Solanum tuberosum L.) Tuber

Maturation of potato (Solanum tuberosum L.) tuber native and wound periderm and development of resistance to periderm abrasion were investigated utilizing cytological and histochemical techniques. Both native and wound periderm consist of three different tissues: phellem, phellogen and phelloderm. It was previously determined that the phellogen walls of immature native periderm are thin and prone to fracture during harvest, leading to periderm abrasion (excoriation). Phellogen walls thicken and become less susceptible to fracture upon maturation of the periderm, leading to resistance to excoriation. We now demonstrate that phellogen cells of immature wound periderm also have thin radial walls and that wound periderm abrasion is due to fracture of these walls. Maturation of the wound periderm is also associated with an increase in the thickness of the phellogen radial walls. Histological analysis with ruthenium red and hydroxylamine-FeCI2, which stain unesterified and highly methyl-esterified pectins, respectively, indicates that the phellogen cell walls of native and wound periderm differ significantly regardless of the stage of maturity. Results obtained by staining with ruthenium red and hydroxylamine-FeCI2 imply that phellogen cell walls of immature native periderm contain methyl-esterified pectin, but are lacking in unesterified (acidic) pectins. Maturation of native periderm is accompanied by an apparent increase in unesterified pectins in the walls of phellogen cells, which may allow for the strengthening of phellogen cell walls via calcium pectate formation. Histological staining of the phellogen walls of wound periderm, on the other hand, implies that these walls are deficient in pectins. Moreover, maturation of wound periderm is not accompanied by an increase in unesterified pectins in these walls. Since peroxidase is known to catalyse the cross-linking of cell wall polymers, we stained native and wound periderm for the presence of peroxidase utilizing guaiacol as a substrate. Peroxidase staining was strong in the phellogen walls of both immature and mature native periderm and we could not detect any differences in staining between them. Peroxidase staining was weak in the phellogen walls of immature wound periderm and was not detectably different in mature wound periderm. Peroxidase data imply that there are distinct differences between native and wound periderm, though our data do not indicate that changes in peroxidase activity are involved in the development of resistance to periderm abrasion that occurs upon maturation of the periderm. However, we cannot rule out the involvement in this process of peroxidase isozymes that have low affinity for the substrates utilized here.     

转BADH基因马铃薯无性繁殖二代耐盐性检测

在0%、0.3%、0.6%NaCl胁迫下,检测转BADH基因马铃薯及其受体亲本‘甘农薯2号’的盆栽植株耐盐性的结果表明:BADH基因的遗传性稳定且转基因植株的耐盐性比非转基因的强。     

Effect of chlorocholine chloride sprays on the carbohydrate composition and activities of sucrose metabolising enzymes in potato (Solanum tuberosum L.)

Chlorocholine chloride (CCC) was sprayed on a potato crop 25 days after sowing (DAS) at 5 day intervals for a total of 7 sprays. Activity of sucrose synthase (SS) in the sucrose cleavage direction was many fold higher than that of acid invertase in all the tissues. The activity of alkaline invertase was negligible. A sharp decline in the starch content of stolons of the CCC-sprayed crop was observed between 60 DAS and 70 DAS. This could divert the carbon towards tubers and thus enhancing its availability for starch synthesis. The CCC-treated crop, in general, had higher SS (cleavage) activity in stem, stolons and tubers. A higher sucrose content in the stem of the CCC-treated crop could be due to the high sucrose phosphate synthase (SPS) activity observed in this plant part. In tubers of CCC-treated crops a higher SS (cleavage) activity along with a high sucrose content in tubers during the active tuber filling stage could lead to better availability of UDP-glucose for its conversion to glucose-1-phosphate, which could enter into the amyloplast leading to higher starch content. High SPS activity in tubers of CCC-treated plants ensures that reducing sugars formed are reconverted efficiently to sucrose. The efficiency of developing tubers from CCC-sprayed plants to convert 14C sucrose fed through stolons into starch was about 2.5 times more than in the control.     

Biochemical and bioassay analysis of resistance of potato (Solanum tuberosum L.) cultivars to attack by the slug Deroceras reticulatum (Muller)

A method is described for using young field slugs Deroceras reticulatum (Muller) in a bioassay study of biochemical resistance of potato (Solanum tuberosum L.) cultivars to slugs. Tuber parts or an artificial diet were provided as food sources. Comparisons were made of feeding, survival and weight gain between the susceptible cultivar Maris Piper and the resistant cultivar Pentland Dell. Biochemical analyses were made of these two cultivars and the resistant cultivars Stormont Enterprise and Majestic. Comparisons of tuber sections and peelings as food sources indicated factors affecting growth were located in the surface layers of the tubers. Phenolics and glycoalkaloids were concentrated in the surface layers but the amounts were similar in the susceptible and resistant cultivars and the bioassays indicated that neither acting alone could explain resistance. The amounts and distribution of free amino acids also did not correlate with resistance although when supplied in the artificial diet they partly inhibited feeding. Proteinaceous inhibitors of slug gut proteolytic enzymes were present throughout the tubers but were not concentrated in the surface layers and the amounts were similar in the different cultivars thus they too did not explain the difference in susceptibility between the cultivars. Bioassays using acetone extracts (low molecular weight substances) and acetone powders (high molecular weight substances) either alone or in combination indicated that the resistant cultivar Pentland Dell contained a high molecular weight substance which together with a low molecular weight substance from either the same cultivar or the susceptible Maris Piper could confer resistance. Bioassays using protein extracts supplied in the presence or absence of chlorogenic acid indicated that this mechanism could comprise enzymic oxidation of phenolics. Assays of phenolase confirmed this since activity was highest in the outer layers of the tubers and was highest in the three resistant cultivars. Thus the chief resistance factor identified was high phenolase activity acting rapidly on phenolics when the slug first bites the tuber surface. The quantity of phenolics per se did not control the resistance. Thus while phenolics must be available, resistance is compatible with low blackening on cutting the tuber.     

Pathways of starch and sucrose biosynthesis in developing tubers of potato (Solanum tuberosum L.) and seeds of faba bean (Vicia faba L.)

Tissue slices from developing potato tubers (Solanum tuberosum L.) and developing cotyledons of faba bean (Vicia faba L.) were incubated with specifically labelled [¹³C]glucose and [¹³C]ribose. Enriched[¹³C]glucose released from starch granules was analysed by nuclear magnetic resonance (NMR). Spectral analyses were also performed on sucrose purified by high-performance liquid chromatography. In both tissues a low degree of randomisation (< 11 % in potato and < 14% in Vicia) was observed between carbon positions 1 and 6 in glucose released from starch when material was incubated with [¹³C]glucose labelled in positions 6 and 1, respectively. Similarly, with [2-¹³C]glucose a low degree of randomisation was observed in position 5. These findings indicate that extensive transport of three-carbon compounds across the amyloplast membrane does not occur in storage organs of either species. This is in agreement with previously published data which indicates that sixcarbon compounds are transported into the plastids during active starch synthesis. When [1-¹³C]ribose was used as a substrate, ¹³C-NMR spectra of starch indicated the operation of a classical pentose-phosphate pathway. However, with [2-¹³C]glucose there was no preferential enrichment in either carbon positions 1 or 3 relative to 4 or 6 of sucrose and starch (glucose). This provides evidence that entry of glucose in this pathway may be restricted in vivo. In both faba bean and potato the distribution of isotope between glucosyl and fructosyl moieties of sucrose approximated 50%. The degree of randomisation within glucosyl and fructosyl moieties ranged between 11 and 19.5%, indicating extensive recycling of triose phosphates.Abbreviation NMR nuclear magnetic resonance We are grateful to Dr. George Ratcliffe for his critical reading of the text and Dr. Bernard Goodman for helpful suggestions on the NMR measurements. The research was funded by a European Economic Community research grant, which the authors duly acknowledge.     

Patatin,a major soluble protein of the potato (Solanum tuberosum L.) tuber is synthesized as a larger precursor

Antibodies were raised against the highly purified glycoprotein patatin. They were used to characterize the product synthesized in a wheatgerm cell-free translation system, programmed with polyadenylated RNA from potato tubers. Sodium dodecylsulfate-polyacrylamide gel electrophoresis revealed that the immunoprecipitated protein had a molecular mass of 43 kDa compared to 40 for the authentic patatin. It is assumed that patatin is synthesized in vivo as a larger precursor which is processed to the mature protein by cleavage of a signal peptide. Our results are in agreement with sequence-analysis data of patatin complementary DNA which indicate a signal peptide of about 23 amino acids (Mignery et al., 1984; Nucleic Acids Res. 12, 7987–8000).Abbreviation Poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis Preliminary results were published in Mitteilungsband, Botaniker Tagung in Wien, p. 180, Wien, September 1984     

Jasmonate effects on in vitro tuberization and tuber bulking in two potato cultivars (Solanum tuberosum L.) under different media and photoperiod conditions

Summary Jasmonic acid (JA) effects on in vitro tuberization of potato nodal explants cvs. Sangre and Russet Burbank were tested under liquid and solid media conditions and 0,8, and 16h photoperiod. Explants taken from stock plants grown on 2.5μM JA-supplemented medium tuberized first, particularly in darkness. The most pronounced benefits of the JA pretreatment were recorded under 16h photoperiod, which is known to inhibit tuberization. Cultivar Sangre benefited from the JA preconditioning of stock plants more than Russet Burbank. Russet Burbank required the JA supplement in tuberization media to reach the same degree of stimulation. Overall, microtubers produced either from JA preconditioned stock plants or on the JA-containing tuberization media were more uniform and larger than from other treatments. Eight hours photoperiod was by far the best treatment for the production of high-quality uniform microtubers. JA conditioning of stock plants prior to taking explants for tuberization is being proposed as a treatment enhancing the quality of microtubers.     

Effects of plant genotype and growth stage on the structure of bacterial communities associated with potato (Solanum tuberosum L.)

The effects of genotype, plant growth and experimental factors (soil and year) on potato-associated bacterial communities were studied. Cultivars Achirana Inta, Désirée, Merkur and transgenic Désirée line DL12 (containing T4 lysozyme gene) were assessed in two field experiments. Cross-comparisons between both experiments were made using Désirée plants. Culture-dependent and -independent approaches were used to demonstrate effects on total bacterial, actinobacterial and Pseudomonas communities in bulk and rhizosphere soils and endospheres. PCR-denaturing gradient gel electrophoresis fingerprints prepared with group-specific primers were analyzed using multivariate analyses and revealed that bacterial communities in Achirana Inta plants differed most from those of Désirée and Merkur. No significant effects were found between Désirée and DL12 lines. Plant growth stage strongly affected different plant-associated communities in both experiments. To investigate the effect of plant-associated communities on plant health, 800 isolates from rhizospheres and endospheres at the flowering stage were tested for suppression of Ralstonia solanacearum biovar 2 and/or Rhizoctonia solani AG3. A group of isolates closely resembling Lysobacter sp. dominated in young plants. Its prevalence was affected by plant growth stage and experiment rather than by plant genotype. It was concluded that plant growth stage overwhelmed any effect of plant genotype on the bacterial communities associated with potato.     

Expression of the IL-2 gene in the potato (Solanum tuberosum cv. Superior)

We intended to examine the expression of the T-cell growth factor (human interleukin-2) so that a binary vector, pSSK-1, carrying the IL-2 gene was constructed and transferred intoA. tumefaciens for the purpose of the transformation of the potato (Solanum tuberosum cv. Superior). All of theAgrobacterium-infected potato explants were regenerated to shoots in modified MS medium and 81% of them rooted on the medium containing kanamycin (200 mg/L). Southern and Northern analysis were performed to verify the transformation events. EL-ISA test indicated that IL-2 protein was produced from IL-2-transformed potatoes. These results suggested expression and production of the IL-2 protein from the transgenic potato.     

Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro

We have examined the ability of a carrot (Daucus carota L.) heat shock protein gene encoding HSP17.7 (DcHSP17.7) to confer enhanced heat tolerance to potato (Solanum tuberosum L.), a cool-season crop. The DcHSP17.7 gene was fused to a 6XHistidine (His) tag to distinguish the engineered protein from endogenous potato proteins and was introduced into the potato cultivar 'Désirée' under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Western analysis showed that engineered DcHSP17.7 was constitutively, but not abundantly, expressed in transgenic potato lines before heat stress. Leaves from multiple regenerated potato lines that contain the transgene exhibited significantly improved cellular membrane stability at high temperatures, compared with wild-type and vector control plants. Transgenic potato lines also exhibited enhanced tuberization in vitro: under a condition of constant heat stress, at 29 degrees C, nodal sections of the transgenic lines produced larger and heavier microtubers at higher rates, compared to the wild type and vector controls. The dry weight and percentages of microtubers that were longer than 5 mm were up to three times higher in the transgenic lines. Our results suggest that constitutive expression of carrot HSP17.7 can enhance thermotolerance in transgenic potato plants. To our knowledge, this is the first study that shows that the thermotolerance of potato can be enhanced through gene transfer.     

The effect of the recombinant human interleukin-2 Gene in potato (Solanum tuberosum cv. superior)

To examine the effect of the T-cell growth factor (human interleukin-2), we constructed a binary vector, pSSK-1, carrying the recombinant human interleukin-2 (rhlL-2) gene, and transferred it intoAsrobacterium tumefaciens. Using this construct, we then transformed potato explants(Solanum tuberosum cv. Superior), achieving 100% regeneration of shoots on a modified MS medium. Of the putative transformed shoots, 81% rooted and were selected on 200 ms/L kanamycin. Both Southern and northern analyses verified the transformation events. An ELISA test also indicated that the rhlL-2 protein was produced from rhlL-2-transformed potatoes. To determine whether this protein was biologically active in the potato cells, we performed a biological assay using the 11.-2 dependent cell line, CTLL-2. The suspension containing extract from the transformants showed significant proliferation of the 11.-2 dependent CTLL-2 cells, whereas cells did not proliferate in the nontransformed potato. We then grew the verified rhlL-2 transgenic potatoes in soil, and compared their performance with that of nontransgenic potatoes as well as those that had been transformed with GUS. Growth rates, as calculated from plant heights, were up to 50% higher than for either the nontrans-genic or the GUS-transformed potatoes. Similar patterns were found withArabidopsis thaliana plants treated in the same manner. All of these results suggest that rhlLo2 may function as a growth factor in potato.     

Effect of physiological age of mother tuber and number of subcultures on in vitro tuberisation of potato (Solanum tuberosum L.)

The physiological age of mother tubers (Solanum tuberosum L. cv. Kennebec) used as a source of material influenced kinetin-induced in vitro tuberisation. Tuberisation significantly increased with physiological age. Kinetin- or ancymidol-induced tuberisation, plantlet and microtuber dry weight decreased with increasing number of subcultures. Single-node segments obtained from tubers stored for more than 9.5 months at 4 °C showed increased kinetin-induced tuberisation rates and earlier tuberisation than those obtained from younger tubers. For any physiological age, material may be safely multiplied using node propagation until the third subculture and bioassayed for tuberisation without variation in the response. Received: 8 July 1996 / Revision received: 28 March 1998 / Accepted: 28 March 1998     

Effects of Physiological Changes in Potato Tubers (Solanum tuberosum L.) after Low Temperature Storage on the Level of Acrylamide Formed in Potato Chips

Acrylamide in potato chips made from tubers stored at 2 or 20°C for two weeks after harvest was analyzed by GC-MS. The acrylamide level in the former chips was higher than ten times of that in the latter, which was highly correlated with both glucose and fructose levels in the tubers.